Targeted next-generation sequencing of 21 candidate genes in hereditary ovarian cancer patients from the Republic of Bashkortostan

Patients had different histology type of tumors but 46 were epithelial ovarian carcinomas. Of these, 30 (65%) were serous tumors; 8 (17%) were mucinous tumors; 2 (4%) were mixed epithelial tumors; 2 (4%) were undifferentiated carcinoma; 1 (2%) was a clear cell tumor; 1 (2%) was an endometrioid tumor; 1 (2%) was a squamous tumor; 1 (2%) was a Brenner’s tumor. Stromal tumors were found in 2 (4%) cases: one granulosa cell tumor (2%) and one tumor from Sertoli-Leydig cells (2%). Tumors were predominantly of a high grade (G3-G4) – 36%. A low-grade tumor (G2) was detected in 32% cases, and grading of cancer cells was not histologically determined in 32% patients. Bilateral ovarian cancer was present in 2 (4%) women with OC. Stage I of disease was established in 16% of patients; II – in 11%; III – in 70% and stage IV – in 3% of cases. Seven patients (15%) also had a personal history of breast cancer, cervical cancer or colon cancer. Metastases were detected in 43% of the patients.

Genomic DNA was isolated from peripheral white blood cells by routine phenol–chloroform extraction. To screen germline variants of the nucleotide sequence, the method of targeted next-generation sequencing was applied on the Illumina MiSeq platform using the AmpliSeq protocol with a custom panel containing primers for the synthesis of 661 amplicons covering the protein coding region of 21 candidate genes, including their UTR regions: BRCA1, BRCA2, BARD1, BRIP1, CDH1, CHEK2, EPCAM, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, PALB2, PMS2, PTEN, RAD50, RAD51C, STK11, TP53. An additional file with used primers shows this in more detail (see Additional file 1). Then, an assessment of the reading quality and secondary processing of data was carried out in the multifunctional online service Base Space Illumina (https://​basespace.​illumina.​com). An individual summary file was obtained for each sample under study, containing information on the number of reads, the percentage of Q30 bases, the percentage of gene coverage, the percentage of aligned reads, the level of autosomal variants colling, the number of identified SNVs, deletions, insertions relative to the reference genome, the gene regions are indicated in which changes were detected, etc. Also, for each sample,.vcf and.bam files were obtained containing complete information about all detected changes in the studied genes. For bioinformatics analysis of the nucleotide sequence variants, Illumina Variant Interpreter, ANNOVAR, SNPeff, ClinVar, gnomAD, ExAC, 1000 Genomes and ALFA services were used. Changes, detected by Targeted Next-Generation Sequencing of 21 candidate genes in hereditary ovarian cancer patients, were annotated in ANNOVAR program, using the summarize_annovar.pl script. It makes possible to compare single nucleotide substitutions with a number of specialized databases and predict the functional significance of the detected changes using in silico tools (SIFT, PolyPhen-2, LRT, Mutation Taster, Mutation Assessor, ClinVar, phyloP, GERP ++ and others) from dbNSFP v.1.3. In addition, the CADD (Combined Annotation Dependent Depletion) program was used. To estimate the population frequencies of the identified variants, we used data from the 1000 Genomes project, the Exome Aggregation Consortium and Allele Frequency Aggregator.

After ANNOVAR annotation, a search for pathogenic variants was conducted that may represent driver mutations in the development of ovarian cancer. This further analysis included the use of custom filters, based on the following criteria:

Functional disease-causing variants (frameshift, stop gain and one deleterious missense variants) were found in 33% of patients. In total, 9 different loss-of-function variants were detected in 16 DNA samples (Table 1). By far the most frequently mutated gene was BRCA1 with the founder mutation c.5266dupC in seven cases, the variant c.3143delG in two patients, two further PVs (c.4035delA, c.3700_3704delGTAAA) in one patient each, and the variant c.181T >G (encoding the RING finger substitution p.Cys61Gly) in one case. In the BRCA2 gene we found one pathogenic variant (c.3751dupA). In the CHEK2 gene we detected one patient with the founder mutation c.1100delC. In the NBN gene we detected a novel truncating variant (c.429G>A, p.W143X). One further truncating variant was identified in MSH6 (c.1299T>G, p.Y433X) (Table 1).

The seven patients heterozygous for the c.5266dupC mutation were diagnosed with serous (5/7), clear cell (1/7) and squamous cell (1/7) carcinomas. Two patients with c.5266dupC additionally had cervical cancer and/or breast cancer, as well as vaginal, omental and liver metastases. Five c.5266dupC carriers were of Russian origin and two carriers of Tatar origin. One of the two patients with BRCA1*c.3143delG also had breast cancer. The patient with the MSH6*p.Y433X truncation also had endometrial cancer, this patient also harbored unclassified variants in MUTYH and BRCA2. The patient with CHEK2*c.1100delC was identified with serous carcinoma. The clinical data of these and the remaining PV carriers are summarized in Table 2.

We additionally identified 15 rare variants of uncertain significance, including six novel missense variants, that were located in BRCA2 (3), PALB2 (2), ATM (3), NBN (2), MRE11 (2), MSH6 (1), and MUTYH (2) genes (Table 3). Six of these variants (BRCA2*p.Cys1348Ser, BRCA2*p.Lys1875Thr, PALB2*p.Asp496His, ATM*p.Arg717Trp, ATM*p.Arg2010Gly, MUTYH*c.985G>A) were found together with truncating variants in BRCA1, CHEK2 or MSH6, respectively, making them less likely to constitute the driver of carcinogenesis. The MUTYH*c.1187G>A variant, encoding p.Gly396Asp (rs36053993), was considered potentially pathogenic in the biallelic state [15] but the patient here was heterozygous only. One patient had three variants of uncertain significance in BRCA2, MRE11 and NBN, illustrating the challenge to identify a causal variant among rare missense substitutions of different DNA repair genes found in the same patient.