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dissertation entitled

HUMAN B-MANNOSIDASE: cDNA CHARACTERIZATION AND
IDENTIFICATION OF MUTATIONS ASSOCIATED WITH 8--
MANNOSIDOSIS

presented by

Aisha Hassan Alkhayat

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HUMAN B-MANNOSIDASE: cDNA CHARACTERIZATION AND
IDENTIFICATION OF MUTATIONS ASSOCIATED WITH B-
MANNOSIDOSIS
By

Aisha Hassan Alkhayat

A DISSERTATION

Submitted to
Michigan State University
in partial fiJlfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Genetics Program

1997

ABSTRACT

HUMAN B-MANNOSIDASE: cDNA CHARACTERIZATION AND
IDENTIFICATION OF MUTATIONS ASSOCIATED WITH B-MANNOSIDOSIS

By

Aisha Hassan Alkhayat

Human B-mannosidosis is an autosomal recessive lysosomal storage disease. The
disorder is caused by the deficiency of the lysosomal enzyme B-mannosidase. The
enzyme is required for the hydrolysis of B-mannoside linkage in the core oligosaccharide
moiety of glycoproteins. The disease clinical manifestations in human are heterogeneous,
with varying degree of neurological deterioration, hearing loss and dysmorphic features.

In order to understand the molecular mechanisms underlying the phenotypic
differences caused by the enzyme deficiency, identification and characterization of the
human B-mannosidase gene and the mutations associated with the enzyme deficiency are
required.

To identify the human B-mannosidase cDNA, the following methods were
performed: cDNA library screening, Reverse Transcription-Ploymerase Chain Reaction,
5’ and 3' Rapid Amplification of cDNA Ends. The consensus human cDNA sequence is
3300 base-pair, consisting of 87 nucleotides 5’untranslated region, 2640 nucleotides
coding region and 573 3’untranslated region. Multiple tissue Northern blot analysis
revealed a unique 3.7 kb transcript that is differentially expressed in pancreas, placenta,
kidney, liver, lung, brain, heart, and muscle. The gene was localized to human

chromosome 4q22-25 using PCR.

The long and accurate PCR method using high fidelity enzyme mixture was used
to amplify the coding region. The RT-PCR product was cloned into pCRII vector. Three
independent clones were isolated and characterized.

Two cell lines from patients affected with B-mannosidosis were obtained for
mutation analysis: the first is from patient from The United States of America whose
parents are from white European ancestry and the second from a gypsy family from the
Czech Republic. Chromosomal and gene mutation analysis for the first cell line by
karyotype analysis, microsatellite markers analysis, RT-PCR, and cDNA sequencing
identified G—>A transition in the coding region at position 1586. However, a diagnostic
AIRS test (Artificial Introduction of Restriction Site) revealed that the patient is
heteroallelic at B-mannosidase locus for the identified G1586A allele.

Mutation analysis for the second the cell line by RT-PCR, cDNA and genomic
DNA sequencing identified an A—>G transition at position -2 of the 3’ splice acceptor
site. The mutation causes utilization of cryptic site, exon skipping and creates a SmaI
restriction site allowing for a diagnostic test for the identified mutant allele. The two

affected siblings from this family were homozygous for the detected mutation.

This work is dedicated to my family especially my father and mother for their unlimited
love and support for me and for my brothers and sisters, for my sister for her courage in
her fight against illness and for my brother for his perseverance.

ACKNOWLEDGMENTS

It gives me a great pleasure to be able to express my feelings of thanks and
gratitiude to those who supported, advised and helped me through my graduate
study in Michigan State University. First, I would like to thank my government
who provided me with the oppurtunity to fulfill my dream and goal to train to be a
scientist. Their trust and provision of moral and finantial support were crutail for
the sucsessfiil completion of this work.

Throughout this work I have been lucky to work with many people that had
a great deal of influence on my life in general and my education in particular. My
mentor, Dr. Karen Friderici, is one of these people whom I will always
remember. I am grateful for her guidence, suppot and help during my studies
which made my stay in this country enjoyable.

I would like to thanks to my Ph.D committee members, Dr. Rachel Fisher,
Dr. Patrick Venta, and Dr. John Fyfe. Their help and guidance especially in
difficult times is very much appreciated.

My sincere thanks to Dr. Margaret Jones and her laboratory staff members.

Her support and encouragement was endless during my graduate studies in the

ii

Department of Pathology. I will always be in debt to Jeff Leipprandt for sharing
his research experience with me especially at the begining of my research project.
I also want to thank Stacey Kreamer for her advice and insights for the research
project and for her remarkable efforts in helping to edit my dissertation.

I want to thank my friends and colleagues, Mark Tway, Eric Olle, Julie
Horvath, Ribka Badilu, and Tim Tesmer for their help and support. Special thanks
to Eric Olle for intellectual stimulation and interesting scientific conversations.

At the end my thanks to my family and friends who were always there for

me when I needed them.

iii

TABLE OF CONTENTS

LIST OF TABLES .............................................................................. vi
LIST OF FIGURES .............................................................................. vii
CHAPTER 1
LITERATURE REVIEW ..................................................................... 1
Introduction .............................................................................. 1
Lysosomal Storage Diseases ................................................... 3
Glycoprotein Catabolism Disorders ............................ 7
Lysosomal Enzyme ................................................................. 8
Transport of Lysosomal Enzymes ................................ 12
B-Mannosidosis ........................................................................ 13
Identification ................................................................. 13
Clinical Cases ................................................................ 15
Disease Pathology ......................................................... 21
Storage Product .............................................................. 23
The Enzyme B-Mannosidase ...................................................... 24
Human Gene Mutations ............................................................ 27
Definition and Relevance to Disease Pathogenesis ........ 27
Genotype-Phenotype Correlation ................................... 30
Types of Mutation Causing Genetic Disease ................. 32
CHAPTER 2
CHARACTERIZATION OF HUMAN B-MANNOSIDASE .............. 35
Introduction ................................................................................ 3 5
Materials and Methods .............................................................. 37
cDNA Cloning ............................................................... 37
RT-PCR .......................................................................... 39
5'Rapid Amplification of cDNA Ends .......................... 40
Northern Blot Analysis .................................................. 40
Chromosomal Localization ............................................ 42
3'Rapid Amplification of cDNA Ends .......................... 42

iv

Results ............................................................................................................ 44

Human B-Mannosidase cDNA Sequence ............................... 44
Expression Analysis ................................................................ 49
Chromosomal Assignment and Regional Localization of
Human B-mannosidase ............................................................ 49
Discussion ........................................................................................... 55
CHAPTER 3
PRODUCTION OF FULL LENGTH cDNA CLONE ................................. 57
Introduction ......................................................................................... 57
Materials and Methods ........................................................................ 60
cDNA Synthesis/Primer Extention ......................................... 60
PCR Cloning of the Full Length RT-PCR Product ................ 65
Results ................................................................................................. 69
Discussion ........................................................................................... 77
CHAPTER 4
MUTATION ANALYSIS OF TWO B-MANNOSIDOSIS PATIENTS ...... 79
Introduction ......................................................................................... 79
Materials and Methods ........................................................................ 81
Mutation Analysis of the Wenger Cell Line ........................... 81
Mutation Analysis of the Kleijer Cell Line ............................. 86
Results .................................................................................................. 88
Discussion ........................................................................................... 98
SUMMARY AND CONCLUSIONS .............................................................. 101
BIBLIOGRAPHY ............................................................................................. 103

Table 1-

Table 2-

Table 3-

Table 4-

Table 5-

Table 6-

LIST OF TABLES

Infmtile Early Onset .................................................................. 17
Cases of Infmtile Early Onset with Relatively early onset ...... l9
Juvenile Onset ........................................................................... 22
Primers for PCR Amplification ................................................. 38
Size Fractionation Results ........................................................ 62
Microsatellite Markers .............................................................. 82

vi

Figure 1-
Figure 2-

Figure 3-

Figure 4-
Figure 5-
Figure 6

Figure 7

Figure 8

Figure 9
Figure 10

Figure 11

LIST OF FIGURES

Pathway for the catabolism of Asn-linked oligosaccharide ..... 9
Sequencing strategy .................................................................. 41

Human B-mannosidase cDNA sequence and predicted

protein translation ...................................................................... 46
Northern blot analysis ............................................................... 52
Chromosomal localization ....................................................... 54
cDNA clones analysis ............................................................... 72

Sequence alignment of the cDNA clones to the 5' end of
human B-mannosidase consensus sequence ............................. 74

Sequence alignment of the cDNA clones to the 3'end of

human B-mannosidase cDNA consensus sequence ................. 76
ClaI AIRS test for the Wenger cell line ................................... 90
Mutation analysis for the Kleijer cell line ............................... 95
Mutation sequence analysis ..................................................... 97

vii

CHAPTER 1
LITERATURE REVIEW

Introduction

Lysosomal storage diseases are a group of inborn errors of metabolism
caused by deficient activity of specific lysosomal enzymes. The metabolic lesions
allow incompletely degraded macromolecules to accumulate in the lysosomes, which
enlarge and perturb the physiology of the cell. The defects affect glycosidases, lipid-
degrading hydrolases and sulphatases, so that complex carbohydrates and lipids
accumulate.

The study of lysosomal storage diseases began in the clinical area with classic
descriptions by Warren Tay (1881) (Tay, 1881), and B. Sachs (1887) (Sachs, 1887),
P.C.E Gaucher (1882) (Gaucher, 1882), and Gerturd Hurler (1919) (Hurler, 1919).
Classic histopathological studies showed cellular inclusions, and that the organs
contained increased amounts of lipids and carbohydrates. The concept of lysosomal
storage diseases was developed by Hers (1965, 1973) (Hers, 1973; Hers, 1965) on
the basis of de Duve’s studies on the biochemistry of lysosomes (de Duve and
Wattiaux, 1966).

More than thirty lysosomal storage diseases have been described and

2

intensive research has been done on the clinical, pathological, biochemical, genetic
and molecular aspects of individual enzyme deficiencies of most of these disorders.
The study of lysosomal enzymes has contributed to the understanding of the normal
processing and cellular roles of the enzymes. Furthermore, the study of the
biochemical and the molecular aspects of the defective enzymes helped us to
understand the basis for the heterogeneity of the clinical manifestation of most
lysosomal storage diseases (Scriver et al., 1995; Watts and Gibbs, 1986;
Conzelmann and Sandhoff, 1987; Gieselmann, 1995; Neufeld. 1991).

The deficiency of the lysosomal B-mannosidase was first discovered in goats
in 1979, (Jones and Dawson, 1981; Healy et al., 1981; Jones and Laine, 1981;
Hartley and Blakemore, 1973) in humans in 1986 (Cooper et al., 1986; Wenger et
al., 1986), and in Salers cattle in 1991 (Jolly et al., 1991; Bryan et al., 1990; Abbitt et
al., 1991). Detailed clinical description of the caprine B-mannosidosis with
histological and biochemical characterization of the enzyme deficiency in various
tissues have been published (Patterson et al., 1991; Lovell et al., 1991; Lovell and
Boyer, 1987; Boyer and Lovell, 1990; Boyer et al., 1990; Lovell and Jones, 1983;
Lovell and Jones, 1985). The caprine and bovine B-mannosidase enzymes have been
purified and characterized (Sopher et al., 1993; Sopher et al., 1992), followed by
cloning and characterization of the cDNA for the enzyme for both species

(Leipprandt et al., 1996; Chen et al., 1995).

3

The human B-mannosidosis cases described so far have a milder disease
manifestation than the ruminant counterpart, however, among these cases there is a
considerable clinical phenotypic variation. To better understand the molecular basis
of this heterogeneity, this research is directed towards the molecular cloning and
characterization of the human B-mannosidase cDNA, tissue specific distribution of
B-mannosidase transcript, and the identification of the mutation causing the disease

in two reported cases (Kleijer et al., 1990; Wenger et al., 1986).

Lysosomal Storage Diseases

The inherited lysosomal storage diseases are caused by deficient activity of
specific lysosomal enzymes. These metabolic lesions allow undegraded and
incompletely degraded macro molecules to accumulate in the lysosomes, causing
progressive increase in the size and number of these organelles. The cellular
pathology will eventually lead to malfunction of the afffected organ (Watts and
Gibbs, 1986). Most of the lysosomal enzymes are exohydrolases acting in sequence,
such that substrates are degraded by stepwise removal of terminal residues. Thus the
deficiency of a single enzyme causes the blockage of the entire pathway, since the
failure to remove terminal residues makes the substrate inaccessible for further
hydrolysis by other lysosomal enzymes (Holtzman, 1989; Conzelmann and

Sandhoff, 1987; Neufeld, 1991; Gieselmann, 1995).

4

The lysosomal storage diseases are classified according to the pathway
affected and the nature of the accumulated substrate. The six main groups of the
inherited lysosomal storage diseases are sphingolipidoses, mucopolysaccharidoses,
glycoproteinoses, acid lipase deficiency, glycogenosis II, and mucolipidoses.
However, there are frequent overlaps of storage material between these groups.
Many lysosomal hydrolases are not specific for a particular compound, but rather for
a terminal residue and linkage which may be identical in different classes of
molecules, for example, gangliosides and glycoprotein, so that both compounds
accumulate when a commonly required enzyme is deficient (Thomas and Beaudet,
1995).

There are more than 30 lysosomal storage diseases and they occur at a rate of
less than 5 per 10,000 births (Watts and Gibbs, 1986). Only rough estimates of the
incidence of any one of the lysosomal storage disease are available. Some of these
disorders are found in certain racial or geographically defined groups. This is
generally thought to reflect heterozygous advantage or a founder effect. Lysosomal
storage diseases are inherited as autosomal recessive traits except for X-linked Fabry
and Hunter (mucopolysaccharidosis ll) disease, and they manifest with considerable
phenotypic and genetic heterogeneity within each disorder.

Most diseases vary with respect to age at onset and progression of symptoms.
The later a disease begins, the more protracted is the development of the symptoms.

Frequently, three different types of disease are distinguished: severe infantile,

5

intermediate juvenile and mild adult forms (Gieselmann, 1995). Although such
classification is useful, it should be realized that the spectrum of clinical severity is a
continuum, and such imposed distinctions are sometimes artificial. Phenotypic
differences between individual patients with the same inborn error of metabolism
become increasingly apparent when a disease is studied in depth. Biochemical and
genetic studies have indicated substantial heterogeneity within each disorder, and
studies of biosynthesis and maturation of the enzymes have provided information on
the molecular basis of this heterogeneity.

The genetic defects leading to deficiency of lysosomal enzymes can be
attributed to the following factors: (1) multiple allelism at the gene locus, (2)
mutations at different gene loci affecting the synthesis of different polypeptide
chains in a single enzyme protein, (3) mutations at different gene loci affecting
different proteins with similar catalytic properties, (4) mutations at loci affecting the
synthesis of protein which either activate the enzyme or its substrate, or protect the
enzyme from abnormal or excessively rapid catabolism or inactivation in vivo, (5)
mutations in non-lysosomal enzymes involved in posttranslational processing.

Study of the lysosomal storage diseases reveal examples from all of the five
genetic factors which relate to specific abnormal gene product. Multiple allelism is
exemplified by a-L-iduronidase deficiency, Hurler, Scheie, and Hurler/Scheie
diseases. The Tay-Sachs and Sandhoff diseases types of GM; gangliosidosis

exemplify variants due to mutations at two loci directing the synthesis of a- and B-

6

chains of the B-hexosaminidase variants. The four different types of Sanfilippo
disease demonstrate how different enzyme lesions produce different biochemical
effects and introduce genetic variation into what appeared to be a single entity on
clinical grounds. The combined defeciency of fi-galactosidase and sialidase, known
as galactosialidosis, is an example of a deficiency in another enzyme that is involved
in stabilization of B-galactosidase and in the activation of sialidase. The I-cell
disease and the pseudoHurler polydystrophy are examples of lysosomal enzyme
deficiency due to a primary deficiency in a non-lysosomal enzyme involved in
posttranslational modification of lysosomal enzymes (reviewed in details in the The
Metabolic and the Molecular Basis of Inherited Disease and in Lysosomal Storage
Diseases: Biochemical and Molecular Aspects) (Watts and Gibbs, 1986; Scriver et
aL,l995)

The phenotypic effects of each mutation can be expressed at the both the
mRNA and the protein levels. Mutations that affect the mRNA stability can
eventually lead to a reduction or absence of detectable precursor protein. On the
protein level, the precursor enzymes can be inactive, retained in either ER or Golgi,
have altered physiochemical and enzymological properties, susceptible to premature
proteolytic degradation, or fail to be glycosylated as have been shown from the study

of mutant lysosomal enzymes.

Glycoprotein Disorders

B-Mannosidosis is a lysosomal storage disease that results from a deficiency
of the lysosomal enzyme B-mannosidase. The enzyme hydrolyses the B-mannoside
linkage which is rare in mammalian systems except at the interbranching core of the
N-linked oligosaccharide of the both acidic and neutral glycoproteins (Dawson.
1987). The disease, together with a-mannosidosis, fiIcosidosis, sialidosis,
aspartylglucosaminuria and carbohydrate deficient glycoprotein syndrome are
classified as disorders of glycoprotein degradation (Thomas and Beaudet, 1995).

Glycoproteins are found within cells and on cell surfaces; therefore, normal
cellular metabolism and turnover involves the degradation of large amounts of this
material. There is considerable cellular and biochemical evidence indicating that this
catabolism normally occurs completely inside the lysosomes of a wide variety of cell
types. As illustrated by disorders resulting from abnormanl glycoprotein catabolism,
an interruption or blockage at any step in the degradation of the glycan portions of
the glycoproteins can cause lysosomal alterations resulting in a cellular pathology
and ultimately, clinical disease (Conzelmann and Sandhoff, 1987).

The catabolism of glycoproteins is accomplished by proteases and
glycosidases. The protein backbone of the glycoprotein is degraded from both ends
and internal points by a series of a lysosomal proteases and peptidases. The

degradation of the oligosaccharide portion of the molecules is accomplished by the

8

ordered removal of sugars from the nonreducing ends and, to a lesser extent,
cleavages at the reducing end of the Asn-linked oligosaccharide. Cleavage at the
nonreducing termini of the oligosaccharide is carried out by a number of
exoglycosidases catalyzing the sequential removal of single sugars from
oligosaccharide branches. The lysosomal enzymes involved in these steps include
neuraminidase (sialidase), B-galactosidase, B-N—acetylhexosaminidase, B-
mannosidase, a-mannosidase, Ot-fucosidase, Figure l (Conzelmann and Sandhoff,
1987; Scriver et al., 1995; Aronson and Kuranda, 1989).

The stored material in these lysosomal diseases appear to be products of
incomplete degradation of known oligosaccharide structure and are compatible with
the metabolites one would expect to result from specific enzyme deficiencies.
Almost all of the storage disease structures are known to occur within typical high—
mannose or complex glycoproteins. Whereas mannosidosis and sialidosis patients
have alterations in oligosaccharides and aspartylglucosaminuria patients accumulate
only glycopeptides, both oligosaccharides and glycopeptides accumulate in the
tissues and fluids of fiIcosidosis patients (Watts and Gibbs, 1986).

Lysosomal Enzymes

Lysosomal hydrolases are enzymes that function optimally in the acid
environment of the lysosomes, and generally are referred to as acid hydrolases.
Collectively, these enzymes are capable of degrading virtually all large cellular

molecules such as nucleic acids, proteins, polysaccharides and lipids. Most

SA SA
a 2,3 or 6 ¢ <' a-Neuraminidase > ¢ 0‘ 2’3 or 6
Gal Gal
[31’4 ¢<r B-Galactosidase >¢ [31,4
GlcNAc GlcNAc
[51,2 “ B-Hexosaminidase > ¢ [512
A or B
Man Man

(lg/[annosidzfi
at 1,3 a 1,6

l 4
iGlcNAc 75—» Man .
[3 1,4* <— B-MannOSIdase

B-Hexosaminidase

A or B
GlCNAC Endo-B-N—Acetyl-

B 1’4 ‘ Glucosaminidase or
B-Hexosaminidase A or B

a 1,6
iFUC WGICNAC
G-Fucosidase B ‘ ‘—AspartylglycosammIdase
—— Asn __

Figure 1 -Pathway for the catabolism of Asn-linked oligosaccharides. The
figure illustrates the enzymes involved in the hydrolysis of spesific. linkages in the
Asn-linked oligosaccharide moiety of glycoprotein.

10

lysosomal enzymes are glycoproteins that have relatively long half-lives and are of
low abundance. Half-lives of a day to a week or so are typical, although some
variation exists between different cell types. The mRNAs thus far identified are also
of low abundance (Holtzman, 1989).Most lysosomal enzymes are processed
essentially as secretory glycoproteins. The hydrolases are synthesized on ribosomes
of the rough endoplasmic reticulum (ER), enter the ER , and pass through the Golgi
apparatus. The signal peptide, a stretch of 15- 30 mainly hydrophobic amino acids
that are present at the N-terminus, directs the nascent polypeptide to the ER (Von
F igura and Hasilik, 1986). This signal polypeptide is cleaved upon the entry of the
polypeptide into the ER. As the nascent polypeptide passes into the ER, precursor
oligosaccharides are attached to the amino groups terminating the side chains of
asparagines found in an Asn-X-Ser/Thr sequence motif; however additional factors,
such as availability of completely assembled and glycosylated lipid-linked
oligosaccharide donor, adequate activity of oligosaccharyltransferase, and a properly
oriented and accessible Asn-X-Ser/T hr sequence in the polypeptide influence
glycosylation (Komfeld and Komfeld, 1985).

Further processing, trimming of the oligosaccharide chains of the
glycoproteins, is done in a series of steps that terminate at different points for
different proteins to produce the high mannose form of the oligosaccharides. Further
trimming and addition to the oligosaccharides of the glycoproteins are carried out by

specific enzymes in the Golgi apparatus to produce the complex oligosaccharides

11

found on many secretory proteins and plasma membrane proteins (Komfeld and
Komfeld, 1985).

Two features of the oligosaccharides moieties of the lysosomal hydrolases are
specific to them. First, the collection of oligosaccharides found on hydrolases
produced by a given cell population often includes some chains of a high mannose-
type, some complex chains, and some of a hybrid variety. Second, lysosomal
hydrolases’ oligosaccharide moieties are unique in their content of mannose-6-
phosphate (M6P) that is added in the Golgi apparatus by a series of enzymatic steps.
This latter characteristic is important for targeting of the enzymes to the lysosomes
(Komfeld and Komfeld, 1985; Von Figura and Hasilik, 1986; Holtzman, 1989).

In addition to the removal of the signal peptide in the ER, the newly
synthesized protein may be subject to additional limited proteolysis during transport
or, more often, afier they have reached the lysosomes. Mature polypeptides found in
lysosomes are smaller than precursors detected in ER and Golgi apparatus.
Proteolytic processing may involve peptide cleavage at the amino- and the carboxy-
terminus, as well as intemal proteolytic cleavage. This process, often called
maturation, is generally deduced from changes in migration in SDS-polyacrylamide
gels under reducing conditions (Holtzman, 1989; Neufeld, 1991). In general, the role
of proteolysis is believed to be associated with protecting cells from undesirable
effects of proteolysis, thus the full activation of proteases is only reached in the

lysosomes. In addition, some lysosomal enyzmes mature into several smaller

12

peptides, suggesting that the precursor form may be important for folding, stability,

or sorting the proteins during their early lives (Hasilik and Neufeld, 1980).

Transport of Lysosomal Enzymes

The efficient delivery of lysosomal enzymes is achieved mainly by mannose-
6-phosphate (M6P) receptor-mediated transport. Phosphorylation of the mannoses in
the newly synthesized proteins is catalyzed by a series of enzymatic reactions that
takes place in the Golgi apparatus. The M6P is the signal that deviates the lysosomal
enzymes from the rest of the secretory glycoproteins. The association with the
receptor is thought to occur in the Golgi apparatus, and the dissociation happens in
the acidic pre-lysosomal compartment. As a fraction of the hydrolases is secreted,
the M6P receptor also participates in the endocytotic retrieval of the lysosomal
enzymes back to the lysosomes. There are, however, some lysosomal enzymes like
glucocerebrosidase, acid phosphatase and a number of lysosomal membrane
proteins, that do not have the M6P recognition marker. The presence of near normal
levels of lysosomal enzymes in some types of tissues in I-cell disease patients, who
are unable to synthesize the M6P recognition marker suggests that there are
alternative mechanisms for delivery of lysosomal enzymes to the lysosome in some
cell types (Dawson and Hancock, 1992; Komfeld, 1990; Komfeld, 1987; Von Figura

and Hasilik, 1986).

13

The selectivity of phosphorylation to lysosomal hydrolases is not yet fully
understood. It is thought that the phosphorylation of lysosomal enzymes is mediated
by a protein site that is specifically recognized by UDP-N-acetylglucosamine (UDP-
GlcNAc): lysosomal enzyme N-acetylamine- 1 -phosphotrans ferase
(phosphotransferase) (Holtzman, 1989). However, a description of the component of
the phosphotransferase recognition site has been complicated by the lack of obvious
sequence homologies among lysosomal enzymes. Another complicating factor has
come from in vitro studies using potentially purified phosphotransferase which have
shown that disrupting the native conformation of lysosomal enzymes by heat
denaturation significantly lowers the efficiency of phosphorylation, and that the
phosphotransferase works better with lysosomal hydrolases such as hexosamine
protein than with proper—looking oligosaccharides not linked to proteins (Cuozzo and
Sahagian, 1994). The enzyme does little with non-lysosomal glycoproteins such as
thyroglobulin or immunoglobulins that have oligosaccharides not too different from
those of the lysosomal enzymes (Holtzman, 1989). This suggests that the site is
probably a surface patch of the protein as opposed to a simple linear sequence of

residues.

B-Mannosidosis
Identification

B-Mannosidosis was the most recent disease to be described in the

14

glycoprotein disorders group. The metabolic nature of the disease was determined by
Jones et al. 1981, (Jones and Laine, 1981) by studying the structure of the
accumulated product and the enzyme activity from tissues of an affected goat.
Composition analysis of the accumulated trisaccharide revealed the presence
of one mole of mannose and two moles Of 2-deoxy-2acetamidoglucose, while the
disaccharide was found to consist of an equimolar composition of mannose and
glucosamine. The anomeric configuration of the glycosidic bonds was suggested as a
B-configuration because both mannose and N-acetylglucosamine were destroyed by
the standard conditions of oxidation, and was confirmed by digestion with glycosyl-
hydrolases. The trisaccharide, when digested with B-mannosidase, released mannose,
and when digested with endo-B-N-acetylgucosaminidase-L cleaved the reducing end
glucosamine and yielded a disaccharide identical to the second storage product that
itself is susceptible to B-mannosidase cleavage. This gave evidence, together with
the confirmation of the presence of a glucosamine reducing end, that the
accumulated substances in the affected goat brain has the following structures:
ManB(1—>4)GlcNAcB(1——>4)GlcNAc and ManB(1—+4)GlcNAc (Figure 1). Enzyme
activity studies showed deficiency of lysosomal B-mannosidase in the brain, kidney
and liver of affected kids, while intermediate levels of the enzyme were found in the

tissues of an obligate carrier animal (Jones and Dawson, 1981; Jones and Laine,

1981).

15

The mode of inheritance of the disease was established first in goats by Jones
et a]. from observations of the breeding program at Michigan State University (Jones
and Laine, 1981). It was observed that both parents were consanguineous and
phenotypically normal, as well as having tissue B-mannosidase activity levels that
were intermediate when compared to control and affected animals. Both males and
females were affected with the disease, and the ratio of the affected Offspring to
normal was 1:3. Similar observations were made for the human cases, which led to
the confirmation of the mode of inheritance to humans.

Clinical Cases

The enzyme deficiency represents two different clinical and biochemical
spectrums between the two ruminant species and human. The disease in goats and in
the Salers cattle is similar with severe neurological deficits and neonatal onset. The
clinical symptoms include facial dysmorphology, joint hyperextention, extensive
dysmyelination of the central nervous system, muscle atrophy, and nerve deafness in
goats (Render et al., 1992; Jones et al., 1983). Histopathological studies of different
goat and cattle cell types from the nervous system, thyroid, muscle, liver, and kidney
revealed various types and levels of cytoplasmic vacuolation (Lovell et al., 1994;
Jones et al., 1983). No enzyme activity was detected in these tissues, however,
higher levels of other lysosomal enzyme activities were detected (Jones and Dawson,
1981). The major excretory and storage product in both ruminant species are the

trisaccharide ManB(1——>4)GlcNAcB(l—->4)GlcNAc with less amounts of disaccharide

l6

ManB(l——>4)G1cNAc. Minor accumulation of tetra- and pentasaccharides have also
been documented (Cavanagh et al., 1982; Jones et al., 1984; Jones et al., 1992;
Matsuura et al., 1981; Jones and Dawson, 1981; Jones and Laine, 1981).

In contrast, human B-mannosidosis presents a milder and heterogeneous
disease. Thirteen cases have been reported, representing a wide range of clinical
symptoms and a wide spread ethnic distribution. The most severe cases are ones that
are associated with mental retardation, developmental delay and neurological signs
(Cooper et al., 1991; Kleijer et al., 1990; Wenger et al., 1986). Milder forms of the
disease involve angiokeratomas (Rodriguez-Sema et al., 1996; Cooper et al., 1986),
and peripheral neuropathy with no mental retardation (Levade et al., 1994).

The patients can be grouped into three groups based on the age of onset and
on whether they had normal development prior to the onset of the symptoms. The
first group include patients described by Dorland et al., 1988, Kleijer et al., 1990,
and Gourrier et al., 1997 (Gourrier et al., 1997; Kleijer et al., 1990; Dorland et al.,
1988) Table 1. These patients had a noticeable early onset during the first months of
life, associated with feeding difficulty and recurrent respiratory infections. Although

manifestations are somewhat different, the patients in this group are severely
affected with the disease. The two brothers described by Dorland et a1. 1988, are
children of a Turkish consanguineous parents. They had feeding difficulties and
recurrent respiratory infections and were hypotonic. Both had speech impairment.

hearing loss, and behavioral problems, but their motor development was considered

Table 1 Infantile early onset.

17

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Family # 1 2 3
Reference Dorland, et. al., 1988 Kleijer, et.a1., 1990 Gourrier, et.a1., 1997
Ethnic origin Turkish Czech Gypsy

Consanguinity yes Founder effect yes

Sex Male Male Female Male Female
Age of onset 3 wk 3wk <14 mo 7 mo
Presenting Feeding difficulty Feeding difficulty
symptom

Mental retardation yes yes yes yes ?
Behavior Troublesome Aggressive ?
Problems

Hearing loss yes yes yes yes

Neurological signs Hypotonia EEG abnormality Hypotonia
Skin Erysipelas

Facial no no yes yes no
dysmophism

Skeletal yes yes no
abnormalities

Respiratory yes yes yes yes yes
infections

Other

Urinary yes yes yes yes yes
disaccharide

Enzyme activity

NmoI/hr/mg

Leukocytes 0/45-150 0 0.9/45-150 ?
Fibroblasts 4/ 5 8-3 89 0/51-95 0.6 ?

 

 

 

 

 

18

normal. The two siblings reported by Kleijer et al. 1990, come from a gypsy family
from the Czech republic. Consanguinity is possible since the parents come from
neighboring villages, but it is not proven. The sister was more severely affected than
her brother. She suffered from psychomotor retardation, bone deformities and
gargoylism and recurrent skin and respiratory infections. She died at age 20 years
from bronchopneumonia. Her older brother have a milder manifestation of facial
dysmorphology, mental retardation, hearing loss and recurrent infections. The
patient described by Gourrier et al. 1997, was a child of consanguineous parents. She
was hypotonic during the first months of life and had abnormalities of swallowing
and esophageal motility resulting in recurring respiratory infections. Her motor
development is retarded, however, no facial dysmorphology was reported.

A second group of patients described by Wenger et al. , 1986, Cooper etal.,

1991, Wijburg et al., 1992, and Poenaru et al., 1992 (Poenaru et al., 1992; Wijburg
et al., 1992; Cooper et al., 1991; Wenger et al., 1986) also had an early onset of
manifestation during the first year, however, they had relatively normal early
development until they were brought to medical attention because of their parents
concern about either speech or locomotor development Table 2. These patients
eventually developed a progressive disease associated with mental retardation and
various neurological signs. The patient described by Wenger et al. 1986, is from the
United States. His parents were not related and they were from white European

ancestry. He had course facial features, mild bone disease, delayed speech

Table 2 Cases of infantile onset with relatively normal early development.

19

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Family # 1 2 3 4
Reference Wenger, et. Cooper, et. Wijburg, et. Poenaru,
al., 1986 al., 1991 al., et.a1.,
1992 1992
Ethnic origin White Jamaican x Turkish White
European Irish European
Consanguinity no no yes no
Sex Male Female Female Male
Age of onset 1 yr 9 mo 15 mo < 3 yr
Presenting symptom Speech & Locomotor Speech Speech
developmen delay delay impairment
tal delay
Mental retardation yes yes yes
Behavior Problems Hyperactive Anorexia Hyperactive
Hearing loss yes no yes no
Neurological signs Epilepsy
Skin
Facial dysmophism yes no yes
Skeletal yes no no no
abnormalities
Respiratory yes yes
infections
Other sulfamidase Ethanolami
deficiency nuria
Urinary yes yes yes yes
disaccharide
Enzyme activity
Nmol/hr/mg
Leukocytes 0/119 0.9/45-150 4.7/160-300 0.5/104-432 I
Fibroblasts 0/103 0/51-95 0/160-300 0/60-208

 

 

 

 

 

 

20

development, hyperactivity and mental retardation. This patient had a combined
enzyme deficiency of B-mannosidase and heparin sulfamidase (Sanfilippo syndrome
type A). The patient described by Wijburg et al.1992, whose parents were first
cousins, suffered from recurrent upper respiratory tract infections and hearing loss.
Her motor and mental development was retarded by age 5 years. This patient had
ethanolaminuria besides the B-mannosidase deficiency. The patient described by
Cooper et al. 1991, was born to unrelated parents, the father being Irish and the
mother was Jamaican in origin. She had a moderate developmental delay,
brachecephaly and suffered from tonic-clonic seizures. No dysmorphic features or
hearing loss were present. The girl died at age 15 months. The patient described by
Poenaru et al, 1992 was of European ancestry whose parents were not related. He
had cranio-facial dysmorphology, mental retardation, speech impairment, increased
susceptibility to bronchial infections and suffered from emotional instability.

Cases with later onset described by Cooper et al.1986, and Lavade et al.
1994, Table 3, were brought to attention, in the first case at 6 years of age because of
impaired intellectual development, and in the latter case because of general apathy
and disinterest (Levade et al., 1994; Cooper et al., 1986). The two brothers reported
by Cooper et al.1986, were Indian Hindus whose parents were not related. They
suffered from mental retardation, angiokeratoma, speech and hearing impairment.
The younger brother had aggressive behavior and was institutionalized. The patient

described by Lavade et al. 1994, was of an African origin of unrelated parents. He

21

suffered mainly from peripheral neuropathy and had no mental retardation nor any
facial or skeletal dysmorphology. Unlike the other patients, only faint band of the
disaccharide in urine was Observed in this mildly affected patient.

A late juvenile onset was reported by Rodriguez-Sema et a1, 1996, Table 3.
The patient was suspected to have lysosomal storage disease because of the presence
of angiokeratoma corporis diffussum which started at age 12 years. This patient had
no mental retardation, on the contrary, her mental development could be considered
normal since she completed high school but she appeared to be somewhat introvert
and scantily communicative (Rodriguez-Sema et al., 1996).

Disease Pathology

Contrary to the extensive and detailed studies of the pathology of the caprine
disease (Patterson et al., 1991; Lovell et al., 1991; Lovell and Boyer, 1987; Boyer
and Lovell, 1990; Boyer et al., 1990; Lovell and Jones, 1983; Lovell and Jones,
1985), only little pathologic information is available about the human B-
mannosidosis. Cytoplasmic vacuoles have been detected in fibroblasts of skin
biopsies from patients described by Cooper et al., 1986, and Levade et al., 1994, and
Rodriguez-Sema et al., 1996 (Rodriguez-Sema et al., 1996; Levade et al., 1994;
Cooper et al., 1990; Wenger et al., 1986). Slight vacuolization and granulation of
bone marrow cells were detected from patients described by Kleijer et al., 1990

(Kleijer et al., 1990). The extent to which specific tissues and organs are affected by

Table 3 Juvenile onset.

22

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Family # l 2 3
Reference Cooper, et.a1., Levade, et.a1., Rodriguez-Sema,
1986 1994 etaL,1996
Ethnic origin Hindu African Spanish
Consanguinity Founder effect no no
Sex male male male Female
Age of onset 5 yr 6 yr 12
Presenting symptom Mental clumsiness Angiokeratoma
retardation
Mental retardation yes yes no no
Behavior Problems Aggressive Disinterest & Scantily
apathy communicative
Hearing loss yes yes no no
Neurological signs Demyelinating
peripheral
neuropathy
Skin Angiokeratoma Angiokeratoma
Facial dysmophism no no no no
Skeletal abnormalities no no no no
Respiratory infections no no
Other
Urinary disaccharide yes yes Faint yes
Enzyme activity
Nmol/hr/mg
Leukocytes 1/245-465 0.2 3/80-170 3/73-186

 

 

Fibroblasts

 

4/58-389

 

 

 

 

23

the storage product and by the increase in the number of lysosomes has not been
investigated yet in human cases.
Storage Product

The disaccharide ManB(l——>4)GlcNAc is the major storage product found in
B-mannosidosis patients. It has been detected from urine analysis of all the described
patients and has been found accumulating in fibroblasts and to a lesser extent in
leukocytes of two patients (Van Pelt et al., 1990). The compound is the expected
product from the metabolic block due to B-mannosidase deficiency and the presence
of an endo-B-N—acetylglucosaminidase that would hydrolyze the linkage between the
two N-acetylglucosamine residues found in most N—linked oligosaccharides chains in
glycoproteins. A sialyated compound, Sia or(2—>6)Man[3(l—a4)GlcNAc, was
identified as a storage product in two patients which seems to result from secondary
enzymatic sialylation of the storage product ManB(1—>4)GlcNAc. A urinary
carbohydrate urea conjugate has also been found in these patients (Hokke et al.,
1990)

The major storage product in the bovine and caprine B-mannosidosis is the
trisaccharide ManB(1—+4)GlcNAcB(1——>4)GlcNAc, and a smaller amounts of the
disaccharide ManB(1—)4)GlcNAc as well as tetra and pentasaccharadise compounds
(Cavanagh et al., 1982; Jones et al., 1984; Jones et al., 1992; Matsuura et al., 1981;

Jones and Dawson, 1981; Jones and Laine, 1981). Reflecting differences in human

24

and ruminant glycoprotein catabolic pathways, the storage oligosaccharides from the
ruminant species has two GlcNAc in a chitobiose linkage, while the storage
oligosaccharide from human B-mannosidosis has only a single GlcNAc. The
significance of the different size of the accumulating storage product for the
development of the clinical symptoms in humans and the ruminant species is not yet

known.

The Enzyme B-Mannosidase

Mammalian B-mannosidase (B-D-mannoside mannohydrolase, E.C.3.2.l.25)
was mentioned for the first time in 1957 among the glycosidic activities of whole
semen, seminal plasma or epididymal secretions in various species: bull, dog,
rabbit, ram, stallion, man (Conchie and Mann, 1957). The lysosomal localization
of acid B-mannosidase was demonstrated in rat liver in 1973 (Labadie and
Aronson, 1973). The enzyme is involved in the catabolism of mannose containing
glycoconjugates (especially glycoproteins) in which it catalyses the hydrolysis of the
B-mannosidic linkage. The mammalian enzyme, i.e. rat, pig, goat, cow, and human
has an Optimal pH range from 4 to 5.5, and has been reported to be heat labile
(Percheron et al., 1992). The enzyme has been purified from guinea pig liver, goat
kidney, bovine kidney, and human placenta and urine (Guadalupi et al., 1996;

Iwasaki et al., 1989; Sopher et al., 1993; Sopher et al., 1992; McCabe and Dawson,

25

1991; Kyosaka et al., 1985) and partially characterized from several other
mammalian sources (Colin et al., 1987; Bernard et al., 1986; Panday et al., 1984). In
all instances the enzyme has been referred to as an acidic form, with a molecular
mass determined in the range of 57-160 kDa.

The goat enzyme preparation consisted of 90 and 100 kDa peptides, both of
which were associated which B-mannosidase activity and were reactive to B-
mannosidase antibodies. Deglycosylation of both polypeptides reduced their size to
86 and 91 respectively. The deglycosylation results suggest different types of
glycosylation for the two forms. The 90 kDA polypeptide contains mainly high
mannose, while the 100 kDa is more heavily glycosylated and contains complex type
oligosaccharides (Sopher et al., 1992). The purified bovine enzyme consisted of
three polypeptides, 100, 110 and a minor 84 kDa, that were associated with B-
mannosidase activity and were immunologically related. Carbohydrate composition
analysis revealed that the bovine polypeptides were similar in size to the goat
enzyme but contained more glycosylation, mainly the complex type (Sopher et al.,
1993)

In humans, attempts to purify the enzyme using the placenta as a source
resulted in contamination by low levels of other lysosomal hydrolases. The
molecular weight of the purified preparation in SDS-polyacrylamide gel was 57 to
98 kDa (Iwasaki et al., 1989). A more recent attempt utilized urine as a source of the

enzyme. Secretion of several lysosomal enzymes in urine have been reported

26

previously (Paigen and Peterson, 1978). From urine, two distinct forms of the
enzyme B-mannosidase were purified and characterized. The two forms differed in
their molecular masses, isoelectric points, thermal stability and subunit composition.
The estimated molecular weight mass was 160 and 135 kDa for the B and A forms.
The two forms were also found in human kidney but with different ratios (Guadalupi
etaL,1996)

In humans B-mannosidase activity has been detected in a variety of different
human tissues and body fluids. It has been detected in fibroblasts, leukocytes,
lymphocytes, chorionic villi, serum, urine and plasma (Colin et al., 1987; Cooper et
al., 1988; Petushkova et al., 1992; Bernard et al., 1986; Panday et al., 1984). The pH
range for optimum enzyme activity was found to be 3~4.5 (Percheron et al., 1992). A
study done by Cooper et al., 1987 to determine the effect of age and sex on enzyme
activity in human plasma found that there was a significant variation in the enzyme
activity with regard to age, with the activity being highest at early age (0-1 year),
then decreasing afierwards. On the other hand, sex differences had no effect on the
enzyme activity (Cooper et al., 1987).

A non-lysosomal B-mannosidase activity was reported by Dawson, 1982 in
goat liver (Dawson, 1982). This form is probably a different gene product since: (i) it
hydrolyzes the synthetic B-mannoside but not the natural substrate, (ii) has a broad

range of pH 5 to 8, and (iii) it does not bind to concanavalin-A indicating that it has a

27

different carbohydrate composition than the lysosomal form. However, the
localization of this form of the enzyme has only been done in goat liver and none of
the studies that reported the enzyme activity from human sources detected a similar
form of the enzyme. The finding of a neutral form is not unique to B-mannosidase,
high residual activity to synthetic substrates was reported in liver homogenates from
patients with a-mannosidosis, Gaucher disease, and to some extent in GM]
gangliosidosis. These forms of the enzymes were reported to be distinct, with
possibly different carbohydrate composition because they do not bind to

concanavalin A-Sepharose affinity columns (Dawson, 1982).

Human Gene Mutations

Definition and Relevance to Disease Pathogenesis

A mutation traditionally has been identified as a stable heritable change on
DNA. This definition does not depend on the functional significance of the change.
It implies a change in the primary nucleotide sequence, and other changes, such as
those involving methylation, are usually referred to as epigenetic events. Mutations
that occur in somatic cells may be most relevant to cancer or aging. Mutations
occurring in germ cells are of greatest importance in terms of their impact on
offspring. The concept that mutations are stable changes remain generally true, but
the discovery of expanding triplet repeat mutations emphasize that mutations can be

unstable either in somatic or germ cell lines (Cooper and Krawczak, 1993).

28

Mutations also represent the driving force behind evolution, the origin of
genetic variation and the ultimate cause of hereditary disease. The study of naturally
occurring mutations is fundamental to understanding these processes. Knowledge of
the nature, relative frequency, and the DNA sequence context of different gene
lesions improves our understanding of the underlying mutational mechanisms and
provides valuable insights into the intricacies of DNA replication and repair. The
effect of mutations on enzyme activity level provide information about functional
aspects of the protein. In addition, understanding the ground rules for assessing and
predicting the relative frequencies and location of specific types of gene lesions may
contribute to improvements in the design and efficacy of mutation research
strategies.

Over the past 15 years, the application of novel DNA technologies has
permitted very rapid progress in the analysis and the diagnosis of human inherited
diseases by the characterization of the underlying gene lesion. Many different types
of mutations, single base-pair substitutions, deletions, insertions, have been detected
and characterized in large number of human genes. Although knowledge of the
nature of a specific gene defect, the disease-genotype, can permit direct detection of
carriers of that lesion, such information can also sometimes also used to make
prognostic predictions about clinical severity, the age of onset and clinical course of
the disease, the disease-phenotype. Detailed analysis of the genotype-phenotype

relationship promises both to improve the accuracy of prognostic predictions and to

29

increase the understanding of the mechanisms and processes of disease pathogenesis.

The incidence and prevalence of human genetic diseases is quite variable;
therefore, it is not surprising that the nature, frequency and location of the
pathological gene lesions in the human genome are non-random. This non-
randomness is largely sequence dependent; thus, some DNA sequences are not only
more mutable than others, but they also mutate in specific ways and at characteristic
frequencies (Cooper and Krawczak, 1990).

Recombinant DNA technology has made possible the study of genetic disease
at the level of the primary lesion, and the in vitro expression of both normal and
mutant genetic information at the mRNA and the protein levels. Undoubtedly, the
most immediate practical importance from recombinant DNA technology in
medical genetics has been in the sphere of improved disease diagnosis, where the
advances has been dramatic (Cooper and Schmidtke, 1991). Indeed, molecular
genetics is now indispensable tool of modern pathology. In combination with
advanced techniques in protein biochemistry, cell biology and immunology, the
foundations have now been laid upon which to build a thorough understanding of
disease pathogenesis. Such understanding will probably help in the correction of
inherited gene defects through the identification of novel therapies (Cooper and
Krawczak, 1993).

Disease diagnosis is an essential basis for causal therapy, and pre-

symptomatic diagnosis is in many instances a prerequisite for successful treatment or

30

prevention. The ability to detect presymptomatically a disease which is at present not
amenable to therapy may, however, present serious ethical problems. Knowledge of
disease susceptibility may be misused in terms of employment opportunities or
access to health insurance.

Genotype-Phenotype Correlation

Phenotypic variation forms the basis for genetic investigation. The range of
variation comprises what is considered to be normal variability and pathological
conditions as well. The genetic approach assumes that phenotypic characters are
either determined or influenced by the genetic contribution of an organism. Thus
either the structure of a single gene or the interaction between several genes is
considered to be responsible for, or contribute to, phenotypic variation (Wolf, 1995).
To understand genotype-phenotype relationship, it is important to define the level of
phenotype with which we are dealing. The total DNA forming the genome is no
doubt the genotype. As far as the DNA is transcribed, though differentially at many
gene loci, and the RNA is translated after its processing, there exist a one-to-one
relationship between DNA and RNA, and between RNA and the amino acid
sequence, while the structure of a functional protein may not be unequivocally
determined by its amino acid sequence. Because of this specification, the phenotypic
level could be defined as commencing with proteins. However, only part of the DNA
is transcribed into RNA, and only part of the transcripts are processed to serve as the

mRNA that is translated into amino acids sequences; other RNA fractions are

31

functional end products, such as tRNA and tRNA, or they become degraded
immediately.

Thus there are clear deviations from one-to-one equivalence between DNA,
RNA or protein, and these deviations vary between cells, organs and individuals.
RNA clearly differs from DNA, despite its complementary nature to DNA, in its
quantitative representation within a given cell, and consequently contributes to
phenotypic variation. The phenotype can thus be defined as the total of markers or
characters of an organism apart from the DNA itself (Wolf, 1995).

The phenotype is a result of ontogenetic development, which holds true also
at the molecular level where genetic and non-genetic factors interact to produce
successive states, each of which is the prerequisite and determines the condition for
the next one. Therefore, genes are necessary but not sufficient component. The
system already presents gradients, threshold values and positional relationships that
are equally essential. Thus, even monofactorial traits can be considered to be of
multifactorial causation, and the varying border line conditions that arise during
development add to the complexity. From this standpoint, it is not to be expected
that a mutation has a consistent phenotypic outcome, and the genotype-phenotype
relationship may be irregular. However, in some cases ontogentic modification
appears to be of minor significance, so that the phenotype of a mutation can be

predicted with considerable accuracy.

32

Types of Mutations Causing Human Disease

Since not all mutations are deleterious, human genetic disease can be
considered as an extreme manifestation of genetic change, superimposed on a
background of normal genetic variability. In broad terms, mutations can be classified
into three categories: (i) genome mutations arising by chromosome missegregation
producing, for example, aneuploidy, (ii) chromosome mutations caused by
chromosome rearrangement leading to, for example, translocations, and (iii) gene
mutation caused by base pair mutations.

Among gene mutations, point mutations and deletions are the most frequent
gene lesions in the human genome (Cooper and Schmidtke, 1991). Mutations occur
in coding regions, regulatory regions, splice sites, within introns, and in
polyadenylation sites, and they interfere with any stage in the pathway for protein
expression. Base-pair substitution in the coding region can lead to a missense
mutation or to a nonsense mutation. Missense mutations can lead to a reduction or
loss of protein activity, alteration in protein structure and stability or sensitivity to
premature proteolytic attack (Pakula and Sauer, 1989), as well as affecting mRNA
stability. Nonsense mutations introduce a premature stop codon to the mRNA
transcript leading to mRNA instability (Maquat, 1996), and to protein truncation.
Mutations at the vicinity of splice site alter the efficiency of mRNA splicing
resulting in either abolished or reduced amounts of the mature mRNA. The effects of

splice site mutations are exon skipping, intron retention or utilization of cryptic sites.

33

These effects rarely occur individually, therefore multiple aberrant transcripts are
usually Observed (Krawczak et al., 1992). Deletions and insertions, when
encountered, often lead to a frame shift.

In general, the pattern of iheritance and segregation ratios of single—gene
disorders are in accord with the principles of Mendelian inheritance. However,
throughout the 20th century exceptions have been encountered. Mutation analysis of
these diseases has identified the molecular mechanisms causing these disorders.
Examples of some of the mechanisms that can affect the transmission or the
expression of single-gene disorders are: genomic imprinting, mosaicism,

mitochondrial inheritance and uniparental disomy.

Objectives and Research Strategy

The Objectives of this research are: (l) to study the molecular biology of the
human B-mannosidase gene by cloning and characterizing the cDNA, (2) to study
mRNA tissue distribution, (3) to determine the chromosomal localization of the
gene, and (4) to identify mutations causing the disease in two described cases
(Kleijer et al., 1990; Wenger et al., 1986).

The methods that were used for the first three objectives involved commonly
used molecular biology techniques, including cDNA library screening, RT-PCR, 5’
and 3’ RACE. northern blotting and PCR. The following factors were taken into

consideration when designing the strategy for mutation detection for the patients: (1)

34

whether the parents were related or not, and (2) the occurrence of another genetic
disease in the same patient.

The strategy for mutation detection for the patient described by Wenger et
al.,l986 was to analyze the cell line for chromosome mutation by doing karyotype
analysis, to investigate the possibility of isodisomy of chromosome 4, to scan cDNA
for size differences and to sequence the entire coding region. The reason for
investigating chromosome abnormalities and isodisomy of chromosome 4, was that
the patient had another genetic lysosomal enzyme deficiency, heparin sulfamidase.
Since the patient’s parents were unrelated it was hypothesized that he was a
compound heterozygous at the B-mannosidase locus. On the other hand, the
possibility of founder effect in the gypsy family described by Kleijer et al., 1990 led
to the hypothesis that the two affected siblings were homozygous at the [3-
mannosidase locus. The mutation in the two siblings was investigated by performing
RT-PCR to scan the cDNA for size differences and by sequencing to identify base-

pair changes.

CHAPTER2

CHARACTERIZATION OF HUMAN B-MANNOSIDASE

Introduction

With the identification of human B-mannosidosis the need to understand the
underlying molecular mechanisms, of the disease in general and of its varying
phenotypic manifestations in particular, became necessary. As a prelude for that, this
research is directed towards the identification, sequencing and characterization of the
human B-mannosidase cDNA which is necessary for screening and identifying the
mutations causing the disease. The identification of the cDNA is important for fiiture
cloning of the coding region for the purpose of expressing the enzyme and
characterizing it.

Earlier work on B-mannosidase deficiency in two ruminant species, the
bovine and the caprine, initiated interest in the enzymology and molecular biology of
the enzyme which led to purification of the goat and bovine enzymes and to the
molecular characterization of the B-mannosidase cDNA from both species
(Leipprandt et al., 1996; Chen et al., 1995; Sopher et al., 1993; Sopher et al., 1992).
The size of the coding region in both genes is the same with 97% sequence identity.

The mRNA size, 4.2 kb, is also identical in both species. The cDNA sequence of the

35

36

two species reveal no sequence homology to any Of the other lysosomal enzymes.
However, a Celegans DNA sequence (accession 278540) revealed 60 % sequence
identity to the bovine cDNA.

The objective of the research described in this section is to characterize the
human cDNA . The bovine cDNA sequence and a human expressed sequence tag
(EST) that showed 80% sequence identity to the bovine sequence, was utilized to
design a DNA probe to screen a human placenta cDNA library (Clontech). The
mRNA levels of B-mannosidase is studied and compared in different human tissues.

The chromosomal location of the gene is also determined.

37

Materials and Methods
cDNA Cloning

A human placental cDNA library constructed in Lambda ZapII (Stratagene) was
screened with a PCR-derived probe according to manufacturer's suggested protocol.
The probe was obtained by amplification of DNA from the human placental cDNA
library, Lambda-ZAPII, using one human based primer BH82 (refer to Table 4 for
all primer sequences) derived from an expressed sequence tag (Genbank accession #
humxt01397) that showed 80% sequence identity to bovine B-mannosidase cDNA,
and one primer based upon the bovine cDNA BB89 (Chen et al., 1995). The
amplified 775 bp DNA fragment, hu 89/82, was labeled to high specific activity
using a random primer labeling kit (Boehringer Mannheim) which was used as a
probe to hybridize to a set of 20 nylon filters (NEN Research Products) containing
total 106 pfu from the human placenta cDNA library. Duplicate filters sets were
prehybridised in 50% forrnamide, 6X SSPE, 5X Denhardts solution, 0.1% SDS and
100 jig/ml denatured herring sperm DNA for 3 hours at 42°C. Hybridization was
performed in the same solution afier adding the denatured probe at 106 cpm/ml, and
was incubated for 16 hours at 42°C. Filters were washed twice with 1X SSC/ 0.1%
SDS at room temperature and once with 0.5X SSC/O.l% SDS for one hour at 42°C.
Positively hybridizing plaques were picked, purified and in vivo excision of the

phagemid pBlueskript (SK-) was performed according to Stratagene protocol.

38

Table 4 Primers for PCR amplification.

 

Primer Orientation and hp position Sequence

33113 Sense493 5' GCAGTGAACATCATTGAGGTGC 3'
33121 Antisense 622 5' TCACATCCCATTCACCCTTC 3'
31-1120 Antigense 647 5' CAACATTCCTCCTTCCCAAC 3'
H8122 Sense, 724 5' TCTCACCTCAACTACTTCACA 3'
BB96 Sense, 957 5' GTCCCCTCATCCACATCCAA 3'
33113 Antisense 1082 5' GCCTCTTCTACAACTTCCACTG 3'
33103 Sense 1272 5' TCACCAGGATCAATTCTACGAACT 3'
311123 Antisense 1431 5' TTCATCATCAGCCCCTCCTC 3'
311139 Antisense 1861 5' TACCACCTTCGTCATCTTGTCC 3'
31182 Sense 1372 5' TGCTTTATCACCCTCCACTTCA 3'
3374 Antisense 2091 5' CCACATCTCATTCACCTCCC 3'
BH138b Sense 2093 5' CTTCACTACGCACCAAACTG 3'
311137 Sense 2152 5' CCTCCACTGTTCCCACTAGC 3'
33141 Sense 2202 5' TCCTCTCTCACATCTTCACTCG 3'
BH160 Sense intron sequence 5' CTTAACCATTTCTGCTAACC 3’
331383 Antisense 2263 5' CCATGTATGGACTCTCAC 3'
311155 Antisense 2330 5' TACACACCCTCTCCTCCTCTCA 3'
33150 Sense 2496 5' CATCACTGCCATCATCTCTCACC 3'
3389 Antisense 2645 5' ACACTCCCTCTCTTCTCACTCA 3'
3H145 Antisense 2745 5' AATACMCCTAGA'I‘"‘CCTTC 3'
311156 Antisense 2771 5' ATCCTTTATTCCCATTGTCC 3'

 

39

XLl-Blue bacteria were infected with the positive phagemids and grown on 100
pg/ml ampicillin-containing LB agar plates. Plasmid DNA was purified from
overnight cultures using Promega's Wizard miniprep kit. Three independent clones
were sequenced at Michigan State University and University of Michigan
sequencing facilities using ABI 373A DNA Sequencer. Sequence analysis and
conti g assembly were done using GCG and Sequencher computer programs.
RT-PCR
Sequence information obtained from the cDNA clones was used to design primers (S
& E sofiware) for use in RT-PCR reactions to complete the cDNA sequencing of the
human B-mannosidase gene. Ten micrograms of human placenta total RNA
(Clontech) were reversed transcribed with M-MLV-RT (GibcoBRL) using the 3’
antisense primer BH145 to synthesize first strand cDNA. The cDNA was ethanol
precipitated and resuspended in 50 pl of H20. PCR reactions were performed using
2 pl from the above reaction as a template in a 20 pl final reaction volume
containing: 1X PCR buffer (GibcoBRL), 0.2 mM each dNTP, 2 mM MgC12, 1 nM
each primer, 1 unit Taq DNA Polymerase (Gibco/BRL). PCR cycling conditions
were: initial denaturation 7 minutes at 94°C, followed by 25 cycles of denaturation
for 45 seconds at 94°C, annealing for 45 seconds at (annealing temperature range
between 55°C and 64°C according to the Tm of the primers) and elongation for 45

seconds at 72°C. A final elongation step was carried out for 10 minutes at 72°C.

40

The total number of cycles was kept at 25 per reaction and reactions were performed
in triplicate to avoid Taq induced errors. The amplified fragments were size
fractionated by 0.5X TBE/ 1% agarose gel electrophoresis and were gel purified
using Promega's Wizard PCR Preps-DNA Purification System. Figure 2 illustrates
the relative position of the fragments, and the primer pairs that were used to produce
them. Table 4 contains a list of the sequence, orientation and the bp positions of the
primers. All fragments were sequenced in both directions using PCR primers and
internal primers where applicable.
5' Rapid Amplification of cDNA Ends
5' Rapid amplification of cDNA (RACE) was performed according to
manufacturer’s protocol (GibcoBRL). Gene specific primers BH120 and BH121
were based on the composite cDNA sequence from RT-PCR and cDNA Clones.
BH120 was used to synthesize first strand cDNA. PCR amplification was
performed using the antisense internal primer BH121 and an anchor primer provided
by the manufacturer using the recommended conditions. Amplified fragments were
cloned in pCRII which was used to transform competent DHSOL. Plasmid DNA was
purified using Promega’s Wizard Midiprep kit and pCRII clones were sequenced as
described above.
Northern Blot Analysis
A human Multiple Tissue Northern Blot obtained from Clontech was hybridized

with probe hu82-89. The probe was labeled with hybridization were performed

41

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42

using Clontech protocol. The same membrane was used for hybridization with a
control B-actin cDNA provided by the manufacturer. The relative gene expression
was determined by quantifying the intensities of the bands in fluorograrns of the
northern blot using a phosphoimager Model 400 B, Molecular Dynamics,
Sunnyvale, CA.

Chromosomal Localization
Primer pair BH150/ BH156 was used to amplify a 276 bp PCR product from a 3'
terminal exon using genomic DNA as a template. The annealing temperature for the
PCR was raised to 64°C to specifically amplify human genomic DNA in
rodent/human cell hybrids. The optimized conditions were applied to DNA isolated
from a series of human-rodent hybrid cell lines (Coriell Cell Repositories) in which
human chromosomes 1-22, X and Y were retained individually in each cell line. To
localize the region of chromosome 4 containing the human B-mannosidase gene, a
second set of cell lines retaining various overlapping deletions of chromosome 4 was
tested (NA10115, NA11447, NA11449, NA13396 and NA13402 (HIGMS Human
Genetic Mutant Cell Repository, Coriell Cell Repositories).

3' Rapid Amplification of cDNA Ends
To verify the 3'UTR sequence obtained from the above methods, 3'RACE was
performed suing the 3’RACE kit (Gibco BRL). The adapter primer was used to

synthesize the first strand cDNA using human placenta total RNA

43

(Clontech)according to the manufacturer protocol. RT-PCR amplification was done
using a sense primer from the coding region, BH150, and the abridged universal
primer (Gibco BRL) as the antisense primer. The resulting PCR product was

sequenced from both directions in Michigan State University sequencing facility.

44

Results

Human B-Mannosidase cDNA Sequence

A AZAPII human placenta cDNA library screened with hu 82/89 probe (see
Materials and Methods) resulted in identification of three independent clones
pHUBM3, pHUBM7 and pHUBM8, with 1.9, 1.15 and 1.49 kb inserts respectively
(Figure 2). The three clones overlapped and had a putative stop codon that matched
the position of the stop codon in the bovine cDNA sequence. However, two of the
clones, pHUBM3, pHUBM7 were artifactual chimeras with other human cDNA
sequences. The third clone, pHUBM8 showed 80% sequence identity with the
bovine B-mannosidase cDNA in the 3' end of the coding sequence, and less
homology following the stop codon. The human sequence has a 38 bp insertion after
which the homology at the 3' UTR dropped to about 73%, with additional stretches
of insertions and deletions. To complete the human B-mannosidase cDNA
sequence, RT-PCR, 5' RACE, and 3'RACE was performed using cDNA synthesized
from total human placenta RNA and primers based on the bovine and human cloned
sequences (Figure 2 and Table l).

The composite human B-mannosidase cDNA, sequenced in both directions, is
3300 nucleotides, and contains an 87 nucleotide 5' UTR, 2640 nucleotides encoding

879 amino acids, and a 573 nucleotide 3' UTR (Figure 3). The predicted translation

45

Figure 3 Human -mannosidase cDNA sequence and the predicted protein
translation. Th esidues are potential glycosylation sites that are unique to the
human protein, residues are shared between the human , bovine and goat proteins
and the Ngis the site shared between the human and the bovine proteins. The X is a
polymorphism found at the site coding either for Valine, which is homologous to the
bovine, or Isoleucine. The bold upper case letters indicate the 5’UTR, and the lower

case letters represent the 3 ’UTR.

46

CTTCCGAGGGGGTGAGTGGCTTCACCGCCGGTCCCTTGCAGCGCTGCCTTTCGATCTCTCCACAICTCGG

TGGCGCGGGATCTCAAGATGCGCCTCCACCTGCTCCTGCTGCTCGCGCTGTGCGGTGCAGGCACCACCGC
M R L H L L L L L A L C G A G T T A

CGCGGAGCTCAGTTACAGCTTGCGTGGCAACTGGAGCATCTGCAATGGGAACGGCTCGCTGGAGCTGCCC

A E L S Y S L I? Gi(:)lW S I C N Gl::]C3 S L E L P

GGGGCGGTCCCTGGCTGCGTGCACAGCGCCTTGTTCCAGCAGGGCCTGATCCAGGATTCTTACTACAGAT
G A V P G C V H S A L F Q Q G L I Q D S Y Y R F

TTAATGACCTTAACCACAGATGGGTCTCTTTGGATAACTGGACCTATAGCAAAGAATTTAAAATCCCCTT
N D L N H R W V S L D [::]W T Y S K E F K I P F

TGAAATTAGCAAATGGCAAAAAGTAAATTTGATTCTTGAGGGAGTGGATACGGTTTCAAAAATCCTGTTC
E I S K W Q K V N L I L E G V D T V S K I L F

AATGAAGTCACTATTGGGGAAACAGACAATATGTTCAATAGATATAGCTTTGATATTACCAACGTGGTCA
N E V T I G E T D N M F N R Y S F D I T N V V R

GGGACGTGAACTCCATTGAGCTGCGTTTCCAGTCAGCGGTGTTGTATGCAGCACAGCAGAGCAAAGCTCA
D V N S I E L R F O S A V L Y A A Q Q S K A H

CACTCSCTACCAGGTTCCCCCAGACTGCCCTCCACTTGTGCAGAAGGGTGAATGCCATGTCAACTTTGTT
T X Y Q V P P D C P P L V Q K G E C H V N F V

CGGAAGGAGCAATGTTCCTTTAGTTGGGACTGGGGGCCTTCCTTTCCTACCCAGGGAATCTGGAAAGATG
R K E Q C S F S W D W G P S F P T Q G I W K D V

TTAGAATTGAAGCCTATAATATTTGTCACCTGAACTACTTCACATTTTCCCCAATATATGATAAGAGTGC
R I E A Y N I C H L N Y F T F S P I Y D K S A

CCAGGAGTGGAATCTGGAAATAGAGTCTACATTTGATGTTGTCAGCTCAAAGCCAGTTGGTGGTCAAGTG
Q E W N L E I E S T F D V V S S K P V G G Q V

ATCNTAGCCATCCCTAAGTTGCAAACACAACAGACATACAGCATTGAACTTCAACCTGGGAAAAGGATTG
I X A I P K L Q T Q Q T Y S I E L Q P G K R I V

TTGAGCTATTTGTGAACATTAGCAAGAATATTACTGTAGAAACTTGGTGGCCTCATGGACATGGAAACCA
E L r v <::>I s K (::>I T v E T w w P H G H G [::]o
GACTGGGTACAACATGACTGTTCTTTTTGAACTGGATGGAGGCTTAAATATTGAAAAATCAGCTAAGGTT
T G Y; Ni M T v L F a L D G G L N I E K s A K v

TATTTTAGGACAGTGGAACTTATAGAAGAGCCTATAAAAGGGTCTCCTGGTTTGAGTTTCTATTTCAAAA
Y F R T V E L I E E P I K G S P G L S F Y F K I

TTAATGGATTTCCCATATTTCTAAAAGGCTCAAACTGGATCCCAGCAGATTCATTCCAGGACCGAGTAAC
N G F P I F L K G S N W I P A D S F Q D R V T

CTCTGAGTTGTTACGGCTCCTTTTACAGTCTGTTGTGGATGCTAATATGAATACTCTTCGGGTTTGGGGA
S E L L R L L L Q S V V D A N M N T L R V W G

GGAGGAATTTATGAGCAGGATGAATTCTATGAACTCTGTGATGAACTAGGAATAATGGTATGGCAGGATT
G G I Y E Q D E F Y E L C D E L G I M V W O D F

TTATGTTTGCCTGTGCCCTTTATCCAACTGATCAGGGCTTCCTGGATTCAGTGACAGCAGAAGTTGCCTA
M F A C A L Y P T D Q G F L D S V T A E V A Y

CCAGATCAAGAGACTGAAATCTCATCCTTCTATCATCATATGGAGTGGCAATAATGAAAATGAGGAGGCG
Q I K R L K S H P S I I I W S G N N E N E E A

CTGATGATGAATTGGTATCATATCAGTTTCACTGACCGGCCAATCTACATCAAGGACTATGTGACACTCT
L M M N W Y H I S F T D R P I Y I K D Y V T L Y

ATGTGAAAAACATCAGAGAGCTCGTACTGGCAGGAGACAAGAGTCGTCCTTTTATTACGTCCAGTCCTAC

70

140

210

280

350

420

490

560

630

700

770

840

910

980

1050

1120

1190

1260

1330

1400

1470

1540

1610

Figure 3 ( cont’d)

47

48

V K N I R E L V L A G D K S R P F I T S S P T

AAATGGGGCTGAAACTGTTGCAGAAGCCTGGGTCTCTCAAAACCCTAATAGCAATTATTTTGGTGATGTA 1680

N G A E T V A E A W V S Q N P N S N Y F G D V

CATTTTTATGACTATATCAGTGATTGCTGGAACTGGAAAGTTTTCCCAAAAGCTCGATTTGCATCTGAAT
H F Y D Y I S D C W N W K V F P K A R F A S E Y

ATGGATATCAGTCCTGGCCGTCCTTCAGTACATTAGAAAAGGTCTCGTCTACAGAGGACTGGTCTTTCAA
G Y Q S W P S F S T L E K V S S T E D W S F N

TAGCAAGTTTTCACTTCATCGACAACATCACGAAGGTGGTAACAAACAAATGCTTTATCAGGCTGGACTT
S K F S L H R Q H H E G G N K Q M L Y Q A G L

CATTTCAAACTCCCCCAAAGCACAGATCCATTACGCACATTTAAAGATACCATCTACCTTACTCAGGTGA
H F K L P Q S T D P L R T F K D T I Y L T Q V M

TGCAGGCCCAGTGTGTCAAAACAGAAACTGAATTCTACCGCCGTAGTCGCAGCGAGATAGTGGATCAGCA
Q A Q C V K T E T E F Y R R S R S E I V D Q Q

AGGGCACACGATGGGGGCACTTTATTGGCAGTTGAATGACATCTGGCAAGCTCCTTCCTGGGCTTCTCTT
G H T M G A L Y W Q L N D I W Q A P S W A S L

GAGTACGGAGGAAAGTGGAAAATGCTTCATTACTTTGCTCAGAATTTCTTTGCTCCACTGTTGCCAGTAG
E Y G G K W K M L H Y F A Q N F F A P L L P V G

GCTTTGAGAATGAAAACACGTTCTATATCTATGGTGTGTCAGATCTTCACTCGGATTATTCGATGACACT
F E N E N T F Y I Y G V S D L H S D Y S M T L

CAGTGTGAGAGTCCATACATGGAGCTCCCTGGAGCCCGTGTGCTCTCGTGTGACTGAACGTTTTGTGATG
S V R V H T W S S L E P V C S R V T E R F V M

AAAGGAGGAGAGGCTGTCTGCCTTTATGAGGAGCCAGTGTCTGAATTGCTGAGGAGATGTGGGAATTGCA
K G G E A V C L Y E E P V S E L L R R C G C T

CACGGGAAAGCTGTGTGGTTTCCTTTTACCTTTCAGCTGACCATGAACTCCTGAGCCCGACCAACTACCA
R E S C V V S F Y L S A D H E L L S P T N Y H

CTTCTTGTCCTCACCGAAGGAGGCCGTGGGGCTCTGCAAGGCGCAGATCACTGCCATCATCTCTCAGCAA
F L S S P K E A V G L C K A Q I T A I I S Q Q

GGTGACATATTTGTTTTTGACCTGGAGACCTCAGCTGTCGCTCCCTTTGTTTGGTTGGATGTAGGAAGCA
G D I F V F D L E T S A V A P F V W L D V G S I

TCCCAGGGAGATTTAGTGACAATGGTTTCCTCATGACTGAGAAGACACGAACTATATTATTTTACCCTTG
P G R F S D N G F L M T E K T R T I L F Y P W

GGAGCCCACCAGCAAGAATGAGTTGGAGCAATCTTTTCATGTGACCTCCTTAACAGATATTTACTGAagg
E P T S K N E L E Q S F H V T S L T D I Y *

aatctaggttgtattttcagtggacaatgggaataaagcatttctaaagcaccgactggagaggaaggca
acagagacaaggagagaagccgagagacatgtctgcgtgctgccacgcatttgagcgattgctctgtgaa
gagttgtacactgaacacttccaggggaggctgtttacccaggcaatgtcctcaaacaagcctgtgccgg
ggttgtcctggaatctgtgccaggactgtgtttttagcccttcacctctcagctttagcaggacatgaac
cagttataacaagatggccctgcagctggttacaagaatgtgacatggcaggatctatggaaccaaatgg
aaggttttgaggtgatgtaggtctttcacagctagctttggggaatacggaatactcaaataaagtgctt
tgttattatttcagagggaatggcgattgaaatgttacaacagagatttcttggtggtagctatttgggt
aaaggtatatgggtatttttctgtacatgtgaattatataaaaataaaagttatataaattacattgaca
actaaaaaaaaaaaaa 3305

1750

1820

1890

1960

2030

2100

2170

2240

2310

2380

2450

2520

2590

2660

2730

2800
2870
2940
3010
3080
3150
3220
3290

49

initiation codon (Kozak, 1987), at nucleotide position 88, was assigned as the
translation initiation site based on homology to the bovine and caprine initiation
sites. Eight potential Asn-linked glycosylation sites are found in the predicted coding
sequence (Figure 3). Three consensus sequences for polyadenylation are found in
pHUBM8, at positions 2762, 3139 and 3263. The AAUAAA sequence at position
3263 is followed by a 12 base poly A stretch.

Expression Analysis

Northern blot analysis of poly A (+) RNA isolated from various human
tissues performed using hu 82/89 probe revealed the presence of a unique 3.7 kb
transcript that was expressed in all the tested tissues, including heart, brain, placenta,
lung, liver, skeletal muscle, kidney and pancreas (Figure 4A). The mRNA levels
varied in the examined tissues. After the hybridization signals were normalized by
probing the same blot for the expression of B-actin (data not shown), B-mannosidase
mRNA found to be most highly expressed in pancreas, followed by placenta and
kidney, with lower expression levels found in liver, lung, brain, heart, and muscle
(Figure 4B). Overexposure of the blot showed no evidence of multiple sized
transcripts in any of the tissues studied.

Chromosomal Assignment and Regional Localization of Human [3-
Mannosidase

To verify previous assignment of the human B-mannosidase gene to

chromosome 4 (Chen et al., 1995; Fisher et al., 1987), a specific PCR test that only

50

amplifies human genomic DNA and not rodent DNA was developed. Amplification
of DNA from a panel of somatic cell hybrid cell lines produced the correct size
fragment only in the cell line that contained human chromosome 4. To further
localize and identify the chromosomal region containing the B-mannosidase gene,
the test was conducted on a series of somatic cell hybrid lines harboring various
deletions in overlapping regions of chromosome 4 (Figure 5). The presence of the
diagnostic PCR product in NA10115, NA11447, NA13402 and not in NA11449,

and NA13396, localized the human B-mannosidase gene to 4q21-25.

51

Figure 4 Northern Blot Analysis. A: Northern Analysis of 2pg/lane poly A+
(Clontech) from pancreas, kidney, muscle, liver, lung, placenta, brain, and heart,
hybridized with hu 82/89. A single 3.7 Kb, is present in all the lanes. B: the relative
amounts of the B-mannosidase messege in the tissues as dterrnined by quantitating
the intensities of the bands in fluorograms of the Northern blot using
phosphoimager.

Kb

9.5 -'
7.5 -‘:

4.4 _‘

2.4 -,

_
5
3.
1

 

 

25-

m A. w 5

=_u0<um_\¢mflfi_m0=cm5_um_

too:
£an
mucous?—
was;
33..
233.2
>22!

23.8ch

53

Figure 5 Localization of the B-mannosidase gene to chromosome 4q21-25.
Human-rodent hybrid cell lines containing various deletions of chromosome 4 were
PCR amplified using primer pair BHlSO/ 156 from the 3’terminal exon. The region
of human chromosome4 retained in each cell line is represented by the vertical lines
parallel to the schematic diagram of chromosome4. The expected size product, 276
bp, is present in lanes NA10115, NA11447, NA13402 and the human genomic
DNA, and absent in lanes NA11449, 13396 and hamster genomic DNA.

NA10115

NA11449
NA11447

NA13396
NA13402

 

54

D 'o

212
39L

Auk); .3 u

wgwwwww N N NNN NM N—I—t—I—I—t—x—k—ta .4 .x
a: wN—IOCO co \1 mouse)». ewwN—swwbgn 91 en

 

 

 

 

Marker

NA10115
NA11449
NA11447
NA13396
NA13402
Human
Hamster
Water

55

Discussion

The composite human B-mannosidase cDNA sequence obtained from cDNA
clones, RT-PCR, 3’RACE and 5' RACE contains 3300 nucleotides, consisting of 87
nt 5' UTR, 2640 nt coding region and 553 nt 3’ UTR. The homology of the coding
region to goat and bovine B-mannosidase identifies the sequence as human [3-
mannosidase. The conserved size of the coding region between the human, the
caprine and the bovine and the 75% protein sequence identity (Leipprandt et al.,
1996; Chen et al., 1995) reflect a high degree of evolutionary conservation for the
enzyme. The human open reading frame codes for 879 amino acid residues starting
with a typical signal peptide sequence (von Heijne, 1986). The predicted human B-
mannosidase sequence contains eight potential N-glycosylation sites, in contrast to
six and four potential sites predicted for the bovine and the caprine protein
(Leipprandt et al., 1996; Chen et al., 1995). Three of these sites are shared between
all three species (Figure 3). The reported sizes for native human B-mannosidase
range from 57 kDa to 160 kDa (Guadalupi et al., 1996; Iwasaki et al., 1989), and
subunit composition is variable. B-Mannosidase purified from bovine kidney is 100
and 110 kDa (Sopher et al., 1993), while the goat kidney enzyme is 90 and 100 kDa
(Sopher et al., 1992). Deglycosylation of the ruminant enzymes yields two peptides
of 91 and 86 kDa in each species (Sopher et al., 1993; Sopher et al., 1992). The

degree of glycosylation of the human enzyme is not known.

56

The 3’ untranslated region (3’UTR) of the human transcript is shorter than
the corresponding region in the bovine mRNA mainly due to stretches of missing
bases in the human 3’UTR. This size difference is consistent with northern analysis
which identifies a 3.7 kb human transcript compared to 4.2 kb in ruminants (Chen et
al., 1995). There is no evidence for multiple transcripts although several potential
polyadenylation sites are present in the human 3’ UTR. The northern analysis also
demonstrates that there is a variation in mRNA levels in these tissues that is
consistent with studies of enzyme activity levels in goat tissues (Lovell et al., 1994).

Previous studies by Fisher et al., 1987 (Fisher et al., 1987), and Chen et al.,
1994 (Chen et al., 1995) indicated that B-mannosidase is located on chromosome 4.
The current study using somatic cell hybrids with deletions of chromosome 4 refines
the chromosomal localization to 4q21-25. This is in agreement with the assignment
of mouse B-mannosidase to chromosome 3 (Lundin, 1987) which is homologous to
human 4q22-28 (O'Brien et al., 1993), and to bovine B-mannosidase on chromosome
6q3 (Schmutz et al., 1996). Homology of synteny between human and mouse would
suggest that the human chromosomal region containing the B-mannosidase gene

could be narrowed to 4q22-25.

CHAPTER 3

PRODUCTION OF FULL LENGTH CDNA CLONE

Introduction

cDNA clones are valuable tools for the characterization of proteins. They are
used for variety of reasons, including confirming the identity of the cloned genes,
expressing large quantities of required proteins, and studying the biosynthesis and
intercellular transport of proteins. The identification of genes encoding various
lysosomal enzymes and the subsequent cloning of their cDNAs followed by protein
expression has been very important in understanding synthesis, maturation and
transport of lysosomal enzymes. The study of mutant enzymes contributed to our
understanding of the molecular basis of the clinical heterogeneity that is commonly
associated with lysosomal storage diseases (Neufeld, 1991). The objective of this
section is to describe methods to produce a full length human B-mannosidase cDNA
clone. Two experimental approaches were chosen for this purpose, cDNA synthesis
using a gene specific primer, and PCR amplification of the entire coding region
using a high fidelity enzyme mixture.

The enzyme Superscript reverse transcriptase (Gibco BRL) was used to
construct a cDNA library with B-mannosidase specific primer. Reverse

transcriptases in general have high fidelity, however, the production of full length

57

58

cDNA is sometimes difficult because of the propensity in vitro of the reverse
transcriptase to stop before it has reached the 5' end of the mRNA (Lewin, 1995).
The enzyme Superscript reverse transcriptase (BRL) is a novel enzyme that has been
genetically engineered from the cloned Moloney murine leukemia virus (M-MLV)
reverse transcriptase. The enzyme lacks the RNase H activity thus improving the
processivity of the enzyme. Previous results from using this enzyme (RT-PCR
section in Chapter 2) indicated that firll length can be achieved.

Amplification of DNA fragments by polymerase chain reaction has become
an important and widespread tool of genetic analysis since the introduction of
thermostable Taq (T hermus aquaticus) DNA polymerase. Two limitations to the
method are the fidelity of the final product and the size of the DNA span that can be
amplified (Saiki et al., 1988). Taq introduces a base change at a rate of about 1.1 x
10’4 (Barnes, 1992; Tindall and Kunkel, 1988). The fidelity problem has been
addressed by the replacement of Taq DNA polymerase with enzymes that exhibit an
integral 3’-5’ exonuclease activity that apparently reduces the mutation per base pair
per cycle, e. g. the enzyme Pfu (Pyrococcus filriosus) DNA polymerase (Lundberg et
al., 1991) and Vent (Thermococcus litoralis) (Cariello et al., 1991). The length
limitation for PCR amplification by Taq polymerase is thought to be caused by low
efficiency of extension at the sites of incorporation of mismatched base pairs
(Barnes, 1994). Enzymes with 3’-5’ exonuclease activity fail to produce long spans

and high yield on their own, however, low levels of 3’-5’ exonuclease are sufficient

59

and optimal for removal of the mismatch to allow the Taq polymerase amplification
to proceed. The optimal low level of 3 ’-5’ exonuclease activity can be set by mixing
and dilution rather than by mutation (Barnes, 1994).

In order to amplify the coding region of human B-mannosidase, LA-PCR (
long and accurate PCR) method was used. This method was originally described by
Barnes, 1994 in which he used a mixture of Pfu and Taq polymerase (Barnes, 1994).
The Expand High Fidelity PCR system (Boehringer Mannheim), a commercially
available LA-PCR system, was used to amplify the coding region. The enzyme
mixture consists of the therrnostable enzymes Pwo (which possesses an intergral 3’-
5’ exonuclease activity) and Taq DNA polymerase and has an improved fidelity of

DNA synthesis in vitro (Barnes, 1994).

60

Materials and Methods

1- cDNA Synthesis/ Primer Extension
All the enzymes and the buffers are supplied by the Superscript TM Choice System
for cDNA Synthesis kit (Gibco BRL). Unless otherwise stated, the procedure was
performed according to the manufacturer’s protocols. The total RNA used for the
cDNA synthesis is a human placenta total RNA (Clontech). The first strand
synthesis was done using Sug of total RNA and 10 pmoles of gene specific primer
BH156 (Table 1), 101 of (1321) dCTP (10 uCi /ul, 3000 Ci /mmol specific activity)
and 1000 units of Superscript II in a 20 ul reaction, incubated at 42°C for 1 hour.
The second strand synthesis was performed by nick translation replacement of the
mRNA. The reaction was catalyzed by E. coli DNA polymerase in combination with
E. coli RNase H and E. coli DNA ligase at 16°C for 2 hours. To insure that the
termini of the cDNA were blunt ends, the reaction mixture was incubated with T4
DNA polymerase, at 16°C for 5 minutes. The reaction mixture was then subjected to
organic extraction with phenol: chloroform: isoamyl alcohol (25:24zl), followed by
ethanol precipitation. The blunt ended product from the first and second strand
synthesis was ligated to EcoRI (Not I) adapters by T4 DNA ligase at 16°C overnight
in a final 50 ul reaction. The EcoRI-adapted cDNA was phosphorylated with T4

DNA polymerase, at 37°C for 30 minutes. The resulting product was then size

61

fractionated by the Superscript choice system l-ml, disposable columns, prepacked
with Sephacryl S-SOO HR. The column chromatography was designed to eliminate

cDNA < 500 and enrich the yield with larger cDNA.

IA- Column preparation

The column was washed four times with 0.8 ml of TEN buffer [IOmM Tris-
HCl (pH 7.5), 0.1 mM EDTA, 25mM NaCl; autoclaved]. The column flow rate was
: (I) wash #1 = 800 pl in 16 minutes, (2) wash #2 = 790 pl in 15 minutes, (3) wash
#3 = 780 pl in l6minutes, (4) wash #4 =790 ul in 13 minutes. Ideally the flow rate
should be 800 ill in 15 minutes.

IB- Size fractionation

The Cerenkov counts (cpm) were estimated using Model 3 survey meter
(Giger counter) from Ludlum Measurements, INC. Table 5 illustrates the size of the
fractions of the cDNA obtained and total counts per minutes for each fraction.
Fractions 7 - 16 were pooled, ethanol precipitated and resuspended in 20 ul H20 for
subsequent vector ligation.

IC- Ligation of cDNA to plasmid vector

Ready-To-Go pUC18 (Pharrnacia), is linearized pUC18 treated with bacterial
alkaline phosphatase (BAP) and formulated with T4 DNA ligase. The mixture is
lyophilized and stable at room temperature. The cDNA from the above reaction was

added to the content of one tube, and incubated for 3-4 minutes at room temperature.

62

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 5 Size fractionation results

Fraction Total Cerenkov Counts

No volume volume (cpm)
(111) (141)

1 78 0

2 144 222 0

3 20 242 0

4 25 267 O

5 36 303 O

6 25 328 0.8

7 22 350 2.2

8 29 379 4

9 30 409 4.2

10 33 442 4,5

11 24 466 4.6

12 23 489 4.6

13 24 513 4.3

14 19 532 3.2

15 28 560 3.5

16 26 586 3

17 14 600

18 15 615

19 31 646

 

 

 

 

 

 

63

The contents of the tube were then centrifuged and incubated at 16°C for 30
minutes.

ID- Transformation and cDNA screening

Seven transformation reactions were performed. In each reaction 2 ul from
the above ligation reaction were used to transform 100 pl of DHSa competent cells
(GibcoBRL) according to the manufacturer’s protocols. The resulting reaction was
diluted 1:10 in SOC media. The cells were plated on IPTG/XGAL LB-Amp 100
11le plates for blue white screening (Sambrook et al., 1989).

3. Initial screening : An initial analysis of one transformation reaction
was done to determine appropriate method for screening the remaining
transformation reactions. About 40% of one transformation reaction was spread on
lPTG/XGAL LB-Amp 100 ug/ml plates, and 20 random recombinant clones were
selected and grown in 3 ml LB/ampicillin 100 rig/ml liquid culture media. Plasmid
DNA purification was performed using Promega’s Wizard miniprep kit. A standard
EcoRl restriction digestion was performed on these clones for insert analysis.

b. Colony screening: The remaining 6 reactions were divided into
two sets, each differed only in the method of colony transfer to the nylon membranes
(S&S Nytran membranes, 0.45 pm pore size, Shleicher & Schuell) . For one set, the
nylon membrane was allowed to come in contact with the bacterial colonies surface

for 3 minutes for the first filter and for 5 minutes for the duplicate filter, with no

64

further amplification for the transferred clones on the membranes. The total number
of the plates from this transfer was 9 (total number of clones was about 500). For the
second set, colonies were manually picked and transferred simultaneously to a
master plate that has been prepared to accommodate 100 clones, and to a duplicate
set of nylon membranes that have been prepared in a similar manner. The master
plates, as well as the nylon filters that had been placed on LB/Amp 100 ug/ml agar
plate, were incubated for 6 hours at 37°C. The total number of plates from this set
was 10 ( total number of clones is 1,000).

DNA isolation and immobilization was done according to manufacturer
protocol (Shleicher & Schuell). Removal of the bacterial debris was performed
according to Sambrook et al. 1989 (Sambrook et al., 1989) with 5X SSC, 0.5X SDS,
1mM EDTA pH 8.0 for 30 minutes at 50°C.

c- Hybridization conditions: Hybridization of the nylon membranes
was done using the following conditions. The filters were prehybridized in 50%
formamide, 3X SSPE, 3X Denhardt’s solution, 0.5% SDS and 100 pg/ml
fragmented, denatured low molecular weight DNA for 2 hours. The probe, hu 82/89,
was labeled with or 32 P dCTP, using Random Priming Labeling kit by (Boehringer
Mannheim) to high specific activity. The probe was denatured and added to the
prehybridization mix and the reaction was incubated at 42°C for 20 hours. The filters

were washed with 2X SSC / 0.1% SDS two times for 15 minutes at room

65

temperature, and one time with 0.5X SSC / 0.1 % SDS for 40 minutes at 48°C. The
filters were exposed to Kodak X-ray films for two days at -80°C.

d- Southern blot analysis: Overnight bacterial liquid cultures 100
ug/ml LB/Amp of the selected putative positive clones were grown in 37°C shaking
water bath. The plasmid DNA was isolated with the Promega’s Wizard Miniprep kit.
An EcoRI standard restriction digestion was performed, including pHUBM8 as a
positive control. The fragments were electrophoresed on 1% agarose gel. The DNA
was transferred to a nylon membrane (Hybond-N, Amersham) using a standard
Southern blot protocol (Sambrook et al., 1989). The DNA was hybridized to
hu82/89, labeled as above. The hybridization mixture contained 50% formamide,
6X SSPE, 5X Denhardts solution, 0.1% SDS and 100 pg/ml herring sperm DNA.
The denatured probe was added to the prehybridization solution after 2 hours at
42°C, and allowed to hybridize for 18 hours at 42°C. The washing conditions were

similar to those described in the above section.

II- PCR Cloning of the Full Length RT-PCR Product
IIA. cDNA Synthesis Using Oligo dT Primer

To prepare a template for the PCR reaction, a first strand cDNA was
synthesized using an oligo d(T) primer. First strand synthesis: 5 ug of human

placenta total RNA (Clontech) and 1 ul oligo (dT) (500 ug/ml), was heated for 10

66

minutes at 70°C and quick chilled on ice. The following reagents were added to the
reaction mixture, 4 pl of 5X first strand buffer (Gibco BRL), 2 pl DTT, and 1 pl of
10 mM dNTPs mix. The temprature was equilibrated to 42°C for two minutes before
adding 5 pl of Superscript RT (1000 units). The reaction was incubated at 42°C for
50 minutes and then the enzyme was inactivated by heating at 70°C for 15 min. The
first strand yield was ethanol precipitated, and resuspended in 40 pl of sterile H20 to
be used directly in the PCR reaction.

[18. Primer Design

To produce a full length human B-mannosidase cDNA that can be directly
cloned with regard to its transcriptional polarity, into some commercially available
expression vectors, i.e., pSVL or pCDNA3, new primers flanking the coding region
were designed with introduced restriction sites. The sense primer, KF177, is a 21
mer and corresponds to the region from bp 47 to 67 in the human B-mannosidase
cDNA (5’ CCT ITC GAI CTC TCC ACA TCT 3’). The underlined nucleotides, T
and T, at position 50 and 55 were changed to C and G respectively to create a XhoI

( CJrTCGAG) restriction site. This site is 32 bp upstream of the translation

initiation codon.

The antisense primer, KF179, was a 23 mer, (5' GTT GCC TTC CTC TCQ
AGI CGG TG 3') and it corresponds to the region from 2802 to 2780 in the human

B-mannosidase cDNA. The underlined bases, C and T were changed to T and A

67

respectively to create a XbaI restriction site (TiCTAGA). This site is 57 bp
downstream of the stop codon.

IIC. PCR Using the Expand High fidelity Enzyme System

PCR reaction conditions: Two reaction mixtures were prepared

separately on ice prior to mixing for the PCR amplification: Mixture #1 contained
200 mM final concentration of dNTPs, 300 nM each primer, KF 177 and 179, 2 pl
of cDNA in 25 pl volume. Mixture #2 contained 5 pl of 10X Expand buffer with 15
mM MgC12, 2 p1 of the Expand High Fidelity PCR system enzyme mix in 25 pl final
volume. PCR amplification cycles: initial elongation cycle for 7 minutes at 95°C,
followed by 10 cycles of 40 seconds at 95°C, annealing for 30 seconds at 60°C,
elongation for 2 mintues at 68°C, followed by 15 cycles with same conditions as the
above in addition to 20 seconds extra in each elongation cycle. A final elongation
cycle was performed for 10 minutes at 68°C (Perkin Elmer 9600 thermocycler). The
PCR Products were analyzed on a 0.8% Agarose gel / 0.5X TBE. The expected size
product was excised and gel purified using Promega’s Wizard PCR Preps Kit,
immediately after the completion of the PCR reaction in order to avoid the 3'-5'
exonuclease activity of the enzyme Pwo. The yield was suspended in 20 p1 of sterile
H20.

IID. Fragment Ligation and TA Cloning

TA cloning is a fast and a convenient method of cloning PCR products for

68

propagation and sequence characterization (Mead et al., 1991; Clark, 1988). The
cloning vector pCRII was supplied by Invitrogen Corporation TA cloning kit. The
lyophilized TA cloning vector was resuspended in 8.8 pl TE buffer to a
concentration of 25 ng/pl. A total of 50 ng of the pCRII vector was used with 6 pl
from the isolated PCR fragment in a ligation reaction with 1 pl of 10X ligation
buffer and 1 pl of T4 DNA ligase (Boehringer Mannheim). The reaction was
incubated at 4°C overnight. Five microliters from the ligation mixture were used to
transform 100 p1 of DHSa (Gibco BRL) competent cells according to the suggested
protocol. After the transformation reaction, the cells were grown in 1 m1 of SOC
media for 1 hour at 37°C, diluted 1:2 with SOC and plated on IPTG/XGAL

LB/Amp plates for blue-white screening. Selected recombinant clones were grown
in 3 ml liquid culture media LB/Amp 50pg/ml at 37°C overnight. Plasmid
purification was performed using Promega’s Wizard miniprep kit. The plasmid
DNA was suspended in 50 p1 sterile H20. A standard EcoRI restriction digestion

was performed. The clones were sequenced in MSU sequencing facility, using ABI

373A DNA sequencer.

69

Results
cDNA synthesis using a gene specific primer

The initial transformation reaction resulted in unexpectedly a high number of
recombinant clones. EcoRI restriction analysis of the random clones revealed that all
the analyzed clones were apparently empty, since only one 2.8 kb fragment
corresponding to pUC18 vector DNA was visible. These results, indicating a
contamination of cDNA preparation with EcoR I linkers, necessitated the need to
screen large number of clones. The primary screening of about 1,500 clones by
hybridization to hu 82/89 probe resulted in the identification of 42 weakly positive
clones. Plasmid purification of these clones and EcoR I digestion analysis revealed
that only 5 clones had inserts ranging in size from 400 to 900 bp. However, Southern
blot analysis of these inserts using the same probe, hu 82/89, was negative. No
further analysis was performed on these clones.
PCR amplification with High Fidelity Enzyme

On the other hand, PCR amplification of the coding region using two 0-
mannosidase specific primers produced an expected size fragment of about 2.7 kb.
Ligation of the PCR product into a pCRII vector and subsequent transformation
resulted in about 40 recombinant clones. Fourteen random recombinant clones were
selected for further analysis. Three clones contained a 2.7 kb insert shown by Xhol
and XbaI restriction digestion (Figure 6). All the other clones were apparently empty.

EcoRI analysis of these three clones resulted in three fragments of approximately

70

1200, 800 and 700 bp long (Figure 6). The size of these fragments correspond to the
expected sizes that would be produced from a human B-mannosidase cDNA based
on the restriction map derived from the consensus sequence.

The three clones were further analysed by sequencing with M13 forward
(Figure 7) and reverse (Figure 8) primers. The initiation start codon and the stop
codon were verified in all three clones. The XbaI restriction site introduced in the
antisense primer was verified in the three clones, whereas the X1101, the site
introduced in the sense primer, was only present in clones #3 and #9 and not in
clone#13. However, the human B-mannosidase coding region in clone #13, is still
encompassed by XhoI and Xbal at the 5' and the 3' end respectively because a vector
XhoI site is found 114 bp upstream from the ATG initiation codon.

Alignment of clone #13 sequence with both vector sequence and human B-
mannosidase cDNA sequence has revealed that there are sequences missing from
both ends of the cDNA sequence, as well as from both ends of the vector arms.
Overall, there are 8 bases missing corresponding to the 5' end of the sense primer,
which are CCT CTC GA, and 3 bases missing from the 3'end of the PCR amplicon.
In the vector, there were 4 bases missing from each end of the vector arms.

The sequence results also reveal sequence variations in the coding region
from the consensus human B-mannosidase. Clone #3 shows G to A transition at

position 2585 that causes an Arginine substitution for Glycine, and a neutral A to G

71

at position 2637 that codes for no amino acid substitution (Figure 7). Clone #13 also
has a neutral C to T at position 331 in the Serine codon (Figure 8). However, the
sequence obtained for clone #9 so far, indicates that there is no sequence variation

from the normal human B-mannosidase coding region consensus sequence.

72

 

Figure 6 cDNA clones analysis Lanes 1, 3, and 5: Xba I, Xho I digest
of clones 3 (1), 9(3) and 13 (5). Lanes 2, 4, and 6: EcoRI digest
of clones 3 (2), 9 (4) and 13(6). MW is 0.25 pg of MW 111 (BMB)

73

Figure 7 Sequence aligment of the cDNA clones with the 5’end of the consensus
human B-mannosidase cDNA sequence. Sequence alignment of the human B-
mannosidase cDNA consensus sequence (from bp 47 to 437) with cDNA clones #3,
#9, and #13 sequenced with M13 forward primer (only the differences are shown).
Hucdna is the human B-mannosidase cDNA consensus sequence, the arrow shows
the position of the sense primer with the mutated sequences shown underneath the
the original sequence to introduce Xho] restriction site, the posistion of the ATG
translation initiation is shown and the circle around the T indicated the base change

inclone#13.

74

 

 

 

 

 

KF177 6O 80 100
mxflna mTTTI ATCTC CCACATCTCGGTGGCGCGGGATCTCAAGATGCG TCCACCTGCTCC
ta#3 G
ta#9 G
ta#13

120 140 160
Sfflifi

11311

'1" J '—
10 '3‘.

- TGCTGCTCGCGCTGTGCGGTGCAGGCACCACCGCCGCGGAGCTCAGTTACAGCTTGCGTG

100 180 200 220
rim“ . . . c o o

3111. l.

“h, - GCAACTGGAGCATCTGCAATGGGAACGGCTCGCTGGAGCTGCCCGGGGCGGTCCCTGGCT
3 All '

#0? 240 260 280

- GCGTGCACAGCGCCTTGTTCCAGCAGGGCCTGATCCAGGATTCTTACTACAGATTTAATG

300 320 340

- ACCTTAACTACAGATGGGTCTCTTTGGATAACTGGACCTATAGCAAAGAATTTAAAATCC

360 380 f 400

- CCTTTGAAATTAGCAAATGGCAAAAAGTAAATTTGATTCTTGAGGGAGTGGATACGGTTT

420

- CAAAAATCCTGTTCAATGAAGTCACTATTGG

75

Figure 8 Sequence alignment of the cDNA clones with the 3’ end of the human
B-mannosidase cDNA consensus sequence Sequence alignment of the human B-
mannosidase cDNA (from bp 2414 to 2802) with cDNA clones #3, #9 and #13
sequenced with M13 reverse primer. Hucdna is the human cDNA sequence, the
arrow shows the position of the antisense primer with the mutated base pairs to
introduce the Xba] restrction site shown underneath the original sequence, the
position of the stop codon TGA is indicated. The base changes in the coding region
are shown in lines 3 and 4

hucdna
ta#3
ta#9
ta#13

76

2430 2450 2470

CAGCTGACCATGAACTCCTGAGCCCGACCAACTACCACTTCTTGTCCTCACCGAAGGAGG

2490 2510 2530

CCGTGGGGCTCTGCAAGGCGCAGATCACTGCCATCATCTCTCAGCAAGGTGACATATTTG

2550 2570 2590

TTTTTGACCTGGAGACCTCAGCTGTCGCTCCCTTTGTTTGGTTGGATGTAGGAAGCATCC

2610 2630 2650

CAGGGAGATTTAGTGACAATGGTTTCCTCATGACTGAGAAGACACGAACTATATTATTTT

2670 2690 2710

ACCCTTGGGAGCCCACCAGCAAGAATGAGTTGGAGCAATCTTTTCATGTGACCTCCTTAA

2730 2750 2770

 

CAGATATTTACTGAAGGAATCTAGGTTGTATTTTCAGTGGACAATGGGAATAAAGCATTT

 

 

 

2790 KF179

A
V

CTAAAGCACC!ACTGGA-AGGAAGGCAAC

 

77

Discussion

Three independent clones that contain the coding region for human [3-
mannosidase were produced by PCR amplification using a high fidelity enzyme mix.
Although the Xho], restriction site in the cDNA insert was missing in clone #13, it
can be replaced by the presence of a vector Xho] site (as shown by the restriction
digestion of clone #13 with XhoI-Xbal Figure 6) making it possible for the
directional cloning of the three cDNA inserts into commercially available expression
vectors. The results of clone 13 sequencing, reveals that it was a blunt end ligation
event rather than a TA ligation. The missing sequences in the cDNA, can be
attributed to the effects of the 3' - 5' exonuclease activity of Pwo enzyme. On the
other hand, the missing sequence from the vector is less likely to be attributed to the
same reason, since the insert was purified by electrophoresis in agarose gel prior to
its isolation. It is possible, however, that the vector sequence was degraded from
both ends as a time related factor, which may not be unusual.

The identified sequence variation in the clones can either be polymorphism or
amplification introduced errors. This may be true for the silent changes, however, it
might not be the case for the significant amino acid substitution in one clone
(clone#3). To fully asses the validity of this method for cDNA cloning, complete
sequence information is necessary as well as expression data from the clones is
needed.

The failure of the method of primer extension for the cDNA synthesis was

78

probably due to contamination of linkers in the final cDNA preparation that was
used for ligation, hence the large number of recombinant clones. The apparently
empty clones also support this hypothesis. The kit was designed for the construction
of cDNA with either oligo d(T) or random hexamers primers, thus most of the
conditions were optimized for higher product concentration. The resulting clones
might have arisen from non-specific annealing of the primer to the mRNA. The

clones were not further analyzed because the Southern blot results were negative.

CHAPTER4

MUTATION ANALYSIS OF TWO B-MANNOSIDOSIS PATIENTS

Introduction

Lysosomal storage diseases have considerable heterogeneity of clinical
symptoms. Advances in mutation detection techniques and in cellular and
biochemical methods, have made evident the molecular basis of disease
heterogeneity. There are thirteen reported cases of human B-mannosidosis revealing
heterogeneous disease manifestations and widespread ethnic distribution. This study
reports mutation analysis for two occurrences of B-mannosidosis, the frrst is a patient
described by Wenger et al. 1986, and the second is a gypsy family with two affected
siblings, described by Klijer et al. 1990.

The patient described by Wenger et al, 1986 was a four year old boy who had
coarsening facial features, mild bone disease, delayed speech development,
hyperactivity and mental retardation. The age of onset was at one and a half years
when his parents became concerned about his speech development. The parents are
from the United States of America, unrelated and both are from European ancestry.
This patient had yet another lysosomal enzyme deficiency, heparin sulfamidase
which causes Sanfilippo syndrome type A. However, complementation studies

showed that the double deficiency was due to mutations in different genes (Hu et al.,

79

80

1991). Because of the combined deficiency, the patient’s karyotype was analysed to
determine whether gross chromosomal rearrangement was present. The possiblity of
chromosome 4 isodisomy was investigated by examining short sequence repeat
(SSR) polymorphic markers covering chromosome 4. The patient’s cDNA was
analyzed for aberrant splicing or for sequence variation in the coding region. As this
patient comes from non-related parents, it was hypothesized that he was a compound
heterozygote at the B-mannosidase locus.

Two affected siblings from a gypsy family reported by Kleijer et al, 1990
have heterogeneous manifestations of the disease, although both showed no
evidence of enzyme activity (Kleijer et al., 1990). The sister suffered from severe
psychomotor retardation, bone deformities and gargoylism and recurrent skin and
respiratory infections. This patient had an early onset, was hypotonic and severely
mentally retarded. However, she had an elder brother who had a milder facial
dysmorphology, mental retardation and hearing loss. These patients’ cDNA was
examined to detect abnormal splicing or sequence variation in the coding region.

The possiblity of leaky mutation was also investigated.

81

MATERIALS AND METHODS:

Mutation analysis:

Cell culture: Fibroblast cell lines were obtained that were derived from the patient
described by Wenger et al., 1986 (will be reffered to as Wenger cell 1ine)(Wenger et
al., 1986), and from a Czech family described Kleijer et al., 1990 (will be reffered to
as Klejier cell lines) (Kleijer et al., 1990): parents, two affected children with B-
mannosidosis confirmed by enzyme activity levels and a heterozygote sister. The cell
lines were grown in F10 media (Gibco-BRL), 15% FCS and 1X P/S antibiotics to
confluency and harvested using 0.125 % trypsin, 1X versene and kept as cell pellets
for further use in -80°C. RNA isolation: RNA from cell lines was extracted using
TriZol reagent (GibcoBRL) following the manufacturer’s protocol and kept in
-80°C. Genomic DNA isolation: Genomic DNA from fibroblasts was isolated using
recommended procedure for Puregene DNA isolation kit (Gentra).

Mutation analysis for the Wenger Cell line:

Karyotype anaylsis: The patient’s fibroblast cells were sent for kayrotype analysis
in Michigan State University Cytogenetics Labrotory (courtesy of Dr. Patrick
Storto).

Microsatellite markers analysis: Primer pairs (MapPairs) were purchased from
Research Genetics, Inc. Table 6 lists the locus, the markers ID, type of sequence

repeat, the expected heterozygosity score, and the expected size of the allele

82

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 6 Microsatellite markers

Locus Marker Type Expected Allele
heterozygosity size/bp
score

D482394 ATA26B08 tri 0.79 235-256

D482369 GATA29G09

D4S23 66 GATA22G05 tetra 0.79 120- 144

D4S2431 GGAA19H07 tetra 0.82 234-258

D4 S236 1 ATA2A03 tri 0.74 149- 164

D4S1652 GATA5B02 tetra 0.71 136-148

D4S1627 GATA7D01 tetra 0.81 177-201

D48 1625 GATA107 tetra 0.74 182-210

D4 82623 GATA62A 12 tetra 0.74 205-241

D4S2639 GATA90B10 tetra 0.85 160-192

 

 

83

(Research Genetics). PCR protocol for microsatellite markers: PCR reactions are
performed in a total volume of 50 p1, containing 40 ng of the patient’s genomic
DNA, 50 pmol of each primer, 1.25 mM dNTPs and 1 unit of Taq polymerase
(Perkin Elmer). The initail denaturation step is performed for 5 minutes at 94°C.
Amplification was carried out during 35 cycles of denaturation (94°C for 40
seconds) and annealing (55°C for 30 seconds). The final elongation step (72°C for 2
minutes) was performed at the end.

Polyacrylamide gel electrophoresis: A 6% polyacrylamide/7M urea gel was
prepared by mixing 22.5 mls of 40% (19:1 bis acrylamide-acrylamide), 15 mls of
10X TBE, 63 gm of urea, and adding ddeO to 150 ml total volume. To polymerize,
75 pl of Temed and 75 pl of 25% Ammonium persulfate was added before the gel
was poured. Sample preparation: 2 pl from the PCR reaction was added to 9 p1 of
the loading buffer (10 mls formamide, 10 mg Xylene Cyanol, 10mg Bromophenol
Blue, 200 pl 0.5 M EDTA pH 8.0). The samples were incubated at 100°C for 10
minutes, then chilled on ice for 10 minutes. The gel is nm at 1,500 Volts for the first
10-15 minutes, between 46 and 48°C. The gel was run for another half hour at 1,200
Volts. The gel was stained by silver staining method using the recommended
protocol from BioRad Silver Stain kit. The gel was exposed to a EDF film (Kodak),

and then developed.

84

Mutation scanning and RT-PCR: cDNA synthesis from the patient's total RNA
isolated from fibroblasts, and normal control RNA (Placental total RNA, Clontech)
was performed as described earlier. Primer pairs covering the entire length of the
coding region were used in RT-PCR reactions to scan for size differences in
patient’s cDNA. The mutant RT-PCR fragments were gel purified using Promega’s
Wizard PCR preps and sequenced at Michigan State University sequencing facility.
The mutant cDNA contig was assembled using Sequencher Program (Version 2.1,
Genes Code Corporation).

Mutation test and population screening: To investigate whether the patient is
homozygous or heterozygous for the identified sequence variation and to rule out a
common polymorphism in the population, an AIRS (Artificial Introduction of
Restriction Site) test was developed. Primer design: The nucleotide sequence from
1610 to 1587 (5’ GTA GGA CTG GAC GTA ATA AAA QGA 3’) in the cDNA
consensus sequence was chosen as an antisense primer (KF 228, ClaI primer). The
underlined sequences (A and G) were mutated to (T and C) respectively. PCR
amplification of the mutant allele will result in the introduction of Ga] site
(AMT) while amplification of the wild type allele will produce the sequence
(A'_1"_C_G_AC) that will not be recognized by the ClaI enzyme. However, PCR
amplification of both alleles will result in the introduction of T an restriction site

(TCGA). This will be used as a control for restriction digestion of the PCR

 

85

amplification reaction.

Mutation test for the cDNA: Primer pair KF228 (Cla I primer) and BB103 (Table
1) are used for AIRS test for the cDNA of both the patient and a normal control.
Mutation test for genomic DNA: Primer pair KF228 (Cla I primer) and an intron
primer KF237 (5’ CAG CTA CTT GGC ATT TCA GG 3’) were used for AIRS test
of the genomic DNA. PCR reaction conditions: 50 ng of template genomic DNA
isolated from fibroblast cell lines of the patient and a normal control was amplified
in the following PCR cycles: initial denatuation cycle at 95°C for 5 minutes, 35
cycles of (94°C for 30 seconds, 65°C for 30 seconds, 72°C for 30 seconds), and a
final elongation cycle at 72°C for 10 minutes. The PCR product is then incubated
with Clal and T aql separately with the appropriate restriction enzyme buffer at 37°C
and 65°C repectively. The PCR products are then sepatated on 4% agarose gel in
0.5X TBE.

Population screening: To test whether the identified sequence variation occurrs as a
polymorphism in the population, 181 random genomic DNA samples from the
general population (provided by Dr. Karen Friderici’s Laboratory at the Department
of Pediatrics and Human Development at Michigan State University) were tested
with Clal test using genomic DNA as a template. They consist of the following
different ethnic groups: 108 Caucasians, 19 Asians, l9 Blacks, 20 Hispanics, and 15

Indians.

86

Mutation analysis of the Kleijer Family:

RT-PCR analysis: Five micrograms of total RNA from the five cell lines and from
normal placenta total RNA (Clontech) were reverse transcribed with BH145 for use
in PCR reactions. The following primer pairs were used for the analysis:
BBll8/113, BB96/ BH139, BH137/BB89 and BH150/145 (refer to Table 1 in
chapter 2 for primers sequence and positions). Refer to section RT-PCR in chapter 2
for reaction conditions.

Genomic structure analysis: Primer pair BH137-155 was used to amplify genomic
DNA in the following PCR conditions: initial denaturation cycle at 94°C for 7
minutes: 30 cycles of 45 seconds denaturation, 40 seconds annealing at 65°C and 60
seconds extension at 72°C: and final extension cycle at 72°C for 10 minutes. The
template concentration was between 10-20 ng for a 20 pl PCR reaction. The PCR
product was sequenced and the intron/ exon borders and intron sequence was
identified. A primer, BH160, based on intron sequence was designed for use in
combination with BH155 for the mutation test in similar PCR reaction conditions as
above except that 62°C was used as the annealing temperature.

Mutation test: The restriction digestion analysis was performed using 10 pl from
the PCR reaction, 1X restriction digestion buffer, 10 units of either SmaI or Xma]
(Boehringer Mannheim) in a 20 pl final reaction volume. The reactions were

incubated at room temperature and at 37°C respectively overnight. The results were

resolved in 1% agarose, 0.5X TBE.

87

88

RESULTS
Mutation analysis of the wenger cell line:

No chromosomal abnormalities were detected from the karyotyope analysis.
The microsatellite markers that were analysed were polymorphic, eliminating the
possibility of isodisomy. RT-PCR analysis of the mutant cDNA revealed no size
difference between the mutant and the normal cDNA indicating the possibility of
either a microdeletion, microinsertion or a base-pair substitution.

Sequencing the entire coding region revealed one sequence variation from the
normal cDNA sequence. The base-pair change is a G to A transition at position 1586
in the coding region. The base change predicts a histidine substitution for the
arginine residue number 500. To exclude the possibility that the above sequence
variation is a Taq polymerase introduced error, the region that contain the sequence
variation was PCR amplified again and sequenced from both directions. The
sequence variation was comfrrmed as G to A transition in the second PCR product.

An AIRS test was developed to test whether the identified base change is
homozygous or heterozygous both at the cDNA and the genomic DNA level.
Artificial introduction of restriction sites (AIRS) is a commonly used technique to
screen for mutations in which an appropriate mismatch primer is utilized in PCR
amplification. This method was first introduced by Cohen and Levinson in 1988
(Cohen and Levinson, 1988). The AIRS test on the genomic DNA indicated that the

patient is heterozygous for the identified G1586A allele (Figure 9B). The cDNA test

89

Figure 9 Clal AIRS test for the Wenger cell line. A: Genomic Clal test: Lane 1
patient’s PCR product uncut, lane 2, patient’s Clal digest , lane 3 , patient’s T an
digest , lane 4 normal control PCR product uncut, lane 5, normal Clal digest , and
lane 6 is normal T an digest. B: cDNA Clal test ; the order of the lanes is similar to

A.

9O

 

A Genomic DNA ClaI test

 

B cDNA Clal test

91

showed a faint band that corresponded to the uncut PCR product (Figure 9A). Two
other PCR reactions with different sense primers were performed on the patient
cDNA, showed the same result. All the restriction digestion analyses were
performed with an excess of the restriction enzyme and with overnight incubation.
The AIRS test was performed on 181 random population samples, from the
following ethnic background: 108 Caucasians, 19 African Americans, 20 Hispanics,

19 Asians, 15 Indians. None of the samples tested positive for the G1586A allele.

Mutation analysis of the Kleijer cell line:

To identify the molecular defect causing B-mannosidosis in a Czech family
reported by Kleijer et a1, 1990 (Kleijer et al., 1990), RNA from two affected siblings,
their heterozygous sister and their parents was analyzed. RT-PCR analysis of the
entire coding region was performed. One region did not yield the expected size
product, but instead two shorter PCR products were observed suggesting aberrant
splicing (Figure 10, lane II-8 and II-2). The corresponding region in genomic DNA
was PCR amplified and sequenced to elucidate the genomic structure (Figure 118).
The exon/intron borders of the normal control were identified and compared to
sequence from the affected siblings. The mutation, an A—>G transition was
identified at a 3 ’ splice acceptor site in both patients.

The PCR products A and B (Figure 10 and Figure 11A) were analyzed to

identify the molecular defect caused by the splice site mutation. The larger PCR

92

product (A) lacked 172 bp starting from nucleotide position 2245, corresponding to
the start of an exon immediately downstream of the mutation. This splicing event is
caused by a utilization of a cryptic acceptor site within the same exon. The shorter
PCR product (B) lacked 258 bp, also starting from the same position as product A,
indicating an exon skipping event. PCR analysis on genomic DNA to elucidate the
genomic structure at this region indicated that the region from 2245 to 2552 is an
exon which confirms that this event is an exon skipping. The 172 bp deletion in the
mRNA causes a frame shift and introduces a stop codon truncating the predicted
protein by 155 residues The 258 bp deletion results in an inframe deletion, so that
the predicted protein lacks 86 internal residues. To investigate the possibility that a
low level of full length mRNA is produced, we performed RT-PCR with one primer,
BH138a, located within the deleted region, paired with BH141 (Figure 12). The
reaction is designed to avoid possible competition by the mutant mRNA species,
which if more abundant, would mask the presence of very low levels of normal
mRNA. We did not detect amplification of the predicted normal product using
cDNA from fibroblasts of the patients (Figure 12) suggesting that leaky splicing
does not occur.

This mutation creates SmaI and Xma] restriction sites. PCR amplification of
genomic DNA using primer pair BH160/ BH155 followed by restriction digestion
and gel electrophoresis shows that both affected individuals are homozygous for the

mutation (Figure 13, lane II-8 and II-2), while the parents and the sister are

93

heterozygous for the same mutation (Figure 13, lanes lI-7, I-1 and 1-2).

94

Figure 10 Mutation analysis of the Kleijer Mutation.

A RT-PCR: PCR product from primer pair BH137-BB89, 495 bp, is detected in
lane C, positive control, I-1 father, [-2 mother, and II-7 sister, but not in lanes II-2 or
II-8. The diagram demostrates the abssence of the normal size product, and the
appearence of shorter mutant bands A and B in the two patients, II-2 and II-8. The
three bands aare found in the other family members confirming their heterozygous
status. B1 is the negative control, and M is Molecular marker V from BMB.

B Absence of normal splicing: Results of BH141-138a RT-PCR. the expected
normal size product is absent from lanes II-2 and II-8 and present in all the other
lanes. Primer BH138a is located in the deletion region.

C Mutation test: SmaI digest of the 194 bp fiagment that is produced by PCR
amplification, HB 160-155, of the region encompassing the intron/exon border
where the mutation was identified. Lanes II-8 and II-2 shows complete digestion and
production of 107 and 85 bp fragment confirming the homozygous status of the
identified mutation in these individuals. Lanes II-7, I-1 and I-2 have the undigested
PCR product as well as the digestion products. No digestion is observed in the
normal control genomic DNA lane.

95

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96

Figure 11 Mutation sequence analysis.

A cDNA Sequence: the figure shows the alignment of sequences from HB 137-
BB89 RT-PCR products, A and B (refer to Figure 10) with C, the normal cDNA.
/..../ represents sequence not shown in the figure. The dotted line extending to panel
B reflects the position of the sequence in relation to the genomic structure at that
region. Arrows represent PCR primers.

C Genomic sequence: Sequence alignment from BH137 from both normal and
mutant sequences. Intronsequence are shown in lower case, aand exon sequence are
in upper case. The Sma I site created by the mutation is shown underlined, and the
nucleotide change is shown with an asterisk underneath.

97

 

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98

DISCUSSION
Mutation Analysis for the Wenger Cell Line:

The examination of the patient’s karyotype and chromosome 4 was normal,
indicating the possibility of gene mutation versus chromosomal abnormalities.
Sequence analysis of cDNA of this patient showed a G—>A transition at position
1586 in the cDNA. However, an AIRS test on genomic DNA, revealed that patient is
heterozygous for the G1586A allele. Although the cDNA AIRS test results reveals
the presence of a faint band at the uncut size product, the expression of the second
allele at the mRNA level cannot be determined from this test. These results may
indicate a possible PCR introduced error that is not recognized by Clal enzyme. The
nature of the uncut product that appears in lane 2 in Figure 9A needs to be
determined and analyzed before conclusions can be drawn from this result.

Population analysis of the identified G1586A allele revealed no evidence for
a common polymorphism. However, site directed mutagenesis of the identified base
pair will be required to confirm the effect of this change on the enzyme activity.

The identified mutation is a G——>A transition at position 1586. The mutation
accurs at CpG dinucleotide which is known for its hypermutability in genomes
(Vogel and Kopun, 1977). Mutations at CpG dinucleotide, either C—-)T or G—>A
transitions, represent 90 % of the total single-base substitutions that cause human
genetic disease (Cooper and Youssoufian, 1988). The base-pair change predicts a

histidine substitution for arginine at residue 500. No conclusions can be drawn from

99

this type of substitution, because no data is available on the structure or active site of
any of the characterized mammalian B-mannosidase at this time. However, based on
hydrophobic cluster analysis studies done by Durand et al., 1997 (Durand et al.,
1997) suggested that residues Arg 388, His 532, Tyr 534, Glu 554, Trp 587 are
involved in the enzyme catalytic activity, the Glu 554 being the nucleophile in the
bovine protein. These sites are conserved in human, but there is no experimental data
to support the above hypothesis.
Mutation Analysis for the Kleijer Cell Line

A 3’ splice site mutation was found to cause B-mannosidosis in this family
with severe but heterogeneous manifestation of the disease (Kleijer et al., 1990). The
affected sister had gargoylism, skeletal abnormalities, severe psychomotor
retardation, and recurrent skin and respiratory infections. Her less affected older
brother had coarse faces, moderate retardation hearing impairment and recurrent
infections. The A—->G transition in the AG acceptor site is congruent with a study of
splice site mutations causing human genetic diseases which found that mutations at
the invariant A at position -2 of the 3’ splice acceptor site occur more ofien than
expected based on the dinucleotide mutability (Krawczak et al., 1992). The length of
the pyrimidine tract adjacent to the AG at the 3’ splice acceptor site has been
implicated in the strength of the effect the mutation has on appropriate splicing

(Krawczak et al., 1992). Although the pyrimidine tract adjacent to the AG in the

100

human B-mannosidase intron is 22 bp long, it was insufficient to alleviate the effect
of the mutation. Splice mutations can result in one or a combination of splicing
defects. These defects are exon skipping, activation of cryptic splice site or intron
retention, reviewed in Maquat (Maquat, 1996). The mutation in this family results in
activation of a cryptic site in the exon immediately downstream of the lesion, and

skipping of the same exon. Skipping of the entire exon resulted in an in-frame
deletion and predicted a protein lacking 86 internal amino acids. Further studies will
be required to assess whether this peptide is expressed in these patients. Low levels
of normal mRNA splicing have been encountered with splice site mutations
associated with genetic diseases, which can reduce the severity of the disease (Raben
et al., 1996). Since the disease phenotype differed between the two siblings, it is
possible that a low level of normal splicing may occur in the brother who had

milder manifestations. However, no evidence for normal splicing was observed in
the fibroblasts. The mutation test performed with PCR amplification of the genomic
DNA flanking the mutation site and restriction digestion with either Sma! or Xma]

confirms homozygosity for the mutation in the two B-mannosidosis patients.

SUMMARY AND CONCLUSION

This research has fulfilled the following purposes: (1) characterized the
human B-mannosidase cDNA sequence , (2) identified the tissue expression pattern
of the mRNA in different human tissues, (3) determined the chromosomal
localization of the human B-mannosidase gene, (4) cloned the coding region into
pCRII vector, and (5) identified two mutations causing B-mannosidosis in two cell
lines.

The identification of the coding region of human B-mannosidase gene reflects
a high degree of evolutionary conservation of the gene among several species, i.e.
bovine, caprine, C. elegans, and mouse (EST sequence data from Genbank).
Elucidation of highly conserved domains (although difficult because of the high
degree of conservation for the whole gene) will aid in the identification of the
catalytic domain of the enzyme.

The expression of mRNA is different in several tested tissues, however, no
conclusion can be drawn as to its biological relevance since no data is available
about the pathology of tissues in individuals affected with B-mannosidosis. In
disorders caused by deficiency of some other lysosomal enzymes that demonstrate

tissue specificity for substrate catabolism, such studies help to understand the disease

101

102

pathology.

The chromosomal localization of the human gene to 4q22-25 is in agreement
with the assignment of the mouse and bovine genes.

The available cDNA clones need to be subcloned into an expression vector,
to examine whether an expression of the enzyme activity can be obtained. Full
sequence data for any expressing clone will be required. The cDNA clone can be
very helpful in future site-directed mutagenesis experiments either to confirm the
effect of identified mutations or for protein structure-function studies.

The two mutations at the B-mannosidase gene associated with the loss of
enzymatic activity confirm the identity of the human B-mannosidase cDNA
sequence obtained. The splice site mutation in the gypsy was homozygous as
predicted. It is difficult to come up with an accurate prediction of the resulting
phenotype of this mutation since one sibling was more severely affected than the
other. As for the clinical phenotype for the patient with the two lysosomal enzyme
deficiencies, it is again a difficult case both because of the allelic heterogeneity at the

B-mannosidase locus and because of the deficiency of another lysosomal enzyme.

BIBLIOGRAPHY

BIBLIOGRAPHY

Abbitt, B., Jones, M.Z., Kasari, T.R., Storts, R.W., Templeton, J .W., Holland, P.,
and Castenson, P. (1991). B—Mannosidosis in twelve Salers calves. JAVMA 198,
109-113.

Aronson, N.N.J. and Kuranda, M.J. (1989). Lysosomal degradation of Asn-linked
glycoproteins. FASEB J 2615

Barnes, W.M. (1992). The Fidelity of Taq polymerase catalizing PCR is improved
by an N-terminal deletion. Genes 112, 29-35.

Barnes, W.M. (1994). PCR amplification of up to 35-kb DNA with high fidelity
and high yield from bacteriophage templates. Proc. Nat. Acad. Sci. USA 91,
2216-2220.

Bernard, M., Sioud, M., Percheron, F., and Foglietti, M.J. (1986). B—Mannosidase
in human serum and urine. A comparative study. Int. J. Biochem. 18, 1065-1068.

Boyer, P.J., Jones, M.Z., Nachreiner, R.F., Refsal, K.R., Common, R.S., Kelley, J .,
and Lovell, KL. (1990). Caprine B—mannosidosis: Abnormal thyroid structure and
function in a lysosomal storage disease. Lab. Invest. 63, 100-106.

Boyer, RI. and Lovell, KL. (1990). In vitro characterization of oligodendrocytes
in caprine B—mannosidosis. Ann. NY Acad. Sci. 605, 424-425.

Bryan, L., Schmutz, S., Hodges, SD, and Snyder, RF. (1990). Bovine [3—
mannosidase deficiency. Biochem. Biophys. Res. Comm. 1 73, 491-495.

Cariello, N.F., Swenberg, J .A., and Skopek, TR. (1991). Fidelity of Thermococcus
Litoralis DNA Polymerase (Vent) in PCR determined by Denaturin g gradient gel
electrophoresis. Nucleic Acids Res 19, 4193-4198.

Cavanagh, K., Dunstan, R.W., and Jones, M.Z. (1982). Plasma or- and B—
mannosidase activities in caprine B—mannosidosis. Am. J. Vet. Res. 43, 1058-
1059.

103

104

Chen, H., Leipprandt, J .R., Traviss, C.E., Sopher, B.L., Jones, M.Z., Cavanagh,
K.T., and F riderici, K.H. (1995). Molecular cloning and characterization of bovine
B—mannosidase. J. Biol. Chem. 270(8), 3841-3848.

Clark, J .M. (1988). Novel non-templated nucleotide addition reactions catalyzed
by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 16, 9677-
9686.

Cohen, J .B. and Levinson, AD. (1988). A point mutation in the last intron
responsible for increased expression and transforming activity of the c-Ha-ras
oncogene. Nature 334, 119-124.

Colin, B., Bernard, M., Foglietti, M.J., and Percheron, F. (1987). B—D-
Mannosidase in human polymorphonuclear leukocytes and lymphocytes: a
comparative study. Int. J. Biochem. 19, 395-397.

Conchie, J. and Mann, T. (1957). Glycosidases in mammalian sperm and seminal
plasma. Nature 179, 1190-1191.

Conzelmann, E. and Sandhoff, K. (1987). Glycolipid and glycoprotein
degradation. In Advances in Enzymology and Related Areas of Molecular
Biology. A. Meister, ed. (New York: John Wiley & Sons), pp. 89-216.

Cooper, A., Sardharwalla, LB, and Roberts, M.M. (1986). Human B-mannosidase
deficiency. N. Engl. J. Med. 315, 1231

Cooper, A., Hatton, C., and Sardharwalla, LB. (1987). Acid B—mannosidase of
human plasma: Influence of age and sex on enzyme activity. J. Inher. Metab.
Dis. 10, 229-233.

Cooper, A., Hatton, C., Thornley, M., and Sardharwalla, LB. (1988). Human B—
mannosidase deficiency: Biochemical findings in plasma, fibroblasts, white cells
and urine. J .Inher.Metab.Dis. 11, 17-29.

Cooper, A., Hatton, C.E., Thornley, M., and Sardharwalla, LB. (1990). a and B—
Mannosidoses. J. Inher. Metab. Dis. 538-548.

Cooper, A., Wraith, J .E., Savage, W.J., Thornley, M., and Noronha, M.J. (1991).
B—mannosidase deficiency in a female infant with epileptic encephalopathy. J.
Inherited Metab. Dis. 14, 18-22.

105

Cooper, D.N. and Krawczak, M. (1990). The mutational spectrum of single
basepair substitutions causing human genetic disease: Patterns and prediction.
Hum. Genet. 55-74.

Cooper, D.N. and Krawczak, M. (1993). Human Gene Mutation (Oxford: Bios
Scientific Publishers).

Cooper, D.N. and Schmidtke, J. (1991). Diagnosis of human genetic disease using
recombinant DNA. Hum. Genet. 519-560.

Cooper, D.N. and Youssoufian, H. (1988). The CpG dinucleotide and human
genetic disease. Hum. Genet. 151-155.

Cuozzo, J .W. and Sahagian, G.G. (1994). Lysine is a common determinant for
mannose phosphorylation of lysosomal proteins. J. Biol. Chem. 269, 14490-
14496.

Dawson, G. (1982). Evidence for two distinct forms of mammalian B—
mannosidase. J. Biol. Chem. 25 7, 3369-3371.

Dawson, G. (1987). Methods in Enzymology 272-284.

Dawson, G. and Hancock, L.W. (1992). Inborn errors of complex carbohydrate
catabolism. In Complex Carbohydrates of Nervous Tissue. R.U. Margolis and R.K.
Margolis, eds. (New York: Plenum Press), pp. 421-447.

de Duve, C. and Wattiaux, R. (1966). Functions of Lysosomes. Annual Review of
Physiology 43 5-492.

Dorland, L., Duran, M., Hoefnagels, F .E.T., Breg, J .N., Fabery de Jonge, H., van
Eeghen-Cransberg, K., Van Sprang, F.J., and van Diggelen, DP. (1988). B—
Mannosidosis in two brothers with hearing loss. J. Inherited Metab. Dis. 11, 255-
258.

Durand, P., Lehn, P., Callebaut, I., Fabrega, S., Henrissat, B., and Momon, J .P.
(1997). Active-site motifs of lysosomal acid hydrolases: Invariant features of clan

GH-A glycosyl hydrolases deduced fi'om hydrophobic cluster analysis.
Glycobiology 7, 277-284.

106

Fisher, R.A., Povey, S., Cavanagh, K., Dupuis, M., and Jones, M.Z. (1987).
Studies to determine the chromosome assignment of human lysosomal B—
mannosidase (MANBA). Am. J. Hum. Genet. 41, A165

Gaucher, P.C.E. (1882). De l'eptheliome primitif de la rate. Thesi de Paris

Gieselmann, V. (1995). Lysosomal storage diseases. Biochim. Biophys. Acta
1270, 103-136.

Gourrier, E., Thomas, M.P., Munnich, A., Poenaru L., Asensi, D., Jan, D., and
Leraillez, J. (1997). A new case of beta-mannosidosis. Archives de Pediatric 4,
147-151.

Guadalupi, R., Bernard, M., Orlacchio, A., Foglietti, M.J., and Emiliani, C. (1996).
Purification and properties of human urinary beta-D-mannosidase. Biochem.
Biophys. Acta 1293, 9-16.(Abstract)

Hartley, W.J. and Blakemore, W.F. (1973). Neurovisceral storage and
dysmyelinogenesis in neonatal goats. Acta Neuropathol. 25, 325-333.

Hasilik, A. and Neufeld, ER (1980). Biosynthesis of lysosomal enzymes in
fibroblast. Synthesis as precursors of higher molecular weight. J. Biol. Chem.
255, 4937-4945.

Healy, P.J., Seaman, J .T., Gardner, LA, and Sewell, CA. (1981). B—mannosidase
deficiency in anglo nubian goats. Aust. Vet. J. 5 7, 504-507.

Hers, H.G. (1965). Inborn lysosomal diseases. Gastroenterology 625-633.

Hers, H.G. (1973). The concept of inborn lysosomal storage diseases. In
Lysosomes and Storage Diseases. H.G. Hers and F. van Hoof, eds. (New York:
Academic Press), pp. 147-171.

Hokke, C.H., Duran, M., Dorland, L., Van Pelt, J ., and Van Sprang, F]. (1990).
Novel storage products in human B—mannosidosis. J. Inherited Metab. Dis. 13,
273-276.

Holtzman, E. (1989). Lysosomes (New York: Plenum).
Hu, P., Wenger, D.A., van Diggelen, DP, and Kleijer, W.J. (1991).

Complementation studies in human and caprine B—mannosidosis. J. Inherited
Metab. Dis. 14, 13-17.

107

Hurler, G. (1919). Uber einen Typ multipler Abartungen, vorwiegend am
Skelettsystem. Zeitschrift fur Kinderheilkunde 220-234.

Iwasaki, Y., Tsuji, A., Omura, K., and Suzuki, Y. (1989). Purification and
characterization of B—mannosidase from human placenta. J. Biochem. 106, 331-
335.

Jolly, R.D., Thompson, K.G., Bayliss, S.L., Vidler, B.M., Orr, M.B., and Healy,
R]. (1991). B—Mannosidosis in a Salers calf: A new storage disease of cattle. J.
New Zealand Vet. Assn 38, 102-105.

Jones, M.Z., Cunningham, J .G., Dade, A.W., Alessi, D.M., Mostosky, U.V.,
Vorro, J .R., Benitez, J .T., and Lovell, KL. (1983). Caprine B—mannosidosis:
clinical and pathological features. J. Neuropath. Exp. Neurol. 42, 268-285.

Jones, M.Z., Rathke, E.J.S., Cavanagh, K., and Hancock, L.W. (1984). B—
mannosidosis: prenatal biochemical and morphological characteristics. J. Inher.
Metab. Dis. 7, 80-85.

Jones, M.Z., Rathke, E.J.S., Gage, D.A., Costello, C.E., Murakami, K., Ohta, M.,
and Matsuura, F . (1992). Oligosaccharides accumulated in the bovine B-
mannosidosis kidney. J. Inherited Metab. Dis. 15, 57-67.

Jones, M.Z. and Dawson, G. (1981). Caprine B—mannosidosis: inherited deficiency
of B—mannosidase. J. Biol. Chem. 256, 5185-5188.

Jones, M.Z. and Laine, RA. (1981). Caprine oligosaccharide storage disease:
accumulation of B—mannosyl (1-4) B—N-acetylglucosaminyl (1-4) [3—
Nacetylglucosamine in brain. J. Biol. Chem. 256, 5181-5184.

Kleijer, W.J., Hu, P., Thoomes, R., Boer, M., Huijmans, J .G.M., Blom, W., van
Diggelen, O.P., Seemanova, E., and Macek, M. (1990). B—Mannosidase

deficiency: heterogeneous manifestations in the first female patient and her
brother. J. Inher. Metab. Dis. 13, 867-872.

Komfeld, R. and Komfeld, S. (1985). Assembly of asparagine-linked
oligosaccharides. Ann. Rev. Biochem. 54, 631-664.

Komfeld, S. (1987). Trafficking of lysosomal enzymes. FASEB J 1, 462-468.

108

Komfeld, S. (1990). Lysosomal enzyme targeting. Biochem. Soc. Trans. 18, 367-
374.

Kozak, M. (1987). An analysis of 5'-noncoding sequences from 699 vertebrate
messenger RNAs. Nucleic Acids Res. 15, 8125-8132.

Krawczak, M., Reiss, J ., and Cooper, D.N. (1992). The mutational spectrum of
single base-pair substitutions in mRNA splice junctions of human genes: causes
and consequences. Hum. Genet. 90, 41-54.

Kyosaka, S., Murata, S., Nakamura, F., and Tanaka, M. (1985). Purification and
kinetic properties of guinea pig liver B—mannosidase. Chem. Pharm. Bull. 33, 256-
263.

Labadie, J .H. and Aronson, J .N.J . (1973). Lysosomal B—D-mannosidase of rat
liver. Biochim. Biophys. Acta 321, 603-614.

Leipprandt, J .R., Kraemer, S.A., Haithcock, B.E., Chen, H., Dyme, J .L.,
Cavanagh, K.T., F riderici, K.H., and Jones, M.Z. (1996). Caprine B—mannosidase:
Sequencing and characterization of the cDNA and identification of the molecular

defect of caprine B—mannosidosis. Genomics 37, 51-56.

Levade, T., Graber, D., F lurin, V., Delisle, M.-B., Pieraggi, M.-T., Testut, M.-F.,
Carriere, J .-P., and Salvayre, R. (1994). Human B—mannosidase deficiency
associated with peripheral neuropathy. Ann. Neurol. 35, 116-119.

Lewin, B. (1995). Genes V (New York: Oxford University Press).

Lovell, K.L., Jones, M.Z., Patterson, J ., Abbitt, B., and Castenson, P. (1991).
Thyroid structure and function in bovine B—mannosidosis. J. Inherited Metab. Dis.
14, 228-230.

Lovell, K.L., Kranich, R., and Cavanagh, K. (1994). Biochemical and
histochemical analysis of lysosomal enzyme activities in caprine B—mannosidosis.
Molec. Chem. Neuropath. 21, 61-74.

Lovell, KL. and Boyer, RI. (1987). Dysmyelinogenesis in caprine B-
mannosidosis: Ultrastructural and morphometric studies in fetal optic nerve. Int. J.
Dev. Neuroscience 5, 243-253.

109

Lovell, KL. and Jones, M.Z. (1983). Distribution of central nervous system
lesions in B-mannosidosis. Acta Neuropathol. 62, 121-126.

Lovell, KL. and Jones, M.Z. (1985). Axonal and myelin lesions in B—
mannosidosis: ultrastructural characteristics. Acta Neuropathol. 65, 293-299.

Lundberg, K.S., shoemaker, D.D., Adams, M.W.W., Short, J .M., Sorge, J .A., and
Mathur, E.J. (1991). Hi gh-fidelity amplification using a thermostable DNA
polymerase isolated from from Pyrococcus furiosus. Gene 108, 1-6.

Lundin, L.-G. (1987). A gene (an) controlling B—mannosidase activity in the
mouse is located in the distal part of chromosome 3. Biochem. Genet. 25, 603-610.

Maquat, LE. (1996). Defects in RNA splicing and the consequence of shortened
translational reading frames. Am. J. Hum. Genet. 59, 279-286.

Matsuura, F., Laine, RA, and Jones, M.Z. (1981). Oligosaccharides accumulated
in the kidney of a goat with B—mannosidosis: mass spectrometry of intact
permethylated derivatives. Arch. Biochem. Biophys. 211, 485-493.

McCabe, NR. and Dawson, G. (1991). A study of the heterogenous structure of

guinea pig lysosomal B—mannosidase using a polyclonal antibody. Biochim.
Biophys. Acta 1077, 133-140.

Mead, D.A., Pey, N.K., Hermstadt, C., Marcil, RA, and Smith, L.M. (1991). A
Universal method for the direct cloning of PCR amplified nucleic acid.
Bio/Technology 9, 657-663.

Neufeld, HF. (1991). Lysosomal Storage diseases. Annu. Rev. Biochem. 60, 257-
280.

O'Brien, S.J., Womack, J .E., Lyons, L.A., Moore, K.J., Jenkins, N.A., and
Copeland, N.G. (1993). Anchored reference loci for comparative genome mapping
in mammals. Nat. Genet. 3, 103-112.

Paigen, K. and Peterson, J. (1978). Coordinacy of lysosomal enzymes excretion in
human urine. J. Clin. Invest. 61, 751-762.

Pakula, AA. and Sauer, RT. (1989). Genetic analysis of protein stability and
function. Annu. Rev. Genet. 23, 289-310.

110

Panday, R.S., van Diggelen, O.P., Kleijer, W.J., and Niermeijer, M.F. (1984). B—
Mannosidase in human leukocytes and fibroblasts. J .Inher.Metab.Dis. 7, 155-156.

Patterson, J .S., Jones, M.Z., Lovell, K.L., and Abbitt, B. (1991). Neuropathology
of bovine B-mannosidosis. J. Neuropathol. Exp. Neurol. 50, 538-545.

Percheron, F ., Foglietti, M.J., Bernard, M., and Ricard, B. (1992). Mammalian [3-
D-mannosidase and B—mannosidosis. Biochimie 74, 5-11.

Petushkova, N.A., Ivleva, TS, and Vozniy, Y.M. (1992). Human chorionic B—
mannosidase: Comparison with B—mannosidase from human cultured fibroblasts.
Prenat. Diagn. 12, 835-839.

Poenaru, L., Akli, S., Rocchiccioli, F ., Eydoux, P., and Zamet, P. (1992). Human
B—mannosidosis: a 3-year-old boy with speech impairment and emotional
instability. Clin. Genet. 41, 331-334.

Raben, N., Nichols, R.C., Martiniuk, F., and Plotz, PH. (1996). A model of
mRNA splicing in adult lysosomal storage disease (glycogenosis type II). Hum.
Mol. Genet. 5, 995-1000.

Render, J .A., Lovell, K.L., and Keller, CB. (1992). The ocular and otic pathology
of bovine B—mannosidosis. J. Vet. Diag. Invest. 4, 96-98.

Rodriguez-Sema, M., Botella-Estrada, R., Chabas, A., Coll, M.-J., Oliver, V.,
F ebrer, M.-I., and Aliaga, A. (1996). Angiokeratoma corporis diffusum associated
with B—mannosidase deficiency. Arch. Derrnatol. 132, 1219-1222.

Sachs, B. (1887). On arrested cerebral development, with special reference to
cortical pathology. J. Nerv. Mental Dis. 541-554.

Saiki, R.K., Gelfand, D.H., Stoffel, S.J., Higuchi, R., Horn, G.T., Mullis, KB, and
Erlich, HA. (1988). Science 239, 487-491.

Sambrook, J ., Fritsch, BF, and Maniatis, T. (1989). Molecular Cloning, A
Labrotory Manual. (Cold Spring Harbour Labrotory Press).

Schmutz, S.M., Moker, J .S., Leipprandt, J .R., and Friderici, K.H. (1996). Beta-
mannosidase maps to cattle chromosome 6. Mamm. Genome 7, 474

111

Scriver, C.R., Beaudet, A.L., SLy, W.S., and Valle, D. (1995). The Metabolic and
the Molecular Basis of Inherited Disease (McGrow-Hill).

S0pher, B.L., Traviss, C.E., Cavanagh, K.T., Jones, M.Z., and Friderici, K.H.
(1992). Purification and characterization of goat lysosomal B—mannosidase using
monoclonal and polyclonal antibodies. J. Biol. Chem. 267, 6178-6182.

Sopher, B.L., Traviss, C.E., Cavanagh, K.T., Jones, M.Z., and Friderici, K.H.
(1993). Bovine kidney B—mannosidase: purification and characterization.
Biochem. J. 289, 343-347.

Tay, W. (1881). Symmetrical changes in the region of the yellow spot in the eye of
an infant. Tans. Opthal. Soc. UK 1, 55-57.

Thomas, OH. and Beaudet, AL. (1995). Disorders of glycoprotein degradation
and structure: or-mannosidosis, B—mannosidosis, fucosidosis, sialidosis,
aspartylglucosaminuria, and carbohydrate-deficient glycoprotein syndrome. In The
Metabolic and Molecular Bases of Inherited Disease. C.R. Scriver, A.L. Beaudet,
W.S. Sly, and D. Valle, eds. (New York: McGraw-Hill, Inc.), pp. 2529-2539.

Tindall, K.R. and Kunkel, TA. (1988). Fidelity of DNA synthesis by the T hermus
Aquaticus DNA polymerase. Biochemistry 27, 6008-6013.

Van Pelt, J ., Hokke, C.H., Dorland, L., Duran, M., Kamerling, J .P., and
Vliegenthart, J .F.G. (1990). Accumulation of mannosyl-B(1—>4)-N-

acetylglucosamine in fibroblasts and leukocytes of patients with a deficiency of B-
mannosidase. Clin. Chim. Acta 187, 55-60.

Vogel, F. and Kopun, M. (1977). Higher frequencies of transitions among point
mutations. J. Mol. Evol. 159-180.

Von F igura, K. and Hasilik, A. (1986). Lysosomal enzymes and their receptors.
Ann. Rev. Biochem. 55, 167-193.

von Heijne, G. (1986). A new method for predicting signal sequence cleavage
sites. Nucleic Acids Res. 14, 4683-4690.

Watts, R.W.E. and Gibbs, DA. (1986). Lysosomal Storage Diseases: Biochemical
and Clinical Aspects (London and Philadelphia: Taylor and Francis).

112

Wenger, D.A., Sujansky, E., Fennessey, P.V., and Thompson, J .N. (1986). Human
B—mannosidase deficiency. N. Engl. J. Med. 315, 1201-1205.

Wijburg, H., De Jong, J ., Wevers, R., Bakkeren, J ., Trijbels, F., and Sengers, R.
(1992). Beta-mannosidosis and ethanolaminuria in a female patient. Eur. J. Pediatr.
151, 311

Wolf, U. (1995). The genetic contribution to phenotype. Hum. Genet. 95, 127-
148.