Evidence for the involvement of proximal chromosome 3P loci in the progression of Von Hippel-Lindau related tumors

Von Hippel-Lindau (VHL) disease is an autosomal dominant disorder characterized by the development of retinal and central nervous system hemangioblastoma, renal cell carcinoma (RCC), pheochromocytoma and pancreatic islet cell tumors (PICT). The VHL gene maps to chromosome 3p25 and has been shown to be mutated in 57% of sporadic cases of RCC, implicating VHL in the genesis of RCC. We report a multigeneration VHL kindred in which four affected female siblings developed PICT at early ages. Analysis of the three coding exons of the VHL gene in this family revealed a single, missense mutation in codon 238. Inheritance of the 238 mutation has been reported to correlate with a 62% risk of pheochromocytoma development. In this kindred, all affected individuals carried the mutation as well as one additional sibling who showed no evidence of disease. Clinical screening of this individual indicated small ($<$1 cm) pancreatic and kidney tumors. Results suggest that inheritance of the codon 238 mutation does not correlate with early onset pheochromocytoma. Rather, the only individual in the pedigree with pheochromocytoma was the proband's mother who developed bilateral pheochromocytoma at the age of 62. Thus, the VHL codon 238 mutation may predispose to late onset pheochromocytoma in this family; however, it does not explain the preponderance of PICT in the third generation since this mutation has not been reported to increase the risk of developing pancreatic lesions. This suggests that inheritance of the codon 238 mutation and subsequent somatic inactivation of the wild type allele of the VHL gene may not be sufficient to explain the initiation and subsequent progression to malignancy in VHL-associated neoplasms. Since the two tumor types that most frequently progress to malignancy are RCC and PICT, we asked whether loss of heterozygosity (LOH) could be detected proximal to the VHL gene on chromosome 3 in distinct regions of 3p previously implicated by LOH and cytogenetic studies to contain tumor suppressor loci for RCC. LOH was performed on high molecular weight DNA isolated from peripheral blood and frozen tumor tissue of family members using microsatellite markers spanning 3p. Results indicated LOH for all informative 3p loci in tumor tissue from affected individuals with PICT. LOH was detected along the entire length of the chromosome arm and included the proximal region of 3p13-14.2 implicated in the hereditary form of renal cell carcinoma.^ If 3p LOH were a critical event in pancreatic islet cell tumorigenesis, then it should be expected that LOH in sporadic islet cell tumors would also be observed. We expanded LOH studies to include sporadic cases of PICT. Consistent LOH was observed on 3p with a highest frequency LOH in the region 3p21.2. This is the first evidence for an association between chromosome 3 loci and pancreatic islet cell tumorigenesis. (Abstract shortened by UMI.) ^

Genetic predisposition to prostate cancer

Prostate cancer (PC) is a significant economic and health burden in the U.S. and Europe but its causes are largely unknown. The most significant risk factors (after gender) are age and family history of the disease. A gene with high penetrance but low frequency on chromosome 1q, HPC 1, has been suggested to cause a proportion of the familial aggregation of PC but other more common genes, conferring less risk, are also thought to contribute to disease predisposition. We have pursued a strategy to study both types of genetic risk in PC. To identify high penetrance genes, affected men from thirteen families have been genotyped for genetic linkage analysis at six microsatellite markers spanning 45 cM of 1q24-25. Both LOD score and non-parametric statistics provide no significant support for HPC1 in this genomic region, although 3 of the families did combine to produce a LOD score of 0.9. These families will be included in a genome wide search for other PC predisposition genes as part of a multinational collaboration.^ For study of common genetic factors in PC development, leukocyte DNA samples from an unselected series of 55 patients and 67 controls have been examined for genetic differences in two other candidate genes, the androgen receptor gene, hAR, at Xq11-12, and the vitamin D receptor gene, hVDR, at 12q12-14. hAR was typed for two trinucleotide repeat length polymorphisms, (CAG)$\rm\sb{n}$ and (GGC)$\rm\sb{n},$ encoding polyglutamine and polyglycine tracts, respectively, which have been implicated in PC susceptibility. These data, combined with similarly processed patients and controls from the U.K. show no consistent association of allele length with PC risk. A novel finding, however, has been a significant association between the number of GGC repeats and the length of time between diagnosis and relapse in stage T1-T4 Caucasian patients irrespective of therapy and age of the patient. Of 49 patients who relapsed out of 108 entering the study, those with 16 or fewer GGC repeats had an average relapse-free-period of 101 (+/$-$7.7) months while for those with more than 16 repeats the period averaged 48 (+/$-$2.9) months, a difference of 2.1 fold or 4.4 years.^ The second gene, hVDR, was genotyped at two polymorphisms, a synonymous C/T substitution in exon 9 identified by differential TaqI enzymatic digestion and a variable length polyA tract in the 3$\sp\prime$ UTR. Although these polymorphisms are in strong linkage disequilibrium only the polyA region showed a possible association with PC risk. Men homozygous for alleles with fewer than 18 A's had an increased risk (OR = 3.0, p = 0.0578) compared to controls. This result is opposite to the findings of others and may either indicate off-setting random errors which together balance out to no significant overall effect or reflect more complex genetic and/or environmental associations.^ Overall, this research suggests that single gene familial predisposition may be less prominent in PC than in other cancers and that the characteristics of PC pathology may be useful in identifying the effects of common genetic factors. ^

Linkage and association of candidate obesity genes in Mexican-Americans from Starr County, Texas

Obesity and related chronic diseases represent a tremendous public health burden among Mexican Americans, a young and rapidly-expanding population. This study investigated the impact of variation within eight candidate obesity genes, which include leptin (LEP), leptin receptor (LEPR), neuropeptide Y (NPY), NPYY1 receptor (NPYY1), glucagon-like peptide-1 (GLP-1), GLP-1 receptor (GLP1R), beta-3 adrenergic receptor (β3AR), and uncoupling protein (UCP1), on variation in human obesity status and/or quantitative traits related to obesity in Mexican Americans from Starr County, Texas. The Trp64Arg polymorphism within β3AR was typed in 820 random individuals and 240 pedigrees (N = 2,044). The Arg allele frequency was significantly greater in obese versus non-obese individuals (0.20 versus 0. 15, respectively). In addition, within the random sample, the Arg allele was associated with significantly greater body weight (p = 0.031) and body mass index (BMI, p = 0.008) than the Trp allele. In the family sample, the Trp64Arg locus was also linked to percent fat (p = 0.045) but not to body weight or BMI. No linkage between obesity, diabetes, hypertension, or gallbladder disease and the Trp64Arg mutation was observed in families using affected sib pair linkage analysis or the transmission disequilibrium test. Microsatellite markers proximate to the remaining seven genes were typed in 302 individuals from 59 families. Sib pair linkage analysis provided evidence for linkage between obesity and NPY within affected sibling pairs (p = 0.042; n = 170 pairs). NPY was also linked to weight (p = 0.020), abdominal circumference (p = 0.031), hip circumference (p = 0.012), DBP (p ≤ 0.005), and a composite measure of body mass/fat (p ≤ 0.048) in all sibling pairs (n = 545 pairs). Additionally, LEP was linked to waist/hip ratio (p ≤ 0.009), total cholesterol (p ≤ 0.030), and HDL cholesterol (p ≤ 0.026), and LEPR was linked to fasting blood glucose (p ≤ 0.018) and DBP (p ≤ 0.003). Subsequent to the linkage analyses, the NPY gene was sequenced and eight variant sites identified. Two variant sites (-880I/D and 69I/D) were typed in a random sample of 914 individuals. The 880I/D variant was significantly associated with waist/hip ratio (p = 0.035) in the entire sample (N = 914) and with BMI (p = 0. 031), abdominal circumference (p = 0.044), and waist/hip ratio (p = 0.041) in a non-obese subsample (BW < 30 kg/m2, n = 594). The 69I/D variant was a rare mutation observed in only one pedigree and was not associated with obesity or body size/mass within this pedigree. Results of this study indicate that variation at or near β3AR, LEP, LEPR, and NPY may exert effects which increase obesity susceptibility and influence obesity-related measures in this population. ^

A novel myeloid leukemia suppressor locus at human chromosome 5q13.3

Molecular mechanisms that underlie preleukemic myelodysplasia (MDS) and acute myelogenous leukemia (AML) are poorly understood. In MDS or AML with a refractory clinical course, more than 30% of patients have acquired interstitial or complete deletions of chromosome 5. The 5q13.3 chromosomal segment is commonly lost as the result of 5q deletion. Reciprocal and unbalanced translocations of 5q13.3 can also occur as sole anomalies associated with refractory AML or MDS. This study addresses the hypothesis that a critical gene at 5q13.3 functions either as a classical tumor suppressor or as a chromosomal translocation partner and contributes to leukemogenesis. ^ Previous studies from our laboratory delineated a critical region of loss to a 2.5–3.0Mb interval at 5q13.3 between microsatellite markers D5S672 and GATA-P18104. The critical region of loss was later resolved to an interval of approximately 2Mb between the markers D5S672 and D5S2029. I, then generated a long range physical map of yeast artificial chromosomes (YACs) and developed novel sequence tagged sites (STS). To enhance the resolution of this map, bacterial artificial chromosomes (BACs) were used to construct a triply linked contig across a 1 Mb interval. These BACs were used as probes for fluorescent in situ hybridization (FISH) on an AML cell line to define the 5q13.3 critical region. A 200kb BAC, 484a9, spans the translocation breakpoint in this cell line. A novel gene, SSDP2 (single stranded DNA binding protein), is disrupted at the breakpoint because its first four exons are encoded within 140kb of BAC 484a9. This finding suggests that SSDP2 is the critical gene at 5q13.3. ^ In addition, I made an observation that deletions of chromosome 5q13 co-segregate with loss of the chromosome 17p. In some cases the deletions result from unbalanced translocations between 5q13 and 17p13. It was confirmed that the TP53 gene is deleted in patients with 17p loss, and the remaining allele harbors somatic mutation. Thus, the genetic basis for the aggressive clinical course in AML and MDS may be caused by functional cooperation between deletion or disruption of the 5q13.3 critical gene and inactivation of TP53. ^

Identification and evaluation of candidate genes for inherited retinal -degenerative diseases

Although more than 100 genes associated with inherited retinal disease have been mapped to chromosomal locations, less than half of these genes have been cloned. This text includes identification and evaluation of candidate genes for three autosomal dominant forms of inherited retinal degeneration: atypical vitelliform macular dystrophy (VMD1), cone-rod dystrophy (CORD), and retinitis pigmentosa (RP). ^ VMD1 is a disorder characterized by complete penetrance but extremely variable expressivity, and includes macular or peripheral retinal lesions and peripappilary abnormalitites. In 1984, linkage was reported between VMD1 and soluble glutamate-pyruvate transaminase GPT); however, placement of GPT to 8q24 on linkage maps had been debated, and VMD1 did not show linkage to microsatellite markers in that region. This study excluded linkage between the loci by cloning GPT, identifying the nucleotide substitution associated with the GPT sozymes, and by assaying VMD1 family samples with an RFLP designed to detect the substitution. In addition, linkage of VMD1 to the known dominant macular degeneration loci was excluded. ^ CORD is characterized by early onset of color-vision deficiency, and decreased visual acuity, However, this retinal degeneration progresses to no light perception, severe macular lesion, and “bone-spicule” accumulations in the peripheral retina. In this study, the disorder in a large Texan family was mapped to the CORD2 locus of 19q13, and a mutation in the retina/pineal-specific cone-rod homeobox gene (CRX) was identified as the disease cause. In addition, mutations in CRX were associated with significantly different retinal disease phenotypes, including retinitis pigmentosa and Leber congenital amaurosis. ^ Many of the mutations leading to inherited retinal disorders have been identified in genes like CRX, which are expressed predominantly in the retina and pineal gland. Therefore, a combination of database analysis and laboratory investigation was used to identify 26 novel retina/pineal-specific expressed sequence tag (EST) clusters as candidate genes for inherited retinal disorders. Eight of these genes were mapped into the candidate regions of inherited retinal degeneration loci. ^ Two of the eight clusters mapped into the retinitis pigmentosa RP13 candidate region of 17p13, and were both determined to represent a single gene that is highly expressed in photoreceptors. This gene, the Ah receptor-interacting like protein-1 (AIPL1), was cloned, characterized, and screened for mutations in RP13 patient DNA samples. ^