NOVEL GENOTYPE IN TWO SIBLINGS WITH 5-α- REDUCTASE 2 DEFICIENCY: DIFFERENT CLINICAL COURSE DUE TO THE TIME OF DIAGNOSIS
Kocova M1,*, Plaseska-Karanfilska D2, Noveski P2, Kuzmanovska M2
*Corresponding Author: Professor Dr. Mirjana Kocova, University Pediatric Clinic, Department of Endocrinology & Genetics, M. Tereza 17, Skopje 1000, Republic of North Macedonia. Tel: +389-70- 242-694. Fax: +389-317-6167. E-mail: mirjanakocova@yahoo.com
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Abstract

Steroid 5-α-reductase-2 (5-ARD) deficiency is a result of mutations of the SRD5A2 gene. It causes the disorder of sexual differentiation (DSD) in 46,XY individuals with a variable genital phenotype. We present two siblings with female external genitalia at birth and bilateral inguinal testes, raised as females. These are the first molecularly characterized patients from the Republic of North Macedonia (RN Macedonia) with a different clinical course due to the time of the diagnosis. Diagnosis of Patient 1 was based upon the detection of bilateral inguinal testes and testosterone/dihidrotestosterone ratio. Sex reversal was initiated by testes removal at the age of 20 months. Breast implantation and vaginoplasty were performed in adolescence and the girl is comfortable with the female sex. Her sibling, Patient 2, raised as a girl, was clinically assessed at 11.5 years due to the growth of phalus, deep voice and Adamís apple enlargement. No change of gender was accepted. Complex molecular analysis including multiplex quantitative fluorescent polymerase chain reaction (PCR) screening for sex chromosome aneuploidies and SRY presence, Sanger sequencing combined with multiplex ligation- dependent probe amplification (MLPA), microarraybased comparative genomic hybridization (aCGH), and real-time PCR analysis for detection of exon copy number changes confirmed a novel c.146C>A (p.Ala49Asp) point mutation in the first exon inherited from the mother, and complete deletion of the first exon and adjacent regions inherited from the father. Novel genotype causing 5-ARD is presented. Genetic analysis is useful for the diagnosis and timely gender assignment in patients with 5-ARD. However, final gender assignment is difficult and requires combined medical interventions. Background. Steroid 5-α-reductase type 2 (5-ARD) deficiency is a rare autosomal recessive genetic condition causing a 46,XY disorder of sexual differentiation (DSD) with unknown incidence [1]. Patients affected by 5-ARD cannot convert testosterone to dihydrotestosterone (DHT), which is a more potent androgen than testosterone and is responsible for male external genitalia phenotype formation. Furthermore, these patients present with under virilized external genitalia. The genital phenotype can vary greatly, from complete female external genitalia on the one hand to overt male pesudohermaphroditism on the other [1-3]. The testes are normal and usually located along the inguinal canal, but can rarely be located in the abdomen or in the scrotum. Internal genitalia are male except for the hypoplastic prostate. Occasionally, patients lack a separate vaginal opening, may have penile hypospadias or even a penile urethra. Due to the appearance of the external genitalia, these individuals are usually considered females at birth. Dihydrotestosterone deficiency is a result of recessive, mostly missense mutations of the steroid 5-α-reductase 2 (SRD5A2) gene located on band 2p23. The SRD5A2 gene consists of five exons and is expressed in the perineal skin and fibroblasts causing virilization in the XY fetus. The first exon of SRD5A2 is a common hotspot for mutations, however, about a dozen different mutations have been described, also involving other exons of the SRD5A2 gene [4,5]. Although the incidence of this disorder has not been established, it is reported with increased prevalence in parts of the world where consanguinity is more frequent in some kindreds (Turkey, Dominican Republic) [6-9]. The sex of rearing of affected individuals varies; both female and male sex have been chosen with a variable outcome. It is worth mentioning that during puberty some virilization appears spontaneously, even if incomplete. A different sex of rearing has been selected in affected individuals even within the same family. The success of functional sex formation and resultant quality of life is variable [2,10]. Here we present the first two molecularly characterized patients with 5-ARD from the Republic of North Macedonia (RN Macedonia), both reared as females, carrying a novel compound heterozygous genotype of SRD5A2, who encountered different problems during growth and development due to the different timing of diagnosis and therapeutic approach. Case Presentations. During this study we complied with the World Medical Association Declaration of Helsinki regarding ethical conduct of research involving human subjects. Informed consent for analyses and data presentation from the parents and Patient 1, and assent from Patient 2, was obtained. The procedures and data analysis were approved by the Institutional Ethics Committee on human research at the University Pediatric Clinic, Skopje, RN Macedonia. Patient 1 was the second child of young, unrelated parents, born at term with a birth weight of 3400 gr and length of 50 cm. Sexual ambiguity was noticed at birth: rugged large labia resembling scrota with 1 mL formation in the middle part of the inguinal channel bilaterally, a phallus of 10 mm in length, and an opening for putative perineal sinus urogenitalis. Blood counts and electrolytes were in the normal range. Ultrasound examination of the labia showed potential testicular tissue 8 × 10 mm (right) and 6 × 10 mm (left). Cytogenetic analysis confirmed a 46,XY karyotype, leading to a clinical diagnosis of disorder of DSD. Urethrocystography showed a male urethra opening in the common sinus urogenitalis. Ultrasound confirmed normal kidneys, ureters and bladder, as well as adrenal glands, and no female internal genitalia in the small pelvis. The testosterone/DHT ratio was 22 suggesting 5-ARD. The girl was followed-up during the first 2 years at 3-month intervals. The parents consulted endocrinologists and psychologists about the possibilities of sex of rearing. Their decision for female sex of rearing was definite. Orchidectomy together with clitoroplasty was performed at the age of 20 months. Hormone levels [17OH progesterone (17OHP), adrenocortiotropic hormone (ACTH), Cortisol, dehydroepiandrosterone sulfate (DHEAS), follicle-stimulating hormone (FSH), luteinizing hormone (LH), thyroxine (T4) and thyroid-stimutating hormone (TSH)], measured yearly afterwards were within the normal range. At the age of 13 years hormonal analyses showed very high LH (31.7 mIU/ml) and FSH (88.5 mIU/ mL) levels, and undetectable estrogen. Her bone age was 10 years, although her height was at the 50th percentile on the growth curve. Induction of puberty with estrogen patches and contraceptives afterwards did not improve the lack of breasts. Pubic and armpit hair was scarce. Breast implants and vaginoplasty were performed at the age of 20 and 22 years, respectively, completing the female sex phenotype [Figure 1(A) and 1(B)]. Patient 2 was born as the third child in the same family with a birth weight of 3000 gr and length of 50 cm. No specific consultations were sought by the parents despite the advice to bring her early due to the diagnosis in her sibling till the age of 7 years when she was brought for a check-up of the genitalia. She had a normal height and weight, but was pretty muscular. Her clitoris was enlarged to 18 mm, along with labial fusion. Other findings were normal. Her karyotype was 46,XY. The FSH, LH, DHEAS, testosterone and estrogen levels were normal. Testes were detected in the labial folds by ultrasound. Parents refused further consultations up to the age of 11.5 years when she was admitted due to a significant virilization: a deep voice, growth of Adamís apple, and growth of the phallus. Puberty stage was M1, P1-2, A1; testes were palpable in the inguinal channels bilaterally, phallus was 4 cm long accompanied by a perineal hypospadia and complete labial fusion [Figure 1(C) and 1(D)]. The same was documented on urethro-cystography. Pelvic ultrasound examination showed no Müllerian structures, and developed testicles (25 × 15 mm) in the upper inguinal channel. Gonadotropins were high, FSH reaching levels of 60 mIU/ mL and LH 22 mIU/mL. The testosterone/ DHT ratio was 20 and although molecular analysis was not possible at that time, the diagnosis of 5-ARD was made. The patient had been reared as a girl since birth and had a female sex identity. The decision of the adolescent and the parents did not change after numerous consultations. Testes removal was performed after written consent and institutional ethical approval together with clitoroplasty. The last checkup at 13.5 years showed decrease in the size of Adamís apple and slight voice improvement. Methods for the Genetic Analyses. Sanger sequencing of exons and exons/introns boundaries of the SRD5A2 gene was performed. Multiple ligation-dependent probe analysis (MLPA) was performed using P334-A3 Gonadal Development Disorder kit (MRC-Holland, Amsterdam, The Netherlands) for detection of the exon copy number changes in the SRD5A2 gene. For the MLPA data analysis, Coffalyser. Net software (https://support.mlpa. com/kb/coffalyser- net) was used. The Sanger sequencing and MLPA reaction products were analyzed on the ABI PRISMģ 3500 Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA). DNA from Patient 2 was analyzed with microarraybased comparative genomic hybridization (aCGH) analysis using Agilent Sure Print G3 Human CGH Microarray, 4 × 180K (Agilent Technologies, Santa Clara, CA, USA). DNA from Patient 2 and the father were studied with real time-polymerase chain reaction (real time-PCR) on 7500 Fast Real-Time PCR System (Thermo Fisher Scientific) using custom designed primers and HOT FIREPolEva Green qPCR Mix Plus (Solis BioDyne, Tartu, Estonia). The pathogenicity of the identified variants was assessed according to the guidelines of the American College of Medical Genetics and Genomics (ACMG) [11]. Results of the Molecular Analyses. Sanger DNA sequencing revealed a novel, likely pathogenic c.146 C>A (p.Ala49Asp) mutation in exon 1 of the SRD5A2 gene, present in a homozygous state in both affected siblings. Following criteria according to the ACMG guidelines were applicable for the classification of the c.146C>A as likely pathogenic: 1) extremely low frequency (0.00000419, 1 in 238,672 alleles) in publically available gnomAD Exomes v.2.1.1 population database (https:/macarthurlab.org/2017/ 02/27/the-genome-aggregation-database-gnomad/) [PM2]; 2) detected in trans with a pathogenic variant [PM3]; 3) cosegregation with disease in two affected family members [PP1]; 4) missense variant in a gene that has a low rate of benign missense variation and in which missense variants are a common mechanism of disease [PP2], and 5) the patientsí phenotypes are highly specific for a disease with a single genetic etiology (5-ARD deficiency due to pathogenic SRD5A2 mutations) [PP4]. Unexpectedly, the sequencing analysis of the parents showed that only the mother was a carrier of the c.146C>A mutation. The father carried a benign c.145G>A mutation in a homozygous state [benign interpretation in ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) and with benign classification according to ACMG criteria] [Figure 2(A)]. These findings could be explained either by non paternity or by the presence of a heterozygous deletion involving exon 1 of the SRD5A2 gene in the father and both affected siblings. Indeed, the MLPA analysis revealed a deletion involving two probes, in exon 1 and intron 1 of the SRD5A2 gene in the father and affected siblings [Figure 2(B)]. Thus, the affected siblings were compound heterozygotes for c.146C>A and c.(?_-71)_(281+1_282-1)del mutations. No pathogenic mutation was present in the healthy sibling, who has inherited the c.145G>A benign mutation from the father. With the aim of more precisely defining the deletion, we performed aCGH and real-time PCR analysis. Array CGH was not successful in determination of the deletion size as a result of insufficient number of probes in the deleted region, and requirement of minimum three deleted probes for copy number variation (CNV) calling (data not shown). Real-time PCR results revealed that five of the eight amplified fragments were amplified at half of the quantity as compared to normal control, indicating the presence of only one copy of targeted DNA sequence (heterozygous deletion) [Figure 3(A)]. Thus, we established that the entire exon 1 and 5í untranslated region (5íUTR) of the SRD5A2 gene were deleted within the region at least approximately 8 kb upstream and approximately 2 kb downstream of exon 1 [Figure 3(B)]. The presence of 64 kb of interspersed repeats and low complexity DNA sequences upstream of the SRD5A2 gene did not allow for a more precise determination of the deletion breakpoints [RepeatMasker (http://www.repeatmasker.org/) track in Figure 3(B)].



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