Mark H. Skolnick

Prevalence of Hemochromatosis among 11,065 Presumably Healthy Blood Donors

There is evidence that iron loading and organ damage can be prevented in patients with hemochromatosis if prophylactic phlebotomy is employed early in the disease--findings emphasizing the importance of early detection before clinical signs occur. This study was designed to determine the efficacy of transferrin saturation as a screening tool for hemochromatosis and to assess the frequency of homozygosity for the HLA-linked hemochromatosis gene in a healthy population. We screened 11,065 presumably healthy blood donors (5840 men and 5225 women). Donors with transferrin saturations of 62 percent or more after an overnight fast were considered potential homozygotes and were asked to undergo liver biopsy and pedigree analysis. The frequency of values for transferrin saturation of 62 or higher in men was 0.008 and in women 0.003. Thirty-eight persons with values higher than 62 were studied in detail; 35 underwent liver biopsy. Liver iron stores ranged from normal to markedly increased. Twelve siblings with an identical HLA match to a proband underwent liver biopsy, and 11 had increased liver iron stores. According to likelihood analysis of the pedigrees, 26 of the 38 probands were homozygotes, and 12 were heterozygotes. The estimated frequency of homozygosity was based on the data in men, because the threshold value of 62 for the transferrin saturation identified only half as many female homozygotes as expected. The frequency of homozygosity was 0.0045, corresponding to a gene frequency of 0.067. The value of population screening is demonstrated in these studies by the detection of homozygotes before clinical manifestations of hemochromatosis occur.

A candidate prostate cancer susceptibility gene at chromosome 17p

It is difficult to identify genes that predispose to prostate cancer due to late age at diagnosis, presence of phenocopies within high-risk pedigrees and genetic complexity. A genome-wide scan of large, high-risk pedigrees from Utah has provided evidence for linkage to a locus on chromosome 17p. We carried out positional cloning and mutation screening within the refined interval, identifying a gene, ELAC2, harboring mutations (including a frameshift and a nonconservative missense change) that segregate with prostate cancer in two pedigrees. In addition, two common missense variants in the gene are associated with the occurrence of prostate cancer. ELAC2 is a member of an uncharacterized gene family predicted to encode a metal-dependent hydrolase domain that is conserved among eukaryotes, archaebacteria and eubacteria. The gene product bears amino acid sequence similarity to two better understood protein families, namely the PSO2 (SNM1) DNA interstrand crosslink repair proteins and the 73-kD subunit of mRNA 3′ end cleavage and polyadenylation specificity factor (CPSF73).

Sequence analysis of BRCA1 and BRCA2: correlation of mutations with family history and ovarian cancer risk.

PURPOSE Previous studies of mutations in BRCA1 or BRCA2 have used detection methods that may underestimate the actual frequency of mutations and have analyzed women using heterogeneous criteria for risk of hereditary cancer. PATIENTS AND METHODS A total of 238 women with breast cancer before age 50 or ovarian cancer at any age and at least one first- or second-degree relative with either diagnosis underwent sequence analysis of BRCA1 followed by analysis of BRCA2 (except for 27 women who declined analysis of BRCA2 after a deleterious mutation was discovered in BRCA1). Results were correlated with personal and family history of malignancy. RESULTS Deleterious mutations were identified in 94 (39%) women, including 59 of 117 (50%) from families with ovarian cancer and 35 of 121 (29%) from families without ovarian cancer. Mutations were identified in 14 of 70 (20%) women with just one other relative who developed breast cancer before age 50. In women with breast cancer, mutations in BRCA1 and BRCA2 were associated with a 10-fold increased risk of subsequent ovarian carcinoma (P = .005). CONCLUSION Because mutations in BRCA1 and BRCA2 in women with breast cancer are associated with an increased risk of ovarian cancer, analysis of these genes should be considered for women diagnosed with breast cancer who have a high probability of carrying a mutation according to the statistical model developed with these data.

Common Inheritance of Susceptibility to Colonic Adenomatous Polyps and Associated Colorectal Cancers

We studied 670 persons in 34 kindreds by flexible proctosigmoidoscopic examination (60 cm) to determine how frequently colorectal adenomas and cancers result from an inherited susceptibility. Kindreds were selected through either a single person with an adenomatous polyp or a cluster of relatives with colonic cancer. The kindreds all had common colorectal cancers, not the rare inherited conditions familial polyposis coli and nonpolyposis inherited colorectal cancer. Likelihood analysis strongly supported the dominant inheritance of a susceptibility to colorectal adenomas and cancers, with a gene frequency of 19 percent. According to the most likely genetic model, adenomatous polyps and colorectal cancers occur only in genetically susceptible persons; however, the 95 percent confidence interval for this proportion was 53 to 100 percent. These results suggest that an inherited susceptibility to colonic adenomatous polyps and colorectal cancer is common and that it is responsible for the majority of colonic neoplasms observed clinically. The results also reinforce suggestions that first-degree relatives of patients with colorectal cancer should be screened for colonic tumors. This evidence of an inherited susceptibility to a cancer with well-recognized environmental risk factors supports the hypothesis that genetic and environmental factors interact in the formation and transformation of polyps.

Haplotype and Phenotype Analysis of Nine Recurrent BRCA2 Mutations in 111 Families: Results of an International Study

Several BRCA2 mutations are found to occur in geographically diverse breast and ovarian cancer families. To investigate both mutation origin and mutation-specific phenotypes due to BRCA2, we constructed a haplotype of 10 polymorphic short tandem-repeat (STR) markers flanking the BRCA2 locus, in a set of 111 breast or breast/ovarian cancer families selected for having one of nine recurrent BRCA2 mutations. Six of the individual mutations are estimated to have arisen 400-2,000 years ago. In particular, the 6174delT mutation, found in approximately 1% of individuals of Ashkenazi Jewish ancestry, was estimated to have arisen 29 generations ago (1-LOD support interval 22-38). This is substantially more recent than the estimated age of the BRCA1 185delAG mutation (46 generations), derived from our analogous study of BRCA1 mutations. In general, there was no evidence of multiple origins of identical BRCA2 mutations. Our study data were consistent with the previous report of a higher incidence of ovarian cancer in families with mutations in a 3.3-kb region of exon 11 (the ovarian cancer cluster region [OCCR]) (P=.10); but that higher incidence was not statistically significant. There was significant evidence that age at diagnosis of breast cancer varied by mutation (P<.001), although only 8% of the variance in age at diagnosis could be explained by the specific mutation, and there was no evidence of family-specific effects. When the age at diagnosis of the breast cancer cases was examined by OCCR, cases associated with mutations in the OCCR had a significantly older mean age at diagnosis than was seen in those outside this region (48 years vs. 42 years; P=.0005).

Hereditary hemochromatosis. Phenotypic expression of the disease.

Previous studies have shown that hemochromatosis is an inherited, autosomal-recessive disease and that the gene is closely linked to the HLA locus on chromosome 6. We obtained a lod score for linkage of +9.8 for a recombination fraction of 0.0 and a gene frequency of 0.056, the frequency estimated in this population. We studied the phenotypic expression of the disease in 261 members of 10 pedigrees. In heterozygotes over 20 years of age, there was an intermediate increase in transferrin saturation and a limited increase in hepatic iron but no clinical manifestations. In male heterozygotes, the average amount of iron in the liver increased from about 0.2 to 1.3 g. Abnormal homozygotes accumulated iron progressively with time, with men accumulating about 18 g in the liver. All measurements of iron status were increased in abnormal homozygotes. Hemochromatosis is inherited as an autosomal-recessive disease, with partial biochemical expression in heterozygotes.

Dominant inheritance of adenomatous colonic polyps and colorectal cancer

Except in the rare polyposis syndromes, the contribution of heritable factors to the genesis of colorectal cancer and adenomatous polyps is not well understood. We examined the inheritance of susceptibility to colonic polyps and cancer in a large Utah pedigree with multiple cases of common colorectal cancer but no recognizable inheritance pattern among them. Inheritance was clarified, however, by systematic screening for colonic polyps in pedigree members and spouse controls, using flexible proctosigmoidoscopy. One or more adenomatous polyps were found in 21 per cent of family members (41 of 191) but in only 9 per cent of controls (12 of 132) (P less than 0.005). Pedigree analysis was performed with likelihood methods that compared random occurrence of cancer and polyps with autosomal recessive and autosomal dominant patterns of inheritance. The analysis suggested that the observed excess of discrete adenomatous polyps and colorectal cancers was the result of an inherited autosomal dominant gene for susceptibility, rather than an inherited recessive gene for susceptibility or a chance occurrence. This type of inheritance of colorectal polyps and cancer may be more common than previously recognized.

Predisposition locus for major depression at chromosome 12q22-12q23.2

Major depression disorder is a common psychiatric disease with a major economic impact on society. In many cases, no effective treatment is available. The etiology of major depression is complex, but it is clear that the disease is, to a large extent, determined genetically, especially among individuals with a familial history of major depression, presumably through the involvement of multiple predisposition genes in addition to an environmental component. As a first step toward identification of chromosomal loci contributing to genetic predisposition to major depression, we have conducted a genomewide scan by using 628 microsatellite markers on 1,890 individuals from 110 Utah pedigrees with a strong family history of major depression. We identified significant linkage to major depression in males at marker D12S1300 (multipoint heterogeneity LOD score 4.6; P=.00003 after adjustment for multiple testing). With additional markers, the linkage evidence became highly significant, with the multipoint heterogeneity LOD score at marker D12S1706 increasing to 6.1 (P=.0000007 after adjustment for multiple testing). This study confirms the presence of one or more genes involved in psychiatric diseases on the q arm of chromosome 12 and provides strong evidence for the existence of a sex-specific predisposition gene to major depression at 12q22-q23.2.

Genetic predisposition to breast cancer

Breast cancer is the most common cancer among American women. Because metastatic breast cancer is an incurable disease, efforts to decrease breast cancer mortality have focused on early detection and improved treatment. Identification and analysis of a specific genetic susceptibility could permit detection of susceptible women and greatly increase the understanding of the initial step that eventually leads to cancer. Because susceptibility loci have been recognized as sites that often are altered during tumor progression, the identification and cloning of such loci could be important in developing cancer therapies. In this article, the progress being made in segregation analysis, linkage analysis, and cloning of breast cancer susceptibility loci is reviewed. The evidence for genetic inheritance is most consistent with dominant inheritance for at least three major susceptibility loci. Proliferative breast disease has been hypothesized to be an inherited lesion in breast cancer kindreds with both pre‐menopausal and postmenopausal probands. Currently, there are many genetic markers for mapping the human genome, Technologic advances have progressed from restriction fragment length polymorphisms to highly polymorphic markers. Using this technology, breast cancer susceptibility in some kindreds with an early onset has been shown to be linked to chromosome 17q. Gene isolation eventually will follow with an increased understanding of the percentage of breast cancer cases that are a result of this genetic locus. Li‐Fraumeni syndrome, which often is expressed as breast cancer, is due to mutations in the p53 gene. Characterization of the syndrome and its relationship to the altered gene should proceed rapidly. There is also a group of families exhibiting a genetic susceptibility that is not due to either of these loci. Together, these findings indicate that there are at least three separate major loci segregating for breast cancer susceptibility. With the current initiative to map and sequence the entire human genome and the advances that recently have been reported, a detailed molecular understanding of breast cancer predisposition can be envisaged.

Analysis of missense variation in human BRCA1 in the context of interspecific sequence variation

Introduction: Interpretation of results from mutation screening of tumour suppressor genes known to harbour high risk susceptibility mutations, such as APC, BRCA1, BRCA2, MLH1, MSH2, TP53, and PTEN, is becoming an increasingly important part of clinical practice. Interpretation of truncating mutations, gene rearrangements, and obvious splice junction mutations, is generally straightforward. However, classification of missense variants often presents a difficult problem. From a series of 20 000 full sequence tests of BRCA1 carried out at Myriad Genetic Laboratories, a total of 314 different missense changes and eight in-frame deletions were observed. Before this study, only 21 of these missense changes were classified as deleterious or suspected deleterious and 14 as neutral or of little clinical significance. Methods: We have used a combination of a multiple sequence alignment of orthologous BRCA1 sequences and a measure of the chemical difference between the amino acids present at individual residues in the sequence alignment to classify missense variants and in-frame deletions detected during mutation screening of BRCA1. Results: In the present analysis we were able to classify an additional 50 missense variants and two in-frame deletions as probably deleterious and 92 missense variants as probably neutral. Thus we have tentatively classified about 50% of the unclassified missense variants observed during clinical testing of BRCA1. Discussion: An internal test of the analysis is consistent with our classification of the variants designated probably deleterious; however, we must stress that this classification is tentative and does not have sufficient independent confirmation to serve as a clinically applicable stand alone method.