Cryptic Genomic Rearrangements in Three Patients with 46,XY Disorders of Sex Development

Background 46,XY disorders of sex development (46,XY DSD) are genetically heterogeneous conditions. Recently, a few submicroscopic genomic rearrangements have been reported as novel genetic causes of 46,XY DSD. Methodology/Principal Findings To clarify the role of cryptic rearrangements in the development of 46,XY DSD, we performed array-based comparative genomic hybridization analysis for 24 genetic males with genital abnormalities. Heterozygous submicroscopic deletions were identified in three cases (cases 1–3). A ∼8.5 Mb terminal deletion at 9p24.1–24.3 was detected in case 1 that presented with complete female-type external genitalia and mental retardation; a ∼2.0 Mb interstitial deletion at 20p13 was identified in case 2 with ambiguous external genitalia and short stature; and a ∼18.0 Mb interstitial deletion at 2q31.1–32 was found in case 3 with ambiguous external genitalia, mental retardation and multiple anomalies. The genital abnormalities of case 1 could be ascribed to gonadal dysgenesis caused by haploinsufficiency of DMRT1, while those of case 3 were possibly associated with perturbed organogenesis due to a deletion of the HOXD cluster. The deletion in case 2 affected 36 genes, none of which have been previously implicated in sex development. Conclusions/Significance The results indicate that cryptic genomic rearrangements constitute an important part of the molecular bases of 46,XY DSD and that submicroscopic deletions can lead to various types of 46,XY DSD that occur as components of contiguous gene deletion syndromes. Most importantly, our data provide a novel candidate locus for 46,XY DSD at 20p13.

Introduction 46,XY disorders of sex development (46,XY DSD) are genetically heterogeneous conditions that result from the impaired production or function of androgens, or from defective organogenesis of external genitalia [1]. To date, several genes such as SRY, AR, SRD5A2, and SOX9 have been identified as causative genes for 46,XY DSD, although mutations in these genes account for only a minor fraction of the molecular causes of these conditions [1], [2].
Recent advances in microarray technology, including comparative genomic hybridization (CGH) analysis and single nucleotide polymorphism (SNP) genotyping, have enabled researchers to identify genomic rearrangements in individuals with apparently normal karyotypes [3]. Cryptic genomic rearrangements can lead to developmental disorders, although they can also occur as benign polymorphisms [4]. To date, CGH analysis and SNP genotyping have been carried out for patients with 46,XY DSD, identifying multiple submicroscopic deletions and duplications [5], [6], [7]. Such rearrangements frequently affected coding exons or regulatory regions of known DSD-associated genes including SF1, SOX9 and DMRT1, or exons of candidate genes including KANK1 and ZEB2 [5], [6], [7]. These data suggest that genomic abnormalities at various chromosomal loci may underlie 46,XY DSD.
To clarify the role of cryptic genomic rearrangements in the development of 46,XY DSD, we performed copy-number analyses for 24 patients. The results provide novel insights into the molecular basis of 46,XY DSD.

Ethics Statement
This study was approved by the Institutional Review Board Committee at the National Center for Child Health and Development. After obtaining written informed consent from the parents, peripheral blood samples were collected from the patients. When possible, blood samples were also obtained from the parents.

Patients
The study population comprised 24 patients with 46, XY DSD, including nine cases with complete female-type external genitalia, five with ambiguous genitalia and 10 with male-type external genitalia with hypospadias ( Table 1). None of the 24 patients had a family history of DSD or a history of prenatal exposure to specific environmental pollutants. G-banding analysis showed a normal 46,XY karyotype in all patients. Mutations in the coding regions of known DSD-causative genes, SRY, AR, SRD5A2, SF1, WNT4, SOX9, WT1, BNC2, DMRT1, HSD17B3, and MAP3K1, were excluded by sequence analyses.

CGH Analysis
Genomic DNA samples were subjected to CGH analyses using a catalog human array (46180 k format, Agilent Technologies, Palo Alto, CA), according the manufacturer's instructions. The sizes and positions of the genomic rearrangements were analyzed using the UCSC genome browser (http://genome.ucsc.edu/; February 2009, hg19, build 37). In the present study, we focused on copy-number alterations with a physical size of more than 1.5 Mb, which have a higher probability of being associated with disease phenotypes [8]. Deletions and duplications registered in the database of genomic variants (http://projects.tcag.ca/ variation/) were excluded as benign polymorphisms.

CGH Analysis
We identified heterozygous submicroscopic deletions in three cases (cases 1-3; Fig. 1). The deletions affected several genes ( Clinical Features of Deletion-positive Patients Case 1 was a genetic male born to non-consanguineous Japanese parents. This patient had complete female-type external genitalia and was raised as a female. This patient exhibited mental retardation and behavioral problems and was diagnosed as having schizophrenia. At 17 years of age, this patient was referred to our clinic because of primary amenorrhea. Clinical analysis detected no dysmorphic facial features or cardiac/renal abnormalities. Abdominal ultrasonography delineated a uterus. Blood endocrine tests indicated primary hypogonadism (Table 3). At 17 years of age, the patient underwent gonadectomy. Histological analyses showed bilateral streak gonads with ovarian ducts. The parents were clinically normal.
Case 2 was a genetic male born to non-consanguineous Vietnamese parents. At birth, this patient exhibited a micropenis, cryptorchidism, and distal hypospadias. Abdominal ultrasonography detected bilateral testes (1266 mm) in inguinal canals. The uterus and ovaries were absent. This patient was raised as a boy and underwent surgical intervention for hypospadias and cryptorchidism at 4 years and 2 months and at 4 years and 3 months of age, respectively. On his visit at 4.5 years of age, the patient showed a penis with a stretched length of 3 cm, and left testis (1269 mm) in the scrotum and right testis (1366 mm) in the inguinal canal (Fig. 2). He had no dysmorphic facial features (Fig. 2). He showed short stature (89 cm, 22.9 SD) and delayed bone age (equivalent to 2 years of age). His mental development was normal. Blood endocrine tests at 4.5 years of age showed low levels of luteinizing hormone and testosterone (Table 3). His growth hormone levels were within the normal range at the baseline, but remained low after physical exercise. His parents were clinically normal and had normal statures.
Case 3 was born to non-consanguineous Vietnamese parents at 40 weeks of gestation with a birth weight of 2.0 kg (23.7 SD). At birth, this patient manifested severe micropenis and hypospadias (Fig. 2). Bilateral testes were palpable in the scrotum, and uterus and ovaries were absent. Thus, this patient was raised as a boy. In addition to genital abnormalities, he exhibited multiple anomalies of the fingers and toes, i.e., camptodactyly and flexion contracture of the proximal interphalangeal joint of the right index and left ring fingers, cutaneous syndactyly of the 2nd and 3rd toes and medial deviation of the 4th toe in the right foot, lateral deviation of the 2nd toe and medial deviation the 4th toe in the left foot, and overriding of the 4th toe on the third toe in both feet (Fig. 2). Furthermore, he showed dysmorphic facial features such as ptosis and micrognathia (Fig. 2). His blood testosterone level at birth was within the normal range (Table 3). On examination at 11 months of age, he showed obvious growth retardation (body weight; 6.0 kg, ,23.0 SD) and developmental delay (DQ ,30). At one year of age, he presented with an episode of febrile convulsion. Brain magnetic resonance imaging detected delayed myelination, hypogenesis of the corpus callosum, and prominent ventricular and CSF spaces (Fig. 2). His parents were clinically normal.  Table 2. doi:10.1371/journal.pone.0068194.g001

Discussion
We identified cryptic heterozygous deletions with physical sizes of more than 1.5 Mb in three of the 24 patients with 46,XY DSD. The results support the notion that submicroscopic genomic rearrangements constitute a portion of causative mechanisms for 46,XY DSD [5], [6], [7]. Furthermore, molecular and clinical data of the three cases imply that cryptic deletions can cause DSD as components of contiguous gene deletion syndromes. Since array-based CGH analysis and SNP genotyping can detect copynumber alterations across the entire genome in a single assay, these methods should be considered for patients with 46,XY DSD, particularly for those with additional clinical manifestations. Case 1 had a ,8.5 Mb heterozygous deletion at 9p involving 40 genes. Of the 40 genes, DMRT1 is known to encode a male specific transcriptional regulator with a conserved zinc finger-like DNAbinding domain [9], [10]. Since mouse Dmrt1 has been implicated in testicular differentiation [11], and intragenic deletions of human DMRT1 have been identified in 46, XY patients with gonadal dysgenesis [6], [12], it appears that DSD in case 1 results from haploinsufficiency of DMRT1. These data argue for the assumption that heterozygous deletions involving the coding exons and/or the upstream region of DMRT1 account for a substantial part of the etiology of complete and partial gonadal dysgenesis in individuals with 46, XY karyotype [6], [12], [13], [14]. Furthermore, our results provide additional information about other disease-associated loci. First, deletions at 9p22.3-23 are known to cause various malformations, such as craniofacial abnormalities, cardiac defects, and dysplastic kidneys, which are collectively referred to as the 9p-syndrome [13]. Lack of clinical manifestations of the 9p-syndrome in case 1 implies that the gene(s) responsible for this syndrome is not located in the ,8.5 Mb terminal region. This is consistent with previous studies which mapped the critical region of this syndrome to a genomic interval approximately 11-15 Mb from the telomere (Fig. 3A) [13]. Second, terminal deletions at 9p have previously been associated with mental retardation [13]. Our data suggest that a gene involved in brain development resides in the ,8.5 Mb terminal region that is deleted in case 1 (Fig. 3A).
Case 2 had a de novo ,2.0 Mb interstitial deletion at 20p13, which has not been identified previously in patients with DSD. Furthermore, none of the 36 genes affected by the deletion have been associated with sex development. These results, in conjunction with previous reports of hypomasculinized external genitalia in a patient with a $6 Mb deletion at 20p13-12.3 [15] and in a patient with a 20p11.2-pter deletion [16], indicate that the genomic interval spanning ,2.7-4.7 Mb from the telomere (deleted in case 2 and in the two aforementioned patients) encompasses a novel causative gene for DSD (Fig. 3B). However, the penetrance of DSD in males with 20p13 deletions appears to be low, because genital abnormalities have been described only in a small percentage of patients with such deletions [16], [17], [18], [19]. It might also be possible that the 20p13 and/or the 2q31.1-32 deletion has unmasked a recessive mutation of the testis development gene(s) on the structurally normal homologous chromosome, leading to DSD. In addition, the deletion of case 2 seems to harbor a gene that is indispensable for growth, because short stature was observed in case 2, as well as in most patients with partial monosomy of 20p [16], [17], [18], [19]. In this regard, although case 2 showed impaired growth hormone secretion after exercise, it remains to be clarified whether short stature in patients with 20p deletions is ascribed to growth hormone deficiency. Furthermore, unlike patients with terminal deletions of 20p [16], [17], [18], [19] case 2 showed no facial dysmorphism or mental retardation. These data indicate that a gene(s) involved in the development of the craniofacial region and brain resides within the 0-2.7 Mb interval from the telomere that is preserved in case 2. Consistent with this, facial abnormalities and developmental delay have been reported in two patients harboring 20p terminal deletions of less than 1.7 Mb [20] (Fig. 3B). Importantly, the deletion in case 2 includes OXT and AVP that are predicted to play a role in social behavior [21]. Lack of social dysfunction in case 2 indicates that haploinsufficiency of OXT and AVP permits normal psychosocial development at least in childhood. However, this notion awaits further investigation. Case 3 had a ,18.0 Mb interstitial deletion at 2q31.1-32.1. Clinical manifestations of case 3 including finger/toe anomalies, mental retardation and facial dysmorphism are compatible with the 2q31 microdeletion syndrome, a well-established contiguous gene deletion syndrome [22]. Notably, abnormal formation of the external genitalia has been reported in both male and female patients carrying 2q31 deletions [23], [24]. Previous studies have attributed the skeletal anomalies of 2q31 microdeletion syndrome to haploinsufficiency of the HOXD cluster [22], [25], and mental retardation and craniofacial abnormalities to deletions of certain genes located within the genomic interval spanning 174-175 Mb from the 2q telomere [22] (Fig. 3C). In this regard, while skeletal abnormalities are obviously milder in case 3 than the previously reported patients with deletions involving HOXD genes [25], this would be consistent with the assumption that haploinsufficiency of developmental genes is frequently associated with a broad phenotypic spectrum [26]. Since mouse Hoxd genes have been shown to play a role in the formation of external genitalia by regulating multiple target genes, genital abnormalities of 2q31 microdeletion syndrome could be associated with haploinsufficiency of HOXD genes [25], [27]. Indeed, the phenotype of case 3, such as hypomasculinized external genitalia without cryptorchidism and a normal blood testosterone value at birth, is indicative of perturbed organogenesis of the external genitalia rather than impaired hormone production in the gonads. However, since DSD has been described for only a small subset of males with 2q31 deletions [22], [23], [24], [25], impaired sex development in case 3 may be caused by other unknown genetic or environmental factors.
In summary, we identified cryptic genomic rearrangements in three of 24 individuals with 46,XY DSD. It appears that the genital abnormalities of case 1 result from gonadal dysgenesis due to haploinsufficiency of DMRT1, while those of case 3 can be ascribed to perturbed organogenesis due to the deletion of the HOXD cluster. These data suggest that submicroscopic deletions can lead to various types of 46,XY DSD that occur as components of contiguous gene deletion syndromes. Moreover, the results obtained from case 2 provide a novel candidate locus for 46,XY DSD at 20p13. Further copy-number analyses on patients with 46,XY DSD and functional assays for genes involved in the genomic rearrangements will help to clarify novel causative mechanisms for 46,XY DSD.