The development of male reproductive system is a complex process controlled by delicate networks that specify sex-specific differentiation, organogenesis and endocrine function. The fragility of these regulatory cascades is illustrated by the high prevalence of genitourinary defects in newborns. These inborn urogenital anomalies present difficult challenges for the parents and the physicians, as care of these children is complicated by surgical, psychological, social and sexual concerns. The gold standard for genetic diagnosis remains a karyotype analysis and an endocrine profile but findings in intersex cases are not always informative. Indeed, only a small portion of these developmental aberrations can be attributed to defects in the synthesis of testosterone or adrenal steroid hormones, receptor alterations, exogenous modifiers or obvious numerical and structural chromosomal alterations, such as Klinefelter syndrome. The underlying causes of the majority of “idiopathic” cases remain to be discovered. In this study, the use of a clinically validated microarray (CMA) revealed the existence of cryptic imbalances strongly associated with defects of urogenital development or recurrently found in patients with DSD. These chromosomal aberrations were mostly too small to be detected by the routinely ordered karyotype, which has a limited resolution of 5–10 Mb, depending on the quality of chromosome preparations. Many of these genomic anomalies went also largely undetected because they were located in subtelomeric loci, which are notoriously difficult to characterize by G-banding. Moreover, mild or isolated cases of hypospadias and cryptorchid patients are usually not referred for genetic testing, while this study proved that this subset of patients harbored structural variation that may convey defective urogenital traits.
Most of the detected chromosomal aberrations encompassed one to a few hundred genes including known gonad-determining genes (SRY
) as well as novel candidate genes such as FGFR2
. Changes in dosage or structure of genes within the affected DNA segments might lead to haploinsufficiency or altered transcription profiles, which may disturb the intricate fine-tuned network of genes controlling the human genital development. Clinically relevant examples of gene dosage alterations have already been documented for factors controlling mammalian sex development. For instance, deletion of the sex-determining gene WNT4
is responsible for the masculinization of XX mouse pups, while its duplication and overexpression in humans leads to XY sex reversal 
. Duplications of large segments of DNA containing DAX1
also cause sex reversal 
. Thus, our findings contribute in a coherent manner to strengthen the emerging concept that sex determination and differentiation are dosage sensitive at multiple steps of their pathways. In addition to dosage effects, imbalances may lead to disruption of regulatory sequences that control the expression of neighboring genes; thus, in some cases, a gene related to genital development may lie adjacent to the detected deletion or duplication. For instance, a submicroscopic 258 Kb deletion, detected 11,320 bp upstream of DAX1
in a 21-year-old 46,XY female, may lead to a loss of regulatory sequences and position-effect upregulation of DAX1
Our findings provide support for the genomic basis of human disorders of sexual development and call for genome-wide CNV screenings which may, due to their extended coverage, reveal a higher proportion of germline mutations associated with urogenital defects. Enrichment in candidate genes for human sexual development is subsequently bound to increase. Our present study using the clinically established CMA platform was motivated by a rapid translation of our findings to the clinical arena. Molecular testing, such as with CMA, could significantly impact patient care by assisting the pediatric urologists and neonatologists in diagnosis. Genetic counseling offered to families based on the identification of pathogenic rearrangements may provide parents with essential clinical information pertaining to the child's diagnosis and permit proper estimates of the risk of recurrence for subsequent pregnancies. De novo imbalances are expected to have a very low risk of recurrence but it may be useful in future pregnancies to check for gonadal mosaicism in the parents. An unbalanced translocation identified by CMA may reveal a balanced translocation in a carrier parent and thus chances for a chromosomally abnormal future pregnancy would be as high as 25%. In vitro fertilization and pre-implantation genetic diagnosis could provide these couples with a possible alternative path to parenthood, specifically in case of severe genital ambiguity.
In conclusion, this study presented structural DNA variation as a potential underlying etiology for human disorders of sexual development. Frequent disease-causing submicroscopic gains and losses of DNA segments were detected across the genome and strongly associated with defective urogenital traits. This has been achieved with significantly higher resolution and greater clinical yield than standard routine karyotype, thus making this array-based CGH screen as a genetic test of choice in diagnosis. While GU defects cases arise among newborns without clear etiology, this study offers novel loci to dissect for determining key genes involved in the human sexual development.