Genetics of diabetes

POS-001-210 THE INSULIN RECEPTOR GENE, INSULIN RESISTANCE AND NON-INSULIN DEPENDENT DIABETES MELLITUS

A REES, R MORGAN, A BISHOP, S LUZIO, DR OWENS Department of Medicine, University of Wales College of Medicine, Heath Park, Cardiff, U.K.

The aetiology of non insulin dependent diabetes mellitus (NIDDM) contains a strong genetic com- ponent, as indicated by nL~nerous pedigree studies. Abnormalities at the insulin receptor gene (IRG) may contribute to the insulin resistance characteristic of NIDDM and we have therefore investigated whether an association exists between certain IRG variants and NIDDM in 77 Welsh caucasian patients compared with 27 non-related matched controls. Southern blotting techniques have revealed restriction fragment length polymorphisms (RFLPs) at the IRG locus with the enzymes Sst I, Bgl II and Rsa I. The Sst I and Bgl II RFLP's appear neutral with respect to NIDDM in our study groups~uttheRsaI~uP~hown by either a 6.7 Kb or 6.2 Kb allele) reveals that homozygosity for the 6.7 Kb allele was significantly more prevalent in the NIDDM group (p <0.01, X2 analysis). We have further investigated whether specific Rsa I IRG genotypes (6.2 Kb homozygotes n = 11, 6.7 Kb homozygotes n = 36, 6.2 Kb 6.7 Kb heterozygotes n = 36) appear to influence metabolic profiles or demography. Each patient prior to any treatment, had a standard meal tolerance test and both oral and intravenous glucose tolerance tests. Peripheral insulin resistance was estimated from serialplasma glucose, insulin and C-peptide levels. Details of renal function, lipid profile,demographic data and glycosylated haemoglobin were obtained on diagnosis. Statistical analyses revealed no significant association between IRG genotype and insulin resistance nor with any other variable measured. Hence we conclude that Rsa I genotype variation does not significantly modify the NIDDM phenotype in our patient group.

POS-001-211 AN HLA-DQ~/DP~-RELATED GENOMIC FRAGMENT DIFFERENTIATES NEWLY DIAGNOSED AND EARLY ONSET IDDM PATIENTS FROM OTHER IDDM AND CONTROL INDIVIDUALS

F J M IRIS, J G H HICKFORD, S M FOUNTAIN, R S SCOTT Department of Medicine, Christchurch School of Medicine, Christchurch, NEW ZEALAND.

The observed strong association of IDDM with the DR3, DR4, DR3/4 HLA class II haplotypes led to the concept of susceptibility genes either in linkage disequilibrium with, or forming part of, the class II genomic region. Accordingly, IDDM patients should share HLA genomic characteristics encountered at low frequency amongst control individuals. Over the past five years, a variety of HLA-associated restriction fragment length poly- morphisms (RFLPs) have been reported in tight correlation with IDDM. These RFLPs have been detected with various DRY, DRy, DQ~ and DQ~ probes. However, none to date have proven to be diagnostically reliable at a population level and the identification of an HLA-associated IDDM marker is still as elusive as ever. We report the detection of a BAM H I RFLP characteristically absent in sub- sets of IDDM patients. This 3.25 to 3.5 kb DQ~-DP~-associated restriction fragment is present in all control and long-term IDDM patients. It is absent in all newly diagnosed and early onset IDDM individuals (diagnosed before age six) analysed to date. We hypothesise that this RFLP could be related to the presence of active autoimmune processes.

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POS-001-212 NO EVIDENCE FOR SEGREGATION DISTORTION FOR HLA GENES IN TYPE I DIABETES

J BERTRAMS*, H KROENKE*, W RATHMANN*, B KUGLIN**, FA GRIES** Diabetes Education and Therapy Center, Elisabeth Hospital Essen FRG* Diabetes Research Institute, University of DUsseldorf, FRG**

A preferential transmission of diabetogenic alleles within the HLA complex was reported by Vadheim et al. (NEJM 1986, 315: 1314-18), but controversially discussed afterwords (NEJM 1987, 316: 1477-78; 317: 768-69). To retest the hypothesis of segregation distortion in diabetic families, we examined parent to offspring transmission of HLA haplotypes and DR alleles in more than 300 families with at least one offspring with well-documented type I diabetes. All individuals were white and completely typed for HLA-A, B,C,DR,C2,C4,BF. The distorted segregation of DR3 and/or DR4 among the diseased individuals can be explained simply be the well-known association of type I diabetes with DR3 and DR4 according to the ascertainment bias. DR3 and DR4 frequencies did not significantly differ from the expected 90-95% among the affected and the expected nearly 50% among the unaffected children. A significant difference, however, was observed between the paternal (92%) and maternal (79%) transmission of DR4 to affected children, also reported by Vadheim et al. and Klitz et al. (GAW V, 1987). Accordingly DR4-positive fathers, possibly due to environmental or non-HLA genetic factors, have a relatively stronger impact on causation of type I diabetes than DR4-positive mothers.

POS-001-213 HLA GENOTYPING BY RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP) ANALYSIS AND IDDM INHERITANCE

JA FLETCHER, C MIJOVIC, E MACKAY, AR BRADWELL, AH BARNETT. Depts of Medicine & Immunology, University of Birmingham, Birmingham, UK.

HLA genotype frequencies among patients may be used to predict the mode of inheritance of IDDM susceptibility. The size of HLA genotype studies using serological typing is limited by the need to perform family studies of patients. Restriction fragment length polymorphism (RFLP) analysis using HLA probes enables direct genotyping without the need to study relatives.

Asian Indian IDDM patients have a high frequency of DR3, with DR4 a secondary associated marker. We have used a D~$-specific gene probe and restriction enzyme TaqI to examine DR~ RFLPs and derive DR genotypes in 41 Asian Indian IDDM patients. Observed genotype frequencies were compared with those predicted by the "antigen genotype frequency among patients" (AGFAP) model. Numbers of patients with each genotype were: 3/4: 12, 3/3: 18, 4/4: 0, 3/X: 8, 4/X: 1 and X/X: 2 ("3" = DR3 RFLP, "4" = DR4 RFLP and "X" = anY9non-DR3, non-DR4 RFLP). The results are inconsistent with simple dominant inheritance (p<10-). When DR3 was analyzed separately, genotype frequencies excluded dominant inheritance for the DR3-associated disease allele (p<0.01) but conformed closely with recessive expectations. We have initiated a similar study on 150- 200 white caucasian IDDM patients. Preliminary results from 35 patients show an excess of DR3/4 heterozygotes (14 patients) over DR3/3 plus DR4/4 homozygotes (7 patients), which is against simple recessive inheritance.

DR~RFLP analysis is a useful new approach for DR genotyping large numbers of patients. Our results support the concept of a synergistic interation between different DR3- and DR4-associated susceptibility genes, but indicate that homozygosity of the DR3 factor also predisposes to IDDM.

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POS-001-214 HLA-D POLYMORPHISM ANALYSIS IN AFRO-CARIBBEAN INSULIN-DEPENDENT DIABETICS IMPLICATES AN HLA-LINKED NON-DR, NON-DQ SUSCEPTIBILITY FACTOR

C MIJOVIC, JA FLETCHER, E MACKAY, O ODUGBESAN, K CRUICKSHANK, AR BRADWELL, AH BARNETT. Depts of Immunology and Medicine, University of Birmingham, Birmingham, UK.

IDDM in white caucasians is positively associated with HLA-DR3 and -DR4. DR7 is negatively associated. Restriction fragment length polymorphism (RFLP) analysis using a DQ~ cDNA probe has shown that DR4 is associated with either a 3.7 or a 12kb Bam HI DNA fragment. In white caucasians, only the 12kb subset of DR4 is associated with IDDM, while the 3.7kb fragment is rare among patients.

We have investigated DR and DQ polymorphism in IDDM patients and control subjects of Afro- Caribbean origin by serological DR typing and DQ~RFLP analysis. _~R4 was increased in frequency in diabetics v controls (15/29 (52%) v 1/53 (2%) (p<2xl~ v). DR3, DR4 or both were present in 25/29 (86%) patients and 17/53 (32%) controls (p<3xl0-). DR3 was present in 14/29 (48%) patients and 16253 (30%) controls (p = NS). DR2 was absent from the diabetics (0/29 (0%) v 18/53 (34%), p<0.002). DR7 was present in 8/29 (28%) patients and 9/53 (17%) controls (p = NS). Combined analysis of our data with those from all published studies of DR antigens in negro IDDM patients showed a significant (p<0.0005) positive association with DR7 (relative risk=l.8).

Among 13 DR4-positive Afro-Caribbean diabetics, 9 (69%) were DQ~ BamHI 12kb-positive, but 4 (31%) were 3.7kb-positive. The 12kb BamHI fragment does not, therefore, mark an IDDM- specific DR4 subset in this group. DR7 in Afro-Caribbean patients was associated with the same DQ~ fragments (4.0+3.2kb) that we have identified in DR7-positive white subjects. Thus

differences between DQ~ genes on DR7 haplotypes in Afro-Caribbean and white subjects are unlikely.

Our data question the postulated primary role of DQ~ in IDDM and implicate an HLA-linked

non-DR, non-DQ susceptibility factor.

POS-001-215 THE IDDM-SUSCEPTIBLE HLA-DR4 HAPLOTYPE IS BEST DEFINED BY A COMBINATION OF DR AND DQ ALLELES

MJ SHEEHY, JR ROWE, BS NEPOM American Red Cross Blood Services and Department of Medicine, University of Wisconsin, Madison, Wisconsin, U.S.A; Virginia Mason Research Center, Seattle, Washington, U.S.A.

Many studies have shown HLA-DR4 to be associated with insulin-dependent diabetes mellitus (IDDM], part icular ly in combination with HLA-DR3. It is now clear, however, that either [a] only certain subtypes of DR4 are diabetogenic, or [b) DR4 is merely a marker for a diabete's allele at a nearby locus (the most l ikely candidate being the HLA-DQB1 [DQ 8] locus]. The present study was designed to test whether the primary IDDM association is with certain DR4 subtypes or with a DQB1 allele, DQ3.2. We find that neither the DR nor the DQ association is pr imary and the other secondary; when one controls for alleles at either locus, the IDDM association at the other locus becomes stronger rather than disappearing. Certain subtypes of DR4 (Dw4 and Dw10) marked a diabetes-associated subset of DQ3.2 haplotypes and vice-versa. In the Southern Wisconsin population studied, relative risks for IDDM were: DR3, 3.8; DR4, 4.2; Dw4 and/or Dwl0, 7.6; DQ3.2, 6.3. The highest relat ive risk was found for persons having a certain combination of DR and DQ alleles. Dw4+DQ3.2 or Dwl0+DQ3.2 [combined RR=14.0). These last two DR4 haplotypes were found in 32/40 (80%] of DR4-positive IDDM patients but only 7/30 [23%) of DR4-positive control subjects (P=1.6 x 10-6]. In the population studied, persons heterozygous for a DR3 haplotype plus one of the diabetes-associated DR4 haplotypes [DR3/Dw4,DQ3.2 or DR3/Dwl0,DQ3.2] have an absolute risk of approximately 1/12 of acquiring IDDM. Thus population-level testing for IDDM susceptibi l i ty is approaching the predict ive power attainable in patients' f irst-degree relatives.

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POS-001-216 INSULIN RECEPTOR AND APOLIPOPROTEIN GENES CONTRIBUTE TO THE DEVELOPMENT OF NON- INSULIN DEPENDENT DIABETES MELLITUS IN CHINESE AMERICANS

K XIANG, NJ COX, N SANZ, P HUANG, JH KARAM, GI BELL HOWARD HUGHES MEDICAL INSTITUTE, THE UNIVERSITY OF CHICAGO, CHICAGO, IL AND METABOLIC RESEARCH UNIT, UNIVERSITY OF CALIFORNIA, SAN FRANCISCO, CA USA

The frequencies of restriction fragment length polymorphisms (RFLPs) as well as RFLP haplotypes at six loci responsible for carbohydrate and lipid metabolism [insulin/ insulin-like growth factor II complex, insulin receptor (INSR), HepG2/erythrocyte-type glucose transporter, apolipoprotein A-II, apolipoprotein B (APOB), and apolipoprotein A-I/C-III/A-IV cluster (APOAI/C3/A4)] were compared between non-diabetic and diabetic Chinese Americans. The disease association data suggest that genetic variation at the INSR, APOB and APOAI/C3/A4 loci contributes to the etiology of non-insulin-dependent diabetes mellitus (NIDDM). The analysis of the INSR locus revealed XbaI-RsaI-KpnI haplotypes, X2R2K2 (X.==I2.06,Pc=O.O06 RI=O.17) and XIR3K2 (X2=8.05,Pc=O.05, RI=O.09) that appear to "protect" individuals from developing diabetes and may be possible to use these two haplotypes as genetic markers to identify individuals having a very low probability of developing NIDDM regardless of the presence of other genes conferring susceptibility to this disorder. The APOB locus appears to contribute to the develop- ment of NIDDM in individuals who are lean/normal weight (X2=5.44,P<O.O2,RI=4.30) and APOA1 locus in individuals who are overweight (×2=8.83,P~O.OO5,RI=3.68). The APOA1 locus may account for about 8% of the difference between baseline and total possible risk of NIDDM in overweight individuals.

POS-001-217 EXPLORING THE RE.ATIONSHIP BETWEEN TYPE I AND TYPE II DIABETES IN ~XICAN A}~RICAN FAMILIES

S SHAW, A ZEIDLER, C VADHEIM, G COSTIN, R PENNY, J ROTTER Division of Diabetes/Clinical Nutrition, Pediatric Endocrinology, USC, Medical Genetics, Cedars-Sinai Medical Center, UCLA, Los Angeles, USA.

Previous studies have indicated an increased prevalence of Type II Diabetes Mellitus (DM) in the Mexican American (MA) population. We have observed a n~mber of apparently classical MA Type II diabetics that were hospitalized for diabetic ketoacidosis. The purpose of this study was to investigate the relationship between Type I and Type II diabetes in this ethnic group. Twenty MA families were ascertained through one offspring (proband) with Type I diabetes (NDDG criteria) and compared to families aged-matched MA non-diabetlc chronic disease controls. A questionnaire was utilized to define the prevalance of Type II DM in various categories of first and second degree relatives of the Type I cases and non-diabetic controls. Histories were available on 69 grandparents (ages 40 to 80 yrs.) and 35 parents (ages 19 to 66 yrs.). 6/69 grandparents (8.7%) and 3/35 (8.6%) of the parents had Type II diabetes. 9/20 families were positive for Type II DM by history. Type II DM appears to be prominent in the relatives of Type I MA probands compared to the published data in the Caucasian population. To test if this prominent occurrence of Type II DM represents a truly increased frequency, data from the control MA families are now being analyzed. If this apparent increase is validated, this would suggest a relationship between the two diseases. If on the otherhand, the prevalance of Type II DM is decreased compared to the MA controls, this could be the result of a disproportionate Caucasian, Native American Indian admixture. We are currently extending the study to include forty families with DM that will be compared with appropriate age-matched MA controls in order to clarify the diabetes types and their interrelationships in this distinct ethnic group.

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POS-001-218 CLINICAL ANALYSIS OF THE PEDIGREE WITH DIABETIC PARENTS

Jinnouchi Hospital. Tomio Jinnouchi,Keizo Kajiwara,Hideaki Jinnouchi. The Dept Qf 2nd Internal Medicine, Ryukyu Univ. Goro Mimura. Eukumitsu Clinic. Sho~i Fukumitsu.

Genetic factors are recognized to be significant as to the incidence of diabetes mellitus(DM), especially in NIDDM. As one of the various approaches, we have studied the incidence of DM in the

children with diabetic parents. Subjects are 64 diabetics parents (32 pairs of couples) and their children. As to the therapy of these parents, diet(D), oral agent(OA) and insulin(Ins.)

are 31, 23 and 10 respectively. The incidence frequency of DM in their children according to the treatment co-

mbination of these parents are 26.9%(7/26) in(Ins.)x(Ins.,OA,D), 26.8%(11/41) in (OA)x(OA,D) and 12.5%(8/24) in (D)x(D) respectively. As to the incidence of DM in every 20 years groups of age are none diabetic in

5 cases under 20 years, 9 cases in 52 cases (17.2%) in 21-40 years and 12 in 32 cases (37.6%) in 41-60 years of age group respectively. Conclusion : (I). The incidence frequency of DM in children with diabetic parents

controlled only with diet is lower compared to the other groups. (2). The incidence frequency of DM in 41-60 years of age group is approximately

twofold higher than 21-40 years of age group.

POS-001-219 GENETIC LOCI CONTROLLING DIABETES SUSCEPTIBILITY IN NOD MICE

E LEITER, M PROCHAZKA The Jackson Laboratory, Bar Harbor, Maine USA

All laboratories studying the genetics of diabetes in the NOD mouse are in agree- ment that (i) the diabetes represents an organ-specific autoimmune disease and (2) control is polygenic. Insulitis is the major histopathological lesion associated with diabetogenesis in NOD mice. The relationship between the genes controlling diabetes and insulitis, however, remains unclear. In (NOD x NON) outcross/back- cross analysis, only one recessive gene controlling insulitis was reported to segregate, whereas at least three are segregating for diabetogenesis. When first and second backcross progeny following outcross of NOD to NON, were studied, 3 diabetogenic recessive genes from NOD were required for insulin-dependent diabetes (Idd). One of these, Idd-l, was tightly linked to M~C on ~hr 17; the resistance allele carried by NON ~ not be separated from the H-2K MHC marker gene after 5 cycles of backcrossing to NOD (Idd-I r congenic, diabete----s resistant). The second gene required for diabetogenesis, Idd-2, has been mapped between the centromere and the Thy-i marker gene on Chr 9. The NON resistance allele at this locus was sepa- rable (by recombination) from the Thy-i a marker gene by 5 cycles of backcrossing to s a . NOD (Idd-i Thy-i congenlc, develops diabetes). We will report the results of histopathological screening for presence or absence of insulitis in 12 month old congenics. The Idd-i r congenic will allow assessment of the role of the NOD M~C in insulitis induction, while the Idd-i s Thy-i a congenic will allow determination of whether "Thy-l" linked genes on Chr 9 other than Idd-i s are involved in insulitis induction. Progress in mapping the third segregating diabetogenic gene, Idd-3 s, will also be reported. No linkage to markers on Chr 1,2,3,4,5,6,7,8,11,14, or 15 has been detected (including the T cell s-receptor gene on Chr 14).

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POS-001-220 GENETIC ASSOCIATION OF GRAVES' DISEASE WITH IDDM.

S HAMADA, T YAGURA, H ISHII, T YOSHIMASA, T ONISHI Department of Endocrinology,Tenri Hospital,Nara,Japan.

Graves' disease, like Hashimoto's thyrolditis, is an autoimmune thyroid disease and is associated with specific types of HLA. Although Hashimoto's thyrolditis is frequently associated with DM, little is known about genetic relation between Graves' disease and DM. We studied intrinsic association of Graves' disease with DM in 28 cases who suffered from both diseases. Age of onset of Graves' disease was closely related with that of DM. Interestingly enough, 6 of 7 cases with Graves' disease at the onset between 12 and 39 years old ( juvenile-onset Graves ) required insulin therapy while only 1 of Ii cases at the onset between 40 and 73 years old (middle-aged Graves) required the same therapy. Furthermore, juvenile- onset Graves had an attack of the disease 3 to Ii years before the onset of DM in 3 cases and at the same time as that of DM in 4 cases. By contrast, middle-aged Graves had the disease 6 to 10 years after the onset of DM in 4 cases and at the same time in 7 cases. All but one case with juvenile- onset Graves had marked decrease in 24-hr urinary CPR excretion and very low CPR response in OGTT, indicating IDDM. On the other hand, all but one case with middle-aged Graves had near-normal 24-hr urinary CPR excretion and near-normal CPR response in OGTT, indicating NIDDM. In addition, 43% of juvenile-onset Graves had the same disease in their family lines, compared with only 18% of the middle-aged Graves. It is concluded therefore that juvenile-onset Graves'disease accompanied by DM is probably predisposed to IDDM by contrast with middle-aged Graves'disease.

POS-001-221 HUMAN LEUKOCYTE ANTIGEN (HLA) DISTRIBUTION IN YOUNG KOREANS WITH INSULIN-USING DIABETES

KB HUH, HC LEE, HM KIM, EJ LEE, SK LIM, DH KIM Department of Internal Medicine and Department of Pediatrics, Yonsei University, Seoul, Korea

Among the young people of Korea, besides maturity-onset diabetes of the young, there are two kinds of diabetes mellitus with clinical similarity which differ in the character of insu- l in dependency, classical insulin-dependent (ketosis-prone) diabetes mellitus (IDDM) and insulin-requiring (ketosis-resistant) diabetes mellitus (IRDM). Furthermore the HLA-DR antigens associated with IDDM in Oriental patients are reported to be different from those in Caucasian patients. HLA-DR antigens were determined in 20 IDDM (mean age at diagnosis 16.6 years, range 1.9-27.0 years) and 30 IRDM (mean age at diagnosis 23.3 years, range II.0- 29.0 years) patients, and compared with 282 unaffected individuals. The first-degree relative family history of the disease was present in 10.0% of IDDM patients and in 23.3% of IRDM patients. In IDDM patients, the frequencies of HLA-DR3 and -DR4 were significantlymcreased (30.0% and 60.0% of patients versus 10.9% and 29.4% of controls, p<O.05 and p<O.Ol, respec- tively) and the HLA-DR2 and -DR5 were total ly absent (23.0% and 12.4% of controls, respecti- vely). In contrast, no HLA-DR specificities were found in the IRDM. These data suggest that the genetic backgrounds of both insulin-using diabetes in young Koreans might be quite diffe- rent each other and accordingly the heterogeneity of etio-pathogenesis in both types could be speculated.

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POS-001-222 HLA CLASS II LIGHT- AND HEAVY-CHAIN SPECIFICITIES OF THE HLA-DQ GEHE REGION CONTRIBUTE TO THE IA-MEDIATED SUSCEPTIBILITY FOR TYPE I (INSULIN-DEPENDENT) DIABETES MELLITUS

K.SCHOFFLING,P.KOHNL ,B.O.BOEHM UNIVERSITY HOSPITAL OF FRANKFURT MEDICAL SCHOOL, CENTER OF INTERNAL MEDICINE,D-60OO FRANKFURT

AM MAIN 70, GERMANY

Human Ia antigens are encoded within the HLA-D gene region of the human major histocompatibility complex. Due to the efforts of the loth International Histocompatibility Workshop, analysis of restriction fragment length polymorphisms of the HLA-DQ genes have been standardized and

linked to serological and cellular forms of HLA typing. In our study of HLA class II RFLPs a panel of 198 Type I diabetics and 3o5 Caucasian controls

without any personal or familiy history of an autoimmune disease, were analyzed with HLA-DQ probes. A HLA-DR4,-DR5 and -DR7 linked DQB split, characterized by the presence of a lo.6kb BamBI fragment, was linked to the disease. This HLA-DQB split, denoted HLA-DQ?3.2, gives a relative risk of 2.2 for the disease. This DQB specific fragment was also found in 44.3% of the controls compared to 61.8% of the IDDM patients. This split was more prevalent with 84.5% of HLA-DR4 positive diabetics and reduced with 61.3% in HLA-DR4 positive controls. In addition, a HLA-DQB split, denoted HLA-DQBI.I, was associated with the disease and describes a relative risk of 2.4 for the disease. Also HLA-DQalpha RFLPs were studied. In this part of our work we applied a nonisotopic technique in using a M13 HLA-DQalpha probe. There is positive evidence that several HLA-DQB and DQalpha combinations are more often found in diabetics than expected by chance alone. A 2.2 kb TaqI DXalpha fragment was more prevalent in HLA-DR3 positive diabetics than in HLA-DR3 positive controls. Also certain DXalpha and DQalpha RFLPs were linked to the HLA-DQB3.2 split. Our results of diabetes linked RFLPs are of considerable interest, since the RFLPs reported herein are all linked to epitopes that are recognized by T-cell clones. Supported by a grant from Deutsche Forschungsgemeinschaft:RO694/l-1 and B0829/I-I.

POS-001-223 DISTINCT BLA-DR52 ALLELES ARE LINKED TO TYPE I (INSULIN-DEPENDENT) DIABETES IN BLA-DE3 BOMOZYGOUS TYPING INDIVIDUALS

B.O. BOEHM, P. KUEHNL AND K. SCHOFFLING University Hospital of Frankfurt Medical School, Frankfurt am Main, U.-Germany

HLA-DR52 (formerly called MT2) is an Ia alloantigen that is expressed in linkage aith BLA-DRS, -.6 and -.8. Since HLA-DR3 is linked to type I diabetes mellitus and since the

adjacent BLA-DR52 gene has recently been split into different subtypes, we have studied a panel of HLA-DR3 homozygoue typing individuals in order to define diabetes-linked NLA-DR52 alleles. Re applied the technique of RFLP analysis and used in addition allele specific oligonueleo- tide probes in order to analyze the panel of DR3 homozygotes (n=42). BamBI and TaqI digested genomic DNAs reveal DR52 splits ehen probed Hith a DR8 specific eDNA

probe. These splits were also seen with a HLA-DR52 specific oligonucleotide. Polymorphic TaqI fragments of 8. okb and 7. Tkb, each corresponding to BIII sequences, were shown to differ in their prevalence in type I diabetics, 6 p~eude-type II diabetics when compared to Caucasian controls and a panel of DR3 homozygous B-lymphoblaetoid cell lines. The 7.7kb TaqI fragment which corresponds to a DR52 a subtype was found with a gene frequency of o. 313 in diabetics and in 0.5 of the controls. Gene frequency of the DR52b allele was 0.688 in diabetics and 0.5 in the controls.

Our data provide evidence that a NLA-DB~ specific polymorphism ,hich maps to the BLA-DR52 gene region is associated with type I diabetes and pseudo-type II diabetes.

This work was supported by a grant from Deutsche Forschungsgemeinschaft, grant B0829/I-I.

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POS-001-224 PERINATAL AND GENETIC PREDICTORS OF AGE AT IDDM ONSET IN CHILDHOOD

A DRASH, M REWERS, L 0'LEARY, M WALCZAK, R LAPORTE

University of Pittsburgh, Pittsburgh, PA USA and Academy of Medicine, Poznan, Poland

The distribution of IDDM in children is bimodal with an early peak at ages 5-6 and a "puber tal" peak at ages 10-13 years. This may reflect a heterogeneity of the disease rather than

action of the same factor(s) at different stages of child development. This study evaluated the perinatal and genetic factors which discriminated early onset cases from adolescent in two diverse populations.

The study group consisted of 1906 Caucasian insulin-dependent diabetic children diagnosed in the Children's Hospital of Pittsburgh, USA (n=1355) and in the Institute of Pediatrics in Poznan, Poland (n=551), during 1965-86. The age distribution of IDDM incidence was compared in population-based samples from the two areas by Poisson regression modelling and was found identical, despite a 4-fold difference in IDDM incidence between the USA and Poland. In both series pooled, 839 and 1067 of the patients were diagnosed, respectively, at ages 3-7 and 9-13 years. Children of diabetic mothers (n=39) were excluded. Data regarding birth order, maternal age and sex were collected in 75% of each of the two age groups. The HLA-DR phenotype was determined in 335 (33%) of the Pittsburgh series.

In stepwlse logistic regression, low birth order (p < 0.003) and advanced maternal age (p < 0.02) were significant predictors of early IDDM onset in both countries. In the smaller Pittsburgh series, the HLA-DR4(+) status (including DR4/4, 4/3 and 4/x) was individually the strongest predictor of early onset (p < 0.001). The effect of birth order, adjusted for HLA-DR status, was borderline significant (p=0.088) and both maternal age and sex effects were negligible.

The results suggest that there are two distinct subsets of diabetic children; those

diagnosed at the pre-school age tend to be first born from older mothers and carry the HLA-DR4 gene.

POS-001-225 POSSIBLE LOCATION OF DIABETOGENIC MHC GENES USING PULSE FIELD

ELECTROPHORESIS

R L DAWKINS

Departments of Clinical Immunology, Royal Perth Hospital, Queen Elizabeth II Medical Centre, Perth, Western Australia.

For many years it has been known that the major histocompatibility complex (MHC) contains genes which influence susceptibility to insulin dependent diabetes mellitus (IDDM). Several MHC supratypes mark these genes and have now been compared in detail using pulse field gel electrophoresis (PHGE).

Two diabetogenic supratypes (1,8,3 and 18,FI,3) are quite different serologically but each has four extensive deletions between HLAB and DQ. Although the deletions differ in length, they do overlap and could result in the loss of the same genes.

A comparison of diabetogenic supratypes in different racial groups suggest that several MHC genes may be involved and that these may be located between HLAB and DQ. Similarly, in the BB rat, there is evidence for involvement of Class II and Class III genes.

Taken together, these data suggest that susceptibility to IDDM may be partly due to the lack of a gene which is yet to be characterised in terms of function and product. (8801)

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POS-001-226 AGE RELATED HETEROGENEITY OF IDDM IN CHILDREN ASSOCIATED WITH TWO DISTINCT HLA-DR3 HAPLOTYPES

BEARING COMPLEMENT FACTOR C4 A, B NULL ALLELES.

I. DESCHAMPS, A. MARCELLI-BARGE, J.C. POIRIER, H. LESTRADET and J. HORS.

HOPITAL HEROLD and HOPITAL St LOUIS, PARIS, FRANCE.

Heterogeneity between two DR3 haplotypes in linkage disequilibrium with B8 and BI8, respecti- vely, with regard to age-at-onset of type I (insulin-dependent) diabetes was investigated in 325 unrelated French patients (146 males, 179 females, age-at-onset before 29 yr) who were HLA-genotyped (225 patients C4 A, B type~). The B8, DR3 haplotype, its smaller segment C4AQOBI, DR3 or C4AQO alone were significantly associated with age-at-onset after 6 years (p < 0,02) ; the BI8, DR3 haplotype, C4A3BQO, DR3 and C4BQO alone were significantly more frequent in patients with onset before 6 years compared with older patients (p < 0,01). These age distributions were sex dependent. Analysis off DR~ and DQ~ restriction fragment polymorphisms showed distinct patterns which correlated with DR3 and/or DQW2 borne by the B8 and the BI8 haplotypes, respectively. These results provide evidence that a heterogeneous background could account for differences in the clinical expression of IDDM.

POS-001-227 CLINICAL CHARACTERISTICS OF DIABETIC PATIENTS HAVING A VERY STRONG HEREDITARY TENDENCY

H KIKUOKA, K NANJO*, M NISHI*, R SOWA*, M MIYANO*, K KUBO, H TABATA, K MACHIDA, M KONDO*, T SANKE*, K MIYAMURA Dept of Med, Hidaka General Hospital, The I Dept of Med, Wakayama Univ of Med Sei~ Wakayama JAPAN

We found two family lines having a very strong hereditary tendency to develop diabetes mellitus (DM) in which three generations were affected, and investigated the clinical characteristics of diabetic patients in these pedigrees (F-DM). Twenty-one patients with F-DM in the two pedigrees, who requi~ed insulin therapy, were classified into generation II and III. The clinical charac- teristics of each generation were compared with those of 58 patients with insulin-requiring non- insulin dependent DM (NIDDM) without a family history (NF-DM) and 9 patients with typical insulin- dependent DM (IDDM). F-DM in generation II was similar to NF-DM in the age of onset, the insulin dose required to obtain a good control and the interval between the onset and the initiation of insulin therapy. On the other hand, F-DM in generation III was similar to IDDM in all of these parameters: The age of onset was younger, the required dose of insulin was larger, and the inter- val between the onset and the initiation of insulin therapy was shorter, all with a significant difference from generation II. In the 75-g oral glucose tolerance test, generation III showed markedly decreased with generation II, despite the shorter duration of illness. In F-DM, no one gave positive reactions to ICA or ICSA either in generation II or in generation III. With regard to HLA typing, the frequency of DR 4 was similar to that in the Japanese in the mass, while the frequency of Bw 54 tended to be slightly higher. It has been considered that in NIDDM a heredi- tary predisposition is an underlying risk and the addition of various environmental factors results in onset of the disease. On the other hand, in IDDM, autoimmune mechanism, viral infection and HLA have been cited as precipitating factors. The present study has revealed that in the family line of NIDDM with a very strong hereditary tendency as in the pedigrees shown here, the clinical characteristics of DM become similar to those of IDDM as generation goes by, without the involvement of the above mentioned factors.

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POS-001-228 OCCURRENCE OF INSULIN-DEPENDENT DIABETES MELLITUS(IDDM), RHEUMATOID ARTHRITIS(RA) AND CRYPTO-

MERIA POLLINOSIS(CP) IN A SINGLE SUBJECT AND INTERFERENCE OF THESE HLA-DR4 RELATED DISEASES.

H NAKANE, Y NAKANE, K TAKAHASHI, T KATAYAMA, H KATO.

Dpt of Medicine, Urawa City Hospital, Urawa, Saitama, JAPAN.

Allergic oculo-rhinitis due to pollen of cryptomeria japonica is seen widely and exclusively in

Japan, and its genetic predisposition has recently been demonstrated on HLA-DR4 locus. The

pathogenesis of CP is attributed to the defect of immunity-related gene which normally sup-

presses the antibody formation against cryptomeria pollen. In Japan, the same HLA haplotype

is incidentally closely related to the susceptibility to IDDM and autoimmune diseases including

RA. The linkage of these three classes of disease has not yet been reported. We found the

occurrence of IDDM, RA and CP in a 27-year-old woman with HLA-DR4 haplotype and investigated

their interrelationship during three years of follow up. The remission and exacerbation of RA

and CP were observed under minimal dosage of non-steroidal, non-cathecholamine drugs. A quite

similar seasonal variation was observed between IDDM and CP, but not between IDDM and RA or

between CP and RA. Furthermore, the study on HLA type of the patient's relatives revealed the

same HLA haplotype and the presence of autoimmune diseases, though neithr IDDM nor CP was dis-

covered. The occurrence of IDDM in the subject who only has the gene setting for both CP and

RA and the close symptomatic relationship between IDDM and CP may indicate that genetic pre-

disposition of IDDM is closer to CP than to RA.

POS-001-229 ~TURITY ONSET DLABETES ~LLITUS IN AN ADOLESCENT DIABETIC CLINIC

R. S~rez, D. ~chado, O. ~teo de Acosta National Institute of ~ndocrinology, havana, Cuba.

We studied twenty-three (23) patients with ~,~turity Onset Diabetes Mellitus (MODY), classified following Tattersall's criteria, from the Adolescent clinic of the Na-

tional Institute of Endocrinology. The frequency found for MODY in a total of 1275 patients attending this service was 1.8%. We also found a strong family history of Diabetes ~ellitus, inasmuch as all patients had a relative with this condition, su~ gesting a possible dominant autosomic inheritance, with 14 parents being affected (60.9%), and the disease manifested along three consecutive generations in 21.7% of all cases. The insulin response to a carbohydrate load was very hetereogeneous.

Obesity was not predominant in this group. Three patients showed lipid alterations

conformed by Type lib }~yperlipoproteinemia in one, and by Type IV in the other. Four patients presented high levels of microalbuminuria in three samples during the same month and without evident renal sepsis, facts interpreted as a possible early kidney lesion (diabetic microangiopathy). One patient had autonomic neuropathy due to a neurogenic bladder, it was demonstrated that Chlorpropamide-Alcohcl-Flushing in this type of non-insulin Diabetes Mellitus in the Young results useful as a pos- sible genetic marker and prognostic index for diabetic microangiopathy. No rela- tion was found between the Histocompatibility Antigen System HLA and body. The re- sults of the study on islet-cell antibodies (ICA) in our patients did not differ from those reported in the medical literature, with a low frequency of ICA. Accord- ing to our results dermatoglyphs may result useful as possible genetic markers in this group of patients.

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POS-001-230 HLA GENETIC DIFFERENCES BETWEEN TYPE 1 DIABETIC FAMILIES IN FINLAND AND ENGLAND

E. WOLF, J. TUOMILEHTO, R. LOUNAMAA AND THE DiMe STUDY GROUP National Public Health Institute, Helsinki, Finland

HLA data are compared between 170 consecutive families with a newly diagnosed Type I diabetic child participating in a recent nationwide study done in Finland and 148 English families recruited to the Bart's Windsor family study before July 1983. The mean age of onset (8.4 yrs vs 9.0 yrs), the proportion of male probands (53 % vs 55 %) and the proportion of simplex (87 % vs 91%) families were not different between the 2 studies.237 Finnish diabetic haplo- types were compared with 282 English diabetic haplotypes, and 214 "healthy" Finnish haplotypes with 256 "healthy" English haplotypes. 70 % of the Finnish and 78 % of the English diabetic haplotypes carried DR3 or DR4, whereas only 34 % of the Finnish and 36 % of the English "healthy" haplotypes did. The HLA-A, B, C, DR antigen combinations and their strength of disequilibrium measured by delta values was different on diabetic and "healthy" haplotypes in both studles.The frequency of DR3 was lower on the Finnish than the English diabetic haplotypes (20.3 % vs 37.2 %). This was compensated by stronger linkage disequilibrium between B8 and DR3 in the Finnish diabetic haplotypes. The delta values between Bw62 and DR4 were only significant for the diabetic (78.8) and not for the "healthy" (19.3) haplotypes in the English study, whereas the delta values were significant for both diabetic and "healthy" haplotypes in the Finnish study. This indicates that not a single HLA locus is important, but the whole HLA haplotype.

POS-001-231 THE HUMAN G~U(X)SE TRANSPORTER GENE: USE OF DNA POLYMDRPHISMTO EVALUATE ITS ROLE IN NIDDM

K KAIOJ, M M~ER, M PER~ Metabolism Division and Cell Biology, Washington University School of Medicine, St. Louis, ME), USA

To evaluate the role o£ inherited defects o£ the glucose transporter gene (GT) in the etiology o£ NIDDM, restriction fragment length polymorphisms (RFLPs) were analyzed in DNA from American Blacks. DNA was digested with a total of 14 enzymes and hybridized with various portions o£ the human erythrocyte Gr cDNA and genomic DNA probes. Three RLFPs were identified; one was detected with a 2.5 kb probe of genomic DNA in Taq 1 digests and the others were observed with a 1.6 kb probe of cDNA in Pst I and Eco RI digests. Allelic frequencies for Taq I and Pst I sites were:

Taq 1 Pat I ÷ - - n @ - - B

C o n t r o l s 49 9 58 4 56 60 (0.84) (0.16) (0.07) (0.93)

N ~ 89 3 92 3 95 98 (0.97) (0.03) (0.03) (0.97)

The ailelie frequencies for the Taq I site differed between NIDI~ and control subjects (X 2= 5.83 with Yates correction, p=0.016); while there was no difference in allelic frequency for the Pst I site. The presence or absence of polymorphism at the two sites defined haplotypes; this analysis revealed no linkage disequilibrium between the sites. Our studies suggest that the Taq I RFLP at the GT locus may be a genetic marker for NIDDM in American Blacks. Further analysis including Eco RI may define specific defective alleles within a subset of NIDOM.

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POS-001-232 SEX LINKAGE IN INHERITANCE OF TYPE 2 DIABETES MELLITUS

M. ATAHARUL ISLAM, A.K. AZAD KHAN, HAJERA MAHTAB, M. IBRAHIM

Department of Statistics, Bangladesh Institute of Diabetes, Endocrine and Metabolic Disorders (BIRDEM), Dhaka, Bangladesh

Genetic factors play an important role in the aetiology of type 2 diabetes mellitus (DM).

The exact nature of the transmission is unknown. This study shows a sex linkage.

All newly registered Type 2 DM patients from 1979 to 1983 were studied. Out of a total of

16145 subjects 12,470 could tell about the family history related to the status of father's DM whereas 13,309 knew about the family history related to mother's DM. It was found that

for every i00 diabetic sons to diabetic fathers there were 61 diabetic daughters. Similarly for every i00 diabetic sons to non diabetic fathers there were 45 diabetic daughters. This

shows that the risk of transmission of diabetes mellitus to daughters increases 1.35 times, as compared to the risk of transmission to sons, for diabetic fathers than that for non diabe- tic fathers (P<0.001). For every i00 diabetic sons to diabetic mothers there were 81 diabetic

daughters whereas for every i00 diabetic sons to non diabetic mothers there were 43 diabetic daughters. This indicates that the risk of having diabetic daughters increases 2.0 times,

as compared to the risk of having diabetic sons, for diabetic mothers than that for non diabe- tic mothers (P~0.001). Furthermore, the difference between the odds ratio for father and

mother is also found to be significant at the same level.

The present findings on the pattern of sex linkage may be helpful for investigating the nature

of transmission of the disease.

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