A novel deleterious c.2656G>T MSH2 germline mutation in a Pakistani family with a phenotypic overlap of hereditary breast and ovarian cancer and Lynch syndrome

Hereditary breast and ovarian cancer syndrome (HBOC) and Lynch syndrome (LS) account for a significant proportion of inherited gynecologic malignancies, mainly caused by pathogenic germline mutations in the BRCA1 and BRCA2 genes or in mismatch repair (MMR) genes, such as MLH1 and MSH2. Women harboring deleterious mutations in these genes have increased life-time risks of developing a number of malignancies including ovarian cancer. Since there is a phenotypic overlap of HBOC and LS, timely identification of individuals at-risk of a particular syndrome is crucial in order to optimize cancer risk management. We report a novel pathogenic MSH2 mutation, c.2656G > T, which was identified in a 67-year-old female patient with breast cancer, who had previously tested negative for a deleterious mutation in the breast cancer susceptibility genes BRCA1, BRCA2, CHEK2 or RAD51C. The patient reported a personal history of endometrial cancer diagnosed at age 48, and a strong family history of breast and ovarian cancer, as well as several other malignancies within the spectrum of LS. The novel mutation was also found in the index patient’s daughter and a niece, who were diagnosed with endometrial and ovarian cancer, respectively. Breast and endometrial tumors from c.2656G > T mutation carriers showed loss of MSH2 and MSH6 protein expression. The mutation was absent in the control population. Our finding suggests that testing for MMR genes may be of benefit to BRCA1/2 negative families with overlapping HBOC and LS phenotype in Pakistan. It is clinically significant to identify individuals harboring mutations in genes linked with a particular syndrome so that they can benefit from targeted life-saving cancer surveillance and preventive strategies.


Background
Genetic testing for hereditary cancer has become an essential part of modern oncologic practice. It is offered to individuals with strong personal and/or family histories of cancer(s). The two most common cancer syndromes with autosomal dominant inheritance among women with gynecologic malignancies include hereditary breast and ovarian cancer (HBOC) and Lynch syndrome (LS; also known as hereditary non-polyposis colorectal cancer (HNPCC)). HBOC is mainly caused by germline mutations in the BRCA1 and BRCA2 genes [1], while LS is due to mutations in mismatch repair (MMR) genes including MLH1, MSH2, MSH6 and PMS2 [2]. Individuals carrying BRCA1/2 mutations face high lifetime risks of breast and ovarian cancer [3]. Individuals with mutations in MMR genes have increased lifetime risks of developing colorectal, endometrial, and ovarian cancers [4][5][6][7][8][9][10][11]. Ovarian cancer is seen in both syndromes. The lifetime risks of developing breast cancer [12][13][14][15][16] or endometrial/colorectal carcinoma [17,18] have also been reported to be elevated among LS-or HBOC-associated mutation carriers, respectively. Hence, there is a possible phenotypic overlap of these syndromes, which makes genetic counseling, screening, preventive and therapeutic decisions more challenging as diverse cancer risk management strategies are offered to individuals affected by HBOC or LS. Appropriate identification of individuals carrying mutations in a particular gene is an important factor in optimizing cancer risk management.
Two families, one from Italy and the other from Canada, with features of HBOC and LS have previously been described [19,20]. In both reports double heterozygotes for BRCA1/2 and MLH1/MSH2 mutations were identified. Here, we report the identification of a novel single (heterozygous) deleterious c.2656G > T MSH2 mutation in a BRCA1/2 negative Pakistani family within the spectrum of HBOC and LS.

Case presentation
A 67-year-old Pakistani woman of Pathan ethnicity presented with a lump in the left breast to the SKMCH & RC, Lahore, Pakistan, in October 2008. Excision biopsy revealed a grade 3 invasive ductal breast carcinoma measuring 2.8 cm in its greatest dimension (pT2) without evidence of lymph node involvement (pN0). Immunohistochemical (IHC) analyses showed that the tumor was negative for estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2), MSH2 and MSH6 expression. She reported a prior diagnosis of endometrial carcinoma at age 48, for which she had undergone a total abdominal hysterectomy and bilateral salpingo-oophorectomy. In the adjuvant setting, she had received radiotherapy at University College Hospital, London, UK.
The index patient (III: 14) reported an extensive family history of malignancy ( Fig. 1). Her sister (III:12), cousin (III:1), and niece (IV:1) were diagnosed with unilateral breast cancer. Another sister (III:10) was diagnosed with bilateral breast cancer. Additionally, six family members had presented with gynecologic malignancies; one sister (III:18) and two nieces (IV:17, IV:20) presented with ovarian cancer; her daughter (IV:23) and two nieces (IV:4, IV:16) were diagnosed with endometrial cancer. Another sister (III:5) and one maternal cousin (III:4) had previously been diagnosed with intestinal cancer, and three descendants (IV:1, IV:3, IV:4) of that sister were affected by colon cancer. The index patient reported several other family members with various malignancies including her mother (II:6) with liver cancer, a brother (III:8) with stomach cancer, one nephew (IV:13) with renal cancer and another (IV:8) with prostate cancer, and one niece (IV:22), one grandniece (V:1) and one grandnephew (V:2) with childhood malignancies including brain tumor, osteoblastoma and leukemia, respectively. The cancer diagnoses of the index patient (III: 14), her daughter (IV:23) and one niece (IV:20) were confirmed by review of medical records and/or pathological reports. All other cancer diagnoses were self-reported or reported by other family members.
Given the strong family history of cancer, the patient was referred to SKMCH & RC for genetic counseling and risk assessment and was enrolled in the study after obtaining written informed consent. The study was approved by the ethical review board of SKMCH & RC. Due to the presence of multiple breast and ovarian cancers in this family, the preliminary diagnosis was HBOC syndrome [1]. The patient had previously tested negative for deleterious small-range BRCA1/2 mutations and large genomic rearrangements (M. U. Rashid, unpublished data) using denaturing high performance liquid chromatography (DHPLC) analysis followed by DNA sequence analysis of variant fragments and multiplex ligation-dependent probe amplification (MLPA) as described [21,22]. The patient had also tested negative for mutations in CHEK2 [23] and RAD51C [24], implying the involvement of other gene(s) contributing to disease risk in this family.
This family also fulfilled the recognized criteria for suspected LS [25] (Fig. 1): that includes (i) among first degree relatives of a colorectal cancer patient (or in himself ) at least 1 colorectal cancer, cancer of the endometrium, small bowel or urinary tract; (ii) at least one of the above cancers diagnosed under age 50; and (iii) familial adenomatous polyposis excluded [25]. The index case was screened for germline mutations in the MLH1, MSH2 and MSH6 genes using DHPLC and DNA sequence analysis as described elsewhere [26]. A novel disease-causative heterozygous nonsense mutation in exon 16 of MSH2, c.2656G > T (p.Glu886*), was identified.
Genetic MSH2 testing was offered to other affected family members. The mutation was also detected in the index patient's daughter (IV:23) who was diagnosed with endometrial cancer, and one niece (IV:20) affected by ovarian cancer. Her nephew (IV:8), who was diagnosed with prostate cancer, tested negative for this mutation. The mutation was also not identified in 100 healthy female controls.

Discussion
We report the identification of a deleterious MSH2 germline mutation in a Pakistani family with a strong family history of malignancy (6 breast cancers, 3 ovarian cancers, 4 endometrial carcinomas, 3 colon cancers, 2 intestinal cancers) within the spectrum of HBOC and LS, who tested negative for BRCA1/2 mutations. Our data highlight the clinical implications of this finding, specifically with respect to genetic counseling, screening, and prophylaxis of mutation carriers in such families.
The index patient harboring the MSH2 mutation was affected by endometrial cancer at age 48 and breast cancer at age 67. The occurrence of breast cancer following endometrial cancer is in line with data from Win and colleagues, who showed that women with LS have an increased risk of breast cancer after endometrial cancer [27]. The mutation, c.2656G > T (p.Glu886*), located in exon 16 of the MSH2 gene, is novel and has not been previously reported in several large variant databases . It is likely to be pathogenic as it generates a premature termination codon resulting in a truncated protein, with loss of 49 C-terminal amino acid residues, a region involved in MSH2 homodimer formation and MSH3/MSH6 interaction [28]. The location of the mutation is in a region of high sequence conservation among humans, grivets, mice and cows and it's absence in 100 healthy controls suggests that it is associated with the disease. Several other pathogenic mutations in this region have previously been described [29,30] (LOVD, http://chromium.liacs.nl/LOVD2/colon_cancer/; UMD database http://www.umd.be/). Moreover, the c.2656G > T mutation segregated with the disease as it was identified in the index patient's daughter, who had herself suffered from endometrial cancer at age 52 and in a niece affected by ovarian cancer at age 43. The latter probably inherited the mutation from her deceased mother, who had been diagnosed with breast cancer at age 58, suggesting that this mutation may predispose to breast cancer. Association of MMR gene mutations with breast cancer has previously been observed in various studies among Australian, Brazilian, Danish, Dutch, and US populations [12][13][14][15][16]. Recently, a four-fold increase in breast cancer risk was reported in MMR gene mutation carriers from Australia, New Zealand, Canada, and the United States compared to the general population [31]. In contrast, initial studies among Finnish and US populations showed no association [4,32]. The variation in observed data may be due to population heterogeneity or involvement of other genetic and/or non-genetic risk factors.
The breast tumor associated with the pathogenic MSH2 mutation in our index patient was negative for ER, PR and HER2 expression, also known as triplenegative breast cancer (TNBC). This observation is in agreement with previous findings that breast tumors linked with mutations in MMR genes are primarily negative for ER and PR expression [33]. However, BRCA1 associated breast tumors are also reported to significantly display TNBC [34], so that TNBC phenotype cannot be utilized to distinguish between BRCA1 and MMR gene mutation carriers. The index patient's breast tumor was also negative for MSH2 and MSH6 protein expression (data not shown). This finding suggests that the MSH2 germline mutation, in addition to endometrial cancer, may also predispose to breast cancer in this family. Given the controversy as to whether breast cancer is a part of LS or not, in the literature, large prospective studies will help to clarify this issue.
Patients with LS have increased lifetime risks of developing ovarian, endometrial, and colorectal cancers, ranging from 6.7-13.5 %, 31.5-62 %, and 50-80 %, respectively [4-6, 9, 11]. In the family reported here, the MSH2 mutation was found to co-segregate with ovarian and endometrial cancer. We were unable to study cosegregation of this mutation in the colon/intestinal cancer cases, since they were either deceased or did not agree to participate in this study. The mean age of colon/intestinal cancer diagnosis in this family was 48.8 years, which is similar to the mean age of 44.8 years previously reported in Caucasians with MSH2 mutations [35].
Previously, bi-allelic germline mutations in MMR genes have been reported with a rare constitutional MMRdeficiency syndrome, characterised by a broad spectrum of childhood onset malignancies mostly among individuals with a history of parental consangunity [36,37]. Among children with this syndrome, MSH2 mutations are less common, while PMS2 mutations are most commonly found [37]. Three paediatric cancers (leukemia, brain tumor, and osteoblastoma) were also observed in our study, with unknown parental consanguinity status. Due to the lack of DNA samples, none of these individuals (IV:22, V:1 and V:2) nor their parents (III:13, IV:21, IV:26 and IV:27) could be tested. It is unlikely that the childhood malignancies in the Pakistani family are linked with the MSH2 mutation, as manifestations of LS in the parents of these children were not observed. Hence, the role of PMS2 or other genes known to be involved in these childhood malignancies cannot be excluded.
Different sets of clinical criteria for identifying patients at high risk of LS include the stringent Amsterdam I/II criteria [38,39], which are based on a family history of at least three relatives with histologically verified colorectal cancer/cancers linked with LS, respectively, and the less stringent Bethesda guidelines, later updated to the revised Bethesda guidelines [40,41]. The revised Bethesda guidelines, based on clinicopathologic parameters, have been developed to identify high-risk patients by evaluation of microsatellite instability (MSI) and/or IHC testing of their tumors. Unfortunately, this strategy could not be applied to the Pakistani index patient due to restraints of normal/tumor tissue. However, the Pakistani family fulfilled the less stringent criteria of suspected LS [25], which is based on a family history of only two LS-linked cancers. Our finding support the notion that the suspected LS criteria may be useful for the identification of Pakistani families [25,42,43].
To our knowledge, this is the first Asian family with a history of cancer within the spectrum of HBOC and LS, in which a novel, single heterozygous mutation in a MMR gene was identified. Different findings have been reported in two other studies among two families, one from Italy and another from Canada, which have a similar phenotype, but different genotypes, with double heterozygous BRCA1/2 and MMR gene mutations, were identified [19,20], which may confer an altered risk. Women with double heterozygous BRCA1/2 and MMR gene mutations were younger at time of diagnosis of breast cancer (range 32-46) than the single heterozygous Pakistani patient(s) (ages: 58 (obligatory carrier), 67) described in this report. The Pakistani patient did not harbor a deleterious mutation in BRCA1/2 and also tested negative for mutations in CHEK2 [23], RAD51C [24], and PALB2 (M. U. Rashid, unpublished data). We cannot exclude the possibility that a mutation in yet another breast cancer susceptibility gene exists, which was not analyzed. While the mean age of breast cancer diagnosis in the Pakistani population is 47 years (range 18-90) [44], Pakistani patients with BRCA1, CHEK2, or RAD51C mutations presented with breast cancer at a mean age of 31 years (range 22-49), 41.5 years (range 30-53), and 50 years (range 49-51), respectively [21,23,24]. In the current pedigree, the mean age of breast cancer onset of 65.7 years (range 58-76) was higher, suggesting the involvement of other susceptibility gene(s). The deleterious mutation in the MSH2 gene reported here may predispose to breast cancer in this family, since it was detected in one family member diagnosed with breast cancer at age 67, while another family member diagnosed with breast cancer at age 58 is likely to be an obligatory carrier, who passed the mutation on to her daughter. The co-segregation of the mutation with breast cancer could not be investigated further, because two relatives were deceased and another refused to participate. Therefore it may still be possible that the overrepresentation of breast cancer in this family is by chance, considering that breast cancer is the most common invasive malignancy in Pakistani women (Globocan 2012; http://globocan.iarc.fr/).

Conclusions
Our findings suggest that MMR gene testing may be beneficial to BRCA1/2 negative families presenting with clinical features and a pedigree chart suggestive of HBOC syndrome, especially if they report other LS-associated cancer(s). It is clinically important to identify individuals with LS, so that they can benefit from targeted life-saving cancer surveillance strategies.