Congenital leptin and leptin receptor deficiencies in nine new families: identification of six novel variants and review of literature.

Early childhood obesity is a real public health problem worldwide. Identifying the etiologies, especially treatable and preventable causes, can direct health professionals toward proper management. Measurement of serum leptin levels is helpful in the diagnosis of congenital leptin and leptin receptor deficiencies which are considered important rare causes of early childhood obesity. The main aim of this study was to investigate the frequency of LEP, LEPR, and MC4R gene variants among a cohort of Egyptian patients with severe early onset obesity. The current cross-sectional study included 30 children who developed obesity during the first year of life with BMI > 2SD (for age and sex). The studied patients were subjected to full medical history taking, anthropometric measurements, serum leptin and insulin assays, and genetic testing of LEP, LEPR and MC4R. Disease causing variants in LEP and LEPR were identified in 10/30 patients with a detection rate of 30%. Eight different homozygous variants (two pathogenic, three likely pathogenic, and three variants of uncertain significant) were identified in the two genes, including six previously unreported LEPR variants. Of them, a new frameshift variant in LEPR gene (c.1045delT, p.S349Lfs*22) was recurrent in two unrelated families and seems to have a founder effect in our population. In conclusion, we reported ten new patients with leptin and leptin receptor deficiencies and identified six novel LEPR variants expanding the mutational spectrum of this rare disorder. Furthermore, the diagnosis of these patients helped us in genetic counseling and patients' managements specially with the availability of drugs for LEP and LEPR deficiencies.

Testis formation in XX individuals resulting from novel pathogenic variants in Wilms' tumor 1 (WT1) gene.

Sex determination in mammals is governed by antagonistic interactions of two genetic pathways, imbalance in which may lead to disorders/differences of sex development (DSD) in human. Among 46,XX individuals with testicular DSD (TDSD) or ovotesticular DSD (OTDSD), testicular tissue is present in the gonad. Although the testis-determining gene SRY is present in many cases, the etiology is unknown in most SRY-negative patients. We performed exome sequencing on 78 individuals with 46,XX TDSD/OTDSD of unknown genetic etiology and identified seven (8.97%) with heterozygous variants affecting the fourth zinc finger (ZF4) of Wilms' tumor 1 (WT1) (p.Ser478Thrfs*17, p.Pro481Leufs*15, p.Lys491Glu, p.Arg495Gln [x3], p.Arg495Gly). The variants were de novo in six families (P = 4.4 × 10-6), and the incidence of WT1 variants in 46,XX DSD is enriched compared to control populations (P < 1.8 × 10-4). The introduction of ZF4 mutants into a human granulosa cell line resulted in up-regulation of endogenous Sertoli cell transcripts and Wt1Arg495Gly/Arg495Gly XX mice display masculinization of the fetal gonads. The phenotype could be explained by the ability of the mutated proteins to physically interact with and sequester a key pro-ovary factor ß-CATENIN, which may lead to up-regulation of testis-specific pathway. Our data show that unlike previous association of WT1 and 46,XY DSD, ZF4 variants of WT1 are a relatively common cause of 46,XX TDSD/OTDSD. This expands the spectrum of phenotypes associated with WT1 variants and shows that the WT1 protein affecting ZF4 can function as a protestis factor in an XX chromosomal context.

Advances in genomic diagnosis of a large cohort of Egyptian patients with disorders of sex development.

Disorders/differences of sex development (DSD) comprise a group of congenital disorders that affect the genitourinary tract and usually involve the endocrine and reproductive system. The aim of this work was to identify genetic variants responsible for disorders of human urogenital development in a cohort of Egyptian patients. This three-year study included 225 patients with various DSD forms, referred to the genetic DSD and endocrinology clinic, National Research Centre, Egypt. The patients underwent thorough clinical examination, hormonal and imaging studies, detailed cytogenetic and fluorescence in situ hybridization analysis, and molecular sequencing of genes known to commonly cause DSD including AR, SRD5A2, 17BHSD3, NR5A1, SRY, and WT1. Whole exome sequencing (WES) was carried out for 18 selected patients. The study revealed a high rate of sex chromosomal DSD (33%) with a wide array of cytogenetic abnormalities. Sanger sequencing identified pathogenic variants in 33.7% of 46,XY patients, while the detection rate of WES reached 66.7%. Our patients showed a different mutational profile compared with that reported in other populations with a predominance of heritable DSD causes. WES identified rare and novel pathogenic variants in NR5A1, WT1, HHAT, CYP19A1, AMH, AMHR2, and FANCA and in the X-linked genes ARX and KDM6A. In addition, digenic inheritance was observed in two of our patients and was suggested to be a cause of the phenotypic variability observed in DSD.

Mutations involving the SRY-related gene SOX8 are associated with a spectrum of human reproductive anomalies.

SOX8 is an HMG-box transcription factor closely related to SRY and SOX9. Deletion of the gene encoding Sox8 in mice causes reproductive dysfunction but the role of SOX8 in humans is unknown. Here, we show that SOX8 is expressed in the somatic cells of the early developing gonad in the human and influences human sex determination. We identified two individuals with 46, XY disorders/differences in sex development (DSD) and chromosomal rearrangements encompassing the SOX8 locus and a third individual with 46, XY DSD and a missense mutation in the HMG-box of SOX8. In vitro functional assays indicate that this mutation alters the biological activity of the protein. As an emerging body of evidence suggests that DSDs and infertility can have common etiologies, we also analysed SOX8 in a cohort of infertile men (n = 274) and two independent cohorts of women with primary ovarian insufficiency (POI; n = 153 and n = 104). SOX8 mutations were found at increased frequency in oligozoospermic men (3.5%; P < 0.05) and POI (5.06%; P = 4.5 × 10-5) as compared with fertile/normospermic control populations (0.74%). The mutant proteins identified altered SOX8 biological activity as compared with the wild-type protein. These data demonstrate that SOX8 plays an important role in human reproduction and SOX8 mutations contribute to a spectrum of phenotypes including 46, XY DSD, male infertility and 46, XX POI.

Pathogenic variants in the DEAH-box RNA helicase DHX37 are a frequent cause of 46,XY gonadal dysgenesis and 46,XY testicular regression syndrome.

PURPOSE: XY individuals with disorders/differences of sex development (DSD) are characterized by reduced androgenization caused, in some children, by gonadal dysgenesis or testis regression during fetal development. The genetic etiology for most patients with 46,XY gonadal dysgenesis and for all patients with testicular regression syndrome (TRS) is unknown. METHODS: We performed exome and/or Sanger sequencing in 145 individuals with 46,XY DSD of unknown etiology including gonadal dysgenesis and TRS. RESULTS: Thirteen children carried heterozygous missense pathogenic variants involving the RNA helicase DHX37, which is essential for ribosome biogenesis. Enrichment of rare/novel DHX37 missense variants in 46,XY DSD is highly significant compared with controls (P value = 5.8 × 10-10). Five variants are de novo (P value = 1.5 × 10-5). Twelve variants are clustered in two highly conserved functional domains and were specifically associated with gonadal dysgenesis and TRS. Consistent with a role in early testis development, DHX37 is expressed specifically in somatic cells of the developing human and mouse testis. CONCLUSION: DHX37 pathogenic variants are a new cause of an autosomal dominant form of 46,XY DSD, including gonadal dysgenesis and TRS, showing that these conditions are part of a clinical spectrum. This raises the possibility that some forms of DSD may be a ribosomopathy.

Biallelic novel missense HHAT variant causes syndromic microcephaly and cerebellar-vermis hypoplasia.

We report two siblings with microcephaly, early infantile onset seizures, and cerebellar vermis hypoplasia, in whom whole exome sequencing revealed a novel homozygous missense (c.770T>C, p.[Leu257Pro]) variant in the hedgehog acyl-transferase gene (HHAT), encoding an enzyme required for the attachment of palmitoyl residues that are critical for multimerization and long and short range hedgehog signaling. There is a report of one family with Nivelon-Nivelon-Mabille syndrome in which HHAT was proposed as the likely candidate gene. The phenotypic overlap with the family we report herein provides further evidence implicating HHAT in cerebellar development and the pathogenesis of this rare spectrum.

GAPO syndrome in seven new patients: Identification of five novel ANTXR1 mutations including the first large intragenic deletion.

GAPO syndrome is a very rare disorder characterized by growth retardation, alopecia, pseudoanodontia and progressive optic atrophy. It is caused by biallelic mutations in the ANTXR1 gene. Herein, we describe the clinical and molecular findings of seven new patients with GAPO syndrome. Our patients presented with the characteristic clinical features of the syndrome except for one patient who did not display total alopecia till the age of two years. Strikingly, optic atrophy and glaucoma were observed in all patients and one patient showed keratopathy in addition. Moreover, craniosynstosis was an unusual associated finding in one patient. Mutational analysis of ANTXR1 gene identified five novel homozygous mutations including two frameshift, two splice site and a large intragenic deletion of exon 3. Our results reinforce the clinical characteristics of the syndrome, expand the mutational spectrum and provide more insights into the role of the ANTXR1 protein in the regulation of extracellular matrix.

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.

Identification of a novel homozygous ALX4 mutation in two unrelated patients with frontonasal dysplasia type-2.

We report two unrelated boys with frontonasal dysplasias type-2 (FND-2) who shared an identical novel homozygous ALX4 mutation c.291delG (p.Q98Sfs*83). Both patients presented with a large skull defect but one had bilateral parietal meningocele-like cysts that lie along with the bony defect and increased in size with age. Scalp alopecia, hypertelorism, and clefted alae nasi were also detected in both of them. Furthermore, impalpable gonads were noted, being unilateral in one and bilateral in the other. Neuroimaging showed small dysplastic occipital lobes with dysgyria and midline subarachnoid cyst. Additional dysplastic corpus callosum and small cerebellar vermis were observed in one patient. Parietal foramina were noted in the parents of one patient. Our findings highlight the dosage effect of ALX4 and underscore the challenges of prenatal genetic counseling. Further, the indirect role of ALX4 in the development of the occipital lobe and posterior fossa is discussed.

Biochemical Analysis of Four Missense Mutations in the HSD17B3 Gene Associated With 46,XY Disorders of Sex Development in Egyptian Patients.

BACKGROUND: Mutations in the HSD17B3 gene are associated with a 46,XY disorder of sexual development (46,XY DSD) as a result of low testosterone production during embryogenesis. AIM: To elucidate the molecular basis of the disorder by chemically analyzing four missense mutations in HSD17B3 (T54A, M164T, L194P, G289S) from Egyptian patients with 46,XY DSD. METHODS: Expression plasmids for wild-type 17ß-hydroxysteroid hydrogenase type 3 (17ß-HSD3) and mutant enzymes generated by site-directed mutagenesis were transiently transfected into human HEK-293 cells. Protein expression was verified by western blotting and activity was determined by measuring the conversion of radiolabeled Δ4-androstene-3,17-dione to testosterone. Application of a homology model provided an explanation for the observed effects of the mutations. OUTCOMES: Testosterone formation by wild-type and mutant 17ß-HSD3 enzymes was compared. RESULTS: Mutations T54A and L194P, despite normal protein expression, completely abolished 17ß-HSD3 activity, explaining their severe 46,XY DSD phenotype. Mutant M164T could still produce testosterone, albeit with significantly lower activity compared with wild-type 17ß-HSD3, resulting in ambiguous genitalia or a microphallus at birth. The substitution G289S represented a polymorphism exhibiting comparable activity to wild-type 17ß-HSD3. Sequencing of the SRD5A2 gene in three siblings bearing the HSD17B3 G289S polymorphism disclosed the homozygous Y91H mutation in the former gene, thus explaining the 46,XY DSD presentations. Molecular modeling analyses supported the biochemical observations and predicted a disruption of cofactor binding by mutations T54A and M164T and of substrate binding by L196P, resulting in the loss of enzyme activity. In contrast, the G289S substitution was predicted to disturb neither the three-dimensional structure nor enzyme activity. CLINICAL TRANSLATION: Biochemical analysis of mutant 17ß-HSD3 enzymes is necessary to understand genotype-phenotype relationships. STRENGTHS AND LIMITATIONS: Biochemical analysis combined with molecular modeling provides insight into disease mechanism. However, the stability of mutant proteins in vivo cannot be predicted by this approach. CONCLUSION: The 17ß-HSD3 G289S substitution, previously reported in other patients with 46,XY DSD, is a polymorphism that does not cause the disorder; thus, further sequence analysis was required and disclosed a mutation in SRD5A2, explaining the cause of 46,XY DSD in these patients. Engeli RT, Tsachaki M, Hassan HA, et al. Biochemical Analysis of Four Missense Mutations in the HSD17B3 Gene Associated With 46,XY Disorders of Sex Development in Egyptian Patients. J Sex Med 2017;14:1165-1174.

Clinical Management of Gender in Egypt: Intersexuality and Transsexualism.

This article reviews the literature on intersexuality (disorders of sex development [DSD]) and transsexuality in Egypt. Egypt's resources for the diagnosis and treatment of rare conditions, such as DSD and transsexualism, are quite limited. The birth of a child with a DSD is likely to be stressful, especially in regard to decisions on gender assignment, and genital abnormalities are associated with stigma and shame. Gender assignment may be biased toward the male gender, because female infertility precludes marriage and female gender adversely affects employment prospects and inheritance. Later gender change in either direction may also carry stigma. Gender reassignment surgery for transsexuals without somatic intersexuality was legalized in Egypt in 2005, but requires permission by a national Sex Identification and Determination Committee.

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 patient's 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.

Clinical and molecular characterization of seven Egyptian families with autosomal recessive robinow syndrome: Identification of four novel ROR2 gene mutations.

Robinow syndrome (RS) is a rare genetic disorder characterized by limb shortening, genital hypoplasia, and craniofacial/orodental abnormalities. The syndrome follows both autosomal dominant and recessive patterns of inheritance with similar phenotypic presentation and overlapping features. Autosomal recessive Robinow syndrome (ARRS) is caused by mutations in the ROR2 gene. Here, we present the clinical, radiological and molecular findings of 11 Egyptian patients from 7 unrelated consanguineous families with clinical features of ARRS. Mutation analyses of ROR2 gene identified five pathogenic mutations distributed all over the gene. The identified mutations included four novel (G326A, D166H, S677F, and R528Q) and one previously reported (Y192D). Our results extend the number of ROR2 mutations identified so far, suggest a founder effect in the Egyptian population, and emphasize the important role of genetic testing in proper counseling and patients' management.

Expanding the phenotypic spectrum of LHCGR signal peptide insertion variant: novel clinical and allelic findings causing Leydig cell hypoplasia type II.

PURPOSE: Leydig cell hypoplasia (LCH) type II is a rare disease with only a few cases reported. Patients presented with hypospadias, micropenis, undescended testes, or infertility. In this study, we report a new patient with compound heterozygous variants in the LHCGR gene and LCH type II phenotype. METHODS: Whole exome sequencing (WES) was performed followed by Sanger sequencing to confirm the detected variants in the patient and his parents. RESULTS: A novel missense variant (p.Phe444Cys) was identified in a highly conserved site and is verified to be in trans with the signal peptide's 33-bases insertion variant. CONCLUSION: Our research provides a more comprehensive clinical and genetic spectrum of Leydig cell hypoplasia type II. It highlighted the importance of WES in the diagnosis of this uncommon genetic disorder as well as the expansion of the genotype of LCH type II.

In vitro cellular reprogramming to model gonad development and its disorders.

During embryonic development, mutually antagonistic signaling cascades determine gonadal fate toward a testicular or ovarian identity. Errors in this process result in disorders of sex development (DSDs), characterized by discordance between chromosomal, gonadal, and anatomical sex. The absence of an appropriate, accessible in vitro system is a major obstacle in understanding mechanisms of sex-determination/DSDs. Here, we describe protocols for differentiation of mouse and human pluripotent cells toward gonadal progenitors. Transcriptomic analysis reveals that the in vitro-derived murine gonadal cells are equivalent to embryonic day 11.5 in vivo progenitors. Using similar conditions, Sertoli-like cells derived from 46,XY human induced pluripotent stem cells (hiPSCs) exhibit sustained expression of testis-specific genes, secrete anti-Müllerian hormone, migrate, and form tubular structures. Cells derived from 46,XY DSD female hiPSCs, carrying an NR5A1 variant, show aberrant gene expression and absence of tubule formation. CRISPR-Cas9-mediated variant correction rescued the phenotype. This is a robust tool to understand mechanisms of sex determination and model DSDs.

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 Homozygous Missense Variant in Hedgehog Acyltransferase (HHAT) Gene Associated with 46,XY Gonadal Dysgenesis.

INTRODUCTION: Disorders of gonadal development represent a clinically and genetically heterogeneous group of DSD, and the etiology in many cases remains unknown, indicating that our knowledge of factors controlling sex determination is still limited. METHODS: We describe a 46,XY DSD patient from Egypt. The patient was reared as female, born to consanguineous parents, and was referred to us at the age of 5 years because of ambiguous genitalia. On examination, the girl was microcephalic (head circumference -3 SD), but her height and weight were normal for her age and sex. RESULTS: Exome sequencing identified a homozygous variant in the hedgehog acyltransferase (HHAT) gene, which encodes an enzyme that is required for multimerization and signaling potency of the hedgehog secreted proteins. The variant is a novel homozygous missense change c.1329C>A (p.N443K), located within transmembrane domain 9, which segregated with the phenotype in the family. DISCUSSION/CONCLUSION: Our results expand the phenotypic spectrum associated with HHAT variants to include 46,XY gonadal dysgenesis and reinforce the role of exome sequencing in unraveling new genes that play a pivotal role in sexual development.

Tissue-specific mosaicism for tetrasomy 9p uncovered by array CGH.

We report on a patient with a mild clinical phenotype, including genital anomalies, with mosaic tetrasomy 9p. Karyotype analysis of peripheral blood lymphocytes detected a supernumerary isochromosome 9p present in every cell, with the initial result being reported as tetrasomy 9p in non-mosaic form. However,array Comparative Genomic Hybridization (aCGH) studies on DNA extracted from peripheral blood lymphocytes and saliva showed that the patient had tissue-specific mosaicism, with a lower level of abnormal cells in the saliva. These results correlate with the patient's clinical features as non-mosaic cases of tetrasomy 9p have a more severe, often lethal, clinical phenotype. If non-mosaic tetrasomy 9p is identified in a peripheral blood culture then examination of a different tissue type should be undertaken. Array CGH may be used as an alternative to karyotype analysis to estimate the level of mosaicism, and may eliminate the need for invasive skin biopsy as samples such as buccal smear and saliva can be used. Array CGH is able to detect mosaicism, establish the euchromatic content of supernumerary marker chromosomes, and identify imbalances elsewhere in the genome allowing more accurate counselling and prognosis for patients.

IGF1R, IGFALS, and IGFBP3 gene copy number variations in a group of non-syndromic Egyptian short children.

BACKGROUND: Insulin-like growth factor-1 (IGF-1) is required for normal intrauterine and postnatal growth, and this action is mediated through IGF1 receptor (IGF1R). IGF1R copy number variants (CNVs) can cause pre- and postnatal growth restriction, affecting an individual's height. In this study, we used multiplex ligation-dependent probe amplification (MLPA) to detect CNVs in IGF1R, IGFALS, and IGFBP3 genes in the diagnostic workup of short stature for 40 Egyptian children with short stature. RESULTS: We detected a heterozygous deletion of IGF1R (exons 4 through 21) in 1 out of the 40 studied children (2.5%). Meanwhile, we did not detect any CNVs in either IGFALS or IGFBP3. CONCLUSION: The diagnostic workup of short stature using MLPA for CNVs of IGF1R and other recognized height-related genes, such as SHOX and GH, in non-syndromic short stature children can be a fast and inexpensive diagnostic tool to recognize a subcategory of patients in which growth hormone treatment can be considered.