Genomic testing for copy number and single nucleotide variants in spermatogenic failure

Abstract

Purpose: To identify clinically significant genomic copy number (CNV) and single nucleotide variants (SNV) in males with unexplained spermatogenic failure (SPGF).

Materials and methods: Peripheral blood DNA from 97/102 study participants diagnosed with oligozoospermia, severe oligozoospermia, or non-obstructive azoospermia (NOA) was analyzed for CNVs via array comparative genomic hybridization (aCGH) and SNVs using whole-exome sequencing (WES).

Results: Of the 2544 CNVs identified in individuals with SPGF, > 90% were small, ranging from 0.6 to 75 kb. Thirty, clinically relevant genomic aberrations, were detected in 28 patients (~ 29%). These included likely diagnostic CNVs in 3/41 NOA patients (~ 7%): 1 hemizygous, intragenic TEX11 deletion, 1 hemizygous DDX53 full gene deletion, and 1 homozygous, intragenic STK11 deletion. High-level mosaicism for X chromosome disomy (~ 10% 46,XY and ~ 90% 47,XXY) was also identified in 3 of 41 NOA patients who previously tested normal with conventional karyotyping. The remaining 24 CNVs detected were heterozygous, autosomal recessive carrier variants. Follow-up WES analysis confirmed 8 of 27 (30%) CNVs (X chromosome disomy excluded). WES analysis additionally identified 13 significant SNVs and/or indels in 9 patients (~ 9%) including X-linked AR, KAL1, and NR0B1 variants.

Conclusion: Using a combined genome-wide aCGH/WES approach, we identified pathogenic and likely pathogenic SNVs and CNVs in 15 patients (15%) with unexplained SPGF. This value equals the detection rate of conventional testing for aneuploidies and is considerably higher than the prevalence of Y chromosome microdeletions. Our results underscore the importance of comprehensive genomic analysis in emerging diagnostic testing of complex conditions like male infertility.

Keywords: Comparative genomic hybridization; Copy number variant; Single nucleotide variant; Spermatogenic failure; Whole-exome sequencing.

Fig. 1

Patient variant size and frequency distribution. Total true variants (n = 2590) unique to individuals with spermatogenic failure (azoospermia n = 41, severe oligozoospermia n = 44, and oligozoospermia n = 17), detected by Agilent 400 K SurePrint G3 Human CGH array and calculated with Cytogenomics ADM-2 or ADM-2 Noisy algorithms, ranged in size from 0.6 kb-2.7 Mb, with > 95% of them less than 100 kb (X axis is shown in log2 scale)

Fig. 2

Patient variant count and distribution per chromosome. Copy number variant detection by Agilent 400 K SurePrint G3 Human CGH array and quantification with Cytogenomics ADM-2 or ADM-2 Noisy algorithms showed no significant difference in average number of true CNVs per condition: azoospermia (n = 28), severe oligozoospermia (n = 28), oligozoospermia (n = 25). Total number of patient deletions (n = 1482) significantly outnumbered total number of patient amplifications (n = 1108) (p < 0.05). Normalization based on chromosome size showed no significant difference in chromosome distribution of copy number variants

Fig. 3

Mosaic X chromosome amplification. A Agilent Cytogenomics chromosome view reveals amplification (blue) extending the entirety of the X chromosome. B Interval table. Highlighted row (gray) shows output for a 1.87-fold (2^0.905) amplification of a 93-Mb region of the X chromosome

Fig. 4

Y Chromosome microdeletions detected by PCR. Agarose gel electrophoresis of amplified DNA shows the presence of SRY gene in eight patients and fertile control (left) and AZFc/sY254 (380 bp) deletions in samples A7, A21, A33, S7, and S25 (right)

Fig. 5

WES confirmation of selected likely pathogenic CNVs. Output from SOPHiA DDM version 5.10.12 showing intragenic deletion of TEX11 (upper) and full deletion of DDX53 (lower) in two separate NOA patients