Three new BLM gene mutations associated with Bloom syndrome

Bloom's syndrome (BS) is a rare autosomal recessive disease predisposing patients to all types of cancers affecting the general population. BS cells display a high level of genetic instability, including a 10-fold increase in the rate of sister chromatid exchanges, currently the only objective criterion for BS diagnosis. We have developed a method for screening the BLM gene for mutations based on direct genomic DNA sequencing. A questionnaire based on clinical information, cytogenetic features, and family history was addressed to physicians prescribing BS genetic screening, with the aim of confirming or guiding diagnosis. We report here four BLM gene mutations, three of which have not been described before. Three of the mutations are frameshift mutations, and the fourth is a nonsense mutation. All these mutations introduce a stop codon, and may therefore be considered to have deleterious biological effect. This approach should make it possible to identify new mutations and to correlate them with clinical information.     

Somatic intragenic recombination within the mutated locus BLM can correct the high sister-chromatid exchange phenotype of Bloom syndrome cells.

Cells from persons with Bloom syndrome feature an elevated rate of sister-chromatid exchange (SCE). However, in some affected persons a minority of blood lymphocytes have a normal SCE rate. Persons who inherit the Bloom syndrome gene BLM identical by descent from a common ancestor very rarely exhibit this high-SCE/low-SCE mosaicism; conversely, mosaicism arises predominantly in persons who do not share a common ancestor. These population data suggested that most persons with Bloom syndrome in whom the exceptional low-SCE cells arise are not homozygous for a mutation at BLM but instead are compound heterozygotes. Following this clue, we carried out a genotype analysis of loci syntenic with BLM in 11 persons who exhibited mosaicism. In five of them, polymorphic loci distal to BLM that were heterozygous in their high-SCE cells had become homozygous in their low-SCE cells, whereas heterozygous loci proximal to BLM remained heterozygous. These observations are interpreted to mean that intragenic recombination between paternally derived and maternally derived mutated sites within BLM can generate a functionally wild-type gene and that low-SCE lymphocytes are progeny of a somatic cell in which such intragenic recombination had occurred.     

Somatic mutations in the neurofibromatosis type 2 gene in sporadic meningiomas

Meningiomas are benign tumors of the central nervous system. Although usually sporadic, they can occur in patients affected by the autosomal dominant syndrome, neurofibromatosis type 2 (NF2). The NF2 gene has recently been isolated from chromosome 22. The presence of germline mutations in NF2 patients and the loss of heterozygosity (LOH) on 22q in NF2 tumors support the hypothesis that the NF2 gene acts as a tumor suppressor. Cytogenetic and LOH studies have suggested that the gene responsible for the development of meningiomas is located in the region of 22q in which the NF2 gene maps. The meningioma gene could therefore be the NF2 gene itself. Recently, somatic mutations of the NF2 gene have been identified in sporadic meningiomas, thus supporting the hypothesis that the NF2 gene is also important in meningioma pathogenesis. In this study, we analyzed sixty-one sporadic meningiomas for LOH of 22q and for mutations in the NF2 gene. LOH was detected in 36 of the 60 informative tumors. Single-strand conformational polymorphism analysis was used to identify nine mutations in five of the eight exons of the NF2 gene studied. The nine tumors with an altered NF2 gene also showed LOH for 22q markers. These results further support the hypothesis that mutations in the NF2 gene are a critical pathogenetic event in at least some meningiomas.     

Structural basis of Bloom syndrome (BS) causing mutations in the BLM helicase domain

BACKGROUND: Bloom syndrome (BS) is characterized by mutations within the BLM gene. The Bloom syndrome protein (BLM) has similarity to the RecQ subfamily of DNA helicases, which contain seven conserved helicase domains and share significant sequence and structural similarity with the Rep and PcrA DNA helicases. We modeled the three-dimensional structure of the BLM helicase domain to analyze the structural basis of BS-causing mutations. MATERIALS AND METHODS: The sequence alignment was performed for RecQ DNA helicases and Rep and PcrA helicases. The crystal structure of PcrA helicase (PDB entry 3PJR) was used as the template for modeling the BLM helicase domain. The model was used to infer the function of BLM and to analyze the effect of the mutations. RESULTS: The structural model with good stereochemistry of the BLM helicase domain contains two subdomains, 1A and 2A. The electrostatic potential of the model is highly negative over most of the surface, except for the cleft between subdomains 1A and 2A which is similar to the template protein. The ATP-binding site is located inside the model between subdomains 1A and 2A; whereas, the DNA-binding region is situated at the surface cleft, with positive potential between 1A and 2A. CONCLUSIONS: The three-dimensional structure of the BLM helicase domain was modeled and applied to interpret BS-causing mutations. The mutation I841T is likely to weaken DNA binding, while the mutations C891R, C901Y, and Q672R presumably disturb the ATP binding. In addition, other critical positions are discussed.     

Germline and somatic mutations in hMSH6 and hMSH3 in gastrointestinal cancers of the microsatellite mutator phenotype

Hereditary and sporadic gastrointestinal cancer of the microsatellite mutator phenotype (MMP) is characterized by a remarkable genomic instability at simple repeated sequences. The genomic instability is often caused by germline and somatic mutations in DNA mismatch repair (MMR) genes hMSH2 and hMLH1. The MMP can be also caused by epigenetic inactivation of hMLH1. The MMP generates many somatic frameshift mutations in genes containing mononucleotide repeats. We previously reported that in MMP tumors the hMSH6 and hMSH3 MMR genes often carry frameshift mutations in their (C)(8) and (A)(8) tracks, respectively. We proposed that these 'secondary mutator mutations' contribute to a gradual manifestation of the MMP. Here we report the detection of other frameshift, nonsense, and missense mutations in these genes in colon and gastric cancers of the MMP. A germline frameshift mutation was found in hMSH6 in a colon tumor harboring another somatic frameshift mutation. Several germline sequence variants and somatic missense mutations at conserved residues were detected in hMSH6 and only one was detected in hMSH3. Of the three hMSH6 germline variants in conserved residues, one coexisted with a somatic mutation at the (C)(8) track and another had a somatic missense mutation. We suggest that some of these germline and somatic missense variants are pathogenic. While biallelic hMSH6 and hMSH3 frameshift mutations were found in some tumors, many tumors seemed to contain only monoallelic mutations. In some tumors, these somatic monoallelic frameshift mutations at the (C)(8) and (A)(8) tracks were found to coexist with other somatic mutations in the other allele, supporting their functionality during tumorigenesis. However, the low incidence of these additional somatic mutations in hMSH6 and hMSH3 leaves many tumors with only monoallelic mutations. The impact of the frameshift mutations in gene expression was studied by comparative analysis of RNA and protein expression in different tumor cell clones with different genotypes. The results show that the hMSH6 (C)(8) frameshift mutation abolishes protein expression, ruling out a dominant negative effect by a truncated protein. We suggest the functionality of these secondary monoallelic mutator mutations in the context of an accumulative haploinsufficiency model.     

Somatic and germline mosaic mutations in the doublecortin gene are associated with variable phenotypes

Mutations in the X-linked gene doublecortin lead to "double cortex" syndrome (DC) in females and to X-linked lissencephaly (XLIS) in males. Because most patients with DC and XLIS are sporadic, representing de novo doublecortin mutations, we considered that some of these patients could be somatic or germline mosaics. Among a population of 20 patients and their families, we found evidence for mosaic doublecortin mutations in 6 individuals. Germline mosaicism was identified in two unaffected women, each with two affected children. Additionally, one affected male with DC was found to be a somatic mosaic, which presumably spared him from the more severe phenotype of lissencephaly. The high rate of mosaicism indicates that there may be a significant recurrence risk for DC/XLIS in families at risk, even when the mother is unaffected.     

Two frameshift mutations in the cystic fibrosis gene

Cystic fibrosis (CF) is a recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. We have identified in exon 7 two frameshift mutations, one caused by a two-nucleotide insertion and the other caused by a one-nucleotide deletion; these mutations--CF1154insTC and CF1213delT, respectively, are predicted to shift the reading frame of the protein and to introduce UAA(ochre) termination codons at residues 369 and 368.     

A novel cell-cycle-regulated interaction of the Bloom syndrome helicase BLM with Mcm6 controls replication-linked processes

The Bloom syndrome DNA helicase BLM contributes to chromosome stability through its roles in double-strand break repair by homologous recombination and DNA replication fork restart during the replication stress response. Loss of BLM activity leads to Bloom syndrome, which is characterized by extraordinary cancer risk and small stature. Here, we have analyzed the composition of the BLM complex during unperturbed S-phase and identified a direct physical interaction with the Mcm6 subunit of the minichromosome maintenance (MCM) complex. Using distinct binding sites, BLM interacts with the N-terminal domain of Mcm6 in G1 phase and switches to the C-terminal Cdt1-binding domain of Mcm6 in S-phase, with a third site playing a role for Mcm6 binding after DNA damage. Disruption of Mcm6-binding to BLM in S-phase leads to supra-normal DNA replication speed in unperturbed cells, and the helicase activity of BLM is required for this increased replication speed. Upon disruption of BLM/Mcm6 interaction, repair of replication-dependent DNA double-strand breaks is delayed and cells become hypersensitive to DNA damage and replication stress. Our findings reveal that BLM not only plays a role in the response to DNA damage and replication stress, but that its physical interaction with Mcm6 is required in unperturbed cells, most notably in S-phase as a negative regulator of replication speed.     

Temporal delineation of sequential HPRT mutations arising in vivo in a T-cell clone with a mutator phenotype

Recurrent mutations in vivo in T-lymphocytes identify clonally restricted genomic instabilities in some individuals. Cell-based assays allow initial recognition of clones with mutator phenotypes, but genotypic selection is required to determine frequencies and temporal sequences of potentially independent mutational events isolated only as complex changes in the same allele. The present work illustrates how two single-base insertions in the HPRT gene recovered only as a double event in a cell-based assay were shown to arise as separate in vivo mutations, being individually present at frequencies of < or =10(-4) and < or =10(-5), respectively, in peripheral blood. Full characterizations of mutator clones will allow elucidation of the earliest events in the emergence of genomic instability in human somatic cells.     

Dominant negative mutator mutations in the mutL gene of Escherichia coli.

The mutL gene product is part of the dam-directed mismatch repair system of Escherichia coli but has no known enzymatic function. It forms a complex on heteroduplex DNA with the mismatch recognition MutS protein and with MutH, which has latent endonuclease activity. An N-terminal hexahistidine-tagged MutL was constructed which was active in vivo. As a first stop to determine the functional domains of MutL, we have isolated 72 hydroxylamine-induced plasmid-borne mutations which impart a dominant-negative phenotype to the wild-type strain for increased spontaneous mutagenesis. None of the mutations complement a mutL deletion mutant, indicating that the mutant proteins by themselves are inactive. All the dominant mutations but one could be complemented by the wild-type mutL at about the same gene dosage. DNA sequencing indicated that the mutations affected 22 amino acid residues located between positions 16 and 549 of the 615 amino acid protein. In the N-terminal half of the protein, 12 out of 15 amino acid replacements occur at positions conserved in various eukaryotic MutL homologs. All but one of the sequence changes affecting the C-terminal end of the protein are nonsense mutations.     

Dominant negative mutator mutations in the mutS gene of Escherichia coli.

The MutS protein of Escherichia coli is part of the dam-directed MutHLS mismatch repair pathway which rectifies replication errors and which prevents recombination between related sequences. In order to more fully understand the role of MutS in these processes, dominant negative mutS mutations on a multicopy plasmid were isolated by screening transformed wild-type cells for a mutator phenotype, using a Lac+ papillation assay. Thirty-eight hydroxylamine- and 22 N-methyl-N'-nitro-N-nitrosoguanidine-induced dominant mutations were isolated. Nine of these mutations altered the P-loop motif of the ATP-binding site, resulting in four amino acid substitutions. With one exception, the remaining sequenced mutations all caused substitution of amino acids conserved during evolution. The dominant mutations in the P-loop consensus caused severely reduced repair of heteroduplex DNA in vivo in a mutS mutant host strain. In a wild-type strain, the level of repair was decreased by the dominant mutations to between 12 to 90% of the control value, which is consistent with interference of wild-type MutS function by the mutant proteins. Increasing the wild-type mutS gene dosage resulted in a reversal of the mutator phenotype in about 60% of the mutant strains, indicating that the mutant and wild-type proteins compete. In addition, 20 mutant isolates showed phenotypic reversal by increasing the gene copies of either mutL or mutH. There was a direct correlation between the levels of recombination and mutagenesis in the mutant strains, suggesting that these phenotypes are due to the same function of MutS.     

Germline missense mutations affecting KRAS Isoform B are associated with a severe Noonan syndrome phenotype

Noonan syndrome (NS) is a developmental disorder characterized by short stature, facial dysmorphia, congenital heart disease, and multiple skeletal and hematologic defects. NS is an autosomal dominant trait and is genetically heterogeneous. Gain of function of SHP-2, a protein tyrosine phosphatase that positively modulates RAS signaling, is observed in nearly 50% of affected individuals. Here, we report the identification of heterozygous KRAS gene mutations in two subjects exhibiting a severe NS phenotype with features overlapping those of cardiofaciocutaneous and Costello syndromes. Both mutations were de novo and affected exon 6, which encodes the C-terminal portion of KRAS isoform B but does not contribute to KRAS isoform A. Structural analysis indicated that both substitutions (Val152Gly and Asp153Val) perturb the conformation of the guanine ring-binding pocket of the protein, predicting an increase in the guanine diphosphate/guanine triphosphate (GTP) dissociation rate that would favor GTP binding to the KRASB isoform and bypass the requirement for a guanine nucleotide exchange factor.     

MECP2 mutations in sporadic cases of Rett syndrome are almost exclusively of paternal origin

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder that apparently is lethal in male embryos. RTT almost exclusively affects female offspring and, in 99.5% of all cases, is sporadic and due to de novo mutations in the MECP2 gene. Familial cases of RTT are rare and are due to X-chromosomal inheritance from a carrier mother. We analyzed the parental origin of MECP2 mutations in sporadic cases of RTT, by analysis of linkage between the mutation in the MECP2 gene and intronic polymorphisms in 27 families with 15 different mutations, and we found a high predominance of mutations of paternal origin in 26 of 27 cases (P<.001). The paternal origin was independent of type of mutation and was found for single-base exchanges as well as for deletions. Parents were not of especially advanced age. We conclude that de novo mutations in RTT occur almost exclusively on the paternally derived X chromosome and that this is most probably the cause for the high female:male ratio observed in patients with RTT. Affected males recently have been described in a few cases of familial inheritance. Identification of the parental origin may be useful to distinguish between the sporadic form of RTT and a potentially familial form. This distinction will allow geneticists to offer more-specific counseling and discriminate between higher (maternal origin) and lower (paternal origin) recurrence risk.     

A novel frameshift mutation in BLM gene associated with high sister chromatid exchanges (SCE) in heterozygous family members

The Bloom syndrome (BS) is an autosomic recessive disorder comprising a wide range of abnormalities, including stunted growth, immunodeficiency, sun sensitivity and increased frequency of various types of cancer. Bloom syndrome cells display a high level of genetic instability, including a 10-fold increase in the sister chromatid exchanges (SCE) level. Bloom syndrome arises through mutations in both alleles of the BLM gene, which was identified as a member of the RecQ helicase family. In this study, we screened a Tunisian family with three BS patients. Cytogenetic analysis showed several chromosomal aberrations, and an approximately 14-fold elevated SCE frequency in BS cells. A significant increase in SCE frequency was observed in some family members but not reaching the BS patients values, leading to suggest that this could be due to the heterozygous profile. Microsatellite genotyping using four fluorescent dye-labeled microsatellite markers revealed evidence of linkage to BLM locus and the healthy members, sharing higher SCE frequency, showed heterozygous haplotypes as expected. Additionally, the direct BLM gene sequencing identified a novel homozygous frameshift mutation c.3617–3619delAA (p.K1207fsX9) in BS patients and a heterozygous BLM mutation in the family members with higher SCE frequency. Our findings suggest that this latter mutation likely leads to a reduced BLM activity explaining the homologous recombination repair defect and, therefore, the increase in SCE. Based on the present data, the screening of this mutation could contribute to the rapid diagnosis of BS. The genetic confirmation of the mutation in BLM gene provides crucial information for genetic counseling and prenatal diagnosis.     

Identification of a mutant DNA polymerase delta in Saccharomyces cerevisiae with an antimutator phenotype for frameshift mutations

We propose that a beta-turn-beta structure, which plays a critical role in exonucleolytic proofreading in the bacteriophage T4 DNA polymerase, is also present in the Saccharomyces cerevisiae DNA pol delta. Site-directed mutagenesis was used to test this proposal by introducing a mutation into the yeast POL3 gene in the region that encodes the putative beta-turn-beta structure. The mutant DNA pol delta has a serine substitution in place of glycine at position 447. DNA replication fidelity of the G447S-DNA pol delta was determined in vivo by using reversion and forward assays. An antimutator phenotype for frameshift mutations in short homopolymeric tracts was observed for the G447S-DNA pol delta in the absence of postreplication mismatch repair, which was produced by inactivation of the MSH2 gene. Because the G447S substitution reduced frameshift but not base substitution mutagenesis, some aspect of DNA polymerase proofreading appears to contribute to production of frameshifts. Possible roles of DNA polymerase proofreading in frameshift mutagenesis are discussed.     

Linkage disequilibrium between the FES, D15S127, and BLM loci in Ashkenazi Jews with Bloom syndrome.

Bloom syndrome (BS) is more common in the Ashkenazi Jewish than in any other population. Approximately 1 in 110 Ashkenazi Jews carries blm, the BS mutation. The locus mutated in BS, BLM, maps to chromosome subband 15q26.1, tightly linked to the proto-oncogene FES. We have investigated the basis for the increased frequency of blm in the Ashkenazim by genotyping polymorphic microsatellite loci tightly linked to BLM in affected and unaffected individuals from Ashkenazi Jewish and non-Ashkenazi populations. A striking association of the C3 allele at FES with blm (delta = .422; p = 5.52 x 10(-7)) and of the 145-bp and 147-bp alleles at D15S127 with blm (delta = .392 and delta = .483, respectively; p = 2.8 x 10(-5) and p = 5.4 x 10(-7), respectively) was detected in Ashkenazi Jews with BS. This linkage disequilibrium constitutes strong support for a founder-effect hypothesis: the chromosome in the hypothetical founder who carried blm also carried the C3 allele at FES and either the 145-bp or the 147-bp allele at D15S127.     

Somatic mutations in the neurofibromatosis 1 gene in human tumors.

The neurofibromatosis 1 (NF1) gene product, neurofibromin, contains a GTPase-activating protein (GAP)-related domain, or NF1 GRD, that is able to down-regulate p21ras by stimulating its intrinsic GTPase. Since p21ras.GTP is a major regulator of growth and differentiation, mutant neurofibromins resulting from somatic mutations in the NF1 gene might interfere with ras signaling pathways and contribute to the development of tumors. We describe an amino acid substitution in the NF1 GRD, altering Lys-1423, that has occurred in three tumor types: colon adenocarcinoma, myelodysplastic syndrome, and anaplastic astrocytoma, and in one family with neurofibromatosis 1. The GAP activity of the mutant NF1 GRD is 200- to 400-fold lower than that of wild type, whereas binding affinity is unaffected. Thus, germline mutations in NF1 that cause neurofibromatosis 1 can also occur in somatic cells and contribute to the development of sporadic tumors, including tumors not associated with neurofibromatosis 1.