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 page: 69 download article in pdf format
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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 Adam’s 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 Adam’s
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 Adam’s 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 PRISM® 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 patients’
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 5’ untranslated region (5’UTR)
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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