$77.52: .0 ”On .A». 9 fix. I 9' 'l ‘ tux: ‘ i "21 T;K‘I'~ I _ ‘ . r8311 _ ".33 u... . . .. advuh "35% c ..—"; ‘ . . .L'Qflg ff:..‘; . L... - . ...-I. _.‘ . _. -.-. 'L‘n‘l’.’.‘.‘:.‘.’.’ S'..'...‘. 2 ' ‘ ‘I. .. ”4...... , . .v J .1. {A ‘. -,.-..—... ...-.... n. ...;..un,». .1. ‘ 40‘IOM o n ‘2-.- _ . flaw-I- J5" in}. - “Ari 4v - _. . .. «o Adun‘ - ‘ ¢uo7.8 milliMolar (mM)) and a random plasma glucose level which exceeds 200 mg/dl (>11.1 mM) (Acevedo). Normal control Puerto Ricans were selected at random and did not have an immediate family member with IDDM. Samples included random insulin dependent diabetic Puerto Rican patients, random normal Puerto Rican controls, and four complete Puerto Rican families from a panel collected and maintained by the Puerto Rican laboratory. Samples were collected to fit into one of seven categories listed in Figure 2. 32 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 33 Random Puerto Rican Diabetics with HLA DR81*03XX and DR81*04XX antigens (n-ll). Random Puerto Rican Diabetics with an HLA DR81*03XX or DR81*04XX antigen and any other HLA antigen including blank (except DR81*03XX and DR81*04XX together)(n-28). Random Puerto Rican Diabetics with no DR81*03XX or DR81*04XX antigens (n-4). Random normal Puerto Rican controls with HLA DRB1*03XX and DRB1*04XX antigens (n82). Random normal Puerto Rican controls with an HLA DR81*03XX or DR81*04XX and any other HLA antigen including blank (except DR81*03XX and DR81*04XX together) (n86) . Random normal Puerto Rican controls with no HLA DR81*03XX or DR81*04XX antigens (n-23). Four complete Puerto Rican families with one diabetic member to evaluate haplotype inheritance (families include both parents and 2-4 children). Figure 2. Population Characterization Random diabetic and control samples were characterized into one of six groups based on their disease state and HLA DR antigens. A set of four families were also included in this study. 34 DNA Isolation of Prosen Whole Blood Samples Frozen whole blood samples were isolated with the QIAampO Blood Kit (QIAGEN Incorporated, Chatsworth, California). A 200 uL aliquot of thawed, previously frozen, whole blood was placed into a 1.5 mL microfuge tube. 25 uL of QIAGEN protease (17.86 mg/mL stock concentration) and 200 uL of Buffer AL, a cell lysis buffer, were added. These samples were immediately vortexed and incubated at 70'C for ten minutes. Then 210 uL of isopropanol (J. T. Baker, Phillipsburg, New Jersey) was added to the samples and vortexed. The lysate was transferred to a QIAamp spin column and centrifuged at 9564 x gravity (9) for one minute. This was a higher g-force than the protocol suggested but was necessary to force all the cell debris through the column as the samples were somewhat old and lysed. The spin column was placed into a clean 2 mL microfuge tube, washed with 500 uL of Buffer AW, a wash buffer, and centrifuged at 9564 x g for one minute. Samples were washed again with 500 uL of Buffer AW and centrifuged at 9564 x g for three minutes. The spin column was placed into a clean UV-treated (90 seconds at 254 nanometers (nm)) (GS Gene Linkern‘tnl Chamber, Bio-Rad, Richmond, California) 1.5 mL microfuge tube and the DNA was incubated with 200 uL of sterile double 35 distilled water (ddHOH) at 70‘C and the DNA was collected by centrifuging at 6000 x g for one minute. The samples were vacuum dried (Savant Instruments Incorporated Speed Vac SC-100, Refrigerated Condensation Trap RT 100, Vacuum Gauge model VG-S, and High Vacuum Pump VP 100, Farmingdale, New York) and resuspended in 10 uL of sterile ddHOH or 10mM Tris (Tris[hydroxymethyl]-aminomethane hydrochloride) pH 9.0 (Sigma Chemical Company, St. Louis, Missouri). Using the GeneQuant RNA/DNA Calculator (Pharmacia LKB Biochrom Limited, Science Park, Cambridge, England), the optical density of a 1:100 dilution of the sample was spectrophotometrically measured using 260 nm, 280 nm, and 230 nm. Also measured and calculated was the protein contamination, purity, and 260/280 ratio. The formula for double stranded DNA, listed in Figure 3, was used to calculate DNA concentration. The concentration was adjusted to 10 ug of DNA per 100 uL of ddHOH. Samples isolated by the QIAGEN method were diluted 1:8 with ddHOH to give a final concentration of 0.0125 ug/uL. DNA was stored at -20°C prior to amplification. Primer Preparation Primers for the Polymerase Chain Reaction (PCR) were synthesized at the Macromolecular Facility at Michigan State 36 WWW (Optical Densityzso)(50ug/mL)(dilution factor)+1000uL-x ug/uL of DNA WWW (Optical Densityzso)(37ug/mL)(dilution factor)(mL resuspended)-Xug DNA WWW 21.122112212251225 [(#dATP)(312.2)]+[(#dCTP)(288.2)]+[(#dGTP)(328.2)]+[(IdTTP)(303.2)] -51 Figure 3. Formulas for the Calculation of DNA Concentration and Primer Preparation The formula for calculation of DNA concentration is based upon optical density. X is the calculated value of the concentration of DNA in ug/ul or ug . An optical densitygag of 1 corresponds to approximately 50 ug of DNA/mL for double stranded DNA (Maniatis, 1992). An optical densitygso of 1 corresponds to approximately 37 ug of DNA/mL for single stranded DNA (GeneQuant User Manual, 1993). In calculating the molecular weight of primers, 61 was subtracted for the absence of the terminal 5' phosphate and the addition of the terminal 3' hydrogen atom. The numbers in parenthesis are the molecular weights of the nucleotides. 37 University. Primers sequences for TAP1 and TAP2 were published sequences (Powis 1993). The 5' flanking and 3' flanking primer sequences for LMP2 were also published (Deng) and the two allele specific primers for LMP2 were developed specifically for this research project. Sequences for these primers are listed in Figure 4. Dried primers were reconstituted in ddHOH. The optical density at 260 nm was measured with a dilution of the primer using the GeneQuant RNA/DNA Calculator (Pharmacia LKB Biochrom Limited, Science Park, Cambridge, England). The primers were vacuum dried (Savant Instruments Incorporated Speed Vac SC- 100, Refrigerated Condensation Trap RT 100, Vacuum Gauge model VG-5, and High Vacuum Pump VP 100 Farmingdale, New York). Using the calculated molecular weight, see Figure 3, and measured amount of recovered DNA, the amount of ddHOH was determined to resuspend the DNA at a concentration of 20 mM. A working solution of 20 microMolar (pH) was prepared from the stock diluting with ddHOH. Typing TAP1, TAP2, and LMP2 Alleles TAP1, TAP2, and LMP2 alleles were typed for using an Amplification Refractory Mutation System (ARMS) method. Typing included the following loci; TAP1 amino acid position 333 (ile/val), TAP1 amino acid position 637 (asp/gly), TAP2 amino acid position 379 (val/ile), TAP2 amino acid position 565 (ala/thr), TAP2 amino acid position 665 (ala/thr), and 38 5 ’CCCTGCACI‘GAGA’I'ITGCAGACCI‘CTGGAGJ ’ 5 ’-GATCAGT GT CCCI‘ CACCATGGT CACCCQGAJ ’ 5 ’GGGCAGMGGAAAAGCAGAGGCAGGGTQAQJ ’ 5 ’-ACCTGGGAACATGGACCACAGGGACAGGGT-3 ’ 5 ’-CATC1TCCCAGAATCTCCCCTATCCAGCTA—3 ’ 5 ’CATCIT GGCCC'I'IT GCI' CT GCAGAGGT AQ_A_—3 ’ 5 '-ACCCCCI‘GACAGCI‘GGCTCCCAGCCI'CQ_Q-3 ’ 5 ’-TGGGGAGGCATCCAATGGAACIGGATITGG-3 ’ 5 ’-TTGGAGGGCTGCAGACCG’ITCGCAGTI'ITG-3 ’ 5 ’-GAGACCI‘ GGAACGCGCC'IT GT ACCT GCQCQ-B ’ 5 ’-ACAACCACTCI'GGTATC'ITACCCTCCI‘§AI—3 ’ 5 ’-ACATAGCI‘CCCCACGC1‘CTCCTGGTAGATC-3 ’ 5 ’CTCACAGTATGAACACI‘GCI‘ACCI‘GCACAG-3 ’ 5’-TG'IT CT CCGG'I'I' Cf GT GAGGAACAACAQT 5:3 ’ 5 ’-ATCATCITCGCAGC1'CTGCAGCCCATAAAQ ’ 5 ’GGAGCMGCITACAATITGTAGAAGATACC-Ii ’ 5 ’-TTGGGGAATGGAATCCGGT 661' GT GAGGGC-3 ’ TAP2/ARMSIO S’CAGTGCI‘GGTGATI‘GCI‘CACAGGCI‘GCAAA- ’ TAPZ/ARMSI l S ’-CACCAGGATCI‘GGTGGGCGCGCTGAACI‘AQ-3 ’ TAP2/ARMS 12 5 ’-TCAGCCGCTGCTGCACCAGGCGGGAATAGA—3 ’ TAP1 TAPl/ARMSI Position 333 TAP1/ARMS2 TAP1/ARMS! TAPl/ARMS4 TAPl TAP1/ARMSS Position 637 TAP1/ARMS6 TAPl/ARMS7 TAPl/ARMS8 TAP2 TAP2/ARMSI Position 379 TAP2/ARMS2 TAP2/ARMS3 TAP2/ARMS4 TAPZ TAP2/ARMSS Position 565 TAP2/ARMS6 TAP2/ARMS7 TAP2/ARM58 TAP2 TAP2/ARMS9 Position 665 LMPZ LMPz-Z Position 60 LMPZ/ARG LMP2/HIS LMPZ-l Figure 4. 5 ’-GTGAACCGAGTG'ITI‘GACAAGC-3 ’ 5 ’-CT GT CCCCGCT GCACGAICQJ ’ 5 ’-AGAGAGTGCACAGTAGAAGT_-3 ’ 5 ’-GCCAGCAAGAGCCGAAACAAG-3 ’ PCR Primers S’flanking 118-333 Val-333 3’flanking 5’ flanking ASHB7 sly-637 3’flanking S’flanking Val-379 Ila-379 3’flanking S’flanking Tin-565 Ala-565 3’flanking S’flanking Thr-665 Ala-665 3’flanking S’flanking Arg-60 His-60 3’flanking PCR primer sequences for TAP1 and TAP2 were obtained from Powis (1993). sequences were obtained from Deng. The LMP2 5' flanking and 3' flanking primer Underlined nucleotides are mismatched or possibly mismatched bases when compared to a consensus sequence. 39 LMP2 amino acid position 60 (arg/his). TAP1 and TAP2 ARMS Polymerase Chain Reaction The volume of the Polymerase Chain Reaction was 10 uL. The volume was doubled to 20 uL if AmpliWaxm PCR Gem 50 (Perkin Elmer, Foster City, California) was used to minimize nonspecific amplification or primer-dimer formation. A master mix containing all the PCR reagents except for the template was prepared for each typing system. These mixes included 0.01875 to 0.025 ug each of four primers (5' flanking, 3' flanking, and two internal allele specific primers listed in Figure 4 needed for a specific loci), 20 uM each of deoxyadenosine triphosphate (dATP), deoxycytosine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and deoxythymine triphosphate (dTTP) (Perkin Elmer, Foster City, California), 1x GeneAmp PCR Buffer (10mM Tris-HCl pH 8.3 [at 25°C ]; 50 mM KCl; 1.5 mM MgC12; 0.001% weight/volume (w/v) gelatin; autoclaved) (Perkin Elmer, Foster City, California), and 0.2 Units of Thermus aquaticus (Tag) DNA polymerase (Perkin Elmer, Foster City, California) (modification of Powis, 1993). DNA template at a concentration of 0.0125 to 0.1 ug was added to the master mix. PCR Reactions were carried out in a 9600 Thermo Cycler (Perkin Elmer, Foster City, California) with an initial 4o denaturation time of 5 minutes at 95I°C. Cycling parameters for TAP1 and TAP2 primers include 11 seconds of denaturation at 94 °C, anneal at 58 °C for 30 seconds, and extension at 72 °C for 30 seconds for 30-35 cycles. A final 72 °C extension for 10 minutes occurs at the end of cycling (modification of Powis, 1993). LMP2 ARMS Polymerase Chain Reaction The PCR conditions were optimized using the 9600 Thermo Cycler (Perkin Elmer, Foster City, California). A master mix containing all the PCR reagents except for the template was prepared for the LMP2 typing system. These mixes included 20 uM each of dATP, dCTP, dGTP, and dTTP (Perkin Elmer, Foster City, California), 1x GeneAmp PCR Buffer (10mM Tris-HCl pH 8.3 [at 25°C]; 50 ml! KCl, 1.5mM ngc12, 0.001% (w/v) gelatin; autoclaved (Perkin Elmer, Foster City, California), 0.4 Units of Thermus equations (Taq) DNA polymerase (Perkin Elmer, Foster City, California), 1uM each of LMP2-1, LMP2-2, LMP2-Arg, and 2uM of LMP2-His. DNA template at a concentration of 0.25 to 0.2 ug was added to this 20 uL master mix. Primer sequences are listed in Figure 4. Initial denaturation was at 95C° for 5 minutes followed by 35 cycles of 94C° for 30 seconds, 56C° for 30 seconds, 720? for 60 seconds, and then a final extension at 72C° for 10 minutes. 41 PCR Controls A negative control has been run with every TAP and LMP PCR reaction. As a negative control, one sample included ddHOH instead of DNA. Samples were typed in duplicate. Controls for TAP1 and TAP2 were obtained from well characterized homozygous typing cell lines (HTCL) of the 10th and 11th International Histocompatibility Workshop purchased from the American Society of Histocompatibility and Immunogenetics cell repository (Powis, 1993 and Carrington). Cell lines are Epstein-Barr virus transformed B-cells for which DNA had been previously harvested by a salt extraction method at the Immunohematology and Serology Laboratory at Michigan State University. One positive homozygous cell was run for each allele with every reaction. A heterozygous sample was also run with each reaction. Because the HTCL are almost always homozygous for every loci in the HLA region, heterozygous control samples were made by making a 1:1 mixture of the appropriate homozygous cell types. Table 4 lists the controls used. As no known types were available at the LMP2 loci, HTCLs were screened with the preliminary ARMS PCR typing method and then sequenced to determine their type. They were then used as controls in the ARMS PCR reaction and are listed in Table 4. Table 4. 42 Control Cell Lines Listed are the control cell lines used for TAP1, TAP2, and LMP2 typing. site. Pos Ce 9080 SPOOlO RAJI B Included are their types at each poymorphic 2821 Pos on 333 Pos on 637 on 379 Pos on 565 Pos a a a a on 665 a a a a a a a B 8 P08 43 Sequencing the LMP2 Controls DNA previously amplified with the 5' flanking and the 3' flanking primers (LMP2-1 and LMP2-2) was diluted 1:10 and 6uL were used in the 20uL fluorescent dye terminator di- deoxy sequencing PCR reaction. The template was sequenced in both directions using 3.2 pmole of the primers individually with 8.0 uL of the ABI PRISM” DNA Ready Reaction Sequencing Kit (ABI/Perkin Elmer, Foster City, California) for 25 cycles of 96C° for 10 seconds, 50C° for 5 seconds, and 60C° for 4 minutes. The samples were then ramped to 4C°. The products were ethanol washed to remove unincorporated labeled nucleotides. The samples were added to 35 uL of 95% alcohol, vortexed, and incubated on ice for 10 minutes. At 4C°, the samples were centrifuged at maximum speed for 15 minutes. After the supernatant was removed, the pellet was vacuum dried and then resuspended in 25 uL of Template Suppression Reagent (ABI/Perkin Elmer, Foster City, California). These samples were vortexed, spun, heated to 95CP, vortexed and spun again before they were sequenced using the ABI PRISMW'310 Genetic Analyzer (ABI/Perkin Elmer, Foster City, California). 44 Electrophoresis of TAP1 and TAP2 Products were electrophoresed using a Mini-PROTEAN® II Cell electrophoresis chamber (Bio-Rad, Richmond, California) and a Model ZOO/2.0 Power Supply (Bio-Rad, Richmond, California). A 6% polyacrylamide gel with the dimensions 80 mm x 73 mm x 0.75 mm was used [1.74% (w/v) acrylamide (Bio- Rad, Richmond, California), 0.06% N,N'-methylene bisacrylamide (Bio-Rad, Richmond, California)], TAE [40 mM Tris(hydroxymethyl)aminomethane (Sigma Chemical Company, St. Louis, Missouri), 0.114% glacial acetic acid (EM Industries Incorporated, Gibbstown, New Jersey), 1 mM EDTA (Sigma Chemical Company, St. Louis, Missouri) pH 8.0], 0.001% N,N,N',N'-Tetramethylethylenediamine (TEMED) (Boehringer Mannheim Corporation, Indianapolis, Indiana), and 0.08% ammonium persulfate (J. T. Baker Incorporated, Phillipsburg, New Jersey). The 10 uL PCR sample was mixed with luL of 6X gel- loading buffer [0.25% bromophenol blue (Sigma Chemical Company, St. Louis, Missouri), 0.25% xylene cyanol (Sigma Chemical Company, St. Louis, Missouri), and 40% (w/v) sucrose (Mallinckrodt Chemical Incorporated, Paris, Kentucky) in ddHOH]. On the gel 5 uL of this mixture was loaded along with one lane of a molecular weight standard, which includes DNA fragments of 1000, 700, 500, 400, 300, 200, 100, and 50 base pairs (Boehringer Mannheim Corporation, Indianapolis, Indiana). A current of 200 volts 45 was applied until the bromophenol blue dye front migrated approximately 2/3 the length of the gel (15-20 minutes). Electrophoresis of LMP2 The 20 uL PCR sample was mixed with 5uL of 6X gel- loading buffer [0.25% bromophenol blue (Sigma Chemical Company, St. Louis, Missouri), 0.25% xylene cyanol (Sigma Chemical Company, St. Louis, Missouri), and 40% (w/v) sucrose (Mallinckrodt Chemical Incorporated, Paris, Kentucky) in ddHOH]. The 6% acrylamide gel with dimensions of 0.5mm x 16cm x 20cm was loaded with 10 uL of sample [1.74% (w/v) acrylamide (Bio-Rad, Richmond, California), 0.06% N,N'-methylene bisacrylamide (Bio-Rad, Richmond, California)], T88 [89 mM Tris(hydroxymethyl)aminomethane (Sigma Chemical Company, St. Louis, Missouri), 89 mM boric acid (J.T. Baker Incorporated, Phillipsburg, New Jersey), 2 mM EDTA (Sigma Chemical Company, St. Louis, Missouri) pH 8.0], 0.001% N,N,N',N'-Tetramethylethylenediamine (TEMED) (Boehringer Mannheim Corporation, Indianapolis, Indiana), and 0.0008% ammonium persulfate (J. T. Baker Incorporated, Phillipsburg, New Jersey)). One lane of a molecular weight standard, which includes DNA fragments of 1000, 700, 500, 400, 300, 200, 100, and 50 base pairs (Boehringer Mannheim Corporation, Indianapolis, Indiana) was also loaded onto the gel. The samples were electrophoresed using a PROTEANG) IIxi Cell electrophoresis chamber (Bio-Rad, Richmond, 46 California) and a Model 1000/500 Power Supply (Bio-Rad, Richmond, California). The gel was cooled with water and electrophoresed at 100-125 volts until the bromophenol blue and xylene cyanol dye fronts separated the gel into thirds (approximately 4 hours). Detection and Analysis The gels were placed into approximately 100-500 mL of ddHOH with one drop of ethidium bromide (10 mg/mL) (Calbiochem-Behring Corporation, La Jolla, California) and incubated with gentle rotation at room temperature for 10 minutes. DNA bands were visualized using the Chromato-Vue transilluminator model 75-36 (UVP Incorporated, San Gabriel, California) at the wavelength of 254 nm. The gels were photographed with type 667 black and white Polaroid® film (Polaroid Corporation, Cambridge, Massachusetts) using a Fotodyne FCR-lo camera (Fotodyne Incorporated, Hartland, Wisconsin). Film was exposed for one second at i=8 and developed for one minute. Statistical Analysis Statistical evaluations used the chi-square method or Fisher's Exact test where appropriate using a 95% confidence interval (p-0.05). Graph Pad Prism (Graph Pad Software Incorporated) was the computer program used to do the chi- square analysis. Comparisons were made between group 1 (di (di DR3 6 ( pol con rec of eva 47 (diabetic DR3, DR4) and group 4 (normal DR3, DR4); group 2 (diabetic with one DR3 or DR4) and group 5 (normal with one DR3 or DR4); and group 3 (diabetic no DR3 or DR4) and group 6 (normal no DR3 or DR4) (refer to Figure 1) for each polymorphic site. A dominant mode of inheritance that considers the frequency of each allele along with a recessive mode of inheritance which considers the frequency of a homozygous type compared to the other outcomes was evaluated. RESULTS Samples for this study included 43 random Puerto Rican IDDM samples and 31 random Puerto Rican normal controls. Also included were 21 samples from four complete first generation Puerto Rican families. Each family included one member with IDDM. Sample populations were divided into three subgroups for a total of six groups. The subgroups were based on the DR allele and include DR3/DR4, DR3 or 4/DRX, and DRX/DRx. The x denotes any allele but DR3 or DR4, however some samples could be homozygous DR3 or homozygous DR4. The subgrouping was done to separate the traditional DR3/DR4 IDDM risk haplotype and to further separate samples with a risk allele (DR3 or DR4). In typing for the TAP and LMP polymorphisms within this population, positive controls were run with every ARMS PCR reaction to verify reaction conditions were sufficient for amplification. As controls were unknown for the LMP2 loci, HTCL were sequenced to obtain appropriate homozygous and heterozygous controls used in further typing analysis. Sequence analysis results of the three HTCL used as controls for LMP2 are shown in Figure 5. Polymorphisms for the TAP and LMP genes at six sites were determined by an ARMS PCR typing method. After amplification with allele specific primers, the PCR products were electrophoresed on acrylamide gels and the DNA was stained with ethidium bromide. The banding patterns 48 49 Figure 5. Electorpherogram Sequence data for the cell lines used as controls in the LMP2 ARMS typing. Cell line 9080 is homozygous for guanosine at codon 60 (AG CGC AT). SPOOlO is homozygous for adenosine at codon 60 (AG CAC AT). RAJI is heterozygous at codon 60 (AG CBC AT). R= adenosine or guanosine (purine). 50 observed were compared to a molecular weight sizing standard and assigned a genotype. See Figure 6-11 for a representation of each of the TAP and LMP polymorphic banding patterns observed on an ethidium bromide stained gel. For TAP1 Amino Acid Position 333, Figure 6, the control band appears at 533bp, the val band appears at 351bp and the ile band appears at 241bp. The blank was negative. For TAP1 Amino Acid Position 637, Figure 7, the control band appears at 429bp, the asp band appears at 307bp and the gly band appears at 180bp. The blank was negative. For TAP2 Amino Acid Position 379, Figure 8, the control band appears at 427bp, the val band appears at 328bp and the ile band appears at 158bp. The blank was negative. For TAP2 Amino Acid Position 565, Figure 9, the control band appears at 400bp, the ala band appears at 298bp and the thr band appears at 161bp. The blank was negative. For TAP2 Amino Acid Position 665, Figure 10, the control band appears at 408bp, the ala band appears at 326bp and the thr band appears at 141bp. The blank was negative. For LMP2 Amino Acid Position 60, Figure 11, the control band appears at 252bp, the Arg band appears at 231bp and the His band appears at 60bp. The blank was negative. Samples were assigned a type based on the size of the PCR fragment. They were assigned either one of two possible homozygous types or a heterozygous type. Samples with only 51 TAP1 Amino Acid 333 val (Are) or ile (are) C4- ‘“ madur 0 co 0 O) 1 blank .5 E control 533bp va|351bp ile 241b Figure 6. TAP1 Amino Acid 333 ARMS Diagram A representation of the ARMS PCR for the TAP1/333 system. Control band appears at 533bp. Valine band is 351bp and the isoleucine band is 241bp. 52 TAP1 Amino Acid 637 asp (GAC) or gly (GQC) ‘l A4- 2 g E Illllllllllllllllllllll ; ,_< E 3 if: " T 3 E E 8 3 2 15 A C 200 corflrol429ln) 100 asp307bp 50 gly 180ln) Figure 7. TAP1 Amino Acid 637 ARMS Diagram A representation of the ARMS PCR for the TAP1/637 system. Control band appears at 429bp. Aspartic acid band is 307bp and the glycine band is 180bp. 53 TAP2 Amino Acid 379 val (QTA) or ile (ATA) (3<¢- r': c t o x .7 ITTIlllllllllllllllllllll g 5 “g g; 'E "’ C 3 a E 8 3 2 1 E G ‘- 500 lllllllllllllllllllllllli, 400 300 "T 200 contn3|429bp 100 va|328bp 50 H0158bp Figure 8. TAP2 Amino Acid 379 ARMS Diagram A representation of the ARMS PCR for the TAP2/379 system. Control band appears at 427bp. Valine band is 328bp and the isoleucine band is 158bp. 54 TAP2 Amino Acid 565 ala (QCT) or thr (ACT) C“- t f llllllllllllllllllllllTT“ '§ ‘5 C’ i -> G 2:28 ~'= £2 : : a) 3 2 1 E C .¢. _llllllllllllllllllllllll 400 300 .‘bA 200 contnfl 400bp 100 ak3298bp 50 thriBlbI Figure 9. TAP2 Amino Acid 565 ARMS Diagram A representation of the ARMS PCR for the TAP2/565 system. Control band appears at 400bp. Alanine band is 298bp and the threonine band is 161bp. 55 TAP2 Amino Acid 665 ala (QCA) or thr (ACA) A‘G- _TTllllllllllllllllllllll -’> T Inadura nIarkcr x r- C E on 500 400 300 200 A ‘0- lllllllllllllllllllllLLL_ -‘>C control 408bp 100 ala 326bp thr141b 50 Figure 10. TAP2 Amino Acid 665 ARMS Diagram A representation of the ARMS PCR for the TAP2/665 system. Control band appears at 408bp. Alanine band is 326bp and the threonine band is 141bp. 56 LMP2 Amino Acid 60 arg (CQC) or his (CAC) (3<¢- Illlllllllllllllllllllll -‘> C SPOO1O Inarkor RAJI 9080 .x P c .E G “- lllllllllllllllllllllllLi I‘VT conno|252bp arg 231bp IflsGObp Figure 11. LMP2 Amino Acid 60 ARMS Diagram A representation of the ARMS PCR for the LMP2/60 system. Control band appears at 252bp. Arginine band is 231bp and the histidine band is 60bp. 57 Table 5. Typing Results Listed are the combined typing results for TAP1 position 333 and 637, TAP2 position 379, 565, and 665, and LMP2 position 60 according to their specific catagories. 12.! . 12.1. 12.1. I! l I! l I! l IHEI DR30r4 [HUK DR3 'DR30r4 IDRXI DR4 .DRX [HU( DR4 IDRX IDRX (Ir-'19 (F28) (F4) (F2) W) (II-=23) TAP1 val,x =2 val,x=3 val,x=0 val,x=0 val,x=l vaLx=l POS 333 val,ilc=5 vaLile=7 vaLile=l vaLile=l val,ilc=l vaLile=6 ilc,x =4 ile,x=18 ile,x=3 ile,x=l ile,x=4 ile,x=16 TAP1 asp,x=4 asp,x=l8 asp,x=3 asp,x=1 asp,x=4 asp,x=l6 P08 637 aspsly=5 aspsly=7 aspsly=l BPS‘Y‘I 881331332 ”PET-'6 x=2 x=3 Bil/1M gyzx=0 fl x=l TAP2 wflx=tl w¢x=Z3 \mbefi VflJFZ uflm=6 wu3=r7 POS 379 val,ile=0 val,ile=5 val,ile=l val,ile=0 val,ile=0_ val,il8=5 ile,x=0 ilc,x=0 ile,x=0 ilc,x=0 ilc,x=0 ilc,x=l TAP2 ahm=ll ShJFQS daa=3 flax=2 ahpfifi» athQZ POS 565 ala,thr=0 ala,thr=3 ala,thr=l ala,tlu=0 ala,thr=0 ala,tht=l flug=0 flua=0 flugFO flu¢=0 flug=0 flmg=0 TAP2 ShJFO AhJFO ahJFO ahJFO ahpfifl ahusfl P08665 daflm=3 danm=7 daflmél Imumfio ahmhfia ahfim=ll thr,x=8 thr,x=21 ther=l ther=2 thr,x=4 thr,x=10 LMP2 arg,x=8 arg,x=l4 arg,x=2 arg,x=l arg,x=5 arg,x=l3 P08 60 arg,his= arg,his=12 arg,his= atg,his=l ' =1 ughis=8 hkm=0 hk¢=2 hkm=0 hmm=0 hkm=0 hkm=2 58 an allele specific band are assumed to be homozygous at that loci. Refer to Table 5 for data collected from these typings. Figure 12 illustrates the family haplotype analysis. Two samples out of 1140 PCR reactions showed aberrant results. Additional typing of these samples was performed and they were assigned a type based on the most prevalent type of that sample. Statistical analysis of the data was done using the Graph Pad Prism statistical program. Results of the chi- square analysis for a dominant affect is shown in Table 6 while analysis for a recessive affect is shown in Table 7. Results for dominant affect compared the frequencies of each amino acid in the population. Recessive affects were tested by comparing the frequency of one homozygous type to other possible outcomes (including homozygosity for the other allele). Then the frequency of the other homozygous allele was compared to the other possible outcomes. 59 Table 6. Dominant Inheritance Results Chi-square analysis for a dominant mode of inheritance (typically found in IDDM protective alleles). Chi-square as well as Fisher's Exact test was performed with a 95% confidence interval (p-0.05). There were no significant outcomes. Chi-Square analysis for Dominant Testinl(p=0.05) Group chi-equatedf p-valuc Outcome Fisher's Exact Outcome p-value TAPl/333 0.36, l 0.55 Not Significant 1.00 Not Significant DR3/4 TAP1/333 0.01751, 1 0.8947 Not Significant 1.00 Not Significant DR3 or 41X TAP1/333 0.1174, 1 0.7319 Not Significant 0.8640 Not Significant DRX/X TAM/637 0.3619, 1 0.5474 Not Significant 1.00 Not Significant DR3/4 TAP1/637 0.2464, 1 0.6196 Not Significant 1.00 Not Significant DR3 or 41X TAP1/637 0.1174, 1 0.7319 Not Significant 1.00 Not Significant DRX/X TAP2/379 NIA N/A Not Significant N/A Not Significant DR3/4 TAM/379 1.156, 1 0.2822 Not Significant 0.5772 Not Significant DR3 or 4/X TAP2/379 0.03987, 1 0.8417 Not Significant 1.00 Not Significant DRX/X TAP2/565 NIA N/A Not Significant N/A Not Significant DPS/4 TAP2/565 0.6725, 1 0.4122 Not Significant 1.00 Not Significant DR3 or 4/X TAP2/565 2.037, 1 0.1535 Not Significant 0.2767 Not Significant DRX/X TAP2/665 0.6166, 1 0.4323 Not Significant 1.00 Not Significant DEM TAP2/665 0.1494, 1 0.6991 Not Significant 0.6541 Not Significant DR3 or 4/X TAP2/665 0.07337, 1 0.7865 Not Significant 1.00 Not Significant DRX/X LMPZJ60 0.3357, 1 0.5623 Not Significant 0.5107 Not Significant DR3/4 LMP2/60 2.159, 1 0.1418 Not Significant 0.2692 Not Significant DR3 or 4/X LMP2/60 0.004193, 1 0.9484 Not Significant 1.00 Not Significant DRX/X Table 7. 60 Recessive Inheritance Results Chi-square analysis for a recessive mode of inheritance (typically found in IDDM susceptibility alleles). Chi- square as well as Fisher's Exact test was performed with a 95% confidence interval (p=0.05). There were no significant outcomes. Chi-Square analysis for Recessive Testin (p=0.05) Group chi-square, df p-value Outcome Fisher's Exact Outcome j-VRIDC TAP1/333-val 0.4298, 1 0.5121 Not Significant 1.00 Not Significant DRy4 TAPl/333-val 0.1687, 1 0.6813 Not Significant 0.5585 Not Significant DR3 or 4/X TAP1/333w 0.1806, 1 0.6709 Not Significant 1.00 Not Significant IMUUX TAP1/333-ile 0.1330, 1 0.7154 Not Significant 1.00 Not Significant DRN4 TAM/333418 0.01227, 1 0.9118 Not Significant 1.00 Not Significant DR3 or 4/X TAP1/333419. 0.04827, 1 0.08261 Not Significant 1.00 Not Significant DRX/X . TAPl/637-asp 0.6300, 1 0.4274 Not Significant 1.00 Not Significant DRN4 TAP1/637418;) 0.01227, 1 0.9118 Not Significant 1.00 Not Significant DR3 or 4/X TAPl/637-asp 0.04827, 1 0.8261 Not Significant 1.00 Not Significant IMUUX TAP1/637-gly 0.2182, 1 0.6404 Not Significant 1.00 Not Significant DRN4 TAPl/637-gly 0.7051, 1 0.4011 Not Significant 1.00 Not Significant DR3 or «UK TAP1/637-gly 0.1806, 1 0.6709 Not Significant 1.00 Not Significant IMUUX TAP2/379m N/A N/A Not Significant N/A Not Significant DRy4 TAPZ/379-ala 1.256, 1 0.2624 Not Significant 0.5585 Not Significant DR3 or 41X TAP2/379m 0.002096, 1 0.9635 Not Significant 1.00 Not Significant IHUUX TAP2/379411: N/A N/A Not Significant N/A Not Significant DRy4 TAP2/3794M N/A N/A Not Significant N/A Not Significant DR3 or 4/X “ran/379411: 0.1728, 1 0.6776 Not Significant 1.00 Not Significant DRX/X Table 7 cont. 61 Group chi-squaredf p-value Outcome Fisher's Exact Outcome 19mm» TARMfiidb IVA NW1 Nu NW1 :Mmsmmmam DR3/4 Significant TAP2/5654111 0.7051, 1 0.4011 Not 1.00 Not Significant DR3 or 4/X Significant TAP2/565w 2.119, 1 0.1455 Not 0.2792 Not Significant DRX/X Significant TARUflfi4hr EMA IVA Na: Nfli Ndfiflmflkmn DR3/4 Significant TARNflfiAhr IMA IVA IV» NW1 Ndfifimflhmn DR3 or 4/X Significant TARNflfi4Mr IMA IVA 1V1 IVA nausmmmcmt DRX/X Significant TARNafiqmn IVA IVA Nu: NHL Ndfihmmkmu DR3/4 Significant Thunkfiiah NW1 IVA Na. IVA IVnflmmfiamt DR3 or 4/X fignificant TAEMafiqh QMDQI «MM91 Na LN) Nmsunmamt DRX/X Significant TAEM%50u QflDLI 03%” Nm LN) imuammmnm: DR3/4 Significant TAP2/6654M 0.1763, 1 0.6746 Not 0.6445 Not Significant DR3 or UK Significant TAP2/6654M 0.4819, 1 0.4876 Not 0.6239 Not Significant DRX/X Significant LMPZ/60-arg 0.4104, 1 0.5218 Not 1.00 Not Significant DR3/4 Significant LMP2/60mg 2.227, 1 0.1356 Not 0.1960 Not Significant DR3 or 4/X Significant LMPZ/60-arg 0.05870, 1 0.8086 Not 1.00 Not Significant DRX/X Significant Inflamonn IVA IVA Nm. Nfll Ndfifimfihuu DR3/4 Significant LMP2/60-his 0.4554, 1 0.4998 Not 1.00 Not Significant DR3 or 4/X Significant LMP2/60-his 0.3757, 1 0.5399 Not 1.00 Not Significant DEX/X Significant Family 1 Father Mother 11 13 (3 I) HLA-A 194 -19 x4 -29 flLA-B 534 -53 X- -44 HLA-DR 3~ -8 4- -7 HLA-DO 2- -3 3- -2 TAP2/665 thr- -thr thrq -thr TAP2/565 alaj -ala thr- -ala TAP2/379 vald -ile val- -val TAP1/637 asp- -asp gly- -asp TAP1/333 ile- -ile val- -ile LMP2/60 his- -his arg~ -arg Diabetic child child child child 13 I) 11 I) 11 (3 l1 (3 194 -29 194 -29 19+ -x 19- -x 53. -44 53d -44 53- -X 53- -X 8- -7 3- -7 3- -4 3d -4 3- -2 2- -2 2q -3 2- -3 thr- -thr thr- -thr thr- -thr thr- -thr ala- -ala ala- -ala ala- -thr ala- -thr ile- -val val- -val val- -val val- -val asp- -asp asp- -asp asp- -gly asp- -gly ile- -ile ile- -i1e ile- -val ile- -val his- -arg his+ -arg his- -arg his~ -arg Figure 12. Extended HLA Haplotypes from Family Studies HLA haplotypes derived from family studies include the class I antigens (HLA-A and HLA-B), class II antigens (HLA- DR and HLA-Do), and TAP1, TAP2, and LMP2 polymorphisms. The large A and B designates paternal haplotypes, while the large C and C designates maternal haplotypes. An undetermined allele is denoted by x. HLA-A HLA-B HLA-DR HLA-D0 TAP2/665 TAP2/565 TAP2/379 TAP1/637 TAP1/333 LMP2/60 child B C X1 -66 X4 -58 11- -15 7‘ ..5 thaw -thr alas -thr val- -val gly- -asp val-n -ile argd -arg Figure 12 cont. 63 Family 2 Father [1 13 -x -18 -11 -7 -thr -ala -val '91Y -val -arg 2- 18- 3.- 21 thr- ala- val- asp- ile- arg~ alaJ val- glyw valJ arg- Mother (3 I) 66- -2 58- -5 15- -11 1- -7 thr- -thr thr- -ala valq -ile asp- -asp ile- -ile arg- -his Diabetic child child 11 I) [1 I) 2- -2 2+--2 184 -5 18- -s 3- -ll 3- -ll 2- -7 2- -7 thr- -thr thr~ -thr ala- -ala ala- -ala val- -ile val~ -ile aspn -asp aspn -asp ile- -i1e ile- -ile arg- -his arg- -his HLA-A HLA-B HLA-DR HLA-D0 TAP2/665 TAP2/565 TAP2/379 TAP1/637 TAP1/333 LMP2/60 64 Family 3 Father [1 13 24- -x 44- -60 1- -13 x- -6 ala- -thr alaw -ala val- -val GSP‘ '91Y ile- -val arg- -arg Diabetic child Child 11 (3 11 (3 24- -24 24+ -24 44- -62 444 -62 l- -4 14 -4 54 -3 5‘ -3 ala- -ala ala- -ala ala: -ala ala- -a1a valn -val val- -val aspd -asp asp- -asp ileJ -ile ile- -ile arg- -arg arg~ -arg Figure 12. cont. Mother (3 24- 62- 4s 31 alaq alaq valq aqu ile- arg- child 24‘ 44- 54 ala- ala- val- asp- ile- argq I) -29 HLA-A HLA-B HLA-DR HLA-DO TAP2/665 TAP2/565 TAP2/379 TAP1/637 TAP1/333 LMP2/60 Figure 12. cont. 65 Family 4 Father [1 I3 21 -24 52- -39 15- -11 sq -7 thrd -ala alad -ala vald -va1 asp- -asp ilen -ile arg- -his Diabetic child 13 (3 24- -3O 39. -35 11- -4 7- -3 ala- -thr ala- -ala val- -val asp- -asp ile- -i1e his- -arg Mother (3 I) 30- -2 35- -52 4- -15 3- -6 thr-t -thr ala- -a1a val- -val aspd -asp ile~ -ile argn -arg child 11 (3 2d -30 52~ -35 15- -4 1- -3 thr4 -thr ala- -ala val- -val asp- -asp ile- -ile argd -arg DISCUSSION Development of LMP: anus PCR ARMS PCR has been a typing method used to detect known mutations in a selected nucleotide sequence. This method used a set of four primers. Two primers flank the mutation site and amplify a control fragment. The other two primers were specific for one of the two known polymorphisms. One allele specific primer was positioned in the sense direction, the other allele specific primer, in the antisense direction. The allele specific primer in the sense direction was paired with the antisense control primer and amplified a fragment of determined length if that allele was present. The other allele specific primer in the antisense direction was paired with the sense control primer and amplified a fragment of determined length if that allele was present (See figure 13). ARMS PCR 3’ m 5’ ‘ihui 51%,. control band (pdunen 1 and 4) dela‘l banflprimeniandS) _ “2mmflnfi2andfl Figure 13. ARMS PCR An illustration of the ARMS PCR method. Two out of four primers serve as controls. The other two primers are allele specific and amplify, with their corresponding control primer, a fragment of a specified length. 66 67 Primers for ARMS PCR amplification of TAP alleles came from published primer sequences. LMP2 allele specific primers were developed specifically for this research project. This enabled the LMP2 polymorphism to also be typed by an ARMS based method. The LMP2 allele specific primers, LMPZArg and LMPZHis, have a 3' terminal primer/template G/T mismatch for the Arg allele and a T/G mismatch for the His allele. It has been shown that Taq polymerase lacks a 3' to 5' exonuclease activity (Newton). The lack of a 3' to 5' repair mechanism is essential to have a refractory PCR amplification system. To increase the specificity of the refractory PCR reaction, the third nucleotide from the 3' end of the allele specific primer was purposefully mismatched (Newton). The mismatches most refractory to amplification were purine/purine or pyrimidine/pyrimidine pairings (Newton). In development of the LMP2 allele specific primers, a deliberate mismatch incorporated into the third nucleotide in from the 3' end of the sequence is primer/template T/C in the Arg specific primer and A/A in the His primer to enhance specificity. Inhibition of PCR The ARMS primer sets were initially screened against HTCL that had been previously typed at the TAP1 and TAP2 loci (Powis, 1993 and Carrington) and sequenced at the LMP2 loci to verify the accuracy of the typing results. While 68 attempting to run initial typings with the Puerto Rican samples, non-amplification was noted in several samples as no allele or control PCR products appeared. The frozen whole blood samples obtained from Puerto Rico were at least five years old. Many of these samples have gone through repeated freeze and thaw cycles. These conditions lead to cell lysis and degradation of DNA. The hemoglobin released from red blood cells is known to be a PCR inhibitor (Wiedbrauk). Because of the less than ideal sample quality, an inhibitor to the PCR process within the non-amplified samples was suspected when non-amplification occurred. There were several ways to reverse the affects of PCR inhibitors. A detergent, solvent, or protein may be added or the inhibitor can be diluted out of the sample. Tween-20 (Polyoxyethylene sorbitan monolaurate) is a detergent that reverses inhibitory effects of 0.01% SDS (Sodium dodecyl sulfate) on Tag polymerase (Varadaraj). Glycerol is an organic solvent that destabilizes double stranded DNA and improves strand separation. Glycerol is thought to either eliminate the formation of secondary structures or affect the thermal activity of Taq polymerase (Lu). Another method to minimize inhibition of PCR is to add bovine serum albumin (BSA). BSA has been shown to suppress the actions of inhibitory factors in DNA samples from mummies dated to 1912 (Lin). Diluting out the inhibitor is another means of 69 eliminating PCR inhibition. PCR inhibitory effects are concentration dependent (Wiedbrauk). To test this hypothesis, Puerto Rican genomic DNA that previously did not amplify, was subject to an additional ARMS PCR with either 2% Tween-20, 2% glycerol, 2% BSA, or DNA dilution with water (1:2, 1:4, and 1:8). Optimal results were obtained with a 1:8 dilution as demonstrated in Figure 14. Therefore, DNA isolated from whole blood samples at Michigan State University were subsequently diluted 1:8 and used for typing. Data Analysis After typing each sample in duplicate, results were analyzed using a chi-square analysis with Fisher's Exact test to correct for small sample numbers. Susceptibility to IDDM is inherited in a recessive mode while genes conferring protection to IDDM are inherited in a dominant mode (She). Statistical analysis of this data revealed no association with any allele that was typed for and IDDM. Analysis considered dominant (presence of an allele) and recessive (homozygosity of an allele) patterns of inheritance. Haplotype analysis Within the four families, there were no haplotypes that predominately associated with IDDM. Even between unrelated diabetics with the same DR antigens, haplotypes including 70 Tween-20 marker to L 3 U to E x C E .Q 9080 388 300 200 100 Inarker Inarker .n: C E .Q bhnk Inadura 9080 2(18) 2(14) 2(12 madura 9080 1(18 1(t4) 1(12) 388 300 200 100 Figure 14. Inhibition of PCR Above are several gels with different methods of overcoming PCR inhibition. 2% glycerol, 2% BSA, 2% Tween-20, 1:2, and 1:4 dilutions were not as effective as a 1:8 dilution of genomic DNA. This gave a final genomic DNA concentration of 0.0125ug/u1 which reduced the amount of DNA in the PCR reaction by 1/8 as compared to the undiluted sample. 71 the A, B, DR, DQ, TAP and LMP alleles, were not common. Within a family there were diabetic and non-diabetic siblings that shared the same haplotypes. These non- diabetic siblings should be noted and watched for IDDM development. conclusions No statistical significance was evident in this Puerto Rican population for the TAP and LMP polymorphisms tested for. There was no TAP or LMP allele associated with IDDM in a dominant or recessive manner. Other studies have found TAP28 to be predisposing to IDDM, however, this was later found to be secondary to linkage disequilibrium to DR4 (Ronningen, 1993). Samples for this study were collected in order to minimize the DR4 and TAP28 linkage. If a TAP or LMP allele was a predisposing or protective factor, then data in all subgroups (DR3/DR4, DR3 or 4/DRX, and DRX/DRX) should have been significant. Because there is no association with TAP or LMP genes in this Puerto Rican population and other studies have shown DP not to be significant, a conclusion based on this and other data is that the IDDM susceptibility gene is telomeric to the TAP and LMP gene region and possibly maps between TAP2 and the DO and DR region. The possibility still exists that susceptibility to IDDM is based upon an interaction of DR and DO and possibly other genes within the MHC region as 72 the relative risk is additive when more loci are included (Huang). SUMMARY Puerto Ricans diabetics and controls were typed for polymorphisms within the TAP and LMP genes. This population was chosen to supplement, in a supportive or refutive manner, the Caucasian population data. The Puerto Rican population with its Hispanic, African, and Native American mixture provided an ideal comparison to facilitate racial haplotype minimization. However, there were no significant findings between IDDM and the TAP and LMP polymorphism typed for in the Puerto Ricans. Sixteen extended HLA haplotypes were described in this population. With further haplotype and disease association information, these haplotypes may be used as a probability predictor for the occurrence of IDDM. If an individual has a haplotype that has been previously associated with IDDM, then that person would be at a higher risk for developing IDDM. 73 RBCOHKBNDATIONS Because of the limited number of samples included in this study, it would be beneficial to repeat the analysis on a larger sampling of this population. Other newly discovered polymorphic loci within the TAP and LMP genes could also be included along with LMP7 polymorphisms. Other studies could focus on the region between TAP2 and DO looking for a yet unknown gene that contributes to IDDM susceptibility or resistance. Promoter region polymorphisms would also be of benefit to study within this region. As promoters regulate transcription and gene expression, a polymorphic or mutant promoter may lead to a less efficient or altered method of gene expression that would be associated with antigen processing or presentation and ultimately lead to disease. Large scale familial haplotype analysis would be beneficial to better determine the extended haplotypes associated with DR3 and/or DR4. From this information, a prediction of the occurrence of the disease can be made. Because genetic factors are not the only influence on the development of IDDM, studies could focus on the peptide presented by the MHC molecule during the onset of the disease. Characterization of this peptide could provide useful information on the target of the immunological response. This knowledge would help in understanding what the autoimmune reaction was actually against and the 74 7s mechanisms behind it. This information could lead to effective treatment or prevention of the disease. LIST 03' REFERENCES LIST 0! RBIIRIICBS Abujoub, A. (1994). Polymorphism of the mitochondrial DNA control region in the Puerto Rican population. Master Thesis. Michigan State University. Acevedo, M. (1995). Personnal Communication. Ponce School of Medicine. Ponce, Puerto Rico. Akiyama, R., Yokota, R., Kagawa, S., et al. (1994). cDNA cloning and interferon-1idown-regulation of proteasomal subunits x and Y. Science 265:1231-1236. Androlewicz, M. and Cresswell, P. (1994). Human transporters associated with antigen processing posses a promiscuous peptide-binding site. Immunity 1:7-14. Beck, 8., Kelly, A., Radley, E., et al. (1992). DNA sequence analysis of 66 kb of the human MHC class II region encoding a cluster of genes for antigen processing. Journal of Molecular Biology 228:433-441. Braud, V., Chevrier, D., Cesbron, A., et al. (1994). Susceptibility to alloimmunization to platelet HPA-la antigens involves TAP1 polymorphsim. Human Immunology 41:141-145. Burney, R., Pile, R., Gibson, R., et al. (1994). Analysis of the MHC class II encoded components of the HLA class I antigen processing pathway in ankylosing spondylitis. Annals of the Rheumatic Diseases 53:58-60. Caillat-Zucman, S., Bertin, E., Timsit, J., et al. (1992). TAP1 and TAP2 transporter genes and predisposition to insulin dependent diabetes mellitus. C.R. Academy of Science, Paris 315:535-539. Caillat-Zucman, s., Bertin, E., Timsit, J., et al. (1993). Protection from insulin-dependent diabetes mellitus is linked to a peptide transporter gene. European Journal of Immunology 23:1784-1788. Cano, P. and Baxter-Lowe, L. (1995). Novel human TAP2*0103 allele shows further polymorphism in the ATP-binding domain. Tissue Antigens 45:139-142. 76 77 Carrington, M., Colonna, M., Spies, T., et al. (1993). Haplotypic variation of the transporter associated with antigen processing (TAP) genes and their extension of HLA class II region haplotypes. Immunogenetics 37:266-273. Cavan, D. and Barnett, A. (1993a). Insulin-dependent diabetes mellitus. In: Leslie R., ed. Q§n§g§_91_pighg§§§; WW3- Chichesterz John Wiley and Sons, 3-24. Cavan, D., Jacobs, R., Penny, M., et al. (1993b). Both DQAl and 0931 genes are implicated in HLA-associated protection from type I (insulin-dependent) diabetes mellitus in a British Causasian population. Diabetologia 36:252-257. Chevrier, D., Giral, M., Braud, V., et al. (1995). Effects of MHC-encoded TAP1 and TAP2 gene polymorphisms and matching on kidney graft rejection. Transplantation 60:292-296. Colonna, M., Bresnahan, M., Bahram, 8., et al. (1992). Allelic variants of the human putative peptide transporter involved in antigen processing. Proceedings of the National Academy of Science, USA 89:3932-3936. Cullen, M., Erlich, H., Kiltz, W., et al. (1995). Molecular mapping of a recombinational hotspot located in the second intron of the human TAP2 locus. American Journal of Human Genetics 56:1350-1358. Davies, J., Kawaguchi, Y., Bennett, 8., et al. (1994). A genome-wide search for human type 1 diabetes susceptibility genes. Nature 371:130-136. Dedeoglu, I. and Feld L. (1996). Insulin-dependent diabetes mellitus and renal complications. Clinical Laboratory Science 9:89-95. Deng, G., Muir, M., Maclaren, N, et al. (1995). Associations of LMP2 and LMP7 genes within the major histocompatibility complex with insulin-dependent diabetes mellitus: population and family studies. American Journal of Human Genetics 56:528-534. Driscoll, J., Brown, M., Finley, D., et al. (1993). MCH- linked LMP gene products specifically alter peptidase activities of the proteasome. Nature 365:262-264. Erlich, H., Zeidler, A., Chang, J., et al. (1993). HLA class II alleles and susceptibility and resistance to insulin dependent diabetes mellitus in Mexican-American families. Nature Genetics 3:358-364. 78 Fakler, J., Schmitt-Egenolf, M., Vejbaesya, S., et al. (1994). Analysis of TAP2 and HLA-DP gene polymorphisms in psoriasis. Human Immunology 40:299-302. Field, L., Tobias, R., and Magnus, T. (1994). A locus on chromosome 15q26 (IDDM3) produces susceptibility to insulin- dependent diabetes mellitus. Nature Genetics 8:189-194. Frazer, T. (1993-1996). Personnal Communications. Ponce School of Medicine. Ponce, Puerto Rico. Gaczynska, M., Rock, H., Spies, T., et al. (1994). Peptidase activities of proteasomes are differentially regulated by the major histocompatibility complex-encoded genes for LMP2 and LMP7. Proceedings of the National Academy of Science, USA 91:9213-9217. GeneQuant RNA/DNA Calculator User Manual. (1993). Issue 1. Pharmacia LKB Biochrom Limited, Science Park, Cambridge, England. Ghosh, P., Amaya, M., Mellins, E., et al. (1995). The structure of an intermediate in class II MHC maturation: CLIP bound to HLA-DR3. Nature 378:457-462. Hashimoto, L., Habita, C., Beressi, J., et al. (1994). Genetic mapping of a susceptibility locus for insulin- dependent diabetes mellitus on chromosome llq. Nature 371:161-164. Howard, J. and Seelig, A. (1993). Peptides and the proteasome. Nature 365:211-212. Huang, H., Peng, J., She, J., et a1. (1995). HLA-encoded susceptibility to insulin-dependent diabetes mellitus is determined by DR and DO genes as well as their linkage disequilibria in a chinese population. Human Immunology 44:210-219. Jackson, D. and Capra, J. (1993). TAP1 alleles in insulin- dependent diabetes mellitus: A newly defined centromeric boundary of disease susceptibility. Proceedings of the National Academy of Science, USA 90:11079-11083. Janeway, C. and Travers, P. (1996). Immunohiglggy; The W. 2nd 130. London: Current Biology, Ltd. Kaufman, D., Clare-Salzler, M., Tian, J., et al. (1993). Spontaneous loss of T-cell tolerance to glutamic acid decarboxylase in murine insulin-dependent diabetes. Nature 366:69-72. 79 Keller-Wood, H., Powis, 8., Gray, J., et al. (1994). MHC- encoded TAP1 and TAP2 dimorphisms in multiple sclerosis. Tissue Antigens 43:129-132. Kelly, A., Powis, 8., Glynne, R., et al. (1991). Second proteasome-related gene in the human MHC class II region. Nature 353:667-668. Lee, G., Shamma, F., Diamond, M., et al. (1992). HLA-D0857 in Hispanic patients with insulin-dependent diabetes mellitus. American Journal of Obstetrics and Gynecology 167:1565-1570. Leslie, R. (1993). Diabetic twin studies. In: Leslie R., ed. WW- Chichester: John Wiley and Sons, 61-82. Lin, 2., Kondo, T., Minamino, T., et al. (1995). Sex determination by polymerase chain reaction on mummies discovered at Taklamakan desert in 1912. Forensic Science International 75:197-205. Lu, Y. and Negre, S. (1993). Use of glycerol for enhanced efficiency and specificty of PCR amplification. Trends in Genetics 9:297. anas, A., Koster, A., and Baumeister, W. (1993). Structural features of the 268 and 208 proteasomes. Enzyme Protein 47:252-273. Maksymowych, W. and Russell, A. (1995). Polymorphism in the LMP2 gene influences the relative risk for acute anterior uveitis in unselected patients with ankylosing spondylitis. Clinical Investigative Medicine 18:42-46. Maniatis, T., Fritsch, E., and Sambrook, R. (1989). W- W. New York: Cold Springs Harbor. Moins-Teisseerenc, H., Bobrynina, V., Loiseau, P., et al. (1994). New polymorphisms within the human TAP1 and TAP2 coding regions. Immunogenetics 40:242. Menaco, J. (1995). Pathways for the processing and presentation of antigens to T cells. Journal of Leukocyte Biology 57:543-547. Morales, P., Martines-Laso, J., Martin-Villa, J., et al. (1991). High frequency of the HLA-DRBl*0405-(Dw15)-DQW8 haplotype in Spaniards and its relationship to diabetes susceptibility. Human Immunology 32:170-175. Newton, C., Graham, A., Heptinstall, L., et al. (1989). Analysis of point mutations in DNA. The amplification 80 refractory mutation system (ARMS). Nucleic Acids Research 17:2503-2516. Nuchtern, J., Biddison, W., and Klausner R. (1990). Class II MHC molecules can use the endogenous pathway of antigen presentation. Nature 343:74-76. One Lambda (1992). One Lambda HLA desktop companion. Canoga Park, California. Penny, M., Mijovic, C., Cavan, D., et al. (1993). An investigation of the associations between HLA-DO heterodimers and type I (insulin-dependent) diabetes mellitus in five racial groups. Human Immunology 38:179- 183. Perl. L. (1979). W. New York: William Morrow and Company. Ploski, R., Flato, B., Vinje, O., et al. (1995). Association to HLA-DRB1*08, HLA-DPB1*0301 and homozygosity for an HLA-linked proteasome gene in juvenile ankylosing spondylitis. Human Immunology 44:88-96. Powis, 8., Deverson, E., Coadwell, W., et al. (1992a). Effects of polymorphism of an MHC-linked transporter on the peptides assembled in a class I molecule. Nature 357:211- 215. Powis, S., Mockridge, I., Kelly, A., et al. (1992b). Polymorphism in a second ABC transporter gene located within the class II region of the human major histocompatibility complex. Proceedings of the National Academy of Science, USA 89:1463-1467. Powis, S., Tanks, 8., Mockridge, I., et al. (1993). Alleles and haplotypes of the MHC-encoded ABC transporters TAP1 and TAP2. Immunogenetics 37:373-380. Powis, 8., Cooper, M., Trowsdale, J., et al. (1994). Major histocompatibility haplotypes associated with immunoglobulin-A deficiency and common bariable immunodeficiency: Analysis of the peptide transporter genes TAP1 and TAP2. Tissue Antigens 43:261-265. Ronningen, K., Gjertsen, H., Iwe, T., et al. (1991). Particular HLA-DO dB heterodimer associated with IDDM susceptibility in both DR4-DQw4 Japanese and DR4-DQw8/DRw*- DQw4 whites. Diabetes 40:759-763. Ronnigen, K., Undlein, D., Ploski, R., et al. (1993). Linkage disequilibrium between TAP2 variants and HLA class II alleles; no primary association between TAP2 variants and 81 insulin-dependent diabetes mellitus. Journal of Immunology 23:1050-1056. Rowe, R., Wapelhorst, B., Bell, G., et al. (1995). Linkage and association between insulin-dependent diabetes mellitus (IDDM) susceptibility and markers near the glucokinase gene on chromosome 7. Nature Genetics 10:240-242. Rubinstein, P., Walker, M., Mollen, N., et al. (1990). No excess of DR*3/4 in Ashkenazi Jewish or Hispanic IDDM patients. Diabetes 39:1138-1143. Savage, D., Hg, 8., Howe, H., et al. (1995). HLA and TAP associations in chinese systemic lupus erythematosus patients. Tissue Antigens 46:213-216. She, J. (1996). Susceptibility to type I diabetes: HLA-DO and DR revisited. Immunology Today 17:323-329. Singal, D., Ye, M., and Quadri, S. (1995). Major histocompatibility-encoded human proteasome LMP2. Journal of Biological Chemistry 270:1966-1970. Solimena, M. and DeCamilli, P. (1993). Spotlight on a neuronal enzyme. Nature 366:15-16. Spies, T., Bresnahan, M., Bahram, S., et a1. (1990). A gene in the human major histocompatibility complex class II region controlling the class I antigen presentation pathway. Nature 348:744-747. Srivastava, P., Udono, H., Blachere, N., et al. (1994). Heat shock proteins transfer peptides during antigen processing and CTL priming. Immunogenetics 39:93-98. Szafer F., Oksengerg, J., and Steinman, L. (1994). New allelic polymorphism in TAP genes. Immunogenetics 39:374. Tajima, M., LaPorte, M., and La Porte, R. (1993). Population studies. In: Leslie R., ed. _Q§Hfi§§_22 Difihfitfifil 9eneti2_and_en21r9nnental_fast2rs- Chichester: John Wiley and Sons, 25-44. Theofilopoulos, A. (1995). The basis of autoimmunity: Part II genetic predisposition. Immunology Today 16:150- 158. Tighe, M., Hall, M., Cardi, E., et al. (1994). Associations between alleles of the major histocompatibility complex-encoded ABC transporter gene TAP2, HLA class II alleles, and Celiac Disease susceptibility. Human Immunology 39:9-16. 82 Tisch, R., Yang, M., Singer, 8., et a1. (1993). Immune response to glutamic acid decarboxylase correlates with insulitis in non-obese diabetic mice. Nature 366:72-75. Todd, J. (1990). Genetic control of autoimmunity in type I diabetes. Immunology Today 11:122-129. Trucco, M. (1992). To be or not to be ASP 57, that is the question. Diabetes Care 15:705-715. Van Endert, P., Lopez, M., Patel, 6., et al. (1992). Genomic polymorphism, recombination, and linkage disequilibrium in human major histocompatibility complex- encoded antigen-processing genes. Proceedings of the National Academy of Science, USA 89:11594-11597. Van Endert, P., Liblau, R., Patel, 8., et al. (1994). Major histocompatibility complex-encoded antigen processing gene polymorphism in IDDM. Diabetes 43:110-117. Varadaraj, K. and Skinner, D. (1994). Denaturants or cosolvents improve the specificity of PCR amplification of a G+C rich DNA using genetically engineered DNA polymerases. Gene 140:1-5. Vicario, J., Martinez-Laso, J., Corell, A., et al. (1992). Comparison between HLA-DRB and DO DNA sequences and classical serological markers as type I (insulin-dependent) diabetes mellitus predictive risk markers in the Spanish population. Diabetologia 35:475-481. Voet, D. and Voet J. (1990). Biggnemigtzy. New York: John Wiley and Sons, 1048. Westman, P., Paranen, J., Leirisalo-Repo, M., et al. (1995). TAP1 and TAP2 polymorphism in HLA-B27-positive subpopulations: No allelic differences in ankylosing spondylitis and reactive arthritis. Human Immunology 44:236-242. Wiedbrauk, D., Werner, J., and Drevon, A. (1995). Inhibition of PCR by aqueous and vitreous fluids. Journal of Clinical Microbiology 33:2643-2646. Wordsworth, B., Pile, K., Gibson, K., et al. (1993). Analysis of the MHC-encoded transporters TAP1 and TAP2 in rheumatoid arthritis: Linkage with DR4 accounts for the association with a minor TAP2 allele. Tissue Antigens 42:153-155. Yoon, J. and Park, Y. (1993). Viruses as triggering agents of insulin-dependent diabetes mellitus. In: Leslie R., ed. W Chichester: John Wiley and Sons, 83-104. "lillllllllllllllllll“