Analysis of the Novel Fanconi Anemia Gene SLX4/FANCP in Familial Breast Cancer Cases

SLX4/FANCP is a recently discovered novel disease gene for Fanconi anemia (FA), a rare recessive disorder characterized by chromosomal instability and increased cancer susceptibility. Three of the 15 FA genes are breast cancer susceptibility genes in heterozygous mutation carriers—BRCA2, PALB2, and BRIP1. To investigate if defects in SLX4 also predispose to breast cancer, the gene was sequenced in a cohort of 729 BRCA1/BRCA2‐negative familial breast cancer cases. We identified a single splice site mutation (c.2013+2T>A), which causes a frameshift by skipping of exon 8. We also identified 39 missense variants, four of which were selected for functional testing in a Mitomycin C‐induced growth inhibition assay, and appeared indistinguishable from wild type. Although this is the first study that describes a truncating SLX4 mutation in breast cancer patients, our data indicate that germline mutations in SLX4 are very rare and are unlikely to make a significant contribution to familial breast cancer.     

German family study on hereditary breast and/or ovarian cancer: Germline mutation analysis of the BRCA1 gene

Women harboring BRCA1 germline mutations carry an 85% lifetime risk of developing breast cancer and a 63% risk of ovarian cancer. In this first systematic study of familial breast and/or ovarian cancer in Germany we investigated 29 families for germline mutations in the BRCA1 gene. We identified mutations in three breast cancer families and in four breast-ovarian cancer families. The mutations include one missense mutation, one frameshift mutation, one splice mutation, and four nonsense mutations cosegregating with breast and/or ovarian susceptibility in five of ten (50%) families showing positive evidence of linkage to chromosome band 17q21 and in two of 19 (11%) families where linkage data was not available. Two apparently unrelated families carried the same nonsense mutation at codon 1835 and three families harbored a C to T transition at nucleotide 49 of the untranslated exon 4. Allelotyping of the markers D17S855, D17S1322, D17S1323, and D17S1327 located within or near BRCA1 revealed that all affected individuals in the two families harboring the mutation at codon 1835 shared at least one allele indicating a founder mutation. With respect to the overall mutation spectrum, no mutations were identified in exon 11 (0/7) in this set of German families. These findings differed significant from those in British (17/32)(P = 0.012) and Southern Swedish (13/15) (P < 0.001) families. The lack of BRCA1 mutations in exon 11 which represents 61% of the entire coding sequence may provide additional insight into BRCA1 associated breast and ovarian tumor development. Genes Chromosom. Cancer 18:126–132, 1997. © 1997 Wiley-Liss, Inc.     

BRCA1 gene-related hereditary susceptibility to breast and ovarian cancer in Latvia

PurposeIn this report, we summarise data on BRCA1 gene analysis in Latvia to characterise criteria of genetic testing for breast and ovarian cancer susceptibility.Material/methodsAnalysis by SSCP/HD, MALDI-TOF mass spectrometry or DNA sequencing was used for mutation detection. Mutations identified were confirmed by direct DNA sequencing.ResultsOut of 1068 breast and 231 ovarian cancer patients from different families: 58 carried the c.5266dupC and 43 carried the c.4035delA mutations. Every 4th patient in our study did not report cancer in the family. The breast cancer was diagnosed earlier in carriers of the c.5266dupC than in carriers of the c.4035delA (p = 0.003). The incidence of breast or ovarian cancer does not differ among the 2 mutation carriers in our patient group. The nature of the c.5266dupC mutation might be more deleterious.ConclusionsWe recommend the screening of 4 founder BRCA1 mutations in all breast and ovarian cancer patients in Latvia at diagnosis of disease regardless of family history or age. The BRCA1 screening can be carried out efficiently using the MALDI-TOF mass spectrometry mutation detection method developed in the Biomedical Research and Study Centre (Riga, Latvia).     

Evaluation of Rare Variants in the New Fanconi Anemia Gene ERCC4 (FANCQ) as Familial Breast/Ovarian Cancer Susceptibility Alleles

Recently, it has been reported that biallelic mutations in the ERCC4 (FANCQ) gene cause Fanconi anemia (FA) subtype FA‐Q. To investigate the possible role of ERCC4 in breast and ovarian cancer susceptibility, as occurs with other FA genes, we screened the 11 coding exons and exon–intron boundaries of ERCC4 in 1573 index cases from high‐risk Spanish familial breast and ovarian cancer pedigrees that had been tested negative for BRCA1 and BRCA2 mutations and 854 controls. The frequency of ERCC4 mutation carriers does not differ between cases and controls, suggesting that ERCC4 is not a cancer susceptibility gene. Interestingly, the prevalence of ERCC4 mutation carriers (one in 288) is similar to that reported for FANCA, whereas there are approximately 100‐fold more FA‐A than FA‐Q patients, indicating that most biallelic combinations of ERCC4 mutations are embryo lethal. Finally, we identified additional bone‐fide FA ERCC4 mutations specifically disrupting interstrand cross‐link repair.     

Whole Exome Sequencing Reveals Uncommon Mutations in the Recently Identified Fanconi Anemia Gene SLX4/FANCP

Fanconi anemia (FA) is a rare genetic disorder characterized by congenital malformations, progressive bone marrow failure (BMF), and susceptibility to malignancies. FA is caused by biallelic or hemizygous mutations in one of 15 known FA genes, whose products are involved in the FA/BRCA DNA damage response pathway. Here, we report on a patient with previously unknown mutations of the most recently identified FA gene, SLX4/FANCP. Whole exome sequencing (WES) revealed a nonsense mutation and an unusual splice site mutation resulting in the partial replacement of exonic with intronic bases, thereby removing a nuclear localization signal. Immunoblotting detected no residual SLX4 protein, which was consistent with abrogated interactions with XPF/ERCC1 and MUS81/EME1. This cellular finding did not result in a more severe clinical phenotype than that of previously reported FA‐P patients. Our study additionally exemplifies the versatility of WES for the detection of mutations in heterogenic disorders such as FA.     

CHEK2基因c.1100delC与中国人遗传性乳腺癌易感性的关联研究

目的研究上海地区非BRCA1／2基因突变的遗传倾向乳腺癌中CHEK2基因c．1100delC突变的携带情况及可能的作用。方法研究对象来自114例遗传倾向性乳腺癌，包括家族性乳腺癌76例，其中8例发病年龄低于40岁；38例单纯早发性乳腺癌（发病年龄〈40岁）。对照组为121名无乳腺癌的健康女性，静脉血中提取基因组DNA，对CHEK2基因的第10～14外显子进行长片段PCR扩增，PCR产物再进行含突变的第10外显子的扩增。突变分析全部由DNA直接测序进行鉴定。结果研究人群和对照人群中都没有发现c．1100delC的突变；在3例（3／114，2．6％）家族性乳腺癌中发现邻近c．1100delC的新的错义突变位点1111C〉T（p-His371 Tyr），对照组中则无此突变发现。结论CHEK2基因c．1100delC突变可能是中国人群罕见的突变位点，在中国人乳腺癌遗传易感性中的作用非常有限；1111C〉T可能与中国上海地区遗传倾向乳腺癌低度外显的易感性有关，需要进行进一步研究确认。     

Genetic testing for RAD51C mutations: in the clinic and community

Much of the observed familial clustering of breast and ovarian cancer cannot be explained by mutations in BRCA1 and BRCA2. Several other cancer susceptibility genes have been identified, but their value in routine clinical genetic testing is still unclear. Germline mutations in RAD51C have been identified in about 1% of hereditary breast and ovarian cancer families. RAD51C mutations are predominantly found in families with a history of ovarian cancer and are rare in families with a history of breast cancer alone. RAD51C is primarily an ovarian cancer susceptibility gene. A mutation is present in approximately 1% of unselected ovarian cancers. Among mutation carriers, the lifetime risk of ovarian cancer is approximately 9%. The average age at onset is approximately 60 years; this suggests that preventive oophorectomy can be delayed until after natural menopause. Under current guidelines, genetic testing for RAD51C is expected to have a limited impact on ovarian cancer incidence at a population level. This is because the penetrance is 9% to age 80; the great majority of families with mutations would be represented by a single case of ovarian cancer, these are potentially preventable through population screening but not through screening of established ovarian cancer families.     

Novel BRCA1/2 mutations in Serbian breast and breast-ovarian cancer patients with hereditary predisposition

Mutations in breast cancer susceptibility (BRCA) genes lead to defects in DNA repair processes resulting in elevated genome instability and predisposing to breast and ovarian cancer. The study was designed to detect mutational spectra of BRCA1/2 genes in a Serbian population. Using automated DNA sequencing, we tested individuals for BRCA mutations, based on positive family history of either breast or ovarian cancer or both. Two novel mutations (c.4765_4784del in BRCA1 exon 15 and c.4367_4368dupTT in BRCA2 exon 11) were detected, in three probands from two different families. These mutations have not been reported previously in the BIC or LOVD databases. Protein products of these mutated alleles lack domains necessary for their DNA repair functions, an indicator that these are deleterious mutations. Neither mutation was found in any proband from 50 other families with hereditary predisposition, so the two mutations are likely family-specific rather than population-specific. Although BRCA1-associated tumors are typically negative for estrogen receptor (ER), progesterone receptor (PR), and ERBB2, the novel BRCA1 mutation identified in this study was detected in a proband with ER- and PR-positive breast cancer. Steroid receptor-positive BRCA-related breast cancer in this proband supports the idea of characteristic pathological features and older age of onset among BRCA1-mutated ER-positive breast cancers.     

High follicular phase luteinizing hormone levels in young healthy BRCA1 mutation carriers: implications for breast and ovarian cancer risk

BRCA1 mutation carriers have up to 80% life-time risk of developing breast cancer and 20-40% risk of developing ovarian cancer. High LH levels have been linked to increased risks of both breast and ovarian cancers in some studies and it is unknown whether gonadotropin levels are associated with BRCA1 mutation status. The aim of the study was to explore whether gonadotropin levels were associated with BRCA1 mutation status among healthy 40-year-old-women from hereditary breast cancer families. All women completed a questionnaire including information on reproductive factors and OC use. We measured height, weight, breast volumes, and plasma levels of LH, FSH, and estradiol (E2) once during menstrual cycle days 5-10 and once again during cycle days 18-23 in 43 non-carriers from BRCA1 families, 20 BRCA1 mutation carriers, and 101 women from non-BRCA1/2 families. The strongest predictors of high LH levels among BRCA1 mutation carriers and non-carriers during cycle days 5-10 were being a BRCA1 mutation carrier (p=0.002), lack of current OC use (p=0.003), and being nulliparous (p=0.01), adjusted for age and menstrual cycle day when the samples were obtained. This association was seen both in non-OC users and current OC users but was only significant in the former group (p=0.005). Because of multiple analyses it is possible that our finding is a result of a Type 1 statistical error. After a permutation test the new adjusted p value in non-OC users was 0.05. FSH and E2 were similar in non-carriers, BRCA1 mutation carriers and women from non-BRCA1/2 families. We found significantly elevated LH levels in the follicular phase among young healthy BRCA1 mutation carriers compared with non-carriers from BRCA1 families. This is a small study and confirmatory studies are warranted to establish whether elevated LH levels are part of the BRCA1 phenotype and may be manipulated in order to reduce cancer risks in BRCA1 mutation carriers.     

Absence of CHEK2*1100delC mutation in families with hereditary breast cancer in North America

The CHEK2*1100delC mutation has been reported to confer a twofold increased risk of breast cancer among carriers. The frequency of the mutation varies among populations. The highest frequency has been described in Northern and Eastern European countries; the frequency may be much lower in North America. In this study, our aim was to determine the frequency of CHEK2*1100delC in members of breast cancer families who tested negative for a deleterious mutation in BRCA1/2 at the University of Michigan Comprehensive Cancer Center. We genotyped 102 members from 90 families for CHEK2*1100delC. Most of these families had several cases of breast cancer or ovarian cancer (or both), as well as multiple members with other cancer types in a single lineage. No CHEK2*1100delC mutations were detected in any of the 102 individuals, including 51 women diagnosed with breast cancer at an early age (<45 years), 8 women with bilateral breast cancer, 3 men with breast cancer, and 8 women with ovarian cancer. Our data are consistent with the reported very low frequency of CHEK2*1100delC mutations in North American populations (compared with Northern Europe), rendering CHEK2*1100delC such an unlikely culprit in BRCA1/2 negative families that routine testing of these families appears unwarranted.     

Paternal or Maternal Uniparental Disomy of Chromosome 16 Resulting in Homozygosity of a Mutant Allele Causes Fanconi Anemia

Fanconi anemia (FA) is a rare inherited disorder caused by pathogenic variants in one of 19 FANC genes. FA patients display congenital abnormalities, and develop bone marrow failure, and cancer susceptibility. We identified homozygous mutations in four FA patients and, in each case, only one parent carried the obligate mutant allele. FANCA and FANCP/SLX4 genes, both located on chromosome 16, were the affected recessive FA genes in three and one family respectively. Genotyping with short tandem repeat markers and SNP arrays revealed uniparental disomy (UPD) of the entire mutation‐carrying chromosome 16 in all four patients. One FANCA patient had paternal UPD, whereas FA in the other three patients resulted from maternal UPD. These are the first reported cases of UPD as a cause of FA. UPD indicates a reduced risk of having another child with FA in the family and has implications in prenatal diagnosis.     

Mutational analysis of RAD51C and RAD51D genes in hereditary breast and ovarian cancer families from Murcia (southeastern Spain)

RAD51C and RAD51D have been defined as susceptibility genes for hereditary breast and ovarian cancer syndrome in several studies. In the present study, a mutation analysis of these genes was performed on non BRCA1/2 families. RAD51C and RAD51D genes were analyzed in 141 and 77 families, respectively. The analysis included direct sequencing and multiple ligation probe analysis. The RAD51C pathogenic variant c.404G?>?A was identified in a breast and ovarian cancer family (0.7%), while the RAD51D pathogenic variant c.694C?>?T was described in an ovarian cancer family (1.3%). Moreover, three unknown clinical significance variants were detected: c.307T?>?G in RAD51C, and c.413A?>?G and c.715C?>?T in RAD51D. No large genomic rearrangements (LGRs) were found. RAD51D carriers suffered from premenopausal ovarian tumors. These results increase our knowledge about the RAD51C and RAD51D mutation spectrum and support the notion that these genes should be included in the gene panel testing performed on patients with hereditary breast and ovarian cancer syndrome.     

Low frequency of BRCA1 germline mutations in 45 German breast/ovarian cancer families.

In this study we investigated 45 German breast/ovarian cancer families for germline mutations in the BRCA1 gene. We identified four germline mutations in three breast cancer families and in one breast-ovarian cancer family. among these were one frameshift mutation, one nonsense mutation, one novel splice site mutation, and one missense mutation. The missense mutation was also found in 2.8% of the general population, suggesting that it is not disease associated. The average age of disease onset in those families harbouring causative mutations was between 32.3 and 37.4 years, whereas the family harbouring the missense mutation had an average age of onset of 51.2 years. These findings show that BRCA1 is implicated in a small fraction of breast/ovarian cancer families suggesting the involvement of another susceptibility gene(s).     

BRCA1 and BRCA2 unclassified variants and missense polymorphisms in Algerian breast/ovarian cancer families

Background: BRCA1 and BRCA2 germline mutations predispose heterozygous carriers to hereditary breast/ovarian cancer. However, unclassified variants (UVs) (variants with unknown clinical significance) and missense polymorphisms in BRCA1 and BRCA2 genes pose a problem in genetic counseling, as their impact on risk of breast and ovarian cancer is still unclear. The objective of our study was to identify UVs and missense polymorphisms in Algerian breast/ovarian cancer patients and relatives tested previously for BRCA1 and BRCA2 genes germline mutations analysis. Methods: We analyzed 101 DNA samples from 79 breast/ovarian cancer families. The approach used is based on BRCA1 and BRCA2 sequence variants screening by SSCP or High-Resolution Melting (HRM) curve analysis followed by direct sequencing. In silico analyses have been performed using different bioinformatics programs to individualize genetics variations that can disrupt the BRCA1 and BRCA2 genes function. Results: Among 80 UVs and polymorphisms detected in BRCA1/2 genes (33 BRCA2 and 47 BRCA2), 31 were new UVs (10 BRCA2 and 21 BRCA2), 7 were rare UVs (4 BRCA2 and 3 BRCA2) and 42 were polymorphic variants (19 BRCA2 and 23 BRCA2). Moreover, 8 new missense UVs identified in this study: two BRCA1 (c.4066C>A/p.Gln1356Lys, c.4901G>T/p.Arg1634Met) located respectively in exons 11 and 16, and six BRCA2 (c.1099G>A/p.Asp367Asn, c.2636C>A/p.Ser879Tyr, c.3868T>A/p.Cys1290Ser, c.5428G>T/p.Val1810Phe, c.6346C>G/p.His2116Asp and c.9256G>A/p.Gly3086Arg) located respectively in exons 10, 11 and 24, show a damaging PSIC score yielded by PolyPhen2 program and could be pathogenic. In addition, 5 new BRCA2 missense UVs out of six that were found to be damaging by PolyPhen2 program, also were deleterious according to SIFT program. The rare BRCA2 UV c.5332G>A/p.Asp1778Asn was found here for the first time in co-occurrence in trans with the deleterious BRCA1 mutation c.798_799delTT/p.Ser267LysfsX19 in young breast cancer patient. Moreover, 10 new identified intronic variants with unknown clinical significance (3 BRCA1 and 7 BRCA2) in the present study, could be considered as benign, because GeneSplicer, SpliceSiteFinder and MaxEntScan prediction programs show no splice site alteration for these variants. Several missense polymorphisms of BRCA1 c.2612C>T/p.Pro871Leu, c.3548A>G/p.Lys1183Arg, c.4837A>G/p.Ser1613Gly and BRCA2 c.865A>C/p.Asn289His, c.1114A>C/p.Asn372His, c.2971A>G/p.Asn991Asp, c.7150C>A/p.Gly2384Lys have been identified with high frequency in patients who were tested negative for BRCA1 and BRCA2 mutations. These missense polymorphisms could have a role as susceptibility breast cancer markers in Algerian breast/ovarian cancer families where pathological BRCA1 and BRCA2 mutations were not present. Conclusions: For the first time, UVs and missense polymorphisms in BRCA1 and BRCA2 genes have been identified in Algerian breast/ovarian cancer families. Evaluation of breast/ovarian cancer risk induced by the eight new missense UVs and common polymorphisms detected in our present work is on going in a larger study.     

BRCA1 mutations in Algerian breast cancer patients: high frequency in young, sporadic cases

Breast cancer rates and median age of onset differ between Western Europe and North Africa. In Western populations, 5 to 10 % of breast cancer cases can be attributed to major genetic factors such as BRCA1 and BRCA2, while this attribution is not yet well defined among Africans. To help determine the contribution of BRCA1 mutations to breast cancer in a North African population, we analysed genomic DNA from breast cancer cases ascertained in Algiers. Both familial cases (at least three breast cancers in the same familial branch, or two with one bilateral or diagnosed before age 40) and sporadic cases less than 38 years of age were studied. Complete sequencing plus quantitative analysis of the BRCA1 gene was performed. 9.8 % (5/51) of early-onset sporadic and 36.4 % (4/11) of familial cases were found to be associated with BRCA1 mutations. This is in contrast 10.3 % of French HBOC families exhibiting a BRCA1 mutation. One mutation, c.798_799delTT, was observed in two Algerian families and in two families from Tunisia, suggesting a North African founder allele. Algerian non-BRCA1 tumors were of significantly higher grade than French non-BRCA tumors, and the age at diagnosis for Algerian familial cases was much younger than that for French non-BRCA familial cases. In conclusion, we observed a much higher frequency of BRCA1 mutations among young breast cancer patients than observed in Europe, suggesting biological differences and that the inclusion criterea for analysis in Western Europe may not be applicable for the Northern African population.     

A PARK2 polymorphism associated with delayed neuropsychological sequelae after carbon monoxide poisoning

Background

The PALB2 c.2323C>T [p.Q775X] mutation has been reported in at least three breast cancer families and breast cancer cases of French Canadian descent and this has been attributed to common ancestors. The number of mutation-positive cases reported varied based on criteria of ascertainment of index cases tested. Although inherited PALB2 mutations are associated with increased risks of developing breast cancer, risk to ovarian cancer has not been fully explored in this demographically unique population.

Methods

We screened the PALB2 p.Q775X variant in 71 families with at least three cases of breast cancer (n=48) or breast and ovarian cancers (n=23) that have previously been found negative for at least the most common BRCA1 and BRCA2 mutations reported in the French Canadian population and in 491 women of French Canadian descent who had invasive ovarian cancer and/or low malignant potential tumors of the major histopathological subtypes.

Results

We identified a PALB2 p.Q775X carrier in a breast cancer family, who had invasive ductal breast carcinomas at 39 and 42 years of age. We also identified a PALB2 p.Q775X carrier who had papillary serous ovarian cystadenocarcinoma at age 58 among the 238 serous subtype ovarian cancer cases investigated, who also had breast cancer at age 52.

Conclusion

Our findings, taken together with previous reports, support adding PALB2 c.2323C>T p.Q775X to the list of cancer susceptibility genes for which founder mutations have been identified in the French Canadian population.     

BRCA1/2 testing in newly diagnosed breast and ovarian cancer patients without prior genetic counselling: the DNA-BONus study

Germline BRCA1/2 testing of breast and ovarian cancer patients is growing rapidly as the result affects both treatment and cancer prevention in patients and relatives. Through the DNA-BONus study we offered BRCA1/2 testing and familial risk assessment to all new patients with breast (N=893) or ovarian (N=122) cancer diagnosed between September 2012 and April 2015, irrespective of family history or age, and without prior face-to-face genetic counselling. BRCA1/2 testing was accepted by 405 (45.4%) and 83 (68.0%) of the patients with breast or ovarian cancer, respectively. A pathogenic BRCA1/2 variant was found in 7 (1.7%) of the breast cancer patients and 19 (22.3%) of the ovarian cancer patients. In retrospect, all BRCA1/2 mutation carriers appeared to fulfill current criteria for BRCA1/2 testing. Hospital Anxiety and Depression Scale (HADS) scores showed that the mean levels of anxiety and depression were comparable to those reported for breast and gynecological cancer patients in general, with a significant drop in anxiety symptoms during a 6-month follow-up period, during which the test result was forwarded to the patients. These results show that BRCA1/2 testing is well accepted in newly diagnosed breast and ovarian cancer patients. Current test criteria based on age and family history are sufficient to identify most BRCA1/2 mutation carriers among breast cancer patients. We recommend germline BRCA1/2 testing in all patients with epithelial ovarian cancer because of the high prevalence of pathogenic BRCA1/2 variants.     

Constant denaturant gel electrophoresis (CDGE) in BRCA1 mutation screening

Screening for mutations in the breast and ovarian cancer susceptibility gene, BRCA1, is complicated by the wide spectrum of mutations found in this large gene. In the present study a constant denaturant gel electrophoresis (CDGE) mutation screening strategy was established for ˜80% of the genomic coding sequence (exons 2, 11, 13–16, 20, 24). This strategy was applied to screen genomic DNA from 50 familial breast and/or ovarian cancer patients who had previously been examined for BRCA1 mutations by SSCP. A total of 14 carriers of 12 distinct disease-associated mutations and 7 carriers of 6 distinct rare substitutions leading to amino acid substitutions were identified. The SSCP failed to detect 40% of the different deletions/insertions (4/10) and 75% (6/8) of the different base substitutions leading to terminating codons or rare amino acid changes. SSCP did, however, identify one rare base substitution that could not be detected in the CDGE screening. To evaluate the CDGE mutation screening strategy further, 25 unrelated patients from Norwegian breast and/or ovarian cancer families were examined for BRCA1 mutations using a combined genomic DNA/cDNA approach covering the entire coding sequence of the gene. A total of six mutation carriers were detected, all of whom had cases of ovarian cancer in their families. Three patients from independent families carried an 1135insA mutation in exon 11, two others had a Gly484ter and an 1675delA mutation, respectively, and the sixth carried a splice mutation (5194-2 a→c) causing deletion of exon 18. CDGE may become an efficient tool in diagnostic and population based screening for BRCA1 mutations. Hum Mutat 11:166–174, 1998. © 1998 Wiley-Liss, Inc.