Mona Mekkawy

Analysis of the gene coding for steroidogenic factor 1 (SF1, NR5A1) in a cohort of 50 Egyptian patients with 46,XY disorders of sex development

OBJECTIVE Steroidogenic factor 1 (SF1, NR5A1) is a key transcriptional regulator of genes involved in the hypothalamic-pituitary-gonadal axis. Recently, SF1 mutations were found to be a frequent cause of 46,XY disorders of sex development (DSD) in humans. We investigate the frequency of NR5A1 mutations in an Egyptian cohort of XY DSD. DESIGN Clinical assessment, endocrine evaluation and genetic analysis of 50 Egyptian XY DSD patients (without adrenal insufficiency) with a wide phenotypic spectrum. METHODS Molecular analysis of NR5A1 gene by direct sequencing followed by in vitro functional analysis of the two novel missense mutations detected. RESULTS Three novel heterozygous mutations of the coding region in patients with hypospadias were detected. p.Glu121AlafsX25 results in severely truncated protein, p.Arg62Cys lies in DNA-binding zinc finger, whereas p.Ala154Thr lies in the hinge region of SF1 protein. Transactivation assays using reporter constructs carrying promoters of anti-Müllerian hormone (AMH), CYP11A1 and TESCO core enhancer of Sox9 showed that p.Ala154Thr and p.Arg62Cys mutations result in aberrant biological activity of NR5A1. A total of 17 patients (34%) harboured the p.Gly146Ala polymorphism. CONCLUSION We identified two novel NR5A1 mutations showing impaired function in 23 Egyptian XY DSD patients with hypospadias (8.5%). This is the first study searching for NR5A1 mutations in oriental patients from the Middle East and Arab region with XY DSD and no adrenal insufficiency, revealing a frequency similar to that in European patients (6.5-15%). We recommend screening of NR5A1 in patients with hypospadias and gonadal dysgenesis. Yearly follow-ups of gonadal function and early cryoconservation of sperms should be performed in XY DSD patients with NR5A1 mutations given the risk of future fertility problems due to early gonadal failure.

Identification of NR5A1 Mutations and Possible Digenic Inheritance in 46,XY Gonadal Dysgenesis.

The phenotypic spectrum of patients carrying NR5A1 mutations ranges from 46,XY gonadal dysgenesis to male infertility. Phenotypic variability could be due to digenic or oligogenic inheritance of pathogenic variants in other testis-determining genes. Here, exome sequencing identified 2 pathogenic de novo NR5A1 mutations in 2 patients with 46,XY gonadal dysgenesis, p.Q206Tfs*20 and p.Arg313Cys. The latter patient also carried a missense mutation in MAP3K1. Our data extend the number of NR5A1 gene mutations associated with gonadal dysgenesis. The combination of an NR5A1 mutation with a MAP3K1 variant may explain the phenotypic variability associated with NR5A1 mutations.

Isodicentric Y chromosomes in Egyptian patients with disorders of sex development (DSD).

Isodicentric chromosome formation is the most common structural abnormality of the Y chromosome. As dicentrics are mitotically unstable, they are subsequently lost during cell division resulting in mosaicism with a 45,X cell line. We report on six patients with variable signs of disorders of sex development (DSD) including ambiguous genitalia, short stature, primary amenorrhea, and male infertility with azoospermia. Cytogenetic studies showed the presence of a sex chromosome marker in all patients; associated with a 45,X cell line in five of them. Fluorescence in situ hybridization (FISH) technique was used to determine the structure and the breakage sites of the markers that all proved to be isodicentric Y chromosomes. Three patients, were found to have similar breakpoints: idic Y(qter→ p11.32:: p11.32→ qter), two of them presented with ambiguous genitalia and were found to have ovotesticular DSD, while the third presented with short stature and hypomelanosis of Ito. One female patient presenting with primary amenorrhea, Turner manifestations and ambiguous genitalia revealed the breakpoint: idic Y (pter→q11.1::q11.1→pter). The same breakpoint was detected in a male with azoospermia but in non‐mosaic form. An infant with ambiguous genitalia and mixed gonadal dysgenesis (MGD) had the breakpoint at Yq11.2: idic Y(pter→q11.2::q11.2→pter). SRY signals were detected in all patients. Sequencing of the SRY gene was carried out for three patients with normal results. This study emphasizes the importance of FISH analysis in the diagnosis of patients with DSD as well as the establishment of the relationship between phenotype and karyotype.

A novel nonsense mutation in exon 1 of HSD17B3 gene in an Egyptian 46,XY adult female presenting with primary amenorrhea.

17-β-Hydroxysteroid dehydrogenase type 3 deficiency is a rare autosomal recessive cause of 46,XY disorder of sex development. Worldwide, about 30 mutations in the hydroxysteroid (17-beta) dehydrogenase 3 (HSD17B3) gene have been reported, involving all exons except exon 1. Herein, we investigated an Egyptian female with 46,XY karyotype and low testosterone/Δ4-androstenedione ratio. Genomic DNA was extracted from blood samples, and then, direct DNA sequencing of HSD17B3 gene was performed. The patient had a homozygous mutation c.198G>A in exon 1 resulting in a stop codon (p.W50X). The study presents the first mutation to be reported in exon 1 of the HSD17B3 gene.

Genotype/phenotype correlation in a female patient with 21q22.3 and 12p13.33 duplications

Many chromosomal rearrangements that lead to copy‐number gains or losses have been shown to cause distinctive and recognizable clinical phenotypes. Conventional cytogenetic analysis can detect many, but not all, rearrangements depending on its power of resolution. The wide use of whole‐genome array‐based comparative genomic hybridization (array‐CGH) techniques has allowed the detection of novel syndromes and to establish genotype–phenotype correlations by delineating at high resolution the regions involved in specific chromosomal aberrations. We report on a two and half‐year‐old female patient with intellectual disability and distinctive phenotypic features resulting from a de novo duplication of about 0.3 Mb in 21q22.3 associated with duplication of about 0.3 Mb in 12p13.33. The patients chromosomal abnormalities were identified at the cytogenetic molecular level, using SNP array analysis, while GTG banding technique revealed a normal karyotype. Clinical findings of the patient were compared with Down syndrome and 12p duplication syndrome. This study suggests that an area of contiguous genes on the distal part of chromosome 21 (21q22.3) contribute to the Down syndrome phenotype and indicates that genes in the distal region of 12p (12p13.33) account for many facial characteristics and hypotonia of trisomy 12p syndrome.

Unique karyotype: mos 46,X,dic(X;Y)(p22.33;p11.32)/ 45,X/45,dic(X;Y)(p22.33;p11.32) in an Egyptian patient with Ovotesticular disorder of sexual development.

Ovotesticular disorder of sexual development (OT-DSD) is an unusual form of DSD, characterized by the coexistence of testicular and ovarian tissue in the same individual. In this report, we present clinical, cytogenetic and molecular data of an Egyptian patient with ambiguous genitalia and OT-DSD, who had a unique karyotype comprising 3 different cell lines: mos 46,X,dic(X;Y)(p22.33;p11.32)/45,X/ 45,dic(X;Y)(p22.33;p11.32). This mosaic karyotype probably represents 2 different events: abnormal recombination between the X and Y chromosomes during paternal meiosis and postzygotic abnormality in mitotic segregation of the dic(X;Y) chromosome, resulting in a mosaic karyotype. The presence of the sex-determining region Y (SRY) gene explains the development of testicular tissue. On the other hand, other factors, including the presence of a 45,X cell line, partial SRY deletion, X inactivation pattern, and position effect, could be contributed to genital ambiguity. Explanation of the patients phenotype in relation to the genotype is discussed with a literature review. We conclude that FISH analysis with X- and Y-specific probes and molecular analysis of the SRY gene are highly recommended and allow accurate diagnosis for optimal management of cases with ambiguous genitalia.

Mutational Profile of 10 Afflicted Egyptian Families with 17-β-HSD-3 Deficiency

This study aimed at the detection of HSD17B3 gene mutations in Egyptian patients with suspected diagnosis of 46,XY DSD due to 17-β-HSD-3 deficiency and at evaluation of phenotype/genotype relationship of these mutations. The study was conducted on 11 patients of 10 families which were provisionally diagnosed to have 17-β-HSD-3 enzyme deficiency. Karyotyping, hormonal evaluation of testosterone, Δ4-androstenedione, and dihydrotestosterone, and sequencing analysis of the 11 exons of the HSD17B3 gene were done. Mutations in HSD17B3 were detected in exons 2, 7, 8, 10, and 11, and 6 novel mutations were determined in exons 1, 2, 7, and 8. Two patients showed compound heterozygous mutations, while 8 families had probands with homozygous mutations. The current study shows that 17-β-HSD-3 deficiency is not an uncommon disorder among Egyptian DSD cases. It was evidenced that the mutational profile of the disease is rather heterogeneous, relatively different from those reported in other populations, and has a high degree of novel genetic defects.

Clinical and Cytogenetic Study of Egyptian Patients with Sex Chromosome Disorders of Sex Development

Disorders of sex development (DSD) are conditions with an abnormal development of chromosomal, gonadal, or anatomical sex. Sex chromosome DSD involve conditions associated with either numerical or structural abnormalities of the sex chromosomes. This study included patients comprising a wide spectrum of presenting features suggestive of DSD and aimed at studying the frequency of sex chromosome abnormalities among 108 Egyptian DSD patients who presented to the Clinical Genetics and Endocrinology Clinics, National Research Centre (NRC) over the 2-year period of 2013 and 2014. The age of the studied patients ranged from 2 months to 39 years. The patients exhibited various presentations, including ambiguous genitalia, undescended testis, hypogonadism, short stature with Turner manifestations, primary or secondary amenorrhea, primary infertility, edema of the dorsum of the hands and feet, and dysmorphic features. The patients were subjected to detailed clinical examination, pubertal staging, and cytogenetic analysis. Our study reported a wide karyotypic diversity and a high frequency of sex chromosome DSD, reaching 44.44% (48/108). In conclusion, we showed a high incidence of sex chromosome DSD among Egyptian DSD patients with wide karyotype/phenotype diversity. The most frequent sex chromosome DSD detected among patients of the present study was Turner syndrome and variants (52.08%; 25/48) followed by Klinefelter syndrome and variants (43.75%; 21/48). Further long term studies are necessary for accurate detection of frequencies of different types of sex chromosomal anomalies and associated phenotypes.

An infertile azoospermic male with 45,X karyotype and a unique complex (Y;14); (Y;22) translocation: cytogenetic and molecular characterization

45,X testicular disorder of sex development (DSD) is a very rare disorder. It usually results from Y/ autosomal translocations or insertion [1–3]. The frequency of Y/ autosome translocations in the general population is generally low occurring in about 1 in 2000 [4]. Y/ autosome (Y;A) translocations are usually balanced and segregate in families with minimal effects on the phenotype [4–6]. On the other hand, unbalanced Y;A reciprocal translocations commonly present with infertility and azoospermia [7–9]. Some patients may present with other presentations related to abnormal sex chromosme dosage as ambiguous genitalia, short stature, and some of the Turner syndrome features . Others may have congeni ta l malformations, and mental subnormality depending on the genes involved in the autosome [2, 3, 10, 11]. Almost all autosomes had been reported, but acrocentrics are the most commonly involved [2, 12–16]. The causative factor of male infertility and spermatogenic failure in Y;A translocation is the disruption of AZF loci by the translocation breakpoint or by a positional effect [17–19]. Another explanation is the occurrence of spermatogenic arrest due to abnormal meiotic pairing with defective X/Y body (sex vesicle) formation and inactivation of autosomal segment and consequently spermatocyte degeneration [13, 20–23].

Unbalanced 14;X Translocation and Pattern of X Inactivation in a Female Patient with Multiple Congenital Anomalies

We report on a female patient who was first evaluated at the age of 6 years with developmental delay, dysmorphic facial features, seizures, and autistic behavior. A brain CT showed complete agenesis of the corpus callosum, and EEG recorded bilateral epileptogenic foci. Karyotype analysis revealed 45,X,psu dic(14;X)(p11;p22). FISH using 14q and Xp subtelomeric probes, combined with a SHOX gene-specific probe, and centromere X and XIST gene analysis revealed ish psu dic(14;X)(D14S1420+; DXYS129-, SHOX-, DXZ1+, XIST+). Array CGH detected a 2-Mb loss at Xp22.33 and a 4.6-Mb gain at Xp22.2p22.12. The deletion contains 34 genes, of which CSF2RA and SHOX are OMIM morbid genes. The duplication also contains some OMIM morbid genes, of which CDKL5, NH5, RPS6KA3, and AP1S2 are the most important. The late replicating chromatin technique was used to detect the pattern of X inactivation in the normal X and in the translocated chromosome. The translocated X was found to be inactive in 70% of the studied blood lymphocytes with patchy extension of inactivation to chromosome 14. In conclusion, the phenotype of the patient may be partially affected by the haploinsufficiency of the genes that are known to escape X inactivation and that lie within the deleted region and by other deleted or duplicated genes on the abnormal X chromosome due to an alternative pattern of X inactivation. The phenotype of the patient was significantly aggravated and complicated by the functional monosomy of some genes on chromosome 14 due to partial spreading of inactivation and silencing of those genes. This case report indicates the importance of structural and functional studies and emphasizes the clinical importance of the follow-up of abnormal microarrays.