Linkage disequilibrium in inbred North African families allows fine genetic and physical mapping of triple A syndrome

Triple A syndrome (Allgrove syndrome, MIM No. 231550) is a rare autosomal recessive disorder characterised by ACTH-resistant adrenal insufficiency, achalasia of the cardia, and alacrimia. The triple A gene has been previously mapped to chromosome 12q13 in a maximum interval of 6 cM between loci D12S1629 and D12S312. Using linkage analysis in 12 triple A families, mostly originating from North Africa, we confirm that the disease locus maps to the 12q13 region (Zmax = 10.89 at theta = 0 for D12S1604) and suggest that triple A is a genetically homogeneous disorder. Recombination events as well as homozygosity for polymorphic markers enabled us to reduce the genetic interval to a 3.9 cM region. Moreover, total linkage disequilibrium was found at the D12S1604 locus between a rare allele and the mutant chromosomes in North African patients. Analysis of markers at five contiguous loci showed that most of the triple A chromosomes are derived from a single founder chromosome. As all markers are located in a 0 cM genetic interval and only allele 5 at the D12S1604 locus was conserved in mutant chromosomes, we speculate that the triple A mutation is due to an ancient Arabian founder effect that occurred before migration to North Africa. Since we also found linkage disequilibrium at D12S1604 in two patients from Southern Europe (France and Spain), the founder effect might well extend to other Mediterranean countries. Taking advantage of a YAC contig encompassing the triple A minimal physical region, the triple A gene was mapped to a 1.7 Mb DNA fragment accessible to gene cloning.     

Fish mapping of a translocation breakpoint at 6q21 (or q22) in a patient with heterotaxia

Summary Heterotaxia is a congenital lateralization defect of visceral organs. As several single-genes that act on the formation of left-right asymmetry during embryogenesis have been identified in animals, a defect in the similar system may play a role in heterotaxia in man. We previously reported a Japanese girl with heterotaxia associated with ade novo balanced translocation (6;18)(q21 or q22;q21.3 or q22). In the present study, based on a hypothesis that one of the putative situs-determining genes is disrupted at a breakpoint of the translocation, we first isolated a yeast artificial chromosome (YAC) clone covering a breakpoint, 6q21 (or q22) of the translocation. Then, using STSs mapped on the YAC, we isolated bacterial artificial chromosome (BAC) clones spanning the breakpoint. FISH analysis using the BAC clones as probes revealed that the breakpoint is confined to a segment between two STS loci, WI-4066 and the CHLC.GATA6B06.192, within a genetic distance of 1.4 cM. The human connexin43 gene was not disrupted in our patient, although mutations of this gene have been reported in patients with complex heart disease and heterotaxia. The molecular localization of the translocation breakpoint in our patient may contribute to the positional cloning of a putative heterotaxia gene.     

A new locus for hereditary haemorrhagic telangiectasia (HHT3) maps to chromosome 5

Patients with hereditary haemorrhagic telangiectasia (HHT, or Osler-Weber-Rendu syndrome) have variable presentation patterns and a high risk of preventable complications. Diagnostic tests for mutations in endoglin (HHT type 1) and ALK-1 (HHT type 2) are available. Some HHT patients are now known to have HHT-juvenile polyposis overlap syndrome due to Smad4 mutations. Families were ascertained following the presentation of probands for embolization of pulmonary arteriovenous malformations. Genome-wide linkage studies using over 700 polymorphic markers, and sequencing of candidate genes, were performed. In a previously described HHT family unlinked to endoglin or ALK-1, linkage to Smad4 was excluded, and no mutations were identified in the endoglin, ALK-1, or Smad4 genes. Two point LOD scores and recombination mapping identified a 5.4 cM HHT3 disease gene interval on chromosome 5 in which a single haplotype was inherited by all affected members of the pedigree. The remainder of the genome was excluded to a 2-5 cM resolution. We are currently studying a further family potentially linked to HHT3. We conclude that classical HHT with pulmonary involvement can result from mutations in an unidentified gene on chromosome 5. Identification of HHT3 should further illuminate HHT pathogenic mechanisms in which aberrant transforming growth factor (TGF)-beta signalling is implicated.     

Genetic refinement and physical mapping of a chromosome 16q candidate region for inflammatory bowel disease.

Crohn's disease (CD) is a complex genetic disorder for which a susceptibility gene, IBD1, has been mapped within the pericentromeric region of chromosome 16. In order to refine the location of IBD1, 77 multiplex CD families were genotyped for 26 microsatellite markers evenly spaced by approximately 1 cM. Nonparametric linkage analyses exhibited a maximum NPL score of 3.49 (P=2.37x10(-4)) in a region centred by markers D16S3136, D16S3117 and D16S770. Simulation studies showed that the probability for IBD1 to be located in a 5 cM region around these markers was 70%. A 2.5 Mb YAC and BAC contig map spanning this genetic region on chromosome band 16q12 was built. TDT analyses demonstrated suggestive association between the 207 bp allele of D16S3136 (P<0.05) and a new biallellic marker hb27g11f-end (P=0.01). These markers were located in the hb27g11 and hb87b10 BAC clones from the contig. Taken together, the present results provide a crucial preliminary step before an exhaustive linkage disequilibrium mapping of putatively transcribed regions to identify IBD1.     

Chromosome imbalances associated with epilepsy

Epilepsy is among the most frequent findings in many, especially autosomal, chromosome aberrations. Its incidence, however, is very variable, and there are very few aberrations in which epilepsy is a constant finding. Even siblings and monozygotic twins with the same aberration are often discordant for seizure disorders. Similar observations can be made for congenital (major) malformations in chromosome aberrations. The common explanation is that in these instances epilepsy is not caused by the action of a single gene in single or triple dose, but is influenced by the combined action of a number of genes within and outside of the aneuploid segment. The situation is comparable to a polygenic model of inheritance. Gene mutations associated with epilepsy are known, to date, only for two disorders: the lissencephaly 1 gene in Miller-Dieker syndrome and mutations in the UBE3A gene in Angelman syndrome. Chromosome aberrations in which epilepsy is a major and consistent finding include Angelman syndrome due to loss of the maternal 15q11.2-q12 segment, tetrasomy of the maternal segment 15pter-q13 due to an additional inv dup chromosome, Miller-Dieker syndrome due to deletion of the 17p13.3 segment including the lissencephaly1 gene, ring chromosome 20, and Wolf-Hirschhorn syndrome due to deletion of at least the 4p16.3 segment.     

Refined genetic mapping of autosomal recessive chronic distal spinal muscular atrophy to chromosome 11q13.3 and evidence of linkage disequilibrium in European families

Chronic distal spinal muscular atrophy (Chronic DSMA, MIM (*)607088) is a rare autosomal recessive disorder characterized by a progressive motor weakness and muscular atrophy, predominating in the distal parts of the limbs. A form of Chronic DSMA gene has been previously mapped to chromosome 11q13 in the 10.3 cM interval defined by loci D11S1889 and D11S1321. By linkage analysis in 12 European Chronic DSMA families, we showed that a disease gene maps to chromosome 11q13.3 (Z(max)=6.66 at theta=0.00 at the DSM4 locus) and suggested that this condition is genetically homogeneous. Recombination events allowed us to reduce the genetic interval to a 2.6 cM region, telomeric to the IGHMBP2 gene, excluding this gene as the disease causing gene in Chronic DSMA. Moreover, partial linkage disequilibrium was found between three rare alleles at loci D11S1369, DSM4 and D11S4184 and the mutant chromosome in European patients. Analysis of the markers at these loci strongly suggests that most Chronic DSMA chromosomes are derived from a single ancestor. Refinement of the Chronic DSMA locus will hopefully allow to test candidate genes and lead to identification of the disease-causing mutations.     

Refinement of the RP17 locus for autosomal dominant retinitis pigmentosa, construction of a YAC contig and investigation of the candidate gene retinal fascin

The RP17 locus for autosomal dominant retinitis pigmentosa has previously been mapped to chromosome 17q by linkage analysis. Two unrelated South African families are linked to this locus and the identification of key recombination events assigned the RP17 locus to a 10 cM interval on 17q22. The work reported here refines the mapping of the locus from a 10 cM to a 1 cM interval between the microsatellite markers D17S1604 and D17S948. A physical map of this interval was constructed using information from the Whitehead/MIT YAC contig WC 17.8. Sequence-tagged site (STS) content mapping of seven overlapping YACs from this contig was employed in order to build the map. A BAC library was screened to cover a gap in the YAC contig and two positive BACs were identified. Intragenic polymorphisms in the retinal fascin gene provided evidence for the exclusion of this candidate as the RP17 disease gene.     

DFNB74, a novel autosomal recessive nonsyndromic hearing impairment locus on chromosome 12q14.2-q15

Autosomal recessive nonsyndromic hearing impairment (ARNSHI) segregating in three unrelated, large consanguineous Pakistani families (PKDF528, PKDF859 and PKDF326) is linked to markers on chromosome 12q14.2-q15. This novel locus is designated DFNB74 . Maximum two-point limit of detection (LOD) scores of 5.6, 5.7 and 2.6 were estimated for markers D 12 S 313, D 12 S 83 and D 12 S 75 at θ = 0 for recessive deafness segregating in these three families. Haplotype analyses identified a critical linkage interval of 5.35 cM (5.36 Mb) defined by D 12 S 329 at 74.58 cM and D 12 S 313 at 79.93 cM. DFNB74 is the second ARNSHI locus mapped to chromosome 12, but the physical intervals do not overlap with one another. A locus contributing to the early onset, rapidly progressing hearing loss of A/J mice ( ahl4 , age-related hearing loss 4) was reported to map to chromosome 10 in a region of conserved synteny to DFNB74 , suggesting that ahl4 and DFNB74 may be due to mutations of the same gene in these two species.     

Smith-Fineman-Myers综合征基因定位于Xq25

目的　定位 Ｓｍｉｔｈ Ｆｉｎｅｍ ａｎ Ｍｙｅｒｓ 综合征基因,为分离该基因奠定基础。方法　应用覆盖 Ｘ染色体全长的、具有多态性的短串联重复序列（ Ｓ Ｔ Ｒ） 对 Ｘ 染色体进行扫查,确定致病基因所在区域和与致病基因连锁的 Ｓ Ｔ Ｒ 位点,再对该位点两侧的 Ｓ Ｔ Ｒ 位点进行分析,确定致病基因的精确位置。结果　用２０个覆盖 Ｘ 染色体全长的、具有多态性的 Ｓ Ｔ Ｒ 位点对该综合征患者家系中的１３ 个能明确提供连锁分析信息的家系成员进行分析,发现位于 Ｘｑ２５ 上的 Ｄ Ｘ Ｓ１００１ 与致病基因紧密连锁,最大两点ｌｏｄｓ 得分为３０１（θ＝ ０） ,对 Ｄ Ｘ Ｓ１００１ 两侧的 Ｓ Ｔ Ｒ 分析证实,该致病基因位于 Ｄ Ｘ Ｓ１００１ 区域,单体型分析表明该致病基因位于 Ｄ Ｘ Ｓ８０６４ 和 Ｄ Ｘ Ｓ８０５０ 之间,区域为１４６ｃ Ｍ。结论　 Ｓｍｉｔｈ Ｆｉｎｅｍ ａｎ Ｍｙｅｒｓ 综合征基因,位于 Ｘｑ２５ 上的 Ｄ Ｘ Ｓ８０６４ 和 Ｄ Ｘ Ｓ８０５０ 之间的１４６ｃ Ｍ 区域,该基因的定位为分离该基因奠定了基础。     

Contiguous deletion of the NDP, MAOA, MAOB, and EFHC2 genes in a patient with Norrie disease, severe psychomotor retardation and myoclonic epilepsy

Norrie disease (ND) is an X-linked disorder, inherited as a recessive trait that, therefore, mostly affects males. The gene responsible for ND, called NDP, maps to the short arm of chromosome X (Xp11.4-p11.3). We report here an atypical case of ND, consisting of a patient harboring a large submicroscopic deletion affecting not only the NDP gene but also the MAOA, MAOB, and EFHC2 genes. Microarray comparative genomic hybridization (CGH) analysis showed that 11 consecutive bacterial artificial chromosome (BAC) clones, mapping around the NDP gene, were deleted. These clones span a region of about 1 Mb on Xp11.3. The deletion was ascertained by fluorescent in situ hybridization (FISH) analysis with different BAC clones located within the region. Clinical features of the proband include bilateral retinal detachment, microcephaly, severe psychomotor retardation without verbal language skills acquired, and epilepsy. The identification and molecular characterization of this case reinforces the idea of a new contiguous gene syndrome that would explain the complex phenotype shared by atypical ND patients.     

Defining a common region of deletion at 13q21 in human cancers.

Previous molecular genetic analyses identified a region of deletion at 13q21 in a variety of human cancers, suggesting the existence of a tumor suppressor gene(s) at this locus. In our earlier study on prostate cancer, the region of deletion was confined to a 3.1 cM interval between D13S152 and D13S162. At present, however, no known gene located in this interval has been firmly implicated in cancer, and the region remains too large for gene identification. To fine-map the area of interest, we established a contig of bacterial artificial chromosome (BAC) clones, narrowed the region of deletion by loss of heterozygosity (LOH) and homozygosity-mapping-of-deletion (HOMOD) analyses in different types of cancers, and tested a candidate gene from the region for mutation and alteration of expression in prostate cancers. The contig consisted of 75 overlapping BAC clones. In addition to the generation of 47 new sequence-tagged-site (STS) markers from the ends of BAC inserts, 76 known STS and expressed sequence tag markers were mapped to the contig (25 kb per marker on average). The minimal region of deletion was further defined to be about 700 kb between markers D13S791 and D13S166 by LOH analysis of 42 cases of prostate cancer, and by HOMOD analysis of eight prostate cancer cell lines/xenografts and 49 cell lines from cancers of the breast, ovary, endometrium, and cervix, using 18 microsatellite markers encompassing the deletion region. A gene that is homologous to the WT1 tumor suppressor gene, AP-2rep (KLF12), was mapped in this region and was analyzed for its expression and genetic mutation. In addition to low levels of expression in both normal and neoplastic cells of the prostate, this gene did not have any mutations in a group of aggressive prostate cancers and cell lines/xenografts, as assessed by the methods of polymerase chain reaction-single strand conformational polymorphism analysis and direct sequencing. These studies suggest that a 700 kb interval at 13q21 harbors a tumor suppressor gene(s) that seems to be involved in multiple types of cancer, and that the AP-2rep gene is unlikely to be an important tumor suppressor gene in prostate cancer. The BAC contig and high-resolution physical map of the defined region of deletion should facilitate the cloning of a tumor suppressor gene(s) at 13q21.