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.

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 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.

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.

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.

Multilocus mapping of the X-linked hypophosphatemic rickets gene.

X-linked hypophosphatemic rickets (HYP), the most common form of familial hypophosphatemic (vitamin D-resistant) rickets, is an X-linked dominant disorder characterized by decreased renal tubular phosphate reabsorption and consequent hypophosphatemia. Despite the application of a wide variety of biochemical and cell biology techniques, controversy exists regarding whether a primary renal abnormality underlies the abnormal phosphate transport or if this defect is secondary to the effects of a hormonal/metabolic factor. Thus localization of the HYP gene and its ultimate cloning may be necessary to elucidate the pathophysiology of the disorder. In order to map the human HYP gene we investigated several new polymorphic probes for linkage to HYP and constructed a map of markers around the gene. The database used to ascertain linkage and perform mapping included 5 large HYP kindreds, 40 Centre d'Etudie Polymorphisms Humain reference pedigrees, and 19 kindreds which had been obtained for other disease linkage studies. Two point LOD scores (odds of linkage, log10) indicate that the probes DXS365, DXS257, DXS451, and DXS41 are tightly linked to the HYP locus. Indeed, there were no cross-overs between DXS365 and HYP with a peak LOD score of 13.98 [recombination fraction (theta) = 0.00]. Moreover, multipoint analysis reveals a probable locus order of: Xtel-DXS315-DXS43-DXS257-HYP-DXS41-DXS4 51-Xcen. The likelihood of HYP occurring between DXS257 and DXS41 is 407:1 over the next most likely position. DXS365 is located between DXS41 and DXS43 but could not be located with respect to HYP and DXS257. Regardless, we have located the HYP gene between the flanking markers DXS257 (telomeric) and DXS41 (centromeric) which are 3.5 centiMorgans apart. Thus, the results of this study will facilitate attempts to further localize and eventually clone the gene.

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.

Early indicators of self-destruction in childhood and adolescence: a survey of pediatricians and psychiatrists.

This paper reports a comparison of pediatricians' and psychiatrists' opinions about screening for children and adolescents at risk for self-destruction. Fifty-nine percent of the members of the Nebraska Chapter of the American Academy of Pediatrics and 69% of the members of the Nebraska District Branch of the American Psychiatric Association completed questionnaires containing selected early indicators for self-destruction and programs for suicide prevention. Principal findings were that pediatricians placed less emphasis than child psychiatrists on major depressive disorder, younger pediatricians placed more emphasis than psychiatrists on psychosocial indicators, physicians from both groups said they usually hospitalize attention-seeking self-destructive children, and pediatricians from smaller communities expressed less confidence in referral to psychiatrists but more confidence in community-based programs. Findings suggest that a small group of children and adolescents at risk for eventual suicide may not be identified as early as the more obvious larger groups of individuals who are accident prone, who ingest toxic substances, and who come from violent families. General indicators of risk for self-destruction include substance abuse, psychosocial problems, and minor depression. Factors indicating more specific risk for suicide include escalating stress, family enmeshment, and major mental illness, particularly major depressive disorder. Postgraduate education, instruments for discriminate screening, and balanced programs for suicide prevention are suggested to improve care.

Genetic/epidemiological findings in a study of smoking-associated tumors.

While the etiology of bronchogenic carcinoma remains enigmatic, an expanding array of carcinogenic pollutants in interaction with host susceptibility factors has been implicated. We have studied family histories of cancer in a consecutive series of cancer patients from two university oncology clinics in Nebraska. Particular attention has been given to carcinoma of the lung and other putative smoking-associated cancers (oral cavity, esophagus, urinary bladder, pancreas). Smoking histories were obtained for relatives of an overlapping series of patients with these tumor sites. Findings revealed that, although a significant cohort effect was observed with respect to smoking habits for both relatives of lung cancer probands and for relatives of probands with other smoking-associated tumors, a corresponding trend for lung cancer frequency was observed only for relatives of lung cancer probands. This result suggests the importance of host factors in combination with environmental exposures in determining lung risk. A cohort trend for lung cancer was also apparent among relatives of breast cancer probands, but not for relatives of colon cancer probands, suggesting the possibility of an intrinsic association between carcinomas of the breast and lung. We believe that further elucidation of host factor susceptibility in lung cancer may have important etiological and preventive implications.

Sex differences in lung cancer incidence: a genetic model.

Family histories pertaining to cancer of all anatomic sites were ascertained on 88 Caucasian patients (61 males, 27 females) with histologically verified lung cancer. Lifetable analysis revealed that relatives of female probands had a significantly higher risk for cancer (all sites) at younger age (p less than .04) compared to relatives of male probands. This trend was peculiar to nonsmoking-associated cancer sites, and was not apparent for smoking-associated cancer sites. We hypothesize that certain components of genetic liability to lung cancer are common to nonsmoking-associated malignant neoplasms, and that females who develop lung cancer are more extreme with respect to genotype than are males who develop lung cancer. Our hypothesis accounts for the lower incidence of lung cancer among females, as well as the apparent increased susceptibility to cancer among their relatives.

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.

Third International Workshop on Human Chromosome 17 Mapping.

Highlights of the meeting this year include progress in merging two independently derived genetic maps, expansion of the composite hybrid breakpoint map, and enhancements in working group communications through the chromosome 17 file server at Baylor. Progress is also being made in developing STS primers for framework markers and reference markers. The task remains of fully reconciling the framework map and composite breakpoint map with the list of chromosome 17 reference markers (Solomon and Ledbetter, 1991). There remain several gaps in the overall map, particularly near the distal end of the long arm, where there has been limited activity.

Characteristics of simple sibship variance tests for the detection of major loci and application to height, weight and spatial performance.

Simple methods have been proposed as screening tests for major loci. These methods rely primarily upon the detection of differences in within sibship variances expected for segregating and non-segregating sibships when a major locus is present. In the present study, computer simulation was used to investigate power and robustness of the test statistics. Power of the analyses depends upon the specific major locus model, but, in general, their application is quite practical for small samples. The test statistics were shown to be sensitive to deviations from normality, but robust under the conditions of either a polygenic or environmental model. Application of the test procedures to sibship data from the Boulder Family Study led to significant results for a three-dimensional spatial rotation test, but results for height and weight were non-significant. The simplest interpretation of the results for the spatial performance test was in terms of a sex-linked major locus.

Models of multilocus recombination: nonrandomness in chiasma number and crossover positions.

Cytological evidence indicates that genetic interference can be partitioned into two empirical components: nonrandomness in the number of chiasmata that occur and nonrandomness in the locations of multiple chiasmata. Previous studies have incorporated the first effect into genetic models for analyzing multipoint data. An extension to this approach is presented which allows for the second component of interference by modeling the probability density function of the locations of multiple crossovers. Results of reanalyses of multilocus data for the Drosophila X chromosome show that models that incorporate only the first effect give a better fit to these data than do standard mapping functions and that the extended model significantly improved the fit by decreasing the predicted frequency of multiple crossovers in nearby regions. Our results demonstrate that chiasma-based models of multilocus recombination, which are unique in incorporating direct estimates of the frequency of multiple crossovers for a chromosome region, can provide a powerful and realistic means of accounting for genetic interference when applied to the problems of gene localization, locus ordering, and exclusion mapping.

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.

Genetic Analysis Workshop II: results of incorporating a linkage disequilibrium parameter.

The corporation of a linkage disequilibrium parameter, delta, into linkage analysis is illustrated for data from Genetic Analysis Workshop II. Points from a joint likelihood surface are calculated and displayed on a recombination fraction-linkage disequilibrium grid using a simple modification of LIPED. The approach is shown to increase the power of linkage analysis and the power of tests for heterogeneity of linkage for the simulated examples.

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.