High Resolution X Chromosome-Specific Array-CGH Detects New CNVs in Infertile Males

<div><h3>Context</h3><p>The role of CNVs in male infertility is poorly defined, and only those linked to the Y chromosome have been the object of extensive research. Although it has been predicted that the X chromosome is also enriched in spermatogenesis genes, no clinically relevant gene mutations have been identified so far.</p> <h3>Objectives</h3><p>In order to advance our understanding of the role of X-linked genetic factors in male infertility, we applied high resolution X chromosome specific array-CGH in 199 men with different sperm count followed by the analysis of selected, patient-specific deletions in large groups of cases and normozoospermic controls.</p> <h3>Results</h3><p>We identified 73 CNVs, among which 55 are novel, providing the largest collection of X-linked CNVs in relation to spermatogenesis. We found 12 patient-specific deletions with potential clinical implication. Cancer Testis Antigen gene family members were the most frequently affected genes, and represent new genetic targets in relationship with altered spermatogenesis. One of the most relevant findings of our study is the significantly higher global burden of deletions in patients compared to controls due to an excessive rate of deletions/person (0.57 versus 0.21, respectively; p = 8.785×10⁻⁶) and to a higher mean sequence loss/person (11.79 Kb and 8.13 Kb, respectively; p = 3.435×10⁻⁴).</p> <h3>Conclusions</h3><p>By the analysis of the X chromosome at the highest resolution available to date, in a large group of subjects with known sperm count we observed a deletion burden in relation to spermatogenic impairment and the lack of highly recurrent deletions on the X chromosome. We identified a number of potentially important patient-specific CNVs and candidate spermatogenesis genes, which represent novel targets for future investigations.</p> </div

List of the 31 patient-specific (not found in normozoospermic controls) CNVs detected by array-CGH and their description according to type, gene location (<i>NO</i> = no gene found within) and occurrence in the Database of Genomic Variants (DGV).

*<p>CNV minimum size.</p

Array-CGH study: Comparison between patients and controls of the mean number and mean extension of CNVs (A) as well as the number of all subjects bearing more than one CNV (B).

<p>sd = standard deviation. OR = odds ratio. CI = confidence interval. <i>p</i>1 refers to the mean number of CNV/subject. <i>p</i>2 refers to the mean DNA change/subject.</p

Case-control study of selected losses preliminarily identified by array-CGH as patient-specific (not found in normozoospermic controls).

<p>Genes inside the CNV minimum size are depicted in bold;</p>*<p>genes inside the CNV maximum size;</p>§<p>the first proximal flanking gene;</p>†<p>the first distal flanking gene; the remaining genes are situated <500 Kb from the minimum size border. Azoosp = Azoospermia; Oligozoosp = Oligozoospermia; Cryptozoosp = Cryptozoospermia; SCOS = Sertoli Cell Only Syndrome; SGA = Spermatogenic Arrest.</p

Array-CGH study.

<p>: distribution of the 73 CNVs according to their size: small (<10 Kb), medium (10–100 Kb) and large (>100 Kb) referred to A) all CNVs (44 gains and 29 losses); B) losses; C) gains. Losses were typically of small/medium size (52%) whereas gains are generally of larger size (48%). On the side, tables display the number of A) all CNVs; B) losses; C) gains of different size and categorized according to their occurrence in patients/controls: i) “patient-specific” when found only in patients; ii) “control-specific” when found only in controls; iii) “patient-enriched” when found predominantly in patients; iv) “control- enriched” when found predominantly in controls; v) “common” when found at a similar frequency in patients and controls.</p

Array-CGH study: comparison of patients' semen parameters according to the number of CNVs.

<p>Sperm concentration and total sperm number are expressed as: mean ± standard deviation (median; range). Significance is depicted by a <i>p</i> value<0.05.</p

List of the 33 control-specific (not found in idiopathic patients) CNVs detected by array-CGH and their description according to type, gene location (<i>NO</i> = no gene found within) and occurrence in the Database of Genomic Variants (DGV).

*<p>CNV minimum size.</p

Schematic representation of the distribution of the 73 CNVs (44 gains and 29 losses) along the X chromosome identified by high resolution X chromosome specific array-CGH analysis.

<p>A) The histogram shows that the 73 CNVs were evenly distributed along the X chromosome but displayed a higher density in the pseudoautosomal region 1, PAR1 (Xp22.33). B) The frequency of gains (upwards) and losses (downwards) per X chromosome region in patients and controls are indicated.</p

Clinical description of the study population.

<p>A) Semen phenotype of the entire study population (array-CGH and case-control study); B) Description of all analyzed patients (array-CGH and case-control study) according to their geographic origin and semen phenotype; C) Hormonal levels and testis volumes of all analyzed patients (array-CGH and case-control study).</p

List of CNVs found by array-CGH considering their occurrence in controls and in patients with their description according to type, gene location (NO = no gene found within) and presence in the Database of Genomic Variants (DGV).

*<p>CNV minimum size.</p