Case of interstitial 12q deletion in association with Wilms tumor.

A 14-month-old boy presenting with Wilms tumor (WT) was found to have a small de novo deletion of the long arm of chromosome 12 (12q11-12q13.11). Microsatellite analysis of this region from constitutional DNA showed that the paternal allele was absent between the markers D12S331 and D12S1713 (inclusive). In the WT there was no evidence of loss of the maternal chromosome. Constitutional chromosome abnormalities can often point to the presence of genes that are important in disease, and the deletion of chromosome 12 in this patient may indicate a gene involved in WT. To determine whether a WT predisposition locus exists at 12q we examined the region in two familial Wilms tumor (FWT) pedigrees unlinked to the known FWT genes on chromosomes 17q (FWT1), 19q (FWT2), and 11p (WT1). In both families WT did not segregate with chromosome 12q markers located within the deletion boundaries.

Evidence for susceptibility genes to familial Wilms tumour in addition to WT1, FWT1 and FWT2.

Three loci have been implicated in familial Wilms tumour: WT1 located on chromosome 11p13, FWT1 on 17q12-q21, and FWT2 on 19q13. Two out of 19 Wilms tumour families evaluated showed strong evidence against linkage at all three loci. Both of these families contained at least three cases of Wilms tumour indicating that they were highly likely to be due to genetic susceptibility and therefore that one or more additional familial Wilms tumour susceptibility genes remain to be found.

Identification of the familial cylindromatosis tumour-suppressor gene.

Familial cylindromatosis is an autosomal dominant genetic predisposition to multiple tumours of the skin appendages. The susceptibility gene (CYLD) has previously been localized to chromosome 16q and has the genetic attributes of a tumour-suppressor gene (recessive oncogene). Here we have identified CYLD by detecting germline mutations in 21 cylindromatosis families and somatic mutations in 1 sporadic and 5 familial cylindromas. All mutations predict truncation or absence of the encoded protein. CYLD encodes three cytoskeletal-associated-protein-glycine-conserved (CAP-GLY) domains, which are found in proteins that coordinate the attachment of organelles to microtubules. CYLD also has sequence homology to the catalytic domain of ubiquitin carboxy-terminal hydrolases (UCH).

Localization to Xq27 of a susceptibility gene for testicular germ-cell tumours.

Testicular germ-cell tumours (TGCT) affect 1 in 500 men and are the most common cancer in males aged 15-40 in Western European populations. The incidence of TGCT has risen dramatically over the last century. Known risk factors for TGCT include a history of undescended testis (UDT), testicular dysgenesis, infertility, previously diagnosed TGCT (ref. 7) and a family history of the disease. Brothers of men with TGCT have an 8-10-fold risk of developing TGCT (refs 8,9), whereas the relative risk to fathers and sons is fourfold (ref. 9). This familial relative risk is much higher than that for most other types of cancer. We have collected samples from 134 families with two or more cases of TGCT, 87 of which are affected sibpairs. A genome-wide linkage search yielded a heterogeneity lod (hlod) score of 2.01 on chromosome Xq27 using all families compatible with X inheritance. We obtained a hlod score of 4.7 from families with at least one bilateral case, corresponding to a genome-wide significance level of P=0.034. The proportion of families with UDT linked to this locus was 73% compared with 26% of families without UDT (P=0.03). Our results provide evidence for a TGCT susceptibility gene on chromosome Xq27 that may also predispose to UDT.

Allelotype of uterine leiomyomas.

Uterine leiomyomas are the most common benign tumor that arise from smooth muscle cells of the myometrium. Little is known about the etiology and pathogenesis of this tumor. To investigate the molecular pathogenesis of these tumors, we have conducted an allelotype of 102 leiomyomas from 12 patients, using 67 fluorescently-tagged oligonucleotide primers amplifying microsatellite loci covering all autosomes. No areas of the genome showed frequent loss of heterozygosity (LOH); however, the highest rate of LOH (9%) was observed on 7q, consistent with previous cytogenetic observations. Uterine leiomyomas are sometimes multiple. In general, multiplicity of other types of neoplasm is associated with genetic predisposition to the disease. Because multiple tumors were available from each of the 12 patients studied, we looked for evidence of allele-specific LOH, which might indicate the presence of an underlying predisposition gene. However, no evidence for allele-specific LOH was detected, indicating that if cases of multiple uterine leiomyoma are due to an underlying predisposition gene, it is unlikely to be a recessive oncogene.

A gene for lymphedema-distichiasis maps to 16q24.3.

Lymphedema-distichiasis (LD) is a dominantly inherited syndrome with onset of lymphedema at or just after puberty. Most affected individuals have distichiasis-fine hairs arising inappropriately from the eyelid meibomian glands-which is evident from birth. A study of three families with LD has shown linkage to chromosome 16q24.3, and subsequent analysis of the region for recombinant genes places the locus between D16S422 and D16S3074, a distance of approximately 16 cM. Possible candidate genes in this interval include the N-proteinase for type 3 collagen, PCOLN3; the metalloprotease PRSM1; and the cell matrix-adhesion regulator, CMAR.

Prevalence of BRCA1 and BRCA2 gene mutations in patients with early-onset breast cancer.

BACKGROUND: Mutations in the BRCA1 and BRCA2 genes are found in most families with cases of both breast and ovarian cancer or with many cases of early-onset breast cancer. However, in an outbred population, the prevalence of BRCA1 and BRCA2 mutations in patients with breast cancer who were unselected for a family history of this disease has not been determined. METHODS: Mutations in the BRCA1 and BRCA2 genes were detected in blood samples from two population-based series of young patients with breast cancer from Britain. RESULTS: Mutations were detected in 15 (5.9%) of 254 women diagnosed with breast cancer before age 36 years (nine [3.5%] in BRCA1 and six [2.4%] in BRCA2) and in 15 (4.1%) of 363 women diagnosed from ages 36 through 45 years (seven [1.9%] in BRCA1 and eight [2.2%] in BRCA2). Eleven percent (six of 55) of patients with a first-degree relative who developed ovarian cancer or breast cancer by age 60 years were mutation carriers, compared with 45% (five of 11) of patients with two or more affected first- or second-degree relatives. The standardized incidence ratio for breast cancer in mothers and sisters was 365 (five observed and 1.37 expected) for 30 mutation carriers and 199 (64 observed and 32.13 expected) for 587 noncarriers. If we assume recent penetrance estimates, the respective proportions of BRCA1 and BRCA2 mutation carriers are 3.1% and 3.0%, respectively, of patients with breast cancer who are younger than age 50 years, 0.49% and 0.84% of patients with breast cancer who are age 50 years or older, and 0.11% and 0.12% of women in the general population. CONCLUSIONS: Mutations in the BRCA1 and BRCA2 genes make approximately equal contributions to early-onset breast cancer in Britain and account for a small proportion of the familial risk of breast cancer.

The gene for cherubism maps to chromosome 4p16.3.

Cherubism is a rare familial disease of childhood characterized by proliferative lesions within the mandible and maxilla that lead to prominence of the lower face and an appearance reminiscent of the cherubs portrayed in Renaissance art. Resolution of these bony abnormalities is often observed after puberty. Many cases are inherited in an autosomal dominant fashion, although several cases without a family history have been reported. Using two families with clinically, radiologically, and/or histologically proved cherubism, we have performed a genomewide linkage search and have localized the gene to chromosome 4p16.3, with a maximum multipoint LOD score of 5. 64. Both families showed evidence of linkage to this locus. Critical meiotic recombinants place the gene in a 3-cM interval between D4S127 and 4p-telomere. Within this region a strong candidate is the gene for fibroblast growth factor receptor 3 (FGFR3); mutations in this gene have been implicated in a diverse set of disorders of bone development.

Inherited susceptibility to colorectal adenomas and carcinomas: evidence for a new predisposition gene on 15q14-q22.

BACKGROUND & AIMS: The aim of this study was to evaluate the role of known colorectal adenoma and carcinoma susceptibility genes and to locate a novel susceptibility gene in an Ashkenazi family (SM1311) with dominantly inherited predisposition to colorectal adenomas and carcinomas. METHODS: Clinicopathologic and family history data were collected. Genetic linkage and mutational analyses were used to investigate the genetic basis of the family's disease. RESULTS: Affected members of SM1311 develop multiple tubular, villous, tubulovillous, and/or serrated colorectal adenomas throughout the large bowel, and some develop colon carcinoma. There are no extracolonic features clearly associated with disease in SM1311. We have shown that the family's phenotype does not result from APC mutations (including the I1307K variant) or from genetic changes in the other known genes that predispose to colon cancer. Using genetic linkage analysis, supplemented by allele loss in tumors, we have provided evidence for a new colorectal cancer susceptibility gene, CRAC1 (colorectal adenoma and carcinoma), mapping to chromosome 15q14-q22. CONCLUSIONS: We provide evidence for a novel colorectal adenoma and carcinoma susceptibility gene on chromosome 15q14-q22. Further studies are needed to confirm this localization and to evaluate the contribution of CRAC1 to this disease.

Heterozygosity for mutations in the ataxia telangiectasia gene is not a major cause of radiotherapy complications in breast cancer patients.

Of patients being treated by radiotherapy for cancer, a small proportion develop marked long-term radiation damage. It is believed that this is due, at least in part, to intrinsic individual differences in radiosensitivity, but the underlying mechanism is unknown. Individuals affected by the recessive disease ataxia telangiectasia (AT) exhibit extreme sensitivity to ionizing radiation. Cells from such individuals are also radiosensitive in in vitro assays, and cells from AT heterozygotes are reported to show in vitro radiosensitivity at an intermediate level between homozygotes and control subjects. In order to examine the possibility that a defect in the ATM gene may account for a proportion of radiotherapy complications, 41 breast cancer patients developing marked changes in breast appearance after radiotherapy and 39 control subjects who showed no clinically detectable reaction after radiotherapy were screened for mutations in the ATM gene. One out of 41 cases showing adverse reactions was heterozygous for a mutation (insertion A at NT 898) that is predicted to generate a truncated protein of 251 amino acids. No truncating mutations were detected in the control subjects. On the basis of this result, the estimated percentage (95% confidence interval) of AT heterozygous patients in radiosensitive cases was 2.4% (0.1-12.9%) and in control subjects (0-9.0%). We conclude that ATM gene defects are not the major cause of radiotherapy complications in women with breast cancer.

A missense mutation in the BRCA2 gene in three siblings with ovarian cancer.

Inherited susceptibility to ovarian cancer has been associated with germline defects at several loci. The major known ovarian cancer susceptibility gene is BRCA1 on chromosome 17q, which confers a risk of approximately 60% by the age of 70 years. Truncating mutations in BRCA2 on chromosome 13q also predispose to ovarian cancer, although they confer a lower risk than mutations in BRCA1. We have studied the molecular basis of ovarian cancer predisposition in a Finnish family with three affected sisters. Analysis of polymorphic markers provided evidence against linkage to BRCA1, but the sibship was consistent with linkage to BRCA2. Conformation-sensitive gel electrophoresis was used to screen the entire coding sequence of BRCA2. A G to A transition at nucleotide 8702 was observed, which is predicted to convert glycine 2901 to aspartate in the encoded protein. This sequence variant was not detected in 220 cancer-free Finnish control individuals, or in several hundred cancer families of many nationalities previously screened for BRCA2 mutations. Taken together with the fact that this amino acid residue and the surrounding region of BRCA2 is identical in mouse and chicken, the data suggest that this alteration is a disease-causing BRCA2 missense mutation. Previously published data indicate that the risks of breast and ovarian cancer conferred by BRCA2-truncating mutations varies with the position of the mutation in the gene. The missense mutation reported here suggests that the BRCA2 domain including and surrounding glycine 2901 may be more important in preventing neoplastic transformation in ovarian epithelium than in breast epithelium.

Low frequency of somatic mutations in the LKB1/Peutz-Jeghers syndrome gene in sporadic breast cancer.

Germ-line mutations in the LKB1 gene on chromosome 19p are responsible for most cases of the Peutz-Jeghers syndrome, in which intestinal hamartomas are associated with elevated risks of several cancer types, including breast cancer. We have evaluated the role of somatic mutations in LKB1 in breast cancer. Of 40 informative primary breast cancers, 3 showed loss of heterozygosity on chromosome 19p in the vicinity of LKB1, and no somatic mutations of LKB1 were observed in 62 primary breast cancers and 17 established breast cancer cell lines. The results indicate that mutations in LKB1 do not play an important role in the development of sporadic breast cancer.

Alleletyping of an oligodendrocyte-type-2 astrocyte lineage derive from a human glioblastoma multiforme.

We have conducted alleletyping of two novel cell lines derived from glioblastoma multiforme, which appear to have arisen from different glial lineages, by using 76 fluorescently labeled oligonucleotide primers amplifying microsatellite loci covering the entire human genome. One cell line, Hu-O-2A/Gb1, expresses antigens and metabolic profiles characteristic of the oligodendrocyte-type-2 astrocyte (0-2A) lineage of the rat central nervous system. This cell line generated, in vitro, cells with characteristics of 0-2A progenitor cells, oligodendrocytes and astrocytes. The second cell line, IN1434, is derived from an astrocyte or a precursor cell restricted to astrocytic differentiation. Hu-O-2A/Gbl cells show allelic losses of loci on chromosomes 2, 5, 6, 7, 8, 9, 10, 11, 13, 15, 16, 17, 20 and 21. IN1434 cells are likely to have allelic losses of loci on chromosomes 1, 3, 8 and 10, although no control DNA is available for this cell line. These results, for the first time, provide a detailed information of the molecular genetic defects occurring in Hu-O-2A/Gb1 and IN1434.

Variation of risks of breast and ovarian cancer associated with different germline mutations of the BRCA2 gene.

The breast cancer susceptibility gene BRCA2 on chromosome 13q12-13 has recently been identified. Germline mutations of BRCA2 are predicted to account for approximately 35% of families with multiple case, early onset female breast cancer, and they are also associated with an increased risk of male breast cancer, ovarian cancer, prostate cancer and pancreatic cancer. Germline mutations of a second cancer susceptibility gene BRCA1 (ref. 5), are associated with a strong predisposition to ovarian cancer as well as female breast cancer. Recent studies have suggested that the phenotype in BRCA1 families with respect to the ratio of breast to ovarian cancer varies with the location of the BRCA1 mutation. To determine whether germline mutations in BRCA2 are associated with a similar variation in phenotypic risk, we have analysed the distribution of mutations in 25 families with multiple cases of breast and/or ovarian cancer ascertained in the United Kingdom and Eire. These mutations all lead to premature truncation of BRCA2 as a result of frameshift deletions/insertions or nonsense mutations. Analysis of the mutation distribution along the length of the gene indicates a significant genotype-phenotype correlation. Truncating mutations in families with the highest risk of ovarian cancer relative to breast cancer are clustered in a region of approximately 3.3 kb in exon 11 (P = 0.0004). Published data on mutations in 45 other BRCA2-linked families provide support for this correlation.

BRCA2 mutations in primary breast and ovarian cancers.

The second hereditary breast cancer gene, BRCA2, was recently isolated. Germline mutations of this gene predispose carriers to breast cancer, and, to a lesser extent, ovarian cancer. Loss of heterozygosity (LOH) at the BRCA2 locus has been observed in 30-40% of sporadic breast and ovarian tumours, implying that BRCA2 may act as a tumour suppressor gene in a proportion of sporadic cases. To define the role of BRCA2 in sporadic breast and ovarian cancer, we screened the entire gene for mutations using a combination of techniques in 70 primary breast carcinomas and in 55 primary epithelial ovarian carcinomas. Our analysis revealed alterations in 2/70 breast tumours and none of the ovarian carcinomas. One alteration found in the breast cancers was a 2-basepair (bp) deletion (4710delAG) which was subsequently shown to be a germline mutation, the other was a somatic missense mutation (Asp3095Glu) of unknown significance. Our results suggest that BRCA2 is a very infrequent target for somatic inactivation in breast and ovarian carcinomas, similar to the results obtained for BRCA1.

The proliferation of multiple myeloma colonies (MY-CFUc) in vitro is independent of prognosis and is not associated with mutated N- or K-ras alleles in human bone marrow aspirates.

During the period September 1987 to March 1993 the proliferation of myeloma cells as colonies (MY-CFUc) in vitro was examined in bone marrow aspirates from 43 patients with multiple myeloma and two patients with Waldenström's macroglobulinaemia. Twenty-four samples from 45 patients, of whom three were at presentation, four were in complete remission (CR), six had achieved a partial response (PR) and 11 had progressive disease (PD), produced MY-CFUc in vitro. The same bone marrow aspirates or one taken within 2 months of that assessed for MY-CFUc were used in the polymerase chain reaction (PCR). Genomic DNA was analysed for mutations in N- and K-ras by slot blotting of the amplified products from the PCR with 32P-labelled probes and by direct sequencing. No mutations were detected in N- or K-ras proto-oncogenes at codons 12, 13 or 61 in any sample. Eleven of the patients from whom MY-CFUc were produced remain alive with a median survival of 73 months (range 15-75 months). MY-CFUc have been cultured from 19 of these 24 patients on subsequent occasions, of whom nine remain alive. Among patients whose cells did not produce MY-CFUc in vitro at the time of sampling for mutated ras alleles, biopsy samples from four patients have produced MY-CFUc in vitro on subsequent occasions, of whom one patient remains alive. The data show that the proliferation of MY-CFUc in vitro occurred independently of disease status and was not indicative of prognosis. The failure to detect mutated N- or K-ras alleles in any sample suggests that if such mutations were present in the cells which form colonies in vitro they represented less than 0.1% of the tumour burden and did not affect the survival of this group of patients.

Neutralising antibodies in patients with multiple myeloma receiving maintenance therapy with interferon alpha 2b.

In a study of 29 patients who were receiving or had received interferon alpha 2b (IFN-alpha 2b) as maintenance therapy for multiple myeloma, antibodies were detected in 58% (17/29) of patients measured by a solid-phase enzyme-linked immunosorbent assay (ELISA). Only 7/17 patients who were positive for antibody in the ELISA had neutralising antibody to IFN-alpha 2b, measured by virus growth inhibition. These patients comprised six who were receiving IFN-alpha 2b at the time of assessment and one who had finished treatment. Among patients who were receiving the cytokine, four had progressive disease, one was in complete remission and one in partial remission. Neutralising activity was also detected to natural human leucocyte IFN-alpha in the same patients. Two patients who were positive for neutralising antibody remain in remission and are continuing to receive IFN-alpha 2b. These two patients have since lost their neutralising titre. No neutralising antibody to IFN-alpha 2b or natural human leucocyte IFN-alpha was detected in serum from six normal donors. The data suggest that neutralising antibody formation in patients with multiple myeloma is not responsible for relapse in patients receiving IFN-alpha 2b. The transient nature of neutralising antibody production in patients who remain in remission suggests that this response to IFN-alpha 2b is not associated with memory B cells.