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.

Three novel patients with epileptic encephalopathy due to biallelic mutations in the PLCB1 gene.

Biallelic mutations in the PLCB1 gene, encoding for a phospholipase C beta isoform strongly expressed in the brain, have been reported to cause infantile epileptic encephalopathy in only four children to date. We report here three additional patients to delineate the phenotypic and genotypic characteristics of the disease. Our three patients were one sporadic case with an intragenic homozygous deletion and two cousins with the homozygous p.(Arg222*) nonsense variant in PLCB1. These patients had severe to profound intellectual disability, epileptic spasms at age 3-5 months concomitant with developmental arrest or regression, other seizure types and drug-resistant epilepsy. With this report, we expand the clinical, radiologic and electroencephalographic knowledge about the extremely rare PLCB1-related encephalopathy. Since the first report in 2010, the overall number of reported patients with our additional patients is currently limited to seven. All seven patients had epileptic encephalopathy, mainly infantile spasms and 6/7 had profound intellectual disability, with one only being able to walk. Truncal hypotonia was the most frequent neurological sign, sometimes associated with pyramidal and/or extrapyramidal hypertonia of limbs. Microcephaly was inconstant. In conclusion, the phenotypical spectrum of PLCB1-related encephalopathy is relatively narrow, comprises infantile spasms and severe to profound intellectual disability, and does not seem to define a recognizable clinical entity.

Consequences of cathepsin C inactivation for membrane exposure of proteinase 3, the target antigen in autoimmune vasculitis.

Membrane-bound proteinase 3 (PR3m) is the main target antigen of anti-neutrophil cytoplasmic autoantibodies (ANCA) in granulomatosis with polyangiitis, a systemic small-vessel vasculitis. Binding of ANCA to PR3m triggers neutrophil activation with the secretion of enzymatically active PR3 and related neutrophil serine proteases, thereby contributing to vascular damage. PR3 and related proteases are activated from pro-forms by the lysosomal cysteine protease cathepsin C (CatC) during neutrophil maturation. We hypothesized that pharmacological inhibition of CatC provides an effective measure to reduce PR3m and therefore has implications as a novel therapeutic approach in granulomatosis with polyangiitis. We first studied neutrophilic PR3 from 24 patients with Papillon-Lefèvre syndrome (PLS), a genetic form of CatC deficiency. PLS neutrophil lysates showed a largely reduced but still detectable (0.5-4%) PR3 activity when compared with healthy control cells. Despite extremely low levels of cellular PR3, the amount of constitutive PR3m expressed on the surface of quiescent neutrophils and the typical bimodal membrane distribution pattern were similar to what was observed in healthy neutrophils. However, following cell activation, there was no significant increase in the total amount of PR3m on PLS neutrophils, whereas the total amount of PR3m on healthy neutrophils was significantly increased. We then explored the effect of pharmacological CatC inhibition on PR3 stability in normal neutrophils using a potent cell-permeable CatC inhibitor and a CD34+ hematopoietic stem cell model. Human CD34+ hematopoietic stem cells were treated with the inhibitor during neutrophil differentiation over 10 days. We observed strong reductions in PR3m, cellular PR3 protein, and proteolytic PR3 activity, whereas neutrophil differentiation was not compromised.

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.

Abnormal profiles of cathepsin C secreted in urine of Papillon Lefevre syndrome patients.

BACKGROUND: Papillon Lefevre syndrome (PLS) is an autosomal recessive disorder that results from a mutated gene that encodes a lysosomal peptidase known as cathepsin C (CTSC). The clinical presentation of PLS involves mainly palmoplantar keratosis and periodontitis with a variable degree of severity. SUBJECTS: and methods: Our study included ten patients with a broad spectrum of palmoplantar keratosis and periodontitis severity. CTSC variants were detected by Sanger sequencing. CTSC protein secreted in urine was detected by western blotting. RESULTS: Five patients have missense variants, Four have nonsense variants, and one has splice variants in CTSC. The activation products of cathepsin C protein (Heavy and light chains) were absent in all patients' urine samples except one with a significantly reduced level compared to the controls. The dimeric form of CTSC protein was found in all the studied cases. The monomeric form was found in five cases. The products of proteolytic activation of CTSC by other cathepsins (L and S) were found in the urine samples of five of the patients. Each patient had a characteristic pattern of accumulated CTSC protein maturation/activation substrates, intermediates, and products. 40% of the patients had the activation products of other lysosomal cathepsins. CONCLUSION: Urinary CTSC in PLS patients could be used as a diagnostic biomarker for the biochemical screening of the disease. Different variants in CTSC result in different profiles of CTSC secreted in the urine of PLS patients. The profiles of secreted CTSC in urine could be correlated to the severity of palmoplantar keratosis.

Gastritis induced by Helicobacter pylori infection in experimental rats.

BACKGROUND: Gastritis, an inflammation of gastric mucosa, may be due to many pathological factors and infection, such as with Helicobacter pylori. The use of experimental models of gastritis is important to evaluate the biochemical changes and study chemotherapeutic intervention. In a previous study we demonstrated an acute gastritis model induced by iodoacetamide. AIMS: Our objective in this study was to evaluate a new gastritis model induced by H. pylori infection in experimental rats in terms of certain biomarkers in serum and mucosal tissues in addition to histopathological examination. METHODS: Gastritis was induced in 20 albino Wistar rats by H. pylori isolated from antral biopsy taken from a 49-year-old male patient endoscopically diagnosed as having H. pylori infection. Another ten rats were used as controls. Serum gastrin, pepsinogen I activity, interleukin-6 (IL-6) and gastric mucosal myeloperoxidase (MPO) activity and prostaglandin E(2) (PGE(2)) were measured. Immunostaining for inducible nitric oxide synthase (iNOS), nitrotyrosine and DNA fragmentation were used to further evaluate H. pylori-induced gastritis. RESULTS: Serum gastrin, IL-6, mucosal MPO activity, and PGE(2) demonstrated significant increases joined with a decreased serum pepsinogen I activity (P < 0.001). Immunohistochemistry demonstrated positive reaction for iNOS, nitrotyrosine and DNA fragmentation. CONCLUSIONS: Helicobacter pylori-induced gastritis models demonstrated massive oxidative stress and pronounced injury in mucosal tissue. Since our model in rats reflected the clinical picture of H. pylori infection, it can be considered as a consistent model to study chemotherapeutic intervention for this type of gastritis.

Assessment of the most common CYP21A2 point mutations in a cohort of congenital adrenal hyperplasia patients from Egypt.

Objectives Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) is a common autosomal recessive disorder caused by defects in the CYP21A2 gene. We aimed to determine the prevalence of the most commonly reported mutations among 21-OHD Egyptian patients and correlate genotype with phenotype. Methods Molecular analysis of the CYP21A2 gene was performed for the detection of the six most common point mutations (p.P30L, p.I172N, p.V281L, p.Q318X, the splice site mutation Int2 [IVS2-13A/C>G], and the cluster of three mutations [p.I236N, p.V237E, and p.M239K] designed as CL6). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was performed on 47 unrelated Egyptian 21α-OH deficiency patients and their available parents to detect the presence of the six most common point mutations. Results Screening for the six most common point mutations in CYP21A2 gene, revealed mutations in 87.2% (82/94) of the studied alleles corresponding to 47 Egyptian patients. The most common mutation among the studied cases was IVS2-13C/A>G that was found to be presented in a frequency of 46.8% (44/94). The genotype/phenotype correlations related to null, A, and B groups were with PPV of 100, 55.5, and 83.3%, respectively. Conclusions The described method diagnosed CAH in 80.8% of the studied patients. Good correlation between genotype and phenotype in salt wasting and simple virilizing forms is determined, whereas little concordance is seen in nonclassical one. Furthermore, studying the carrier frequency of 21-OHD among the normal population is of great importance.

Turmeric-based diet can delay apoptosis without modulating NF-kappaB in unilateral ureteral obstruction in rats.

The unilateral ureteral obstruction (UUO) model of renal injury in rat is characterized by nuclear factor kappaB (NF-kappaB) activation and tumour necrosis factor alpha (TNF-alpha) production, which induces apoptosis via activation of caspase 8 resulting in cell death. Curcumin, the major component found in turmeric spice, has been reported to provide protection against fibrosis and apoptosis elicited by UUO. This study examined the effects of a turmeric-based diet (5% w/w) on the apoptotic pathway induced by UUO in rats after 30 days of ligation. Administration of a turmeric-based diet demonstrated a significant decrease (P<0.05) in mRNA expression of TNF-alpha and caspase 8, but not NF-kappaB, expression, which may contribute to the protective role of the turmeric-based diet. We conclude that a turmeric-based diet can delay apoptosis without modulating NF-kappaB, so as not to sensitize the mesangial cells to the apoptotic stimuli.

Platelet rich plasma enhances the immunohistochemical expression of platelet derived growth factor and vascular endothelial growth factor in the synovium of the meniscectomized rat models of osteoarthritis.

This study was carried out on a rat model of surgically-induced osteoarthritis (OA) to assess the histological and immunohistochemical changes in the synovial membrane and to evaluate the effects of intra-articular injection of platelet rich plasma (PRP) in such cases. Forty five male albino rats were divided into 3 equal groups; control, surgically-induced OA and surgically-induced OA followed by intra-articular injection of PRP. Knee joints were processed for histological and immunohistochemical staining with anti-platelet derived growth factor (PDGF-A) and anti-vascular endothelial growth factor (VEGF) and the area percentages of immunostaining were measured by digital image analysis. Serum levels of PDGF-A and VEGF were analyzed by ELISA. The osteoarthritis research society international (OARSI) score was significantly higher in OA (2433.8±254) than in control (230.4±37.8; p<0.001) and in PRP-treated tissues (759.7±45.8; p<0.001). The immunostained area percentages for PDGF-A was significantly higher in PRP-treated tissues (20.6±2.4) than in OA (11.06±1.3; p=0.007) and in control tissues (4.1±0.78; p<0.001). Likewise, the immunostained area percentage for VEGF was significantly higher in PRP-treated tissues (22.5±1.6) than in OA (14.9±1; p=0. 002) and in control tissues (6.5±0.7; p<0.001). ELISA analysis revealed a significant increase in serum levels of the PDGF-A and VEGF after intraarticular PRP injection when compared to the other groups (p<0.000). The present study concluded that intra-articular injection of PRP could produce optimizing effects in surgically induced OA in the form of; decreasing the OARSI score, improving the inflammatory events in synovium and modulating the PDGF-A and VEGF serum levels and synovial tissue immunoexpression. These effects could be reflected positively on the associated chondral defect.

Light and electron microscopic study on the effect of antischizophrenic drugs on the structure of seminiferous tubules of adult male albino rats.

INTRODUCTION: Sexual dysfunction and infertility are symptoms which have been rarely studied in patients treated with antischizophrenic drugs, aripiprazole and olanzapine, for long period. This work aimed to investigate the effects of aripiprazole and olanzapine on the structure of seminiferous tubules of rats at both light microscopic and ultrastructural levels. MATERIAL AND METHODS: Sixty adult male rats were divided into 3 groups (n = 20): control group (Group I) and two experimental ones (II and III). Rats in Group II received 2 mg/kg/day aripiprazole while rats in Group III received 0.5 mg/kg/day olanzapine for 14 weeks. Thereafter, testis were removed and processed for both light and electron microscopic study. Qualitative morphological analyses and histomorphometric measurements of seminiferous tubules were performed. RESULTS: Rats in Group II showed reduction of testicular weight, seminiferous tubules' diameter, epithelial height, spermatogenic count, spermatogenic index and spermatogenic score whereas Sertoli cells count was increased. Olanzapine-treated rats also showed epithelial desquamation, separation and apoptotic changes of germ cells. Sertoli cells showed vacuolization, dilatation of smooth endoplasmic reticulum and accumulation of lipid droplets. Abnormality in the shape and structure of late spermatids and presence of giant cells were also demonstrated. Aripiprazole induced less adverse histological changes in rat testis than olanzapine. CONCLUSIONS: Olanzapine followed by aripiprazole had adverse histological effects on the structure of the seminiferous tubules, which may affect spermatogenesis.

A novel mutation (c.2735_2736delTC) in the androgen receptor gene in 46,XY females with complete androgen insensitivity syndrome in an Egyptian family.

BACKGROUND: Androgen insensitivity syndrome (AIS) results from resistance of the target tissues to the effect of the androgenic hormones producing a phenotype with varying degrees of feminization ranging from male infertility to completely normal female external genitalia. Androgen receptor (AR) is a transcription factor that interacts with the androgenic steroids that act as ligands activating the AR, and via different cellular mechanisms, the activated AR binds to the DNA of target tissues to induce the desired biological changes. To date, more than 800 different mutations in the AR gene have been identified in patients with AIS and the majority of these mutations are localized in the ligand-binding domain. METHODS: Here we describe an Egyptian family with 7 affected 46,XY females with complete androgen insensitivity. RESULTS: Mutational analysis of the AR gene revealed a novel frameshift mutation in exon 8 of the gene c.2735_2736delTC. CONCLUSION: This study extends the number of AR gene mutations identified so far. Further, it confirms that AR gene mutations are the most frequent cause of 46,XY disorder of sexual development, with higher frequency in the complete phenotype.

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.

Oxytocin inhibits NADPH oxidase and P38 MAPK in cisplatin-induced nephrotoxicity.

Oxidative stress significantly contributes to cisplatin (CP)-associated cytotoxicity, and use of antioxidants could counteract such cytotoxic effects of CP. The major biochemical pathway for reactive oxygen species (ROS) formation proceeds through O2⁻ production, which is generated by NADPH oxidase, such oxidative stress can activate p38 MAPK to intensify the cytotoxic effect of CP. We mainly aimed to study the protective effect of oxytocin (OT) on CP-induced nephrotoxicity whereas; it was previously shown to have anti-inflammatory effects in different inflammation models. Administration of OT significantly decreased the gene expression of both NADPH oxidase and P38 MAPK, nitric oxide (NO), myloperoxidase (MPO), and TBARS, furthermore it increased the renal tissue levels of antioxidants; reduced glutathione (GSH), and superoxide dismutase (SOD). Histologically, OT reduced the monocellular infiltration as well as the tubular damage in CP-induced nephrotoxicity. In conclusion OT has a powerful antioxidant effect that can alleviate the CP-induced nephrotoxicity through inhibition of NADPH oxidase and P38 MAPK resulting in improvement of kidney functions.

Interleukin-10 to tumor necrosis factor-alpha ratio is a predictive biomarker in nonalcoholic fatty liver disease: interleukin-10 to tumor necrosis factor-alpha ratio in steatohepatitis.

OBJECTIVES: Fatty liver disease is commonly associated with diabetes mellitus (DM). Insulin resistance (IR) as an investigative biomarker is only concerned with fatty liver that results from DM type 2 associated with metabolic syndrome. Irrespective of IR, DM is generally characterized by overproduction of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha), whereas action of the latter is modulated by the anti-inflammatory cytokine interleukin-10 (IL-10). The aim of this study was to investigate the efficacy of using TNF-alpha alone or IL-10/TNF-alpha ratio compared to IR, as a promising biomarker for fatty liver assessment in DM. Furthermore, we hypothesized that using garlic as an immunomodulator may decrease TNF-alpha and increase IL-10 production to improve steatohepatitis. METHODS: DM was induced metabolically by a high-fat diet to bring about IR, or chemically by alloxan, producing insulin deficiency, in male albino rats. Garlic powder was supplemented (15 mg/kg per day) for 3 weeks. Fatty liver was depicted histologically and biochemically (aspartic aminotransferase, alanine aminotransferase, HOMA-IR, TNF-alpha, IL-10, IL-10/TNF-alpha ratio). RESULTS: We found that, in contrast to obese rats, garlic decreased IL-10/TNF-alpha ratio, despite decreasing TNF-alpha in alloxan diabetic rats in agreement with the histology, which revealed more prominent improvement in the obese group. Moreover, the effect of garlic was not linked to improvement of IR in obese rats. CONCLUSION: We conclude that IL-10/TNF-alpha ratio may be considered as a convenient biomarker for investigation of fatty liver of different grades, apart from being associated with IR, and immunomodulation of this ratio in favor of increasing it may exert significant improvement.