Prostate cancer susceptibility genes on 8p21-23 in a Dutch population.

BACKGROUND: Prostate cancer is the most commonly diagnosed cancer in men in Europe and the United States. Numerous studies have indicated genetics to have a major role in the aetiology of this disease; as much as 42% of the risk may be explained by heritable factors. Genome-wide association studies have detected an association between prostate cancer and chromosome 8p21-23. In this study, we analysed eight microsatellite (MS) markers in that region in order to confirm previous results and narrow down the location of candidate prostate cancer genes. METHODS: 292 cases and 278 controls were selected from the Netherlands Cohort Study (NLCS). The following MSs were used in the analyses: D8S136, D8S1734, D8S1742, D8S261, D8S262, D8S351, D8S511 and D8S520. Associations were evaluated using a χ(2) test and logistic regression. We checked for any effects on the association by tumour stage. RESULTS: Associations that were found confirmed previous research that pointed to the 8p21-23 region. Two MSs: D8S136 (odds ratio (OR), 0.69; P=4.00 × 10(-28)), and D8S520 (OR, 0.80; P=3.37 × 10(-11)), were consistently and strongly related with prostate cancer. Genotype analysis showed an additive effect for D8S136 (P-trend=6.22 × 10(-03)) and D8S520 (P-trend=2.62 × 10(-22)), suggesting an increased risk for people with a short number of repeats on both alleles at those markers. CONCLUSIONS: This study provides strong evidence that the 8p21-23 region is likely to harbour prostate cancer genes.

The impact of Converso Jews on the genomes of modern Latin Americans.

Modern day Latin America resulted from the encounter of Europeans with the indigenous peoples of the Americas in 1492, followed by waves of migration from Europe and Africa. As a result, the genomic structure of present day Latin Americans was determined both by the genetic structure of the founding populations and the numbers of migrants from these different populations. Here, we analyzed DNA collected from two well-established communities in Colorado (33 unrelated individuals) and Ecuador (20 unrelated individuals) with a measurable prevalence of the BRCA1 c.185delAG and the GHR c.E180 mutations, respectively, using Affymetrix Genome-wide Human SNP 6.0 arrays to identify their ancestry. These mutations are thought to have been brought to these communities by Sephardic Jewish progenitors. Principal component analysis and clustering methods were employed to determine the genome-wide patterns of continental ancestry within both populations using single nucleotide polymorphisms, complemented by determination of Y-chromosomal and mitochondrial DNA haplotypes. When examining the presumed European component of these two communities, we demonstrate enrichment for Sephardic Jewish ancestry not only for these mutations, but also for other segments as well. Although comparison of both groups to a reference Hispanic/Latino population of Mexicans demonstrated proximity and similarity to other modern day communities derived from a European and Native American two-way admixture, identity-by-descent and Y-chromosome mapping demonstrated signatures of Sephardim in both communities. These findings are consistent with historical accounts of Jewish migration from the realms that comprise modern Spain and Portugal during the Age of Discovery. More importantly, they provide a rationale for the occurrence of mutations typically associated with the Jewish Diaspora in Latin American communities.

The frequency of GJB2 and GJB6 mutations in the New York State newborn population: feasibility of genetic screening for hearing defects.

In the US, approximately one in every 1000 children has hearing loss sufficiently severe to interfere with the acquisition of normal speech [Ann NY Acad Sci 630 (1991) 16]. The causes of non-syndromic hearing loss (NSHL) are known to be heterogeneous, with genetic factors accounting for 50-75%[Am J Med Genet 46 (1993) 486]. Often individuals with NSHL thought to be caused by mutations in GJB2 have only one detectable mutant allele [Am J Hum Genet 62 (1998) 792, Hum Mol Genet 6 (12) (1997) 2173]. Another gene that has been identified as a possible cause of NSHL is GJB6 that codes for the gap junction protein, connexin 30. A consecutive series of anonymous newborn dried blood specimens (n = 2089) was tested for two GJB2 mutations: (i) 35delG, a pan-ethnic mutation; and (ii) 167delT, a mutation more frequently found in individuals of Ashkenazi Jewish and Mediterranean descents. Mutation detection was validated using allele-specific oligonucleotide hybridization in single wells. Once the positive samples had been identified, the samples were pooled and retested. All positives in the individual experiment were correctly identified in the pooled experiment. The same random set of anonymous newborn dried blood specimens plus some additional samples were tested (n = 2112) for the 342-kb deletion in the GJB6 gene.

Mendelian diseases among Roman Jews: implications for the origins of disease alleles.

The Roman Jewish community has been historically continuous in Rome since pre-Christian times and may have been progenitor to the Ashkenazi Jewish community. Despite a history of endogamy over the past 2000 yr, the historical record suggests that there was admixture with Ashkenazi and Sephardic Jews during the Middle Ages. To determine whether Roman and Ashkenazi Jews shared common signature mutations, we tested a group of 107 Roman Jews, representing 176 haploid sets of chromosomes. No mutations were found for Bloom syndrome, BRCA1, BRCA2, Canavan disease, Fanconi anemia complementation group C, or Tay-Sachs disease. Two unrelated individuals were positive for the 3849 + 10C->T cystic fibrosis mutation; one carried the N370S Gaucher disease mutation, and one carried the connexin 26 167delT mutation. Each of these was shown to be associated with the same haplotype of tightly linked microsatellite markers as that found among Ashkenazi Jews. In addition, 14 individuals had mutations in the familial Mediterranean fever gene and three unrelated individuals carried the factor XI type III mutation previously observed exclusively among Ashkenazi Jews. These findings suggest that the Gaucher, connexin 26, and familial Mediterranean fever mutations are over 2000 yr old, that the cystic fibrosis 3849 + 10kb C->T and factor XI type III mutations had a common origin in Ashkenazi and Roman Jews, and that other mutations prevalent among Ashkenazi Jews are of more recent origin.

TbetaR-I(6A) is a candidate tumor susceptibility allele.

We have previously described a type I transforming growth factor (TGF)-beta receptor (TbetaR-I) polymorphic allele, TbetaR-I(6A), that has a deletion of three alanines from a nine-alanine stretch. We observed a higher than expected number of TbetaR-I(6A) homozygotes among tumor and nontumor DNA from patients with a diagnosis of cancer. To test the hypothesis that TbetaR-I(6A) homozygosity is associated with cancer, we performed a case-control study in patients with a diagnosis of cancer and matched healthy individuals with no history of cancer and who were identical in their gender and their geographical and ethnic background to determine the relative germ-line frequencies of this allele. We found nine TbetaR-I(6A) homozygotes among 851 patients with cancer. In comparison, there were no TbetaR-I(6A) homozygotes among 735 healthy volunteers (P < 0.01). We also observed an excess of TbetaR-I(6A) heterozygotes in cancer cases compared to controls (14.6% versus 10.6%; P = 0.02, Fisher's exact test). A subset analysis revealed that 4 of 112 patients with colorectal cancer were TbetaR-I(6A) homozygotes (P < 0.01). Using mink lung epithelial cell lines devoid of TbetaR-I, we established stably transfected TbetaR-I and TbetaR-I(6A) cell lines. We found that, compared to TbetaR-I, TbetaR-I(6A) was impaired as a mediator of TGF-beta antiproliferative signals. We conclude that TbetaR-I(6A) acts as a tumor susceptibility allele that may contribute to the development of cancer, especially colon cancer, by means of reduced TGF-beta-mediated growth inhibition.

Mutation and haplotype studies of familial Mediterranean fever reveal new ancestral relationships and evidence for a high carrier frequency with reduced penetrance in the Ashkenazi Jewish population.

Familial Mediterranean fever (FMF) is a recessive disorder characterized by episodes of fever with serositis or synovitis. The FMF gene (MEFV) was cloned recently, and four missense mutations were identified. Here we present data from non-Ashkenazi Jewish and Arab patients in whom we had not originally found mutations and from a new, more ethnically diverse panel. Among 90 symptomatic mutation-positive individuals, 11 mutations accounted for 79% of carrier chromosomes. Of the two mutations that are novel, one alters the same residue (680) as a previously known mutation, and the other (P369S) is located in exon 3. Consistent with another recent report, the E148Q mutation was observed in patients of several ethnicities and on multiple microsatellite haplotypes, but haplotype data indicate an ancestral relationships between non-Jewish Italian and Ashkenazi Jewish patients with FMF and other affected populations. Among approximately 200 anonymous Ashkenazi Jewish DNA samples, the MEFV carrier frequency was 21%, with E148Q the most common mutation. Several lines of evidence indicate reduced penetrance among Ashkenazi Jews, especially for E148Q, P369S, and K695R. Nevertheless, E148Q helps account for recessive inheritance in an Ashkenazi family previously reported as an unusual case of dominantly inherited FMF. The presence of three frequent MEFV mutations in multiple Mediterranean populations strongly suggests a heterozygote advantage in this geographic region.

Mutations in the connexin 26 gene (GJB2) among Ashkenazi Jews with nonsyndromic recessive deafness.

BACKGROUND: Mutations in the GJB2 gene cause one form of nonsyndromic recessive deafness. Among Mediterranean Europeans, more than 80 percent of cases of nonsyndromic recessive deafness result from inheritance of the 30delG mutant allele of GJB2. We assessed the contribution of mutations in GJB2 to the prevalence of the condition among Ashkenazi Jews. METHODS: We tested for mutations in GJB2 in DNA samples from three Ashkenazi Jewish families with nonsyndromic recessive deafness, from Ashkenazi Jewish persons seeking carrier testing for other conditions, and from members of other ethnic groups. The hearing of persons who were heterozygous for mutations in GJB2 was assessed by means of pure-tone audiometry, measurement of middle-ear immittance, and recording of otoacoustic emissions. RESULTS: Two frame-shift mutations in GJB2, 167delT and 30delG, were observed in the families with nonsyndromic recessive deafness. In the Ashkenazi Jewish population the prevalence of heterozygosity for 167delT, which is rare in the general population, was 4.03 percent (95 percent confidence interval, 2.5 to 6.0 percent), and for 30delG the prevalence was 0.73 percent (95 percent confidence interval, 0.2 to 1.8 percent). Genetic-linkage analysis showed conservation of the haplotype for 167delT but the existence of several haplotypes for 30delG. Audiologic examination of carriers of the mutant alleles who had normal hearing revealed subtle differences in their otoacoustic emissions, suggesting that the expression of mutations in GJB2 may be semidominant. CONCLUSIONS: The high frequency of carriers of mutations in GJB2 (4.76 percent) predicts a prevalence of 1 deaf person among 1765 people, which may account for the majority of cases of nonsyndromic recessive deafness in the Ashkenazi Jewish population. Conservation of the haplotype flanking the 167delT mutation suggests that this allele has a single origin, whereas the multiple haplotypes with the 30delG mutation suggest that this site is a hot spot for recurrent mutations.

Dating the origin of the CCR5-Delta32 AIDS-resistance allele by the coalescence of haplotypes.

The CCR5-Delta32 deletion obliterates the CCR5 chemokine and the human immunodeficiency virus (HIV)-1 coreceptor on lymphoid cells, leading to strong resistance against HIV-1 infection and AIDS. A genotype survey of 4,166 individuals revealed a cline of CCR5-Delta32 allele frequencies of 0%-14% across Eurasia, whereas the variant is absent among native African, American Indian, and East Asian ethnic groups. Haplotype analysis of 192 Caucasian chromosomes revealed strong linkage disequilibrium between CCR5 and two microsatellite loci. By use of coalescence theory to interpret modern haplotype genealogy, we estimate the origin of the CCR5-Delta32-containing ancestral haplotype to be approximately 700 years ago, with an estimated range of 275-1,875 years. The geographic cline of CCR5-Delta32 frequencies and its recent emergence are consistent with a historic strong selective event (e.g. , an epidemic of a pathogen that, like HIV-1, utilizes CCR5), driving its frequency upward in ancestral Caucasian populations.

Severe factor XI deficiency in an Arab family associated with a novel mutation in exon 11.

We investigated an 8-year-old Arab girl with severe factor XI deficiency; one sibling and her father also have severe factor XI deficiency. Her parents and her father's parents are first cousins. Restriction analysis and DNA sequencing excluded the type I, II, III and IV mutations. We demonstrated a previously undescribed C-->A mutation at nucleotide 1254 in exon 11 resulting in a threonine to asparagine (T-->N) substitution at amino acid 386. We postulate that this substitution interferes with folding and secretion of the molecule.

Familial colorectal cancer in Ashkenazim due to a hypermutable tract in APC.

Approximately 130,000 cases of colorectal cancer (CRC) are diagnosed in the United States each year, and about 15% of these have a hereditary component. Two well-defined syndromes, familial adenomatous polyposis (FAP) and hereditary non-polyposis colorectal cancer (HNPCC), account for up to 5% of the total new cases of CRC. Truncating APC mutations are responsible for FAP, and defective mismatch repair genes cause HNPCC. However, the genes responsible for most of the familial cases are unknown. Here we report a mutation (T to A at APC nucleotide 3920) found in 6% of Ashkenazi Jews and about 28% of Ashkenazim with a family history of CRC. Rather than altering the function of the encoded protein, this mutation creates a small hypermutable region of the gene, indirectly causing cancer predisposition.

The carrier frequency of the BRCA2 6174delT mutation among Ashkenazi Jewish individuals is approximately 1%.

Certain germline mutations in either BRCA1 or BRCA2 confer a lifetime risk of developing breast cancer that may approach 90%. The BRCA1 185delAG mutation was found in 20% and the BRCA2 6174delT mutation in 8% of Ashkenazi Jewish women with early-onset breast cancer. The 185delAG mutation was observed in 0.9% of 858 Ashkenazi Jews unselected for a personal or family history of cancer. Assuming comparable age-specific penetrances, a carrier frequency of 0.3% was estimated for the 6174delT BRCA2 mutation. To test this hypothesis, we performed a population survey of 1,255 Jewish individuals. In two independent groups, a prevalence of approximately 1% (C.I. 0.6-1.5) was observed for the 6174delT mutation. The relative risk of developing breast cancer by age 42 was estimated to be 9.3 (C.I. 2.5-22.5) for 6174delT, compared to 31 (C.I. 11-77) for 185delAG. Analysis of 107 Ashkenazi Jewish women with breast cancer and a family history of breast or ovarian cancer confirmed a four-fold greater prevalence for the BRCA1 185delAG mutation compared to the BRCA2 6174delT mutation. Our findings suggest a difference in cumulative life-time penetrance for the two mutations. Genetic counseling for the one in 50 Ashkenazi Jewish individuals harbouring specific germline mutations in BRCA1 or BRCA2 must be tailored to reflect the different risks associated with the two mutations.

High-level inducible expression of visual pigments in transfected cells.

A method for high-level expression of a functionally active, recombinant human red cone opsin was developed by adding the coding sequence for the C-terminal epitope of bovine rhodopsin onto the C terminus of the cone opsin and cloning the resulting construct into the vector pMEP4 beta. The recombinant pMEP4 beta vector was transfected stably into 293-EBNA cells, and expression of the cone opsin was induced by the addition of CdCl2 into the medium. The recombinant cone opsin was reconstituted with 11-cis retinal and purified by immunoaffinity chromatography. Spectral analysis prior to and following photobleaching confirmed its identity as a red cone opsin. The protein was targeted to the cell membrane and activated bovine transducin.

Hearing loss in Turner syndrome.

A retrospective study was undertaken to answer the following questions: Is the sensorineural hearing loss (SNHL) in Turner syndrome progressive? Can the occurrence of hearing loss be explained by the parental origin of the intact X chromosome? Twenty-four individuals recruited through the Turner Syndrome Society completed a questionnaire and submitted sufficient medical records to determine their otologic status. The majority (21/24) have had problematic otitis media (OM), and two thirds (16/24) have SNHL. In seven of the Turner subjects (age range: 12 to 51 years), gradual progressive SNHL began in late childhood or early adulthood. Molecular techniques showed no correlation between parental origin of the retained X chromosome and hearing status in 17 Turner subjects and at least one of their parents. SNHL and frequent OM appear to be independent variables that are both present in Turner syndrome. It is postulated that the presence of unpaired genes on the X chromosome may account for hearing loss and other phenotypic abnormalities seen in this syndrome.

Prenatal diagnostic testing for familial dysautonomia using linked genetic markers.

Familial dysautonomia (FD), a recessively inherited disease, has been mapped to chromosome 9q31. Highly polymorphic dinucleotide repeat markers flanking the genetic locus and at the same genetic location have been identified. We describe the prenatal diagnosis of FD using linkage and linkage disequilibrium analyses with these markers. Twelve families were analysed for informativeness and of these, seven went on to have prenatal testing (a total of eight fetuses tested). All of these fetuses were predicted to be heterozygous unaffected (FD carriers). Seven fetuses have come to term and are normal. In the absence of a recombinant proband, a panel of three proximal and three distal markers is sufficient to provide informative flanking markers and an 87-96 per cent likelihood of a highly predictive test. In an additional family at 1:4 risk for FD, no DNA was available from the propositus. This family was analysed using linkage disequilibrium to the #18 allele of the tightly linked marker D9S58 in conjunction with linkage analysis using data from two unaffected children. Prenatal diagnosis in this family indicated an affected fetus.

Renin gene promoter activity in GC cells is regulated by cAMP and thyroid hormone through Pit-1-dependent mechanisms.

Transcriptional activity of human renin gene (hREN) 5'-flanking DNA sequences in pituitary cells is highly dependent on binding of the pituitary-specific transcription factor Pit-1. Pit-1 has been implicated in cAMP regulation of a number of pituitary genes and has also been shown to interact with thyroid hormone (T3) receptors in mediating T3 responsiveness of the rat growth hormone gene. In the present study we examine the effects of forskolin and T3 on the expression of luciferase hybrid genes containing hREN 5'-flanking DNAs (hREN.luc) transiently transfected into the pituitary cell line GC. Basal activities of all hREN.luc constructs transfected into cells grown in media containing serum stripped of hormones were low. Addition of forskolin stimulated expression up to 48-fold, depending on the hREN sequences present. The hREN sequence -148 to +18 was sufficient for both maximal expression and maximal stimulation by forskolin. Mutagenesis of the Pit-1 site between -82 and -58 reduced forskolin induction 4-5-fold. In addition to the Pit-1 site, the sequence between -148 and -98 was also required for maximal activity and forskolin induction. T3 on its own had no effect on hREN promoter activity in GC cells, but suppressed the effects of forskolin. Gel mobility shift and Western blot analyses indicated that forskolin treatment had no effect on Pit-1 DNA binding or Pit-1 levels. However, T3 reduced Pit-1 levels which was reflected in lower DNA binding under the conditions employed. Taken together, these findings emphasize the importance of cAMP-dependent mechanisms in directing renin gene expression.

Pituitary-specific transcription factor (Pit-1) binding site in the human renin gene 5'-flanking DNA stimulates promoter activity in placental cell primary cultures and pituitary lactosomatotropic cell lines.

Renin gene expression is limited to a number of specific tissues, including the kidney, adrenal glands, reproductive organs (of particular relevance to this study, the placenta), and the pituitary gland. In the present study, we investigated the human renin (hRen) 5'-flanking DNA sequences required to drive the expression of a luciferase reporter gene in placental and pituitary cells and in two cell lines, 293 and JEG-3, which have been proposed as model systems with which to study transcriptional regulation of renin genes. The activities of specific sequences in the hRen 5'-flanking DNA sequences in human placental cell primary cultures were very similar to those that we previously reported in pituitary cells, suggesting the involvement of common promoter elements and related transcription factors. Accordingly, the binding site for the pituitary-specific transcription factor (Pit-1) was the major determinant of renin promoter activity in both pituitary and placental cells. Gel mobility shift analysis showed a placental nuclear factor with a gel mobility different from that of Pit-1. However, Northern blot analysis failed to demonstrate abundant Pit-1-related mRNAs in renin-expressing cultures of chorionic and decidual cells, suggesting that the placental factor is not closely related to Pit-1. Although a factor from 293 cells also bound to the Pit-1 site, it had gel mobility shift characteristics different from Pit-1 and the placental factor. Moreover, the low promoter activity in 293 cells was independent of this site or, indeed, of sequences upstream from the TATA box. In JEG-3 cells, renin 5'-flanking DNA sequences showed virtually no transcriptional activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Fibrinogen assembly: insights from chicken hepatocytes.

In all vertebrate species studied, the complex, disulfide-linked structure of fibrinogen is essentially the same: a hexamer assembled from three different subunits (A alpha, B beta, gamma)2. This study utilized species differences in fibrinogen subunit monomer pools to address the question of how these surplus subunit pools may affect the assembly process. We used a chicken model system in which B beta and gamma-subunits are present in excess, in contrast to the A alpha and gamma-subunit surplus found in human model systems. Analysis was based on pulse-chase experiments with electrophoretic separation of intracellular forms and secreted fibrinogen on reducing and nonreducing gels. The chicken liver-derived cells employed for this purpose, primary hepatocytes and a hepatoma cell line with a fortuitous defect in fibrinogen synthesis, together offer advantages over human systems for resolving the complexes formed in the early stages of assembly. The results demonstrate that in chicken hepatocytes there is an initial binding of gamma to A alpha subunits rather than to B beta subunits, as occurs in human hepatoma cells. Nevertheless, the presence of similar intracellular fibrinogen-related forms in both chicken- and human-derived cells, in the context of their differing subunit monomer pools, suggests an assembly pathway common to both species, with the versatility to be regulated by limitation of A alpha or B beta subunit production.