Clustering of missense mutations in the ataxia-telangiectasia gene in a sporadic T-cell leukaemia.

Ataxia-telangiectasia (A-T) is a recessive multi-system disorder caused by mutations in the ATM gene at 11q22-q23 (ref. 3). The risk of cancer, especially lymphoid neoplasias, is substantially elevated in A-T patients and has long been associated with chromosomal instability. By analysing tumour DNA from patients with sporadic T-cell prolymphocytic leukaemia (T-PLL), a rare clonal malignancy with similarities to a mature T-cell leukaemia seen in A-T, we demonstrate a high frequency of ATM mutations in T-PLL. In marked contrast to the ATM mutation pattern in A-T, the most frequent nucleotide changes in this leukaemia were missense mutations. These clustered in the region corresponding to the kinase domain, which is highly conserved in ATM-related proteins in mouse, yeast and Drosophila. The resulting amino-acid substitutions are predicted to interfere with ATP binding or substrate recognition. Two of seventeen mutated T-PLL samples had a previously reported A-T allele. In contrast, no mutations were detected in the p53 gene, suggesting that this tumour suppressor is not frequently altered in this leukaemia. Occasional missense mutations in ATM were also found in tumour DNA from patients with B-cell non-Hodgkin's lymphomas (B-NHL) and a B-NHL cell line. The evidence of a significant proportion of loss-of-function mutations and a complete absence of the normal copy of ATM in the majority of mutated tumours establishes somatic inactivation of this gene in the pathogenesis of sporadic T-PLL and suggests that ATM acts as a tumour suppressor. As constitutional DNA was not available, a putative hereditary predisposition to T-PLL will require further investigation.

Does 77C-->G in PTPRC modify autoimmune disorders linked to the major histocompatibility locus?

A 77G allele of the gene encoding CD45, also known as the protein tyrosine phosphatase receptor-type C gene (PTPRC), has been associated with multiple sclerosis (MS). Here we determine allele frequencies in large numbers of MS patients, primary immunodeficiencies linked to major histocompatibility complex (MHC) locus and over 1,000 controls to assess whether aberrant splicing of PTPRC caused by the 77C-->G polymorphism results in increased susceptibility to these diseases. Our results show no difference in the frequency of the 77G allele in patients and controls and thus do not support a causative role for the polymorphism in the development of disorders with a strong autoimmune component in etiology.

Colocalization of X-linked agammaglobulinemia and X-linked immunodeficiency genes.

Mice that bear the X-linked immunodeficiency (xid) mutation have a B lymphocyte-specific defect resulting in an inability to make antibody responses to polysaccharide antigens. A backcross of 1114 progeny revealed the colocalization of xid with Bruton's agammaglobulinemia tyrosine kinase (btk) gene, which is implicated in the human immune deficiency, X-linked agammaglobulinemia. Mice that carry xid have a missense mutation that alters a highly conserved arginine near the amino-terminus of the btk protein, Btk. Because this region of Btk lies outside any obvious kinase domain, the xid mutation may define another aspect of tyrosine kinase function.

Trichoepitheliomas contain somatic mutations in the overexpressed PTCH gene: support for a gatekeeper mechanism in skin tumorigenesis.

The nevoid basal cell carcinoma (Gorlin) syndrome (NBCCS) is an autosomal dominant disorder characterized by multiple developmental defects and cancer susceptibility. NBCCS is caused by mutations in the human homologue (PTCH) of the Drosophila patched gene, a developmental regulator implicated in signaling of hedgehog and smoothened. The PTCH gene was found to contain somatic mutations also in sporadic basal cell carcinomas and medulloblastomas, tumors seen in NBCCS, consistent with PTCH acting as a tumor suppressor. Because basal cell carcinomas have been observed to develop in association with benign trichoepitheliomas (TEs) in the same lesions, patients, and families and may share the same cell of origin, we have analyzed PTCH for mutations and expression in TEs. We report frameshift and in-frame somatic deletions in this gene and a consistent overexpression of PTCH mRNA in TEs. These findings provide the first evidence of a gene mutation in TEs and identify a common pathogenic pathway for histopathologically similar but prognostically distinct skin tumors. Moreover, these results support the presence of a gatekeeper mechanism in multistep skin tumorigenesis exerted by the altered PTCH signaling pathway.

ATM mutations in cancer families.

Ataxia-telangiectasia (A-T) is a multisystem recessive disease characterized clinically by cerebellar ataxia, oculocutaneous telangiectasias, immunodeficiency, sensitivity to radiomimetic agents, and cancer predisposition. This pleiotropic disorder is caused by mutations in the ATM (mutated in A-T) gene, which is located in the human chromosomal region 11q22-q23. The ATM gene product is a member of a novel family of large proteins implicated in the regulation of the cell cycle and response to DNA damage. Heterozygosity for A-T was previously suggested to be associated with an increased risk of tumors, particularly female breast cancer. Because of loss of constitutional heterozygosity at 11q22-q23 is a frequent event in breast and other tumors, suggesting the presence of a tumor suppressor gene(s) in this region, we screened blood DNA samples from 88 unrelated breast cancer patients of Swedish cancer families for ATM mutations using single-strand conformation polymorphism analysis. All patients had a family history of tumors previously associated with A-T heterozygosity or homozygosity. We demonstrate the first three germ-line mutations in ATM identified by screening of breast cancer patients. Two mutations were previously found in A-T homozygotes and one mutation was a 1-bp insertion. All mutations were found in families with a large number of tumors, however, they did not cosegregate with malignancies. Although the proportion of A-T carriers in this sample seems to be higher than expected by chance, larger studies and pooled data sets will be required to establish that an A-T allele confers cancer susceptibility in heterozygotes.

The FHIT gene at 3p14.2 is abnormal in breast carcinomas.

Chromosome 3p deletions in breast cancer have been detected at 3p12-p21 by cytogenetic and loss of heterozygosity studies. Recently, we have cloned the FHIT (fragile histidine triad) gene, located at 3p14.2. Abnormalities of the FHIT locus were found in many established cancer cell lines, and the gene was abnormally transcribed in primary tumors of the digestive tract and lung. In this report, we describe the analysis of breast cancer, cell lines, and primary tumors for alterations in transcription of the FHIT gene; about 20% of the samples exhibited altered transcripts. In most of the cases, aberrant transcripts were missing exons. Lack of expression of FHIT mRNA was observed in another 10% of primary tumor samples. These results suggest that alterations in the FHIT gene may play an important role in breast cancer tumorigenesis and suggest that the MIT gene product functions in the control of the tumorigenic phenotype in a large variety of human neoplasms.

Patched (ptch)-associated preferential expression of smoothened (smoh) in human basal cell carcinoma of the skin.

The discovery of specific overexpression of a gatekeeper gene, ptch, in basal cell carcinoma (BCC) led to a hypothesis that the human homologue of patched (PTCH) normally functions as a negative regulator of the signaling pathway that is initiated by hedgehogs (HHs) and activated by the human homologue of smoothened (SMOH); however, no evidence for the involvement of smoh and hhs has been provided. Here, we show novel evidence that smoh is also preferentially overexpressed in BCC, together with ptch (P < 0.002), and that Sonic hh was expressed in only some BCCs. Our data, therefore, indicate that such overexpression of smoh may be associated with overexpression or mutation of PTCH and that this overexpression subsequently stimulates the PTCH/SMOH signaling pathway. In an investigation of a possible regulation of ptch and smoh, we demonstrated that expression of exogenous p21WAF1 in immortalized keratinocytes down-regulates both ptch and smoh and that the down-regulation is accompanied by growth arrest, which suggests the involvement of p21WAF1 in regulation of the PTCH/SMOH signaling pathway.

Mutations in the human homologue of Drosophila patched (PTCH) in basal cell carcinomas and the Gorlin syndrome: different in vivo mechanisms of PTCH inactivation.

The nevoid basal cell carcinoma (Gorlin) syndrome (NBCCS) is an autosomal dominant disorder characterized by multiple developmental defects and cancer susceptibility, in particular to basal cell carcinoma. The human homologue of Drosophila patched (PTCH) was recently identified, mapped to the NBCCS locus on chromosome 9q22.3, and found mutated in patients with NBCCS and also in sporadic basal cell carcinomas. Here we show germ-line PTCH mutations in three families with NBCCS. We demonstrate that a germ-line PTCH frameshift deletion in one patient with NBCCS was accompanied by loss of the normal copy of PTCH in a tumor developed in the same patient. Another basal cell carcinoma from this patient did not show the loss of the normal copy of PTCH, instead a missense mutation in a highly conserved residue was identified in the nondeleted allele, illustrating two different mechanisms of PTCH inactivation in different tumors derived from the same NBCCS patient. We also show somatic PTCH mutations in 4 basal cell carcinomas identified by analyzing 18 non-NBCCS patients with sporadic tumors. These data provide further support for PTCH as an important tumor suppressor gene in the development of the most common human cancer.

The ATM gene and susceptibility to breast cancer: analysis of 38 breast tumors reveals no evidence for mutation.

Heterozygosity for ataxia-telangiectasia (A-T), a cancer-prone recessive syndrome, has been associated with an increased risk of breast cancer. The gene for A-T (ATM) is located at chromosomal region 11q22-q23, a region of frequent loss of constitutional heterozygosity in breast and other tumors. Loss of constitutional heterozygosity at 1lq22-q23 was found in 47% of informative cases in the series of primary tumors analyzed in this study. To investigate the role of ATM in breast cancer, we have determined the complete genomic organization of the gene, developed an exon-scanning PCR single-strand conformation polymorphism (PCR-SSCP) assay for mutation detection of ATM, and screened 38 consecutive breast tumors for mutations using both genomic DNA- and cDNA-based assays. In addition to common ATM polymorphisms detected both in the coding sequence and in flanking introns, seven unique SSCP alleles were identified in six tumor DNAs. Sequence analysis of these alleles revealed rive nucleotide substitutions that were predicted to change the encoded amino acid. However, PCR-SSCP and nucleotide sequencing analysis of the paired blood samples and of an extended sample size of a total of 224 chromosomes indicated that these SSCP patterns represent constitutional rare polymorphisms with a frequency between 0.005 and 0.023. Because the majority of A-T mutations are null mutations and none of the ATM alleles found in breast cancer samples would lead to the truncation of the translation product, we conclude that, in this initial sample of sporadic breast cancer patients, there was no evidence for an increased number of A-T carriers. In addition, because no somatic mutations were found, our study rules out the ATM gene as the frequently altered tumor suppressor gene at 11q23.

Ataxia-telangiectasia and T-cell leukemias: no evidence for somatic ATM mutation in sporadic T-ALL or for hypermethylation of the ATM-NPAT/E14 bidirectional promoter in T-PLL.

The ATM gene deficient in ataxia-telangiectasia, a recessive multisystem disease associated with a high risk of lymphomas and leukemias, was found previously to be inactivated in a rare sporadic malignancy, T-cell prolymphocytic leukemia (T-PLL), which is often associated with cytogenetic aberrations of chromosome 14. The ATM gene was shown to sustain frequent loss-of-function mutations in T-PLL tumor cells, consistent with functioning as a tumor suppressor gene in this leukemia. To investigate the possibility of nonmutational or nonrecombinational mechanisms of T-PLL development, we have used bisulfite genomic sequencing to analyze DNA methylation in the putative bidirectional promoter region of the closely linked ATM and NPAT/E14 genes within the CpG island at 11q22-q23. We show that this region is completely demethylated in lymphocytes expressing ATM; however, no extensive hypermethylation was found in 9 T-PLL tumor DNA samples without evidence of ATM/p53 mutations. Because acute T-cell lymphoblastic leukemias (T-ALL) were also observed in ataxia-telangiectasia patients and T-ALL tumor cells contain chromosome 14 abnormalities, 19 presentation samples of T-ALL patients were analyzed for ATM mutations. Although T-ALL patients exhibited rare nucleotide substitutions not previously found in ATM, all were identified in the germ-line, indicating constitutional polymorphisms, potentially confined to ethnic subpopulations. The absence of somatic nucleotide changes in ATM in T-ALL as compared with T-PLL suggests a distinct pattern of genetic events in the development of the two leukemias.

Somatic mutations in the human homologue of Drosophila patched in primitive neuroectodermal tumours.

The naevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by multiple developmental defects and cancer susceptibility, in particular to basal cell carcinomas (BCCs). Medulloblastomas, primitive neuroectodermal tumours (PNETs) arising in childhood, occur in about 3-5% of NBCCS patients and a subset of PNETs was previously found with allelic imbalance at 9q22-q23, the region containing the gene for NBCCS (PTCH). We have analysed tumour DNA samples from 37 unrelated patients with sporadic PNETs and five medulloblastoma cell lines for PTCH mutations using an exon-by-exon single strand conformation polymorphism assay. We found three missense mutations, which affect conserved residues in transmembrane domains of the gene product and in the extracellular loop implicated in binding sonic hedgehog, one 2 bp deletion and an exon skipping splice site mutation. Most mutations were associated with the absence of the wild-type allele and were found in tumours exhibiting loss of heterozygosity (LOH) at loci flanking PTCH. The finding of LOH at 9q22-q23 in most mutated tumours while present in only three out of 26 tumours, in which a mutation was not identified, implicates PTCH as the target gene in PNETs with LOH at 9q22-q23 and deficient PTCH in the development of a subset of these tumours. Since all observed mutations were absent in the germ-line, a sporadic medulloblastoma developing as the first symptom of NBCCS is likely to be a very uncommon event.

Refinement of the LOH region 1 at 11q23.1 deleted in human breast carcinomas and sublocalization of 11 expressed sequence tags within the refined region.

Loss of constitutive heterozygosity at 11q23 has been detected in various human solid tumors. Here, we described the analysis of a series of normal and tumor pairs from 110 breast carcinomas for the presence of loss of heterozygosity at 11q23 loci. The overall frequency of LOH was 48%, confirming the importance of deletions at 11q23 in breast tumorigenesis. Previously, we have identified two independent regions of LOH at 11q23, the LOH region 1 at 11q23.1 and the LOH region 2 at 11q23.3. The most telomeric region was recently refined between loci D11S1345 and D11S1316, a region of about 1 Mb. However, the LOH region 1, most centromeric, was still not finely refined: the boundaries were defined by loci D11S2000 and D11S897, separated by about 8 Mb. Here, we refined its boundaries between loci D11S1347 and D11S927, a region of about 2 Mb. We have mapped 11 expressed sequence tags (ESTs) within this region and excluded another 20. This study represents a further step toward the identification of the putative tumor suppressor gene found within the LOH region 1 at 11q23.1.

ATM is usually rearranged in T-cell prolymphocytic leukaemia.

T-prolymphocytic leukaemia (T-PLL) is a rare, sporadic leukaemia similar to a mature T-cell leukaemia seen in some patients with Ataxia Telangiectasia (A-T), a recessive multisystem disorder caused by mutations of the ATM gene at chromosome 11q23. ATM sequence mutations have been reported in 46% of T-PLL cases, but some cases also have karyotypic abnormalities at 11q, including 11q23. This led us to investigate the structure of the ATM locus in a panel of eight cases, two of which had 11q23 abnormalities. As expected, nucleotide changes were detected in some samples. Two remission samples were wild type. To test for structural lesions, DNA fibres were hybridized with a contig of four labelled cosmids spanning the ATM locus. In all samples there were structural lesions and in four samples both alleles were affected. This provides strong evidence for our suggestion that ATM acts as a tumour suppressor during T-PLL tumorigenesis. Some additional role for ATM during T-PLL tumorigenesis is possible since nucleotide changes were present in addition to structural lesions disrupting both alleles. The mechanism of inactivation appeared to be unusual because multiple structural lesions on one allele were often observed.

UV mutation signature in tumor suppressor genes involved in skin carcinogenesis in xeroderma pigmentosum patients.

Molecular analysis of p53 and patched (PTCH), two candidate tumor suppressor genes for non-melanocytic skin cancer, was performed in skin tumors from six patients affected by the cancer-prone disease xeroderma pigmentosum (XP). UV-specific p53 mutations were detected at a frequency of 38-50% in all the tumor types analysed, including melanomas. Additional analysis of PTCH mutations in the subset of eight basal call carcinomas (BCC) revealed a very high mutation frequency of this gene (90%) which exceeded that detected in the p53 gene in the same tumors (38%). PTCH mutations were predominantly UV-specific C>T transitions. This mutation pattern is different from that reported in BCC from normal donors where PTCH mutation frequency is 27% and mutations are frequently deletions and insertions. These findings suggest that PTCH mutations represent an earlier event in BCC development than p53 alterations and that the inability of XP patients to repair UV-induced PTCH mutations might significantly contribute to the early and frequent appearance of BCC observed in these patients.

Low frequency of alterations of the alpha (PPP2R1A) and beta (PPP2R1B) isoforms of the subunit A of the serine-threonine phosphatase 2A in human neoplasms.

The phosphatase 2A (PP2A) is one of the major cellular serine-threonine phosphatases. It was recently shown that the gene encoding for the beta isoform of its subunit A, PPP2R1B, is altered in human lung and colorectal carcinomas, suggesting a role in human tumorigenesis. Here, we report the detection of mutations in breast, lung carcinomas and melanomas in the genes of both alpha (PPP2R1A) and beta isoforms. Mutations affecting PPP2R1B were found in four breast carcinomas, while mutations in PPP2R1A were found in carcinomas of the breast and of the lung and in one melanoma. Most of the mutations affecting PPP2R1B were exons deletions, suggesting abnormal splicing. These splicing abnormalities were detected in tumor samples in the absence of the normal splicing product, and were not found in several normal controls. In one case, a homozygous deletion present in tumor DNA, and not in the matched normal control was demonstrated. Mutations affecting the PPP2R1A gene were nucleotide substitutions changing highly conserved amino acids and one frame-shift. Although the frequency of alterations is low, the inclusion of both isoforms of subunit A in the genes mutated in human cancer and the addition of breast cancer to the list of neoplasms in which PPP2R1B is altered, strengthen the potential role of PP2A in human tumorogenesis.

Deletion and reduced expression of the Fanconi anemia FANCA gene in sporadic acute myeloid leukemia.

Fanconi anemia (FA) is an autosomal recessive chromosomal instability disorder caused by mutations in one of seven known genes (FANCA,C,D2,E,F,G and BRCA2). Mutations in the FANCA gene are the most prevalent, accounting for two-thirds of FA cases. Affected individuals have greatly increased risks of acute myeloid leukemia (AML). This raises the question as to whether inherited or acquired mutations in FA genes might be involved in the development of sporadic AML. Quantitative fluorescent PCR was used to screen archival DNA from sporadic AML cases for FANCA deletions, which account for 40% of FANCA mutations in FA homozygotes. Four heterozygous deletions were found in 101 samples screened, which is 35-fold higher than the expected population frequency for germline FANCA deletions (P<0.0001). Sequencing FANCA in the AML samples with FANCA deletions did not detect mutations in the second allele and there was no evidence of epigenetic silencing by hypermethylation. However, real-time quantitative PCR analysis in these samples showed reduced expression of FANCA compared to nondeleted AML samples and to controls. These findings suggest that gene deletions and reduced expression of FANCA may be involved in the promotion of genetic instability in a subset of cases of sporadic AML.

Short tandem repeat (STR) haplotypes in HLA: an integrated 50-kb STR/linkage disequilibrium/gene map between the RING3 and HLA-B genes and identification of STR haplotype diversification in the class III region.

We present a dense STR/linkage disequilibrium(LD)/gene map between the RING3 and HLA-B loci, reference allelic sizes on the most prevalent HLA haplotypes and their allelic frequencies in pedigree founders. This resource will facilitate LD, evolution and gene mapping studies, including comparisons of HLA and STR haplotypes and identification of HLA recombinants. The map was constructed by testing novel and previously reported STRs using a panel of 885 individuals in 211 families and 60 DNA samples from cell lines and bone marrow donors homozygous in the HLA-A, -B and -DR loci selected from over 15 000 entries into the registry of Swedish bone marrow donors. We have also analysed the variability of STR alleles/haplotypes on the most prevalent HLA haplotypes to identify STRs useful for fine mapping of disease genes in the region previously implicated in susceptibility to many disorders. The analysis of 40 HLA-A*01, B*0801, DRB1*03011, DQB1*0201 haplotypes in homozygous donors showed a surprising stability in 23 STRs between the class II recombination hot spot and HLA-B, with the average of 1.9% (16/838) variant alleles. However, 40% variant alleles were found at the D6S2670 locus in intron 19 of the tenascin-X gene both in the families and homozygous donors. The nucleotide sequence analysis of this STR showed a complex polymorphism consisting of tetra- (CTTT)(8-18) and penta-nucleotide (CTTTT)(1-2) repeats, separated by an intervening non-polymorphic sequence of 42 bp. The HLA-A1, B*0801, DRB1*03011, DQB1*0201 haplotypes had five (CTTT)(14-18)/(CTTTT)(2) variants with a predominant (CTTT)(16) allele, implicating the tetranucleotide component as the source of this ancestral haplotype diversification, which may be due to the location of D6S2670 in the region of the highest GC content in the human MHC.

Exon-scanning mutation analysis of the ATM gene in patients with ataxia-telangiectasia.

Using a polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) assay, which amplifies individually all coding exons of the ATM gene deficient ataxia-telangiectasia (A-T), we have analyzed 10 patients with A-T for ATM mutations. Mutation were detected in 9 patients. We describe the first ATM mutation in the splice junction found in the 5' splice site of intron 17, leading to exon skipping. However, most mutations were small deletions or insertions resulting in premature termination of the translation product. The development of DNA-based methods for detection of unknown mutations and further characterization of ATM mutation pattern will facilitate identification of A-T carriers and assessment of their cancer risk.

The patched/hedgehog/smoothened signalling pathway in human breast cancer: no evidence for H133Y SHH, PTCH and SMO mutations.

The patched/hedgehog/smoothened signalling pathway has been implicated in the development of sporadic tumours associated with the naevoid basal cell carcinoma (Gorlin) syndrome (NBCCS). Mutations in sporadic basal cell carcinomas (BCCs) of the skin and medulloblastomas have been found in genes encoding all three proteins of the pathway. A substantial proportion of breast carcinomas has recently been suggested to contain missense mutations in the human patched (PTCH) and sonic hedgehog (SHH) homologues. However, an independent study showed that the implicated mutation in SHH (H133Y) was absent in a large number of BCCs, medulloblastomas, breast, ovary and colorectal tumours. We searched for the H133Y SHH mutation in 84 primary breast carcinomas, but did not detect this change in any sample. In addition, a subset of 45 primary breast tumours was analysed for mutations in the PTCH coding region and 48 samples in previously implicated exons of human smoothened, but no mutations were found. Although our results do not exclude the presence of clonal alterations of these genes in a small proportion of breast carcinomas, these data do not support the existence of frequent mutations in genes encoding major protein partners of this signalling pathway. The absence of nucleotide changes in PTCH may point to another linked gene in the chromosome region 9q22-q23, previously suggested to contain a breast cancer susceptibility gene.

Genetics of IgA deficiency.

IgA deficiency is the most common humoral defect in man and results in an increased susceptibility to respiratory tract and gastrointestinal infections. Both clinical and genetic data support a close relationship with common variable immunodeficiency, a disease which involves not only IgA and IgG production, but also, in half of the patients, IgM. It is likely that the two disorders represent an allelic condition with a variable expression of a common gene defect which is thought to be involved in the regulation of immunoglobulin class switching. It is possible that a single, autosomally inherited gene with a limited penetrance is responsible for the development of both these defects.