A novel allele, HLA-DPB1*296:01, identified by sequence-based typing in a Thai bone marrow donor.

A novel HLA-DPB1 allele, named DPB1*296:01, was identified in the Thai mother of a hematologic patient.

Reconstructing the genealogy of a BRCA1 founder mutation by phylogenetic analysis.

Estimating the age of founder mutations may contribute to improve our knowledge of population genetics and evolutionary history of diseases. Previous haplotype analysis suggested that the BRCA1*1499insA mutation was a founder allele, probably originated in Tuscany (Italy). Here, we collected additional pedigrees carrying this mutation, and applied a phylogenetic method for estimating mutation age. A chromosome segment of about 25 cM, including 37 short tandem repeats (STRs) on both sides of the BRCA1 gene (DeCode map), was typed in 50 subjects (28 mutation carriers) from 14 unrelated families. The time to the most recent common ancestor (MRCA) of the mutation carriers was estimated by the length of the shared haplotype between all possible pairs of individuals. A function relating the length of the shared haplotype to the time to the MRCA was obtained by a computer simulation. This approach gives results comparable with those of other existing mutation-dating methods, but does not depend explicitly on population-specific parameters such as allele frequencies, provides narrower confidence intervals (CI), and allows one to build an extended genealogical tree of all mutation carriers. The 1499insA mutation shared by the investigated subjects was estimated to be present in an individual living about 30 generations ago (95% CL 22-56), or 750 years (95% CL 550-1,400).

A genetic model for determining MSH2 and MLH1 carrier probabilities based on family history and tumor microsatellite instability.

Mutation-predicting models can be useful when deciding on the genetic testing of individuals at risk and in determining the cost effectiveness of screening strategies at the population level. The aim of this study was to evaluate the performance of a newly developed genetic model that incorporates tumor microsatellite instability (MSI) information, called the AIFEG model, and in predicting the presence of mutations in MSH2 and MLH1 in probands with suspected hereditary non-polyposis colorectal cancer. The AIFEG model is based on published estimates of mutation frequencies and cancer penetrances in carriers and non-carriers and employs the program MLINK of the FASTLINK package to calculate the proband's carrier probability. Model performance is evaluated in a series of 219 families screened for mutations in both MSH2 and MLH1, in which 68 disease-causing mutations were identified. Predictions are first obtained using family history only and then converted into posterior probabilities using information on MSI. This improves predictions substantially. Using a probability threshold of 10% for mutation analysis, the AIFEG model applied to our series has 100% sensitivity and 71% specificity.

Microgeographic variation of HLA-A, -B, and -DR haplotype frequencies in Tuscany, Italy: implications for recruitment of bone marrow donors.

HLA-A/B haplotype frequencies were estimated in a sample of 2355 bone marrow donors born in a subregion of Tuscany (Italy), and the HLA-A, -B, -DR haplotype frequencies were estimated in a subset of 809 individuals. This area was divided in 10 subsamples (two-locus haplotypes), or six subsamples (three-locus haplotypes), all with sample size >50, based on administrative boundaries. A considerable level of heterogeneity of haplotype frequency was present among subsamples; this heterogeneity was associated to a large variation (up to 4-fold) of the number of new donors that must be typed in order to reach 50% chance of finding an HLA-A, -B phenotype of intermediate frequency. Knowledge of the genetic structure of the population at a microgeographic level may be useful in directing the search of specific bone marrow donors.

HRPT2, encoding parafibromin, is mutated in hyperparathyroidism-jaw tumor syndrome.

We report here the identification of a gene associated with the hyperparathyroidism-jaw tumor (HPT-JT) syndrome. A single locus associated with HPT-JT (HRPT2) was previously mapped to chromosomal region 1q25-q32. We refined this region to a critical interval of 12 cM by genotyping in 26 affected kindreds. Using a positional candidate approach, we identified thirteen different heterozygous, germline, inactivating mutations in a single gene in fourteen families with HPT-JT. The proposed role of HRPT2 as a tumor suppressor was supported by mutation screening in 48 parathyroid adenomas with cystic features, which identified three somatic inactivating mutations, all located in exon 1. None of these mutations were detected in normal controls, and all were predicted to cause deficient or impaired protein function. HRPT2 is a ubiquitously expressed, evolutionarily conserved gene encoding a predicted protein of 531 amino acids, for which we propose the name parafibromin. Our findings suggest that HRPT2 is a tumor-suppressor gene, the inactivation of which is directly involved in predisposition to HPT-JT and in development of some sporadic parathyroid tumors.

Association between canine malignant lymphoma, living in industrial areas, and use of chemicals by dog owners.

A case-control study was carried out to determine whether residential exposure to environmental pollutants increased risk for canine lymphoma in pet dogs. One hundred one cases with cytologically or histologically confirmed lymphoma diagnosed at a veterinary teaching hospital between the middle of 1996 and the middle of 1998 were examined. Controls were obtained by choosing twice the number of dogs without neoplastic disease, with overlapping distributions of province of residence, age, sex, and breed. Information regarding animal management, residence type, professional or hobby use of chemicals by owners, and treatment with herbicides or other pesticides in the area frequently visited by the dogs was obtained with a multiple-choice questionnaire by telephone interview. Two variables were positively and independently associated with the disease, namely residency in industrial areas (odds ratio [OR]; = 8.5; 95% confidence interval [CI], 2.3-30.9) and use of chemicals by owners, specifically paints or solvents (OR = 4.6; 95% CI, 1.7-12.6). A significantly lower value of the mean age of disease onset was found in the group of dogs at risk in comparison with the group of all other dogs (6.1 +/- 0.4 years, n = 36 versus 7.5 +/- 0.4 years, n = 65, respectively; P = .008). Variables describing animal care and pesticide use were either not associated with the disease or were uninformative. We suggest that canine lymphoma may be considered a sentinel of potentially hazardous situations for humans, because of the relatively short latency between exposure and disease onset.

Microsatellite instability in colorectal-cancer patients with suspected genetic predisposition.

Hereditary non-polyposis colorectal cancer (HNPCC) is a dominantly inherited syndrome linked to DNA-mismatch-repair (MMR) gene defects, which also account for microsatellite instability (MSI) in tumour tissues. Diagnosis is based mainly on family history, according to widely accepted criteria (Amsterdam Criteria: AC). Aim of this work was to assess MSI in colorectal-cancer patients with suspected genetic predisposition, and to verify whether MSI represents a tool to manage MMR gene (hMSH2 and hMLH1) mutation analysis. We investigated 13 microsatellites (including the 5 NCI/ICG-HNPCC markers) in 45 patients with suspected hereditary predisposition (including 16 subjects from HNPCC families fulfilling the AC). We found MSI-H (high frequency of instability, i.e., in > or =30% of the markers) in 85% of the HNPCC patients and in 16% of the non-HNPCC subjects. The 5 NCI/ICG-HNPCC microsatellites proved to be the most effective in detecting MSI, being mononucleotide repeats the most unstable markers. We investigated the association between hMSH2- and hMLH1 gene mutations and MSI. Our results indicate that AC are highly predictive both of tumour instability and of MMR-gene mutations. Therefore, as the most likely mutation carriers, HNPCC subjects might be directly analyzed for gene mutations, while to test for MSI in selected non-HNPCC patients and to further investigate MMR genes in MSI-H cases, appears to be a cost-effective way to identify subjects, other than those from kindred fulfilling AC, who might benefit from genetic testing.

Different expressivity of two adjacent mutations of the APC gene.

AIMS AND BACKGROUND: The phenotypic expression of different APC mutations in familial adenomatous polyposis (FAP) is variable: two to three variants of the disease have been defined based on the severity of colonic manifestations. Age of onset and number of polypectomies per person-year of post-surgical follow-up were compared in two FAP families with very close mutation sites in the APC gene, in order to ascertain mutation-specific variation of expressivity. FAMILIES AND APC MUTATIONS: Family A (5 patients) carried a newly characterized mutation, a four bp deletion at codon 843. Family B (5 patients) carried a previously identified mutation at codon 835. RESULTS: Mean age of onset was 49.7 years in family A and 30.5 years in family B; number of polypectomies per person-year of follow-up was 1.05 for family A and 10.1 for family B (P < 0.001). CONCLUSIONS: There is significant variation of expressivity (allelic heterogeneity) in FAP between two mutations separated by only eight codons, located at the 5' extremity of APC gene exon 15.

Screening for mutations of the APC gene in 66 Italian familial adenomatous polyposis patients: evidence for phenotypic differences in cases with and without identified mutation.

Germline mutations in the APC gene are responsible for familial adenomatous polyposis (FAP), a dominantly inherited syndrome characterized by the development of hundreds to thousands of polyps in the colon and in the rectum of affected individuals and by variable extracolonic manifestations (gastric and duodenal polyps, osteomas, retinal lesions, and desmoid tumors). Through the combined use of single-strand conformation polymorphism (SSCP) analysis and the protein truncation test (PTT), we have screened 66 Italian FAP patients and found 29 different APC mutations in a total of 34 cases. Of the identified mutations, 15 were nonsense, 12 were 1- to 5-bp deletions or insertions and two were complex rearrangements, all leading to the formation of premature stop codons. Only 10 mutations had been already previously described at the germline level, confirming the high heterogeneity of the APC mutational spectrum. The mean age of diagnosis in mutation positive cases and their affected relatives was significantly lower than in cases without identified mutation (30.6 vs 39.1 years, respectively; p = 0.003). In addition, among patients without a family history of polyposis, all mutation-positive cases displayed at least one of the extracolonic manifestations usually associated with FAP, whereas in one-half of the cases without identified mutation, none of these phenotypes was observed. Although a fraction of apparently mutation-negative cases were likely to be due to limitations of the mutation screening strategy, our results suggest, in agreement with previous reports, that allelic and/or genetic heterogeneity might be responsible for the phenotypic variability observed in FAP patients.

Survival of patients with hereditary colorectal cancer: comparison of HNPCC and colorectal cancer in FAP patients with sporadic colorectal cancer.

Conflicting data exist on the prognosis of hereditary colorectal cancer. HNPCC patients, in particular, are often reported to have a better survival. We examined 2,340 colorectal-cancer patients treated in our Institution: 144 HNPCC patients (Amsterdam Criteria), 161 FAP patients and 2,035 patients with sporadic cancer. Data on hereditary-cancer patients treated between 1980 and 1995 was collected in a registry. The 2,035 sporadic colorectal-cancer patients (controls) included all new cases treated in the Department of Gastrointestinal-Tract Surgery during the same period. Observed survival was estimated using the Kaplan-Meier method. Cumulative survival probability was estimated at 5 years within each group and stratified by various clinical and pathological variables. The age distribution at diagnosis of sporadic patients was significantly higher than that of FAP and HNPCC patients (median 60 years vs. 43 and 49 years; p < 0.0001). In the HNPCC group, 40% had a right cancer location, vs. 14% in the FAP group and 13% in the sporadic-cancer group. In the sporadic group, 51% were early-stage cancers (Dukes A or B) vs. 48.4% and 52.1% in the FAP and HNPCC groups respectively. In the HNPCC, FAP and sporadic-cancer groups, the 5-year cumulative survival rate was 56.9%, 54.4% and 50.6% respectively. Survival analysis by the Cox proportional-hazards method revealed no substantial survival advantage for HNPCC and FAP patients compared with the sporadic group, after adjustment for age, gender, stage and tumor location. The hazard ratio for HNPCC was 1.01 (95% CI 0.72-1.39) and 1.27 (95% CI 0.95-1.7) for FAP patients compared with the sporadic-colorectal-cancer group.

Rapid assessment of replication error phenotype in gastric cancer.

Forty gastric tumors were investigated for microsatellite instability at the D2S119 and L-myc loci. These tumors and 143 other gastrointestinal cancers were previously analyzed for instability at several different microsatellites. By evaluating previous and present results, repeated sequences were selected that frequently underwent replication errors (RERs). To coamplify these sequences, the following multiplex polymerase chain reactions (PCRs) were performed: 1) D2S119/L-myc/D18S59; 2) D2S119/L-myc/D3S1076; and 3) D2S177/L-myc/BAT-RII. Therefore, the 40 gastric tumors in the present survey were rescreened using multiplex PCRs. Each multiplex allowed detection of nearly all RER+ tumors (80% for multiplex 3 and 87% for multiplexes 1 and 2) that had been previously identified by amplifying 9 different loci with independent reactions. Moreover, for multiplexes 1 and 2, the size differences between normal and RER alleles were sufficient to be detected by electrophoresis on conventional polyacrylamide gels after DNA staining with ethidium bromide. This approach allows a rapid and easy assessment of RER phenotype in gastric tumors.

Chain-terminating mutations in the APC gene lead to alterations in APC RNA and protein concentration.

APC mutations introduce premature stop codons into the open reading frame of the gene, leading to the formation of truncated tumor suppressor proteins. Both RNA and protein levels are likely to be profoundly altered by such nonsense mutations. To test this hypothesis, Western blotting and RT-PCR strategies were used to characterize mutant and normal APC protein and APC RNA concentrations in lymphoblastoid cell lines from 22 unrelated polyposis patients carrying different APC mutations. Variable levels of truncated APC peptides were observed in 14 of 14 cell lines with APC mutations within exon 15. No truncated APC protein was detected in six of eight cell lines with APC mutations located 5' of exon 15. Mutations located in exon 15 showed mutant RNA underrepresentation in four of eight cell lines, whereas mutations located 5' of exon 15 showed RNA reduction in five of six cell lines. These findings indicate that a two- to threefold decrease in RNA concentration is common when APC alleles carry chain-terminating mutations. They also suggest that the severe decrease of truncated APC protein observed in some cell lines is due to mechanisms acting at the protein level.

Mean age of tumor onset in hereditary nonpolyposis colorectal cancer (HNPCC) families correlates with the presence of mutations in DNA mismatch repair genes.

Fourteen Italian families affected with hereditary nonpolyposis colorectal cancer (HNPCC) were screened for germline mutations at three DNA mismatch repair (MMR) genes, MSH2, MLHI, and GTBP, by using a combination of different methods that included an in vitro synthesized protein assay, single-strand conformation polymorphism analysis, and direct sequencing. DNA alterations were observed in six instances, including a single base deletion in MSH2 exon 14, an A-to-G transition in the splice donor site of MLHI exon 6, and two missense mutations in MLHI exons 5 and 9. A previously reported common mutation affecting the splice donor site of MSH2 exon 5 was identified in two families. No mutations were detected in the GTBP gene. In total, eight of 16 Italian HNPCC families (50%), including two previously reported kindreds, were found to carry a mutation in MMR genes. We compared the mean age of colorectal cancer onset in the index cases (three patients for each family) between the two groups of kindreds, those with identified mutation vs. those without, and found that the first had a significantly lower value (43.0 vs. 53.7 years, P = 0.014). This finding suggests that HNPCC families with a more advanced age of tumor onset are less likely to be associated with known MMR genes.

Genetic epidemiology of colorectal cancer.

Starting from a survey of the studies on familial aggregation of colorectal cancer, we introduce the aims of genetic epidemiology. One of its main goals is to assess population frequency of cancer susceptibility genes and to determine the age-specific risks for carriers with respect to non-carriers. In section two, segregation analysis investigations are reviewed, and inferences on the relevance of genetic components of susceptibility to colorectal cancer are drawn. In section three, the HNPCC paradigm is discussed in the light of the Knudson model of tumorigenesis and recent advances of molecular research. In the last section we show an example of genotype/environment interaction in the etiology of a particular cancer and present a conceptual framework for studies on cancer genetic epidemiology in terms of attributable and relative risk.

Forensic evaluation of HUMCD4: an Italian database.

The YTTTC pentanucleotide short tandem repeat polymorphism HumCD4 was studied in an Italian population sample. PCR products were compared to an allelic ladder by manual PAGE and silver staining. A total of 6 alleles ranging from 5 to 12 repeats were represented in the analysed sample, of which 3 alleles (10, 6 and 5 repeats) were predominant and displayed a combined frequency of 0.91. Successful amplification was obtained from different sources such as blood and urine stains, teeth and paraffin embedded tissues. Results were also determined in cases of severely degraded DNA. We consider that the HUMCD4 polymorphism may be a useful tool for individual identification, paternity testing, population studies and have also employed this locus to monitor engraftment of bone marrow transplantation.

Age of onset in familial adenomatous polyposis: heterogeneity within families and among APC mutations.

Heterogeneity among and within FAP pedigrees for the age of symptom onset and the age at death from colorectal cancer was studied in a sample of 583 patients of the Italian Polyposis Registry. The among pedigree variation was largely explained by clustering of families in two groups, 'early FAP' (most colorectal cancer deaths below 45 years of age) and 'late FAP' families (most deaths above age 45). The within-family variation was explained by a marked phenomenon of anticipation (15 years per generation, on the average), possibly not due to ascertainment bias. We then considered the pedigrees with identified mutation in the APC gene. Six families shared a common deletion at codon 1309 and showed the early FAP phenotype. Two families shared a mutation at codon 1061 and revealed the late FAP phenotype. Another two families (codons 453 and 302) clustered with the late FAP group, whereas a family with mutation at codon 835 clustered with the early FAP group. We suggest that there are at least two classes of mutations in the APC gene with different consequences at the phenotypic level. It seems that there are several critical points within the APC protein sequence at which truncation causes a more aggressive disease than truncation at other points.

[Familial neoplasms: investigation of genealogic trees of patients surgically treated for colonic adenocarcinoma]. / Familiarità dei tumori: indagine sugli alberi genealogici di pazienti operati per adenocarcinoma del grosso intestino.

In order to study the familial aggregation of colorectal cancer we investigated the pedigrees of the patients with adenocarcinoma of the large bowel who underwent a surgical operation between november 1990 and october 1992 at the Istituto di Chirurgia Generale e Sperimentale Of Pisa University. For each proband, information was obtained on his/her four grandparents and all their second generation descendants. The final sample included 99 probands and 1455 relatives. Only two cases with diagnosis of familial adenomatous polyposis were excluded. As a control group, we applied the same methodology to the spouses of probands, collecting in the end a sample of 72 families including 1163 individuals. The frequency of both colorectal and extracolonic cancer was higher in the relatives of cases than in the control group, for all the relationships. Among the first degree relatives, the empirical risk of colorectal cancer was 1/30 among the case families and 1/139 among the control families, for a 4.6 fold increase in risk. For cancers at all sites (colorectal excluded), the corresponding risk were 1/8 and 1/12. We computed the posterior probability of dying from cancer for a random individual, given the known affection status of one or more of his/her relatives of specified relationship. For an individual with one first degree relative affected by colorectal cancer the posterior risk of the same tumor was 1/15, compared to a value of 1/70 for the entire control population. Considering all cancers, colorectal excluded, we obtained the result that for a person with at least three affected relatives, one of first, one of second and one of third degree, the probability of dying from colorectal cancer was 6%. The distribution of the number of affected individuals for kindred was highly skewed, with a few families responsible of a large part of the observed familial aggregation. This was true for both the cases of colorectal cancer and for all-site cancer. However, no family fulfilled the criterion of hereditary nonpolyposis colorectal cancer (Lynch syndromes I or II).