Physical and genetic mapping of IDDM8 on chromosome 6q27.

Genome-wide mapping studies have provided evidence of a type 1 diabetes susceptibility gene (IDDM8) that is located on chromosome 6q27. However, association studies of IDDM8 have so far been negative. The purpose of this investigation was to determine a linkage disequilibrium (LD) map in the chromosome 6q27 region and to better localize IDDM8. A physical map of nearly 1 Mb containing the chromosome 6 telomere was constructed, and polymorphic markers spanning this region were defined. Haplotypes composed of the markers in LD were tested for association with type 1 diabetes in 266 families. A microsatellite marker allele and multiple haplotypes were associated with IDDM8, which suggests localization of this type 1 diabetes susceptibility gene to the terminal 200 kb of chromosome 6.

Linkage analyses in type I diabetes mellitus using CASPAR, a software and statistical program for conditional analysis of polygenic diseases.

We have developed software and statistical tools for linkage analysis of polygenic diseases. We use type I diabetes mellitus (insulin-dependent diabetes mellitus, IDDM) as our model system. Two susceptibility loci (IDDM1 on 6p21 and IDDM2 on 11p15) are well established, and recent genome searches suggest the existence of other susceptibility loci. We have implemented CASPAR, a software tool that makes it possible to test for linkage quickly and efficiently using multiple polymorphic DNA markers simultaneously in nuclear families consisting of two unaffected parents and a pair of affected siblings (ASP). We use a simulation-based method to determine whether lod scores from a collection of ASP tests are significant. We test our new software and statistical tools to assess linkage of IDDM5 and IDDM7 conditioned on analyses with 1 or 2 other unlinked type I diabetes susceptibility loci. The results from the CASPAR analysis suggest that conditioning of IDDM5 on IDDM1 and IDDM4, and of IDDM7 on IDDM1 and IDDM2 provides significant benefits for the genetic analysis of polygenic loci.

Analysis of candidate genes for susceptibility to type I diabetes: a case-control and family-association study of genes on chromosome 2q31-35.

Recent genome searches suggest a putative linkage of many loci to susceptibility to type I diabetes. The chromosome 2q31-35 region is reported to be linked to susceptibility to type I diabetes and is thought to contain several diabetes susceptibility loci. These candidate genes include the HOXD gene cluster, BETA2, CTLA4, CD28, IGFBP2, and IGFBP5. Association studies in populations and families are required to confirm and/or identify the actual susceptibility loci. We hereby report several previously unknown DNA polymorphisms for HOXD8, BETA2, and IGFBP5, which we have used along with previously known polymorphisms of HOXD8 and CTLA4 to test whether these candidate loci are the susceptibility genes on chromosome 2q31-35. Using a case-control design with a subsequent family-association approach to confirm associations, we find no evidence that these candidate genes are associated with susceptibility to type I diabetes.

The search for IDDM susceptibility genes: the next generation.

Two human chromosomal regions, the HLA region on chromosome 6p2l and the insulin gene region on chromosome 11p15, have been investigated in detail for more than 10 years for the presence of IDDM susceptibility genes. Recent genome searches indicate the possible existence of many additional susceptibility genes in IDDM. The lengthy and protracted studies to prove the linkage and identity of the susceptibility genes in the HLA and insulin gene regions provide a perspective and background for understanding the complexities and time course for characterization of the putative additional IDDM susceptibility genes uncovered by genome searches.

Molecular abnormalities of the 21-hydroxylase gene in hyperandrogenic women with an exaggerated 17-hydroxyprogesterone response to short-term adrenal stimulation.

OBJECTIVE: Our purpose was to establish the incidence of point mutations of the 21-hydroxylase gene (CYP21) in hyperandrogenic women with and without a 17-hydroxyprogesterone response to corticotropin stimulation above normal but below those levels associated with nonclassic adrenal hyperplasia. STUDY DESIGN: We studied 22 patients with hirsutism or hyperandrogenic oligoovulation: eight with an exaggerated net increase in 17-hydroxyprogesterone (i.e., change in 17-hydroxyprogesterone between 8.8 and 36 nmol/L) and 14 with a normal change in 17-hydroxyprogesterone. Large deletions of the 21-hydroxylase gene were evaluated by laser densitometry. Point mutations were detected with the polymerase chain reaction and dot blot hybridization analysis and included 30 Leu, intron-2 (G), 8 bp deletion exon-3, 172 Asn, 236-237-239 exon-6, 281 Leu, 318 stop, 339 His, 341 Trp, 356 Trp, and 453 Ser. RESULTS: Four patients with an increase in 17-hydroxyprogesterone carried a 281 Leu mutation, one patient had an intron-2 (G) mutation, and one had a complete deletion of CYP21. Only two of these patients demonstrated no obvious abnormality of CYP21. In contrast, only one of the control patients demonstrated a CYP21 abnormality, a significant difference (p < 0.001). CONCLUSIONS: These findings suggest that the majority of hyperandrogenic women with an exaggerated 17-hydroxyprogesterone response to corticotropin stimulation are heterozygotes (carriers) for inherited defects of CYP21. Whether these mutations are incidental to the androgen excess or predispose to the development of this disorder remains to be determined.

The HOXD8 locus (2q31) is linked to type I diabetes. Interaction with chromosome 6 and 11 disease susceptibility genes.

Type I diabetes susceptibility genes have been identified within the major histocompatibility complex (MHC) on chromosome 6p21.3 and near the VNTR/insulin region on chromosome 11p15.5. We have used polymorphic dinucleotide repeat markers to search the human genome for additional susceptibility genes in 162 type I diabetic families with an affected sibling pair. We report that an additional susceptibility gene is located on chromosome 2q31 near HOXD8 (P < 10(-5), maximum logarithm of odds score = 4.8) in an analysis of affected sibling pairs having specific human leukocyte antigen (HLA) and hypervariable nucleotide tandem repeat (VNTR)/insulin gene haplotypes (absence of high-risk HLA-DR3/4 haplotypes and presence of homozygous high-risk class I VNTR alleles). These results suggest the interaction of a minimum of three genes in the pathogenesis of type I diabetes in humans.

Linkage of the VNTR/insulin-gene and type I diabetes mellitus: increased gene sharing in affected sibling pairs.

Ninety-six multiplex type I diabetic families were typed at the 5' flanking region of the insulin gene by using a PCR assay that better resolves the VNTR into multiple alleles. Affected sibling pairs shared 2, 1, and 0 VNTR alleles--identical by descent--at a frequency of .47, .45, and .08, respectively, a ratio that deviated from the expected 1:2:1 ratio (P < .001). These results confirm linkage of the chromosome 11p15.5 region with type I diabetes mellitus susceptibility.

Localization of a type I diabetes susceptibility locus to the variable tandem repeat region flanking the insulin gene.

A susceptibility gene for type I diabetes is present on chromosome 11p15.5, but its location, identity, and mechanism of action are unknown. We have sequenced 14 kilobases of DNA flanking the human insulin gene and found new DNA polymorphisms and determined their frequencies in the general population and in families of type I diabetic subjects. A DNA polymorphism located 3123 base pairs downstream from the initiation site of transcription of the insulin gene, when present in the homozygous state, provides a relative risk for type I diabetes of 5.2 (P = 0.006). However, this DNA polymorphism as well as other diabetes-associated 3' markers are in linkage-disequilibrium with the actual susceptibility region, because these polymorphisms are found on haplotypes both positively and negatively associated with type I diabetes susceptibility. Nucleotide sequence analysis of the variable tandem repeat region flanking the 5' end of the insulin gene shows variable tandem repeat elements associated with these haplotypes to differ greatly in composition, i.e., an ATAGGGGTGTGGGG repeat element is absent on a haplotype associated with type I diabetes susceptibility, but is found in 6-10 copies on two haplotypes negatively associated with the disease. These findings suggest that the type I diabetes susceptibility locus on chromosome 11p15.5 is probably located in the 5' variable tandem repeat region rather than in the 3' region of the insulin gene.

DNA analysis of HLA-DR, DQ, and DP alleles in children with polyarticular juvenile rheumatoid arthritis.

HLA-DR, DQ and DP alleles were determined by restriction fragment length polymorphism analysis and oligonucleotide probe hybridization of polymerase chain reaction amplified genomic DNA in 94 Caucasian children with polyarticular juvenile rheumatoid arthritis (JRA) [13 rheumatoid factor (RF)+ and 81 RF-] and 100 healthy controls. HLA-DRw8, DQw4, DQA1*0401, DQB1*0402 were increased in frequency in those patients with RF seronegative disease, with highest frequencies seen in patients with young age at onset (< 5 years of age). These findings were similar to what we observed in children with pauciarticular JRA, especially those with young age at onset. DPB1*0301 was also found in increased frequency in the RF- group, and in particular those seronegative for antinuclear antibody. In contrast to what is observed in patients with pauciarticular JRA, the frequency of DPB1*0201 was not increased in any polyarticular JRA patient group. These data suggest that polyarticular JRA shares many genetic features with pauciarticular JRA.

Pro-453 to Ser mutation in CYP21 is associated with nonclassic steroid 21-hydroxylase deficiency.

Steroid 21-hydroxylase deficiency is the leading cause of impaired cortisol synthesis in congenital adrenal hyperplasia (CAH), with the nonclassic form (NC) comprising approximately 1% of the Caucasian population. The structure of the CYP21 gene was studied in 13 unrelated NC-CAH patients, three affected siblings, and 55 blood donors using polymerase chain reaction. In addition to the Leu-281 and Leu-30 mutations previously associated with NC-CAH, the finding of a Pro-453 to Ser mutation in exon-10 of CYP21 in the NC-CAH patients is reported. Ser-453 was found in 46.2% of unrelated NC-CAH patients, but only 7.7% and 3.6% of salt-wasting CAH patients and blood donors, respectively. In contrast to the Leu-281 and Leu-30 mutations, Ser-453 has not been previously detected in the CYP21 pseudogene (CYP21P) and, therefore, has not likely arisen by gene conversion.

Association of polar amino acids at position 26 of the HLA-DQB1 first domain with the anticentromere autoantibody response in systemic sclerosis (scleroderma).

HLA class II alleles (detected by DNA typing) were determined in 116 Caucasians with systemic sclerosis positive and negative for anticentromere autoantibodies (ACA). Significantly increased frequencies of HLA-DR5(DRw11) (P = 0.009) and the Dw13(DRB1*0403, *0407) subtypes of DR4 (probability corrected, Pc = 0.005) were seen in ACA positive patients, and HLA-DR1 and DRw8 were also increased. These findings appeared to reflect linkage disequilibrium of DR5(DRw11) and many DR4(Dw13) haplotypes with HLA-DQw7 and DR1 with DQw5. In fact, the presence of a DQB1 allele having a polar glycine or tyrosine at position 26 of the DQB1 first domain versus a hydrophobic leucine accounted for 100% of ACA positive Caucasian systemic sclerosis patients compared to 69% of the ACA negative SS patients (P = 0.0008) and 71% of Caucasian controls (P = 0.0003) as well as all 7 ACA patients of non-Caucasian background. Furthermore, the genotype frequency of DQB1 alleles lacking leucine at position 26 was 73% in ACA positive SS patients, compared to 42% of ACA negative patients (P = 1.2 x 10(-5)) and 38% of controls (P = 5.8 x 10(-7)). These data, then, suggest that the second hypervariable region of the HLA-DQB1 chain may form the candidate epitope associated with the ACA response.

Prenatal diagnosis of 21-hydroxylase deficiency congenital adrenal hyperplasia using the polymerase chain reaction.

We present an improved method for the prenatal diagnosis of congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency. The polymerase chain reaction (PCR) was used to analyze DNA from an affected index case, the parents, and a cultured chorionic villus sample, for point mutations in the steroid 21-hydroxylase (CYP21) gene. We can predict that the fetus is an unaffected carrier.

Salt-wasting congenital adrenal hyperplasia: detection and characterization of mutations in the steroid 21-hydroxylase gene, CYP21, using the polymerase chain reaction.

We have characterized mutations in the steroid 21-hydroxylase gene (CYP21) in salt-wasting congenital adrenal hyperplasia (SW-CAH) subjects, healthy control subjects, and affected sibling pairs with SW-CAH. To identify point mutations in CYP21, we have used an improved polymerase chain reaction methodology that allows analysis of the entire CYP21 gene. In addition, we have used polymerase chain reaction to search for abnormally spliced mRNAs resulting from putatively abnormal CYP21 genes transfected into COS1 cells. We found that all 26 SW-CAH subjects from whom DNA could be completely analyzed, had mutations that could account for the 21-hydroxylase enzyme deficiency. These mutations included CYP21 gene deletion, conversion to the inactive CYP21P form, point mutations leading to amino acid substitutions or stop codons, small gene deletions, and a point mutation in intron-2 that leads to an abnormally spliced mRNA. The point mutation in intron-2 was directly shown to activate a cryptic splice site 19 basepairs from exon-3 of CYP21 and thereby cause a reading frame mutation. This CYP21 mutation was frequently found in our white SW-CAH subjects, while the frequency of this mutation was extremely low in a racially matched control population. Furthermore, affected sibling pairs shared this mutation in all cases examined. The results presented should have important applications for the prenatal diagnosis of CAH.

DNA analysis of HLA-DR, DQ and DP genes in pauciarticular juvenile rheumatoid arthritis.

HLA-DR, DQ, and DP alleles were determined by restriction fragment length polymorphism (RFLP) and oligonucleotide hybridization analysis in 50 Caucasian children with pauciarticular juvenile rheumatoid arthritis (PaJRA) and 82 controls. There was an increased frequency of DR5, DRw8, and DQw4, as well as individual DQ alpha and beta chains, DQA*0401 and DQB1*0402, respectively, in this group of patients. There was an absolute association between DRw8, DQw4, DQA1*0401, and DQB1*0402 in the patient population. HLA-DPw2.1 was also increased in frequency. There was little evidence of linkage disequilibrium found between DPw2.1 and DR5, DRw8, or DQw4. These MHC Class II associations were more characteristic of those patients with young age of onset (less than 5 years), rather than those with onset greater than or equal to 5 years of age. Our data confirmed the previous associations of HLA-DR5, DRw8, and DPw2.1 with PaJRA and suggested a new association for DQ alpha and beta genes in the clinical expression of this disease.

Multigenic basis for type I diabetes. Association of HRAS1 polymorphism with HLA-DR3, DQw2/DR4, DQw8.

We analyzed extended haplotypes composed of DNA loci on the short arm of chromosome 11 for segregation with insulin-dependent (type I) diabetes mellitus. The markers for these loci are tyrosine hydroxylase, insulin, and c-Ha-ras-1 proto-oncogene (HRAS1). We report, in a study of 27 families, that a specific haplotype (H), containing a 3-kilobase (kb) HRAS1-Taq I DNA polymorphism, segregated differentially in diabetic and nondiabetic siblings (P = 0.005). A parallel population study showed that the 3-kb HRAS1-Taq I polymorphism is increased in frequency in type I patients having two strong HLA-susceptibility haplotypes compared with other type I patients or healthy control blood donors (P less than 0.010 and P less than 0.025, respectively). The polymorphic variable, enhancer, and promoter regions flanking the human insulin gene on the H haplotype were not associated with type I diabetes. These results indicate that the HRAS1 locus or genes in linkage disequilibrium with this locus are involved in the pathogenesis of HLA-DR3/4 type I diabetes mellitus.

Direct analysis of CYP21B genes in 21-hydroxylase deficiency using polymerase chain reaction amplification.

Steroid 21-hydroxylase deficiency is the leading cause of impaired cortisol synthesis in congenital adrenal hyperplasia (CAH). We have studied the structure of the CYP21B gene in 30 unrelated CAH patients using the polymerase chain reaction (PCR) to differentiate the active CYP21B gene from its highly related CYP21A pseudogene. The PCR approach obviates the need to distinguish the CYP21A and CYP21B genes by restriction endonuclease digestion and electrophoresis before analysis with labeled probes. Furthermore, direct nucleotide sequence analysis of CYP21B genes is demonstrated on the PCR-amplified DNA. Gene deletion of CYP21B, gene conversion of the entire CYP21B gene to CYP21A, frame shift mutations in exon 3, an intron 2 mutation that causes abnormal RNA splicing, and a mutation leading to a stop codon in exon 8 appear to be the major abnormalities of the CYP21B gene in our patients. These mutations appear to account for 21-hydroxylase deficiency in 22 of 26 of our salt-wasting CAH patients.

Primary association of HLA-DQw8 with type I diabetes in DR4 patients.

DNA from 164 Caucasian type I (insulin-dependent) diabetic patients and 200 Caucasian nondiabetic control blood donors were analyzed by the polymerase chain reaction technique for HLA-DR4 and the associated Dw and DQB subtypes of DR4. The DQw8 subtype of HLA-DR4 was associated with type I diabetes in all DR4 subgroups (DR4/3 and DR4/non-3). Dw subtypes of DR4 other than DW10 did not confer additional association with type I diabetes. Thus, the DQ region appears to provide the primary major histocompatibility association with type I diabetes in most DR4 patients.

Oligonucleotide probes for HLA-DQA and DQB genes define susceptibility to type 1 (insulin-dependent) diabetes mellitus.

We have typed 27 Caucasoid families for DNA restriction fragment length polymorphisms and specific sequences using HLA class II specific cDNA, genomic and oligonucleotide probes. DNA haplotypes were identified by restriction fragment length polymorphism analysis that correlated with previously serologically-defined extended major histocompatibility haplotypes. These DNA haplotypes sort into positive, neutral or negative associations with Type 1 (insulin-dependent) diabetes mellitus. The DNA susceptibility haplotypes are even more simply and specifically defined by oligonucleotide probes for sequences of DQA and DQB genes. Our oligonucleotide probes define variabilities in nucleotide sequences coding for amino acid residues 26, 37 and 38 in the DQ beta-chain. Probes defining DQA sequences are also important for defining susceptibility since certain DQA genes appear to modify DQB susceptibility by conferring resistance. Thus, major histocompatibility conferred susceptibility to diabetes cannot be adequately explained by an amino acid change at a single position in the DQ beta-chain. These probes allow the direct identification of major histocompatibility susceptibility genes in Type 1 diabetes without the necessity of determining full haplotypes.

Tissue-specific expression of transfected human insulin genes in pluripotent clonal rat insulinoma lines induced during passage in vivo.

The pluripotent rat islet tumor cell line MSL-G2 expresses primarily glucagon or cholecystokinin and not insulin in vitro but changes phenotype completely after prolonged in vivo cultivation to yield small-sized hypoglycemic tumors composed almost entirely of insulin-producing beta cells. When a genomic DNA fragment containing the coding and upstream regulatory regions of the human insulin gene was stably transfected into MSL-G2 cells no measurable amounts of insulin or insulin mRNA were detected in vitro. However, successive transplantation of two transfected clones resulted in hypoglycemic tumors that efficiently coexpressed human and rat insulin as determined by human C-peptide-specific immunoreagents. These results demonstrate that cis-acting tissue-specific insulin gene enhancer elements are conserved between rat and human insulin genes. We propose that the in vivo differentiation of MSL-G2 cells and transfected subclones into insulin-producing cells reflects processes of natural beta-cell ontogeny leading to insulin gene expression.