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.

Chromosome 9p terminal deletion in nine Egyptian patients and narrowing of the critical region for trigonocephaly.

BACKGROUND: This study aimed to delineate the clinical phenotype of patients with 9p deletions, pinpoint the chromosomal breakpoints, and identify the critical region for trigonocephaly, which is a frequent finding in 9p terminal deletion. METHODS: We investigated a cohort of nine patients with chromosome 9p terminal deletions who all displayed developmental delay, intellectual disability, hypotonia, and dysmorphic features. Of them, eight had trigonocephaly, seven had brain anomalies, seven had autistic manifestations, seven had fair hair, and six had a congenital heart defect (CHD). RESULTS: Karyotyping revealed 9p terminal deletion in all patients, and patients 8 and 9 had additional duplication of other chromosomal segments. We used six bacterial artificial chromosome (BAC) clones that could identify the breakpoints at 17-20 Mb from the 9p terminus. Array CGH identified the precise extent of the deletion in six patients; the deleted regions ranged from 16 to 18.8 Mb in four patients, patient 8 had an 11.58 Mb deletion and patient 9 had a 2.3 Mb deletion. CONCLUSION: The gene deletion in the 9p24 region was insufficient to cause ambiguous genitalia because six of the nine patients had normal genitalia. We suggest that the critical region for trigonocephaly lies between 11,575 and 11,587 Mb from the chromosome 9p terminus. To the best of our knowledge, this is the minimal critical region reported for trigonocephaly in 9p deletion syndrome, and it warrants further delineation.

Clinical and Cytogenomic Characterization of De Novo 11p14.3-p15.5 Duplication Associated with 18q23 Deletion in an Egyptian Female Infant.

Paternal microduplication of 11p14.3-p15.5 causes the clinical manifestations of Beckwith-Wiedemann syndrome (BWS), while microdeletion of 18q23-ter is clinically characterized by short stature, congenital malformations, and developmental delay. We describe a 15-month-old girl presenting with protruding tongue, dysmorphic facial features, moderate developmental delay, umbilical hernia, hypotonia, mild-to-moderate pulmonary hypertension, small patent ductus arteriosus, and mild ventricular septal hypertrophy. Brain magnetic resonance imaging showed mild atrophic changes. Chromosomal analysis revealed 46, XX, add(18)(q23). Fluorescence in situ hybridization using subtelomere 18q and whole chromosome painting 18 showed subtelomere deletion in 18q, and the add segment was not derived from chromosome 18. Microarray-based comparative genomic hybridization detected a 22 Mb duplication of chromosome 11p15.5p14.3 and a 3.7 Mb deletion of chromosome 18q23. The phenotype of the chromosomal rearrangements is probably resulted from a combination of dosage-sensitive genes. Our patient had clinical manifestations of both 18q deletion and BWS.

Clinical and genetic characterization of ten Egyptian patients with Wolf-Hirschhorn syndrome and review of literature.

BACKGROUND: Wolf-Hirschhorn syndrome (WHS) (OMIM 194190) is a multiple congenital anomalies/intellectual disability syndrome. It is caused by partial loss of genetic material from the distal portion of the short arm of chromosome. METHODS: We studied the phenotype-genotype correlation. RESULTS: We present the clinical manifestations and cytogenetic results of 10 unrelated Egyptian patients with 4p deletions. Karyotyping, FISH and MLPA was performed for screening for microdeletion syndromes. Array CGH was done for two patients. All patients exhibited the cardinal clinical manifestation of WHS. FISH proved deletion of the specific WHS locus in all patients. MLPA detected microdeletion of the specific locus in two patients with normal karyotypes, while array CGH, performed for two patients, has delineated the extent of the deleted segments and the involved genes. LETM1, the main candidate gene for the seizure phenotype, was found deleted in the two patients tested by array CGH; nevertheless, one of them did not manifest seizures. The study emphasized the previous. CONCLUSION: WHS is a contiguous gene syndrome resulting from hemizygosity of the terminal 2 Mb of 4p16.3 region. The Branchial fistula, detected in one of our patients is a new finding that, to our knowledge, was not reported.

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.

Phenotypic and Molecular Cytogenetic Analysis of a Case of Monosomy 1p36 Syndrome due to Unbalanced Translocation.

Monosomy 1p36 syndrome is one of the most common submicroscopic deletion syndromes, which is characterized by the presence of delayed developmental milestones, intellectual disability, and clinically recognizable dysmorphic craniofacial features. The syndrome comprises 4 cytogenetic groups including pure terminal deletions, interstitial deletions, complex rearrangements, and derivative chromosomes 1 due to unbalanced translocations, where unbalanced translocations represent the least percentage of all cases of monosomy 1p36 (7%). Most patients with monosomy 1p36 due to an unbalanced translocation can be cytogenetically diagnosed using conventional techniques. However, chromosomal microarray analysis is mandatory in these cases to detect copy number variance and size of the deletion and allows for setting a phenotype-genotype correlation. Here, we studied a 1.5-year-old female patient who showed intellectual disability, delayed milestones, hypotonia, seizures, and characteristic dysmorphic features including brachycephaly, straight eyebrows, deep-set eyes, downslanting palpebral fissures, midface hypoplasia, depressed nasal bridge, long philtrum, and pointed chin. Conventional cytogenetic analysis (CCA), microarray study, and fluorescence in situ hybridization (FISH) analysis were performed. CCA showed a translocation involving chromosomes 1 and 21, 45,XX,der(1)t(1;21)(p36.32;q21.1)dn. Microarray analysis revealed copy number losses at both 1p36 and proximal 21q. FISH confirmed the presence of the 1p36 deletion, but was not performed for 21q. We have concluded that phenotype-genotype correlation for monosomy 1p36 syndrome can be performed for the fundamental clinical manifestations; however, the final aspect of the syndrome depends on composite factors. Monosomy 1p36 due to unbalanced translocation may present either classically or with additional altered features of various severity based on the copy number variations involving different chromosomes.

Cytogenetic Spectrum of Ovotesticular Difference of Sex Development (OT DSD) among a Large Cohort of DSD Patients and Literature Review.

Ovotesticular difference of sex development (OT DSD) is a rare genetic disorder with an incidence of about 1/100,000 live births. The majority of OT DSD patients show a 46,XX karyotype, others may have 46,XX/46,XY chimerism or exhibit various mosaic sex chromosome combinations, and less commonly they may have a 46,XY karyotype. The aim of this work is to report the clinical, pathological, and karyotypic variations in OT DSD patients diagnosed among a large cohort of DSD patients. The study included 10 patients thoroughly evaluated for clinical, genital, and hormonal abnormalities and subjected to imaging studies, laparoscopy with gonadal biopsy, karyotype, and FISH analysis. The current study revealed a greater percentage of mosaic cell line combinations than previously reported and showed variable cytogenetic abnormalities, including the rare isodicentric (Y)(p11.32) abnormality and X;Y translocation. The study also revealed a unique pattern of gonadal type and combination frequencies. To our knowledge, this is the first study on OT DSD patients among a large cohort of DSD patients in Egypt and the Middle East.

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.

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.

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.

Aromatase Deficiency due to a Homozygous CYP19A1 Mutation in a 46,XX Egyptian Patient with Ambiguous Genitalia.

Aromatase deficiency (AD) is a very rare disorder resulting from mutations in the CYP19A1 gene encoding aromatase, a cytochrome P450 enzyme that plays a pivotal role in androgen conversion to estrogens. AD is inherited in an autosomal recessive trait, and to date only 35 cases have been described in the literature. Herein, we depict a new patient reared as a male, who presented at the age of 21 years with no palpable testis, hypoplastic scrotum, penis-like phallus (3 cm), and penoscrotal hypospadias. The patient was born to consanguineous parents, his karyotype was 46,XX, and SRY was negative. Pelvic sonar showed a small hypoplastic uterus, and no testis could be identified. Serum testosterone was within the reference range of females along with high gonadotropins. Pathology of gonadal biopsy showed ovarian stroma negative for oocytic follicle consistent with streak gonads. All these data were suggestive of AD, which was subsequently confirmed by molecular investigation of the CYP19A1 gene. A homozygous splice site mutation in the donor splice site of exon 9 was identified, c.1263 + 1G>T. This is the first report of such a rare disorder in an Egyptian patient. Our results reinforce the importance of considering AD in patients with 46,XX disorders of sex development after ruling out congenital adrenal hyperplasia.

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.

Sex Chromosome Mosaicism in the Gonads of DSD Patients: A Karyotype/Phenotype Correlation.

Sex chromosome mosaicism results in a large clinical spectrum of disorders of sexual development (DSD). The percentage of 45,X cells in the developing gonad plays a major role in sex determination. However, few reports on the gonadal mosaic status have been published, and the phenotype is usually correlated with peripheral lymphocyte karyotypes, which makes the phenotype prediction imprecise. This study was conducted on 7 Egyptian DSD patients to demonstrate the effect of sex chromosome constitution of both blood lymphocytes and gonadal tissues on the phenotypic manifestations. Conventional cytogenetic and FISH analyses of blood lymphocytes were conducted, and laparoscopy with gonadal biopsy was performed for histopathologic examination and FISH analysis. Gonosomal mosaicism was detected in 3 patients who had a non-mosaic chromosome pattern in blood lymphocytes. Two patients showed the same type of sex chromosome mosaicism in both the blood and gonadal tissues but with different distributions. Two other patients revealed a non-mosaic pattern in both tissues. The present study elucidates the importance of examining sex chromosome mosaicism in gonadal tissues of DSD patients and highlights the critical role of 45,X mosaicism which can lead to serious effects during early gonadal organogenesis.

46,XY disorder of sex development in a sudanese patient caused by a novel mutation in the HSD17B3 gene.

In this study, we present a Sudanese 46,XY patient raised as a female and diagnosed at the age of 20 years with having 17ß-hydroxysteroid dehydrogenase type 3 (17ß-HSD3) deficiency. She presented with primary amenorrhea, undeveloped breasts and a male pattern of secondary sexual characteristics. Examination of her external genitalia showed type IV genital circumcision. Steroid measurements both in urine and serum pointed to 17ß-HSD3 deficiency. A novel homozygous splice-site mutation [c.524 + 2T>A] was detected in intron 7 of the HSD17B3 gene. In this patient, steroid concentration clearly supported both the clinical diagnosis of 17ß-HSD3 deficiency and the functional relevance of the mutation. Interestingly, despite of the type IV genital circumcision, the patient expressed her interest in reassigning her sex from female to male.

Constitutional retinoblastoma gene deletion in Egyptian patients.

BACKGROUND: Retinoblastoma is a neuroblastic tumor of childhood with an incidence of 1: 20 000. Retinoblastoma gene (Rb1) is a tumor suppressor gene that is located on the long arm of chromosome 13 at region 14. This study was to evaluate the constitutional monoallelic Rb1 deletion among retinoblastoma families. METHODS: Nine families with an affected Rb proband were evaluated. Clinical examination, pedigree analysis, and complete eye examination were given to patients, their sibs and parents. Standard cytogenetic and fluorescence in situ hybridization (FISH) analyses were carried out for all of them. Also, two sib fetuses were tested for Rb1 deletion. RESULTS: No dysmorphic features were detected in any patient. Developmental milestones were within normal limit except in one proband who had a mild delay. The age of onset ranged from one month to 4 years. Positive family history was found in two families. In one, the father and 3 sibs had retinoblastoma, and in the other, 2 sibs were affected, but the parents were free. Chromosomal study revealed no abnormalities in all parents and sibs. Two patients had mosaic chromosome 13 abnormalities, 46,XY/46,XY,del(13)(pter-->q14:) and 46,XX/46,inv(13)(q14q22). FISH analysis detected mosaic Rb1 deletion in two patients and excluded Rb1 deletion in two fetuses. CONCLUSIONS: The detection of genetic alterations affecting the Rb1 locus is important for the establishment of carriers, and prenatal and presymptomatic diagnosis. The search for deleted Rb1 mosaic cell lines is important for genetic counseling. Germline mutation may be considered as genetic transmission method of the Rb1 gene.