Deletions of the long arm of chromosome 10 in progression of follicular thyroid tumors

Previous studies of follicular thyroid tumors have shown loss of heterozygosity (LOH) on the short arm of chromosome 3 in carcinomas, and on chromosome 10 in atypical adenomas and carcinomas, but not in common adenomas. We studied LOH on these chromosomal arms in 15 follicular thyroid carcinomas, 19 atypical follicular adenomas and 6 anaplastic (undifferentiated) carcinomas. Deletion mapping of chromosome 10 using 15 polymorphic markers showed that 15 (37.5%) of the tumors displayed LOH somewhere along the long arm. Thirteen of these tumors showed deletions involving the telomeric part of chromosome 10q, distal to D1OS 187. LOH on chromosome 3p was found in 8 (20%) cases. Seven of these also showed LOH on chromosome 10q. In eight cases LOH was seen on chromosome 10q but not 3p. In comparison, the retinoblastoma gene locus at chromosome 13q showed LOH in 22% of the tumors. Most of these also had deletions on chromosome 10q. The results indicate that a region at the telomeric part of 10q may be involved in progression of follicular thyroid tumors.     

A Common Region of Deletion on Chromosome 17q in Both Sporadic and Familial Epithelial Ovarian Tumors Distal to BRCA1

Linkage analysis in familial breast and ovarian cancer and studies of allelic deletion in sporadic ovarian tumors have identified a region on chromosome 17q containing a candidate tumor-suppressor gene (referred to as BRCA1) of likely importance in ovarian carcinogenesis. We have examined normal and tumor DNA samples from 32 patients with sporadic and 8 patients with familial forms of the disease, for loss of heterozygosity (LOH) at 21 loci on chromosome 17 (7 on 17p and 14 on 17q). LOH on 17p was 55% (22/40) for informative 17pl3.1 and 17pl3.3 markers. When six polymorphic markers flanking the familial breast/ovarian cancer susceptibility locus on 17ql2-q21 were used, LOH was 58% (23/40), with one tumor showing telomeric retention. Evaluation of a set of markers positioned telomeric to BRCA1 resulted in the highest degree of LOH, 73% (29/40), indicating that a candidate locus involved in ovarian cancer may reside distal to BRCA1. Five of the tumors demonstrating allelic loss for 17q markers were from individuals with a strong family history of breast and ovarian cancer. More important, two of these tumors (unique patient number [UPN] 57 and UPN 79) retained heterozygosity for all informative markers spanning the BRCA1 locus but showed LOH at loci distal to but not including the anonymous markers CMM86 (D17S74) and 42D6 (D17S588), respectively. Deletion mapping of seven cases (two familial and five sporadic) showing limited LOH on 17q revealed a common region of deletion, distal to GH and proximal to D17S4, that spans −25 cM. These results suggest that a potential tumor-suppressor gene involved in both sporadic and familial ovarian cancer may reside on the distal portion of chromosome 17q and is distinct from the BRCA1 gene.     

Loss of NOTCH2 positively predicts survival in subgroups of human glial brain tumors

The structural complexity of chromosome 1p centromeric region has been an obstacle for fine mapping of tumor suppressor genes in this area. Loss of heterozygosity (LOH) on chromosome 1p is associated with the longer survival of oligodendroglioma (OD) patients. To test the clinical relevance of 1p loss in glioblastomas (GBM) patients and identifiy the underlying tumor suppressor locus, we constructed a somatic deletion map on chromosome 1p in 26 OG and 118 GBM. Deletion hotspots at 4 microsatellite markers located at 1p36.3, 1p36.1, 1p22 and 1p11 defined 10 distinct haplotypes that were related to patient survival. We found that loss of 1p centromeric marker D1S2696 within NOTCH2 intron 12 was associated with favorable prognosis in OD (P = 0.0007) as well as in GBM (P = 0.0175), while 19q loss, concomitant with 1p LOH in OD, had no influence on GBM survival (P = 0.918). Assessment of the intra-chromosomal ratio between NOTCH2 and its 1q21 pericentric duplication N2N (N2/N2N-test) allowed delineation of a consistent centromeric breakpoint in OD that also contained a minimally lost area in GBM. OD and GBM showed distinct deletion patterns that converged to the NOTCH2 gene in both glioma subtypes. Moreover, the N2/N2N-test disclosed homozygous deletions of NOTCH2 in primary OD. The N2/N2N test distinguished OD from GBM with a specificity of 100% and a sensitivity of 97%. Combined assessment of NOTCH2 genetic markers D1S2696 and N2/N2N predicted 24-month survival with an accuracy (0.925) that is equivalent to histological classification combined with the D1S2696 status (0.954) and higher than current genetic evaluation by 1p/19q LOH (0.762). Our data propose NOTCH2 as a powerful new molecular test to detect prognostically favorable gliomas.     

Construction of a 5-Mb YAC Contig from the Putative 10q25 Tumor-Suppressor Region for Glioblastomas

During the final step of the malignant progression to glioblastoma multiforme (GBM), the most frequent and malignant of primary brain tumors, more than 90% of the cases exhibit loss of genetic material on chromosome 10. We previously identified a 4-cM deletion interval in the 10q24–qter region that is common to all the GBM we have examined. A contig of 20 YACs spanning the 5 Mb of chromosomal DNA in the region has been assembled. Overlaps between YACs have been verified by STS content, fingerprinting analysis, and/orAlu–AluPCR. The contig contains 17 known microsatellite markers, 15 new STSs derived from the insert ends of YACs, 9 ESTs, and 11 other STSs, for a total of 52 STSs (average marker density 1/100 kb). The physical map of this region will facilitate the search for a candidate tumor-suppressor gene(s) that is inactivated during the formation of GBM.     

A sequence-ready BAC clone contig of human chromosome 10p15 spanning the loss of heterozygosity region in glioma

Deletion of chromosome 10 is one of the most common chromosomal alterations in glioma. At 10p15, the telomeric region of the short arm of chromosome 10, loss of heterozygosity (LOH) has been frequently observed by microsatellite analysis, suggesting the presence of a tumor suppressor gene. We examined LOH in 34 gliomas on chromosome 10, and frequent LOH on 10p was detected on 10p15, in agreement with deletion mapping studies on chromosome 10. We then constructed a bacterial artificial chromosome (BAC) clone contig covering the critical region, which spanned the interval between D10S249 and D10S533 on 10p15. The map contained 68 BAC clones connected by 74 sequenced tag sites (STSs) and covered approximately 2.7 Mb, with one gap. A total of 74 STSs, including 6 microsatellite markers, 29 expressed sequenced tags (ESTs), and 39 BAC end STSs, were physically arranged. Twenty-eight ESTs were mapped in the interval between D10S249 and D10S559 (approximately 1200 kb), and another EST was mapped in the interval between D10S559 and D10S533 (approximately 1300 kb). This sequence-ready BAC clone contig map will be a basic resource for high-quality sequencing and positional cloning of the putative tumor suppressor gene at 10p15 in glioma.     

Linkage disequilibrium of three polymorphic RFLP markers in the apolipoprotein AI-CIII gene cluster on chromosome 11

A panel of glial tumors consisting of 11 low grade gliomas, 9 anaplastic gliomas, and 29 glioblastomas were analyzed for loss of heterozygosity by examining at least one locus for each chromosome. The frequency of allele loss was highest among the glioblastomas, suggesting that genetic alterations accumulate during glial tumor development. The most common genetic alteration detected involved allele losses of chromosome 10 loci; these losses were observed in all glioblastomas and in three of the anaplastic gliomas. In order to delineate which chromosome 10 region or regions were deleted in association with glial tumor development, a deletion mapping analysis was performed, and this revealed the partial loss of chromosome 10 in eight glioblastomas and two of the anaplastic gliomas. Among these cases, three distinct regions of chromosome 10 were indicated as being targeted for deletion: one telomeric region on 10p and both telomeric and centromeric locations on 10q. These data suggest the existence of multiple chromosome 10 tumor suppressor gene loci whose inactivation is involved in the malignant progression of glioma.     

Physical Mapping of a Commonly Deleted Region, the Site of a Candidate Tumor Suppressor Gene, at 12q22 in Human Male Germ Cell Tumors

A candidate tumor suppressor gene (TSG) site at 12q22 characterized by a high frequency of loss of heterozygosity (LOH) and a homozygous deletion has previously been reported in human male germ cell tumors (GCTs). In a detailed deletion mapping analysis of 67 normal-tumor DNAs utilizing 20 polymorphic markers mapped to 12q22–q24, we identified the limits of the minimal region of deletion at 12q22 between D12S377 (proximal) and D12S296 (distal). We have constructed a YAC contig map of a 3-cM region of this band between the proximal marker D12S101 and the distal marker D12S346, which contained the minimal region of deletion in GCTs. The map is composed of 53 overlapping YACs and 3 cosmids onto which 25 polymorphic and nonpolymorphic sequence-tagged sites (STSs) were placed in a unique order. The size of the minimal region of deletion was approximately 2 Mb from overlapping, nonchimeric YACs that spanned the region. We also developed a radiation hybrid (RH) map of the region between D12S101 and D12S346 containing 17 loci. The consensus order developed by RH mapping is in good agreement with the YAC STS-content map order. The RH map estimated the distance between D12S101 and D12S346 to be 246 cR₈₀₀₀and the minimal region of deletion to be 141 cR₈₀₀₀. In addition, four genes that were previously mapped to 12q22 have been excluded as candidate genes. The leads gained from the deletion mapping and physical maps should expedite the isolation and characterization of the TSG at 12q22.     

Homozygosity and linkage-disequilibrium mapping of the urofacial (Ochoa) syndrome gene to a 1-cM interval on chromosome 10q23-q24.

The urofacial (Ochoa) syndrome (UFS) is a rare autosomal recessive disease characterized by congenital obstructive uropathy and abnormal facial expression. The patients present with enuresis, urinary-tract infection, hydronephrosis, and voiding dysfunctions as a result of neurogenic bladders. To map the UFS gene, a genome screen using a combination of homozygosity-mapping and DNA-pooling strategies was performed in 20 selected patients, one patient pool, and three control pools (unaffected relatives). After analyses of 36 randomly chosen markers, D10S677 was identified as being linked to and associated with UFS, as suggested by a significant excess of homozygosity in patients compared with that in unaffected relatives (P < 10(-6)), as well as by the allelic-frequency differences between the patient pool and control pools. Ten additional markers flanking D10S677 and covering a 22-cM region then were analyzed to fine-map the UFS gene by use of haplotype (linkage disequilibrium) analysis. All 31 patients were found to be homozygous for two closely linked markers (D10S1726 and D10S198) located approximately 5 cM telomeric to D10S677, whereas only 12% of the unaffected relatives were homozygous for both markers (P < 10(-19)). Several patients are heterozygous at two markers immediately flanking D10S1726/D10S198, one on the centromeric side (D10S1433) and the other on the telomeric side (D10S603). These recombinational events place the UFS gene near D10S1726/D10S198 and within a 1-cM interval defined by D10S1433 and D10S603 on chromosome 10q23-q24.     

A novel form of "central pouchlike" cataract, with sutural opacities, maps to chromosome 15q21-22

Congenital cataract is a clinically and genetically highly heterogeneous eye disorder, with autosomal dominant inheritance being most common. We investigated a large seven-generation family with 74 individuals affected by autosomal dominant congenital cataract (ADCC). The phenotype in this family can be described as "central pouchlike" cataract with sutural opacities, and it differs from the other mapped cataracts. We performed linkage analysis with microsatellite markers in this family and excluded the known candidate genes. A genomewide search revealed linkage to markers on chromosome 15, with a maximum two-point LOD score of 5.98 at straight theta=0 with marker D15S117. Multipoint analysis also gave a maximum LOD score of 5.98 at D15S117. Multipoint and haplotype analysis narrowed the cataract locus to a 10-cM region between markers D15S209 and D15S1036, closely linked to marker D15S117 in q21-q22 region of chromosome 15. This is the first report of a gene for a clinically new type of ADCC at 15q21-22 locus.     

Genomic profiling maps loss of heterozygosity and defines the timing and stage dependence of epigenetic and genetic events in Wilms' tumors

To understand genetic and epigenetic pathways in Wilms' tumors, we carried out a genome scan for loss of heterozygosity (LOH) using Affymetrix 10K single nucleotide polymorphism (SNP) chips and supplemented the data with karyotype information. To score loss of imprinting (LOI) of the IGF2 gene, we assessed DNA methylation of the H19 5' differentially methylated region (DMR). Few chromosomal regions other than band 11p13 (WT1) were lost in Wilms' tumors from Denys-Drash and Wilms' tumor-aniridia syndromes, whereas sporadic Wilms' tumors showed LOH of several regions, most frequently 11p15 but also 1p, 4q, 7p, 11q, 14q, 16q, and 17p. LOI was common in the sporadic Wilms' tumors but absent in the syndromic cases. The SNP chips identified novel centers of LOH in the sporadic tumors, including a 2.4-Mb minimal region on chromosome 4q24-q25. Losses of chromosomes 1p, 14q, 16q, and 17p were more common in tumors presenting at an advanced stage; 11p15 LOH was seen at all stages, whereas LOI was associated with early-stage presentation. Wilms' tumors with LOI often completely lacked LOH in the genome-wide analysis, and in some tumors with concomitant 16q LOH and LOI, the loss of chromosome 16q was mosaic, whereas the H19 DMR methylation was complete. These findings confirm molecular differences between sporadic and syndromic Wilms' tumors, define regions of recurrent LOH, and indicate that gain of methylation at the H19 DMR is an early event in Wilms' tumorigenesis that is independent of chromosomal losses. The data further suggest a biological difference between sporadic Wilms' tumors with and without LOI.     

An autosomal recessive nonsyndromic-hearing-loss locus identified by DNA pooling using two inbred Bedouin kindreds.

Autosomal recessive nonsyndromic hearing loss (ARNSHL) is the most common form of severe inherited childhood deafness. We present the linkage analysis of two inbred Bedouin kindreds from Israel that are affected with ARNSHL. A rapid genomewide screen for markers linked to the disease was performed by using pooled DNA samples. This screen revealed evidence for linkage with markers D9S922 and D9S301 on chromosome 9q. Genotyping of individuals from both kindreds confirmed linkage to chromosome 9q and a maximum combined LOD score of 26.2 (recombination fraction [theta] .025) with marker D9S927. The disease locus was mapped to a 1.6-cM region of chromosome 9ql3-q2l, between markers D9S15 and D9S927. The disease segregates with a common haplotype in the two kindreds, at markers D9S927, D9S175, and D9S284 in the linked interval, supporting the hypothesis that both kindreds inherited the deafness gene from a common ancestor. Although this nonsyndromic-hearing-loss (NSHL) locus maps to the same cytogenetic interval as DFNB7, it does not overlap the currently defined DFNB7 interval and may represent (1) a novel form of NSHL in close proximity to DFNB7 or (2) a relocalization of the DFNB7 interval to a region telomeric to its reported location. This study further demonstrates that DNA pooling is an effective means of quickly identifying regions of linkage in inbred families with heterogeneous autosomal recessive disorders.     

Allelic loss is frequent in tuberous sclerosis kidney lesions but rare in brain lesions.

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterized by seizures, mental retardation, and hamartomatous lesions. Although hamartomas can occur in almost any organ, they are most common in the brain, kidney, heart, and skin. Allelic loss or loss of heterozygosity (LOH) in TSC lesions has previously been reported on chromosomes 16p13 and 9q34, the locations of the TSC2 and TSC1 genes, respectively, suggesting that the TSC genes act as tumor-suppressor genes. In our study, 87 lesions from 47 TSC patients were analyzed for LOH in the TSC1 and TSC2 chromosomal regions. Three findings resulted from this analysis. First, we confirmed that the TSC1 critical region is distal to D9S149. Second, we found LOH more frequently on chromosome 16p13 than on 9q34. Of the 28 patients with angiomyolipomas or rhabdomyomas, 16p13 LOH was detected in lesions from 12 (57%) of 21 informative patients, while 9q34 LOH was detected in lesions from only 1 patient (4%). This could indicate that TSC2 tumors are more likely than TSC1 tumors to require surgical resection or that TSC2 is more common than TSC1 in our patient population. It is also possible that small regions of 9q34 LOH were missed. Lastly, LOH was found in 56% of renal angiomyolipomas and cardiac rhabdomyormas but in only 4% of TSC brain lesions. This suggests that brain lesions can result from different pathogenic mechanisms than kidney and heart lesions.     

DFNA25, a novel locus for dominant nonsyndromic hereditary hearing impairment, maps to 12q21-24

Using linkage analysis, we identified a novel dominant locus, DFNA25, for delayed-onset, progressive, high-frequency, nonsyndromic sensorineural hearing loss in a large, multigenerational United States family of Czech descent. On the basis of recombinations in affected individuals, we determined that DFNA25 is located in a 20-cM region of chromosome 12q21-24 between D12S327 (centromeric) and D12S84 (telomeric), with a maximum two-point LOD score of 6.82, at recombination fraction.041, for D12S1030. Candidate genes in this region include ATP2A2, ATP2B1, UBE3B, and VR-OAC. DFNA25 may be the human ortholog of bronx waltzer (bv).     

A sequence-ready BAC/PAC contig and partial transcript map of approximately 1.5 Mb in human chromosome 17q25 comprising multiple disease genes

Hereditary neuralgic amyotrophy (HNA) is an autosomal dominant recurrent neuropathy mapped to a 4-cM interval on chromosome 17q25 between the short tandem repeat (STR) markers D17S1603 and D17S802. Chromosome 17q25 in general and the 4-cM HNA region in particular are also implicated in the pathogenesis of a number of tumors (tylosis with esophageal cancer, sporadic breast and ovarian tumors) and harbor a psoriasis susceptibility locus. Initial attempts to construct a yeast artificial chromosome contig failed. Therefore, we have now constructed a complete P1 artificial chromosome (PAC) and bacterial artificial chromosome (BAC) contig of the region flanked by the STR markers D17S1603 and D17S802. The contig contains 22 PAC and 64 BAC clones and covers a physical distance of approximately 1. 5 Mb. A total of 83 sequence-tagged site (STS) markers (10 known STSs and STRs, 56 STSs generated from clone end-fragments, 12 expressed sequence tags, and 5 known genes) were mapped on the contig, resulting in an extremely dense physical map with approximately 1 STS per 20 kb. This sequence-ready PAC and BAC contig will be pivotal for the positional cloning of the HNA gene as well as other disease genes mapping to this region.     

An 800-kb region of deletion at 13q14 in human prostate and other carcinomas

Deletions of regions at 13q14 have been detected by various genetic approaches in human cancers including prostate cancer. Several studies have defined one region of loss of heterozygosity (LOH) at 13q14 that seems to reside in a DNA segment of 7.1 cM between genetic markers D13S263 and D13S153. To define the smallest region of overlap (SRO) for deletion at 13q14, we first applied tissue microdissection and multiplex PCR to detect homozygous deletion and/or hemizygous deletion at 13q14 in 134 prostate cancer specimens from 114 patients. We detected deletions at markers D13S1227, D13S1272, and A005O48 in 13 (10%) of these tumor specimens. Of the 13 tumors with deletions, 12 were either poorly differentiated primary tumors or metastases of prostate cancer. To fine-map the deletion region, we then constructed a high-resolution YAC/BAC/STS/EST physical map based on experimental and database analyses. Several markers encompassing the deletion region were analyzed for homozygous deletion and/or hemizygous deletion in 61 cell lines/xenografts derived from human cancers of the prostate, breast, ovary, endometrium, cervix, and bladder, and a region of deletion was defined by duplex PCR assay between markers A005X38 and WI-7773. We also analyzed LOH at 13q14 in the 61 cell lines/xenografts using the homozygosity mapping of deletion approach and 26 microsatellite markers. We found 24 (39%) of the cell lines/xenografts to show LOH at 13q14 and defined a region of LOH by markers M1 and M5. Combination of homozygous or hemizygous deletion and LOH results defined the SRO for deletion to be an 800-kb DNA interval between A005X38 and M5. There are six known genes located in or close to the SRO for deletion. This region of deletion is at least 2 Mb centromeric to the RB1 tumor-suppressor gene and the leukemia-associated genes 1 and 2, each of which is located at 13q14. These data suggest that the 800-kb DNA segment with deletion contains a gene whose deletion may be important for the development of prostate and other cancers. This study also provides a framework for the fine-mapping, cloning, and identification of a novel tumor-suppressor gene at 13q14.     

Genetic and radiation hybrid mapping of the hyperekplexia region on chromosome 5q.

Hyperekplexia, or startle disease (STHE), is an autosomal dominant neurologic disorder characterized by muscular rigidity of central nervous system origin, particularly in the neonatal period, and by an exaggerated startle response to sudden, unexpected acoustic or tactile stimuli. STHE responds dramatically to the benzodiazepine drug clonazepam, which acts at gamma-aminobutyric acid type A (GABA-A) receptors. The STHE locus (STHE) was recently assigned to chromosome 5q, on the basis of tight linkage to the colony-stimulating factor 1-receptor (CSF1-R) locus in a single large family. We performed linkage analysis in the original and three additional STHE pedigrees with eight chromosome 5q microsatellite markers and placed several of the most closely linked markers on an existing radiation hybrid (RH) map of the region. The results provide strong evidence for genetic locus homogeneity and assign STHE to a 5.9-cM interval defined by CSF1-R and D5S379, which are separated by an RH map distance of 74 centirays (roughly 2.2-3.7 Mb). Two polymorphic markers (D5S119 and D5S209) lie within this region, but they could not be ordered with respect to STHE. RH mapping eliminated the candidate genes GABRA1 and GABRG2, which encode GABA-A receptor components, by showing that they are telomeric to the target region.     

Differential loss of heterozygosity in familial, sporadic, and uremic hyperparathyroidism

Various genetic loci harboring oncogenes, tumor suppressor genes, and genes for calcium receptors have been implicated in the development of parathyroid tumors. We have carried out loss of heterozygosity (LOH) studies in chromosomes 1p, 1q, 3q, 6q, 11q, 13q, 15q, and X in a total of 89 benign parathyroid tumors. Of these, 28 were sporadic parathyroid adenomas from patients with no family history of the disease, 41 were secondary parathyroid tumors, 5 were from patients with a history of previous irradiation to the neck, 12 were from patients with a family history of hyperparathyroidism, and 3 were parathyroid tumors related to multiple endocrine neoplasia type 1 (MEN1). In addition, we determined the chromosomal localization of a second putative calcium-sensing receptor, CaS, for inclusion in the LOH studies. Based on analysis of somatic cell hybrids and fluorescent in situ hybridization to metaphase chromsomes, the gene for CaS was mapped to chromosomal region 2q21-q22. The following results were obtained from the LOH studies: (1) out of the 24 tumors that showed LOH, only 4 had more than one chromosomal region involved, (2) in the tumours from uremic patients, LOH of chromosome 3q was detected in a subset of the tumors, (3) LOH of the MEN1 region at 11q13 was the most common abnormality found in both MEN1-related and sporadic parathyroid tumours but was not a feature of the other forms of parathyroid tumors, (4) LOH in 1p and 6q was not as frequent as previously reported, and (5) tumor suppressor genes in 1q and X might have played a role, particularly on the X chromosome, in the case of familial parathyroid adenomas. We therefore conclude that the tumorigenesis of familial, sporadic, and uremic hyperparathyroidism involves different genetic triggers in a non-progressive pattern. Received: 28 October 1996 / Revised: 16 November 1996     

Molecular genetic investigations of the mechanism of tumourigenesis in von Hippel-Lindau disease: analysis of allele loss in VHL tumours

Von Hippel-Lindau (VHL) disease is a dominantly inherited familial cancer syndrome characterised by the development of retinal and central nervous system haemangioblastomas, renal cell carcinoma (RCC), phaeochromocytoma and pancreatic tumours. The VHL disease gene maps to chromosome 3p25-p26. To investigate the mechanism of tumourigenesis in VHL disease, we analysed 24 paired blood/tumour DNA samples from 20 VHL patients for allele loss on chromosome 3p and in the region of tumour suppressor genes on chromosomes 5, 11, 13, 17 and 22. Nine out of 24 tumours showed loss of heterozygosity (LOH) at at least one locus on chromosome 3p and in each case the LOH included the region to which the VHL gene has been mapped. Chromosome 3p allele loss was found in four tumour types (RCC, haemangioblastoma, phaeochromocytoma and pancreatic tumour) suggesting a common mechanism of tumourigenesis in all types of tumour in VHL disease. The smallest region of overlap was between D3S1038 and D3S18, a region that corresponds to the target region for the VHL gene from genetic linkage studies. The parental origin of the chromosome 3p25-p26 allele loss could be determined in seven tumours from seven familial cases; in each tumour, the allele lost had been inherited from the unaffected parent. Our results suggest that the VHL disease gene functions as a recessive tumour suppressor gene and that inactivation of both alleles of the VHL gene is the critical event in the pathogenesis of VHL neoplasms. Four VHL tumours showed LOH on other chromosomes (5q21, 13q, 17q) indicating that homozygous VHL gene mutations may be required but may not be sufficient for tumourigenesis in VHL disease.     

A physical and transcript map based upon refinement of the critical interval for PPH1, a gene for familial primary pulmonary hypertension. The International PPH Consortium

Primary pulmonary hypertension (PPH), an often fatal disorder, is characterized by sustained elevation of pulmonary artery pressure of unknown cause. In its familial form (FPPH), the disorder segregates as an autosomal dominant and displays markedly reduced penetrance. A gene for FPPH was previously localized to a 25-cM interval on the long arm of chromosome 2 (2q31-q33). We now report a complete yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC)/P1 artificial chromosome contig (PAC), assembled by STS content mapping, across a newly identified minimum nonrecombinant interval containing the gene designated PPH1. The physical map has served to establish polymorphic marker order unequivocally, enabling the establishment of detailed haplotypes for the region. Together with the identification of novel recombination events in affected individuals from six newly ascertained kindreds, these data have allowed the significant reduction of the minimum PPH1 critical interval to a 4.8-cM region. The region, flanked by the polymorphic markers D2S115 (centromeric) and D2S1384 (telomeric), corresponds to a minimum physical distance of 5.8 Mb at 2q33. Numerous expressed sequence tags and known genes were placed on the YAC/BAC contig spanning the PPH1 gene critical region.     

Localization of multiple melanoma tumor-suppressor genes on chromosome 11 by use of homozygosity mapping-of-deletions analysis

Loss-of-heterozygosity (LOH) studies have implicated one or more chromosome 11 tumor-suppressor gene(s) in the development of cutaneous melanoma as well as a variety of other forms of human cancer. In the present study, we have identified multiple independent critical regions on this chromosome by use of homozygosity mapping of deletions (HOMOD) analysis. This method of analysis involved the use of highly polymorphic microsatellite markers and statistics to identify regions of hemizygous deletion in unmatched melanoma cell line DNAs. Regions of loss were defined by the presence of an extended region of homozygosity (ERH) at > or =5 adjacent markers and having a statistical probability of < or =.001. Significant ERHs were similar in nature to deletions identified by LOH analyses performed on uncultured melanomas, although a higher frequency of loss (24 [60%] of 40 vs. 16 [34%] of 47) was observed in the cell lines. Overall, six small regions of overlapping deletions (SROs) were identified on chromosome 11 flanked by the markers D11S1338/D11S907 (11p13-15.5 [SRO1]), D11S1344/D11S11385 (11p11.2 [SRO2]), D11S917/D11S1886 (11q21-22.3 [SRO3]), D11S927/D11S4094 (11q23 [SRO4]), AFM210ve3/D11S990 (11q24 [SRO5]), and D11S1351/D11S4123 (11q24-25 [SRO6]). We propose that HOMOD analysis can be used as an adjunct to LOH analysis in the localization of tumor-suppressor genes.