Next Generation Sequencing Technology For Susceptible Gene Screening In Familial Non-medullary Thyroid Carcinoma

Objective:Thyroid carcinoma is the most common malignancy of endocrine organs,and its prevalence is increasing rapidly.It includes medullary thyroid carcinoma originating from the calcitonin-secreting parafollicular thyroid C cells and non-medullary thyroid carcinoma arising from the follicular thyroid epithelium cells.Non-medullary thyroid carcinoma accounts for more than 95% of all thyroid cancers.5%~10% of non-medullary thyroid carcinomas are hereditary.Some genes likely related to familial non-medullary thyroid cancer(FNMTC)have been reported while the responsible genes for FNMTC are unclear.The purpose of this study was to design a panel for susceptibility screening of FNMTC on the basis of next-generation sequencing(NGS)and investigate the application of NGS in thyroid carcinoma.Methods:A panel of NGS including whole exon regions from 31 genes and one intronic region from RET was designed.Sequencing was performed on a Miseq for DNA samples extracted from peripheral blood leukocytes of 47 FNMTC patients and 16 sporadic non-medullary thyroid carcinoma(SNMTC)respectively.Sequencing was performend by Illumina MiSeq.Local alignment optimization and variant calling and annotation were performed using GATK 3.2.DNA translocation analysis was performed using both Tophat2 and Factera 1.4.3.The high-confidence non-synonymous mutations were filtered with both the ExAC and ClinVar databases.Mutations were also validated by Sanger Sequencing.In addition,the clinic-pathological characteristics were compared between patients with or without mutations by SPSS 19.0 statistical software.A p-value< 0.05 was considered statistically significant.Results:1.We performed deep sequencing with the FNMTC susceptibility panel on 63 NMTC patients.We obtained an average of 2.5 million reads for each sample.On av-erage 98.9% of all reads can be mapped back to the genome and 54.3% of all reads are mapped to our designed target regions.The average coverage depth appears to be 500×,with a minimum of 157× and a maximum of 1505×.All samples have greater than 96.0% of all regions with coverage depth greater than 200×.We then assessed the repeatability of probe capturing efficiency by measuring the correlation of coverage depth in each target region between different samples.Most pairs have a correlation coefficient greater than 0.8.2.There were 45 clinically meaningful germline variants identified in 63 NMTC patients,including 38 SNVs,one short 4-bp frame-shift deletion,one splice donor variant and five in-frame insertions or deletions.Thirty-seven mutations were detected in FNMTC patients and eight in SNMTC cases.The percentage of the mutation carriers was 61.7%(29/47)in the familial group and 37.5%(6/16),a marginal but not statistically significant(P=0.092).Notably,ten germline mutations from eight genes were found matching between paired FNMTC patients from the same family,including APC L292 F and A2778 S,BRAF D22 N,MSH6 G355 S and A36 V,MSH2 L719 F,MEN1 G508 D,BRCA1 SS955 S,BRCA2 G2508 S,and a GNAS inframe insertion.3.Based on sequencing results,patients were divided into two groups: mutation positive and mutation negative.The clinical features of mutation positive patients were compared with that of negative patients.There was a significant difference in central lymph nodes involvement of the two groups(68.6% vs.30.8%,P=0.003).Among the FNMTC patients,central lymph nodes involvement occurred more frequently in the offsprings than in the first generation(100% vs.42.9%;P=0.019).Conclusion:The capturing efficiency of the probe was stable.The quality assessment of the targeted sequencing data demonstrated that the data accorded with the typical distribution of germline variants and had a high reliability.This panel of NGS is helpful for us to diagnose and elucidate the genetic cause of the FNMTC patients.A few mutational loci that haven’t been reported in FNMTC patients were found in this study.The findings would also be a basis for individualized treatment of patients with thyroid cancer and provide insights on predicting the genetic risk of potential second cancers for NMTC patients.