Biallelic mutation of MSH2 in primary human cells is associated with sensitivity to irradiation and altered RAD51 foci kinetics.

BACKGROUND: Reports of differential mutagen sensitivity conferred by a defect in the mismatch repair (MMR) pathway are inconsistent in their conclusions. Previous studies have investigated cells established from immortalised human colorectal tumour lines or cells from animal models. METHODS: We examined primary human MSH2-deficient neonatal cells, bearing a biallelic truncating mutation in MSH2, for viability and chromosomal damage after exposure to DNA-damaging agents. RESULTS: MSH2-deficient cells exhibit no response to interstrand DNA cross-linking agents but do show reduced viability in response to irradiation. They also show increased chromosome damage and exhibit altered RAD51 foci kinetics after irradiation exposure, indicating defective homologous recombinational repair. DISCUSSION: The cellular features and sensitivity of MSH2-deficient primary human cells are broadly in agreement with observations of primary murine cells lacking the same gene. The data therefore support the view that the murine model recapitulates early features of MMR deficiency in humans, and implies that the variable data reported for MMR-deficient immortalised human cells may be due to further genetic or epigenetic lesions. We suggest caution in the use of radiotherapy for treatment of malignancies in individuals with functional loss of MSH2.

Fanconi anaemia complementation group B presenting as X linked VACTERL with hydrocephalus syndrome.

BACKGROUND: The VACTERL with hydrocephalus (VACTERL-H) phenotype is recognised to be a severe manifestation of autosomal recessive Fanconi anaemia. Several families have been described in which the VACTERL-H phenotype segregates as an X linked syndrome. The mutations which cause X linked VACTERL-H syndrome are not known. OBJECTIVE: To determine if mutations in FANCB, which are known to cause Fanconi anaemia complementation group B, are a cause of X linked VACTERL-H syndrome. METHODS: A three generation pedigree with X linked VACTERL-H syndrome was investigated. X inactivation was tested in carrier females, and fibroblasts from an affected male fetus were analysed for increased sensitivity to diepoxybutane. FANCB coding exons and flanking splice sites were screened for mutations by direct sequencing of polymerase chain reaction (PCR) fragments amplified from genomic DNA. cDNA from affected fetal fibroblasts was analysed by PCR and direct sequencing using specific exonic primers. RESULTS: A FANCB mutation which results in a premature stop codon by causing skipping of exon 7 was identified. Chromosomes from the affected fetus showed increased sensitivity to diepoxybutane, and carrier women were found to have 100% skewed X inactivation in blood. CONCLUSIONS: Mutations in FANCB are a cause of X linked VACTERL-H syndrome. The data presented are of relevance to the genetic counselling of families with isolated male cases of VACTERL-H and Fanconi anaemia.

The G2 chromosomal radiosensitivity assay.

Although requiring stringent experimental conditions to achieve good reproducibility, the G2 assay has potential as a sensitive marker for cancer susceptibility, and is particularly useful in population studies. Immediate culture of blood is preferable, but overnight storage of blood either at ambient temperature or at 4 degrees C does not appear significantly to affect G2 scores. Transport of blood may lead to additional variability in assay results and should be well controlled. Although reproducibility is generally good, G2 scores on blood from certain individuals appear to show significant variability in repeat samples. Thus, determination of an individual's radiosensitivity may require multiple assays on different occasions. While it is recognized that the distinction between aligned and mis-aligned discontinuities has no scientific basis, some laboratories have decided for the purpose of record-keeping to score all aligned discontinuities as gaps, and mis-aligned discontinuities as breaks. In all cases the final G2 score should comprise the sum of all gaps and breaks.

Is telomere length in peripheral blood lymphocytes correlated with cancer susceptibility or radiosensitivity?

Mean terminal restriction fragment (TRF) lengths in white blood cells (WBCs) have been previously found to be associated with breast cancer. To assess whether this marker could be used as a test for breast cancer susceptibility in women, TRF length was measured in 72 treated female breast cancer patients and 1696 unaffected female controls between the ages of 45 and 77 from the Twin Research Unit at St Thomas' Hospital, as well as 140 newly diagnosed breast cancer cases and 108 mammographically screened unaffected controls from Guy's Hospital. Mean TRF was also tested for correlation with chromosome radiosensitivity and apoptotic response in the Guy's Hospital patients. After adjusting for age, smoking and body mass index, there was no significant difference in TRF lengths between the treated breast cancer patients and unaffected controls (P=0.71). A positive correlation between age-adjusted apoptotic response and mean TRF in newly diagnosed untreated breast cancer patients (P=0.008) was identified but no significant difference in TRF lengths between breast cancer patients and unaffected controls was detected (P=0.53). This suggests that TRF lengths in WBC, is not a marker of breast cancer susceptibility and does not vary significantly between affected women before and after treatment.

Novel mutations and polymorphisms in the Fanconi anemia group C gene.

Fanconi anemia (FA) is an autosomal recessive disorder associated with hypersensitivity to DNA cross-linking agents and bone marrow failure. At least four complementation groups have been defined, and the FA group C gene (FAC) has been cloned. We have screened 76 unrelated FA patients of diverse ethnic and geographic origins and from unknown complementation groups for mutations in the FAC gene either by chemical cleavage mismatch analysis or by single-strand conformational polymorphism (SSCP). Five mutations were detected in four patients (5.3%), including two novel mutations (W22X and L496R). Nine polymorphisms were detected, seven of which have not been described previously (663A-->G, L190F, IVS6 + 30C-->T, I312V, V449M, Q465R, and 1974G-->A). Six of the nine polymorphisms occurred in patients or controls from the Tswana or Sotho chiefdoms of South Africa and were not found in 50 unrelated European controls. Restriction site assays were established for all 8 pathogenic mutations identified in the FAC gene to date and used to screen a total of 94 unrelated FA patients. This identified only one other group C patient, who was homozygons for the mutation IVS4 + 4A-->T. This study indicates that the proportion of FA patients from complementation group C is generally likely to be less than 10%. Guidelines for the selection of FA patients for FAC mutation screening are proposed.