Yield of Lynch Syndrome Surveillance for Patients With Pathogenic Variants in DNA Mismatch Repair Genes.

BACKGROUND & AIMS: Patients with Lynch syndrome are offered the same colorectal cancer (CRC) surveillance programs (colonoscopy every 2 years), regardless of the pathogenic DNA mismatch repair gene variant the patient carries. We aimed to assess the yield of surveillance for patients with these variants in MLH1, MSH2, MSH6, and PMS2. METHODS: We analyzed data on colonoscopy surveillance, including histopathology analysis, from all patients diagnosed with Lynch syndrome (n = 264) at a single center. We compared the development of (advanced) adenomas and CRC among patients with pathogenic variants in the DNA mismatch repair genes MLH1 (n = 55), MSH2 (n = 44), MSH6 (n = 143), or PMS2 (n = 22) over 1836 years of follow-up (median follow-up of 6 years per patient). RESULTS: At first colonoscopy, CRC was found in 8 patients. During 916 follow-up colonoscopies, CRC was found in 9 patients. No CRC was found in patients with variants in MSH6 or PMS2 over the entire follow-up period. There were no significant differences in the number of colonoscopies with adenomas or advanced adenomas among the groups. The median time of adenoma development was 3 years (IQR, 2-6 years). There were no significant differences in time to development of adenoma. However, patients with variants in MSH6 had a significant longer time to development of advanced neoplasia (advanced adenoma or CRC) than patients in the other groups. Six carriers died during follow up (5 from cancer, of which 3 from pancreatic cancer). CONCLUSIONS: No CRC was found during follow-up of patients with Lynch syndrome carrying pathogenic variants in MSH6; advanced neoplasia developed over shorter follow-up time periods in patients with pathogenic variants in MLH1 or MSH2. The colonoscopy interval for patients with pathogenic variants in MSH6 might be increased to 3 years from the regular 2-year interval.

Gain-of-Function Mutations in ZIC1 Are Associated with Coronal Craniosynostosis and Learning Disability.

Human ZIC1 (zinc finger protein of cerebellum 1), one of five homologs of the Drosophila pair-rule gene odd-paired, encodes a transcription factor previously implicated in vertebrate brain development. Heterozygous deletions of ZIC1 and its nearby paralog ZIC4 on chromosome 3q25.1 are associated with Dandy-Walker malformation of the cerebellum, and loss of the orthologous Zic1 gene in the mouse causes cerebellar hypoplasia and vertebral defects. We describe individuals from five families with heterozygous mutations located in the final (third) exon of ZIC1 (encoding four nonsense and one missense change) who have a distinct phenotype in which severe craniosynostosis, specifically involving the coronal sutures, and variable learning disability are the most characteristic features. The location of the nonsense mutations predicts escape of mutant ZIC1 transcripts from nonsense-mediated decay, which was confirmed in a cell line from an affected individual. Both nonsense and missense mutations are associated with altered and/or enhanced expression of a target gene, engrailed-2, in a Xenopus embryo assay. Analysis of mouse embryos revealed a localized domain of Zic1 expression at embryonic days 11.5-12.5 in a region overlapping the supraorbital regulatory center, which patterns the coronal suture. We conclude that the human mutations uncover a previously unsuspected role for Zic1 in early cranial suture development, potentially by regulating engrailed 1, which was previously shown to be critical for positioning of the murine coronal suture. The diagnosis of a ZIC1 mutation has significant implications for prognosis and we recommend genetic testing when common causes of coronal synostosis have been excluded.

Substantial evidence for the clinical significance of missense variant BRCA1 c.5309G>T p.(Gly1770Val).

PURPOSE: Classification of rare BRCA1 missense variants presents a major challenge for the counseling and treatment of patients. Variant classification can be complicated by conflicting lines of evidence. BRCA1 c.5309G>T p.(Gly1770Val) has been shown to abrogate BRCA1 protein homologous DNA repair; however, multiple sequence alignment demonstrates a lack of sequence conservation at this position, suggesting that glycine at position 1770 may not be essential for cellular maintenance in humans. We analyzed clinical information to resolve the classification of BRCA1 c.5309G>T p.(Gly1770Val). METHODS: We performed multifactorial likelihood analysis combining segregation data for 14 informative families, and breast tumor histopathological data for 17 variant carriers, ascertained through the ENIGMA consortium. RESULTS: Bayes segregation analysis gave a likelihood ratio of 101:1 in favor of pathogenicity. The vast majority of breast tumors showed features indicative of pathogenic variant carrier status, resulting in a likelihood ratio of 15800794:1 towards pathogenicity. Despite a low prior probability of pathogenicity (0.03) based on bioinformatic prediction, multifactorial likelihood analysis including segregation and histopathology analysis gave a posterior probability of > 0.99 and final classification of Pathogenic. CONCLUSIONS: We provide evidence that BRCA1 c.5309G>T p.(Gly1770Val), previously described as a Moroccan founder variant, should be treated as a disease-causing variant despite a lack of evolutionary conservation at this amino acid position. Additionally, we stress that bioinformatic information should be used in combination with other data, either direct clinical evidence or some form of clinical calibration, to arrive at a final clinical classification.

Acute myeloid leukaemia in a case with Tatton-Brown-Rahman syndrome: the peculiar DNMT3A R882 mutation.

BACKGROUND: Recently a novel syndromic form of overgrowth with intellectual disability and distinct facial features was identified caused by constitutional mutations in the epigenetic regulator DNA-methyltransferase 3A (DNMT3A), referred to as Tatton-Brown-Rahman syndrome (TBRS). Somatically acquired mutations in DNMT3A occur in haematological malignancies and are frequently present in acute myeloid leukaemia (AML) affecting in more than 50% the arginine residue at position 882 (R882). To date, additional cases with TBRS have been published but so far none of the reported cases with TBRS developed AML. METHODS AND RESULTS: Here we present the first case of TBRS who developed AML at the age of 15 years. Whole-exome sequencing identified a constitutional heterozygous DNMT3A R882C mutation. Our case exhibits macrocephaly, intellectual disability, distinct facial dysmorphism and other recurrent features fitting with the TBRS phenotype. The AML of the myelomonocytic subtype harboured only few additional somatically acquired mutations, that is, an aberrant karyotype and a recurrent PTPN11 mutation. DISCUSSION: The peculiarity of the specific R882 mutation in contrast to other DNMT3A mutations is discussed, including the hypothesis of the more aggressive nature of this variant.Our case represents the first evidence of the possible increased risk of the development of haematological malignancies in particular AML in cases with TBRS.

Type 1 papillary renal cell carcinoma in a patient with schwannomatosis: Mosaic versus loss of SMARCB1 expression in respectively schwannoma and renal tumor cells.

In schwannomatosis, germline SMARCB1 or LZTR1 mutations predispose to the development of multiple benign schwannomas. Besides these, other tumors may occur in schwannomatosis patients. We present a 45-year-old male patient who developed multiple schwannomas and in addition a malignant type 1 papillary renal cell carcinoma (pRCC1). We identified a duplication of exon 7 of SMARCB1 on chromosome 22 in the constitutional DNA of the patient (c.796-2246_986 + 5250dup7686), resulting in the generation of a premature stop codon in the second exon 7 copy (p.Glu330*). The mutant SMARCB1 allele proved to be retained in three schwannomas and in the pRCC1 of the patient. Loss of heterozygosity analysis demonstrated partial loss of the wild-type SMARCB1 allele containing chromosome 22, suggesting loss of that chromosome in only a subset of tumor cells, in all four tumors. Immunohistochemical staining with a SMARCB1 antibody revealed a mosaic SMARCB1 expression pattern in the three benign schwannomas, but absence of expression in the malignant tumor cells of the pRCC1. To our knowledge, this difference in SMARCB1 protein expression has not been reported before. We conclude that a germline SMARCB1 mutation may predispose to the development of pRCC1, thereby further widening the spectrum of tumors that can develop in the context of schwannomatosis.

Identification of Intragenic Exon Deletions and Duplication of TCF12 by Whole Genome or Targeted Sequencing as a Cause of TCF12-Related Craniosynostosis.

TCF12-related craniosynostosis can be caused by small heterozygous loss-of-function mutations in TCF12. Large intragenic rearrangements, however, have not been described yet. Here, we present the identification of four large rearrangements in TCF12 causing TCF12-related craniosynostosis. Whole-genome sequencing was applied on the DNA of 18 index cases with coronal synostosis and their family members (43 samples in total). The data were analyzed using an autosomal-dominant disease model. Structural variant analysis reported intragenic exon deletions (of sizes 84.9, 8.6, and 5.4 kb) in TCF12 in three different families. The results were confirmed by deletion-specific PCR and dideoxy-sequence analysis. Separately, targeted sequencing of the TCF12 genomic region in a patient with coronal synostosis identified a tandem duplication of 11.3 kb. The pathogenic effect of this duplication was confirmed by cDNA analysis. These findings indicate the importance of screening for larger rearrangements in patients suspected to have TCF12-related craniosynostosis.

Cost-effectiveness of routine screening for Lynch syndrome in colorectal cancer patients up to 70 years of age.

PURPOSE: To assess the cost-effectiveness of routine Lynch syndrome (LS) screening among colorectal cancer (CRC) patients ≤70 years of age. METHODS: A population-based series of CRC patients ≤70 years of age was routinely screened for LS. We calculated life years gained (LYG) and incremental cost-effectiveness ratios (ICERs) for different age cutoffs and comparing age-targeted screening with the revised Bethesda guidelines. RESULTS: Screening 1,117 CRC patients identified 23 LS patients, of whom 7 were ≤50 years of age, 7 were 51-60, and 9 were 61-70. Additionally, 70 LS carriers were identified among relatives (14, 42, and 14 per age category). Screening amounted to 205.9 LYG or 43.6, 118.0, and 44.3 LYG per age category. ICERs were [euro ]4.226/LYG for screening CRC patients ≤60 years of age compared with those ≤50 years and [euro ]7.051/LYG for screening CRC patients ≤70 years compared with those ≤60 years. The revised Bethesda guidelines identified 70 of 93 (75%) LS carriers. The ICER for LS screening in CRC patients ≤70 years of age compared with the revised Bethesda guidelines was [euro ]7.341/LYG. All ICERs remained less than [euro ]13.000/LYG in one-way sensitivity analyses. CONCLUSION: Routine LS screening by analysis of microsatellite instability, immunohistochemistry, and MLH1 hypermethylation in CRC patients ≤70 years of age is a cost-effective strategy with important clinical benefits for CRC patients and their relatives.Genet Med 18 10, 966-973.

The CYLD p.R758X worldwide recurrent nonsense mutation detected in patients with multiple familial trichoepithelioma type 1, Brooke-Spiegler syndrome and familial cylindromatosis represents a mutational hotspot in the gene.

BACKGROUND: Multiple familial trichoepithelioma type 1 (MFT1; MIM 601606), a rare monogenic skin disease with autosomal dominant inheritance, is characterized by the development of multiple skin-colored papules on the central area of the face, frequently occurring in the nasolabial area. The disease is associated with various mutations in the cylindromatosis (CYLD; MIM 605018) gene that are also responsible for familial cylindromatosis (FC) and Brooke-Spiegler syndrome (BSS). METHODS: Recently we have identified a Spanish MFT1 pedigree with two affected family members (father and daughter). Direct sequencing of the CYLD gene revealed a worldwide recurrent heterozygous nonsense mutation (c.2272C/T, p.R758X) in the patients. RESULTS: This mutation has already been detected in patients with all three clinical variants - BSS, FC and MFT1 - of the CYLD-mutation spectrum. Haplotype analysis was performed for the Spanish patients with MFT1, Dutch patients with FC and an Austrian patient with BSS, all of whom carry the same heterozygous nonsense p.R758X CYLD mutation. CONCLUSIONS: Our results indicate that this position is a mutational hotspot on the gene and that patients carrying the mutation exhibit high phenotypic diversity.

Identification of a BRCA2-specific modifier locus at 6p24 related to breast cancer risk.

Common genetic variants contribute to the observed variation in breast cancer risk for BRCA2 mutation carriers; those known to date have all been found through population-based genome-wide association studies (GWAS). To comprehensively identify breast cancer risk modifying loci for BRCA2 mutation carriers, we conducted a deep replication of an ongoing GWAS discovery study. Using the ranked P-values of the breast cancer associations with the imputed genotype of 1.4 M SNPs, 19,029 SNPs were selected and designed for inclusion on a custom Illumina array that included a total of 211,155 SNPs as part of a multi-consortial project. DNA samples from 3,881 breast cancer affected and 4,330 unaffected BRCA2 mutation carriers from 47 studies belonging to the Consortium of Investigators of Modifiers of BRCA1/2 were genotyped and available for analysis. We replicated previously reported breast cancer susceptibility alleles in these BRCA2 mutation carriers and for several regions (including FGFR2, MAP3K1, CDKN2A/B, and PTHLH) identified SNPs that have stronger evidence of association than those previously published. We also identified a novel susceptibility allele at 6p24 that was inversely associated with risk in BRCA2 mutation carriers (rs9348512; per allele HR = 0.85, 95% CI 0.80-0.90, P = 3.9 × 10(-8)). This SNP was not associated with breast cancer risk either in the general population or in BRCA1 mutation carriers. The locus lies within a region containing TFAP2A, which encodes a transcriptional activation protein that interacts with several tumor suppressor genes. This report identifies the first breast cancer risk locus specific to a BRCA2 mutation background. This comprehensive update of novel and previously reported breast cancer susceptibility loci contributes to the establishment of a panel of SNPs that modify breast cancer risk in BRCA2 mutation carriers. This panel may have clinical utility for women with BRCA2 mutations weighing options for medical prevention of breast cancer.

Complete APTX deletion in a patient with ataxia with oculomotor apraxia type 1.

BACKGROUND: Ataxia with oculomotor apraxia type 1 is an autosomal-recessive neurodegenerative disorder characterized by a childhood onset of slowly progressive cerebellar ataxia, followed by oculomotor apraxia and a severe primary motor peripheral axonal motor neuropathy. Ataxia with oculomotor apraxia type 1 is caused by bi-allelic mutations in APTX (chromosome 9p21.1). CASE PRESENTATION: Our patient has a clinical presentation that is typical for ataxia with oculomotor apraxia type 1 with no particularly severe phenotype. Multiplex Ligation-dependent Probe Amplification analysis resulted in the identification of a homozygous deletion of all coding APTX exons (3 to 9). SNP array analysis using the Illumina Infinium CytoSNP-850 K microarray indicated that the deletion was about 62 kb. Based on the SNP array results, the breakpoints were found using direct sequence analysis: c.-5 + 1225_*44991del67512, p.0?. Both parents were heterozygous for the deletion. Homozygous complete APTX deletions have been described in literature for two other patients. We obtained a sample from one of these two patients and characterized the deletion (156 kb) as c.-23729_*115366del155489, p.0?, including the non-coding exons 1A and 2 of APTX. The more severe phenotype reported for this patient is not observed in our patient. It remains unclear whether the larger size of the deletion (156 kb vs 62 kb) plays a role in the phenotype (no extra genes are deleted). CONCLUSION: Here we described an ataxia with oculomotor apraxia type 1 patient who has a homozygous deletion of the complete coding region of APTX. In contrast to the patient with the large deletion, our patient does not have a severe phenotype. More patients with deletions of APTX are required to investigate a genotype-phenotype effect.

A novel mutation in FGFR2.

Craniosynostosis is a congenital anomaly that can occur as an isolated condition or as part of a syndrome. Although several genes are known to cause syndromic craniosynostosis, only 24% can be attributed to known genes. Therefore, it is likely that more mutations and other genes are involved. We present the identification of a novel point mutation in fibroblast growth factor receptor 2 (FGFR2), c.812G>T, p.(Gly271Val) or c.1851G>C, p.(Leu617Phe). Furthermore, we describe a mutation that has been identified just recently, c.812G>T, (p.Gly271Val) or c.1851G>C, (p.Leu617Phe). In addition, we describe findings from a sequence analysis of all coding exons and exon/intron boundaries of FGFR2 performed on 124 patients with syndromic craniosynostosis.

Somatic aberrations of mismatch repair genes as a cause of microsatellite-unstable cancers.

Lynch syndrome (LS) is caused by germline mutations in mismatch repair (MMR) genes, resulting in microsatellite-unstable tumours. Approximately 35% of suspected LS (sLS) patients test negative for germline MMR gene mutations, hampering conclusive LS diagnosis. The aim of this study was to investigate somatic MMR gene aberrations in microsatellite-unstable colorectal and endometrial cancers of sLS patients negative for germline MMR gene mutations. Suspected LS cases were selected from a retrospective Clinical Genetics Department diagnostic cohort and from a prospective multicentre population-based study on LS in The Netherlands. In total, microsatellite-unstable tumours of 40 sLS patients (male/female 20/20, median age 57 years) were screened for somatic MMR gene mutations by next-generation sequencing. In addition, loss of heterozygosity (LOH) of the affected MMR genes in these tumours as well as in 68 LS-associated tumours and 27 microsatellite-unstable tumours with MLH1 promoter hypermethylation was studied. Of the sLS cases, 5/40 (13%) tumours had two pathogenic somatic mutations and 16/40 (40%) tumours had a (likely) pathogenic mutation and LOH. Overall, LOH of the affected MMR gene locus was observed in 24/39 (62%) tumours with informative LOH markers. Of the LS cases and the tumours with MLH1 promoter hypermethylation, 39/61 (64%) and 2/21 (10%) tumours, respectively, demonstrated LOH. Half of microsatellite-unstable tumours of sLS patients without germline MMR gene mutations had two (likely) deleterious somatic MMR gene aberrations, indicating their sporadic origin. Therefore, we advocate adding somatic mutation and LOH analysis of the MMR genes to the molecular diagnostic workflow of LS.

Functional analysis of MSH2 unclassified variants found in suspected Lynch syndrome patients reveals pathogenicity due to attenuated mismatch repair.

BACKGROUND: Lynch syndrome, an autosomal-dominant disorder characterised by high colorectal and endometrial cancer risks, is caused by inherited mutations in DNA mismatch repair (MMR) genes. Mutations fully abrogating gene function are unambiguously disease causing. However, missense mutations often have unknown functional implications, hampering genetic counselling. We have applied a novel approach to study three MSH2 unclassified variants (UVs) found in Dutch families with suspected Lynch syndrome. METHODS: The three mutations were recreated in the endogenous Msh2 gene in mouse embryonic stem cells by oligonucleotide-directed gene modification. The effect of the UVs on MMR activity was then tested using a set of functional assays interrogating the main MMR functions. RESULTS: We recreated and functionally tested three MSH2 UVs: MSH2-Y165D (c.493T>G), MSH2-Q690E (c.2068C>G) and MSH2-M813V (c.2437A>G). We observed reduced levels of MSH2-Y165D and MSH2-Q690E but not MSH2-M813V proteins. MSH2-M813V was able to support all MMR functions similar to wild-type MSH2, whereas MSH2-Y165D and MSH2-Q690E showed partial defects. CONCLUSIONS: Based on the results from our functional assays, we conclude that the MSH2-M813V variant is not disease causing. The MSH2-Y165D and MSH2-Q690E variants affect MMR function and are therefore likely the underlying cause of familial cancer predisposition. Since the MMR defect is partial, these variants may represent low penetrance alleles.

Apparently synonymous substitutions in FGFR2 affect splicing and result in mild Crouzon syndrome.

BACKGROUND: Mutations of fibroblast growth factor receptor 2 (FGFR2) account for a higher proportion of genetic cases of craniosynostosis than any other gene, and are associated with a wide spectrum of severity of clinical problems. Many of these mutations are highly recurrent and their associated features well documented. Crouzon syndrome is typically caused by heterozygous missense mutations in the third immunoglobulin domain of FGFR2. CASE PRESENTATION: Here we describe two families, each segregating a different, previously unreported FGFR2 mutation of the same nucleotide, c.1083A>G and c.1083A>T, both of which encode an apparently synonymous change at the Pro361 codon. We provide experimental evidence that these mutations affect normal FGFR2 splicing and document the clinical consequences, which include a mild Crouzon syndrome phenotype and reduced penetrance of craniosynostosis. CONCLUSIONS: These observations add to a growing list of FGFR2 mutations that affect splicing and provide important clinical information for genetic counselling of families affected by these specific mutations.

Disruption of TUFT1, a Desmosome-Associated Protein, Causes Skin Fragility, Woolly Hair, and Palmoplantar Keratoderma.

Desmosomes are dynamic complex protein structures involved in cellular adhesion. Disruption of these structures by loss-of-function variants in desmosomal genes leads to a variety of skin- and heart-related phenotypes. In this study, we report TUFT1 as a desmosome-associated protein, implicated in epidermal integrity. In two siblings with mild skin fragility, woolly hair, and mild palmoplantar keratoderma but without a cardiac phenotype, we identified a homozygous splice-site variant in the TUFT1 gene, leading to aberrant mRNA splicing and loss of TUFT1 protein. Patients' skin and keratinocytes showed acantholysis, perinuclear retraction of intermediate filaments, and reduced mechanical stress resistance. Immunolabeling and transfection studies showed that TUFT1 is positioned within the desmosome and that its location is dependent on the presence of the desmoplakin carboxy-terminal tail. A Tuft1-knockout mouse model mimicked the patients' phenotypes. Altogether, this study reveals TUFT1 as a desmosome-associated protein, whose absence causes skin fragility, woolly hair, and palmoplantar keratoderma.

Common variants of the BRCA1 wild-type allele modify the risk of breast cancer in BRCA1 mutation carriers.

Mutations in the BRCA1 gene substantially increase a woman's lifetime risk of breast cancer. However, there is great variation in this increase in risk with several genetic and non-genetic modifiers identified. The BRCA1 protein plays a central role in DNA repair, a mechanism that is particularly instrumental in safeguarding cells against tumorigenesis. We hypothesized that polymorphisms that alter the expression and/or function of BRCA1 carried on the wild-type (non-mutated) copy of the BRCA1 gene would modify the risk of breast cancer in carriers of BRCA1 mutations. A total of 9874 BRCA1 mutation carriers were available in the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) for haplotype analyses of BRCA1. Women carrying the rare allele of single nucleotide polymorphism rs16942 on the wild-type copy of BRCA1 were at decreased risk of breast cancer (hazard ratio 0.86, 95% confidence interval 0.77-0.95, P = 0.003). Promoter in vitro assays of the major BRCA1 haplotypes showed that common polymorphisms in the regulatory region alter its activity and that this effect may be attributed to the differential binding affinity of nuclear proteins. In conclusion, variants on the wild-type copy of BRCA1 modify risk of breast cancer among carriers of BRCA1 mutations, possibly by altering the efficiency of BRCA1 transcription.

Yield of routine molecular analyses in colorectal cancer patients ≤70 years to detect underlying Lynch syndrome.

Although early detection of Lynch syndrome (LS) is important, a considerable proportion of patients with LS remains unrecognized. We aimed to study the yield of LS detection by routine molecular analyses in colorectal cancer (CRC) patients until 70 years of age. We prospectively included consecutive CRC patients ≤70 years. Tumour specimens were analysed for microsatellite instability (MSI), immunohistochemical mismatch-repair protein expression and MLH1-promoter methylation. Tumours were classified as either: (a) likely caused by LS; (b) sporadic microsatellite-unstable (MSI-H); or (c) microsatellite-stable (MSS). Predictors of LS were determined by multivariable logistic regression. A total of 1117 CRC patients (57% males, median age 61 years) were included. Fifty patients (4.5%, 95% CI 3.4-5.9) were likely to have LS, and 71 had a sporadic MSI-H tumour (6.4%, 95% CI 5.1-8.0). Thirty-five patients likely to have LS (70%) were aged > 50 years. A molecular profile compatible with LS was detected in 10% (15/144) of patients aged ≤50, in 4% (15/377) of those aged 51-60 and in 3% (20/596) of patients > 61 years. Compared to MSS cases, patients likely to have LS were significantly younger (OR 3.9, 95% CI 1.7-8.7) and more often had right-sided CRCs (OR 14, 95% CI 6.0-34). In conclusion, molecular screening for LS in CRC patients ≤70 years leads to identification of a molecular profile compatible with LS in 4.5% of patients, with most of them not fulfilling the age criterion (≤50 years) routinely used for LS assessment. Routine use of MSI testing may be considered in CRC patients up to the age of 70 years, with a central role for the pathologist in the selection of patients.

BRCA1 R1699Q variant displaying ambiguous functional abrogation confers intermediate breast and ovarian cancer risk.

BACKGROUND: Clinical classification of rare sequence changes identified in the breast cancer susceptibility genes BRCA1 and BRCA2 is essential for appropriate genetic counselling of individuals carrying these variants. We previously showed that variant BRCA1 c.5096G>A p.Arg1699Gln in the BRCA1 transcriptional transactivation domain demonstrated equivocal results from a series of functional assays, and proposed that this variant may confer low to moderate risk of cancer. METHODS: Measures of genetic risk (report of family history, segregation) were assessed for 68 BRCA1 c.5096G>A p.Arg1699Gln (R1699Q) families recruited through family cancer clinics, comparing results with 34 families carrying the previously classified pathogenic BRCA1 c.5095C>T p.Arg1699Trp (R1699W) mutation at the same residue, and to 243 breast cancer families with no BRCA1 pathogenic mutation (BRCA-X). RESULTS: Comparison of BRCA1 carrier prediction scores of probands using the BOADICEA risk prediction tool revealed that BRCA1 c.5096G>A p.Arg1699Gln variant carriers had family histories that were less 'BRCA1-like' than BRCA1 c.5095C>T p.Arg1699Trp mutation carriers (p<0.00001), but more 'BRCA1-like' than BRCA-X families (p=0.0004). Further, modified segregation analysis of the subset of 30 families with additional genotyping showed that BRCA1 c.5096G >A p.Arg1699Gln had reduced penetrance compared with the average truncating BRCA1 mutation penetrance (p=0.0002), with estimated cumulative risks to age 70 of breast or ovarian cancer of 24%. CONCLUSIONS: Our results provide substantial evidence that the BRCA1 c.5096G>A p.Arg1699Gln (R1699Q) variant, demonstrating ambiguous functional deficiency across multiple assays, is associated with intermediate risk of breast and ovarian cancer, highlighting challenges for risk modelling and clinical management of patients of this and other potential moderate-risk variants.

Prospective evaluation of molecular screening for Lynch syndrome in patients with endometrial cancer ≤ 70 years.

OBJECTIVE: Lynch syndrome (LS) is a hereditary syndrome that predisposes to multiple malignancies including endometrial cancer (EC). We aimed to evaluate a diagnostic strategy for LS based on routine analysis of microsatellite instability (MSI) and immunohistochemical (IHC) staining for mismatch repair (MMR) proteins in tumour tissue of all newly diagnosed EC patients ≤ 70 years. METHODS: Consecutive EC patients ≤ 70 years were included prospectively in eight Dutch centres. EC specimens were analysed for MSI, IHC of four MMR proteins, MMR gene methylation status and BRAF-mutations. tumours were classified as; 1) likely to be caused by LS, 2) sporadic MSI-H, or 3) microsatellite stable (MSS). RESULTS: Tumour specimens of 179 patients (median age 61 years, IQR 57-66) were analysed. In our study 92% of included patients were over 50 years of age. Eleven EC patients were found likely to have LS (6%; 95% CI 3-11%), including 1 patient suspected of an MLH1, 2 of an MSH2, 6 of an MSH6 and 2 of a PMS2 gene defect. Germline mutation analyses revealed 7 MMR gene germline mutations. Ten patients likely to have LS (92%) were older than 50 years. In addition, 31 sporadic MSI-H tumours with MLH1 promoter hypermethylation (17%; 95% CI 13-24%) were identified. CONCLUSIONS: Molecular screening for LS in patients with EC diagnosed ≤ 70 years, leads to identification of a profile likely to have LS in 6% of cases. New screening guidelines for LS are needed, including recommendations for EC patients older than 50 years of age.

CHEK2*1100delC homozygosity is associated with a high breast cancer risk in women.

BACKGROUND: Mutations in the CHEK2 gene confer a moderately increased breast cancer risk. The risk for female carriers of the CHEK2*1100delC mutation is twofold increased. Breast cancer risk for carrier women is higher in a familial breast cancer setting which is due to coinheritance of additional genetic risk factors. This study investigated the occurrence of homozygosity for the CHEK2*1100delC allele among familial breast cancer cases and the associated breast cancer risk. METHODS AND RESULTS: Homozygosity for the CHEK2*1100delC allele was identified in 8/2554 Dutch independent familial non-BRCA1/2 breast cancer cases. The genotype relative risk for breast cancer of homozygous and heterozygous familial breast cancer cases was 101.34 (95% CI 4.47 to 121 000) and 4.04 (95% CI 0.88 to 21.0), respectively. Female homozygotes appeared to have a greater than twofold increased breast cancer risk compared to familial CHEK2*1100delC heterozygotes (p=0.044). These results and the occurrence of multiple primary tumours in 7/10 homozygotes indicate a high cancer risk in homozygous women from non-BRCA1/2 families. CONCLUSIONS: Intensive breast surveillance is therefore justified in these homozygous women. It is concluded that diagnostic testing for biallelic mutations in CHEK2 is indicated in non-BRCA1/2 breast cancer families, especially in populations with a relatively high prevalence of deleterious mutations in CHEK2.