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Brca1 and Brca2 Families and the Risk of Skin Cancer

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BRCA1/2 mutations are not a common cause of malignant melanoma in the Polish population

  • Rodney J. Scott,
  • Bohdan Górski,
  • Bartłomiej Masojć,
  • Andrzej Kram,
  • Romuald Maleszka,
  • Cezary Cybulski,
  • Katarzyna Paszkowska-Szczur,
  • Aniruddh Kashyap,
  • Dawid Murawa,
  • Karolina Malińska,
  • Magdalena Kiedrowicz,
  • Emilia Rogoża-Janiszewska,
  • Helena Rudnicka,
  • Jakub Deptuła,
  • Paweł Domagała,
  • Wojciech Kluźniak,
  • Marcin R. Lener,
  •  [ ... ],
  • Jan Lubiński
  • [ view all ]
  • [ view less ]

BRCA1/two mutations are non a mutual cause of cancerous melanoma in the Polish population

  • Tadeusz Dębniak,
  • Rodney J. Scott,
  • Bohdan Górski,
  • Bartłomiej Masojć,
  • Andrzej Kram,
  • Romuald Maleszka,
  • Cezary Cybulski,
  • Katarzyna Paszkowska-Szczur,
  • Aniruddh Kashyap,
  • Dawid Murawa

PLOS

x

  • Published: Oct 4, 2018
  • https://doi.org/10.1371/journal.pone.0204768

Abstruse

The association of BRCA1/2 mutations with melanoma is not completely determined; the interpretation of variants of unknown significance is as well problematic. To evaluate these issues we explored the molecular ground of melanoma risk by performing whole-exome sequencing on a cohort of 96 unrelated Smooth early-onset melanoma patients and targeted sequencing of BRCA1/two genes on additional 30 melanoma patients with familial aggregation of breast and other cancers. Sequencing was performed on peripheral blood. We evaluated MutationTaster, Polyphen2, SIFT, PROVEAN algorithms, analyzed segregation with cancer affliction (in both families with identified BRCA2 variants) and in i family performed LOH (based on ii primary tumors). We plant neither pathogenic mutations nor variants of unknown significance within BRCA1. Nosotros identified 2 BRCA2 variants of unknown significance: c.9334G>A and c.4534 C>T. Illness allele frequency was evaluated past genotyping of 1230 consecutive melanoma cases, 5000 breast cancer patients, 3500 prostate cancers and 9900 controls. Both variants were found to exist absent among unselected cancer patients and healthy controls. The MutationTaster, Polyphen2 and SIFT algorithms point that c.9334G>A is a damaging variant. Due to lack of tumour tissue LOH assay could not be performed for this variant. The variant segregated with the disease. The c.4534 C>T variant did not segregate with disease, there was no LOH of the variant. The c.9334G>A variant, classified as a rare variant of unknown significance, on current bear witness may predisposes to cancers of the breast, prostate and melanoma. Functional studies to draw how the DNA change affects the protein role and a big multi-heart written report to evaluate its penetrance are required.

Citation: Dębniak T, Scott RJ, Górski B, Masojć B, Kram A, Maleszka R, et al. (2018) BRCA1/2 mutations are not a common cause of malignant melanoma in the Polish population. PLoS ONE 13(10): e0204768. https://doi.org/ten.1371/journal.pone.0204768

Editor: Alvaro Galli, CNR, ITALY

Received: June 8, 2018; Accepted: September 13, 2018; Published: October 4, 2018

Copyright: © 2018 Dębniak et al. This is an open up admission article distributed under the terms of the Creative Eatables Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: We have uploaded the minimal data set for this study, including the sequencing data, to the Harvard Dataverse (https://doi.org/x.7910/DVN/MACR3E).

Funding: This research was supported past the Polish National Science Centre grant number 2014/13/B/NZ2/03888 to TD. The funders had no role in written report design, information drove and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Melanoma is 1 of the nearly aggressive human malignancies. The incidence of malignant melanoma (MM) of the pare is increasing worldwide, with the near rapid increment observed in Caucasian populations [1]. In Poland the number of affected individuals has increased by more threescore% over the past eleven years and at present accounts for over 2600 new diagnosed annually. The etiology of melanoma is complex, involving both genetic and ecology factors. Meaning heritability of this malignancy was reported from large twin studies [two]. Family studies provide strong back up for heterogeneous mechanisms involved in an inherited susceptibility to melanoma, likely equally a result of polygenic factors that influence both inherited high-hazard and low-risk alleles.

To date one major melanoma susceptibility gene: CDKN2A has been identified that is responsible for only upwardly to 20% of melanoma cases [3]. Mutations within other MM high hazard genes, such as CDK4, ACD, CXC, TERT, TERF2IP, POT1 or BAP1 are extremely rare and to date they take simply been identified in a few families worldwide [iii, 4, 5]. The list of intermediate- to low-penetrance risk alleles include variants in the MC1R [6, seven, 8], XPD [ix], VDR [ten], and MITF [11]. Even so, in the vast bulk of MM cases the molecular groundwork remains unclear. A possible association between melanoma and breast cancer has been reported in some studies, which suggest an independent involvement of BRCA2 in melanoma development [12]. Although the association of BRCA1 and BRCAtwo mutations with breast and ovarian cancer take a chance is well-defined, information technology has not been fully explored and 2 pregnant questions remain; one. the potential association of BRCA1 and BRCA2 mutations with other malignancies such as melanoma is not completely determined; and 2. the estimation of variants of unknown significance on cancer take chances is remains problematic.

To evaluate these issues we explored the molecular background of melanoma past performing whole-exome sequencing on a cohort of 96 unrelated Polish patients who were diagnosed with disease before the historic period of 40 yrs. All patients have previously been tested and found to be negative for any deleterious alter in CDKN2A. Targeted sequencing of BRCA1/2 in the 96 MM patients and an boosted 30 melanoma cases with a familial aggregation of cancers that included chest cancer. The variants identified in the MM discovery phase were validated by genotyping them using a large series of 1230 consecutive MM cases and 1700 melanoma controls, 5000 sequent breast cancer patients and 3500 prostate cancer patients and compared to 9900 controls. Prostate cancer patients were included since a modest proportion of them are associated with causative germline variants in BRCA1 or BRCA2 [xiii]. For those patients who were found to comport a BRCA1 or BRCA2 variant segregation assay was undertaken in their respective families.

Materials and methods

Study subjects

We studied three not-overlapping groups of melanoma patients. The first grouping included 96 unrelated patients with early-onset melanoma; the second group 30 unrelated MM patients with a familial cancer aggregation amidst 1st and iind degree relatives that included breast cancer—both of these groups were used for the discovery phase of the study. The 3rd grouping consisted of 1230 unselected (for age, gender, cancer family history, grading or other characteristics) MM patients for the validation phase. In the validation phase we as well studied a group of 5000 women with unselected breast cancer, 3500 men with unselected prostate cancer and 9900 good for you controls. Overall, patient participation rates exceeded 75%. All patients and command subjects were of European ancestry and ethnic Poles. The Polish report was approved by the ethics committee of Pomeranian Medical Academy (Szczecin, Poland). All study subjects provided a signed consent course for participation in the written report.

Discovery phase

In the first phase of this study we have performed whole exome sequencing in a cohort of 96 unrelated patients with early-onset (<40 yrs) melanoma from CDKN2A-negative families (65 females, mean historic period 30,viii; range 15–40; 31 males, hateful age 30,ix, range xviii–39). 29 out of this 96 cases were sporadic early onset melanoma patients with negative cancer family unit history, 38 patients were characterized by a familial aggregation of melanoma amidst starting time- or second-degree relatives, 29 were early on onset melanoma patients with familial aggregation of other cancers (1 or more cancers among 1st or twond degree relatives). Cases were diagnosed between 2006 and 2015 and selected from cancer registries in six Polish cities (Szczecin, Opole, Bialystok, Zielona Gora, Gorzow Wielkopolski and Poznan).

They were asked to participate at the fourth dimension of diagnosis or during an outpatient visit to an oncology dispensary and were selected for their age. In the 2nd stage we performed targeted BRCA1/two sequencing in accomplice of 30 unrelated MM patients (28 females, mean age of diagnosis of 50.eight years, range 47–70; 2 males diagnosed at 47 and 70) with a familial aggregation of cancers that were not melanoma among 1st and 2nd degree relatives. In all these families breast cancer was one of the malignancies diagnosed amidst the affected relatives. Iv female person MM probands too developed breast cancer. Patients were diagnosed between 2003 and 2015 in Szczecin. They were asked to participate at the time of diagnosis or during an outpatient visit to an oncology clinic and were selected for their family history.

All patients had previously been screened and found not to behave CDKN2A-causative variants.

Validation phase

We verified the role of any detected variants using a large instance control-report of 1,230 unselected melanoma patients (760 females, hateful age 53,9, range fifteen–92; 470 males, mean age 55, range 18–84) from Poland that came from two cohorts. The first consisted of 748 unselected MM cases (464 women, hateful age 53.vii y; 284 men) diagnosed between 2002 and 2006 and identified from cancer registries in five Polish cities (Szczecin, Opole, Bialystok, Zielona Gora, and Gorzow Wielkopolski). The registries capture more than 95% of all diagnosed melanomas. The second group consisted of 482 unselected MM cases (296 females,186 males) diagnosed in Szczecin between 2010 and 2016.

The breast instance series consisted of 5000 prospectively ascertained unselected female patients with invasive breast cancer (age range of xviii–92 years; mean historic period of 53.two years) who were diagnosed from 2008 to 2013 at 18 different hospitals in Poland. All patients diagnosed with invasive breast cancer at participating centers were eligible.

Patients were unselected for family history. The patient participation rate was 76.i%. Women with a previous contralateral breast cancer or with a current diagnosis of bilateral cancer were considered to exist bilateral.

The prostate example series consisted of 3500 men with unselected prostate cancer (historic period range: 41–96 years; mean historic period: 68.8), who were diagnosed between 1999 and 2012 in 14 centers situated throughout Poland. This study was initiated in Szczecin in 1999 and was extended to include Bialystok, Olsztyn in 2002 and Opole in 2003. Other centers began recruiting between 2005 and 2008 (Koszalin, Gdansk, Lublin, Lodz, Warszawa, Wroclaw, Poznan, Rzeszow, Bydgoszcz, Zabrze). All men with prostate cancer were invited to participate. Study subjects were asked to participate at the time of diagnosis or during an outpatient visit to an oncology clinic and were unselected for age or family history. The participation rate was 86.4%.

Controls

Melanoma command group consisted of 1696 salubrious adults: 943 women (mean age, 64 years) and 753 men (mean age, 67 years) with no cancers diagnosed in their families. The healthy adults were deemed as having a negative cancer family history (commencement- and second-degree relatives included) after answering a questionnaire about their family's medical history which was part of a population-based written report of the ane.five 1000000 residents of Due west Pomerania (northwest Poland) to identify familial aggregations of malignancies amid first and second caste relatives from 1.258 million residents (87%) who were registered with the Westward Pomeranian Regional Health Foundation.

During the interview, the goals of the study were explained, informed consent was obtained, genetic counseling was given and a blood sample was taken for Dna analysis.

Individuals affected with any malignancy or with cancers diagnosed amongst beginning- or second-caste relatives were excluded from our study command grouping.

The chest control group included 4,702 cancer-free women aged 18 to 94 years (mean age of 53.0 years). These controls were derived from four sources. The starting time subgroup consisted of 979 women from the region of Szczecin (age range of 24 to 84 years) who were chosen for this study to be matched by age and geographical region with a series of patients with incident breast cancer diagnosed in Szczecin between 1996 and 2004. These women were role of a population-based written report of the i.v million residents of West described in a higher place. The second control series consisted of one,707 unselected women (age range of 32–72 years) who participated in breast ultrasonography screening at eight different centers beyond Poland between 2009 and 2011 and provided blood samples for DNA analysis. Women with breast cancer and women with a positive family history of breast cancer were excluded from this group. The tertiary command group included 1,031 unselected women (age range of 20–94 years) selected at random from the computerized patient lists of family practices located in the region of Opole (s Poland). These women were invited to participate past mail and participated in 2012 and 2013. The 4th series included 985 Smoothen women (age range of l–66 years) who participated in the population colonoscopy screening program for colorectal cancer between 2007 and 2010 in Szczecin, Bialystok and Lodz, who were negative for polyps and or cancer and provided blood samples for DNA analysis.

The prostate command group consisted of 3500 cancer-gratuitous men aged 23–90 years (mean historic period: 61.viii years) selected from a part of a population-based survey of ane.5 million residents of West described above.

The first subgroup included 1,026 cancer free males (historic period range: 23–87 years; mean historic period: 61.6 years) with no family history of whatsoever cancer in a first-degree relative.

These men were selected at random from a registry of patients who participated in the population-based study and were invited for an interview in 2007. Individuals with a first-degree relative diagnosed with cancer were excluded from this control group. The second serial of prostate controls consisted of 2474 unselected men at age higher up 45 (age range: 45–90 years; hateful age: 61.nine years). These men were likewise selected past random from a database of the population-based study and interviewed between 2010 and 2012. During the interview family history of cancer was collected and detailed family unit history was taken. A claret sample was provided from all men for Dna analyses. Men with a positive family unit history of prostate cancer were excluded from this grouping. In total, the control group included cancer-complimentary men age 23–90 years (mean age: 61.8 years).

Whole exome sequencing

DNA has been isolated using standard methods from peripheral blood leukocytes taken from participants in Department of Genetics and Pathology in Szczecin.

The Illumina Nexter Rapid Capture Expanded Exome kit (target region size = 62 MB, coding exons, UTRs and miRNAs included) was used for capturing sequence target regions. The kit captured 62 Mbp of the human genome covering coding exons in CCDS and RefSeq databases besides every bit exons annotated by GENCODE project. The captured regions for each sample were barcoded and every two samples were pooled and used for paired-terminate sequencing for 100 cycles (generating 100 bp reads) on a single lane of Illumina HiSeq2000's period-jail cell.

The 100 meg reads for each exome sequence for each individual were aligned to the reference sequence of the homo genome using Burrows-Wheeler transform algorithm[14] SAMtools [fifteen] and GATK [xvi] packages were used for calling variants.

Sanger sequencing

The new BRCA2 variant identified past whole-exome sequencing in the discovery phase was confirmed past Sanger straight sequencing. The entire coding regions of BRCA1 (NG_005905) and BRCA2 (NG_012772) were sequenced in 63 amplicons in discovery stage. Sequencing reactions were performed using the BigDye Terminator v3.i Cycle Sequencing kit (Life Technologies) according to the manufacturer'south protocol (dx.doi.org/10.17504/protocols.io.ta7eihn). Sequencing products were analyzed on the ABI Prism 3500XL Genetic Analyzer (Life Technologies). All sequences were compared to the BRCA1 and BRCA2 RefSeq sequence for variant detection using Mutation Surveyor software (SoftGenetics).

LOH analysis

Loss of heterozygosity was evaluated in 2 primary tumors from patient carrying germline BRCA2 variant of unknown significance (c.4534 C>T) identified by targeted sequencing.

Using methane series-embedded tissues we performed microdissection and isolated DNA from primary tumors and healthy tissues for the control. Sanger sequencing of the Dna fragments containing two germline BRCA2 variants of unknown significance identified by WES and targeted sequencing was performed to evaluate potential loss of heterozygosity.

Unstained formalin-fixed, paraffin-embedded (FFPE) slides were microdissected post-obit pathology revision to select tumor and normal areas. DNA from skin FFPE tissues from melanoma patients conveying ii identified BRCA2 variants of unknown significance was isolated using the Qiagen DNeasy FFPE Tissue kit.

DNA (50 ng) from normal and tumor areas was amplified, and Sanger sequencing was carried out in parallel. Primers and reaction weather are available upon request.

TaqMan genotyping

Deoxyribonucleic acid was isolated from 5 to 10 ml of peripheral claret. The BRCA2 c.4534 C>T and c.9334G>A variants were genotyped using a TaqMan assay (Applied Biosystems/Life Technologies) and the LightCycler Existent-Fourth dimension PCR 480 organisation (Roche Life Scientific discipline) according to the manufacturer'southward protocol (dx.doi.org/10.17504/protocols.io.ta4eigw). The primer and probe sequences are available upon request. Laboratory technicians were blinded to case-control condition. The overall genotyping call rate was 99.iii%. The presence of each variant detected past Taqman analysis was confirmed past Sanger sequencing as described above.

Software prediction of Dna variants.

PROVEAN (Protein Fiveariation Effect Analyzer) is a software tool which predicts whether an amino acid exchange or indel has an affect on the biological function of a protein. A delta alignment score is computed for each supporting sequence. The scores are then averaged within and across clusters to generate the concluding PROVEAN score. If the PROVEAN score is equal to or below a predefined threshold (due east.g. -ii.5), the protein variant is predicted to have a "deleterious" result. If the PROVEAN score is above the threshold, the variant is predicted to take a "neutral" result [17].

SIFT (Sorting Intolerant From Tolerant) uses sequence homology to predict whether an amino acid substitution will affect protein function and hence, potentially alter phenotype. The score is the normalized probability that the amino acid change is tolerated. SIFT predicts substitutions with scores less than 0.05 as deleterious [18].

PolyPhen-two (Polymorphism Phenotyping v2), available equally software and via a Web server, predicts the possible bear upon of amino acrid substitutions on the stability and role of human proteins using structural and comparative evolutionary considerations [19]. The PolyPhen-2 Web interface can be reached at http://genetics.bwh.harvard.edu/pph2/.

MutationTaster is a gratis web-based application to evaluate DNA sequence variants for their disease-causing potential. The software performs a battery of in silico tests to estimate the impact of the variant on the gene product / protein. Tests are made on both, poly peptide and DNA level [20].

In 2008 IARC (International Agency for Enquiry on Cancer) proposed a standardized classification organisation of 5 classes of variants based on the degree of likelihood of pathogenicity for application to sequence-based results for cancer predisposition genes (Ref). With the term "pathogenic mutation" we herein hateful either course v variant (defininitely pathogenic, probability of being pathogenic >0.99) or class iv variants (probable pathogenic, probability of pathogenic 0.95–0.99) according to IARC classification. With the term variant of unknown significance we hateful form 3 variant (uncertain, probability of being pathogenic 0.05–0.949) co-ordinate to IARC classification [21].

Results

Discovery phase

We institute neither pathogenic mutations nor variants of unknown significance within BRCA1 sequence. We found no unequivocal pathogenic mutations in BRCA2.

WES identified one BRCA2 variant of unknown significance: NM_000059.three(BRCA2):c.9334G>A (p.Asp3112Asn, rs759851035).

The variant was found in a female patient diagnosed with MM at the age of 34. Her master tumor was localized to abdominal skin, Clarks grade III, 0.6mm Breslow thickness, mitotic index 1/mm2, ulceration absent. Evaluation of family unit history revealed occurrence of cancers of prostate, chest, colon, thyroid and leukemia among family members (Fig ane).

thumbnail

Fig i. Pedigree of the family with c.9334G>A detected variant.

* age. Abbreviation: Pr, prostate cancer; Leu, leukemia; Br, chest cancer; Thr, thyroid cancer; CRC, colorectal cancer; Liv, liver cancer; (+), mutation positive; (-), mutation negative; (ne), non examined.

https://doi.org/ten.1371/periodical.pone.0204768.g001

Targeted sequencing of 30 MM cases with cancer familial aggregation identified a second BRCA2 variant of unknown significance: NM_000059.3(BRCA2):c.4534C>T (p.Arg1512Cys, rs80358684). This variant was found in a female patient diagnosed with synchronous MM at the age of 50 ((https://doi.org/x.7910/DVN/MACR3E).

Her first primary tumor was localized on the skin of her lower leg, Clarks form, 1.4mm Breslow thickness, mitotic index iv/mm2, ulceration absent. Her second primary tumor was localized on the skin of the popliteal fossa, Clarks grade IV, 5.6mm Breslow thickness, mitotic index 1/mm2, ulceration absent. Evaluation of family history revealed occurrence of colorectal cancer affecting the mother of the proband (Fig 2).

thumbnail

Fig 2. Pedigree of the family with c.4534 C>T detected variant.

* age. Abridgement: Leu, leukemia; Br, chest cancer; Lu, lung cancer; CRC, colorectal cancer; (+), mutation positive; (-), mutation negative; (ne), not examined.

https://doi.org/10.1371/journal.pone.0204768.g002

Clan written report

C.9334G>A modify was nowadays in none of the 1230 unselected MM cases and 1696 melanoma controls, none of the 4802 chest cancer cases and 4251 breast controls, none of the 3268 prostate cancer cases and 3478 prostate controls.

C.4534 C>T change was present in none of the 1230 unselected MM cases and 1696 controls. It was present in none of the 4888 breast cases and 4436 chest controls (https://doi.org/10.7910/DVN/MACR3E).

LOH assay

We plant no loss of heterozygosity of c.4534 C>T in melanoma Deoxyribonucleic acid isolated from the carrier. Due to the absence of neoplasm tissue available for analysis we were unable to make up one's mind if the c.9334G>A variant co-occurred with a second deleterious change.

Segregation analysis

The c.9334G>A variant segregated with malignancies among the relatives of the proband (Fig one). Such stiff segregation was not observed for the c.4534 C>T variant (Fig 2).

Software prediction

The c.9334G>A is predilected to affect the BRCA2 protein and thus to be pathogenic according to MutationTaster, Polyphen2 and SIFT algorithms.

The c.4534 C>T variant is considered to exist a neutral variant co-ordinate to MutationTaster, Polyphen2, SIFT and PROVEAN algorithms.

Discussion

Testify suggesting possible association betwixt BRCA1 mutations and melanoma is weak. Deleterious BRCA1 mutations have been identified in ii patients of European origin with master breast cancer and melanoma [22].

A trend of increasing incidence of melanoma in BRCA1 mutation carriers (SIR iii.312, 95% CI 1.511–vi.288, p = 0.004) was reported by Mersch et al [12]. All other large familial studies signal to no such association [23–26]. The lack of BRCA1 mutations in our melanoma patients supports the notion that it is non involved in MM development.

In contrast to BRCA1, many studies have suggested an clan between BRCA2 mutations and melanoma. Suspected BRCA2 carriers have shown an increased melanoma risk in the majority (but not all) of reports [27]. As a result of the largest study, the Breast Cancer Linkage Consortium (BCLC) reported an approximate 2.6-fold increase in the adventure for CMM amid BRCA2 mutant carrier families [28]. Several studies have evaluated the prevalence of BRCA2 mutations among MM patients.

The BRCA2 rare nonsense variant (rs11571833) has previously been associated with moderately (OR ranging between 1.26–6.0) increased susceptibility to lung, pancreatic, esophageal and breast cancer [29, thirty, 31, 32] was also reported to be significantly over-represented in melanoma patients (OR = ii.80, p = 0.035) [33]. We have previously reported the significantly higher prevalence of the BRCA2 N991D variant in melanoma patients when compared to control subjects (OR = 1.8, p = 0.002), in contrast to T1915M and N372H variants [34]. An association between SNVs in BRCA2 and melanoma specific overall survival has been suggested by Yin et al [35].

In a recent written report of 82 patients with primary melanoma and breast cancers two carriers of BRCA2 variants were identified: 1 with a deleterious mutation the second with a variant of unknown significance [fifteen].

In contrast, genotyping of Italian families with an assemblage of melanoma and chest cancer revealed the absence of BRCA2 mutations, which does not exclude the link between BRCA2 and melanoma but suggests it is not common [36]. The rarity of founder BRCA1 and BRCA2 mutations in Askenazi patients with CMM supports the thesis [37]. Consistently our results point at the rarity of BRCA2 mutations amongst Polish melanoma patients. We identified only two BRCA2 variants: c.9334G>A in 1 out of 96 MM early-onset families and c.4534 C>T in ane out of 30 families with aggregation of MM and breast cancer.

BRCA2 mutations are responsible for up to 1% of unselected UK prostate cancer cases [38] and for only up to 0.two% of unselected Polish breast cancer cases [39]. Minor allele frequency of both variants in the Exome Assemblage Consortium (EXAC) population is simply 0.00000824. Our results confirm both variants are extremely rare in Smoothen population too. The identification of ane person inside the family unit harbouring the c.9334G>A variant does, notwithstanding, lends support to the notion that this specific allele is causative and confers a like adventure to a diversity of malignancies.

Both alterations are recognized as variants of unknown clinical significance. Nosotros evaluated these variants with algorithms adult to predict the consequence of missense changes on poly peptide structure and function such as MutationTaster, Polyphen2, SIFT and PROVEAN. C.9334G>A (rs759851035, Asp3112Asn, POS 32394766) is predicted to exist deleterious by three algorithms. The c.9334G>A variant is situated in BRCA2 OB3 domain (oligonucleotide/oligosaccharide-binding, domain 3). The OB3 domain spans between 3052–3186 amino acids, consists of a curved v-stranded beta-sheet that forms a beta-barrel. OB3 domain has a grooves situated between beta one and beta 2 sheets and between beta four and beta v sheets, which let for stiff ssDNA binding) [xl].

Although, Asp3112 amino acid is situated in a curlicue structure between beta three and beta four sheets and does not bind ssDNA directly, the residuum is conserved across mammalian species, suggesting that alter might adversely bear upon protein part. Nevertheless, these predictions take not been however confirmed by published functional studies.

The prediction that c.9334G>A is deleterious is supported by segregation analysis thereby pointing information technology towards pathogenicity. The variant was detected in a patient affected with MM, 2 prostate cancer patients, patient with 3 master tumours and a female with T-cell leukemia (Fig 1). The spectrum of cancers affecting carriers of c.9334G>A variant is broad- with the exception of leukemia, thyroid and colorectal cancer all other malignancies are part of the BRCA2 phenotype [38]. The about usually reported cancers with BRCA2 mutations include breast, pancreas, prostate, and melanoma [26, 28, 41, 42, 43]. Nosotros cannot exclude a possibility of an association between T-cell leukemia and BRCA2 in our patient- in a recent prospective written report of 7243 women with a BRCA1 or a BRCA2 mutation a college risk of developing leukemia amid women with a BRCA2 mutation and breast cancer was reported [44].

The c.4534 C>T variant is situated in NHii-terminal BRC-repeat (between third and fourth BRC repeat) and does non represent any known functional domain. Although there is a big physico-chemical divergence between arginine and cysteine, the arginine residue is weakly conserved, and cysteine amino acid rest is found in multiple mammalian species, suggesting that this missense modify does not adversely affect poly peptide office. All 4 algorithms predict c.4534 C>T (rs80358684, Arg1512Cys, POS 32338889) to be a missense alter that does not affect poly peptide function. Genotyping of large cohorts of our melanoma and breast cancer patients and healthy controls revealed that both variants are extremely rare in the Smooth population.

We were unable to perform LOH analysis in tumor DNA equally information technology was unavailable from the patient carrying c.9334G>A variant. Still, the variant segregated with BRCA2-related cancer illness in the family and in silico prediction algorithms indicated it affected BRCA2 protein function. It strongly suggests that this BRCA2 variant is pathogenic, and carriers of this change are at an increased risk of BRCA-related cancers, that include chest, prostate and melanoma. In contrast the c.4534 C>T variant did not segregate with cancer and was nowadays in many unaffected individuals. Furthermore, LOH of this variant was not detected. Together, these findings point towards c.4534 C>T beingness a rare benign polymorphism that should exist re-classified as a likely beneficial variant.

In conclusions, BRCA2 mutations are infrequent among early-onset melanoma patients or MM cases with a familial aggregation with chest cancer. The c.9334G>A variant of BRCA2, classified as variant of unknown significance, on current evidence is suggestive of pathogenicity and predisposes to cancers of the breast, prostate and melanoma. Functional studies to describe how the Deoxyribonucleic acid change affects the protein part are needed and due to its rarity in the Caucasian population a large multi-center study to evaluate its penetrance is required.

Acknowledgments

This inquiry was supported by the Polish National Scientific discipline Eye grant number 2014/xiii/B/NZ2/03888 to TD. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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