Julian Barwell

Phenotypic expression of double heterozygosity for BRCA1 and BRCA2 germline mutations

Mutations in two major cancer susceptibility genes, BRCA1 and BRCA2 , predispose to early onset breast and ovarian cancer. Since 1994, genetic testing for germline mutations in these genes has been carried out in many countries in both diagnostic and research settings. Mutation analysis is usually done on the basis of a (family) history of breast or ovarian cancer—for example, (very) early age of onset, multiple affected close relatives, multiple tumours in one patient, and breast cancer in men.1–3 Ethnic background may also play a role in decisions about DNA testing, as in some populations founder BRCA1 or BRCA2 mutations are known to occur at relatively high prevalence (for example, 185delAG and 5382insC in BRCA1 and 6174delT in BRCA2 in Ashkenazi Jews4). In recent years several families have been described in which more than one BRCA mutation segregated, predominantly involving Ashkenazi Jewish founder mutations. These reports describe families that harbour two pathogenic BRCA1 mutations,5 one BRCA1 and one BRCA2 mutation,6,7,8,9,10,11,12,13,14,15,16,17 or even three pathogenic mutations in BRCA genes.18 Some of these families were uncovered because the index case appeared to carry two (founder) mutations, and only rarely was co-segregation of two different mutations suspected beforehand on the basis of the family history. This has led to the recommendation that one should always test for all three founder mutations in individuals of known Jewish ancestry.19 However, DH has also been reported without prior knowledge of Jewish ancestry.10,12,13,16,17,20 In this paper we present four new cases with mutations in both BRCA1 and BRCA2 and review and update the 30 cases reported in the literature, in order to investigate the phenotypic consequences of double heterozygosity (DH)—that is, the …

Is chromosome radiosensitivity and apoptotic response to irradiation correlated with cancer susceptibility

Purpose: Individuals who have been treated for breast cancer have been reported to have increased lymphocyte chromosomal sensitivity to ionizing radiation and a significantly lower apoptotic response to irradiation compared to controls. We set out to test these findings using a substantial number of cases sampled before treatment (which could alter the parameters measured), compared to age-matched controls with normal mammograms. Material and methods: We used the G2 chromosome breakage, and apoptotic response assays of peripheral blood lymphocytes to ionizing radiation to compare 211 unselected newly diagnosed and untreated breast cancer patients, with 170 age, sex and ethnically matched controls. Results: We found no significant differences between breast cancer patients and their matched controls in the G2 assay or apoptotic response. However, there was some evidence that both cases and controls with a strong family history of breast cancer had higher radiosensitivity than those without. Conclusions: rThis is the largest and best controlled study of its kind, but it has not replicated previous reports of differences between chromosome breakage or apoptotic response in breast cancer cases vs. controls. However there was a suggestion of increased radiosensitivity in patients with a strong family history, which may indicate a heritable cancer susceptibility trait, warranting further study.

Lymphocyte radiosensitivity in BRCA1 and BRCA2 mutation carriers and implications for breast cancer susceptibility

There is conflicting evidence as to whether individuals who are heterozygous for germ‐line BRCA1 or BRCA2 mutations have an altered phenotypic cellular response to irradiation. To investigate this, chromosome breakage and apoptotic response were measured after irradiation in peripheral blood lymphocytes from 26 BRCA1 and 18 BRCA2 mutation carriers without diagnosed breast cancer, and 38 unaffected age, ethnically and sex‐matched controls. To assess the role of BRCA1 and BRCA2 in homologous recombination, an S phase enrichment chromosome breakage assay was used. BrdUrd incorporation studies allowed verification of the correct experimental settings. We found that BRCA1 mutation carriers without cancer had increased chromosome breaks as well as breaks and gaps per cell post irradiation using the classical G2 assay (p = 0.01 and 0.004, respectively) and the S phase enrichment assay (p = 0.01 and 0.01, respectively) compared to age‐matched unaffected controls. BRCA2 mutation carriers without cancer had increased breaks as well as breaks and gaps per cell post irradiation using the S phase enrichment assay (p = 0.045 and 0.012, respectively). No difference was detected using the G2 assay (p = 0.88 and 0.40 respectively). BRCA1 and BRCA2 mutation carriers had normal cell cycle kinetics and apoptotic response to irradiation compared to age‐matched controls. Our results show a demonstrable impairment in irradiation induced DNA repair in women with heterozygous germline BRCA1 and BRCA2 mutations prior to being diagnosed with breast cancer.

What hinders minority ethnic access to cancer genetics services and what may help

Ethnic disparities in use of cancer genetics services raise concerns about equitable opportunity to benefit from familial cancer risk assessment, improved survival and quality of life. This paper considers available research to explore what may hinder or facilitate minority ethnic access to cancer genetics services. We sought to inform service development for people of South Asian, African or Irish origin at risk of familial breast, ovarian, colorectal and prostate cancers in the UK. Relevant studies from the UK, North America and Australasia were identified from six electronic research databases. Current evidence is limited but suggests low awareness and understanding of familial cancer risk among minority ethnic communities studied. Socio-cultural variations in beliefs, notably stigma about cancer or inherited risk of cancer, are identified. These factors may affect seeking of advice from providers and disparities in referral. Achieving effective cross-cultural communication in the complex contexts of both cancer and genetics counselling, whether between individuals and providers, when mediated by third party interpreters, or within families, pose further challenges. Some promising experience of facilitating minority ethnic access has been gained by introduction of culturally sensitive provider and counselling initiatives, and by enabling patient self-referral. However, further research to inform and assess these interventions, and others that address the range of challenges identified for cancer genetics services are needed. This should be based on a more comprehensive understanding of what happens at differing points of access and interaction at community, cancer care and genetic service levels.

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.

A new neocentromere locus on chromosome 13 resulting in mosaic tetrasomy for distal 13q and an asymmetric phenotype

An 8‐year‐old girl was referred to the Genetics Centre with mild developmental delay, mild dysmorphic features, and a head circumference on the 98th centile. She was noted to have large irregular ear lobes, torticollis, and mild hemihypertrophy. Karyotype analysis of cultured peripheral lymphocytes and skin fibroblasts revealed the presence of a symmetrical supernumerary marker chromosome in 13% of cells from both tissue types. Further analysis showed that this marker chromosome originated from the distal region of chromosome 13 and contained no centromeric α‐satellite DNA. The marker chromosome was not found in blood from the parents. This case represents a novel symmetrical structure with a previously unreported neocentromere locus, leading to an unusual phenotype. Similar cases of individuals with a chromosome 13 with a neocentromere have been reported. They are reviewed and compared with the current case. The importance of scanning metaphases for abnormalities in individuals presenting with asymmetry is emphasized.

Evidence against a major genetic basis for combined breast and colorectal cancer susceptibility

To the Editor: There is debate as to whether there is an inherited susceptibility to both breast cancer (BC) and colorectal cancer (CRC), distinct from the relatively site-specific Mendelian conditions known to cause a high risk of BC (BRCA1/2 mutations) and CRC (hereditary non-polyposis colorectal cancer, HNPCC) (1–10). CHEK2 mutations, including the common 1100delC, cause moderately increased risks, and are candidates for this role (11–23). We ascertained individuals diagnosed with both primary BC and CRC, through British Society of Human Genetics colleagues and the Thames Cancer Registry, with ethical approval. We contacted patients through their physicians. Participants completed a family history questionnaire; diagnoses were confirmed in probands (but not in relatives, for confidentiality); and blood was obtained from all cases and tumour from both the cancers wherever possible. DNA was extracted from peripheral blood lymphocytes using NUCLEON BACC3 Genomic kit (Nucleon Biosciences, UK) and from paraffin-embedded tumour using QIAmp DNA Mini-Kit (Qiagen Inc., Chatsworth, CA, USA). Tumour DNA was tested for microsatellite instability (MSI) using fluorescence-labelled nearmonomorphic, mononucleotide markers BAT25 and BAT26. Tumours MSI1 at either marker were tested by immunohistochemistry for MLH1, MSH2 and MSH6 proteins. Constitutional DNA from individuals with negatively staining tumours was screened for germline mutations in the relevant gene using denaturing high-performance liquid chromatography (dHPLC) and multiplex ligation-dependant probe amplification (MLPA) (all exons). All constitutional DNA samples were screened for germline mutations in exons 2, 20 plus end fragments of exon 11 of BRCA1, and exon 9 plus end fragments of exons 10 and 11 of BRCA2 using dHPLC. Exon 10 of CHEK2 was analysed in all samples, and exons 1–8 in 77 samples using single-stranded conformational polymorphism (24). Any abnormalities were sequenced using ABI 1300 (Applied Biosystems, Warrington, UK). Positive controls were analysed alongside test samples for the CHEK2 1100delC variant. We ascertained 97 participants and 97 anonymous cancer-free UK-wide controls already available. Tumour blocks were obtained from 67 individuals; and both BC and CRC were obtained from 36 individuals. Average age at diagnosis of BC was 62.1 years (37–87 years) and CRC, 63.5 years (35–83 years). Fourteen women had more than two cancers (six a second BC, one CRC, three endometrial cancer). Two pedigrees were Amsterdam criteria (AC) positive for HNPCC and six were modified AC1 (2). Four fulfilled National Institute for Clinical Excellence guidelines for tertiary referral for BRCA1/BRCA2 testing (www.nice.org.uk/ pdf/CG014Fullguideline.pdf). None of 52 BC tested showed MSI. Six of 57 (10.5%) CRCs were MSI1. In three of them, BC tested negative for MSI (Table 1). No germline MLH1 mutation was detected in these women, but a protein-truncating MSH6 mutation was found in a woman with four cancers (modified AC1). We could not perform further testing to establish whether the MSH6 mutation detected segregated with BC. Because HNPCC accounts for 2–3% of all CRCs, this data does not support mismatch repair mutations as a common cause of breast/colon cancer susceptibility. We detected two polymorphic variants in BRCA1 (Q356R in 15 cases) and one in BRCA2 (Table 2) (25). BRCA1 Q356R was detected in 9 of 97 controls (difference not significant). One pathogenic BRCA2 mutation (c755delCAGT) was detected in a woman diagnosed with BC at 66 years, CRC at 75 years. Her daughter had BC at 35 years. The pathogenic germline BRCA2 mutation detected was in a woman whose daughter had

Class II neocentromeres: a putative common neocentromere site in band 4q21.2.

Neocentromeres are rare functional centromeres formed within noncentromeric chromosomal regions. We report the finding of a neocentromere in a very rare class II analphoid chromosome. This neocentromere was detected prenatally in a fetus with the karyotype: 47,XY,del(4)(p15.3q21.1),+r(4)(p15.3q21.1).ish del(4)(D4S3360+,WHS+,D4Z1−,4qsubtel+),r(4)(D4S3360−,WHS−,D4Z1+,4qsubtel−)de novo. The fetus was missing one normal chromosome 4 but had a ring chromosome, consisting of the pericentromeric region of chromosome 4, and a deleted chromosome 4, the reciprocal product of the ring formation. In situ hybridization established that the chromosome 4 pericentromeric heterochromatin was located on the ring chromosome, while the Wolf-Hirschhorn critical region and chromosome 4 subtelomeric regions were present on the deleted chromosome. A C-band-negative constriction was observed in band 4q21.2 of the deleted chromosome 4, indicating that a neocentromere had been formed in this band, allowing stable segregation during cell division. This chromosome abnormality was detected in cultured amniocytes from a 20-week pregnancy presenting with intrauterine growth retardation and echogenic bowel. The pregnancy resulted in intrauterine death at 33–34 weeks. Despite the apparently balanced karyotype, the fetus is likely to have been phenotypically impaired due to disruption of genes by the neocentromere, rearrangement and ring chromosome formation. There has been one previous report of neocentromere formation in band 4q21; the observation presented here might refine a putative common neocentromeric site to sub-band 4q21.2.