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This is to certify that the

dissertation entitled

The Linkage Analysis of Cystic Fibrosis
and Red and White Cell Loci

presented by

Janel Susan Rehm Hector

has been accepted towards fulfillment
of the requirements for

PhD degree in Genetics

MMQC

E
Major professor

 

Date June 21, 1982

 

MS U it an Affirmaliw Action/Equal Opportunity Institution 0- 12771

 

 

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THE LINKAGE ANALXSIS OF CYSTIC IilBROSIS
AND RED AND WHITE CELL LOCI

By

Janel Susan Rehm Hector

A DISSERTATION

Submitted to
Nflchigan State University
in partial fulfillment of the requirements
fOr the degree of

DOCTOR OF PHILOSOPHY

Department of Natural Science

19 82

ABSTRACT

THE LINKAGE ANALYSIS OF CYSTTC FIBROSIS
AND RED AND WHITE CELL LOCI

By

Janel Susan Rehm Hector

Cystic fibrosis of the pancreas causes severe Chronic pulmonary disease,
dietary insufficiencies and delayed growth in infants, children and
young adults. Inheritance is autosomal recessive. Heterozygote
individuals cannot be distinguished currently from.individuals
homozygous for the nonaffected allele. The cystic fibrosis locus has
not yet received Chromosomal assignment, and is not known to be

linked with any other locus of the human genome.

Affected individuals and their siblings, parents and grandparents
were typed for antigens from the histocompatibility complex and.nine
of the red blood cell loci (ABO, Rhesus, MNSs, Kell, Kidd, P, Duffy,
Lewis and Secretor). The resulting family data.were analyzed for
association of cystic fibrosis with the tested antigens (chi-square
method), and linkage of the cystic fibrosis locus with the test loci

(lod scores of Morton).

Linkage analysis excludes the cystic fibrosis locus from within .20 or
less map units from.the ABO locus, .13 or less units from the HLA
loci, .09 or less units from the Rhesus loci, .08 or less units from
the MNSs loci, .04 or less units frcnlthe Duffy and P loci and .01

or less units from the Kidd, Lewis and Secretor loci. Possible

locations of the cystic fibrosis loci are at zero map units from the

Kell locus (10d scores of 2.0727) with a probability of 118 to l.and
.30 map units from.the MNSs loci (lod score of 1.781) with a.pr0b-

ability of 60 to 1.

Association analysis indicated.a.very high association between the HLA—B7
antigen of the histocompatibility complex and the occurrence of

cystic fibrosis. 0f 67 cystic fibrosis children, 13 had the HLA—B?
antigen and 5“ did not have the antigen; 25 of a total of 51 non—
afflected siblings possessed the HLA-B? antigen. (Chi-square value of
11.6339, prObability between .0010 and .0005, highly significant.)

The HLA—B27 antigen was not significantly associated.with the

occurrence of cystic fibrosis. Four of the 67 cystic fibrosis
individuals possessed HLA-B27; 7 of 51 full siblings were HLA-B27
positive (chi-square value of 2.060“, prObability between .2 and .1),

no significance .

Dedicated to
my husband
David Alphonso Hector, M.D.
with love

ii

ACIWOWIEDGMENTS

I would like to express thanks to those members of my graduate
committee, each of whom assisted in one or more aspects of research
design, patient contact, clinical or laboratory work and in the
writing of this dissertation: Dr. Janice Lindstrom, Dr. Ronald J.
Patterson, Dr. Richard E. Honicky and Dr. James V. Higgins. A very
special thanks goes to my advisor and committee chairman, Dr. Emanuel

Hackel, who directed my research from beginning to end.

I would also like to extend gratitude to the technicians of the
American Red Cross Great Lakes Regional Blood Center for the red
blood cell typing and to Dr. Charles Pelzer for the o<1-antitrypsin

quantitations .

Third, I would like to thank Drs. Wortley, Kellogg and Howatt, clinic
directors of the cystic fibrosis clinics who most graciously allowed

me to enter their clinics and work with their patients and staff.

And finally, a very special thanks goes to all of the children with

cystic fibrosis and their families who consented to blood donations

and then offered even more .

iii

I.

II.

III.

TABLE OF CONTENTS

BACKGROUND

A.
B.
C.

D.

G.

INTRODUCTION

CYSTIC FIBROSIS OF THE PANCREAS

ANALYSIS OF LINKAGE

DETECTION OF CARRIERS OF THE CYSTIC FIBROSIS
ALLELE

CHROMOSOMAL ASSIGNMENT 0F CYSTIC FIBROSIS
THE TEST LOCI

1. The Human Histocompatibility Region

2. The Red Blood Cell Loci

THE HUMAN GENOME

METHODS

A.

[11.0003

TESTING OF CYSTTC FIBROSIS PATIENTS AND THEIR
FAMILIES

TYPING OF THE HISTCOMPATIBILITY ANTIGENS
TYPING OF THE RED BLOOD CELL ANTIGENS

TESTING OF o<l ANTITRYPSIN

LINKAGE ANALYSIS

RESULTS

A.
B.

C.

PARTICIPATION BY CYSTIC FIBROSIS FAMILIES
SEX RATIO

ANALYSIS OF DATA OF THE RED AND WHITE BLOOD
CELL ANTIGENS

iv

Page
Page
Page
Page

Page
Page
Page
Page
Page
Page
Page

Page
Page
Page
Page
Page
Page
Page

Page

Page

1
l

1

17
2O
22
22
2A
26
27

27
31
33
35
35
38
38
38

39

LIST OF CONTENTS (Continued)

D.

l. The Histocompatibility Complex

2. The ABC Blood Group

3 The Rhesus Blood Group

A. The MNSS Blood Group

5. The Lewis and.Secretor Blood Groups

6. The Kell,. P, Duffy and Kidd Blood Groups
RESULTS OFCXI17ANTTTRYPSIN TESTING

IV. DISCUSSION

A.

ASSOCIATION OF BLOOD GROUP ANTTCENS AND CYSTTC
FIBROSIS

o<1.ANTITRYPSIN

LINKAGE RESULTS BETWEEN CYSTTC FIBROSIS AND THE
TEST LOCI

COMBINED DOD SCORES OF THIS STUDY WITH SCORES OF

OTHER PUBLISHED STUDIES

'V. CONCLUSION

APPENDICES

APPENDIX A. Family and individual identification

numbers and intrafamilial relationships.

APPENDIX B. HLA haplotypes of each individual.

APPENDIX C. Results of red cell analysis.

LIST OF REFERENCES

Page
Page

Page

39
145
50

Page 58

Page

58

Page 66

Page

Page

Page

Page

Page

Page

Page
Page

Page

Page

75
75

75
78

78

82
87

94
100

107

TABLE

TABLE

TABLE

TABLE A

TABLE
TABLE
TABLE
TABLE
TABLE
TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

13.

1A.

15.

LIST OF TABLES

INHERITANCE OF CYSTIC FIBROSIS.
RISK OF A NORMAL SIBLING (OF A CYSTIC FIBROSIS IN-
DIVIDUAL) TO PRODUCE A CHILD AFFECTED WITH CYSTIC
FIBROSIS.
POPULATTON FREQUENCIES OF CYSTIC FIBROSIS
PARENTAL MATING TYPES AND Z SCORES.
Z SCORE FORMULAS.
TYPE A AND B ERRORS.
LOCI TESTED FOR LINKAGE WITH CYSTIC FIBROSIS.
MAP LOCATIONS OF TEST LOCI.
CYSTIC FIBROSIS CLINICS.
RED CELL ANTIGENS TYPED.
CHI-SQUARE ANALYSIS OF ASSOCIATION OF WHITE
BLOOD CELL ANTIGENS WITH CYSTIC FIBROSIS.
IOD SCORES OF CYSTIC FIBROSIS AND THE
HISTOCOMPATIBILITY COMPLEX.
CHI-SQUARE ANALYSIS OF ASSOCIATION OF RED BLOOD
CELL ANTIGENS WITH CYSTIC FIBROSIS.
LOD SCORES OF CYSTIC FIBROSIS AND THE ABO
BLOOD GROUP.
LOD SCORES OF CYSTIC FIBROSIS AND THE RHESUS

BLOOD GROUP.

Page

Page

Page

Page

Page

Page

Page

Page

Page

Page

Page

Page

Page

Page

3

3

5
1’4

15
18
21
28
29
3A

143

146

51

52

55

LIST OF TABLES (Continued)

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

l6.

17.

18.

19.

20.

21.

22.

23.
214.

LOD SCORES OF CYSTIC FIBROSIS AND THE.MNSS BLOOD
GROUP

LOD SCORES OF CYSTIC FIBROSIS AND THE LEWIS BLOOD
GROUP.

LOD SCORE OF CYSTIC FIBROSIS AND THE SECRETOR
BLOOD GROUP.

LOD SCORES OF CYSTIC FIBROSIS AND THE KELL BLOOD
GROUP.

LOD SCORES OF CYSTIC FIBROSIS AND THE P BLOOD
GROUP.

LOD SCORES OF CYSTIC FIBROSIS AND THE DUFFY

BLOOD GROUP.

LOD SCORES OF CYSTIC FIBROSIS AND THE KIDD

BLOOD GROUP.

ASSOCIATION OF HLA-B7 WITH CYSTIC FIBROSIS.
TOTALS OF LOD SCORES OF THE BLOOD GROUP LOCI

FROM THIS STUDY AND FROM OTHER PUBLISHED STUDIES.

Vii

Page

Page

Page

Page

Page

Page

Page

Page

Page

59

62

614

67

69

71

73
77

83

FIGURE 1.

FIGURE 2.

FIGURE 3.

FIGURE A.

FIGURE 5.

FIGURE 6.

FIGURE 7.

FIGURE 8.

FIGURE 9.

FIGURE 10.

FIGURE 11.

LIST OF FIGURES

PEDIGREE OF FAMILY A16 ILLUSTRATING A RECOMBINANT

EVENT AT THE HLA REGION.

LOD SCORE TOTALS BETWEEN CYSTIC
HISTOCOMPATIBILITY COMPLEX.

LOD SCORE TOTALS BETWEEN CYSTIC
ABO BLOOD GROUP.

LOD SCORE TOTALS BETWEEN CYSTIC
RHESUS BLOOD GROUP.

LOD SCORE TOTALS BETWEEN CYSTIC
MNSS BIOOD GROUP.

LOD SCORE TOTALS BETWEEN CYSTIC
LEWIS BLOOD GROUP.

LOD SCORE TOTALS BETWEEN CYSTIC
SECRETOR BLOOD GROUP.

LOD SCORE TOTALS BETWEEN CYSTIC
KELL BLOOD GROUP.

LOD SCORE TOTALS BETWEEN CYSTIC

P BLOOD GROUP.

LOD SCORE TOTALS BETWEEN CYSTIC FIBROSIS AND THE

DUFFY BIOOD GROUP.

LOD SCORE TOTALS BETWEEN CYSTIC FIBROSIS AND THE

KIDD BLOOD GROUP.

viii

FIBROSIS AND THE

FIBROSIS AND THE

FIBROSIS AND THE

FIBROSIS AND THE

FIBROSIS AND THE

FIBROSIS AND THE

FIBROSIS AND THE

FIBROSIS AND THE

Page

Page

Page

Page

Page

Page

Page

Al

’49

5A

57

61

63

65

68

70

72

7A

umwmmmmmM)
FIGURE 12. LCD SCORE TOTAL FOR 17 FAMILIES SOORED FOR

SEGREGATION OF THE FATHERS ONLY, BY EDWARDS . Page 80
FIGURE 13. LOD SCORE TOTALS BETWEEN CYSTTC FIBROSIS AND THE

DUFFY , KELL AND KIDD BLOOD GROUPS—COMBINED

RESULTS OF THIS STUDY WITH OTHER PUBLISHED

STUDIES. Page 84
FIGURE 14. LOD SCORE TOTALS BETWEEN CYSTIC FIBROSIS AND THE

MNSS, HLA AND ABO BLOOD GROUPS—COMBINED RESULTS

OF THIS STUDY WITH OTHER PUBLISHED STUDIES. Page 85

ix

I . BACKGROUND

A . INTRODUCTION

The purpose of this study was to identify any antigen association or‘
linkage relationship between cystic fibrosis and ten of the red and
white blood cell loci: HLA (the human histocompatibility complex),

ABO, Rhesus, MNSS, Kell, Duffy, Kidd, P and Lewis-Secretor).

Association of cystic fibrosis with the blood cell antigens was tested
by chi-square analysis, with the full siblings of the prOpositus
(cystic fibrosis) individuals as a control group.

Linkage was tested between cystic fibrosis and the ten test loci
using the log—odd (10d) score method of Morton; grandparents were tested
to aid in identification of recessive alleles in the parental genotypes.

A genetic linkage of a cystic fibrosis locus with a test locus could
provide the basis for heterozygote identification and the consequent

prenatal diagnosis of cystic fibrosis in affected families.
B. CYSTTC FIBROSIS OF THE PANCREAS

Cystic fibrosis was recognized as a separate disease entity in the
1920's based on characteristic findings at autopsy, including fibrosis
of the pancreas. In the 1930's cystic fibrosis was defined clinically.1
Until 1915, the average age of death of children diagnosed as having

cystic fibrosis was one year.1

2
Cystic fibrosis is an inherited autosomal recessive disease
characterized by chronic pulmonary disease, chronic maldigestion
and malabsorption and an elevated sweat chloride level. Males
and females are equally afflected.1 Ratios of affected to normal
children in sibships correspond to expected Mendelian ratios of
one to three when a correction is made for those sibships where no

cystic fibrosis individuals occur.2

The child with cystic fibrosis has two recessive genes that produce
the disease state. The phenotypically normal parents are carriers,
or heterozygotes at the cystic fibrosis locus. Any child of these
parents has a one-in-four chance of receiving a cystic fibrosis

gene from both parents and having clinical disease. A phenotypically
normal child from the same mating may be a carrier for the allele or
may have two nonaffected alleles. Each phenotypically normal child
has a two to one probability of being a carrier of the cystic fibrosis
allele, and one chance in 120 of producing a cystic fibrosis child
from a random mating with another phenotypi cally normal individual.
Attempts at heterozygote assays are currently unable to distinguish
between the normal and carrier siblings. (Tables 1 and 2)

As a result of the life-limiting nature of the disease process, few
cystic fibrosis individuals live long enough to reproduce. Those

children born to cystic fibrosis individuals may be homozygous for
cystic fibrosis or may be heterozygous depending on the. genotype of
the other parent. Children of an affected individual and a carrier

will have a fifty. percent chance of being affected; all phenotypically

3
TABLE 1. INHERITANCE OF CYSTIC FIBROSIS.

 

Mating Type Genotype of OffSpring
1. Cfo X Cfo 100% Cfo
2. Cfo X Cfcf 50% Cfo + 50% Cfcf
3. Cfo X Cfo 100% Cfcf
A. Cfcf x Cfcf 25% Cfo + 50% Cfcf + 25% cfcf
5. Cfcf x cfcf 50% Cfcf + 50% cfcf
6. cfcf x cfcf 100% cfcf

Cfo represents a nonaffected individual at the cystic fibrosis locus,
Cfcf is a.heterozygote or carrier, and cfcf is an individual affected
with cystic fibrosis.

TABLE 2. RISK OF A NORMAL SIBLING (OF A CYSTIC FIBROSIS INDIVIDUAL)
TO PRODUCE A CHILD AFFECTED WITH CYSTIC FIBROSIS.

 

Mating Type Offspring Probability of
Mating
1. Sibling of Cfo, mate is Cfo 100% Cfo (1/3) (19/20)

2. Sibling is Cfo, mate is Cfcf 50% Cfo, 50% Cfcf (1/3) (1/20)

3. Sibling is Cfcf, mate is crcr 50% Cfo, 50%Cfcf (2/3) (19/20)

’4. Sibling is Cfcf, mate is Cfcf 25% Cfo, 50% Cfcf (2/3) (1/20)
25% cfcf

An affected Child can occur only in mating type A. The prObability
for a sibling of a cystic fibrosis individual to have an affected
child is: (2/3) x (1/20) x (l/A) = 1/120

normal children will be carriers. A mating with an individual
homozygous for the nonaffected allele will produce only heterozygous
children. Based on racial gene frequencies, the probability of a
cystic fibrosis offspring for an unknown mating has been approximated

3

at two and one—half percent. The phenotype of each successive

child will give evidence of the genotype of the normal mate.

A mating between two affected individuals has never been reported
but would be expected to result in all affected children. (This
mating is mlikely not only because of the limited life Span of
affected individuals but also because affected males are usually

sterile.)

Cystic fibrosis occurs primarily in Caucasian populations. (Table 3)
The incidence of cystic fibrosis in Caucasians is estimated to be
from one in 1000 to one in 3500 live births.1 Based

on the frequency of cystic fibrosis births, and using the Hardy-
Weinberg law, p + q = 1, it can be estimated that approximately one

person in twenty is a carrier.

In non—Caucasian pOpulations, cystic fibrosis occurs rarely; the
incidence of cystic fibrosis in Orientals residing in Hawaii is only
one in 90,000 live births}4 The incidence of cystic fibrosis in
Native African Blacks is even lower; a search of world literature
revealed only eighteen reported cases of cystic fibrosis in these

non—American Blacks.5 (These comparisons assume that diagnostic

criteria for cystic fibrosis is the same in medical facilities

5
TABIE 3. POPULATION FREQUENCIES OF CYSTIC FIBROSIS.

 

Parental Ethnic Group Affected.neWborns/1ive births
1. Caucasian 1/ 1000 - 1/ 3500
2. Mixed Caucasian - Oriental l/lA,000
3. Oriental l/90,000
A. NoneAmerican Blacks rare*
5. .Mixed Caucasian - Black 1/17,000

*Only eighteen cases reported in the world literature.

6
worldwide as it is in the clinics of the United States, and that

homozygous cystic fibrosis alleles superimposed on different genetic
backgrounds and in different geographic areas will still be expressed
and clinically identifiable.)

Populations with mixed genetic backgrounds express intermediate
frequencies of cystic fibrosis. For example, Hawaiian matings of
mixed ancestry (i.e. both Oriental and Caucasian ancestors) have an

average frequency of cystic fibrosis of one in 115000 live births. 6

It has been estimated, based on studies of the Rh blood groups, that
approximately thirty percent of the alleles now present in the
American Black population are of Caucasian origin.2 The incidence of
cystic fibrosis in a Black population in Washington, D.C. is about
one in 17,000 live births;5 this is intermediate between the incidence
of cystic fibrosis in Caucasian populations and non-American Black

populations .

For the Caucasian population, this information results in a genetic
paradox. The cystic fibrosis allele is an almost complete genetic
lethal - very few affected children survive to reproductive age, and
of the survivors, most males are sterile. According to the Hardy-
Weinberg law, p2 + 2pq + q2 = 1; if the q2 or homozygous recessive
individuals of each generation do not survive to reproduce, then the
succeeding generations will result from matings within the remaining
(p2 + 2pq) population representing homozygous normal and heterozygous
individuals . With no other factors operating than normal random

mating, the incidence of cystic fibrosis would be expected to be low,

7
and the cystic fibrosis allele would be maintained in the population

by mutation. This may be the situation represented in the Oriental
and non-American Black populations. However in the Caucasian
population, the incidence of cystic fibrosis is too high to be explained

simply by normal mutation rates.

Average mutation rates have been estimated from spontaneous new
mutations of a number of dominant disorders, and may be approximated
at from one to five per 100,000 genes.7 For a recessive allele such
as cystic fibrosis, the mutation rate, u, is equal to (l - f)(x),7
where f is the relative fitness of cystic fibrosis individuals, and

x is the frequency of cystic fibrosis. The fitness of cystic fibrosis
individuals in terms of reproduction may be estimated as zero, and the
frequency of cystic fibrosis in Caucasian populations may be taken as
one in 1600. Therefore, the mutation rate of the normal allele at the
cystic fibrosis locus to becore a cystic fibrosis allele may be
estimated at one in 1600, which is from 12.5 to 62.5 times greater

than the average mutation rate of one to 5 in 100,000.

Possible explainations of the high frequency of cystic fibrosis in
Caucasians include an unusually high mutation rate that is not
present in other pOpulations.7 Alternatively, there may be a heter-
ozygote advantage at the cystic fibrosis locus in Caucasians, not

present in other pOpulations.

It has been suggested that a selective advantage of heterozygotes
8
may be manifested by increased numbers of offspring. Greater numbers

of offspring resulting from some undetermined reproductive advantage,

8

would cause greater numbers of cystic fibrosis children, and

carrier Siblings, thus increasing the frequency of the cystic fibrosis
allele. Another suggested heterozygote advantage may be the reduction
of blood pressure, due to sweat electrolyte ’ loss, . resulting in a
decreased probability of hypertension in carriers9 and therefore a
longer reproductive period. Increased sibship size may also be
attributed to compensation, by the parents, for the loss of affected

offspring.

Clinically, affected individuals manifest a number of symptoms;
whether these result from the presence of two recessive alleles for
cystic fibrosis or from the absence of a dominant nonaffected allele
at the cystic fibrosis locus is not yet known. A basic triad of
symptoms - decreased pancreatic excretory flinction, pulmonary
obstructive disease and increased sweat electrolytes - along with

pedigree information, is considered diagnostic for cystic fibrosis.

The prenatal period is generally uneventful, and general physical
examination of the neonate does not reveal any Specific signs of
cystic fibrosis. The first clinical indication of cystic fibrosis
may be failure of the newborn to pass meconium. Usually the meconium
is viscid and is unable to proceed past the terminal portion of the
ileum (hence the term "meconium ileus"). Large amounts of protein in
the meconium of affected individuals 10: ll. 12 may be related to
obstruction as well as the metabolic defect of cystic fibrosis.

Not only do 5 to 10 percent of cystic fibrosis patients have a

history of neonatal meconium ileus3, obstruction may also occur in

9
the older child (meconium ileus equivelant)13. The trapped meconium

sometimes has to be removed surgically, however complete bowel
obstruction seldom recurs .

Pulmonary disease is present in almost all affected individuals,3
and may appear weeks or years after birth. The pulmonary disease
results from continuous and viscid mucous production. Mild symptoms
include a nonproductive cough; subsequent respiratory infections may
result in the first swigis of pulmonary obstruction. Repeated
respiratory infections increase the obstruction and the compensatory
emphysema results in the typical barrel Shaped chest. Cor pulmonale
and cardiac failure are often the end results of the chronic lung
disease; other pulmonary complications include lobar atelectasis,
lung abcesses, hemoptysis and pneumothorax.3 Antibiotics are used to
minimize respiratory infections and their consequences, and daily
pulmonary drainage helps eliminate much of the mucous produced,

slowing the progressive airway obstructive process.

Pancreatic insufficiency is present in eighty percent of cystic
fibrosis patients .3 Although endocrine function is generally intact,
pancreatic enzyme production is severely limited, again due to

mucoid obstruction of the pancreatic ducts. This results in
maldigestion and malabsorption of food. Stools are large, bowel
movements frequent, and the abdomen protruberant. These children

may eat great amounts of food and yet be malnourished, exhibiting
"failure-to—thrive" or stunted growth. Pancreatic enzymes can be
given orally to increase intestional absorption although the pancreatic

obstruction continues. Dietary supplements are also available

10
containing necessary calories and protein that do not require

pancreatic enzymes for digestion.1u’ 15’ l6 Eventually, the fibrosis
of the pancreas due to the mucous obstruction of the pancreas may

hinder endocrine function and result in secondary diabetes mellitus.

Sweat electrolytes are often used as a diagnostic test for cystic
fibrosis. The skin is salty to the taste due to increased sodium,
chlorine and potassium. Excretory regulation of electrolytes is
normal, however electrolyte loss from profuse sweating may result
in cardiovascular collapse in hot weather (heat prostration).3
A possible beneficial result of electrolyte loss is lowered blood

pressure in affected individuals.9

Other less frequent clinical problems of cystic fibrosis children
include rectal prolapse, abdominal adhesions, intussusception,
hypoalbuminemia, lactase deficiency, neonatal obstructive Jaundice,
ocular lesions and infertility.3 Cystic fibrosis may also coexist
with other disease entities such as Kartagener syndrome”, Silver-
Russell dwarfismlB, leukemialg, respiratory allergy/2O, and chromosomal

deletion syndromele.

Along with physical symptoms, patients with cystic fibrosis may have
difficulties in dealing with personal, family and social problems due
to the severity of the physical disease and the poor prognosis.22

Intelligence of affected individuals is. normal22

, and a high compliance
rate during antibiotic treatment suggests that patients understand

the seriousness of their disease process.23 Pulmonary drainage is an

11

important part of treatment requiring assistance; this makes separation

of the young adult patient from the parents and family difficult.2u

The prognosis for patients with cystic fibrosis has always been poor.
It is now estimated that twenty percent of all cystic fibrosis patients
are fifteen years or olderl; this increase in life expectancy may be
attributed to a.number of medical advances, and especially to the use
of antibiotics in the treatment of respiratory infections, surgical

intervention of meconium ileus and improved diet.

For the infant, meconium ileus may result in very early death. For
survivors, respiratory Obstruction and complications are the most
significant threat to life, and the severity of the pulmonary
disease generally determines the prognosis for the child. Early
diagnosis and initiation of respiratory therapy offer the best hOpe
at this time, and.are aided by factors of general health sudh as

diet and exercise.

Pancreatic insufficiency is now seldom a direct cause of death,
because of enzyme supplements and artificial diets, however poor
nutrition.mey predispose to other complications and decrease the

patient's resistance to infections or other fOrms of stress.26

Occasional deaths occur due to other complications such as salt

loss in hot weather, portal hypertension or diabetes.26

Increasing numbers of individuals with cystic fibrosis are now

surviving into adulthood. This relatively long lifespan of these

 

12

individuals may be due to one or more of several factors -_ genetic
make-up, environment, corpliance and nature of treatment, nutrition
and general health and emotional status. Males have a slightly
longer life expectancy than females for unknom reasons. If cystic
fibrosis is corposed of more than one disease entity, as has been

suggested25, one form may be milder than another.

Reproduction of cystic fibrosis individuals is still rare due to

the limited lifespan of affected individuals.3

Although both males
and females acheive sexual maturation, the time of maturation is
frequently delayed in the more severely affected individuals. Most
males (97 to 98%26) are sterile due to aspermia resulting from
abnormal development of the epididymis, vas deferens and seminal
vesicles3; however, several cases of fertile males have been
reported.3 Females are fertile and a number of successful pregiancies
have been recorded, all resulting in normal offspring except one.3’ 25
However, pregiancy and delivery in cystic fibrosis females may be
corplicated by their pulmonary disease, and difficulties with the
pregnancy are directly correlated with the degree of pulmonary

involvement .3
C. ANALYSIS OF LINKAGE

The preferred method currently available for the analysis of human
linkage data is the log-odd ratio or lod score develOped by Morton.27
These scores were developed based on two concepts: first, that the

analysis of human linkage data is a sequential process, i.e. that

13
many families may be tested and each family yields a little more

information, and second, that the best method for analyzing such a

sequential sample is a sequential probability ratio test.28

A lod score (z score) is calculated for each family using a formula
chosen based on the mating type of the parents. (Tables LI and 5)

A correction score (S score) may also be added to the lod score if

the genotype of one or both of the parents was known because of the
phenotype of any one of the children. Z and 0 scores are calculated
for each family added to the study. The equations for determining

lod scOres have two variables: first, the numbers of each possible
kind of offspring in that family (a through h), and second, the
chromosome map distance (theta) the researcher wishes to test. The
scores of many families are additive at a given map distance. Further,
the scores may be added at arbitrary intervals during the course of
the linkage study. If, at a given map distance, the additive scores
of a series of families is greater than +3, linkage is considered
present between the test locus and the main locus. If the additive
scores are less than —2, linkage is considered absent from the two
loci for that map distance. Linkage is not excluded, however, from the
two loci for greater or smaller map distances. The third possibility,
that the additive score for a given map distance falls between -2 and
+3, conceeds only that more data is needed, before a conclusion may be

reached.

Each cumulative ("total") score contains three component parts:

1)) male scores, 2) female scores and 3) both or combined scores.

111

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16
Families in which segregation is visible in the mother but not the
father are viewed independently of families in which segregation
of alleles are visible in the father only. This segregation results
from.linkage data (suCh as of the Lutheran and Secretor loci in
man) indicating that recombination events occur more frequently in
fémales than in.males, and therefore that linkage might be detected
more easily in males.31’ 32 In the families where segregation was
visible in both parents, scores were also broken down into scores

for female and for male segregation.

The null hypothesis being tested is that the recombination fraction
of the two loci in the population is one-half. This situation would
be present if: 1) the two loci were on different chromosomes (not
linked), or 2) if the two loci were on the same chromosome but far
enough apart so that the frequency of recombination events make the
loci appear to assort independently (syntenic). Any variation from
the appearance of no linkage, such as incomplete penetrance,

biased family selection or ascertainment, non-random segregation of
loci on nonhomologous Chromosomes, or the presence of linkage
between the two tested loci, may result in rejection of the null
hypothesis.27 Assumptions necessary for the application of the lod
score test for linkage are: 1) the parental genotypes are known
(except for phase), 2) the segregation ratios are not altered by
incomplete penetrance or differential visibility, and 3) families
are selected zand ascertained randomly.27 The truncate method of
selection used to ascertain cystic fibrosis families is corrected

for in the calculations.

17

When linkage is shown to be present at a tested.map distance, the
final step is to find the map distance of the two loci. The lod
score sums may be graphically portrayed to visually find the most
likely peak value; then the families may be scored at smaller
intervals (sudh as one centimbrgan map distance) to determine the
peak value. Linkage distance varies between male, female, combined
and total scores, and would be expected to be closer inlnale than

in female scores.

While it is desirous to extend the human linkage map as quickly as
possible, only those linkages which are certain should be added to
the human gene map. On the other hand, there is less danger in
incorrectly excluding a positive linkage due to nonrandom sampling
or other statistical error. The limiting values of +3 and -2
correspond to type I and type II errors, respectively. The prOb-
ability of a type I error (accepting a linkage that is in fact not
present) is .001. The prObability of a type II error (rejecting a

linkage that is in fact present) is .0127 (Table 6)

D. DETECTION OF CARRIERS OF THE CYSTIC FIBROSIS ALLELE

Finding a consistent cystic fibrosis heterozygote assay is difficult
because the biochemical defect fer cystic fibrosis is unknown. The
best known assay to date is the ciliary inhibition test, based on

the observation that a.protein in the serum of affected individuals
and heterozygotes inhibits the beating action of cilia of rabbit
traChea or oyster gills. This assay has been extremely difficult to

standardize due to the prOblems associated with using a live organism

18

TABLE ‘6; ' 'TYPE'A:AND'B'ERRORS.27

O<=

,3:

LogA

LogA

LogB

Log B

.001, the probability of establishing a linkage which
does, in fact, not exist.

(.01, the probability of excluding a linkage which does,

in fact, exist .

Log [(1-fl )/°< ]

+3, the minirmm lod score accepted to confirm the presence
of linkage.

Log [/8 /(l-°< )]

—2, the minimum lod score accepted to confirm the absence
of linkage.

l9

assay. The results have not been consistent when performed by
different technicians or in different laboratories, thus limiting
its potential diagnostic power for heterozygotes. Nor is ciliary
dyskinesis specific to cystic fibrosis - other diseases of pulmonary
or ilmnmologic origin also inhibit ciliary movement,33 thus com—

plicating the diagnosis of a possibly heterozygous individual.

Other heterozygote assays include the staining patterns of leukocytes
and fibroblasts. One study noted two types of families, those in
which both affected individuals and parents leukocytes stained, and
those in which cells of neither parents nor affected children stained.3u
No distinction was made between heterozygotes and nonaffected.

Another study showed that cellular staining of fibroblasts also occurs
in an array of different diseases, thus further reducing its effective-
ness as a tool to diagnose cystic fibrosis heterozygotes.35 Studies
of leukocyte degranulation36, isoelectic focusing of plasma37 and of
serum-38, beta—glucuronidase activity of sweat39, and sperrnidine to
spermine ratios in bloodLl0 also showed no significant difference

between affected individuals and heterozygotes.

A number of other assays have been able to show a range of results,
with heterozygotes reacting intermediately between affected and
normal groups, but have been unable to specify accurately the
genotype for any given individual. Some of these assays are based on
determination of serum alpha-fetoprotein levelsul, serum complement
three levels‘Q, plasma kallikrein activity‘B, serum isoelectric
focusing in thin-layer polyacrylamide gels“, white cell glutathione

reductase activityu5, and trypsin binding by alpha-2-macroglobulinu6.

20

E. CHROMOSOMAL ASSIGNMENT OF CYSTIC FIBROSIS

At the present time, the chromosomal location (of the cystic fibrosis
locus is unknown; nor is the cystic fibrosis locus known to be

linked to any other locus of the human genome.

Chromosomal assignment of currently known loci depends on three

basic techniques: 1) cell hybridization, 2) chromosomal rearrangements
and 3) linkage studies. None of these techniques have yet been
successful with the cystic fibrosis locus. First, cell hybridization
studies provide assignments when specific chemical characteristics of

a locus are known; however, no such metabolic alteration has yet

been recognized in cystic fibrosis. Second, individuals emressing
cystic fibrosis and a chronosoral anomaly simultaneously may give

clues to the chromosomal location of the cystic fibrosis locus. One
such child elcpressed both cystic fibrosis and Cri-du—chat syndrome.2l’u7
Both parents and two siblings had normal phenotypes and karyotypes.

A third sibling died at two days of age from respiratory insufficiency,
but did not have meconium ileus and was not diagnosed as having cystic
fibrosis. The determination must be whether the deletion child was
"hemizygous" (because of the deletion) or homozygous for cystic fibrosis,
and without a consistent heterozygote assay to test the parents, or

a positive family history of cystic fibrosis such a distinction is not
possible. Lastly, the cystic fibrosis locus may be shown to be linked

to another locus which has already received chromosomal assignment,

Several studies have tested linkage of the cystic fibrosis locus

to other loci. (Table 7) Fifty-eight families were scored for

21

 

TABLE 7. LOCI TESTED FOR LINKAGE WITH CYSTIC FIBROSIS,29’ 30. “8
Test Locus # Families Visible a, Map Combined
Segregation Distance Lod Score
ABO 75 Male 0.10 -2.605
0.30 -0.l77
Duffy 56 Male 0.10 —0.290
0.30 +0.222
HLA 33 Male 0 . 10 +0 . 5814
0.30 * +0.439
Kidd 33 Male 0.10 s0.06u
0030 -00020
Kell 17 the 0.10 -l.l5u
0.30 —0.225
MNSSl 32 Male 0.10 +0.096
0.30 +0.315
MNSS2 7 Male 0.10 +0.058
0.30 +0.019
MNSs3 58 Male 0.05 -l.u26u
0.10 -0.5130
0.30 +0.1163
Female 0.05 +0.5l33
0.10 +0.7713
0.30 +0.4303
Both 0.05 -0.9131
0.10 +0.2583
0.30 +0.5u66
Total 0.05 —2.6306
0.10 -0.2741
0.30 +0.5279

1. These families were typed for the M, N and S antigens of the
MNSS blood group.

2. These families were typed for the M, N, S and s antigens of
the MNSS blood group.

3. All of these families were typed for the M and N antigens, and

sore were also tested for the S antigens.

22
linkage between the cystic fibrosis and the MNSS blood group loci;
linkage was excluded for a map distance of less than five Centimorgans,

but the data was inconclusive for greater distance.295 30

A collection of computer analyzed data has also been made available.‘18
(Table 7) Linkage between the cystic fibrosis and ABO blood group
loci was excluded at ten map units but inconclusive at thirty map
units. Linkage was inconclusive at both ten and thirty map units for
the cystic fibrosis locus and the Duffy locus (fifty-six families),
the HLA loci (thirty-three families), the Kidd locus (thirty-three
families), the Kell locus (seventeen families) and the MNSS locus
(thirty-nine families). All of the above families were scored for
segregation of the father only. (Data for map distances other than

10 and 30 map units were not published).

F. THE TEST IDCI

l. The Human Histocompatibility Region

The HLA loci are located on the short arm of the sixth human
chromosome.“9a 50 The three loci to be tested in this study, A, B and C,
are located along a 1.0 centimorgan chromosomal segment £499 51: 52: 53
the order of these loci from the centromere is HLA-B - HLA-C - HLA-A.
All three loci are polymorphic and the A, B and C loci may be typed for
18, 32 and 8 alleles, respectively.5u On a single chromosome, the
alleles occurring at the A, B and C loci are called a haplotype; each
individual has a pair of number six chromosomes, and therefore two

HLA haplotypes. Typing of a single individual will result in six

HLA antigens, two for each of the three loci. These six alleles may

23

be defined as two haplotypes by also typing other family members and
correlating information on like and unlike antigens in each person.
For example, a child has HLA antigens Al, A2, B5, B7, C1 and CA. The
mother has antigens A1, A30, B5, B12, 03 and oh; the father has
antigens A2, A33, B7, B35, Cl and C3. The child and mother have HLA
antigens A1, B5 and CA in common, and share the BS-Cu—Al haplotype.
The father and child share the HLA haplotype B7—Cl—A2. The mother
has HLA antigens A30, 1312 and C3 not shared by the child; these three
antigens form her second HLA haplotype B12-C3—A30. The second

chromosore of the father has the HLA haplotype B35-C3—A33.

Confusion in HLA typing may arise from several sources. Recombination
events between the HLA-B and HLA-A loci are infrequent, but when
present may cause some difficulty in assigning haplotype arrange—
ments in family members. Occasionally, an individual has a homozygous
phenotype at one or more of the loci; i.e., that person may have two
identical alleles at the HLA-A locus. This possibility may be con-
firmed by typing of the parents and finding that both parents also
have that particular allele. If one parent lacks that allele, the
child may have a "blank" - an allele not yet identifiable by currently
available antisera. Unidentified alleles represent approximately 1,

A and 20 percent of the total alleles present in the Caucasian
population for the HLA-A ,1 B and C loci, respectively.55 Other

racial groups share many of the same alleles with Caucasians but

have larger percentages of blanks at each locus.’49 An alternative

to the presence of an unidentified allele or blank, is the possibility

(of a chromosomal deletion of that locus.

2A
Although the HLA ailleles are generally co-dominant in their’behavior,
a few antigens show enough cross-reactivity with antibodies of other
alleles to be considered sUbgroups of a common antigen. Those
similar alleles can be distinguished by using non-crossreacting

antibodies.

Cystic fibrosis has not at this time been consistently associated
with any one HLA antigen. Statistically significant increases in
the frequency of the HLA-B18 antigen (in a population of 12 cystic
fibrosis patients and 32 heterozygotes) in one study suggest a
possible association.56 However, this conclusion has not been
supported by other larger studies. HLA frequencies of 28 patients

and 2H0 unrelated blood (ionors showed no significant difference.57a 58

2. The Red Blood Cell Loci

The Rhesus system is composed of three loci, D, C and E (in that
order). These loci are located on the distal end of the short anm
of the first human chromosome.50 TWO common antigens, "D" and "d",
determine whether a.person is Rh positive or negative; the d

allele is recessive and is manifested as the absence of "D" antigen.
A third allele "Du" occurs infrequently and represents a weak
production of the D antigen. "Du" is recessive to "D" but dominant
to "d". The "C" and "c" alleles of the C locus and the "E" and "e"
alleles of the E locus are codominant. As in the HLA system, the
arrangement of D, C and E alleles on eadh first Chromosome represents

a Rhesus "haplotype" which can be confirmed.by typing of other

25

family members. The "haplotypes" are often abbreviated by symbols
such as R1 (alleles D, C and e) or R2 (alleles D, c and E).
Recombination events occur very rarely between the three alleles,
and the three Rhesus genes are considered inherited as a single

complex. 59

The ABO locus is located on the distal end of the long arm of the
ninth human chromosore.50’ 60 Four common alleles are known: A1, A2,
B and O. The 0 allele is recessive to the other three and the B
allele is codominant with Al and A2. The A1 allele is dominant to the

A2 allele, and A2 directs a decreased production of the A1 antigen.

The Kell locus has two common codominant alleles, K and k. The

chromosomal location of the Kell locus is unknown.

The Kidd locus has two common codominant alleles J‘ka and ka, and
is located on the long arm of chromosome 7.61 A third allele Jk is

recessive and rare in the Caucasian population.

The Duffy locus also has two common codominant alleles, Fya and Fyb,
and a recessive allele Fy which is not uncommon in the Caucasian
population. The Duffy locus is located in the center of the long
arm of the first chromosome.62 Although Duffy and Rhesus are both
located on the first human chromosome and therefore syntenic,

they appear to be unlinked by family studies as a result of the

map distance of more than fifty centimorgans (the limiting distance

for family linkage studies).

26
The lewis and Secretor loci are not linked but do interact to

produce a single phenotypic response. The Lewis and Secretor loci
both have two alleles which are of simple dominant-recessive
inheritance. The alleles Le, 1e, Se and se may be present in an
individual in one of nine combinations. The presence of dominant
alleles of both loci (LeLeSeSe, LeLeSese, leleSeSe or LeleSese) is
represented by a phenotype of Le(a-b+). The presence of the dominant
allele of the Lewis locus only (Lelesese or Lelesese) is represented
by the Le(a+b—) phenotype. The absence of dorinance at the Lewis
locus (leleSeSe, leleSese or lelesese) is represented by the

1e(a-b-) phenotype. Neither the Lewis nor the Secretor loci have

received chromosomal assignment.

The MN and Ss loci are closely linked and all four alleles (M, N,
S and s) behave as codominants. As for the HLA and Rhesus systems,
a chromosomal type may be defined after typing informative family

members. The MNSS loci are located on the long arm of chromosore “.60

The P locus is located on human chromosore six and is syntenic with

60

(but not linked to) the HLA locus. Two common alleles are present,

P1 and P2; P2 is recessive to P1.

G. THE HUMAN GENOME

The total autosomal map distance has been estimated at 27.5 Morgans
in the human male and 38.5 Morgans in the female.63 In this study,
the linkage of the cystic fibrosis locus will be tested with ten

unlinked test linkage groups. The test loci to be typed in this

27

study are the HLA system (A, B and C loci), the ABO, Kell, Kidd, Rhesus,
Duffy, Lewis (and Secretor), MNSS and.P blood group loci, and the alpharl-
antitrypsin locus. (Table 8) Linkage will be tested at the practical
limit of 0.40 Morgans map distance from.ea0h test locus. Thus a

total of'8 Morgans map distance of the human autosome will be

tested for presence of the cystic fibrosis locus. This represents a
total of 29.1% and 20.8% of the total autosomal map distance in the

male and female, respectively. The total.mep distance these test

loci have excluded from linkage with cystic fibrosis from prior studies is
only 0.30 Morgans, or 1.1% and 0.8%, respectively of the total linkage
map. In addition to the linkage analysis, an association analysis

will also be carried out on the data collected.

II. METHODS
A. TESTING OF CYSTIC FIBROSIS PATIENTS AND THEIR FAMILIES

Cystic fibrosis individuals and their families were contacted through
three pulmonary and chest clinics in Lansing and Ann Arbor, Michigan
and Cincinnati, Ohio. Contact was made with the director(s) of

eaCh clinic: Drs. wortley and Honicky in Lansing, Drs. Kellogg

and Ingberg in Cincinnati, and Drs. Howatt and Roloff in Ann Arbor.

(Table 9)

With the permission.of the clinic directors, cystic fibrosis
individuals and accompanying family members were contacted at the
time of their regular clinic visit. The majority of cystic fibrosis

patients were children or teenagers, and most came to the clinic with

28

TABLE 8. MAP IOCATTONS OF TEST LOCI.61

 

Test Locus or Loci Map Location

1. HLA system. 6p2

2. Rhesus lp3

3. Duffy lql3

LI. ABO 9q3

5. P 6q2

6. MNSS _ ”q

7. Lewis unknown
8. Secretor unknown
9. Kidd 7q
10. Kell unknown

11. Alpharl-antitrypsin possibly 2 or 12

29

TABLE'9."‘CYSTTC'FIBROSIS'CLINICS.

1.

Chest Clinic

Drs. WOrtley and Honicky
Ingham.Medical Center
Lansing, MiChigan

Chest Clinic

Drs. Kellogg and Ingberg and Miss Lisa.Richter
Children's Hospital of Cincinnati

Cincinnati, Ohio

Cystic Fibrosis Clinic
Drs. Howatt and Roloff
Mbtt Children's Hospital
University of.MiChigan
Ann Arbor, Michigan

30

one or both parents and frequently with siblings as well. The
presence of a minimum of two family members, one affected individual
and one relative (either parent or sibling) was the first pre-
requisite for contacting the family. If consent to participate in
this study was obtained from two or more of the family members
present at this clinic visit, then arrangements were made, either
during the same visit or during a follow—up visit, to contact

other family members .

Each participating individual was asked to sign a consent form.

The consent form was approved for use by the committees on research
in valuing human subjects of Michigan State University and the
University of Michigan. Consent forms for all minors were co-signed
by a parent or other accompanying adult. Any questions regarding
the consent form were answered at the time of signing, and families
were offered copies of the consent form to keep for their personal

records .

Blood was obtained for typing the red and white blood cell antigens.
A physician was present to supervise all venipunctures. Those
venipunctures performed in the clinics were often performed by one
of the directors or a member of the nursing staff at the clirnic. A
few families requested to be drawn at the histocompatibility lab

at Michigan State University and simultaneously receive a brief tour
of the research facilities; for these individuals, a physician was
also present. A large number of families requested home visits to
include additional family members, and again, a physician was present

to supervise and/or perform the venipuncture.

31

Blood was drawn either directly into a heparin vacutainer tube, or
was drawn into a syringe and immediately transferred to a heparin
tube to prevent clot formation. The blood typing was initiated
within twenty-four hours, the outside limit fer’lymphocyte viability
for the histocompatibility antigen testing. Needles chosen for the
venipuncture were 18 guage or larger, so that the cellular components
of the blood would not be destroyed. All laboratory work was
performed in the histocompatibility laboratory at Michigan State
University.

B. TYPING OF THE HISTCOMPATTBILITY ANTIGENS

Typing of the human histocompatibility system was perfbrmed.by the

microcytotoxicity method.6u

Antigens tested were A1, A2, A3, A9
(A23 and.A2A), A10 (A25 and A26), A33, A3”, A11, A28, A29, A30,
A31, A32 and A25 at the HLAeA locus, BS, B7,B8, B12, B13, Blu,
815, B16 (B38 and B39), 817, B18, B21, B22, B27, B35, B37 and BAG
at the HLA—B locus, and Cl, C2, C3 and CA at the HLA-C locus.

Eadh sample was tested against all available antisera. The HLA-C
antisera'were not available until the end of this study, thus many

individuals were not typed fer the HLA-C alleles.

TWO lymphocyte separation proceedures were used. In the first
proceedure, the heparinized sample was centrifhged for ten minutes
at 500 g. The buffy.coat was mixed with McCoy's medium and layered
on Ficoll, then centrifuged twelve minutes at 500 g. The interface

was transferred to a clean tube and centrifhged 1.5 minutes at 3000 g.

32
The resulting cell pellet was suspended in McCoy's medium.and three

drops of thrombin (100 NIH units per milliliter) was added to the tube
(thrombin.agglutinates platelets and granulocytes). The tubes were
rotatedtwo to five minutes, centrifuged two to three seconds at

1000 g, and the supernatant was transferred to a clean tube. The
lymphocytes in the supernatant were then washed twice with MCCoy's

medium and adjusted to 2.5 — 5 x 106 cells per milliliter.6u

Alternatively, one.milliliter of whole blood was rotated with a
pipette tip full of carbonyl iron fer 15 to 30 minutes. The blood
was mixed.with feur milliliters of GEPS solution (EDTA phosphate
saline), layered over Ficoll and centrifuged for 30 minutes at A00 g.
The interface was centrifuged for 10 minutes at 325 g and the cell
pellet was washed twice with GEPS and twice with McCoy's medium.

The lymphocytes in McCoy's medium were adjusted on the hemocytometer

to 2.5 — 5 x 106 cells per milliliter.6“

The lymphocytes were then dispensed in one microliter amounts onto
prepared.antisera trays to test fer antigenic specificities. The

trays each contained 60 to 72 wells (Terasaki or Michigan State
University trays, respectively), and each well held a different anti-
serum. EaCh tray had two wells designated to act as positive and
negative controls. Five microliters of complement was then mixed in
each well and the trays were incubated for 60 minutes. Eosin was
mixed in eadh well, the trays were incubated for 5 minutes, and

then formalin was added to each well to halt the reaction. The

trays were sealed under a glass coverslip‘withrparaffin.and read under

an inverted.microscope: l = negative, the same percentage of cells

33
living as in the negative control of McCoy's medium, 2 = doubtful
negative, ’4 = doubtful positive, 6 = positive, viability noticeably
different from controls, 8 = strong positive, more than 90% of cells
killed (comparable to the positive control), and O = not readable.
When a tray did not result in a satisfactory typing, another tray was
analyzed. Each individual was assigned two haplotypes, based on the

typing results and the pedigree analysis. (Appendices A and B).
C. TYPING OF THE RED BLOOD CELL ANTIGENS

The ABO blood group was also typed and recorded at the time of the

HLA typing. Red cell fractions from each sample were then sent to

the American Red Cross Great Lakes Regional Blood Center of Lansing,
Michigan for typing of the ABO, Rhesus, MNSS, lewis—Secretor, P,

Kell, Duffy and Kidd blood groups. The following red cell antigens
were tested in all samples: A and B (ABO), D, C, E and c (Rhesus),

M, N, S and s (MNSS), Lea and Leb (Lewis-Secretor), P1 (P), Fya
(Duffy) and J'ka (Kidd). Some individuals were also tested for A1
(ABO), Du and e (Rhesus), k (Kidd) and Eyb (Duffy) antigens. (Table 10)
Each of the above loci contain a number of rare antigens or alleles

as well as the common alleles listed above. Since the cost of typirng
these rare antigens would probably not have outweighed the information

gained, these rare alleles were not typed.

3A

 

TABLE 10. ‘RED CELL ANTIGENS TYPED.
' BEEN} ‘ 'Antige' ns
1. ABO A1, A2, B
2. Rhesus D, C, E, c, e, Du
3. MNSS M, N, S, s
A. Lewis-Secretor Lea, Leb
5. Kell K, k
6. P P1
7. Duffy mamyb
8. Kidd Jka

35

D. TESTING OF o<l ANTITRYPSTN

Alpha-l—antitrypsin was quantitatively determined by a starch—gel
electrophoresis method. Between 1/4 to 2 milliliters of serum was
extracted from each sample and stored at -lI° C until electrophoresis
was performed. Since qualitative studies were not performed, the
specific genotype of any individual could not be determined by

this method; however, this test would differentiate any individual
with a deficiency of serum alpha-l—antitrypsin. Since the most
common cause of a deficiency of serum alpha-l-antitrypsin is the
presence of the Z allele, this test served as an inexpensive screen

for the NZ genotype in cystic fibrosis family members.

E . LINKAGE ANALYSIS

In relation to the cystic fibrosis or main locus, all parents were
assumed to be heterozygous. The mating type of the parents is thus
Cfcf x Cfcf. It was assumed that no new mutations occurred at the
cystic fibrosis locus. For the test loci, the mating type of each
family at each test locus was determined from the pedigree and typing
information available. For example, at the ABO locus, a type 0
child born to type A parents indicates a parental mating type of
AOxAO. Matingtypes suchasOOxOOorAAxBBgiveno linkage
information because no segregation is visible. One parent only shows
visible segregation in matings such as A0 x 00 or B0 x AA, and both
parents show segregation in such matings as AB x AB. A few matings

could not be scored because of insufficient information such as if

36

both parents are type A and all tested Children are type A: the
mating type of the parents may have been A0 x AO, however lacking
the presence of an 00 Child or infbrmative grandparents, the mating
type remains unknown and yeilds no linkage information. Grandparents
were typed when possible, to provide additional information on the

parental genotypes.

For each family, lod scores were Obtained for testing linkage
between the cystic fibrosis locus and each test locus, at the
fbllowing map distances (values of theta): 0.00, 0.05, 0.10, 0.20,
0.30 and 0.A0 centimorgans. The scores of all families fer eaCh test
locus and eaCh map distance were then added and cumulative scores
were Obtained. Linkage is considered present if a total lod score

of +3 is attained for all families tested, fer any given map
distance. The value +3 represents odds of 1000 to 1 that the con-
clusion of linkage is correct. Similarly, a lod score of -2 or less
indicates that linkage is absent between 2 loci at a given map

distance, with a prObability of 100 to l of accuracy.

Lod scores for each family at a single test locus may be calculated
in three ways. For example, at the HLA locus, the father and mother
have haplotypes A, B and.C, D, respectively. Their Children.may
have cystic fibrosis and HLA genotypes CfLA/C, cfcf.A/C and cfcf A/D.
First, a lod (z) score may be calculated for segregation of cystic
fibrosis and HLA alleles from.the father to the children. The 22
formula is used.(mating type 2, Tables A and 5). All three Children

received haplotype A from.the father and two are affected. Thus, the

37
progeny phenotypes are a (Cle), b (chl) and b (chl), respectively.
No correction (e) score is needed since the father would express
both haplotypes serologically. Second, a lod (z) score may be
calculated for segregation of the cystic fibrosis and HLA alleles
from.the mother. Mating type 2 is used again, and.progeny
phenotypes are a (Cle),‘b (chl) and d (chlT2), respectively.
Third, since both parents show visible segregation, a "both" or
combined score may be calculated using mating type 6 and z score
formula Z7. Progeny phenotypes are a (CleT3), b (chlT3) and
f (chlTM).

Some matings cannot be scored fer both parents because segregation
is visible in only one parent. An example is mating type AB x 00
(ABO blood group). A z score may be calculated fer segregation of
the A and B alleles of the father, but not fer segregation of the

OO alleles of the mother.

Finally, the scores may be added or totalled. Male scores of all
families with visible segregation of the alleles of the male may be
totalled at each test locus. Similarly, all families at that test
locus with visible segregation of alleles in the mother may be
added for a cumulative female score. And third, a cumulative
combined score is obtained by adding the combined 2 scores of all
families with visible segregation of the alleles of both parents.
And last, a "total" score fer all families at that test locus may
be obtained by adding the most infermative score for eaCh family (a

score for both parents is more infermative than either a female or

38

a.male score). Any family not showing any visible segregation in
either parent is scored as zero (for example, an 00 x 00 mating at

the ABO locus).

III. RESUIJS

A. PARTICIPATION BY CYSTIC FIBROSIS FAMILIES

A total of fifty—nine families participated in this study. Seventeen
. families were from the Lansing area and seen at the Lansing clinic;
thirty-three families were seen at the Cincinnati clinic and.nine
families at the Ann Arbor clinic. None of the families are known

to be related.

A total of 2““ individuals were typed. Of these, 67 individuals
had cystic fibrosis, 51 individuals were unaffected siblings and A
were unaffected.half-siblings. Eighty-nine parents, 32 grandparents
and l great-grandparent were typed. Some family members.suCh as
young Children and infants were not typed because of technical

difficulties in Obtaining adequate blood samples.

B. SEX RATIO

The sex ratio of cystic fibrosis and unaffected children is consistent
with expected values fer an autosomal recessive trait. Of the 118
Children in the study, 67 were affected and 51 were unaffected.
Thirty-five of the affected Children were males and 32 were females;
30 of the unaffected Children were males and 21.were females.

(Chi-square = .5075, no significant difference.)

39

C. ANALYSIS OF DATA OF THE RED AND WHITE BLOOD CELL ANTIGENS

A total of fifteen different loci were tested: the A, B and C loci
of the HLA system, the D, C and E loci of the Rhesus system, the
MN and Ss loci, and the P, Kell, Kidd, Lewis , Secretor, ABO and
Duffy loci. These fifteen loci represent ten separate linkage
groups: the MNSS, HLA, P, Kell, Kidd, Lewis, Secretor, ABO,

Rhesus and Duffy linkage groups. (Table 8)

1. The Histocorrpatibility Complex

HLA typing was performed on each individual by a microcytotoxicity
method as described above. The HLA-A, HLA-B and HLA-C loci

antigens possessed by each individual were then combined into two
haplotypes based on the HLA antigens of other family members.

For example, in family B02, mother has HLA alleles A2 , A2“, B7

and 812. Father has alleles Al, A2, B8 and B1”. Two dnildren, B023
and 8025 have alleles A2 , B12 and B1“. These children each received
an A2/Bl2 haplotype from the mother, and a A2/Blll haplotype from

the father. Child B0214 received an A2/BlLl haplotype from the father
and an A24/B7 haplotype from the mother. Children B026, B027 and
B028 share the haplotype Al/B8 with the father, and have respective

A2LI/B7, A2/B12 and A2/812 haplotypes in common with their mother.

Haplotypes of each individual are listed in Appendix A. Three
families are listed by individual antigens rather than by haplotypes ,

since family evidence was insufficient to determine haplotype

140

grouping (families B09, A43 and A119). For example, in family A119,
the mother has HLA alleles A2, A3 and B7. The affected son has
alleles A2, A29, B12 and B15. Since the mother did not type for
either 1312 or B15, no discernable haplotype is shared by the mother
and affected son; however, it may be assumed that his A2 allele came
from the mother. The unaffected son has alleles A2 , A3, B7 and B15.
He may share either A2 or A3 with the mother, at the HLA—A locus.

At the B locus, he may share B7 with the mother, or both he and his
affected sibling may share 815 with the mother. It is not possible

in any of these three individuals to identify the HLA haplotypes.

Occasionally, a family occurred with a discrepancy in HLA typing.
In family B32, the child B325 appears to be a half—sibling to the
affected child B321. The haplotype A2/B5 is transmitted from the
mother, but the reported father B323 has neither the A series
antigen A26 or the B series B27. Also, the MNSS and Duffy antigens
present in the child support nonpaternity. The child B325 was
therefore assumed to be a half-sibling in the data analysis arnd

computation of lod scores for linkage.

One family (A16) appeared to have a recombinant individual in the
HLA-A system. (Figure 1) Based on the grandmother A162, the father
A1611 must have haplotypes A2/B35 and A2/B7. Each child received one
intact haplotype from the father: A163 has A2/B35 and A165 has
A2/B7. Thus Child A163 must have haplotype A30/B12 and child A165
has A30/B35 from the mother. If the haplotypes of the mother are

A28/Bl2 and A30/B35 then child A163 is a recombinant; if haplotypes

A162

female
unaffected
A2/B35, A19/Buo

A164
male

H1

 

unaffected
A2/B35, A2/B7

A163

male

affected
A2/B35, A30/Bl2

 

A161

female

unaffected

A28, A30, B12, B35

A165
female
unaffected

A30/B35, A2/B7

FIGURE '1.‘ ‘ 'PEDIGREE ‘OF FAMILY 'A16 7 ILLUSTRATING ‘A "RECOMBINANT EVENT

"AT'THE'HLA'REGION.'

A2
of the mother are A28/B35 and A30/B12 then child A165 is a recombinant.

The maternal grandparents were not available fer typing.

Families B30 and AA7 both have individuals demonstrating more than

two alleles at a single locus. Individual B303 reacts positively to
antisera for alleles B7, B12 and BAO; individual AA71 reacts to
antisera fer alleles A2, A25, and A32. Individual AA71 passed these
specificities to one Child; in family B30 it is unclear if the

doUble allele was passed from the mother or from the father. This

type of reaction is prObably the result of a typing error. Alternative
explainations are due to an inserted allele (duplication) in the
genome, or an allele which was combined with another allele (an
intragenic recombinant event) so that the resulting molecule has

receptor sites for antibodies of both type of alleles.

Last, individual 0582 was not typed for HLA antigens because the

blood sample obtained from her was of insufficient quantity.

All cystic fibrosis individuals and their full siblings were tested
for any significant variation in the expected frequencies of eaCh
HLA antigen of'both the A and B series alleles. (Table 11) Only one
antigen, B7, manifested a.highly significant difference (chi-square
value between .0005 - .0010). Thirteen affected individuals out of
a total of 67 carried this allele; 25 normal full siblings out of a
total of 51 had the allele. The reason for this association of the
B7 allele with unaffected siblings may be a Chance occurrence or

may be attributed to a relatively small sample size.

A3

' TABLE '11. ' 'CHI-'-SQUARE ANALYSIS 'OF ASSOCIATION 'OF 'WHITE BLOOD CELL
' ANTIGENS WITH CYSTIC FIBROSIS.

 

Antigen Present Antigen Absent
Not Not Chi-
Antigen Affected Affected Affected Affected Square Alpha

A1 15 13 52 38 0.15A0 .6 - .7
A2 A1 3A 26 17 0.37AA .5 — .6
A3 13 13 5A 38 0.62A6 .A — .5
A9 11 12 56 39 0.9333 .3 - .A
(A23) A 1 63 50 1.1A72 .2 — .3
(A2A) 6 11 61 A0 3.7361 .05 - .1
A10 6 A 61 A7 0.0A62 .8 - .9
(A25) 3 0 6A 51 2.3A32 .1 - .2
(A26) 1 3 66 A8 1.7039 .1 - .2
All 2 2 65 A9 0.0775 .7 - .8
A28 6 5 61 A6 0.02A7 .8 — .9
A29 13 7 5A AA 0.6631 .A - .5
A30 3 2 6A A9 0.0221 .8 - .9
A31 2 0 65 51 1.5A86 .2 - .3
A32 8 5 59 A6 0.13A8 .7 - .8
A33 2 2 65 A9 0.0775 .7 - .8
B5 7 7 60 AA 0.2975 .5 - .6
B7 13 25 5A 26 11.6339 .0005 — .0010
B8 8 10 59 A1 1.3169 .2 - .3
B12 28 13 39 38 3.3937 .05 - .10
B13 5 2 62 A9 0.6507 .A - .5
BlA 5 A 62 A7 0.0059 .90 - .95
B15 13 6 5A A5 1.2506 .2 - .3
B16 A 2 63 A9 0.2518 .6 - .7
(B38) 1 1 66 50 0.0381 .8 - .9
(B39) 3 1 6A 50 0.5601 .A—- .5
B17 A 1 63 50 1.1A72 .2 - .3
B18 3 1 6A 50 0.5601 .A - .5
B21 3 5 6A A6 1.2999 .2 - .3
B22 3 3 6A A8 0.118A .7 - .8
B27 A 6 63 A5 1.2536 .2 - .3
B35 10 5 57 A6 0.68A5 .A - .5
837 A 0 63 51 3.1516 .05 - .10
BAO 11 7 56 AA 0.162A .6 - .7

Example of calculation for the HLA—B7 Antigen:

d.f. = l
2

)( '[(13‘X'26)'4'(5A'x 25)l2'(118) = 11.6339

(67) (5D (38) (80)
(X between .0010 - .0005

AA

The histocompatibility complex was recognized by the haplotype derived
from typing of the A, B and C loci and by correlation of the segregation
of those alleles in each family. In 3 families, haplotypes could

not be assigned to every individual because of insufficient information
and lod scores were calculated based on the segregation of one locus

instead of the entire haplotype. (Families B09, A143 and AA9).

In family Al6 (Figure 1), one of the two children received a recombinant
haplotype from the mother, however information is not sufficient to
determine which child is a recombinant and which received an intact
haplotype from the mother. (The recombinant event took place between
the A and B loci of the histocompatibility complex.) Although lod
scores cannot be computed for cystic fibrosis and the HLA complex,

lod scores can be computed for cystic fibrosis and a single HLA

locus. Computing 27 scores for this family using segregation of the

A locus of the mother gives lod scores of +.12L19 , +.O859, +.0556,
+.Ol8A, +.OO37 and +.OOO2 for map distances of 0, .05, .10, .20, .30
and .40 centimorgans, respectively. Calculation of Z7 scores using
segregation of the HLA-B locus of the mother gives respective lod
scores of +.l2ll9, +.l219, +.llO6, +.O780, +.OAO6 and +.Oll2. Both

sets of scores are positive (give evidence for the presence of linkage).
Lod scores of segregation of the HLA-A locus were arbitrarely

chosen in this family for information of linkage with the cystic

fibrosis locus.

Family B30 was not scored for linkage at the HLA locus. One child
has both A series alleles in common with the mother and the other

child has two B series alleles in common with the mother. (Appendix

“5

A and B) The father and other siblings were not available and
scoring of one child for A alleles and the other for B alleles would

negate a recombinant event, if present.

All other families with two or more children were scored for linkage
of the cystic fibrosis locus and the histocompatibility complex.

(See Table 12 for lod scores, using the Z7 formula, Table 5). All
families were scored for segregation of both parents using the Z7
formula. Each parent was also scored separately using the Z2 lod
score formula. linkage was excluded (total lod score value was less
than -2) for 0.00, 0.05 and 0.10 centimorgans from the cystic
fibrosis locus, using the cumulative lod scores of both parents for
all families. (Figure 2) Linkage was excluded from 0.00 and 0.05
centimorgans when lod scores were calculated for segregation of the
father; segregation of the mother excluded linkage of the HLA complex
and cystic fibrosis from 0.00 centimorgans. One family could not be
scored for the individual parents; family B22, two children were typed
but consent was not obtained for the parents; since the parents were

not typed, it is not known which parent had which haplotypes.

2. The ABC Blood Group

The ABO blood group antigens were typed twice, first at the histo—
compatibility lab at Michigan State University and then at the
American Red Cross Great Lakes Regional Blood Center. For each
individual the ABO type obtained from the two laboratories was the
same. The ABC types were also consistent within each family

(Appendix A). Results of the association analysis showed no significant

TABLE '12.” LOD'SCDRES ’OF'

CYSTIC'FIBROSIS'AND'THE'HISTOCOMPATIBILITY

46

 

'COMPEEX.
0.00 0.05 0.1 0.2 0.3 0.4

B01 Z7 Both -00 —.6174 -.3597 -.1446 -.0532 -.0120
Z2 Female —.0512 —.0410 -.0320 -.0177 -.0078 -.0019

Z2 Male -.3522 —.2596 —.1908 —.O969 -.0404 -.0098

B02 Z7 Both —00 -.4687 -.2522 -.0947 -.0347 -.0079
Z2 Female -.2784 -.2l86 -.1683 —.0910 -.0394 —.0096

Z2 Male .0226 .0219 .0199 .0138 .0070 .0019

B03 Z7 Both .1249 .0859 .0556 .0184 .0037 .0002
22 Female .1249 .1038 .0840 .0492 .0226 .0058

Z2 Male —.1761 —.1367 —.1042 -.0555 -.0238 —.0058

A06 Z7 Both .4998 .4333 .3653 .2310 .1130 .0302
22 Female .4998 .4354 .3703 .2407 .121) .0335

Z2 Male -.1023 -.O819 -.0641 -.0355 -.0156 —.OO39

A14 Z7 Both —00 —1.2399 -.7297 -.3110 -.1410 -.0729
Z2 Female -.4033 -.3006 -.2229 —.1146 —.O482 -.Oll7

Z2 Male -.4033 -.3006 -.2229 -.ll46 -.0482 —.0117

A15 Z7 Both ~ ~-oo -.6174 -.3597 -.l446 -.0532 -.0120
22 Female -oo -.7212 -.4437 -.l938 -.0757 -.0177

Z2 Male .1249 .1038 .0840 .0492 .0226 .0058

A16 27 Both ..12A9 .0859 .0556 .018A .0037 .0002
Z2 Female -.l761 —.1367 -.1042 -.0555 -.O238 -.OO58

Z2 Male .1249 .1038 .0840 .0492 .0226 .0058

B19 Z7 Both -00 —.4965 -.2491 -.0666 -.0125 -.0008
Z2 Female -00 -.6838 -.4139 -.l768 —.0681 9.0158

Z2 Male .2499 .2167 .1828 .1158 .0567 .0151

B21 Z7 Both .1249 .1210 .1106 .0780 .0406 .0112
Z2 Female .1249 .1038 .0840 .0492 .0226 .0058

Z2 Male .1249 .1038 .0840 .0492 .0226 .0058

B22 Z7 Both .1249 .0859 .0556 .0184 .0037 .0002
B23 27 Both .7270 .6181 .5100 ,.3059 .1398 .0349
Z2 Female .1249 .1038 “0840 .0492 .0226 .0058

' Z2 Male .4260 .3711 .3153 .2049 .1027 .0280
B24 Z7 Both -00 —.9617 -.4907 -.l395 -.0282 -.0019
z2 Female .2725 .2482 .2192‘ .1509 .0793 .0222

TABLE'l2“(Continued)

 

A26

B27

B31

B32

B36

B37

B38

B39

B41

B42

A47

A48

Z7
Z2
Z2

Z7
Z2
Z2

Z7
Z2
Z2

Z7
Z2

Z7
Z2
Z2

Z7
22
Z2

Z7
Z2
Z2

Z7
Z2
22

Z7
Z2
Z2

Z7
Z2
Z2

Z7
Z2
Z2

Z7
Z2
Z2

Both
Female
Male

Both
Female
Male

Both
Female
Male

Both
Female
Male

Both
Female
Male

Both
Female
Male

Both
Female
Male

Both
Female
Nble

Both
Female
Male

Both
Female
Nble

Both
Female
Male

Both
Female
Male

0.00
-00
-.3522
-.3522

-00
.4260

-00

.1249
.1249

.1249
.1249
.1249

-00
-.0512

.1249
—.1761
.1249

.3748
.3748
.3748

.4998
.1987

.1249
.1249
.1249

-00
-oo
.1249

0.05
-.7622
-.2596
-.2596

.3711
-.7212

.0859
.1038
-.1367

.1210
.1038
.1038

-l.0747

-.0492

.0859
-.1367
.1038

.3651
.3231
.3231

.3935
.1712

-.O8l9

.1210
.1038

—.6l74
-0 7212
.1038

—1.4425

-07212
-0 7212

-.6174
-.1367
-.1367

47

0.1
-.4757
-.1908
-.1908

-.1237
.3153
-.4437

.0556
.0840
-.1042

.1106
.0840

—.5775
-.0442
-.0442

.0556
-.1042
.0840

.3394
.2715
.2715

.2949
.1434
-.0641

.1106
.0840
.0840

-0359?
.0840

-.8874
"'0 ””37

-0 3597
"'0 10142
-.1042

0.2
-.2116
-.0969
-.0969

.0307
.2041
-.1938

.0184
.0492
-.0555

.0780
.0A92
.0A92

-.l905
-.0295
-.0295

.0184

-.0555
.0492

.2544
.1717
.1717

.1373
.0895
-.0355

.0780
.0492
.0492

.0492

"olg38

—.l446

0.3
-.0835
-.0404
-.0404

.0734
.1027

-00757

30226

.0406
.0226
.0226

‘.Ol44
-.0144

.0037
-.0238
.0226

.1444
.0843
.0843

.0461
.0431
-.0156

.0226
.0226

-.0532
-.0757
.0226

-.1514
-.0757

-.0532
-.0238
—.O238

0.4
-.0197
-.0098

.0866
.0280

.0002
.0058
-.0058

.0112
.0058
.0058

-.0092
-.0038
-.0038

.0002
-.0058
.0058

.0432
.0226
.0226

.0087
.0114
-.0039

.0112
°0058

—.0120
-.0177
.0058

-.0120
-.0058
—.0058

TABLE'12"(COntinued)

 

TOTALS:

0.00
Female —00
Male -00
Both —00
Total —00

48

0.05 0.1
—1.9547 -.8921
-2.0971 -1.l392
-6.8341 —3.1054
_6.8341 -3.1054

0.2
-.1223
-.3602
—.7314
-.7314

0.3

.0501
—.0876
-.0610

0.4
.0294
-.0110

.0423

.0423

49

+2

+1

 

-1

 

 

Lod Score

FIGUBE'2. LOD SCORE TOTALS BETWEEN‘CYSTIC FIBROSIS 'AND‘THE
' 'HIS‘IOCOMPA’IIBM'I‘I COMPLEX.

 

m

‘o're.

mol-

.7 .‘o-nv

--O

n--

.-

 

 

0.0144! s.‘\

 

V4...

 

 

..q»

a

50
association of any of the ABO antigens with cystic fibrosis (Table 13).
The combined lod scores of all families and those showing segregation
of alleles by the father exclude linkage from 0.00 and 0.05 centimorgans.
Segregation by the mother and those families where segregation could not
be separated in the parents (both or combined scores) exclude linkage

from 0.00 centimorgans. (Table 14, Figure 3).

3. The Rhesus Blood Group

Typing of the alleles of the Rhesus system yielded one discrepancy.
In family A15, child A157 has alleles D, C, c and e. The parents
A154 and A151 each have alleles D, C and e but do not have the c
allele. Typing of all other antigen systems is consistent beWeen
parents and child; there are no conflicts in antigens present in
two affected siblings. Since only one allele is unexplained, the
pedigree is assumed to be accurate. The c allele in individual
A157 may be the result of inaccurate typing, or a result of some

other irregularity in genetic transmission or expression.

There is no significant association between possession of any of

the Rhesus antigens and expression of cystic fibrosis. (Table 13).

Linkage between the Rhesus and cystic fibrosis loci is also negative.
Lod scores exclude linkage at 0.00 and 0.05 centimorgans for all
families; and from 0.00 map units for those families scored for

segregation of male, female and combined alleles. (Table 15,

Figure 4).

51

TABLE 13. 'CHI—SQUAR ‘ANALYSI ‘OF'ASSOCIATTON'OF RED BLOOD CELL
'ANTIGENS WITH CYSTIC FIBROSIS.

Antigen Antigen
Present Absent
Trait Antigen (2)3 3 Chi-
d' d‘ Square Alpha
m: m, E: :>
:2 a w :2
m m m m
o o g. o
8 8' 9 8
o. a. 9* :1
ABC A 34 19 33 32 2.1303 .1-.2
B 6 6 61 45 0.2502 .6-.7
00 29 27 38 24 1.0831 .2—.3
A1 21 13 43 37 0.6224 .4-.5
Rhesus D 56 39 11 11 0.5844 .4—.5
C 45 27 22 23 2.0964 .l-.2
E 19 13 48 37 0.0801 .7-.8
c 52 A2 15 8 0.7398 .3-.4
e 65 47 2 3 0.6362 .4-.5
MNSS M’ 50 44 17 6 3.2422 .05-.1
N 46 31 21 19 0.5713 .4-.5
S 29 3O 38 20 3.2006 .05-.1
s 58 42 9 8 0.1519 .6-.7
Lewis Le(apb— 8 6 59 44 0.0001 .975-.990
Le(a+b- 1A 6 53 44 1.5987 .2-.3
Le(apb+) 45 38 22 12 1.0843 .2-.3
Kell K 7 6 60 AA 0.0699 .7-.8
P P1 56 41 ll 9 0.0506 .8-.9
Duffy Fyg 41 23 26 27 2.6676 .1-.2
Fy 40 14 4 1 0.0848 .7—.8
Kidd Jka 55 42 12 8 0.0737 .7-.8
Sex Female 32 21 35 30 0.5075 .4—.5

52

TABLE’14."LOD'SCORES'OF'CYSTIC'FIBROSIS'AND‘THE'ABO'BLOOD'GBOUP.

B01

B02

A15

B19

B21

B23

A26

B31

B36

B37

B38

B39

Z2
e2

23

Z2
e2

Z2
e2

Z2
e2

Z7
e2
22
22
e2

22
e2

22
e2

Z7
e2
22
e2
Z2

22
e2

22
e2

Male

Female

Both

Male
Female
Male
Both
Female
Male
Male
Male
Both
Female
Nble

Female

Male

0.0 0.05

.0226 .0219
-.0060 -.0045
.2499 .2076

.0738 .0628
-.0032 -.0031

.1249 .1038
-.0458 -.0374

.0171

—00
.0212

.2499 .2076
-.0332.55027l
-.0512 -.0410

.2499 .2122
—.0332 —.0271

.1249 .1038
-.0458 -.0374

-00 -.7622
-.0032 —.0031

.12A9 .1210
-.0458 -.0374
.1249 .1038
-.0458 -.0374
.1249 .1038

-.5283 -.3608

-.4033 -.3006
-.0111 -.0087

0.1

.0199
-.0033

.1680

.0519
-.0028

.0840
-.0298

-.4437
.013A

.1680
—.0215
-.0320

.1754
-.0215

.oauo
“.0298

-.4757
-.0028

.1106
—.0298
.0840
—.0298
.0840

-.2532
-.0126

-.2229
-.0067

0.2
-.0177

.0138
-.0017

.0984

.0315
—.0019

.0492

-.1938
.0075

.0984
-.0122
-.0177

.1072
-.0122

.0492
-.0170

.0780
-.0170
.0492
-.0170
.0492

-.1219’

-.0070

-.1146
-.0036

0.3
-.0078

.0070
-.0007

.0451

.0148
-.0009

.0226

-.0757
.0033

.0451
-.0055
-.0078

.0509
-.0055

.0226
—.0077

-.0835
-.0009

.0406
-.0077
.0226
-.0077
.0226

-.O494
-.0031

—.0482
-.0015

0.4
-.0019

.0019
""0 0002

.0115

.0038
-.0003

.0058
-.0019

.0008

.0115
-.0014
-.0019

.0133
-.0014

.0058
-.0019

-.0197
-.0003

.0112
—.0019
.0058
-.0019
.0058

TABLE'14."(Continued)

 

A47 27
e3
22
e2
22
e2

TOTALS:

Both
Female
Male

Female
Nble
Both

Total

-00

.0631

-00

.0212

-00

.0212

-l.4425
.0443
-.7212

.0171
-.7212
.0171

-.9717
-2.1088
-.9265

-2.9131

53

-.887A
.0305
-.4437
.0134
-.4437
.013A

-.6031
-1.2595

-l.7237

-.3876
.0132
-.1938
.0075
-.1938

.0075

-.2564
-.5159
-.1288

-.6872

-.1514
.0047
-.0757
.0033
-.0757
.0033

-.0966
-.l899
-.0291

-.2459

’00355
.0010

-.0177
.0008
-.0177
.0008

—.O219
“50427

-.0541

54

+3

+2

+1

 

 

 

Lod Score

FIGURE 3. ' LOD SCORE TOTALS BETWEEN CYSTIC FIBROSIS AND THE 'ABO
‘ BLOOD GROUP.

55

 

TABLE 15. LOD SCORES OF CYSTIC FIBROSIS AND THE RHESUS BLOOD GROUP.
0.0 0.05 0.1 0.2 0.3 0.4
B01 Z2 Female -.0512 —.0410 -.0320 -.Ol77 -.0078 -.0019
B03 Z2 Female -.l76l -.1367 -.1042 -.0555 -.O238 -.0058
A06 Z7 Both .4998 .4873 .4547 .3471 .2036 .0635
22 Female .4998 .4354 .3703 .2407 .1219 .0335
22 Male .4998 .4354 .3703 .2407 .1219 .0335
A14 Z2 Male -.4033 -.3006 —.2229 —.ll46 —.O482 -.0117
B19 Z2 Male -oo -.7622 -.4757 -.2115 -.O835 -.0197
B21 Z2 Female -.l761 -.l367 -.1042 -.0555 -.O238 -.OO58
B27 Z2 Male .1249 .1038 .0840 .0492 .0226 .0058
B31 Z7 Both .1249 .1210 .1106 .0780 .0406 .0112
Z2 Female .1249 .1038 .0840 .0492 .0226 .0058
Z2 .Male .1249 .1038 .0840 .0492 .0226 .0058
B32 Z7 Both .1249 .0895 .0556 .0184 .0037 .0002
Z2 Female .1249 .1038 .0840 .0492 .0226 .0058
22 Male -.l761 -.l367 -.1042 -.0555 -.0238 -.0058
B36 22 Male -00 -.7622 -.4757 —.2115 -.0835 -.0197
B37 22 Female .1249 .1038 .0840 .0492 .0226 .0058
B38 27 Both -00 -.6666 -.4039 -.1741 -.0676 -.0158
Z2 Female -.2272 -.1776 -.l362 -.O732 -.O3l6 -.OO78
Z2 Male —.2272 -.l776 -.1362 -.0732 -.0316 -.0078
B39 24 Both .1987 .1747 .1472 .0886 .0300 -.0218
Z2 Female .7270 .1038 .0840 .0492 .0226 .0058
Z2 Male .7270 .1038 .0840 .0492 .0226 .0058
B41 Z7 Both .1249 .0859 .0556 .0184 .0037 .0002
Z2 Female .1249 .1038 .0840 .0492 .0226 .0058
B42 22 Female -oo -.7212 -.4437 —.1938 -.0757 -.0177
A47 Z7 Both -00 —.3502 -.l284 .0103 .0296 .0866
22 Female -00 -.7212 -.4437 -.1938 -.0757 -.0177
Z2 Male .4260 .3711 .3153 .2041 .1027 .0280
A48 Z2 male .1249 .1038 .0840 .0492 .0226 .0058

 

TABLE 15. Continued)
0.0

TIHALS: Female -00
Male -00

Both -00

Total —00

56

0.05 0.1

-.9800 -.4737
-.9l76 -.3931
-.6045 -.0308
.3.1501 —1.6372

0.2

-.1028
-.0247

.2510
-.4615

0.3

-.0035
.0444
.1910

-.O875

0.4

.0058
.0200
.1046
.0397

57

+3

+2

+1

 

-2

 

 

FIGURE 9. 'LOD SCORE TOTALS BETWEEN CYSTIC FIBROSIS AND THE RHESUS
"BLOOD'GROUP.

 

-i- _7-

v

-1... _

 

58

4. The MNSS Blood GrOLp

The MNSS blood grOLp shows no significant association of any single
antigen with the cystic fibrosis locus.

Linkage between the MNSS and cystic fibrosis loci was excluded at
0.00 centimorgans for all four categories: families in which
segregation is visible in males only, families in which segregation
is visible in females, families in which segregation is visible

in both parents, and all families. (Table 16, Figure 5).

5. The Lewis and Secretor Blood Groups

There was no significant association evident between the expression

of Lewis-Secretor phenotype and cystic fibrosis (Table 13).

Linkage with cystic fibrosis was tested separately between the Lewis
and the Secretor loci (Tables 17 and 18). For the Lewis and cystic
fibrosis loci, linkage was excluded from 0.00 map distance for families
with segregation visible in the female and for all families. Linkage
was inconclusive for families with segregation visible in the male

and for families with segregation visible in both parents. (Figure 6).

At the secretor locus, lod scores excluded linkage at 0.00 centimorgans
for only one family with visible segregation in both parents. Linkage
was inconclusive when scored for male and for female segregation.

(Figure 7).

TABLE'16."LOD'SCORES'OF'CYSTTC‘FIBROSIS'AND'THE‘MNSS'BLOOD'GROUP.

B01

B02

A14

A15

B19

B21
B23
B24
A26
B27
B30
B31
B32

B36

B37

B38

B41

Z7
Z2
Z2

Z2

Z7
Z2
Z2

Z7
Z2
Z2

24
Z2
Z2
24
22
Z2
Z4
22
Z2
Z4
Z2
Z4

22
Z2

Z7
Z2
Z2

Z2

Z7
22
22

Both
Female
Nble
Female
Both
Female
Male
Both
Female
Male
Both
Female
Male
Both
Female
Male
Both
Female
Nble
Both
Male
Both
Female
Male
Both
Female
Male
Female
Both

Female
Male

0.0

.2499
—.0512
.2499

-.2784

.4998
.1987
-.1023

.1249

-00
-.1761
-.1761

.1249
-.1761
.1249

.0738
.1249

.1249
.1249

0.05

.1866
-.0410
02122

-.2l86

.3935
.1712
—.0819

-1.4425

-.7212
-07212

.1774

.2122
.2122

.1079
-.l367
“05332
-.0347
—.7212

.1038

.1079
..1038
-.7246
-.1367

.0859
-.1367

.1038

.0628

.1210

.1038
.1038

59

0.1
.1279
—.0320

-.1683

.2949
.1434
-.8874
.3922
.1754
.0987
—.1042
-.4205
-.0266
—.4437
.0840
.0987
.0840

-.1042
-.1042

.0556
-.1042
.0840

.0519

.0840
.0840

0.2

.0297
-.0177
.1072
-.0910

.1373
.0895
-.0355

-.3876
-.l938
’11938

.5100
.1072
.1072
.0962
-.0555
-.2264
—.0257
-.1938
.0492
.0962
.0492
-.l638
.0184
-.0555
.0492
.0780

.0492
.0492

0.3
-.0354
-.0078

.0509
-.0394

.0461

.0431

-.1514
-.0757
“00757

.4770
.0509
.0509
.1015
-.0238
-.0962
-.0757
.0226
.1015
.0226
-.0238
-.0238
.0037
-.0238
.0226
.0148
..0406

.0226
.0226

0.4
-.0638
—.0019

.0133
-.0096

.0087

.0114
-.0355
-.0177
-.0177

.3726

.0133
-.0058
—.0234
-.0177

.0058

.1036

.0058
-.0122
-.0058
-.0058

.0002
-.0058

.0058

.0038

.0112

.0058
.0058

TABLE 16. (Continued)

 

B42 22 Male .1249 .1038

A47 22 Female .4260 .3711

TOTALS Female —00 —1.1910
Both —00 —1.0216

Total. -00 -1.8860

60

.0840
.3153

-.6303
—.2617
-.1589
-.6764

.0492

.2041

—.1813
-.0508
.3887
.2052

.0226

.1027

-.0859
.0035
.A91A
.3916

.0058
.0280

-.0020
.0048
.AA07
.4334

61

+3

+2

+1

 

 

 

-l

-2

 

Lod Score

' FIGURES. ' ~1001300BEV'1011A1s‘BE'IWEEN ”CYSTIC ”FIBROSIS 'AND'THE MNSS
' BLOOD 'GROUP. ‘

 

"TABTE'17."LOD'SCORES'OF'CYSTIC'FTBROSIS'AND‘THE'LEWIS'BLOOD'GROUP.

B01 22

e2

B02 23
e3
22
e2
22
e2
B19 22
e2
B23 22
e2

TOTALS:

Male

Both
Female

Male

'Female

Male

Female
Male
Both

Total

0.0

.2725
-.O400
.1249
.0212
.1249
.0212

-00

.4260
.0212

.8100
.2325

-00

0.05
.2122

.2296
-.0275
.1038
.0171
.1038
.0171

-.6838
-.0031

.3711
.0171

-.5660
.6942
.2021

.0885

62

0.1
-.0215

.1882
-.0184
.0840
.0134
.0840
.0134

-0u139
-.0028

.3153
.0134

-.3l93
..5800
.1698
.2357

0.2
.1072
-.0122

01123
-.0075
.0492

.0075
.0492
.0075

-.l768
-.0019

.2041
.0075

-.1220
.3633
.1048
.2327

0.3
.0509

.0518
-.0025
.0226
.0226
.0033

-.0681
-.0009

.1027
.0033

—.0431
.1773
.0A93
.1317

0.4

.0133
-.0014

.0132
-.0005
.0058
.0008
.0058
.0008

-.0003

.0280
.0008

-.0095
.0473
.0127

.0373

63

+3

+2

+1

 

 

 

 

Lod Score

‘ FIGURE '6. ' 'LOD'SCORE TOTALS BETWEEN CYSTIC FIBROSIS 'AND THE LEWIS
' 'BLOOD'GROUP.

 

64

'TABLE 18. 'LOD SCORE OF CYSTI ‘FIBROSIS'AND'THE'SECRETOR LEAKEDGROUP.

0.0 0.05 0.1 0.2 0.3 0.4

B36 Z3 Both -00 -.7877 -.4859 -.2092 -.O801 -.0185
e2 -.0032 -.0031 -.OO28 -.0019 -.0009 -.0003

Z2 Female -.1761 -.l367 -.1042 -.0555 -.O238 -.0058

Z2 Male -.1761 -.l367 -.1042 -.0555 -.0238 -.OO58

e2 -.O458 -.0374 -.O298 -.0170 -.0077 -.0019
TOTALS: Female -.l761 -.1367 -.1042 -.0555 -.0238 —.0058
Male —.2219 —.1741 —.1340 -.O725 -.O315 -.OO77

Both -00 -.7908 -.4887 -.2111 -.O810 —.0188

Total -00 -.7908 -.4887 -.2111 -.0810 -.Ol88

65

+3

+2

+1

 

 

 

Lod Score

FIGURE‘7: 'LOD'SCORE'TOTALS'BETWEEN ,CYSTIC FIBROSIS‘AND‘THE'SECRE‘IOR
' BLOOD GROUP.

 

66

6. The Kell, P, Duffy and Kidd Blood Groups

There is no significant association of antigens of the Kell, P,

Duffy or Kidd loci with the cystic fibrosis locus (Table 13).

Linkage at the Kell locus was inconclusive (greater than -2 and less
than +3) for all categories: male scores, female scores, both or

combined scores and total scores for all.famfilies (Table 19,

Figure 8).

Linkage of the P blood group locus and the cystic fibrosis locus was
excluded at 0.00 map units by female, both and total scores. Linkage
was inconclusive based on the scores of the families segregating
visibly in the fathers (Table 20, Figure 9).

At the Duffy and Kidd loci linkage was excluded at 0.00 map units fer
families with segregation by the fathers and fer all families; scores
for the females and the families with segregation at both loci was

inconclusive (Table 21 and 22, Figures 10 and 11).

One individual (A165) was drawn at the completion of the study and
was not typed for the red.cell blood group antigens by the American
Red Cross Great Lakes Regional Blood Center. However, she was typed
fer the histocompatibility antigens and fer the ABO blood group
alleles. Since only one other sibling in her family was included in
this study, the family (A16) was scored in the linkage analysis for
onlyiimaABO and HLA loci. She was included only in the association
analysis fer ABO and HLA antigens and for sex distribution.

67

TABLE‘l9.’"LOD'SCORES'OF'CYSTTC‘FIBROSIS‘AND’THE'KELL'BLOOD'GROUP.
0.0 0.05 0.1 0.2 0.3 0.4
A06 22 Male .1987 .1712 .1434 .0895 .0431 .0114
A15 22 Male .1249 .1038 .0840 .0492 .0226 .0058
B24 22 Nhle .8746 .7853 .6911 .4889 .2756 .0847
B30 22 Female .1249 .1038 .0840 .0492 .0226 .0058
B31 Z2 Female .1249 .1038 .0840 .0492 .0226 .0058
B37 22 Male .1249 .1038 .0840 .0492 .0226 .0058
B39 22 Female .4998 .4354 .3703 .2407 .1219 .0335

TOTALS: Female .7496 .6430 .5383 .3391 .1671 .0A51
Male 1.3231 1.16A1 1.0025 .6768 .3639 .1077
Both 0.0 0.0 0.0 0.0 0.0 0.0

' Total 2.0727 1.8071 1.5408 1.0159 .5310 .1528

68

+3

+1

........................

..................................

 

Lod Score

‘ FIGURE '8. ' LOD 'SCORE TOTALS BETWEEN 'CYSTIC FIBROSIS ‘AND THE KELL

'TABLE‘20."LOD'SCORES'OF'

B01

B02

A15

A26

B31

B32

B36

B37

B41

B42

A48

A49

Z3
e3
22
e2
22
e2

22
e2

Z3

Z3
e3

22
e2

22
e2

23
e2

22
e2

22
e2

22
e2

22
e2

22
e2

TOTALS:

Both
Female

Male

Nble

Both

Both

Male

Female

Both

Nble

Female

Female

Male

Female

Female

Male
Both

e’Total

0.0

.2499
—.0619
.0457
.0162
.0457
.0162

-.5794
-.0060

-00

.2499
-.0332

.1249
-.0458

012,49
-.0458

.1987
-.0032

-.l76l
—.O458

-00
.0212

.1249
-.O458

-.1761
-.0458
-00
- o 5 872
-00

-OO

69

CYSTIC'FTBROSIS'AND'THE'P'BLOOD'GROUP.

0.05
.2076

-.0470
.0374
.0132
.0374
.0132

-.4018
—.0045

.1993

.1038
-.0374

.1038
-.0374

.1632
-.0031

-.1367
-.0374

"o 7212
.0171

.1038
-.0374

-.l367
-.0374

"'0 3970

-l.5188

0.1
.1680

—.0348
.0298
.0104
.0298
.0104

—.2853
-.OO33

'0 3270

.1543
-.0215

.0840
-00298

.0840
—.0298

.1298

—.0298

-.0298

.0134

.0840

-.10A2
—.0298
-.6039
-.2740

.0660
-.8923

0.2
.0984

-.0173
.0170
.0058
.0170
.0058

-.l397
-.0017

.0824
-.0122

.0492

.0492
-.0170

.0727
-.0019

-.0555
-.0170

-.0170

-.1039
.0075

.0492
-.0170

-.0555
-.0170
-.1864
-.1267

.0977
-.3594

0.3
.0451
-.0069

.0077
.0026

.0077
.0026

-.0572
-.0007

-.0435

.0346

.0226

.0226
-.0077

.0317
-.0009

"'0 0238
-0007?

-.0757
.0033

.0226
-.0077

-.0238
-.0077
.0546

0.4
.0115

-.0016
.0019
.0006
.0019
.0006

—.0094

.0083

.0058
-.0019

.0058

.0078
—.0003

-.0058
-.0019

—.0019

.0008

.0058
-.0019

-.0058
-.0019
-00259
-.0113

.0149

-.0196

70

+3

+2

+1

Morgans

 

-2

 

 

Lod Score

FIGURE 9. ' LOD SCORE TOTALS BETWEEN CYSTIC FIBROSIS AND THE P
' BLOOD GROUP.

 

71

 

TABLE 21. LOD SCORES OF CYSTIC FIBROSIS AND THE DUFFY BLOOD GROUP.
0.0 0.05 0.1 0.2 0.3 0.4

B02 22 Female -.5794 -.4018 -.2853 —.l397 -.0572 -.0137
e2 - -.0060 —.OO45 -.0033 -.0017 —.0007 -.0002
A06 Z2 Female -.1023 -.0819 -.0641 -.0355 -.0156 -.0039
e2 -.0111 -.0087 -.0067 -.OO36 —;0015 -.0004
A14 Z2 Female .1987 .1712 .1434 .0895 .0431 .0114
e2 -.0111 -.0087 -.0067 -.0036 —.0015 —.0004
A15 26 Both .7270 .6288 .5301 .3377 .1671 .0450
Z2 Female .3010 .2577 .2148 .1335 .0645 .0170
Z2 Male .3010 .2577 .2148 .1335 .0645 .0170
B19 Z2 Male -00 -.7622 -.4757 -.2115 -.O835 -.Ol97
B21 Z2 Female .1249 .1038 .0840 .0492 .0226 .0058
A26 Z2 Male -.0512 -.O410 -.O320 -.0177 -.0078 -.0019
e2 -.0332 -.O27l -.0215 -.0122 -.0055 -.0014
B31 Z2 Female -.1761 -.1367 -.1042 -.0555 -.0238 -.0058
B37 Z2 Female .1249 .1038 .0840 .0492 .0226 .0058
B39 Z2 Male -.4033 -.3006 -.2229 -.1l46 -.0482 -.0117
TOTALS: Female -.1365 .0153 .0559 .0818 .0525 .0136
Male -00 -.8732 -.5373 -.2225 -.0805 —.0177
Both .7270 .6288 .5301 .3377 .1671 .0450
Total -00 —.7445 -.3809 -.0700 .0101 .0089

72

+3

+2

+1

 

 

 

Lod Score

' "FIGURE ‘10. ' 100 SCORE TOTALSBETWEEN 'CYSTTC FIBROSIS ”AND THE DUFFY
' BLOOD GROUP. ’

 

TABLE'22."LOD'SCORES'OF'

B02

A06

A14

B23

B27

B32

B39

A48

Z3
e3

Z3
e3

22
e2

22
e2

22
e2

22
e2

22
e2

22
e2

TOTALS:

Both

Both

Female

Female

Male

Male

Male

Female

Female
Male

Both

'Total

0.0
.6247
-.0400

-.0285
-.0480

-.1761
-.0458

.3010
.1761

-00
.0212

-.l761
-.0458

-.4033
-.0111

-.l761

.0333

.5082

~00

CYSTTC'FIBROSIS'AND'THE'

0.05
.5165
-.0275

;.0322
-.0345

-.0374

.2577
.1367

-.7212
.0171

-.l367
-.0374

-.3006
-.0087

-.l367
-.0374

.0462

-.7931
.4223

-0 7190

73

0.1
.4135
-.0184

-.0242

—.1042

.2148
.1042

-.4437
.013A

-.1042

-.2229
-.OO67

-.1042
-.0298

.0510
-.4749
.3402

—.4027

0.2
.2327
-.0075

-.0208

-.0170

.1335
.0555

-.1938
.0075

-.0555

-.1146
—.0036

-0 0555
-.0170

.0440
—.1880
.1936
-.1394

KIDD'BLOOD'GROUP.

0.3
.1002

-.0100

-.O238

.0645
.0238

-.0757
.0033

-.0238

-.0482
-.0015

-.0238
-.0077

.0253
-.0653
.0838
-.OAA5

0.4
.0240
-.0005

—.OO58
-.0019

.0170
.0058

.0008

-.0058
-.0019

—.0117

-.0019

.0074
.0201

74

+3

+2

+1

MOrgans

 

 

 

Lod Score

~FIGURE'll.’’LOD’SCORE'TOT‘ALS'BETWEEN'CYSTIC'FTBROSIS'AND'THE'KIDD
"BLOOD'GROUP. ‘

 

75
D. RESULTS OF (X ANTITRYPSIN TESTING

1
Alpha-l-antitrypsin was quantitatively determined in each individual
to show the presence of any of the deficiency alleles at the alpha-l-
antitrypsin locus. Only two individuals exhibited a decreased amount
of alpha-l-antitrypsin. Individuals A154 and A162 both had half the
normal amount of alpha-l-antitrypsin in their serum. Individual
A154 is the father of two cystic fibrosis children; individual A162
is a paternal grandmother of one cystic fibrosis child. These two
individuals probably have one normal (M) and one deficiency (Z)
allele at the alpha-l-antitrypsin locus. All other individuals in

this study had normal amounts of alpha-l—antitrypsin.

IV. DISCUSSION

The ideal approach to diagnosis and treatment of cystic fibrosis
depends on the ascertainment of the basic biochemical defect causing
the disease. However, this biochemical alteration has continued to
elude investigators and remains Imknown. Since cystic fibrosis
individuals do not yet benefit from the specific treatment given
other genetic diseases, such as phenylketonuria, another approach

must be sought .

A. ASSOCIATION OF BLOOD GROUP ANTIGENS AND CYSTIC FIBROSIS

Understanding of other diseases has benefited from association analyses,

such as the association of the HLA-827 allele of the histocompatibility

complex with ankylosing spondylitis. In a general Caucasian population,

76
the B27 antigen occurs in about 5% of the individuals; the B27
antigen occurs in as many as 95% of patients having ankylosing
spondylitis.26’ 65 This association is so strong that determination
of the 827 antigen is now used.as a diagnostic tool to differentiate

between the many causes of lower back pain.

This study found no significant association of cystic fibrosis and
HLA-B27. Four affected children and seven unaffected siblings were HLA-

B27 positive ()i2 value of 2.060A, alpha between .2 and .1).

A statistically significant increase in the frequency of the HLA-B18
allele of the histocompatibility complex has been feund in cystic
fibrosis individuals,56 by Kaiser, Laszlo and.Gyurkovits. Another
study of 28 patients and 240 unrelated blood donors, by GotZ, Ludwig
and Polymenidis, found no significant association of any HLA allele
with cystic fibrosis.579 58 In this study, three affected individuals
and one normal sibling carried the B18 allele and the difference is

not significant.

However, a.highly significant association.was fbund between cystic
fibrosis and the HLA-B7 allele. Thirteen affected individuals out of
a total of 67 carried this allele, while 25 normal siblings out of a
total of 51 had the allele (prObability between .0010 and .0005).

(Table 23)..

The conflicting results of these three studies may be due to
statistical error’present in small samples. Alternatively, these

results may be explained if "cystic fibrosis" is composed of more

77

TABLE '23. ' ASSOCIATION 'OF HLA-4B7 WITH CYSTIC FIBROSIS.

 

HLA—B7 Positive HLA—B7 Negative
Affected 13 54
Non-affected 25 26
Totals 38 80

x2 ="[<13 x 26) '-"(54’x-25)12‘(118)
(38)(80)(67)(51)

Degrees of Freedom = 1

X2 = 11.6339

alpha between .0010 —- .0005

Totals

67

51

118

78
than one disease entity. Inheritance as an autosomal recessive trait
seems well established. However, two different autosomal recessive
diseases with similar clinical findings would be distinguishable only by
l) biochemical methods, or 2) a mating of two affected individuals

resulting in normal offspring.
B. C>< l-ANTITRYPSIN

At the alpha-l-antitrypsin locus, the ZZ genotype is associated with

emphysema, a pulmonary disorder.66

Only two individuals in this

study have decreased amounts of alpha-l-antitrypsin, a father and
a.paternal grandmother, in two different families; they probably

have the MZ genotype. Thus the alpha-l-antitrypsin deficiency

alleles appear to have no association with the occurrence of cystic

fibrosis.
C. LINKAGE RESULTS BETWEEN CYSTIC FIBROSIS AND THE TEST LOCI

Another approach to improve diagnosis and treatment of cystic fibrosis
individuals is to identify a linkage of the cystic fibrosis locus
with another locus. Typing of the HLA loci is already used for
diagnosis of a number of other inherited disorders. Several
components of the complement system, including C2, C4 and C8 are
known to be closely linked to the HLA 1001.67’ 68 Juvenile diabetes
mellitus692 70, hemochromatosis71 and 21-hydroxylase deficiency53

are also known to be closely linked to the HLA system.

79
The highest total lod score found in this study was +2.0272, for

0.00 map units distance, between the cystic fibrosis and Kell loci.
This lod score represents a probability of 118 to 1 that linkage is
present between the cystic fibrosis and Kell loci. The accepted
"cut-off" value for linkage is +3; this represents odds of 1000 to

1 that the conclusion of linkage is correct. At odds of 118 to l,
the 1 represents .85% chance that linkage is not present between the
two loci, but only appears to be present due to some unknown bias or

error in population selection or experimental design.

Data for linkage of the cystic fibrosis and Kell loci is also of
interest because absolute linkage cannot be excluded by this study.
Absolute linkage would indicate that the cystic fibrosis and Kell
loci occupy the same location of the human genome, or that they
occupy different locations but are controlled by a single expressor
region. However, absolute linkage may be eliminated by the study of
Edwards (Table 7). Scores for 17 families scored for segregation of
the father gave total lod scores of —l.154 and —0.225 at map distances
of 0.10 and 0.30 Morgans, respectively. At 0.00 Morgans map distance,
the score of these 17 families would approach negative infinity.

(Figure 12).

A known linkage of the cystic fibrosis locus to a test locus such as
Kell has a variety of practical uses. 1) In families where a cystic
fibrosis individual has previously occurred, at-risk infants could
be screened at birth for the test locus, and therapy initiated
immediately for affected individuals.2) Siblings of patients could

be screened fOr carrier status; Mendelian risks of sibling carriers

+3

+2

+1

..... _. A. . . "Morgans‘"

 

 

 

Lod Score

FIGURE '12. ’ ‘LOD‘SCORE TOTAL 'FOR'17 FAMILIES 'SCORED 'FOR 'SEGREGATION
' 'OF THE FATHERS ONLY, BY 'EDWARDSllb

81

are 67%; risks based on determination of a linked test locus would
approach 0% or 100% fer many siblings. 3) At risk parents would have
the Option of in utero testing and subsequent abortion of high risk
fetuses for a test locus that could be determined by amniocentesis.
4) A known linkage of‘cystic fibrosis would permit further linkage
studies, first to localize the cystic fibrosis locus on the human
genome and second with the cystic fibrosis locus asza relatively

common test locus.

Limitations of the use of a test locus fer diagnosis of cystic

fibrosis and identification of carriers are l) the alleles of the

test locus must visibly segregate in the family (i.e. at the Kell

locus, a kk x kk mating gives no diagnostic information), 2) the

test locus must be close to the cystic fibrosis locus; the greater

the distance separating the two loci, the greater the prObability

of a recombinant event occurring between them, and 3) a highly
polymorphic locus, such as the HLA locus will yield.more information
than a test locus such as Kell with only two common alleles; fer example
at the HLA loci, it can be determined exactly which alleles of each

child came from eaCh parent in almost all families.

A number of areas of the human linkage map may be excluded from

linkage with the cystic fibrosis locus by this study. (I.e., when

the lod score for all families is less than —2, linkage is excluded
with a probability of 100 to 1.) The cystic fibrosis locus is excluded
from a distance of .14 map units or less on either side of the

histocompatibility complex,. .09 map units or less from the'ABO locus

82
and the Rhesus loci, .05 map units or less from the MNSS loci, .04
map units or less from the P locus and .01 map units or less from the
Lewis, Secretor, Kidd and Duffy loci. In all a total of .90 map

units are shown not to contain the cystic fibrosis locus.

Absolute linkage (linkage at 0.00 map units distance) is excluded for
each of the above loci. However, any of these loci may be linked to
the cystic fibrosis locus at a greater map distance. For example,
although linkage of the cystic fibrosis and HLA loci was excluded
from 0.00 to 0.14 map units, they may be linked at .30 map units.
Linkage could be either excluded or confirmed at .30 map units by
scoring additional families until a total lod score of either -2

or +3, respectively, was reached.

These chromosomal regions where linkage can neither be confirmed nor
denied are "grey" areas, where the cystic fibrosis locus may or may
not be located. All other areas of the human autosome not tested by
this study also remain as "grey" areas until data is gathered to

either confirm or deny linkage with the cystic fibrosis locus.

D. COMBINED LOD SCORES OF THIS STUDY WITH SCORES OF OTHER

PUBLISHED STUDIES

Addition of lod scores of this study with lod scores of other published
studies increases the information available.29’ 30’ L18 (Tables 7 and 24,
Figures 13 - 14). Combining lod scores of all families for this study

and the other studies does not result in a confirmed linkage of the

83

 

TABLE 24. TOTALS 'OF LOD SCORES 'OF THE BLOOD GROUP LOCI FROM THIS
”STUDY 'ANDFROM'OIHER'PUBLISHED'STUDIES. ' (SEE TABLE '7.)
Map Distances, Centimorgans
0.00 0.05 0.10 0.20 0.30
HLA Male -2.521 .378
ABC Male -4.329 - .423
MNS Female -00 - .6777 .1410 .5503 .3444
Male -00 —1.9560 - .7747 .0052 .1198
Both -00 -1.9347 .0994 1.1763 1.0380
Total ~00 —4.5166 — .9505 .9214 .9195
Female - .1644 .9123 .7747
Male -3.3824 -l.2877 .2361
Both —2.8478 .3577 1.5846
Total —7.l472 -l.2246 1.4474
Total -l.07l 1.781
Kell Male .387 .306
Kidd Male - .467 - .065
Duffy Male - .671 .232

 

0.40

84

+3

+2

+1

Kell

 

 

 

 

 

Lod Score

' FIGURE -' 13.‘ ' "LODSCORE TOTALS BETWEEN 'CYSTI_C FIBROSIS ANDTHE ”DUFFY
' KELL .- ‘KIDD BLOOD 'GROUPS-AJ-COMBIPJED—FRES‘ITLT—TS =O'F"‘TTi' 's“"'vSTUDY
-- cm "W: I. -. .1.

 

  

  
  

  
  

     

 

85

+3

+2

+1

..................

 

-1

—2

 

m
I

 

Lod Scores

’FTGURE'14.LOD SCORE TOTALS BETWEEN CYSTIC FIBROSIS AND THE MNSs‘ HLA
AND ABO BLOOD GROUPS-«COMBINED RESUL OF
OTHER PUBLISHED STUDIES.

 
 

 

86
cystic fibrosis locus. However, the cystic fibrosis locus can be
excluded from greater areas of the human gene map ; from .20 map units
or less on either side of the ABO locus, .04 map units or less from
the Duffy locus and .08 map units or less from the MNSS locus. These
combined studies exclude the cystic fibrosis locus from 1.22 map units

of the total human genome.

This study indicates a possible linkage of the cystic fibrosis and
Kell loci. After combining lod scores of this study and the study of

Edwards ’48

at the Kell locus, the total lod score at 0.10 map units
was 0.387, and at 0.30 map units was 0.306. (Graphically the scores
of Edwards indicate exclusion of absolute linkage between Kell and

cystic fibrosis.) (Figure 13).

The possibility of more than one cystic fibrosis locus (or allele)

has still not been excluded. If "cystic fibrosis" is a composite

of more than one disease process, then any linkage studies would

have to be modified to address linkage of several cystic fibrosis

7 1001 or of a multiallelic cystic fibrosis locus. While current

data still support autosomal recessive inheritance, conclusive proof
depends on either a confirmed mating of two cystic fibrosis individuals
or identification of the basic metabolic defect responsible for the

clinical symptoms of cystic fibrosis.

V} CONCLUSION

A total of fifty-nine families were studied fer linkage and for
association of the cystic fibrosis locus with 15 red and.white blood
cell loci. Linkage was not confirmed between cystic fibrosis and
any of the test loci. Areas of the human linkage map that may be
excluded from.1inkage with the cystic fibrosis locus by this study
are: .14 map units or less fromlthe histocompatibility loci,-

.09 map units or less from.the ABO locus and the Rhesus loci, .05
map units or less from the MNSS loci, .04 map units or less from
the P locus and .01 map units or less from.the Lewis, Secretor,

Kidd and.Duffy loci. In all, a total of .90 map units are shown not
to contain the cystic fibrosis locus. A significant association

was feund.between the cystic fibrosis locus and the HLA-B7 antigen.
(CX between .0010 and .0005). No association was found between

cystic fibrosis and deficiency of alphaplsantitrypsin.

87

APPENDICES

APPENDIX A

Family and individual identification numbers and intrafamilial
relationships.
Family Number

Individual Number Relation to Affected Child (*)

B01 B011 * affected female
B012 mother:
B013 father
B014 brother
B015 brother
B02 B021 father
B022 mother
B023 * affected female
B024 brother
B025 brother
B026 brother
8027 brother
B028 brother
B03 B031 * affected female
B032 mother
B033 sister
B04 B041 * affected female
B042 mother
B05 B051 mother
B052 * affected female
B053 maternal grandmother
B054 halfebrother (same mother as *)
B055 maternal grandfather
A06 A061 * affected male
A062 father
A063 sister
A064 sister
A065 sister
A066 brother
A067 mother
B07 B071 * affected male
B072 mother
B073 father
B074 maternal grandmother
808 B081 father
B082 * affected female

88

B09

A10

A11

A12

A13

A14

A15

A16

B17

B18

819

BO91
B092

A101
A102

A111
A112

A121
A122

A131
A132
A133

A141
A142
A143
A144
A145
A146

A151
A152
A153
A154
A155
A156
A157
A158
A159

A161
A162
A163
A164
A165

B171
B172
B173

B181
B182

B191
B192
B19 3
B194
B195
B196
B197
B198

89

* affected male
mother

father
* affected female

* affected female
mother

mother
* affected.male

* affected male
mother
father

* affected male
brother

father

sister

sister

brother

mother

* affected female

* affected female
father

paternal grandmother
paternal grandfather
brother

maternal grandfather
maternal grandmother

mother

paternal grandmother
* affected male
father

sister

mother
* affected male
father

* affected female
mother

* affected female
mmmher

* affected male
sister

father

brother

paternal grandmother
maternal grandmother

B20

B21

B22

B23

B24

A25

A26

B27

B287

B29

B201
B202

B211
B212
B213
B214

B221
B222

B231
B232
B233
B234

B241
B242
B243
B244
B245
B246
B247
B248
B249

B241-l
B242-l
B243-1

A251
A252

A261
A262
A263
A264
A265
A266
A267

B271
B272
B273
B274
B275

B281
B282

B291
B292
B293
B294
B295

90

mother
* affected male

* affected.male
mother
sister
father

sister
* affected.ma1e

* affected female
mother

brother

* affected.ma1e

* affected female
* affected male
mother

brother

brother

brother

sister

sister
maternal.grandmother
brother

father

brother

mother
* affected female

* affected female
mother

maternal grandfather
maternal grandmother
father

brother

brother

mother
* affected.male
* affected.male
father
sister

mother
* affected.male

* affected female
mother

father

maternal grandfather
maternal,grandmother

B30

B31

B32

B33

A34

A35

B36

B37

B301
B302
B303

B311
B312
B313
B314
B315
B316

B321
B322
B323
B324
B325

B331
B332
B333
B334
B335

A341
A342
A343
A344
A345
A347
A348

A351
A352
A353

B361
B362
B363
B364
B365
B366
B368
B369

B371
B372
B373
B374
B375

91

* affected.male
mother
brother

* affected male
father

mother

brother

maternal grandmother
maternal grandfather

* affected.male

mother

father

brother

halfLsister (same mother as *)

* affected male
maternal grandmother
mother

father
maternal.gmandfather

* affected female

mother

maternal grandmother
paternal grandmother

father
maternal.gmandmother's mother
maternal grandfather

* affected male
father
mother

father

sister

* affected.male

* affected male
mnmher

sister

maternal grandfather
maternal grandmother

mother

* affected female
brother

father

paternal grandmother

B38

B39

B40

B41

B42

A43

B44

B45

B46

B381
B382
B383
B384
B385
B386
B387

B391
B392
B393
B394
B395
B396
B397

B401
B402

B411
B412
B413

B421
B422
B423
B424
B425

A431
A432

B441
B442
B443

B451;

B452
B453
B454
B455
B456
B457

B461
B462
B463

92

brother

sister

sister

mother

father

* affected male
maternal grandmother

mother
brother
father
brother
sister
brother
* affected male

mother
* affected male

* affected female
brother
mother

* affected female
* affected male
father

mother

sister

mother
* affected male

* affected female
father
paternal grandmother

* affected.fema1e
mother

father

maternal grandfather
maternal grandmother
paternal grandmother
paternal.grandfather

* affected.ma1e
mother
father

A47

A48

A49

A50

C51

C52

C53

C54

C55

C56

C57

C58

C59

A471
A472
A473
A474
A475

A481
A482
A483
A484

A491
A492
A493

A501
A502
A503

C511
C512

C521
C522

C531
C532

C541
C542

C551
C552
C553

C561
C562

C571
C572

C581
C582
C583

C591
C592
C593
C594
C595

93

mother

father

brother

* affected female
* affected.male

* affected female
mother
sister
father

brother
* affected male
mother

mother
father
* affected female

* affected female
father

mother
* affected female

* affected male
father

* affected male
father

mother
* affected female
maternal grandmother

* affected female
mother

* affected female
father

father
mother
* affected.male

* affected female

father

mother

half-sister (mother same as *)
halfesister (mother same as *)

HLA haplotypes of eaCh individual.

Individual Number

B011*
B012
B013
B014
B015

BO21
B022
B023*
8024
B025
BO26
B027
8028

B031*
BO32
B033

B041*
B042

8051
B052*
B053
B054
B055

A061*
A062
A063
A064
A065
A066
A067

B071*
B072

B073
B074

B081
B082*

BO91*
BO92

APPENDIX B

First Haplotype

A3/B40
A32/B12
A1/B15
A3/B40
A3/B40

Al/B8
A2/B12
A2/B12
A2/Bl4
A2/B12
Al/B8
Al/B8
Al/B8

A24/B27
A2/B7
A2/B7

Al/B7
A2/B15

A2/B39
A2/B39
A2/B37
A-/B12
A2/B39

A2/B12
A3/B7
A2/B27/C2
A2/B27/C2
A2/B27/C2
A2/B27/O2
A2/Bl2

Al/B7
Al/B7
A2/B5
A3/Bl5

Al/B8
A-/B35

Al, A2, B15, B22
A1, A2, B—, B22

94

(* affected Child).

Second Haplotype

A32/B27
A32/B27
A3/B40

A32/B27
A32/B12

A2/B1A
A24/B7
A2/Bl4
A24/B7
A2/Bl4
A24/B7
A2/Bl2
A2/Bl2

A32/B14
A32/B14
A24/B27

A2/B15
A3/B35

A23/Bl4
A25/B40
A23/Bl4
A2/B39
A26/B7

A33/Bl4
A33/B14
A33/Bl4
A3/B7
A3/B7
A33/Bl4

A2/B27/C2

A2/B5
A3/Bl5
All/B39
A24/Bl2

A2/Bl5
A2/B15

C4

C2

c2, C3

C4

A101

A102*.

A111*
A112

A121
A122*

A131*
A132
A133

A141*
A142
A143
A144
A145
B146

A151
A152*
A153*
A154
A155
A156
A157
A158
A159

A161
A162
A163*
A164
A165

8171
B172*
3173

B181*
B182

B191*
B192
B193*
B194
B195
B196

B197
B198

95

As/B—
A2/B15

Al/B8
A2/B16

A—/B8
A2/B15

A10/B7
A10/B7
All/327

A2/B15
112/815
A2/B2l
A2/Bl5/C3
A2/B15/C3
A2/B35/C4

Al/Bl7
Al/Bl7
A23/Bl2
A2/B7
A23/B12
A2/B7
Al/B17
A3/B18
Al/Bl7

A28, A30, B12, B35
A2/B35
A2/B35
A2/B7
A2/B7

A2/BS
A2/B5
A2/B2l

A2/Bl3
43/37

A2/B40
A2/Bl2
A2/Bl2
A2/B40
A2/B12
A2/Bl2
A—/B7

A2/312

A29/B17
A29/B17

A28/B35
A28/B35

A2/B15
A3/B40

A32/B12
All/B12
A32/B12

A24/B21
A2/B2l

A24/B2l
A24/B21
A24/B21
A24/B2l

A28/B35
A2 3/B12
A28/B35
A2 3/B12
A28/B35
A28/840
A23/Bl2
A28/B35
A3/B35

A19/B40
A30/B12
A2/B35

A30/B35

A24/B40
A2/B2l
43/317

A3/B7
A28/B-

A28/B5
A28/B5
A2/BA0
A28/B5
A2/BAO
A28/B5‘
A2/B12
A3/Bl8

C4
C4

C1

B201
B202*

B211*
B212
B213
B214

B221
B222*

B231*
B232
B233
B234*

B241*
B242*
B243
B244
B245
B246
B247
B248
B249
B241-1
B242-1
B243-1

A251
A252*

A261*
A262
A263
A264
A265
A266
A267

B271
B272*
B273*
B274
B275

B281
B282*

96

A2/B27
Al/B8

A2/B7
A2/B7

All/B27

A2/B12

A2/B15
A10/B8

A2/315
All/B8
A2/B8

A2/B15

A2/B7
Al/B8
A2/B7
A2/B7
Al/B8
Al/B8
Al/B8
Al/B8
A2/B7
A2/B7
A1/B8
A3/B7

A2/Bl5
A2/Bl5

A2/B12
A2/Bl2
Av/B12
A2/B12

A2/B35/C4

A2/B12
A2/B12

41/37
A2/B12
A23/B8
A2/813
Al/B7

A2/Bl3
A—/B27

A24/B35
A24/B35

A2/Bl2

A24/B15
A24/B15
All/B27

A10/B8
A24/B35

A30/B13
A30/813
A30/813
A30/813

A29/B5
A2/B7
A3/B7
A29/B5
A2/B7
A3/B7
A3/B7
A3/B7
A—/B15
A29/B5
A29/B5
A29/B5

A28/B13/C4
A—/B18

A3/B7
A-/B15
A-/B15/C3
A28/B15/C3
A3/B7
A3/B7
A3/B7

A23/B8
A23/B8
A28/BB9
A28/B39
A28/B39

All/B7
A2/B13 C1

8291*
8292
B293
B294
B295

8301*
8302
B303

8311*
8312
8313
B314
B315
B316

8321*
B322
B323
B324
B325

8331*
8332
B333
8334
B335

A341*
A342
A3A3
A3AA
A345
A347
A348

8351*
A352
A353

8361
B362
8363*
8364*
B365
B366
B368
B369

97

A2/812
Al/B8
A-/840
A2/Bl2
Al/B8

A29, A30, 814, 840
A29, A32, B7, 840
A28, A29, B7, 812, B40

A2/812
83/335/C4
A2/Bl2
A3/B35
A2/814
Al/817

Al/B7
Al/B7
A3/B7
A2/B5
A2/B5

A29/Bl2
A1/88
A1/B8
A2/B4O
A29/B27

A2/B22
A2/822/CB
A2/822/C3
A2/A32/Bl5/C3
A25/B39
A2/B27/C2
All/B27

Al/837
Al/B37
A28/B12

A2/B5

A2/812
A2/B12
A2/812
A2/812
A2/812
A2/B2l
A2/812

A2/Bl2
A2/Bl2
A2/812
A3/B7

A2/813

A3/815/C4
A29/812
A24/821
A24/B21
A24/821
A2/812

A3/B7
A2/B5
A24/87

A24/87
A26/B27

A32/840
A2/Bl2

A32/840
A29/Bl2
A32/840

A32/B40
All/B27
A2/B27/C2
A25/B39
A32/B40
A32/B35/04
A24/B8

A31/B21
A2/B38
A31/B21

A29/812
A29/812
A29/812
A29/812
A2/B2l
A29/812
A24/812/C1
A3/B7

98

8371 A3/812
A 2
8373* 22/822/03 Ag/g835/CA
B37“ figgg C A3/B12
B375 / 3 BB?
A-/B35/'CA 83/87
8381
B382 23% A26/B22
B383 A2/B7 A26/B22
B38“ A2/B AZ6/822
B385 A2/B'377 A26/B22
B38“ A2/B37 A3/B39
B387 A26 A3/B39
”322 429/818
13391
B392 11117887 A32/B40
B393 111/1335 A32/840
B394 Al/B35 A24/B7
B395 121/35 A32/840
B396 All/B; figi/BAO
B397* Al/B35 Alljgg
B401
1129/1312 A
8402* A3/BAO ASS/€315
8411*
B412 21371318 1233/87
B1113 112/318 113/6153
8421*
Bug” 1533/1135 A29/812
B423 A24/B7 A29/812
B424 A2/B12 25%ng
842 35
5 112/812 A29/Bl2
A431 A2 A B
9 '3 12, B15, C2, C
B441!
8442 212888 - 2215122
8443 [124/815 A26/B27/C2
8451*
81452 8225812 A31/1335/cA
B453 A10/887 A29/812
B145“ A25/Bl A31/835/CA
BASS Al/B. 7 A32/B40
8456 A2/812 A29/812
B457 Al/Bl A10/88
7 A31/B35/OA
8461*
B462 fiéA/B37 A24/840
8463 /B12 A2A/B37
A24/BAO A26/B39

C4

A471
A472
A473
A474*
A475*

A481*
A482
A483
A484

A491
A492*
A493

A501
A502
A503*

C511*
C512

C521
C522*

C531*
C532

C541*
C542

C551
C552*

C553

C561*
C562

C571*
C572

C581
C582
0583*

C591*
C592
C593
C594
C595

99

A2/8- A25A32/817
A1/B38 A2/Bl2
Al/B38 A2/B-
A2/Bl2 A25A32/Bl7
Al/B38 A2/B—
Al/B8 A2/B5
A2/85 A-/B-
Al/88 A2/B5
Al/B8 A3/B22/C3
A2, A3, 87, B15, C4

A2, A29, 812, 815

A2, A3, B7, 8-

A2/Bl2 A29/815
Al/Bl7 A2/Bl2
Al/Bl7 A29/Bl5
A2/B5 A25/818
Al/Bl2 A25/818
A2/Bl2 A3/B5
A—/Bl4 A3/B5
A-/B- A24/B40/C3
A2/Bl2 A24/B40/C3
A2/812 A32/B35/C4
A2/Bl2 A24/B21/Cl
A2/812 A3/B7
A-/B~ A2/812
A2/Bl2 A24/B7
Al/B37 A28/812
Al/B37 A2/B27/C1
A2/Bl2 A3/B7
A-/B40 A2/Bl2
A1/Bl3 All/B7
Insufficient sample to type

A3/B— All/B7
A26/827/Cl A29/Bl2
A2/Bl8 A26/B27/Cl
A2/B15/C3 A29/Bl2
A2/Bl5/C3 A24/B35/CA
A24/B35 A29/Bl2

APPENDIX C

Results of red cell analysis.

m%&xk a Fwwbma

DuMNSS

h DCEce

A B

+++++

NNNNN
_____

+++__

NNNNN
___._

N ++++
N +
N +
N +_+-
N ++—+

+++++
+
+
++—++

+
+
N
N
+

++_+++++

NNNNNNNN
_+ _ _ _ _ _+

+_+++++.

NNNNNNNN
_______.

_+

+.+.+.
....__

+—

_+__+.
++++++
1+__+_
++++++

+_+_
++++

++NNNNN
+++++++
____.._
__._.__
_..____

—--++

N++NN++N

_++__++_
123u5678
meanness

+ _

+—-+

++—+N

+_ ..
-+ N
_+ N

*8031
8032
B033

- N +

— N +

- N +
_+-++ — ++-+ +
— -+—+ _+

—+—++

+
N
+
+

8051
*8052
m8
m%
8055

++++_++

++NNNNN
+_+__++

+++++++

N++N++N
_++_++

+ _
+
+ -

++++++
+_++++

._____
++++++

+-+—

NNNNNNN

++NNNNN
+++++++

______.
+__..++
+++++++

NNNNNNN

iloaauhchKuvl
roro/o/o/oro/o
nonvnonununvno
MAAAAAA

++++

+++N
+_++

+++_

NNNN
..__

_+

+_
—+

+..+
++++
++++

_+++

N_N_

+++N
++++
++_.
+_+_
+.+_

_N_N

+ -
+ _

m8

mfl

*8071
8072

++
._

++

++

_+

+_
++

_+
++

++
++

._
++
++

NN

—+
-+

N +—++
N +—++

+—+++
+_+++

NN

*8091
8092

100

101

leaLeb K k Pl FyaFyb Jka

Du- M N S 8

Al D C E c e

A B

— +
+ _

+ + - + + N + — + +
N + + + +

N
N

A101
*A102

+ _ + + +

+ +

+ + + + _

N
N

+ _ _ + + + + +

*Alll

—+

_ + _ +

N + + _ _ +

A112

— +
— +

+ _ + +
+ _ _ +

++--+ N

+ -

A121
*A122

N

N + + _ _ +

N - + — + + _

+ + — + +
_ _ - + +

+ + _ _ +

*A131

.+ +

..+ .—

+ -

- + _ +

A132

- +

- N +

+ + — +

N

+ _ +

8133
*A141
A142

_ +
+

— + + +
+ + + _

_ _ _ + +
_ _ + + +
_ _ + + +

N
N

+—

+ + + —

+ — N

A143
A144

+ + _ + —
+ + - -
+ _ _ — + N + + + + — +

A145
A146

N ++++
N
N
N
N
N
N
N
N

+ + — — +

+ _
+
N

+N
.N
_N
+N

+ — - +

+ + _ + +
N

+ _

+ + + — + + N + + - +
— — — + +
— — — + +

+ _

8171
*8172

+ — + +
+ + + +

+ -

— +

+ _ +

3173
*8181

—+

+ _ + —

N
N

- - N + — + + +
—+N

8182

+ +

- N +

+ + + +

+ + _ + +

+ + _ + _ + + +
_ _ _ _ .

+.+ _
+ + +

LeaLeb K k Pl FyaFyb Jka

— +
_ +

102
DuMNSs
+ + — +

- + — +

N
N

A 8 Al D C E c e
+ — + + - — +
+ — + + + — _ +

8201
*8202

++NN
.++_

+ +.+ +
NNNN

+.+.+ _

+ + — +
+
+
+

N
N
N
N

+"NHN
+ + _
_ . _
+.+.+
+.+ +

+ + - + +

— +
_ +
+ -
+ _
_ +
- +
— +
_ +
— +

+ — + +
+ + + +
+ _ + +
+ + + +
+ + — +
+ + + +
+ + + +
+ + + +
_ + _ +
+ +.+ +
+ + + +

N
N
N
N
N
N
N
N
N
N
N

+ + _ _ +
+ + _ + +
+ — + + -
+ — + + +
+ — + + +
+ _ + + +
+ + — — +
+ + - — +
+ + _ - +
+ + — _ +
+ + — — +

N
N
+
+

-+
+ _

B221
*8222
*8231

8232

B233
*8234
*8241
*8242

B243

8244

8245

8246

B247

B248

B249

+.+ + +

NNNNNNN

.+ +.+

. +.+ +

NNNNN+N

+ + +

_ _ _
+.+.+
+.+.+

—+

__+_

+ + _ +
_ _ + _

+ +.+ +
+ +.+ +
+.+.+ +
+.+_+.+

+.+.+ +
_ _ . _
_ _ _ _
+.+ + +
+ +.+.+

NNNNNNNNNNNN

B241P1 — -
B242-1 - -

_ +
— +

+ — + +
+ + — +
+ + — +

N
N
N

+ + — + +
+ + + + +
+ + - + N

N
N
N

+ -
— +
— +
+ _

B243—l — -
A263

A251
*A261
A262
A264
A265

*A252

_ _ _
+.+.+
+ +.+

+NN

-+

A266
A267

+

—+
—+
-+
—+
-+
-+
—+

+ + + +
— + _ +
+ + + +
— + _ +
+ + + +
— + _ +
- + — +

N
N
N
N
N
N

_ _ _ + +
+ + _ + +
+ + — + +
+ + — + +
+ + - + +
+ + - _ +
+ + + + +

+
+
+
+
+
N
N

+ -
+ _
+ _
— +
—+

B271
*8272
*B273
B274
B275
B281
*8282

Fyapyb Jka

Lea-Leb K k Pl

—+
—+
+ _
—+
_+

103
DuMNSs
++—+
++++
—+—+
—+—+
+++—
++_+

N
N
N
N
N

Al D C E c e
++—-+
++__+
++-++
+ + - + N
++—++

+
+
N
+
N

A 8
+ _
+ _
+

*8301
B302
B303

*8311

+++++ ._+_+ +++++ +_++++_ ++

+NNNN ++NNN +++NN _+++_N+ __
_+++. .___+ _+___ ++++++_ +++
+_+++ ++_.+ +++++ +++_+++ +++

+ N
+ N

+

N+NN+ NNNNN NNNNN NNNNNNN NNN

_+._+ _.___ ___._ ___..__ __
+_.++ .++++ ____+ __+___+ _+
__+__ +____ ___._ ++__+__ +_
+++++ ++__+ +++++ +++++++ ++
_._._ __++_ ___._ ___._._ _.
+++_+ ____+ +++++ ___+__+ _+
++_+_ +++++ _++._ +++++++ ++
NNNNN NNNNN NNNNN NNN__N_ NN
+N++N ++++N +++++ ++NN++N ++
+++++ ++++_ _+__+ ++_++_+ ++
+_++_ +.++_ ____+ __.____ ++
_+_++ +++_+ +++++ +++._+_ +_
+++++ +++++ +++++ +++__+. ++
+N+N. +++++ NNN_+ ++_NN_+ ++
__.__ ___._ _..__ __._.__ __
+_+_+ +++++ _._++ +++__++ ++
MOSH—.96 123u5 l23hfi5 123.4578 12
1111 22222 32333 uuuuuuu 55
33333 33333 33333 3333333 33
88888 @8888 @8888 MAAAAAA MA

++
- N

+ _
—+
—+
_+
+ _
+ _
+.—
_+

++—+
++_+
-+-+
++—+
_+-+
++—+
++—+

N
N
N
N
N
N

+—+++
+++++
++—++
+++++
++——+

+ + - - N

+
+

+ _
+ -

A353
B361
B362
*8363
*8364
B365
B366
B368
B369

Wagyb Jka

LeaLeb K k Pl

_ +
— +
_ +
_ +
— +

M.N S s
+ + + +
+ + _ +
— + _ +
+ + _ +
+ _ _ +

10M

Du
N
N
N

Al D C E c e
+ + — + N
— — — + N
+ + — + N
— — — + N
+ — + + +

N
N
N

A B
_ +
— +

B371
*B372
B373
B37“
B375

_ +

—+-+

N_NNN_N

+ +.+ +.+
+ +.+ +.+
. _ _ + _
_ +.+ _ _
_ +.+ + _

+ - + + +
+ + + + +

___+N_+

_ . _ _ . _
+.+ + + _ + +

1‘9L3Jhwugéuvl
oooooooooooooo
QJQJQJQJQJQJQJ
BBBBBWHB

+ _
_ +
_ +
_ +

_ + _ +
+ + + +
+ + + +
+ + + +
+ + + +

N

N +_++
N + + + —

+ + _ + N
+ + + + +

NN

+ +

BUOl
*BUOZ

+ _
+ -

+ +_+++ N -+-+
+ + + — + N N + — + +
+ + + + + + N + + + +

+ -
+ _
+ -

*BUll
Bul2
B413

—+

— + — +

+.Nu+
+ + +
+ . +
_ _ _
+ _ +

— +

N - + — +

+ — + + +

+ _
_ +

+ + + —
+ — + +
+ + + +

N
N
+ + - + N N

+ — + + —
+ — + + +

N
N
N

*Buul
BMH2
BUU3

+ N
N
N
N
N

+ N

+ N

+
+
+
+
+
+
+

+—
_ +

+
— +

_ + — +
_ + _ +
- + _ +
_++—
— + _ +
+ + + +
_ + _ +

___+_. .—
+ + — + - N
+ + — + N N
_ _ - + N _
+ + — + N N
++-+N N

+
+
+
N
+
+
N

____+._

1.952ancjauvt
:chchchchJ
“Huahqnqnqnqnq
umuBBBBBB

LeaLeb K k Pl FyaFyb Jka

105
DuMNSs

Al D C E c e

A B

unnumn
+ + +

+ + +
Human“

+ _
+ -

*Bu6l
BU62
8&63

+.+ + +

nwmunumn
+.+ + +

— +
- +
_ +
_ +

— + N + + — + + N + + _ +
N - _
N - + + — +

Au71
Au72
A473
*Au7u
*Au75

1T

+N

N + + — +

+ + _ + +

+ +

NNNN

+ +"NnN
. . + +
. _ _ .
+ +.+ +
+ + + +

*AM81
Au82
Au83
AMBH

+

+N
+N
+N

+
+
+

- N

- +
— +
_ +
+ _

+ + — —
+ + _ _
+ — — +
+ + — +
+ — + —
+ + + —
+ + + +
— + _ +

N
N
N
N
N
N

+ + - _ +
+ + — — +
+ + — + +
+ + _ + +
+ + _ - N
+ + - — N
— — — + N
+ + - + N

+
+
N
+
N

— +
—+
+ _
+ _
+ _
+ -

Au9l
*Au92
AU93
A501
A502
*A503
*C511
C512
0521
*0522

+.+
. +
+.+
+ +

_ N

mum"
+ +
_ .
_ +
_ +

_+_

NNN
__—

. + +
_ _ _

+ + +

C551
*0552

C553
*0561
0562

+ .
+ +
+ +
_ +

_ N

+ _

+ -
— +

+ + + _
+ — + +
+ + + +

N
N
N

+ + _ + N

+ + - + N
+ + — + N

N
N
N

— +
_ +

C581
C582
*0583

*0591
C592
0593
CS9“
C595

NOTE:

11>

+ + + I +

Zl+ak

03
3D
U

I
+ I +-2:I
+ +1+ +-+

indicates
indicates
indicates
indicates

106

CEce Du MNSS LeaLeka Pl FyaFbe'ka
+—+N N +—++ —+ —N+ +N +
_++— N ++—+ _+ -N+ +N +
+_+N N +—++ ..+ —N+ —N +
+-.—N N +-+.- —+ -N+ -N -
+—+N N +_++ -+ -N+ -N +

an individual with cystic fibrosis.

a.positive result, i.e. the antigen is present.

a negative result, i.e. the antigen is not present.
the sample was not tested for that particular antigen.

LIST OF REFERENCES

10.

ll.

13.

LIST OF REFERENCES

The Professional Education Commdttee of the Cystic Fibrosis
Foundation: ' 'GUide to 'Diagms‘is' 'and management 'Of‘ 'Cystic

‘ FibroSis. Cystic FibroSis‘Foundation, Atlanta, 197“

Thompson, J. S. and Thompson, M. W.: ‘Genetics in Medicine.
‘W. B. Saunders Company, Philadelphia, 1967.

 

Nelson ‘Iextbook‘of‘Pediatrics, edited by Vaughan, v. c., and
.McKay, R. J. 7W. B. Saunders Company, Philadelphia, 1975.

 

wright, S. W. and Merton, N. E.: Genetic Studies on Cystic
Fibrosis in Hawaii. Amt J. Hum. Genet., 20, 157, 1968.

Kulczycki, L. L. and Sohauf,'V.: Cystic Fibrosis in Blacks
in washington, D.C. Am. J. Dis. Child., 127, 64, 1974.

Wright, S. W. and Morton, N. E.: The Incidence of Cystic
Fibrosis in Hawaii. Hawaii Med. J., 27, 229, 1968.

Stern, C.:' Principles 'of HUman GenetiCs, W. H. Freeman and
Company, San Francisco, 1973.

 

Knudson, A. G., Wayne, L. and Hallett, W. Y.: On the Selective
Advantage of Cystic Fibrosis Heterozygotes. Am. J. Hum. Genet.,
19, 388, 1967.

Lieberman, J. and Rodbard, 8.: Low Blood Pressure in Young
Adults with Cystic Fibrosis. Ann. Int. Med., 82, 806, 1975.

Green, M. N., Clarke, J. T. and SchwaChman, H.: Studies in
Cystic Fibrosis of the Pancreas: Protein Pattern in Meconium
Ileus. Peds. 21, 635, 1958.

Stephan, U., BUSCh, E. W., Kollberg, H. and Hellsing, K.:
Cystic Fibrosis Detection by Means of a.Test-Strip. Peds.,
55, 35, 1975.

Antonowicz, 1., Ishida, S. and Schwachman, H.: Studies in
meconium: Disacdharidase Activities in Meconium from Cystic
Fibrosis Patients and Controls. Peds. 56, 782, 1975.

Perman, J., Breslow, L. and Ingall, D.: Nonoperative Treatment
of Meconium Ileus Equivalent. Am. J. Dis. Child., 129, 1210,1975.

107

108

LIST OF REFERENCES (Continued)

1’4. Allan, J. D. Mason, A., Moss, A. D.:' Nutritional Supplementation
in Treatment of Cystic Fibrosis of the Pancreas. Am. J. Dis.
Child., 126, 22, 1973.

15. Dodge, J. A., Salter, D. G. and Yassa, J. G.: Essential Fatty
Acid Deficiency Due to Artificial Diet in Cystic Fibrosis.
British J. Med., 1975.

16. Berry, H. K., Kellogg, F. W., Hunt, M. M., Ingberg, R. L.,
Richter, L. and Gutjahr, 0.: Dietary Supplement and Nutrition
in Children with Cystic Fibrosis. Am. J. Dis. Child., 129, 165,
1975.

17. Burnell, R. H. and Robertson, E. F.: Cystic Fibrosis in a
Patient with Kartagener Syndrome. Am. J. Dis. Child., 127,
746, 197a.

18. Taussig, L. M., Braunstein, G. D., White, B. J. and Christiansen,
R. L.: Silver-Russell Dwarfism and Cystic Fibrosis in a Twin.
Am. J. Dis. Child., 125, L195, 1973.

19. Pennington, J. E., Reynolds, H. Y., Wood, R. E., Robinson, R. A.
and Levine, A. 8.: Use of a Pseudomonas Aeruginosa Vaccine in
Patients with Acute Leukemia and Cystic Fibrosis. Am. J. Med. ,
58, 629, 1975.

20. Rachelefsky, G. S., Osher, A., Dooley, R. E., Ank, B. and Stiehm,
E. R.: CoeJC'Lstent Respiratory Allergy and Cystic Fibrosis. Am. J.
Dis. Child., 128, 355, 19711.

21. Smith, D. W., Docter, J. M., Ferrier, P. E., Frias, J. L. and
Spock, A.: Possible Localization of the Gene for Cystic Fibrosis
of the Pancreas to the Short Arm of Chromosome 5. The Lancet,
1968, ii, 309.

22. Gayton, W. F. and Friedman, S. B.: Psychosocial Aspects of Cystic
Fibrosis. Am. J. Dis. Child., 126, 856, 1973.

23. Meyers, A., Dolan, T. F. and Mueller, D.: Compliance and Self-
medication in Cystic Fibrosis. Am. J. Dis. Child., 129, 1011,

1975 .

211. Rosenlund, M. L. and Lustig, H. 3.: Young Adults with Cystic
Fibrosis, Ann. Int. Med., 78, 959, 1973.

25. Di Sant'Agnese, P. A. and Davis, P. B.: Research in Cystic
Fibrosis. N. Eng. J. Med., 295, 1181, 1976.

26. Textbook‘Of'NEdiCine, edited by Beeson, P. B. and McDermott, W.
W. B. Saunders Company, Philadelphia, 1975.

 

109

LIST OF REFERENCES (Continued)

27.

28.

29.

30.

31.

32.

33.

3“.

35.

36.

37.

38.

39.

140.

Morton, N. E. : '. Sequential-Tests for the Detection of Linkage.
Am. J. Hum. Genet., 7, 277, 1955.

Wald, A.: ' Sequential Analysis. Wiley, New York, 19147.

 

Steinberg, A. G., Shwachman, H., Allen, F. H. and Dooley, R. R.:
Linkage Studies with Cystic Fibrosis of the Pancreas. Am. J.
Hum. Genet., 8, 162, 1956.

Steinberg, A. G. and Morton, N. E.: Sequential Test for Linkage
Between Cystic Fibrosis of the Pancreas and the MNS Locus.
Am. J. Hum. Genet., 8, 177, 1956.

Cook, P. J. L.: The Lutheran-Secretor recombination fraction in
mar61: a possible sex difference. Ann. Hum. Genet., Lond., 28, 393,
19 5.

Cook, P. J. L., Noades, J., Hopkinson, D. A. and Robson, E. B.:
Demonstration of a sex difference in recombination fraction in the -
loose linkage, Rh and PGMl. Ann. Hum. Genet., Lond., 35, 239, 1972.

Conover, J. H., Conod, E. J. and Hirschhorn, K.: Ciliary-Dyskinesia
Factor in Immunological and Pulmonary Disease. The Lancet, 1973,
1, 119A.

McManus, S. P. and Masterson, J .: Cellular Metachromasia with
Toluidine Blue 0 in Cultured White Cells of Cystic Fibrosis
Heterozygotes. J. Med. Genet., 11, 216, 19714.

Danes, B. S., Scott, J. E. and Bearn, A. G.: Further Studies on
Metachromasia in Cultured Human Fibroblasts. J. Exp. Med. , 132,
765, 1970.

Conod, E. J., Conover, J. H. and Hirschhorn, K.: Demonstration of
Human Leukocyte Degranulation Induced by Sera from Homozygotes and
Heterozygotes for Cystic Fibrosis. Pediat. Res., 9, 7211, 1975.

Wilson, G. B. and Fudenberg, H. H.: Studies on Cystic Fibrosis
Using Isoelectric Focusing. II. Demonstration of Deficient
Proteolytic Cleavage of a1pha-2—macroglobuljn in Cystic Fibrosis.
Plasma. Pediat. Res. 10, 87, 1976.

Forstner, G., Koheil, A. and Forstner, J .: Isoelectric Focusing of
serum in Cystic Fibrosis: Failure to Distinguish Between Homozygote
and Heterozygote Sera. Clin. Chim. Act., 70, 1159, 1976.

Evans, B. W., Kelsch, R. C. and Howatt, W. F.: Beta-Glucuronidase
Activity in the Sweat of Children with Cystic Fibrosis. Am. J. Dis.
Child., 127, 660, 197A.

Lundgren, D. W., Farrell, P. M. and Di Sant'agnese, P. A.:
Polyamine Alterations in Blood of Male Homozygotes and Heterozygotes
for Cystic Fibrosis. Clin. Chim. Act., 62, 357, 1975.

llO

LIST OF REFERENCES (Continued)

“1.

142.

“3.

All.

45.

L16.

47.

I48.

“9.

50.

51.‘

52.

53.

5“.

Chandra, R. K.,‘Madhavankutty, K. and Way, R. 0.: Serum
alpha-FetOprotein Levels in Patients with Cystic Fibrosis and
their Parents and Siblings. Brit. Med. J ., '1, 7111, 1975.

Conover, J. H., Conod, E. J. and Hirschhorn, K.: Complement
Components in Cystic Fibrosis. The Lancet, 1973, ii, 1501.

Rao, G. J. S., Posner, L. A. and Nadler, H. L.: Deficiency of
Kallikrein Activity in Plasma of Patients with Cystic Fibrosis.
Science, 177, 610, 1972.

Wilson, G. B., Jahn, T. L. and Fonseca, J. R.: Demonstration of
serum protein differences in Cystic Fibrosis by Isoelectric
Focusing in Thin-Layer Polyacrylamride Gels. Clin. Chim. Act. , 149,
79, 1973.

Shapiro, B. L., Smith, Q. T. and Martinez, A.: White—Cell
Glutathione Reductase in Cystic Fibrosis. The Lancet, 1971!
ii, 1020.

Shapira, E., Ben-Yoseph, Y., Rao, G. J. S., Wessel, H. U. and
Nadler, H. L.: Decreased Trypsin Binding by Alphaa2—Macroglobu1in
from Cystic Fibrosis Patients and Obligate Heterozygotes. Int.
Cong. of Hum. Genet., Mex., 48, 1976.

Danes, B. S. and Bearn, A. G.: Localization of the Cystic-Fibrosis
Gene. The Lancet, ii, 1303, 1968.

Edwards, J. H.: Total Lods for Linkage Analyzed by the New York
Blood Center and Birmingham Computer Programs. ' Human Gene Mapping,
New Haven Conference, edited by Bergsma, D. , National Foundation,
March of Dimes, 10, 187, 1974.

Manual of Clinical Immunology, edited by Rose, N. R. and Friedman,
H., Am. Soc. for Microbiol., Wash., D.C., 1976.

 

McKusick, V. A. and Ruddle, F. H.: The Status of the Gene Map of
the Human Chromosomes. Science, 196, 390, 1977.

Human Gene Mapping” 6. Birth Defects: Original Article Series:
18, No. 2, 1982.

 

Klouda, et. al. J. Med. Genet., 19, 337, 1980.

Levine, L. S., Zachmann, M., New, M. I., Prader, A., Pollack, M. S.,
O'Neill, G. J., Yang, S. Y., Oberfield, S. E. and Dupont, B.:
Genetic Mapping of the 2l-Hydroxy1ase—Deficiency Gene within the
HLA Linkage Group. New Eng. J. Med., 299, 911, 1978.

Histocompatibility Testing 1980. Terasaki, P. 1., Editor. Report
of the Eighth International Histocompatibility WorkshOp, U.C.L.A.
Tissue Typing Laboratory, Los Angeles, California, 1980.

lll

LIST OF REFERENCES (Continued)

55.

56.

57.

58.

59.

60.

61.

62.

63.

614.

65.

66.

67.

68.

69.

Bodmer, W. F. and CavallisSforza, L. L.: ‘ Genetics, 'EVOlutiOn,

 

"and'Man. W. H. Freeman and Company, San Fransisco, 1976.

Kaiser, G. 1., Laszlo, A. and Gyurkovits, K.: HLA Antigens in
Cystic Fibrosis: An Association of B18 with the Disease. Inserm.
Abstracts, 58, 252, 1976.

Polymenidis, Z., Ludwig, H. and Gotz, M.: Cystic Fibrosis and
HLA Antigens. The Lancet, ii, 11452, 1973.

Gotz, M.,Ludwig, H. and Polymenidis, Z.: HLA Antigens in
Cystic Fibrosis. Z. Kinderheilk, 117, 183, 1971!.

‘An Introduction ‘tO'ImImInOhematology, Neville J. Bryant. W. B.

Saunders Company, Philadelphia, 1976.

McKusick, V. A., The Anatomy of the Human Genome. Hospital
Practice, 16, 82, 1981.

McKusick, V. A.: The Anatomy of the Human Genome. Am. J. of
Med., 69, 267, 1980.

Donahue, et. al. Proc. National Acad. Sci., USA, 61, 9H9, 1968.

Elston, R. C .: An Approximation for the Prior Probability of
Autoscmal Linkage. Human Gene Mapping-2, Rotterdam Conference,
edited by Bergsma, D. , National Foundation, March of Dimes, 11,
120 (290), 1975.

Laboratory Notebook, Histocompatibility Lab, B—228, Life Sciences
Building, Michigan State University, East Lansing, Michigan.

Calin, A.: HLA-B27: To Type or Not to Type? Ann. Int. Med.,
92, 208, 1980.

The Metabolic Basis of Inherited Disease, edited by Stanbury, J. B. ,
Wyngaarden, J. B. and Fredrickson, D. S. McGraw—Hill Book Company,
New York, 1972.

Olaisen, B. and Teisberg, P.: Structural polymorphism of human Cu,
and linkage of its locus to the HLA complex of chromosome 6.
International Cong. of Hum. Genet. , Mex. , 115, 1976.

Day, N. K., L'Esperance, B, Good, R. A., Michael, A. F., Hansen,
J. A., Dupont, B. and Jersild, C.: Hereditary C2 Deficiency:
Genetic Studies and Association with the HLA System. J. Exp.
Med., 141, 1164, 1975.

Cudworth, A. G. and Woodrow, J. C.:‘ Evidence for HLA-Linked
Genes in "Juvenile" Diabetes Mellitus. Brit. Med. J ., 3, 133,
1975.

112

LIST OF REFERENCES (Continued)

70. Rubinstein, P. Suciu-Foca, N. and Nicholson, J. F. :' Close
Genetic Linkage of HLA and Juvenile Diabetes Mellitus. New

Eng. J. Med., 297, 1036, 1977.

71. Simon, M. Bourel, M., Genetet, B. and Fauchet, R.: Hemochromatosis:
Recessive Inheritance and Early Detection by HLA Typing. New Eng.
J. Med., 297, 1017, 1977.

HICHI

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