Balkan Journal of Medical Genetics

NEXT-GENERATION SEQUENCING INFERTILITY PANEL IN TURKEY: FIRST RESULTS
Ikbal Atli E1*, Yalcintepe S1, Atli E1, Demir S1, Gurkan H1
*Corresponding Author: Corresponding Author: Associate Prof. Emine Ikbal Atli, Trakya University, Faculty of Medicine, Department of Medical Genetics, Edirne, Turkey Balkan Campus, Highway D100 ORCID ID: 0000- 0001-9003-1449; Postal code: 22030; Phone: 0(284) 235-76-41/2330; Email: emine.ikbal@gmail.com / eikbalatli@trakya.edu.tr
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METHODS

Patients and Samples The research included 85 individuals with a clinical diagnosis of male infertility who had tested negative on diagnostic genetic testing. 84 individuals were thought to have primary spermatogenic failure, while one individual was thought to have central hypogonadism. Following the elimination of female factor infertility and acquired reasons of male infertility, main spermatogenic failure was suspected with a history of couple infertility longer than two years (e.g. male accessory gland infection, varicocele, testicular trauma, etc.). All patients are cases of infertil- ity for two or more years. The patient group consists of patients who do not have any known additional disease or malignancy. Additionally, individuals included in this study tested negative for early genetic anomalies such karyotype ab- normalities, Y chromosome AZF microdeletions. MLPA technique was performed using the SALSA MLPA probe- mix P360 version B1 (MRC Holland, Amsterdam, The Netherlands) kit following the manufacturer’s instructions. The kit contained 55 probes, of which 12 were located in autosomal chromosomes (for internal control reaction), and 43 were located in Y-chromosome AZF regions (16 AZFa, 15 AZFb, and 12 AZFc regions). Each patient provided written consent after being fully briefed. The study was conducted in accordance with the principles outlined in the Declaration of Helsinki, and the local Ethics Committee approved the study. Conventional G-banded karyotype analysis from pe- ripheral blood was performed as part of the initial screening tests. The study included peripheral lymphocyte culture by a standard method using the Leishman-banding technique, centromere-banding (C-banding) and nucleolar organizing region staining performed as needed according to the AGT Cytogenetics Laboratory Manual. The best metaphases were karyotyped, and the total chromosome count was usually determined in 25 cells. The International System for Human Cytogenetic Nomenclature (ISCN) was used for the nomenclature of human chromosomes. Patients with no anomalies as a result of karyotype analysis were included in the NGS analysis. Eighty five samples were sequenced using QIAseq Targeted DNA Custom Panel (Qiagen, Hilden, Germany). 2 mL of peripheral blood were collected and then preserved in anticoagulation tubes. Genomic DNA was isolated from peripheral whole blood using the EZ1 DNA Investiga- tor Kit (Qiagen, Hilden, Germany). After DNA extrac- tion, target sequences were enriched by using customized capture probes chips (Illumina, San Diego, CA). This kit included 132 genes targeting disease. Libraries covering the target genes were prepared according to the QIAseq Targeted DNA Panel protocol (Qiagen, Hilden, Germany). Following the target enrichment process, libraries were sequenced on the MiSeq System (Illumina, San Diego, CA, USA). OCI analysis (Qiagen, Hilden, Germany) was used for Quality control and Variant Call Format file genera- tion. In silico evaluation of the pathogenicity of nucleotide changes in exons was performed using Polymorphism Phenotyping v2 (PolyPhen-2, http:// genetics.bwh.harvard. edu/pph2/), Sorting Intolerant from Tolerant (SIFT, https:// sift.bii.a-star.edu.sg/), and MutationTaster (http://www. mutationtaster.org). Minor allele frequencies (MAF) were checked in the Genome Aggregation Database gnomAD (http://gnomad.broadinstitute.org/).Variant analysis was performed with Ingenuity software (Qiagen, Hilden, Ger- many). Variants were interpreted according to the Ameri- can College of Medical Genetics and Genomics (ACMG) recommended standard. Sanger sequencing was performed for confirmation when the target region coverage was less than 15 reads. Nucleotide alterations were analyzed and validated by Sanger sequencing. After confirmation, each variant was classified as a pathogenic, likely pathogenic, variant of unknown significance (VUS), likely benign, or benign, according to the American College of Medical Genetics (ACMG) guidelines. Coding genomic regions (CDS) that were sequenced with coverage less than 15X were eventually re-sequenced using Sanger technology.

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