Allelic Deletion of VHL Gene Detected in Papillary Tumors of the Broad Ligament, Epididymis, and Retroperitoneum in von Hippel-Lindau Disease Patients

Although clinically associated with von Hippel-Lindau (VHL) disease, the pathogenesis of papillary tumors of the broad ligament, epididymis, and peritoneum arising in patients with VHL disease is not clear. The "classic" VHL-associated neoplasms, including hemangioblastoma and renal ceil carcinoma, have been found to be associated with the inactivation of both VHL gene copies. It is not known whether a similar inactivation of the VHL gene is also responsible for the development of these uncommon VHL-associated lesions. The authors performed PCR (polymerase chain reaction) and PCR-based SSCP (single-strand conformation polymorphism) analysis on five predominantly papillary tumors in five VHL patients (one papillary cystadenoma of the broad ligament, one endometrioid cystadenoma of the broad ligament, two papillary cystadenomas of the epididymis, one papillary tumor of the retroperitoneum) with four polymorphic markers of VHL gene (D3S1038, D3S1110, D3S2452, 104/105). All five tumors showed allelic loss of VHL gene. The results provide the first genetic evidence for the role of VHL gene in the tumorigenesis of these rare benign neoplasms and confirm these tumors as phenotypic manifestations of VHL disease. Int J Surg Pathol 8(3):207-212, 2000     

The isolation of a yeast artificial chromosome (YAC) contig extending for 2 megabases in the vicinity of the Von Hippel Lindau disease gene

Von Hippel LIndau disease (VHL) is a rare autosomal dominantdisease associated with tumors and cysts in multiple organ systems.The VHL disease gene is tightly linked to the polymorphic DNAmarker 233E2 (D3S720) and flanked by 479H4 (D3S719) on its telomericand RAF1 on its centromeric side. Two additional markers, D3S1038and D3S601, have also been identified, and these markers, likeD3S720, are very tightly linked to VHL. Previously 93 cosmidclones were mapped to the larger region, 3p24.2 - pter, surroundingthe VHL disease gene (1). Using a Southern-based screening strategyon pools of YAC clones we have Isolated a contig of overlappingYAC clones that extends about 0.7 megabase centromeric, andabout 1.3 megabases telomeric of D3S720 and contains all threetightly linked VHL markers. Individual YACs In this contig werehybridized to grids containing cosmids localized between 3p24.2-pterand to several cosmids localized by fluorescent in situ hybridization(FISH) to 3p25. A total of 28 cosmids were positioned on thiscontig of overlapping YAC clones. We have also identified homologousYAC clones to many additional cosmid clones localized between3p24.2–p25, although these have not yet been preciselylocalized relative to the contig of YAC clones. This contigof YAC clones probably contains the VHL disease gene and shouldfacilitate the isolation and characterization of this gene.     

Prenatal prediction of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a common cause of inherited morbidity and mortality in childhood. The wide range of phenotypes in SMA, uncertainty regarding its mode of inheritance, and the suggestion of linkage heterogeneity have complicated the genetic counselling of parents of affected children. The locus responsible for autosomal recessive SMA has been mapped to 5q11.2-q13.3. The most likely order of loci is cen-D5S6-(SMA,D5S125)-(JK53CA1/2,D5S112)-D5S3 9-qter, with highly polymorphic loci being identified at JK53CA1/2 and D5S39. We describe linkage studies with another highly polymorphic locus, D5S127, that is closely linked to D5S39. This genetic map can be used as the basis for genetic counselling in families with autosomal recessive SMA. Appropriate allowance can be made for sporadic cases owing to non-inherited causes and for linkage heterogeneity or misdiagnoses.     

Detailed genetic mapping of the von Hippel-Lindau disease tumour suppressor gene.

Von Hippel-Lindau (VHL) disease is an autosomal dominant inherited familial cancer syndrome characterised by a predisposition to the development of retinal, cerebellar, and spinal haemangioblastomas, renal cell carcinoma, and phaeochromocytoma. The gene for VHL disease has been mapped to chromosome 3p25-p26 and flanking markers identified. We report the detailed genetic mapping of the VHL disease locus in 38 families. Significant linkage was detected between VHL disease and D3S601 (Zmax = 18.86 at theta = 0.0, CI 0.0-0.025), D3S18 (Zmax = 11.42 at theta = 0.03, CI 0.005-0.08), RAF1 (Zmax = 11.02 at theta = 0.04, CI 0.007-0.01), and D3S1250 (Zmax = 4.73 at theta = 0.05, CI 0.005-0.15). Multipoint linkage analysis mapped the VHL disease locus between D3S1250 and D3S18 close to D3S601. There was no evidence of locus heterogeneity. This study has (1) confirmed the tight linkage between VHL disease and D3S601, (2) identified D3S1250 as the first marker telomeric to RAF1 which maps centromeric to the VHL disease gene, and (3) narrowed the target region for isolation of the VHL disease gene by positional cloning techniques to a 4 cM interval between D3S1250 and D3S18. These findings will improve the clinical management of families with VHL disease by improving the accuracy of presymptomatic diagnosis using linked DNA markers, and will enhance progress towards isolating the VHL disease gene.     

Genetic linkage between Von Hippel--Lindau disease and three microsatellite polymorphisms refines the localisation of the VHL locus

Von Hippel—Lindau (VHL) disease is a dominantly inheritedfamilial cancer syndrome characterised by the development ofretinal and central nervous system haemangioblastomas, renalcell carcinoma and phaeochromocytoma. The gene for VHL diseasehas been mapped to chromosome 3p25–p26 and presymtomaticdiagnosis using linked DNA markers is available. We have previouslymapped the VHL disease gene to a 4 cM interval between D3S1250and D3S18. To increase access to presymptomatic diagnosis andto accelerate progress towards isolating the VHL disease genewe attempted to identify microsatellite DNA markers linked tothe disease gene by genetic linkage analysis in 29 families.We found significant linkage between the VHL disease gene anddinucleotide (CA) repeat polymorphisms at D3S1038 (Zmax = 22.24at     

Mapping of human interferon regulatory factor 3 (IRF3) to chromosome 19q13.3–13.4 by an intragenic polymorphic marker

We have identified a highly polymorphic intragenic marker composed of two dinucleotide repeats in an intron of the human interferon regulatory factor ( IRF3 ) gene. This polymorphic marker has allowed us to map IRF3 to the boundary of 19q13.3–13.4 between the polymorphic markers D19S604 and D19S206 close to KLK1 . An intron is present in a homologous position in the mouse Irf3 gene, but lacks the dinucleotide repeats present in the human intron. Syntenic conservation between this region of 19q13.3–13.4 maps Irf3 to either mouse chromosome 7 or 17.     

A linkage map of 10 loci flanking the Marfan syndrome locus on 15q: results of an International Consortium study.

Members of an International Consortium for Linkage Analysis of the Marfan Syndrome (MFS1) have pooled data for joint analysis in an attempt to determine the precise location of the MFS1 gene and the order of 10 DNA markers on 15q. Five laboratories performed a total of 2111 genotypes in 22 families consisting of 225 affected and 248 normal subjects. For each marker a mean of 98 meioses was informative. D15S48 and D15S1 were identified as the closest linked markers with 99% upper confidence intervals of 12% and 13% respectively. We have used the CRI-MAP program to construct the most likely order as: D15S24-D15S25-D15S1-MFS1-D15S48-D15S49+ ++-(D15S45/S51)-(D15S29/S38). Placement of D15S2 in relation to -D15S1-D15S48- cannot be determined with certainty. The genetic map of these markers extends 53.6 cM in males and 65.0 cM in females with a sex averaged map of 60.7 cM. The sex difference was statistically significant (p = 0.005). Linkage heterogeneity between 22 MFS1 families was documented (p = 0.009) necessitating the exclusion of one family from the analysis. However, comparison of the remaining 21 families for two point and multipoint lod scores showed no evidence for linkage heterogeneity of the MFS1 locus.     

Allelic deletion and mutation of the von Hippel-Lindau (VHL) tumor suppressor gene in pancreatic microcystic adenomas.

An association between pancreatic microcystic (serous) adenomas (MCAs) and von Hippel-Lindau (VHL) disease has been suggested. However, genetic alterations of the VHL gene in MCAs of the pancreas have never been reported. In this study, we performed genetic analysis of 12 pancreatic MCAs. In 2 cases, VHL disease was documented clinically, and 10 cases were sporadic. For LOH analysis, tumor and normal pancreatic cells were procured from formalin-fixed, paraffin-embedded material using tissue microdissection. After DNA extraction, the samples were amplified by polymerase chain reaction using the polymorphic markers D3S2452, D3S1110, D3S192, and D3S656. In addition, the sporadic tumors were analyzed for VHL gene mutations using probes 3b/10b and K55/K56. Both MCAs associated with VHL disease showed LOH with at least one of the microsatellite markers tested. Among the 10 sporadic cases, 7 tumors showed LOH at the VHL gene locus. A somatic VHL gene mutation on exon 2 was documented in one sporadic case. The study provides the first direct genetic evidence for the role of the VHL gene in MCA tumorigenesis. Furthermore, VHL gene alterations may be detected in both VHL-associated and sporadic pancreatic MCAs.     

A second locus (GLC3B) for primary congenital glaucoma (Buphthalmos) maps to the 1p36 region

Primary congenital glaucoma (gene symbol: GLC3) is an ocular disorder that occurs for 0.01-0.04% of blind people. In the majority of familial cases reported so far, this condition is inherited as an autosomal recessive trait. We have recently used a group of 17 GLC3 families with a minimum of two affected offspring and consanguinity in most of the parental generation and mapped the first GLC3 locus (GLC3A) to the 2p21 region. Six families did not show any linkage to the GLC3A locus and thus provided evidence for genetic heterogeneity of this disorder. A total of eight families unlinked to the 2p21 region were used to search for the chromosomal location of the second GLC3 locus. Herein, we describe mapping of a new locus (designated GLC3B) for primary congenital glaucoma to the short arm of chromosome 1 (1p36.2-36.1) that is situated centromeric to the neuroblastoma and Charcot-Marie-Tooth type 2A (CMT2A) loci. A total of 17 DNA markers were genotyped from this region of chromosome 1. Four families showed no recombination with the two markers D1S2834 and D1S402 with a maximum lod score of 4.510 and 4.157 respectively. Pairwise and multipoint linkage analysis and inspection of the haplotypes revealed that the remaining four families are not linked to this part of chromosome 1, thus providing further evidence that at least one more locus for the autosomal recessive form of GLC3 must exist in the genome. Based on the recombination events, the overall linkage map of this region is: tel-D1S1192-D1S1635-D1S1193 - (D1S1597/-D1S489/D1S228)- [GLC3B/D1S2834/D1S402] - (D1S1176/D1S507/D1S407) - D1S2728-(MFAP2/D1S170) - D1S1368 - D1S436- D1S1592-cen.      

Physical mapping of the human chromosome 11q23 region containing the ataxia-telangiectasia locus

Two breakpoints within chromosome 11q23 were characterized with 29 DNA probes to establish a physical map of the region. This region is notable in that it contains at least 14 functional genes which are also syntenic in the mouse (chromosome 9). Chromosome 11q23 includes these markers: STMY, CLG, NCAM, DRD2, APOA1, APOC3, APOA4, CD3E, CD3D, CD3G, PBGD, THY1, ets-1, and cbl-2. The two breakpoints, herein called "X;11" and "4;11," defined a region of approximately 8 cM containing the APO and CD3 complexes as well as the polymorphic marker D11S29. DRD2 localized centromeric to the X;11 breakpoint despite evidence for close genetic linkage to D11S29, suggesting that DRD2 lies close to the X;11 breakpoint. THY1, PBGD, and cbl-2 localized telomeric to the 4;11 breakpoint and thus to the [D11S29--APO--CD3] grouping as well. The physical map helps to correlate the cytogenetic and linkage maps of this region. It also suggests that the human 11q23 syntenic grouping is inverted with respect to its murine counterpart. Based on this physical map and on our primary linkage map of the 11q23 region, we are able to confirm a preliminary localization of the gene for ataxia-telangiectasia group A (ATA) to a region centromeric to the interval defined by D11S144 (pYNB3.12) and THY1.     

A fine-structure deletion map of human chromosome 11p: Analysis of J1 series hybrids

Deletion analysis offers a powerful alternative to linkage and karyotypic approaches for human chromosome mapping. A panel of deletion hybrids has been derived by mutagenizing J1, a hamster cell line that stably retains chromosome 11 as its only human DNA, and selecting for loss of MIC1,a surface antigen encoded by a gene in band 11p13. A unique, self-consistent map was constructed by analyzing the pattern of marker segregation in 22 derivative cells lines; these carry overlapping deletions of 11p13, but selectively retain a segment near the 11p telomere. The map orders 35 breakpoints and 36 genetic markers, including 3 antigens, 2 isozymes, 12 cloned genes, and 19 anonymous DNA probes. The deletions span the entire short arm, dividing it into more than 20 segments and define a set of reagents that can be used to rapidly locate any newly identified marker on 11p, with greatest resolution in the region surrounding MIC1.The approach we demonstrate can be applied to map any mammalian chromosome. To test the gene order, we examined somatic cell hybrids from five patients, whose reciprocal translocations bisect band 11p13; these include two translocations associated with familial aniridia and two with acute T-cell leukemia. In each patient, the markers segregate in telomeric and centromeric groups as predicted by the deletion map. These data locate the aniridia gene (AN2)and a recurrent T-cell leukemia breakpoint (TCL2)in the marker sequence, on opposite sides of MIC1.To provide additional support, we have characterized the dosage of DNA markers in a patient with Beckwith-Wiedemann syndrome and an 11p15-11pter duplication. Our findings suggest the following gene order: TEL-(HRAS1, MER2, CTSD, TH/INS/IGF2, H19, D11S32)-(RRM1, D11S1, D11S25, D11S26)-D11S12-(HBBC, D11S30)-D11S20-(PTH, CALC)-(LDHA, SAA, TRPH, D11S18, D11S21)-D11S31-D11S17-HBVS1-(FSHB, D11S16)-AN2-MIC1-TCL2-J-CAT-MIC4-D11S9-D11S14-ACP2-(D11S33, 14L)-CEN.We have used the deletion map to show the distribution on 11p of two centromeric repetitive elements and the low-order interspersed repeat A36Fc.Finally, we provide evidence for an allelic segregation event in the hamster genome that underlies the stability of chromosome 11 in J1. The deletion map provides a basis to position hereditary disease loci on 11p, to distinguish the pattern of recessive mutations in different forms of cancer and, since many of these genes have been mapped in other mammalian species, to study the evolution of a conserved syntenic group.Deceased.     

Exclusion of BMP6 as a candidate gene for cleidocranial dysplasia

Cleidocranial dysplasia (CCD) is an autosomal dominant, generalized skeletal dysplasia in humans that has been mapped to the short arm of chromosome 6. We report linkage of a CCD mutation to 6p21 in a large family and exclude the bone morphogenetic protein 6 gene (BMP6) as a candidate for the disease by cytogenetic localization and genetic recombination. CCD was linked with a maximal two-point LOD score of 7.22 with marker D6S452 at θ = 0. One relative with a recombination between D6S451 and D6S459 and another individual with a recombination between D6S465 and CCD places the mutation within a 7 cM region between D6S451 and D6S465 at 6p21. A phage P1 genomic clone spanning most of the BMP6 gene hybridized to chromosome 6 in band region p23–p24 using FISH analysis, placing this gene cytogenetically more distal than the region of linkage for CCD. We derived a new polymorphic marker from this same P1 clone and found recombinations between the marker and CCD in this family. The results confirm the map position of CCD on 6p21, further refine the CCD genetic interval by identifying a recombination between D6S451 and D6S459, and exclude BMP6 as a candidate gene. Am. J. Med. Genet. 71:292–297, 1997. © 1997 Wiley-Liss, Inc.     

A novel autosomal recessive nonsyndromic hearing impairment locus (DFNB42) maps to chromosome 3q13.31-q22.3

A consanguineous family with autosomal recessive nonsyndromic hearing impairment (NSHI) was ascertained in Pakistan and displayed significant evidence of linkage to 3q13.31-q22.3. The novel locus (DFNB42) segregating in this kindred, maps to a 21.6 cM region according to a genetic map constructed using data from both the deCode and Marshfield genetic maps. This region of homozygosity is flanked by markers D3S1278 and D3S2453. A maximum multipoint LOD score of 3.72 was obtained at marker D3S4523. DFNB42 represents the third autosomal recessive NSHI locus to map to chromosome 3.     

Physical mapping and YAC-cloning connects four genetically distinct 4qter loci (D4S163, D4S139, D4F35S1 and D4F104S1) in the FSHD gene-region

We have constructed a long-range restriction map of the regionon chromosome 4q that contains the gene for facioscapulohumeralmuscular dystrophy (FSHD). This region contains the linkagegroup cen...D4S163-D4S139-D4F35S1-D4F104S1-FSHD...4qter, whichspans a genetic distance of about 5 cM. Pulse field gel electrophoresis(PFGE) mapping indicated that these loci span a region not morethan 1 Mb. STSs were developed for several of these loci, whichserved to isolate four overlapping yeast artificial chromosomes(YACs). These YACs confirmed the PFGE map and have allowed usto generate a more detailed restriction map using cosmid contigmapping (1). The physical distances were smaller than was expectedon the basis of the genetic map. Two potential HTF islands havebeen detected within the cloned region. One HTF island mapsabout 100 kb centromeric from the tandem repeats involved inthe FSHD mutation, whereas the other maps within these tandemrepeats.     

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.     

Estimation of the size of the chromosome 17p11.2 duplication in Charcot-Marie-Tooth neuropathy type 1a (CMT1a). HMSN Collaborative Research Group.

We have previously shown a duplication in 17p11.2 with probe pVAW409R3 (D17S122) in 12 families with hereditary motor and sensory neuropathy type I (HMSN I) or Charcot-Marie-Tooth disease type 1 (CMT1). In this study we aimed to estimate the size of the duplication using additional polymorphic DNA markers located in 17p11.2-p12. Two other 17p11.2 markers, pVAW412R3 (D17S125) and pEW401 (D17S61), were found to be duplicated in all HMSN I patients tested. Furthermore, all HMSN I patients showed the same duplication junction fragment with probe pVAW409R3. On the genetic map the duplicated markers span a minimal distance of 10 cM while on the physical map they are present in the same NotI restriction fragment of 1150 kb. The discrepancy between the genetic and physical map distances suggests that the 17p11.2 region is extremely prone to recombinational events. The high recombination rate may be a contributing factor to the genetic instability of this chromosomal region.     

Association of HLA genes with ankylosing spondylitis in Han population of eastern China

Ankylosing spondylitis (AS) is a chronic inflammatory disorder with a multifactorial genetic basis. HLA-B27 was reported with the greatest susceptibility to AS but did not act alone. The aim of this study was to search for other gene(s) associated with AS independently of HLA-B27 using 13 microsatellite markers spanning 1.5 Mb from locus TAP1 to HLA-Cw and a single-nucleotide polymorphism marker within NFkappaBIL1 gene promoter. Genotyping for microsatellites was performed in 175 AS patients of eastern Chinese and 219 ethnically matched healthy controls using polymerase chain reaction with fluorescence-labelled primers, whereas the SNP marker was genotyped by direct DNA sequencing. Allele as well as haplotype frequencies were compared between cases and controls, and a linkage disequilibrium analysis was performed to estimate the LD relationship between the candidate regions. The frequencies of alleles D6S2811*128, STR_MICA*A5.1 and D6S2672*109, as well as haplotypes D6S2811*128-D6S2927*213-D6S2810*340, D6S2927* 221-D6S2810*350-MICA*A5.1, and D6S2810*350-MICA*A5.1-D6S2800* 136 were significantly increased in B27-positive AS patients when compared with B27-positive controls. The results indicated that there may be other gene(s) within the HLA region, especially around locus HLA-B or HLA-Cw, with susceptibility to AS independently of HLA-B27.     

Mutation in PVRL4 gene encoding nectin-4 underlies ectodermal-dysplasia-syndactyly syndrome (EDSS1)

Ectodermal-dysplasia-syndactyly syndrome (EDSS1) is a rare form of ectodermal dysplasia (ED), affecting skin and its appendages mainly hair, teeth and nails. In the present study, we have investigated a large consanguineous Pakistani family with 10 individuals showing features of EDSS1. Human genome was screened using highly polymorphic microsatellite markers to identify the gene causing EDSS1. The disease locus for EDSS1 was assigned to chromosome 1q23.1-q23.3. This region corresponds to 5.63?Mb according to the sequenced based physical map (Build 36.2) of the human genome and flanked by markers D1S1653 and D1S1677. A maximum two-point LOD score of 5.05 was obtained with the marker D1S484. Sequence analysis revealed a homozygous missense mutation (c.635C>G; p.Pro212Arg) in the recently reported PVRL4 gene causing EDSS1. The involvement of mutant nectin-4 in causing EDSS1 may open up interesting prospectives into the role of cell adhesion molecules in causing syndromic forms of EDs.     

Charcot-Marie-Tooth neuropathy type 1A mutation: apparent crossovers with D17S122 are due to a duplication.

A locus for the slow conducting form of Charcot-Marie-Tooth neuropathy (CMT1A) was localised to the proximal short arm of chromosome 17, in band p11.2, distal to D17S58. Linkage studies of CMT1A in 3 large Australian families with the marker loci D17S58, D17S71, and D17S57 suggested the order, pter-CMT1A-D17S71-D17S58-centromere-D17S57. However, the estimate of the recombination fraction between CMT1A and D17S122, also assigned to p11.2, was incompatible with known map distances. The impasse was resolved when the D17S122 genotypes were revised to take into account a dosage effect due to a duplication. After correction of the genotypes, the maximum lod score between CMT1A and D17S122 increased from 0.53 at a recombination fraction of 0.3 to 34.28 at zero recombination. This result emphasizes that genotypes for markers in the p12-p11.2 region should be examined very carefully as ignoring the duplication changes the linkage results dramatically. The fact that no crossovers were found between CMT1A and D17S122 suggests that the duplication may cause the disease phenotype.     

High-resolution genetic and physical map of the Lgn1 interval in C57BL/6J implicates Naip2 or Naip5 in Legionella pneumophila pathogenesis

Prior genetic and physical mapping has shown that the Naip gene cluster on mouse chromosome 13D1-D3 contains a gene, Lgn1, that is responsible for determining the permissivity of ex vivo macrophages to Legionella pneumophila replication. We have identified differences in the structure of the Naip array among commonly used inbred mouse strains, although these gross structural differences do not correlate with differences in L. pneumophila permissiveness. A physical map of the region employing clones of the C57BL/6J haplotype confirms that there are fewer copies of Naip in this strain than are in the physical map of the 129 haplotype. We have also refined the genetic location of Lgn1, leaving only Naip2 and Naip5 as candidates for Lgn1. Our genetic map suggests the presence of two hotspots of recombination within the Naip array, indicating that the 3' portion of Naip may be involved in the genomic instability at this locus.