Diagnosis and staging of HCV associated fibrosis, cirrhosis and hepatocellular carcinoma with target identification for miR-650, 552-3p, 676-3p, 512-5p and 147b.

BACKGROUND: Chronic HCV infection progresses to fibrosis, cirrhosis and hepatocellular carcinoma (HCC). The latter represents the third most common cause for cancer mortality. Currently, there is no reliable non-invasive biomarker for diagnosis of HCV mediated disorders. OBJECTIVE: Profiling expression signature for circulatory miRNAs in the plasma of 167 Egyptian patients (40 healthy, 48 HCV fibrotic, 39 HCV cirrhotic and 40 HCV-HCC cases). METHODS: QRTPCR was used to quantify expression signature for circulatory miRNAs. RESULTS: MiR-676 and miR-650 were powerful in discriminating cirrhotic and late fibrosis from HCC. MiR-650 could distinguish mild (f0-f1) and advanced (f2-f3) fibrosis from HCC cases. MiR-650 and miR-147b could distinguish early fibrosis from healthy controls meanwhile miR-676 and miR-147b could effectively distinguish between mild chronic and (f1-f3) cases from healthy individuals. All studied miRNAs, except miR-512, can differentiate between (f0-f3) cases and healthy controls. Multivariate logistic regression revealed three potential miRNA panels for effective differentiation of HCC, cirrhotic and chronic liver cases. MiR-676-3p and miR-512-5p were significantly correlated in (f1-f3) fibrosis meanwhile miR-676 and miR-512 could differentiate between cirrhosis and (f0-f3) cases. Both miR-650 and miR-512-5p were positively correlated in the cirrhotic group and in (f0-f4) group. Putative targets for investigated miRNAs were also determined. CONCLUSIONS: Investigated miRNAs could assist in staging and diagnosis of HCV associated disorders.

Deep analysis of the LRTOMTc.242G>A variant in non-syndromic hearing loss North African patients and the Berber population: Implications for genetic diagnosis and genealogical studies.

Autosomal recessive non-syndromic hearing loss (ARNSHL) is the most common inherited sensory impairment. It is particularly frequent in North African populations who have a high rate of consanguineous marriage. The c.242G>A homozygous variant in LRTOMT gene was previously established as pathogenic and is associated with NSHL in both humans and mice. The aim of this study is to determine the carrier frequency for the LRTOMT c.242G>A variant and also to estimate its age in addition to evaluating its diagnostic potential as a deafness biomarker among various populations and ethnicities in Northern African countries. A total of 179 Tunisian and 34 Libyan unrelated deafness patients were screened for this variant. The homozygous c.242G>A variant was found in 5.02% and 2.94% in Tunisian and Libyan families, respectively. Subsequent screening for this variant in 263 healthy controls of various ethnicities (136 Tunisian Berbers, 32 Andalusian and 95 Tunisian from undefined ethnic origin) revealed higher frequency for the heterozygous state among Tunisians of Berber origin only (19.11%). Genotyping 7 microsatellite markers nearby the variant location in ARNSHL patients who had the homozygous variant revealed the same haplotype suggesting a common founder origin for this variant. The age of this variant was estimated to be between 2025 and 3425 years (this corresponds to 3400 years when the variant rate was set at 10-3 or 2600 years when the variant rate is set at 10-2 ), spreading along with the Berber population who migrated to North Africa. In conclusion, the LRTOMT c.242G>A homozygous variant could be used as a useful deafness biomarker for North African ARNSHL patients meanwhile the heterozygous variant could be utilized in genealogical studies for tracing those of the Berber ethnic group.

Circulatory miR-221 & miR-542 expression profiles as potential molecular biomarkers in Hepatitis C Virus mediated liver cirrhosis and hepatocellular carcinoma.

Chronic hepatitis C virus (cHCV) is a leading cause for liver cirrhosis (LC) and hepatocellular carcinoma (HCC) globally. So far, there is no optimal non-invasive biomarker for diagnosing HCV associated hepatic disorders. Circulatory miRNAs have drawn great attention as potential non-invasive biomarkers in various diseases. We quantified miR-221 and miR-542 levels in the plasma of 153 Egyptian patients (38 healthy controls (HC), 36 cHCV, 39 HCV-LC and 40 HCV mediated HCC groups) using qRT-PCR. All diseased groups exhibited significant upregulation in miR-221 expression (P < 0.001) with an increasing trend towards late stages (HCV-LC+HCV-HCC) as compared to early stages (cHCV). MiR-221 could significantly discriminate HCC patients from cHCV and HCV-LC with (AUC=0.698; P = 0.002) and (AUC=0.644; P = 0.032) respectively. Furthermore, miR-221 could significantly discriminate between HCC and non-HCC groups (AUC=0.670, P<0.001). HCV-LC & cHCV groups showed significant upregulation in miR-542 with remarkable downregulation in HCC group (P = 0.004). MiR-542 exhibited diagnostic power of (AUC=0.640; P = 0.044) and (AUC= 0.644; P = 0.040) for discriminating HCV-LC from HCC and cHCV groups respectively. Both miR-221 and miR-542 were significantly upregulated in cirrhotic group (HCV-LC) (P = 0.046 and P = 0.002 respectively) as compared to non-cirrhotic group (cHCV+HC). Combining both miRNAs in a panel significantly improved diagnostic performance as follows; HC and HCC (AUC=0.714, P < 0.001); HCC and LC (AUC=0.714, P = 0.001); HC and LC (AUC=0.710, P = 0.002) and also cHCV and HCC (AUC=0.672, P = 0.006). In conclusion, both miR-221 & miR-542 could stand as a standalone biomarker for staging various HCV associated disorders. Combining them would greatly enhance their diagnostic potential.

SRD5A3-CDG: 3D structure modeling, clinical spectrum, and computer-based dysmorphic facial recognition.

Pathogenic variants in Steroid 5 alpha reductase type 3 (SRD5A3) cause rare inherited congenital disorder of glycosylation known as SRD5A3-CDG (MIM# 612379). To date, 43 affected individuals have been reported. Despite the development of various dysmorphic features in significant number of patients, facial recognition entity has not yet been established for SRD5A3-CDG. Herein, we reported a novel SRD5A3 missense pathogenic variant c.460 T > C p.(Ser154Pro). The 3D structural modeling of the SRD5A3 protein revealed additional transmembrane α-helices and predicted that the p.(Ser154Pro) variant is located in a potential active site and is capable of reducing its catalytic efficiency. Based on phenotypes of our patients and all published SRD5A3-CDG cases, we identified the most common clinical features as well as some recurrent dysmorphic features such as arched eyebrows, wide eyes, shallow nasal bridge, short nose, and large mouth. Based on facial digital 2D images, we successfully designed and validated a SRD5A3-CDG computer based dysmorphic facial analysis, which achieved 92.5% accuracy. The current work integrates genotypic, 3D structural modeling and phenotypic characteristics of CDG-SRD5A3 cases with the successful development of computer tool for accurate facial recognition of CDG-SRD5A3 complex cases to assist in the diagnosis of this particular disorder globally.

Design and novel synthetic approach supported with molecular docking and biological evidence for naphthoquinone-hydrazinotriazolothiadiazine analogs as potential anticancer inhibiting topoisomerase-IIB.

A novel synthetic approach was developed for the synthesis of 3-hydrazinotriazolothiadiazines in just one step from Purpald and phenacyl bromides. They were then selectively tethered to naphthoquinone fragments through hydrazine moiety generating novel Naphthoquinone-hydrazinotriazolothiadiazine analogues. In vitro cytotoxicity for the synthesized chemical entities was validated against HepG2 and MCF-7 cell lines and recorded IC50 inhibitory profile range of 0.07-19.68 µM and 1.19-67.32 µM respectively. Among the synthesized series, compound 4c had maximal cytotoxicity against HepG2 and was therefore selected for further downstream biological investigations. Caspase 3 apoptotic marker was significantly upregulated in cells treated with compound 4c with induction of apoptosis at Pre-G1 phase and cell death at G2/M phase. Compounds 4a, 4c and 4d exhibited the most powerful inhibitory range (0.55-0.64 µM) against Topo IIB. Molecular docking study revealed potential interactions of those compounds within the ATP catalytic binding domain of Topo-IIB with high scores. In conclusion, the novel Naphthoquinone-hydrazinotriazolothiadiazine analogues could serve as promising anticancer agents through inhibition of Topoisomerase-IIB.

CDH23 Methylation Status and Presbycusis Risk in Elderly Women.

Introduction: Presbycusis, an age-related hearing impairment (ARHI) disease, is the most common cause for HI in adults worldwide. One of the best candidate genes for ARHI susceptibility is Cadherin 23 (CDH23) which encodes stereocilia tip-links of the inner ear sensory hair cell. Although alterations in the methylation status of CpG dinucleotides across various genes were reported to be associated with HI, methylation changes in CDH23 gene have not been reported previously. Objectives: This study aimed at investigating whether DNA methylation level of CDH23 gene at intragenic CpG island overlapping an exonic-intronic region at position chr10:73565570-73565827 (GRCh37/hg19) could be risk factor associated with ARHI. Materials and Methods: We screened for methylation changes in this particular position for CDH23 gene in 50 blood samples of elderly women affected with presbycusis and healthy control cohort. Methylation of CpG sites were assessed using Quantitative methylation-specific PCR (qMSP) following sodium bisulfite DNA conversion chemistry. Methylation levels were normalized against TSH2B reference gene. Results: DNA methylation analysis for the common CpG islands in CDH23 gene revealed 3.27-folds significant increase (p < 0.0001) in methylation profile for ARHI women as compared to healthy controls with an elevated risk odds ratio (OR) of 2.219 [95% CI 1.071-4.597]. Conclusion: Our study is the first of its kind to prove that higher CpG site methylation levels in CDH23 gene are likely to be associated with ARHI.

Down-expression of P2RX2, KCNQ5, ERBB3 and SOCS3 through DNA hypermethylation in elderly women with presbycusis.

CONTEXT: Presbycusis, an age-related hearing impairment (ARHI), represents the most common sensory disability in adults. Today, the molecular mechanisms underlying presbycusis remain unclear. This is in particular due to the fact that ARHI is a multifactorial complex disorder resulting from several genomic factors interacting with lifelong cumulative effects of: disease, diet, and environment. OBJECTIVE: Identification of novel biomarkers for presbycusis. MATERIALS AND METHODS: We selectively ascertained 18 elderly unrelated women lacking environmental and metabolic risk factors. Subsequently, we screened for methylation map changes in blood samples of women with presbycusis as compared to controls, using reduced representation bisulfite sequencing. We focused on hypermethylated cytosine bases located in gene promoters and the first two exons. To elucidate the related gene expression changes, we performed transcriptomic study using gene expression microarray. RESULTS: Twenty-seven genes, known to be expressed in adult human cochlea, were found in the blood cells to be differentially hypermethylated with significant (p < 0.01) methylation differences (>30%) and down-expressed with fold change >1.2 (FDR <0.05). Functional annotation and qRT-PCR further identified P2RX2, KCNQ5, ERBB3 and SOCS3 to be associated with the progression of ARHI. DISCUSSION AND CONCLUSION: Down-expressed genes associated with DNA hypermethylation could be used as biomarkers for understanding complex pathogenic mechanisms underlying presbycusis.

Genetic diversity and haplotype structure of 21 Y-STRs, including nine noncore loci, in South Tunisian Population: Forensic relevance.

Y chromosome STRs (Y-STRs) are being used frequently in forensic laboratories. Previous studies of Y-STR polymorphisms in different groups of the Tunisian population identified low levels of diversity and discrimination capacity (DC) using various commercial marker sets. This definitely limits the use of such systems for Y-STRs genotyping in Tunisia. In our investigation on South Tunisia, 200 unrelated males were typed for the 12 conventional Y-STRs included in the PowerPlex® Y System. Additional set of nine noncore Y-STRs including DYS446, DYS456, DYS458, DYS388, DYS444, DYS445, DYS449, DYS710, and DYS464 markers were genotyped and evaluated for their potential in improving DC. Allele frequency, gene diversity, haplotype diversity (HD), and DC calculation revealed that DYS464 was the most diverse marker followed by DYS710 and DYS449 markers. The standard panel of 12 Y-STRs (DC = 80.5%) and the nine markers were combined to obtain DC of 99%. Among the 198 different haplotypes observed, 196 haplotypes were unique (HD = 99.999). Out of the nine noncore set, six Y-STRs (DYS458, DYS456, DYS449, DYS710, DYS444, and DYS464) had the greatest impact on enhancing DC. Our data provided putative Y-STRs combination to be used for genetic and forensic applications.

NADf chip, a two-color microarray for simultaneous screening of multigene mutations associated with hearing impairment in North African Mediterranean countries.

Hearing impairment (HI) is the most frequent sensory defect. Genetic causes are involved in two thirds of prelingual cases. Moreover, the autosomal recessive HI frequency is increased in countries where there is a high rate of consanguinity, such as in North African Mediterranean countries. This population shares several features, including history and social behavior, that promote the spread of founder mutations. HI is characterized by tremendous heterogeneity in both the genetic and clinical aspects. The identification of the causal mutation is important for early diagnosis, clinical follow-up, and genetic counseling. Addressing the extreme genetic heterogeneity of HI using classic molecular methods would be expensive and time-consuming. We designed a cost-effective North African Deafness chip for rapid and simultaneous analysis of 58 mutations using multiplex PCR coupled with dual-color arrayed primer extension. These mutations are found in North African HI patients and are distributed over 31 exons and five introns in 21 distinct genes. Assay specificity was initially optimized using 103 archived DNA samples of known genotypes. Blind validation of HI-unrelated patients revealed mutant alleles in 13 samples, and these mutations were confirmed by Sanger sequencing. The North African Deafness chip allows for simultaneous genotyping of eight different samples, at a minimal cost and in a single day, and is therefore amenable to large-scale molecular screening of HI in North Africa.