Dina Mehaney

Validation of a semiconductor next‐generation sequencing assay for the clinical genetic screening of CFTR

Genetic testing for cystic fibrosis and CFTR‐related disorders mostly relies on laborious molecular tools that use Sanger sequencing to scan for mutations in the CFTR gene. We have explored a more efficient genetic screening strategy based on next‐generation sequencing (NGS) of the CFTR gene. We validated this approach in a cohort of 177 patients with previously known CFTR mutations and polymorphisms. Genomic DNA was amplified using the Ion AmpliSeq™ CFTR panel. The DNA libraries were pooled, barcoded, and sequenced using an Ion Torrent PGM sequencer. The combination of different robust bioinformatics tools allowed us to detect previously known pathogenic mutations and polymorphisms in the 177 samples, without detecting spurious pathogenic calls. In summary, the assay achieves a sensitivity of 94.45% (95% CI: 92% to 96.9%), with a specificity of detecting nonvariant sites from the CFTR reference sequence of 100% (95% CI: 100% to 100%), a positive predictive value of 100% (95% CI: 100% to 100%), and a negative predictive value of 99.99% (95% CI: 99.99% to 100%). In addition, we describe the observed allelic frequencies of 94 unique definitely and likely pathogenic, uncertain, and neutral CFTR variants, some of them not previously annotated in the public databases. Strikingly, a seven exon spanning deletion as well as several more technically challenging variants such as pathogenic poly‐thymidine‐guanine and poly‐thymidine (poly‐TG‐T) tracts were also detected. Targeted NGS is ready to substitute classical molecular methods to perform genetic testing on the CFTR gene.

Selective screening for inborn errors of metabolism by tandem mass spectrometry in Egyptian children: A 5 year report

OBJECTIVE In order to enhance awareness and promote registry for inborn errors of metabolism (IEMs) in Egypt, we aimed to evaluate the prevalence and main clinical findings of IEMs detectable by tandem mass spectrometry (MS/MS) among high risk pediatric patients presenting to our tertiary care facility at Cairo University Childrens Hospital over a period of 5 years and to compare the disease burden in Egypt in the absence of a national screening program for inherited metabolic disorders with other populations. METHODS During this period 3380 Egyptian children were suspected of having IEMs based on clinical/laboratory presentation and were analyzed by MS/MS. Confirmatory testing was performed according to flagged analyte by MS/MS using a different sample type such as plasma or urine or by a different technique such as GC/MS. RESULTS A relatively high number of patients (203/3380 (6%)) were confirmed with 17 different types of IEMs. Averages for age at diagnosis for different disorders ranged from 2.5 months to 6.6 years with general developmental delay and irreversible neurological damage being the most common presenting features (75.9% and 65.5%, respectively). Amino acid disorders (127/203 (62.6%)), mainly phenylketonuria (100/203 (49.3%)), were the most encountered, followed by organic acidemias (69/203 (34%)), while fatty acid oxidation defects (7/203 (3.4%)) were relatively rare. 88% of patients were born to consanguineous parents. CONCLUSIONS The development of a nationwide screening program for IEMs is mandatory for early detection of these potentially treatable disorders, prompt and properly timed therapeutic intervention and prevention of the devastating neurological outcomes.

Analysis of oxidative stress status, catalase and catechol-O-methyltransferase polymorphisms in Egyptian vitiligo patients.

Vitiligo is the most common depigmentation disorder of the skin. Oxidative stress is implicated as one of the probable events involved in vitiligo pathogenesis possibly contributing to melanocyte destruction. Evidence indicates that certain genes including those involved in oxidative stress and melanin synthesis are crucial for development of vitiligo. This study evaluates the oxidative stress status, the role of catalase (CAT) and catechol-O-Methyltransferase (COMT) gene polymorphisms in the etiology of generalized vitiligo in Egyptians. Total antioxidant capacity (TAC) and malondialdehyde (MDA) levels as well as CAT exon 9 T/C and COMT 158 G/A polymorphisms were determined in 89 patients and 90 age and sex-matched controls. Our results showed significantly lower TAC along with higher MDA levels in vitiligo patients compared with controls. Meanwhile, genotype and allele distributions of CAT and COMT polymorphisms in cases were not significantly different from those of controls. Moreover, we found no association between both polymorphisms and vitiligo susceptibility. In conclusion, the enhanced oxidative stress with the lack of association between CAT and COMT polymorphisms and susceptibility to vitiligo in our patients suggest that mutations in other genes related to the oxidative pathway might contribute to the etiology of generalized vitiligo in Egyptian population.

Inborn errors of metabolism detectable by tandem mass spectrometry in Egypt: The first newborn screening pilot study.

Objectives To estimate the burden of metabolic disorders detectable by tandem mass spectrometry in Egypt, through a pilot expanded newborn screening programme at Cairo University Childrens Hospital in 2008, and examining the results of 3,900 clinically at-risk children, investigated at Cairo University Children’s Hospital for the same disorders over the past 7 years using the same technology. Methods Dried blood spots of 25,276 healthy newborns from three governorates in Upper, Middle, and Lower Egypt were screened, to give a representative sample of the Egyptian newborn population. Based on the pilot study outcomes and the results of clinically suspected children, we estimated the total birth prevalence of tandem mass spectrometry detectable metabolic disorders, and the relative frequency of several individual disorders. Results Among the healthy newborns, 13 metabolic disorder cases (five phenylketonuria [1:5,000], two methylmalonic acidemia, and isovaleric acidemia [1:12,500], one each of maple syrup urine disease, propionic acidemia, β-ketothiolase deficiency, and primary carnitine deficiency [1:25,000]) were confirmed, giving a total birth prevalence of 1:1944 live births. Among the clinically suspected children, 235 cases were diagnosed, representing a much wider disease spectrum. Conclusions Egypt has one of the highest reported birth prevalence rates for metabolic disorders detectable by tandem mass spectrometry. Early diagnosis and management are crucial for the survival and well-being of affected children. A nationwide NBS programme by tandem mass spectrometry is recommended.

Association of chitotriosidase enzyme activity and genotype with the risk of nephropathy in type 2 diabetes.

OBJECTIVE The immune-inflammatory system has been implicated in the pathogenesis of diabetic nephropathy; however, many of the mechanisms involved remain unclear. Chitotriosidase enzyme is an active human chitinase and a major protein product of activated macrophages. Although playing an important role in innate and acquired immunity, chitotriosidase involvement in the development of diabetic nephropathy is unknown. DESIGN AND METHODS Chitotriosidase enzyme activity and the presence of the functional 24-bp duplication mutation of the chitotriosidase gene (CHIT1) were assessed in 262 Egyptian type 2 diabetic patients with and without nephropathy and 90 non-diabetic controls. In diabetic patients, multiple linear regression models were adapted to assess the association of chitotriosidase activity with two important measures of renal disease progression: urinary albumin/creatinine ratio and eGFR, while the association of the CHIT1 genotype with the incidence of nephropathy was evaluated by multiple logistic regression. RESULTS In diabetic patients, chitotriosidase enzyme activity showed a statistically significant elevation as compared to controls and correlated positively with the progression of nephropathy. A significant association of chitotriosidase activity with both urinary albumin/creatinine ratio and eGFR was detected after adjusting for age, gender, duration of diabetes, body mass index, hypertension status, total cholesterol, triglycerides and HbA1c levels, P<0.001. We also identified a protective association between the CHIT1 mutated genotype and diabetic nephropathy after adjusting for the same confounders (odds ratio: 0.517, 95% CI: 0.289-0.924, P=0.026). CONCLUSIONS This study demonstrates for the first time that the immunomodulatory effects of chitotriosidase enzyme could be implicated in the development of nephropathy in type 2 diabetes.

Profile of cystic fibrosis in a single referral center in Egypt

It was generally believed that Cystic fibrosis (CF) is rare among Arabs; however, the few studies available from Egypt and other Arabic countries suggested the presence of many undiagnosed patients. The aim of the present study was to determine the frequency of CF patients out of the referred cases in a single referral hospital in Egypt. A total of 100 patients clinically suspected of having CF were recruited from the CF clinic of the Allergy and Pulmonology Unit, Children’s Hospital, Cairo University, Egypt, throughout a 2 year period. Sweat chloride testing was done for all patients using the Wescor macroduct system for collection of sweat. Quantitative analysis for chloride was then done by the thiocyanate colorimetric method. Patients positive for sweat chloride (⩾60 mmol/L) were tested for the ΔF508 mutation using primer specific PCR for cystic fibrosis transmembrane conductance regulator (CFTR) gene. Thirty-six patients (36%) had a positive sweat chloride test. The main clinical presentations in patients were chronic cough in 32 (88.9%), failure to thrive in 27 (75%), steatorrhea in 24 (66.7%), and hepatobiliary involvement in 5 (13.9%). Positive consanguinity was reported in 50% of CF patients. Thirty-two patients were screened for ΔF508 mutation. Positive ΔF508 mutation was detected in 22 (68.8%) patients, 8 (25%) were homozygous, 14 (43.8%) were heterozygous, and 10 (31.3%) tested were negative. CF was diagnosed in more than third of patients suspected of having the disease on clinical grounds. This high frequency of CF among referred patients indicates that a high index of suspicion and an increasing availability of diagnostic tests lead to the identification of a higher number of affected individuals.

Serum Vitamin D and Vitamin D Receptor Gene Polymorphism in Mycosis Fungoides Patients: A Case Control Study

Background Vitamin D has been considered a key player in various malignancies including cutaneous cancers. To date, mycosis fungoides (MF) has been the least studied in relation to vitamin D. Furthermore, the vitamin D receptor (VDR) single nucleotide polymorphisms (SNPs) have not been tackled before in the context of MF, despite their incrimination in numerous diseases. Aim of study To assess the role of vitamin D in MF by measuring its serum level, and studying VDR SNPs (TaqI, BsmI, FokI) in different stages of MF. Patients and Methods 48 patients with various stages of MF, and 45 healthy controls were included. Complete history, full clinical examination and a five mm punch skin biopsy were performed to all recruited patients. Venous blood samples were withdrawn from both patients and controls to determine the serum vitamin D level and VDR gene polymorphisms. Results Serum vitamin D level was significantly lower in patients (5.3–33.7 nmol/L)] compared to controls (8.3–90.1 nmol/L)] (P<0.001). A significant difference was observed between patients and controls regarding the FokI polymorphism only, being higher in patients (P = 0.039). Also Vitamin D serum levels differed significantly in patients with FokI genotypes (P = 0.014). No significant correlations were detected between any of the studied parameters and the demographic and clinical data of the included subjects. Conclusion Depressed vitamin D and FokI polymorphism are potentially involved in the context of MF. VDR gene polymorphisms warrant further larger scale investigations to detect the exact genes involved in the pathogenesis of such an enigmatic disease.

Triple Test Screening for Down Syndrome: An Egyptian-Tailored Study

Background The incidence of Down syndrome (DS) in Egypt varies between 1∶555 and 1∶770 and its screening by triple test is becoming increasingly popular nowadays. Results, however, seem inaccurate due to the lack of Egyptian-specific information needed for risk calculation and a clear policy for programme implementation. Our study aimed at calculation and validation of the triple marker medians used in screening Egyptian females as well as to recommend programme conventions to unify screening in this country. Methods The study was conducted on 668 Egyptian women, in weeks 15–20 of pregnancy as proven by sonar. Chorionic gonadotropin (CG), α-fetoprotein (AFP) and unconjugated oestriol (uE3) were measured on Siemens Immulite analyzer. Medians of the three parameters were calculated, regressed against gestational age (GA) and weighted by the number of participants/week. Equations were derived to adjust each parameter to the maternal weight and were centered on the median Egyptian weight. Prisca software was fed with the above data, multiples-of-median (MoM) and DS risks were calculated and the screening performance was evaluated at a mid-trimester risk cutoff of 1∶270. Results Log-linear [AFP/uE3 = 10(A+B*GA)] and exponential equations [CG = A*e (B*GA)] were derived and the regressed medians were found to follow similar patterns to other Asian and Western medians. Oestriol was always lowest (even halved) while CG and AFP were intermediate. A linear reciprocal model best fitted weight distribution among Egyptians and successfully adjusted each parameter to a weight of 78.2 kg. Epidemiological monitoring of these recommendations revealed satisfactory performance in terms of 6.7% initial positive rate and 1.00 grand MoM. Conclusions Adoption of the above recommendations is hoped to pave the way to a successful DS screening programme tailored to Egyptian peculiarities.

AB0270 Serum Sclerostin Level among Egyptian Rheumatoid Arthritis Patients: Relation to Bone Mineral Density, Disease Activity and Radiological Grading

Background Bone loss in rheumatoid arthritis (RA) is caused by increased bone resorption, however, there is no increased bone formation1. The Wnt pathway is important in the control of bone formation through regulation of osteoblast activity2. Sclerostin is an important regulator of the Wnt pathway by blocking Wnt binding to its receptor and thereby inhibiting bone formation3. An increased sclerostin expression in synovial tissues of RA was found compared to osteoarthritis patients4. Objectives This work aimed to study serum sclerostin level in a group of Egyptian rheumatoid arthritis patients and to correlate its level with bone mineral density (BMD), disease activity and radiological grading. Methods Forty RA patients, 26 (65%) were females and 14 (35%) males. Their ages ranged from 21 years to 68 years with a mean of 48.9±11.6 years, their mean value of disease duration was 8±6.4 years and 40 age and sex matched apparently healthy subjects were included. Routine laboratory investigations and testing for serum sclerostin level were done. Plain radiographs of hands & feet and dual-energy x-ray absorptiometry (DXA) test were done for all patients. Results In RA patients, serum level of sclerostin ranged from 0.1 to 1.1 ng/ml with a mean of 0.4±0.2 ng/ml. In the controls, it ranged from 0.2 to 2.3 ng/ml with a mean value of 0.5±0.4 ng/ml. No significant difference was found between RA patients & healthy controls as regard mean value of serum sclerostin level. Postmenopausal RA patients had higher levels of serum sclerostin than premenopausal RA patients (mean value 0.46±0.26 and 0.29±0.18 ng/ml repectively). However, it was statistically significant on comparing healthy postmenopausal to healthy premenopausal with mean values 0.5±0.39 and 0.32±0.14 ng/ml respectively and P value =0.02. Serum sclerostin had significantly positive correlations with age of RA onset (r=0.328, P=0.039), weight of RA patients (r=0.32, p=0.043) and negative correlation with ESR in RA patients (r= -0.34, P=0.03). Forteen (35%) of RA patients had osteoporosis on DXA test. There was no statistically significant correlation between serum sclerostin and BMD, disease activity or radiographic grading. Conclusions Serum sclerostin level in RA patients did not differ significantly from healthy subjects. Serum sclerostin levels have no correlation to disease activity, radiographic joints damage or BMD in RA. For better identification of the role of sclerostin on bone loss in RA, larger sample size is needed. More studies on serum sclerostin levels among different grades of RA activity are encouraged. References Eggelmeijer F, Papapoulos SE, Westedt ML, et al. Bone metabolism in rheumatoid arthritis; relation to disease activity. Br J Rheumatol 1993; 32: 387–391. van Bezooijen RL, Svensson JP, Eefting D, et al. Wnt but not BMP signaling is involved in the inhibitory action of sclerostin on BMP-stimulated bone formation. J Bone Miner Res 2007; 22:19–28. Weidauer SE, Schmieder P, Beerbaum M, et al. NMR structure of the Wnt modulator protein Sclerostin. Biochem Biophys Res Commun 2009; 380:160–165 Kim J. H., Liu X., Wang J., et al. Wnt signaling in bone formation and its therapeutic potential for bone diseases. Ther Adv Musculoskel Dis 2013; 5(1), 13–31. Acknowledgements I would like to express my deepest gratitude and thankfulness; first to Allah for giving me the will and strength to fulfill this work then to my family, Dr. Somaya Anwar, Dr. Sahar Fakhreldin and Dr. Dina Mehaney. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.1169

The effect of CYP3A5 polymorphism on cyclosporine plasma level in Egyptian renal transplant recipients

Cyclosporine (CsA) and tacrolimus are immunosuppressants used for the prevention of rejection of transplanted organs. The genes encoding cytochrome (CYP) P450 enzymes, CYP3A4, and CYP3A5 are the main ones involved in the pharmacokinetics of calcinurin inhibitors (CNI). Several single nucleotide polymorphisms were identified in these genes such as CYP3A5*3 (6986A>G). The association of the CYP3A5*3/*3 genotype with decreased clearance of its substrates was reported among different ethnic populations. This study aims to evaluate the effect of CYP3A5*3 polymorphism on CsA plasma levels in Egyptian renal transplant patients at the first week and first month of transplantation. A total of 44 renal transplant recipients receiving CsA were genotyped for CYP3A5*3 polymorphism. The C0 and C2 of CsA were measured and their relationships with CYP3A5*3 genotypes were investigated. CYP3A5*3 allele was present in six patients and the CsA level didn’t differ significantly between the CYP3A5*3 the CYP3A5*1 allele carriers at the first week and the first month post transplantation. Large-scale studies with the involvement of multiple genetic markers claimed to affect the CsA pharmacokinetics are highly recommended to elucidate their pharmacogenetic role in renal transplant patients.