Detection of Germline Mutation in Hereditary Breast and/or Ovarian Cancers by Next-Generation Sequencing on a Four-Gene Panel.

Mutation in BRCA1/BRCA2 genes accounts for 20% of familial breast cancers, 5% to 10% of which may be due to other less penetrant genes which are still incompletely studied. Herein, a four-gene panel was used to examine the prevalence of BRCA1, BRCA2, TP53, and PTEN in hereditary breast and ovarian cancers in Southern Chinese population. In this cohort, 948 high-risk breast and/or ovarian patients were recruited for genetic screening by next-generation sequencing (NGS). The performance of our NGS pipeline was evaluated with 80 Sanger-validated known mutations and eight negative cases. With appropriate bioinformatics analysis pipeline, the detection sensitivity of NGS is comparable with Sanger sequencing. The prevalence of BRCA1/BRCA2 germline mutations was 9.4% in our Chinese cohort, of which 48.8% of the mutations arose from hotspot mutations. With the use of a tailor-made algorithm, HomopolymerQZ, more mutations were detected compared with single mutation detection algorithm. The frequencies of PTEN and TP53 were 0.21% and 0.53%, respectively, in the Southern Chinese patients with breast and/or ovarian cancers. High-throughput NGS approach allows the incorporation of control cohort that provides an ethnicity-specific data for polymorphic variants. Our data suggest that hotspot mutations screening such as SNaPshot could be an effective preliminary screening alternative adopted in a standard clinical laboratory without NGS setup.

Comprehensive spectrum of BRCA1 and BRCA2 deleterious mutations in breast cancer in Asian countries.

Approximately 5%-10% of breast cancers are due to genetic predisposition caused by germline mutations; the most commonly tested genes are BRCA1 and BRCA2 mutations. Some mutations are unique to one family and others are recurrent; the spectrum of BRCA1/BRCA2 mutations varies depending on the geographical origins, populations or ethnic groups. In this review, we compiled data from 11 participating Asian countries (Bangladesh, Mainland China, Hong Kong SAR, Indonesia, Japan, Korea, Malaysia, Philippines, Singapore, Thailand and Vietnam), and from ethnic Asians residing in Canada and the USA. We have additionally conducted a literature review to include other Asian countries mainly in Central and Western Asia. We present the current pathogenic mutation spectrum of BRCA1/BRCA2 genes in patients with breast cancer in various Asian populations. Understanding BRCA1/BRCA2 mutations in Asians will help provide better risk assessment and clinical management of breast cancer.

The importance of analysis of long-range rearrangement of BRCA1 and BRCA2 in genetic diagnosis of familial breast cancer.

Germline BRCA gene mutations are reportedly associated with hereditary breast and ovarian cancers. Identification of BRCA mutations greatly improves the preventive strategies and management of breast cancer. Sanger sequencing has been the gold standard in identifying these mutations. However, 4-28% of inherited BRCA mutations may be due to large genomic rearrangements (LGRs), which could be missed by using Sanger sequencing alone. Our aim is to evaluate the pick-up rate of LGRs in our cohort. A total of 1,236 clinically high-risk patients with breast and/or ovarian cancers were recruited through The Hong Kong Hereditary Breast Cancer Family Registry from 2007 to 2014. Full gene sequencing (either Sanger or next generation sequencing) and multiplex ligation-dependent probe amplification (MLPA) were performed. We identified 120 deleterious BRCA mutations: 57 (4.61%) were in BRCA1 and 63 (5.10%) were in BRCA2. LGRs accounted for 6.67% (8 of 120) of all BRCA mutations, whereas 8.77 % (5 of 57) were BRCA1 mutations and 4.76% (3 of 63) were BRCA2 mutations. Through this integrated approach, both small nucleotide variations and LGRs could be detected. We suggest that MLPA should be incorporated into the standard practice for genetic testing to avoid false-negative results, which would greatly affect the management of these high-risk families.

Elevation of methylated DNA in KILLIN/PTEN in the plasma of patients with thyroid and/or breast cancer.

Around 80% of mutations in the PTEN gene have been reported to be associated with diseases such as Cowden syndrome, which is an autosomal dominant disorder associated with an increased risk of developing breast, thyroid, and endometrial neoplasms. Recent studies have also demonstrated that KILLIN, which is located proximally to PTEN, shares the same transcription start site, and is assumed to be regulated by the same promoter, but is transcribed in the opposite direction. In this regard, we postulate that there may be a connection between KILLIN/PTEN genes and breast and thyroid cancers. Using real-time quantitative polymerase chain reaction (qPCR), we found that expression of KILLIN, but not PTEN, was significantly decreased in 23 Chinese women with a personal history of breast and thyroid cancer or a personal history of breast cancer and a family history of thyroid cancer, or vice versa, and at least two persons in the family with thyroid cancer or at a young age <40 years, when compared with healthy controls (P<0.0001). No PTEN mutations were found in these 23 patients. We then developed a simple methylation-sensitive restriction enzyme digestion followed by real-time quantitative assay to quantify plasma methylated KILLIN/PTEN DNA in these patients. Plasma levels of methylated KILLIN/PTEN DNA were significantly increased in these patients when compared with healthy controls (P<0.05). This study shows that plasma methylated KILLIN/PTEN DNA was significantly elevated, suggesting hypermethylation of the KILLIN/PTEN promoter in breast and thyroid cancer patients.

A novel de novo BRCA1 mutation in a Chinese woman with early onset breast cancer.

Germline mutations in the two breast cancer susceptibility genes, BRCA1 and BRCA2 account for a significant portion of hereditary breast/ovarian cancer. De novo mutations such as multiple exon deletion are rarely occurred in BRCA1 and BRCA2. During our mutation screening for BRCA1/2 genes to Chinese women with risk factors for hereditary breast/ovarian cancer, we identified a novel germline mutation, consisting of a deletion from exons 1 to 12 in BRCA1 gene, in a patient diagnosed with early onset triple negative breast cancer with no family history of cancer. None of her parents carried the mutation and molecular analysis showed that this novel de novo germline mutation resulted in down-regulation of BRCA1 gene expression.

Aberrant promoter hypermethylation and silencing of the critical 3p21 tumour suppressor gene, RASSF1A, in Chinese oesophageal squamous cell carcinoma.

3p21 is an important locus harbouring critical tumour suppressor genes (TSG), which are implicated in the pathogenesis of multiple tumours, including oesophageal carcinoma. RASSF1A is a 3p21.3 candidate TSG frequently inactivated by promoter methylation in multiple tumours. We investigated RASSF1A promoter methylation and gene expression in Chinese oesophageal squamous cell carcinoma (ESCC) to compare it to data from Japanese patients. Methylation-specific PCR (MSP) showed that RASSF1A was partially methylated in 3/7 (43%) cell lines; 22/64 (34%) primary tumours and 3/64 (5%) corresponding non-tumour samples; and was not methylated in 2 immortalized normal oesophageal epithelial cell lines and 6 normal oesophageal epithelium samples. Bisulfite genome sequencing confirmed the MSP results. Promoter hypermethylation correlated well with RASSF1A mRNA down-regulation. Treatment of cell lines with 5-aza-2'-deoxycytidine activated RASSF1A mRNA expression along with promoter demethylation. RASSF1A hypermethylation in the Chinese cohort was much lower than in a published report of Japanese ESCC patients (52%) and cell lines (74%). Our own analysis of Japanese ESCC cell lines for direct comparison also detected a high frequency of RASSF1A hypermethylation (8/10; 80%) and high levels of hypermethylation at each CpG site. No significant association between RASSF1A hypermethylation and histological differentiation (p=0.953), tumour staging (p=0.117), or survival (p=0.7571) was found in Chinese ESCC, unlike the results of Japanese patients. The incidence of oesophageal cancer shows marked variation by geographic area and ethnic group; it is almost three times higher in China than in Japan, indicating possible different pathogenetic mechanisms. Our results show that RASSF1A hypermethylation in ESCC has epidemiological/ethnic differences, and suggest that Chinese ESCC may result from different pathogenetic mechanisms.