Identification of a Comprehensive Spectrum of Genetic Factors for Hereditary Breast Cancer in a Chinese Population by Next-Generation Sequencing

<div><p>The genetic etiology of hereditary breast cancer has not been fully elucidated. Although germline mutations of high-penetrance genes such as BRCA1/2 are implicated in development of hereditary breast cancers, at least half of all breast cancer families are not linked to these genes. To identify a comprehensive spectrum of genetic factors for hereditary breast cancer in a Chinese population, we performed an analysis of germline mutations in 2,165 coding exons of 152 genes associated with hereditary cancer using next-generation sequencing (NGS) in 99 breast cancer patients from families of cancer patients regardless of cancer types. Forty-two deleterious germline mutations were identified in 21 genes of 34 patients, including 18 (18.2%) BRCA1 or BRCA2 mutations, 3 (3%) TP53 mutations, 5 (5.1%) DNA mismatch repair gene mutations, 1 (1%) CDH1 mutation, 6 (6.1%) Fanconi anemia pathway gene mutations, and 9 (9.1%) mutations in other genes. Of seven patients who carried mutations in more than one gene, 4 were BRCA1/2 mutation carriers, and their average onset age was much younger than patients with only BRCA1/2 mutations. Almost all identified high-penetrance gene mutations in those families fulfill the typical phenotypes of hereditary cancer syndromes listed in the National Comprehensive Cancer Network (NCCN) guidelines, except two TP53 and three mismatch repair gene mutations. Furthermore, functional studies of MSH3 germline mutations confirmed the association between MSH3 mutation and tumorigenesis, and segregation analysis suggested antagonism between BRCA1 and MSH3. We also identified a lot of low-penetrance gene mutations. Although the clinical significance of those newly identified low-penetrance gene mutations has not been fully appreciated yet, these new findings do provide valuable epidemiological information for the future studies. Together, these findings highlight the importance of genetic testing based on NCCN guidelines and a multi-gene analysis using NGS may be a supplement to traditional genetic counseling.</p></div

Characteristics of 99 probands and their families, and proportions of probands with germline deleterious mutations.

<p>This figure consisted of two aspects. The left column presented the overview of characteristics of the 99 probands and their families. It provided the numbers and proportions of each subgroup. The right color lumps reflected the proportions of probands with different gene mutations in each subgroup. The numbers on the right side of the color lumps represented the proportions. Proportions of probands with one gene mutation are shown in pure color squares, and proportions of probands with mutations in two or more genes are shown in two-color squares. Abbreviations and definitions are as follows: HR, hormone receptor; Her-2, human epidermal growth factor receptor 2; BC, breast cancer; HBOCS, hereditary breast and ovarian cancer syndrome; LFS, Li-Fraumeni syndrome; LS, Lynch syndrome; FA, Fanconi anemia genes, including <i>RAD50</i>, <i>PALB2</i>, <i>FANCD2</i>, <i>FANCI</i>, <i>SLX4</i>, and <i>RAD51C</i>. <i>MMR</i>, mismatch repair genes, including <i>MLH1</i>, <i>MLH3</i>, and <i>MSH3</i>. OTHER, other genes, including <i>RGSL1</i>, <i>CDKN2A</i>, <i>SPINK1</i>, <i>TNFRSF13B</i>, <i>FGFR3</i>, <i>WRN</i>, <i>MUTYH</i>, and <i>CYP17A1</i>.</p

All putative deleterious germline mutations and proband characteristics.

<p>Mutation nomenclature follows the recommended guidelines of the Human Genome Variation Society, with the nucleotide numbering based on the GenBank reference sequence indicated by its accession NCBI number. Details are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125571#pone.0125571.s002" target="_blank">S2 Table</a>.</p><p>Abbreviations: HCS, hereditary cancer syndrome; HBOCS, hereditary breast and ovarian cancer syndrome; LFS, Li-Fraumeni syndrome; LS, Lynch syndrome; U/A, unavailable; D, damaging; B, benign; C, conserved; NC, not conserved; and PD, probably damaging. BIC, breast cancer information core, URL: <a href="http://research.nhgri.nih.gov/bic/" target="_blank">http://research.nhgri.nih.gov/bic/</a>. p53 mutation database, URL: <a href="http://www-p53.iarc.fr" target="_blank">http://www-p53.iarc.fr</a>, version R16.</p><p>^a Results of segregation studies marking positive individuals out of the total number available for validation.</p><p>^b All variants were queried against 1000 Genomes (1000G) data using the 1000 Genomes Browser (<a href="http://browser.1000genomes.org/index.html" target="_blank">http://browser.1000genomes.org/index.html</a>) which integrates SNP and InDel calls from 1,092 individuals (data released 2012 April). The minor allele frequency (MAF) is provided here.</p><p>^c<i>In sillico</i> prediction (missense prediction software and evolutionary conservation) were used for the determination of pathogenicity of missense mutations. Detailed criteria were described in <i>Materials and Methods</i>.</p><p>All putative deleterious germline mutations and proband characteristics.</p

Filtering of variants.

<p>Detailed procedure was described in <i>Materials and Methods</i>. The URLs for the locus-specific databases used were as follows: <i>BRCA1/2</i>, <a href="http://research.nhgri.nih.gov/bic/" target="_blank">http://research.nhgri.nih.gov/bic/</a>; <i>TP53</i>, <a href="http://www-p53.iarc.fr/" target="_blank">http://www-p53.iarc.fr/</a>; DNA <i>MMR</i> genes, <a href="http://www.insight-group.org/" target="_blank">http://www.insight-group.org/</a>.</p

Details of patients carrying mutations in two or more genes.

<p>Mutation nomenclature follows the recommended guidelines of the Human Genome Variation Society with the nucleotide numbering based on GenBank reference sequence indicated by its accession NCBI number. Details were listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125571#pone.0125571.s002" target="_blank">S2 Table</a>. Abbreviations and definitions are as follows: FH, family history; HBOCS, hereditary breast and ovarian cancer syndrome; Br, breast; FA, Fanconi anemia; Lym, lymphoma; LS, Lynch syndrome; FAP2, familial adenomatous polyposis, 2; WS, werner syndrome; and CVID2, common variable immune deficiency,2.</p><p>Details of patients carrying mutations in two or more genes.</p

Expression of Msh3 protein in normal and tumor tissues of <i>MSH3</i> mutation carriers.

<p>Arrows indicate the location of the expression of Msh3 protein. A, the normal ovarian tissue of the proband from family No. 49. B, the ovarian tumor tissue of the proband from family No. 49. The loss of Msh3 expression was observed in the tumor tissue. C, the normal breast tissue of the sister of the proband from family No. 75. D, the breast tumor tissue of the sister of the proband from family No. 75. Msh3 is expressed strongly in the normal tissue and weakly in the tumor tissue.</p

Results of IHC and MSI analysis in tissues of <i>MSH3</i> mutation patients.

<p>For MSH3 IHC, “3+” indicates strong staining, “2+” indicates intermediate staining, “1+” indicates week staining, and “0” indicates no staining. For MSI profiles, it includes MSI data for five consensus National Cancer Institute (NCI) microsatellite markers (two mono-A repeats, BAT25 and BAT26, and three dinucleotide repeats, D2S123 through D17S250), two additional dinucleotide (D) markers (D18S69 and D18S64), and seven EMAST markers (MYCL1 through D8S321). For MSI data, “-” indicates the absence of the mutation, and “+” indicates the presence of the mutation. For MSI using the NCI panel (NCI), S indicates MSS. For MSI using the NCI panel and two additional dinucleotide markers (NCI and D status), S indicates MSS, and L indicates MSI-L. For EMAST status, E indicates EMAST positive, and non-E indicates EMAST negative.</p><p>Results of IHC and MSI analysis in tissues of <i>MSH3</i> mutation patients.</p