Differential effects of DRB1*0301 and DQA1*0501-DQB1*0201 on the activation and progression of islet cell autoimmunity.

Autoimmune diabetes shows extreme variation in age of onset and clinical presentation, although most studies have been done in children with the most severe subtype. Disease risk is strongly associated with HLA-DRB1*0301-DQA1*0501-DQB1*0201 (DR3-DQ2), but it has not been possible to separate the effects of the DR and DQ alleles. We have identified a large Bedouin kindred in which a high prevalence of islet autoimmunity is associated with two different DR3 haplotypes, one carrying the usual DQ2 and the other carrying DQA1*0102-DQB1*0502 (DQ5). Results of prospective follow-up studies indicate that DR3 is associated with the initial activation of islet autoimmunity whereas DQ2 is associated with early-onset and severe clinical disease. The association signals map to a 350-kb interval, thus implicating primary effects for DR3 and DQ2. Overall, our results emphasize the importance of prospective genetic studies that examine the full range of variation in the initiation, progression and expression of autoimmune disease.

Conserved extended haplotypes discriminate HLA-DR3-homozygous Basque patients with type 1 diabetes mellitus and celiac disease.

The major susceptibility locus for type 1 diabetes mellitus (T1D) maps to the human lymphocyte antigen (HLA) class II region in the major histocompatibility complex on chromosome 6p21. In southern European populations, like the Basques, the greatest risk to T1D is associated with DR3 homo- and heterozygosity and is comparable to that of DR3/DR4, the highest risk genotype in northern European populations. Celiac disease (CD) is another DR3-associated autoimmune disorder showing certain overlap with T1D that has been explained by the involvement of common genetic determinants, a situation more frequent in DR3-rich populations, like the Basques. As both T1D- and CD-associated HLA alleles are part of conserved extended haplotypes (CEH), we compared DR3-homozygous T1D and CD patients to determine whether CEHs were equally distributed between both disorders or there was a differential contribution of different haplotypes. We observed a very pronounced distribution bias (P<10(-5)) of the two major DR3 CEHs, with DR3-B18 predominating in T1D and DR3-B8 in CD. Additionally, high-density single nucleotide polymorphism (SNP) analysis of the complete CEH [A*30-B*18-MICA*4-F1C30-DRB1*0301-DQB1*0201-DPB1*0202] revealed extraordinary conservation throughout the 4.9 Mbp analyzed supporting the existence of additional diabetogenic variants (other than HLA-DRB1*0301-DQB1*0201), conserved within the DR3-B18 CEH (but not in other DR3 haplotypes) that could explain its enhanced diabetogenicity.

Genetics of type 1A diabetes.

Type 1A diabetes is an autoimmune disease with genetic and environmental factors contributing to its etiology. Twin studies, family studies, and animal models have helped to elucidate the genetics of autoimmune diabetes. Most of the genetic susceptibility is accounted for by human leukocyte antigen (HLA) alleles. The most-common susceptibility haplotypes are DQA1*0301-DQB1*0302 and DQA1*0501-DQB1*0201. Less-common haplotypes such as DQA1*0401-DQB1*0402 and DQA1*0101-DQB1*0501 are associated with high risk for diabetes; however, large study populations are needed to analyze their effect. The DQA1*0102-DQB1*0602 haplotype is associated with diabetes resistance. DR molecules, such as DRB1*1401, confer protection from diabetes. Monozygotic twins of patients with type 1A diabetes have a diabetes risk higher than that for HLA-identical ordinary siblings, suggesting that non-HLA genes contribute to diabetes risk. Polymorphisms in the regulatory region of the insulin gene (designated IDDM2), polymorphisms in cytotoxic T lymphocyte antigen-4 (CTLA-4) gene (IDDM12), and other genes are likely to contribute to diabetes risk and susceptibility in some individuals. In selected families, major diabetogenes (e.g., IDDM17, autoimmune regulator gene (AIRE)) are likely to be of importance. Other factors--either noninherited genes (i.e., somatic mutations and T-cell receptor or immunoglobulin rearrangements) or environment--may have a role in progression to diabetes. This is suggested by the finding that the risk for monozygotic twins of patients with type 1A diabetes is not 100 percent. Studying the genetics of type 1A diabetes will allow us to better define this disease, to improve our ability to identify individuals at risk, and to predict the risk of associated disorders.

X-Linked syndrome of polyendocrinopathy, immune dysfunction, and diarrhea maps to Xp11.23-Xq13.3.

We describe genetic analysis of a large pedigree with an X-linked syndrome of polyendocrinopathy, immune dysfunction, and diarrhea (XPID), which frequently results in death during infancy or childhood. Linkage analysis mapped the XPID gene to a 17-cM interval defined by markers DXS8083 and DXS8107 on the X chromosome, at Xp11. 23-Xq13.3. The maximum LOD score was 3.99 (recombination fraction0) at DXS1235. Because this interval also harbors the gene for Wiskott-Aldrich syndrome (WAS), we investigated mutations in the WASP gene, as the molecular basis of XPID. Northern blot analysis detected the same relative amount and the same-sized WASP message in patients with XPID and in a control. Analysis of the WASP coding sequence, an alternate promoter, and an untranslated upstream first exon was carried out, and no mutations were found in patients with XPID. A C-->T transition within the alternate translation start site cosegregated with the XPID phenotype in this family; however, the same transition site was detected in a normal control male. We conclude that XPID maps to Xp11.23-Xq13.3 and that mutations of WASP are not associated with XPID.

No evidence for linkage of a novel CA repeat polymorphism for apoptosis antigen 1 (APO-1 or fas) with type I diabetes in a Caucasian population.

A novel microsatellite marker was found within 48.5 kb of the Fas gene. The observed heterozygosity in 160 healthy unrelated controls was 0.78. There was no evidence of linkage to type I diabetes mellitus in 120 diabetic children using the transmission disequilibrium test.

Evidence for oligogenic inheritance of type 1 diabetes in a large Bedouin Arab family.

Based on a genomic search for linkage, a locus contributing to type 1 diabetes in a large Bedouin Arab family (19 affected relatives) maps to the long arm of chromosome 10 (10q25; nonparametric linkage = 4.99; P = 0.00004). All affected relatives carry one or two high-risk HLA-DR3 haplotypes that are rarely found in other family members. One chromosome 10 haplotype, the B haplotype, was transmitted from a heterozygous parent to 13 of 13 affected offspring compared to 10 of 23 unaffected siblings. Recombination events occurring on this haplotype place the susceptibility locus in an 8-cM interval between markers D10S1750 and D10S1773. Two adjacent markers, D10S592 and D10S554, showed evidence of linkage disequilibrium with the disease locus. A 273-bp allele at D10S592 was transmitted to 8 of 10 affected offspring compared to 3 of 14 unaffected siblings, and a 151-bp allele at D10S554 was transmitted to 15 of 15 affected offspring compared with 10 of 24 unaffected siblings. D10S554 and D10S592 and the closest flanking markers are contained in a 1,240-kb yeast artificial chromosome, a region small enough to proceed with positional cloning.

A high resolution CEPH crossover mapping panel and integrated map of chromosome 11.

High resolution (0.1 cM) CEPH crossover mapping panels were constructed for chromosome 11. These panels will facilitate a transition from top-down physical and genetic mapping strategies to integrated breakpoint mapping strategies. Novel methods, which differ from other methods in overcoming the limitations of incomplete heterozygosity and variable marker density, were developed for creating the panels and integrated maps. This made it possible to identify and sublocalize the majority of crossovers in 61 families. The panels were used to map 139 microsatellite markers. A semi-integrated map and a fully-integrated map were constructed by combining these data with data from CEPH 7.1 and then integrating data from the radiation hybrid (RH) map. Genetic lengths estimated from the mapping panels were similar to the estimates obtained when all recombinant and non-recombinant offspring were included (189.4 cM in females and 126.1 cM in males), indicating that genetic distances are stable at this high marker density. The maps have a cM density of 0.62. The distance between ordered markers is 1.39-2.92 cM depending on the criterion for order and the extent of map integration. The 2D maps provide the resolution and flexibility needed to enhance current applications such as positional cloning and mapping complex disorders; while the mapping panels will greatly improve the resolution, reliability and efficiency of future genetic mapping.

A mutation causing Alport syndrome with tardive hearing loss is common in the western United States.

Mutations in the COL4A5 gene, located at Xq22, cause Alport syndrome (AS), a nephritis characterized by progressive deterioration of the glomerular basement membrane and usually associated with progressive hearing loss. We have identified a novel mutation, L1649R, present in 9 of 121 independently ascertained families. Affected males shared the same haplotype of eight polymorphic markers tightly linked to COL4A5, indicating common ancestry. Genealogical studies place the birth of this ancestor >200 years ago. The L1649R mutation is a relatively common cause of Alport syndrome in the western United States, in part because of the rapid growth and migratory expansion of mid-nineteenth-century pioneer populations carrying the gene. L1649R affects a highly conserved residue in the NC1 domain, which is involved in key inter- and intramolecular interactions, but results in a relatively mild disease phenotype. Renal failure in an L1649R male typically occurs in the 4th or 5th decade and precedes the onset of significant hearing loss by approximately 10 years.

Refined genetic mapping and proteolipid protein mutation analysis in X-linked pure hereditary spastic paraplegia.

X-linked hereditary spastic paraplegias (HSP) present with two distinct phenotypes, pure and complicated. The pure form is characterized by spasticity and gait difficulties but lacks the additional features (nystagmus, dysarthria, mental retardation) present in the complicated form. The complicated form is heterogeneous, caused by mutations of the L1CAM gene at Xq28 (SPG1) or the PLP gene at Xq22 (SPG2) that is allelic to Pelizaeus-Merzbacher disease (PMD). Since in one kindred (K313) the pure form of HSP was also mapped to Xq22, this raises the issue as to whether a pure form of HSP exists that is allelic to X-linked complicated HSP (SPG2) and PMD. To answer this question, we carried out linkage analysis in a new pedigree with pure HSP (K101) and refined linkage in pedigree K313. The PLP gene was also screened for mutation by direct sequencing and reverse-transcriptase polymerase chain reaction (RT-PCR). In both families, the disease locus mapped to Xq22 with Lod scores at zero recombination of 5.3 for COL4A5 2B6 in K313 and 2.4 for DXS101 in K101. A T to C transition in exon 5 of the PLP gene was identified from affected individuals of K313. This transition causes a Ser to Pro mutation in the major extracellular loop of PLP/DM20. This finding demonstrates that a form of X-linked pure spastic paraplegia, X-linked complicated HSP (SPG2) and PMD are allelic disorders. There was no evidence of mutations in either coding sequences or the intron/exon junctions of PLP in pedigree K101, suggesting that the disease-producing mutation may be in the noncoding portions of PLP or in a nearby gene.

BRCA1 R841W: a strong candidate for a common mutation with moderate phenotype.

BRCA1 mutations cause increased risk for breast and ovarian cancer, frequently of early onset. Many different mutations occur in BRCA1, including several examples of recurrent mutations, each of which accounts for a significant number of families with heritable cancer predisposition. These common mutations have an etiological role in many breast and ovarian cancer cases and provide the opportunity to examine genotype-phenotype correlations and genotype-environment interactions in individuals with the identical BRCA1 lesion. We report a novel missense change in BRCA1, 2640 C-->T (R841W), found in 3 cases from a subject group of 305 breast and 79 ovarian cancer cases from Orange County, CA. These are consecutive, population-based cases not selected for age or family history. In all three cases, there is a strong family history of breast, ovarian, or other cancers possibly related to a BRCA1 defect and family members showed a high concordance of cancer incidence with the presence of R841W. The age of cancer onset was not always distinct from typical sporadic cases. Testing of a sample of 413 unrelated individuals to examine the hypothesis that R841W might be a rare polymorphism detected one additional instance in a woman with breast cancer diagnosed at age 77 years, and cancer in one parent. R841W is likely to be an etiologically significant lesion with involvement in close to 1% (95% confidence interval of 0-1.7%) of all breast and ovarian cancers in this population.

Consortium fine localization of X-linked Charcot-Marie-Tooth disease (CMTX1): additional support that connexin32 is the defect in CMTX1.

Charcot-Marie-Tooth (CMT) disease is the most common form of inherited motor and sensory neuropathy. X-linked CMT (CMTX1) has been localized to the pericentric region of the X chromosome. Recently, mutations have been defined in the connexin32 gene that cosegregate with the CMTX1 phenotype in several families. The present paper presents the results of an international consortium to fine map the gene for CMTX1 to a small segment of Xq12-13. The linkage data, together with the molecular genetic studies, support the hypothesis that connexin32 is the genetic defect in CMTX1.

A 2D crossover-based map of the human X chromosome as a model for map integration.

We have constructed a two-dimensional map of 243 markers on the X chromosome. The average distance between markers ordered by two recombinants is 5.4 centiMorgans (cM), which is reduced to 3.2 cM using a less stringent criterion of one recombinant. Map resolution is enhanced by replacing the usual reference marker format with a 2D format, and the two-recombinant rule is more conservative than the lod 3.0 criterion for order. Taken together, crossover mapping and the 2D format produces maps with greater reliability and higher resolution than maps constructed using currently accepted standards. This first high-density crossover-based map of an entire human chromosome provides a model for integrating physical and genetic maps.

Fine structure mapping of the human X-linked hypophosphatemic rickets gene locus.

X-linked hypophosphatemic rickets (HYP) is an X-linked dominant disorder characterized by decreased renal tubular phosphate reabsorption and consequent hypophosphatemia. Renal cross-transplantation studies in Hyp mice indicate that the disorder is secondary to the elaboration of an as yet unidentified humoral factor. A full understanding of the pathophysiology of the disease and the nature of this factor will be facilitated by identification of the HYP gene. Efforts to isolate the HYP gene have been deterred by limited precision in the map of the Xp22.1 region and the consequent distance between DXS365 and DXS274, the previously discovered flanking markers for the HYP gene. To map the HYP region precisely, HYP family resources from two groups of investigators were combined, and several newly available microsatellite repeat probes were tested for linkage to HYP. Our data indicate that DXS365, DXS3424, DXS443, DXS1052, DXS274, and DXS1683 are tightly linked to the HYP gene and suggest a locus order of: Xtel-DXS315-(GLR/DXS43)-DXS257-(DXS443+ ++-DXS3424)-DXS365-HYP-DXS1683-DXS1052-DXS 274-(DXS41/DXS92)-DXS451-Xcen. The HYP gene is located in the 350- to 650-kilobase region between DXS365 and DXS1683. These results will provide a basis for the isolation of candidate genes from the region.

A germline 2.35 kb deletion of p53 genomic DNA creating a specific loss of the oligomerization domain inherited in a Li-Fraumeni syndrome family.

The primary genetic cancer predisposing event in many Li-Fraumeni syndrome families is a germline mutation in the p53 gene. We describe an extended Li-Fraumeni family with a germline mutation in the p53 gene involving a deletion of exon 10. The mutation is a 2.35 kilobase intragenic deletion encompassing exon 10, which results in the specific loss of the entire p53 oligomerization domain. This mutation segregates with the cancer phenotype. A lymphoblastoid cell line developed from a mutation carrier shows accumulation of mutant p53 protein by immunoblotting. However, tumor tissues from two affected carriers are negative by immunohistochemical staining. A major structural alteration specifically involving the oligomerization domain of a germline p53 gene has not been previously described and occurs in a region rarely mutated in sporadic tumors. The oligomerization domain is dispensable for many wild-type p53 functions, including transactivation, sequence-specific DNA binding, and suppression of oncogenic transformation. However, the domain appears to be required for transcriptional repression, and DNA strand reassociation. The identification of this mutation in an LFS family may yield insights into the importance of the oligomerization domain for suppressor function of the p53 tumor suppressor gene.

Refined genetic mapping of X-linked Charcot-Marie-Tooth neuropathy.

Genetic linkage studies were conducted in four multigenerational families with X-linked Charcot-Marie-Tooth disease (CMTX), using 12 highly polymorphic short-tandem-repeat markers for the pericentromeric region of the X chromosome. Pairwise linkage analysis with individual markers confirmed tight linkage of CMTX to the pericentromeric region in each family. Multipoint analyses strongly support the order DXS337-CMTX-DXS441-(DXS56,PGK1).

Definition and mapping of STSs at STR and RFLP loci in Xp11-Xq22.

New primer pair sequences specific for 25 loci in the Xp11-q22.1 region are described. Eighteen of the pairs span segments containing significant CA dinucleotide repeats, with 9 of these revealing polymorphisms of greater than 50% heterozygosity. Four of the CA-containing segments occur in probes previously reported to detect RFLPs, while the remaining 14 are from newly isolated clones. STSs were also developed for 7 other RFLP-only loci. All of these 25 STSs plus 11 other published STR markers have been fine-mapped with respect to chromosomal breakpoints, defining 15 subintervals in Xp11-Xq22. This map of 36 STSs, nearly all of which are associated with markers that are genetically mapped and/or highly polymorphic, will significantly aid efforts to construct a complete physical map of this region and to correlate it with the high-density genetic map.

Flanking markers define the X-linked hypophosphatemic rickets gene locus.

X-linked hypophosphatemic rickets (HYP) is an X-linked dominant disorder characterized by decreased renal tubular phosphate reabsorption and consequent hypophosphatemia. The defect in tubular phosphate reabsorption is probably secondary to an unidentified humoral factor. Identification of the humoral factor and a full understanding of the pathophysiology of the disease await the identification of the HYP gene. Previously we demonstrated that DXS257 and DXS41 are flanking markers for the HYP gene. Two markers, DXS365 and DXS274, are tightly linked to the HYP gene, but investigators have been unable to determine whether they are centromeric or telomeric to the disease gene. Since tightly linked flanking markers are necessary prerequisites to obtain the gene by positional cloning techniques, we sought to determine the relative positions of these markers to the HYP gene by expanding our data base for linkage studies. We also investigated a new polymorphic probe for linkage to HYP to construct a more detailed genetic map around the HYP locus. Our data indicate that the markers DXS365, DXS274, and DXS92 are tightly linked to the HYP locus and suggest a locus order of Xtel-(DXS444/DXS315)-DXS43-(DXS257/DXS3 65)-HYP-(DXS274/DXS41/DXS92)-DXS-451- DXS319-Xeen. These results will facilitate attempts further to localize and clone the HYP gene.