Balkan Journal of Medical Genetics

DUPLICATION OF THE SOX3 GENE IN AN SRY-NEGATIVE 46,XX MALE WITH ASSOCIATED CONGENITAL ANOMALIES OF KIDNEYS AND THE URINARY TRACT: CASE REPORT AND REVIEW OF THE LITERATURE
Tasic V1, Mitrotti A2, Riepe FG3, Kulle AE3, Laban N1, Polenakovic M4, Plaseska-Karanfilska D4, Sanna-Cherchi S2, Kostovski M1, Gucev Z1,*
*Corresponding Author: Professor Dr. Zoran Gucev, University Children’s Hospital, Medical Faculty Skopje, ul. Majka Tereza 17, 1000 Skopje, Republic of Macedonia. Mobile: +389-70-279-742. E-mail: gucevz@ gmail.com
page: 81

CASE PRESENTATION

We were consulted on a 11-year-old white Caucasian male for the findings of hypoplasia of the right kidney and coronal moderate hypospadias, after surgical correction of the urethra anomaly. He was the first child of a non consanguineous couple. His parents and younger sister were healthy. His intelligence was normal (IQ 92) and he had no other anomalies. The behavior, growth and development were all normal. His testes volume was >4 mL and the penis length was 5 cm. Abdominal ultrasound and magnetic resonance imaging (MRI) did not show internal female genitalia, and confirmed right kidney hypoplasia (Figure 1, Table 1). The left kidney size was 80 × 32 mm, while the right kidney size was 57 × 23 mm. The patient was investigated as part of a study approved by the institutional review board at our International Centre for genetic Engineering and Biotechnology in Skopje (Republic of Macedonia) and at the Department of Nephrology, Columbia University, New York, NY, USA. This patient was already reported as part of our prior study on copy number variations (CNVs) in kidney malformations [28]. An additional 23 patients were selected to perform targeted Sanger resequencing of SOX3. We selected 23 males affected by urinary tract developmental defects (10 renal hypodysplasia; three vescicoureteral reflux; two posterior urethral valve; four obstructive uropathy; one bladder anomaly, one ectopic, one accessory kidney and one horseshoe kidney) and associated DSD (11 hypospadias, nine cryptorchidism, one epispadia and one congenital hidrocele). Endocrine Analysis. Plasma concentrations of steroid hormones, comprising mineralocorticoids, glucocorticoids and androgens, were determined using UPLC Quattro Premier/Xe system (Waters, Milford, MA, USA) as previously described [29-31]. In brief, aliquots of plasma samples, calibrator and controls with a volume of 0.1 mL were combined with an internal standard mixture to monitor recovery. All samples were extracted using Oasis MAX SPE system Plates (Waters). Genetic Analyses. After receiving informed consent, collected according to the Ethics Board of the Macedonian Academy of Sciences and Arts (Skopje, Republic of Macedonia), genomic DNA was obtained from peripheral blood samples using standard methods. Genome wide genotyping was conducted on patient MCD_13 using Illumina 610-Quad chip (Illumina Inc., San Diego, CA, USA) [32]. Copy number variation analysis was performed as previously described and data were compared to 21,575 multiethnic controls [28,33-35]. Briefly, genotype calls and quality-control analyses were conducted using GenomeStudio v.2010.3 (Illumina Inc.) and PLINK software [36]. Standardized genotyping methods implemented by the PennCNV program [37] were used for genome-wide CNV calls. The human reference genome hg18 (NCBI build 36.1, March 2006) was the reference assembly used to map the CNVs. The annotation of the CNVs was then performed using the UCSC RefGene and RefExon (CNVision program) [38]. Specific primers were designed to direct polymerase chain reaction (PCR) at the exon and exon-intron boundaries of SOX3, and bidirectional Sanger sequencing was performed by BigDye® terminator (Nimagen BV, Nijmegen, The Netherlands) reaction followed by a run on an automatic capillary DNA sequencer. Sequence and alignment was conducted using Sequencer 5.4 software (Gene Codes Corp., Ann Arbor, MI, USA). An adreno corticotropic hormone (ACTH) test showed normal basal and stimulated 17OH-progesterone excluding a form of 46,XX DSD due to 21-hydroxylase deficiency. The 11-deoxycorticosterone (DOC) and 11-deoxycortisol were normal at both baseline and after ACTH stimulation, excluding 11-hydroxylase deficiency. Cortisol levels were in the mid-normal range at baseline and responded to stimulation, excluding primary adrenal insufficiency. The hCG (human chorionic gonadotrpin) test found testosterone in the low-normal range for male sex and age at baseline. After stimulation, it raised up to 146.0 ng/mL indicating the presence of functional Leydig cells targeted by hCG. The stimulated ratio A:T was below 1, not supporting 17-β-hydroxysteroid dehydrogenase type 3 deficiency. The stimulated ratio T:DHT was 5.6, not supporting 5 α-reductase insufficiency. Microarray-based copy number analysis was previously performed in this patient as part of a larger study on congenital kidney defects [28]. In our 11-year-old male patient affected by renal dystrophy (RHD) and DSD (MCD_13), the microarray analysis showed an unique duplication of about 550 kb of the chromosome region Xq27, involving multiple genes and transcripts: SOX3, RP1-177G6 and CDR1, the non coding RNA LINC00632, and the miRNA MIR320D2 [28] (Figure 2). None of the genes within the duplication locus has previously been reported to be in association with kidney and urinary tract phenotypes [39,40]. The chromosomal microarray analysis confirmed the 46,XX female karyotype. Parental DNA material was not available to test segregation; therefore, we could not verify if the Xq27 duplication was a de novo or inherited genomic imbalance. No causal mutations were detected in the 23 male patients selected for targeted resequencing indicating that SOX3 coding variants might be a very rare cause of urinary tract malformations associated with DSDs.

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