Vincent McCann

Ancestral haplotypes reveal the role of the central MHC in the immunogenetics of IDDM

The major histocompatibility complex (MHC) contains multiple and diverse genes which may be relevant to the induction adn regulation of autoimmune responses in insulin dependent diabetes mellitus (IDDM). In addition to HLA class I and II, the possible candidates include TNF, C4, and several other poorly defined polymorphic genes in the central MHC region. This study describes two approaches which take advantage of the fact that the relevant genes are carried by highly conserved ancestral haplotypes such as 8.1 (HLA-B8, TNFS, C4AQO, C4B1, DR3, DQ2). First, three “diabetogenic” haplotypes (two Caucasoid and one Mongoloid) have been compared and it has been shown that all three share a rare allele of BAT3 as well as sharing DR3, DQ2. In 43 sequential patients with IDDM the cross product ration for BAT3S was 4.8 (p<0.01) and 6.9 for HLA-B8 plus BAT3S (p<0.001). Second, partial or recombinant ancestral haplotypes with either HLA class I (HLA-B8) or II (HLA-DR3, DQ2) alleles were identified. Third, using haplotypic polymorphisms such as the one in BAT3, we have shown that all the patients carrying recombinants of the 8.1 ancestral haplotype share the central region adjacent to HLA-B. These findings suggest that both HLA and non-HLA genes are involved in conferring susceptibility to IDDM, and that the region between HLA-B and BAT3 contains some of the relevant genes. By contrast, similar approaches suggest that protective genes map to the HLA class II region.

Clinical Criteria that Reflect C-Peptide Status in Idiopathic Diabetes

Mako, M. E., Reynolds, C., and Molnar, G. D.: C-peptide suppression test for insulinoma. J. Lab. Clin. Med. 1977; 90:180-86. 5 Service, F. J., Rubenstein, A. H., and Horwitz, D. L: Cpeptide analysis in diagnosis of factitial hypoglycemia in an insulindependent diabetic. Mayo Clin. Proc. 1975; 50:697-701. 4 Service, F. J., and Palumbo, P. J.: Factitial hypoglycemia— three cases diagnosed on the basis of insulin antibodies. Arch. Intern. Med. 1974; 134:336-40. 5 Scarlett, J. A., Mako, M. L , Rubenstein, A. H., Blix, P. M., Goldman, J., Horwitz, D. L , Tager, H., Jaspan, J. B., Stjernholm, M. R., and Olefsky, J. M.: Factitious hypoglycemia—diagnosis by measurement of serum C-peptide immunoreactivity and insulinbinding antibodies. N. Engl. J. Med. 1977; 297:1029-32. 6 Walfish, P. G., Kashyap, R. P., and Greenstein, S.: Sulfonylurea-induced factitious hypoglycemia in a non diabetic nurse. Can. Med. Assoc. J. 1975; 112:71-72. 7 Jordan, R. M., Kammer, H., and Riddle, M. R.: Sulfor.ylureainduced factitious hypoglycemia: a growing problem. Arch. Intern. Med. 1977; 137:390-93. 8 Ahlquist, D. A., Nelson, R. L., and Callaway, C. W.: Pseudoinsulinoma syndrome from inadvertent tolazamide ingestion. Ann. Intern. Med. 1980; 93:281-82.

Susceptibility to IDDM is marked by MHC supratypes rather than individual alleles.

Abstract107 patients with insulin-dependent diabetes mellitus (IDDM) were typed for HLA A, B, C, and DR antigens, and for complement C4A, C4B, and Bf alleles, and the results were compared with those of a combined reference group of 332 appropriately matched healthy subjects. Supratypes (allelic combinations) were identified from the phenotype of each group, and it was shown that the frequency of several supratypes is increased in patients with IDDM, in particular supratypes (A1 Cw7) B8 C4AQ0 C4B1 BfS DR3 (P = 0.0001), (A30 Cw-) B18 C4A3 C4BQ0 BfF1 DR3 (P = 0.0003), (A2 Cw3) B62 C4AR C4B2.9 BfS DR4 (P = 0.0002), and three other supratypes including DR4. It was also shown that increases in the frequency of individual alleles are secondary to increases in supratype frequency. Moreover, supratypes appeared to interact; the presence of two relevant supratypes being particularly important. The absolute risk of IDDM was approximately 0.5 in subjects who were homozygous for B18 C4A3 C4BQ0 BfF1 DR3. We concluded that genetic susceptibility is best recognized by MHC supratypes rather than isolated alleles, and that supratype combinations make the identification of even greater disease risk possible.

HLA and complement allotypes in Type 1 (insulin-dependent) diabetes.

SummaryA group of patients with Type 1 (insulin-dependent) diabetes mellitus was investigated for HLA-A, B and DR antigens as well as C4 and factor B polymorphism. A significant excess of DR3/DR4 heterozygotes was observed (27% versus 17% by Hardy-Weinberg expectation). The factor B allele BfF1 was present in 13% of patients with Type 1 diabetes (gene frequency of 0.08 versus 0.01 in control subjects). A rare C4 B allele, C4 B2.9, was found in 18% of patients with Type 1 diabetes (n=63) compared with 1.1% of control subjects (n=176). Total C4 deficiency at the C4A locus (C4AQ0,0) was present in 10% of patients with Type 1 diabetes compared with 0% of control subjects. Examination of HLA, C4 and Bf phenotypes in patients with Type 1 diabetes suggested that three high risk supratypes, HLA-A1 B8 BfS C4AQ0 C4 B1 DR3; HLA-B18 BfF1 C4A3 C4BQ0 DR3; HLA-A2 CW3 BW62 BfS C4A3 C4 B2.9 DR4 are markers for susceptibility alleles.

Localization of central MHC genes influencing type I diabetes

The contribution of MHC class II haplotypes to susceptibility to type I diabetes has been clearly established, and interest has now focused on the effects of additional genes in the MHC region. We have investigated the central MHC alleles on 8.1 ancestral haplotype (HLA-A1, B8, DR3, DQ2), as it is well conserved in Caucasian populations. The HLA-DR3-DQ2 genotype is a recognized risk factor for type I diabetes. Single nucleotide polymorphisms and microsatellites in the MHC were used to map segments of the 8.1 ancestral haplotype carried by type I diabetic and control subjects expressing either HLA-B8 or DR3, but not both these markers. In this way we controlled for the diabetogenic effect of carriage of DR3. Alleles of the 8.1 ancestral haplotype between TNFA-308/D6STNFa and HLA-B were carried with significantly greater frequency in B8(-), DR3(+) type I diabetic patients compared with B8(-), DR3(+) controls. This interval was marked by a BAT1 gene polymorphism and a MIB microsatellite allele.

Homocysteine, folate, methylene tetrahydrofolate reductase genotype and vascular morbidity in diabetic subjects.

In the present study, the determinants of fasting plasma homocysteine in diabetic subjects were examined; whether plasma homocysteine and vascular disease are related and the influence of the C677T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene on serum and erythrocyte folate, plasma homocysteine and vascular disease. Diabetic clinic subjects (Type I, n=354; Type II, n=392) were recalled for a cross-sectional survey. Standard methods were used to measure biochemical variables and to characterize vascular disease and MTHFR genotype. Plasma homocysteine was significantly and directly related to age, male sex and serum urea, and inversely related to serum folate and vitamin B12, independently in stepwise regression. When corrected for age and sex, homocysteine was significantly related to hard end points of coronary artery disease and stroke (each P<0.01), remaining significant when additionally adjusted for serum folate (P=0.043 and P=0.019 respectively). Serum folate was not clearly related to these events, although there was a trend to associate with the lower quintile of serum folate. The MTHFR genotype was not a determinant of plasma homocysteine, even in those in the lowest quintile of serum folate, nor of vascular disease. TT homozygosity at residue 677 was associated with elevation of total erythrocyte folate compared with both other genotypes (P<0.0001), almost certainly due to the diversion of 5,10-methylenetetrahydrofolate into derivates subsequent to the partial metabolic block that results from the MTHFR enzyme defect. In conclusion, in this clinic cohort of people with diabetes, vascular disease is related to plasma homocysteine, which is correlated with serum folate. The MTHFR genotype does not significantly influence either plasma homocysteine or vascular disease, despite it being a determinant of erythrocyte folate, which reflects its effect on folate metabolism.

Does a central MHC gene in linkage disequilibrium with HLA-DRB1 * 0401 affect susceptibility to type 1 diabetes?

Subtypes of HLA-DR4 are associated with susceptibility or protection against type 1 diabetes (T1DM). We addressed whether this reflects linkage disequilibrium with the true susceptibility locus by studying broader MHC haplotypes marked by alleles of HLA-B, IKBL (adjacent to TNFA) and complement C4. The study used a largely Caucasian cohort from Western Australia. HLA-DRB1*0401 and HLA-DRB1*0405 marked susceptibility to T1DM. In Caucasians, DRB1*0401 occurs predominantly in the 44.1 ancestral haplotype (AH; HLA-A2,B44, DRB1*0401,DQB1*0301) and the 62.1AH (HLA-A2,B15(62),DRB1*0401,DQB1*0302). HLA-B15 marked susceptibility and HLA-B44 marked with resistance to T1DM in patients and controls preselected for HLA-DRB1*0401. A gene between TNFA and HLA-B on the 8.1AH (HLA-A1,B8,;DR3,DQ2) modifies the effects of the class II alleles. Here, alleles characteristic of the 62.1AH (C4B3, IKBL+446*T and HLA-A2,B15) were screened in donors preselected for HLA-DRB1*0401. C4B3 was associated with diabetes, consistent with a diabetes gene telomeric of MHC class II. However, increases in carriage of IKBL+446*T and HLA-A2,B15 were marginal, as too few control subjects were available with the diabetogenic alleles. However, with these tools, selection of HLA-DRB1*0401, DQB1*0302 donors who are positive and negative for C4B3 will allow bidirectional mapping of diabetes genes in the central MHC.191 words

Pyridoxine and diabetic neuropathy: a double-blind controlled study.

Insulin Treatment and the Postreceptor Defect The article by Scarlett et al. demonstrating that insulin treatment may successfully reverse the postreceptor defect in type II diabetes is provocative. Although the patients in this study were mildly overweight (mean relative weight: 1.12 ± 0.8), they still required substantial amounts of insulin (mean dose of 110 ± 15 U/day) to overcome the insulin resistance so as to achieve a lower mean fasting blood glucose value (125 ± 13 mg/dl). In contrast to these results, clinical experience often demonstrates the failure of insulin therapy in type II diabetic individuals to reduce blood glucose levels despite the fact that the doses used may frequently be in a range similar to that employed in the present study. It is also commonly appreciated that the use of oral agents or insulin has the best chance of succeeding in the regulation of diabetes when the patient is compliant with a suitable diet and with weight loss when indicated. It is therefore conceivable that other factors may have contributed to the improvement in diabetes control observed by Scarlett et al. aside from insulin use alone. Although the study patients were placed on weight-maintaining diets and were exercised, qualitative differences in food intake before and after admission to a Clinical Research Center may have accounted for the enhanced blood glucose regulation. For example, a 45% carbohydrate diet consisting of complex rather than simple sugars could significantly have altered the nature of the blood glucose response. It is thus possible that the consumption of an isocaloric-controlled diet alone for 2 wk could have resulted in an improvement in the insulinresistant state and that treatment with insulin may have served to enhance these effects. If a qualitative dietary modification could be shown to modify the nature of the postreceptor defect, this would help in elucidating a common observation that type II diabetic patients frequently require much less insulin when hospitalized even for very brief periods in the absence of significant weight loss. It would also be of interest to discover if insulin would be equally successful in treating substantially more obese type II diabetic patients with presumably a greater degree of insulin resistance than in those subjects currently reported by Scarlett et al. Finally, outpatient follow-up studies are needed to investigate whether insulin therapy of type II diabetes ultimately results in progressive weight gain and loss of the shortterm benefits with the potential for further deterioration in diabetic control due to worsening of the insulin-resistant state.

Can MHC class II genes mediate resistance to type 1 diabetes

Numerous studies have associated carriage of HLA‐DRB1∗1501, DQA1∗0102 and DQB1∗0602 (DR15, DQ6) with dominant resistance to type 1 diabetes and have concluded that one or more of the component HLA class II molecules mediate this effect. Mechanisms for MHC class II‐mediated resistance to diabetes have been proposed from studies of transgenic mice, usually using the diabetes‐prone non‐obese diabetic (NOD) strain. However, these studies have not reached any consensus on a plausible mechanism. In this study we question why the role of central MHC genes in resistance to diabetes has not been addressed, as the central MHC carries markers of susceptibility to diabetes in linkage disequilibrium with several genes with known or putative immunoregulatory functions. To illustrate the type of studies required to address this issue, we selected diabetes patients and control subjects for carriage of HLA‐DR15 and the C allele at position +738 in the inhibitor of kappa B‐like gene (IKBL). These alleles mark the 7.1 haplotype (HLA‐A3, B7, IKBL738∗C, DR15, DQ6). HLA‐DR15 was the most effective marker of resistance, but an effect may be evident with IKBL738∗C in a larger study. Moreover, carriage of the entire haplotype was particularly rare in patients. The best explanation for this is that the critical gene lies between IKBL and HLA‐DRB1, and is more closely linked to HLA‐DRB1. Candidate genes at the centromeric end of the central MHC are reviewed, highlighting the need for further study.

HLA and complement genetic markers in diabetic retinopathy

Dear Sir, In a recent publication Dornan et al. [1] reported an association between HLA-DR4 and retinopathy in Type I (insulin-dependent) diabetic patients and concluded that genetically determined factors appeared to influence susceptibility to retinopathy. We have also examined the relationship between HLA and retinopathy in a smaller series of patients with Type 1 diabetes with onset before the age of 40years and duration > 10years. Patients were divided into three groups; those with no retinal changes, those with background retinopathy and those with severe retinopathy (proliferative changes or vascular changes requiring laser therapy). HLA antigens, properdin factor B(Bf) and complement C4 allotypes were determined in these patients. Table 1 shows the frequencies of HLA-B8, B15, DR3, DR4 and the complement variant C4B2.9 in the three patient groups examined. Although B 15 and DR4 are more frequent in patients with severe retinopathy, the differences were not statistically significant. The rare factor B allele BfFI was equally prevalent among the patients with and without retinopathy. On the other hand, the C4B allele C4B2.9 was present in 28% with severe retinopathy and only in 10% of patients with no retinopathy (NS). This variant is nearly always found on a DR4 haplotype and is also associated with rheumatoid arthritis.