Bioinformatic analysis of KIT juxtamembrane domain mutations in Syrian GIST patients: jigsaw puzzle completed.

BACKGROUND: The huge number of detected somatic KIT mutations highlights the necessity of in silico analyses that are almost absent in the relevant medical literature. The aim of this study is to report the mutation spectrum analysis of exon 11 encoding the juxtamembrane (JM) domain of the KIT gene in a group of Syrian GIST patients. METHODS: Forty-eight formalin-fixed paraffin-embedded GIST tissue samples, collected between 2006 and 2016, were retrieved from the pathological archives and analyzed for KIT exon 11 mutations by DNA sequencing. Structural/functional impact of detected variants was predicted using several bioinformatic tools. RESULTS: Twenty-one different variants have been detected in intron 10, exon 11, and intron 11 of the KIT gene, eight of which were novel changes. Mutations in exon 11 of the KIT gene were detected in 28 of 48 (58.3%) GIST patients and predicted to be pathogenic and cancer promoting. Specifically, age above 60 was very significantly associated with the negative selection of deletion mutations (p = .007), a phenomenon that points to deletion severity. CONCLUSIONS: Six bioinformatic tools have proved efficient in predicting the impact of detected KIT variations in view of published structural, experimental, and clinical findings.

Salivary-Based Cell-Free Mitochondrial DNA Level Is an Independent Prognostic Biomarker for Patients with Head and Neck Squamous Cell Carcinoma.

Changes in the copy numbers of cell-free nuclear DNA (cf-nDNA) and cell-free mitochondrial DNA (cf-mtDNA) have shown promising diagnostic utilities among patients with head and neck squamous cell carcinoma (HNSCC). Considering the absence of objective prognostic tools for HNSCC surveillance, this study aimed to assess the utility of saliva-based cf-nDNA and cf-mtDNA in predicting the overall survival of patients with HNSCC. The study included ninety-four patients with a confirmed HNSCC diagnosis with a mean follow-up time of 32.04 months (±19.1). A saliva-based liquid biopsy was collected from each patient. A multiplex quantitative PCR was used to determine the absolute number of cf-nDNA and cf-mtDNA. The Kaplan-Meier estimator and Cox proportional hazards regression models were used to assess overall survival. The absolute copy numbers of cf-nDNA and cf-mtDNA were statistically significantly higher among the deceased patients than among the censored ones (p < 0.05). Individuals with elevated levels of cf-nDNA or cf-mtDNA were associated with a significantly poorer overall survival (p ≤ 0.05). A univariate analysis showed that only the absolute copy number of cf-mtDNA was the sole predictor of overall survival. However, the multivariate analysis showed that all the absolute copy numbers of cf-nDNA, the absolute copy numbers of cf-mtDNA, and the stage of HNSCC were predictors of overall survival. Our study confirms that saliva is a reliable and non-invasive source of data that can be used to predict the overall survival of patients with HNSCC, where cf-mtDNA levels act as the sole predictor.

Association Between STAT4 rs7574865 Polymorphism and Rheumatoid Arthritis: Debate Unresolved.

BACKGROUND: STAT4 rs7574865 polymorphism has been evidently associated with susceptibility to Rheumatoid Arthritis (RA) in European and Eastern Asian populations, whereas studies in other countries reported otherwise. OBJECTIVE: We investigated the distribution of STAT4 rs7574865 polymorphism in a group of Syrian RA patients. METHODS: Eighty-one RA patients and forty healthy controls were enrolled and STAT4 rs7574865 was genotyped by direct sequencing. RA patients were stratified according to Anti-Citrullinated Protein Antibodies (ACPA) status for analysis. RESULTS: Minor T allele frequencies were 30.4%, 16.7%, and 23.8% in ACPA-positive RA patients, ACPA-negative RA patients, and healthy controls, respectively. No significant differences in STAT4 rs7574865 allele/genotype frequencies were found between ACPA-positive RA patients, ACPA-negative RA patients, and healthy controls (P>0.05). CONCLUSION: STAT4 rs7574865 TT genotype showed a potential impact on ACPA positivity in Syrian RA patients. However, STAT4 rs7574865 effect on RA onset and severity is minor compared to other genetic factors such as HLA-DRB1 shared epitope alleles.

Seropositivity of Hepatitis B and C among Syrian Multi-transfused Patients with Hemoglobinopathy.

BACKGROUND AND OBJECTIVES: Blood transfusion is a lifesaving therapy for patients with hemoglobinopathies. However, the need of frequent transfusion carries the risk of transmitting hepatitis B and C infections which are intermediately prevalent in Syria. Despite screening blood donations with sensitive methods, the risk of transmission is still present when infectious blood is donated within the window period. This study aimed to investigate the incidence of HBV and HCV seropositivity, and its association with multiple transfusions among Syrian hemoglobinopathies patients. MATERIALS AND METHODS: HBsAg, anti-HBc, anti-HBs and anti-HCV were tested for 159 Syrian multi-transfused patients by Enzyme-Linked Immunosorbent Assay (ELISA). RESULTS: Thirty-nine of 159 (24.5%) multi-transfused patients were HBsAg/anti-HBc or anti-HCV positive, 26 (16%) of which never visited the dentist, and they either tested postsurgically negative for HBsAg and anti-HCV or never underwent a surgical procedure. On the contrary of anti-HCV seropositivity, HBsAg/anti-HBc seropositivity was significantly associated with the number of blood transfusions, number of blood units and age (P < 0.001). CONCLUSION: About one-sixth of our patients most likely acquired HBV/HCV infection via blood transfusion. Administering HBV vaccine, ensuring the immune status, and monitoring hepatitis markers might considerably minimize the incidence of viral hepatitis among multi-transfused patients.

Frequency of BCR-ABL Transcript Types in Syrian CML Patients.

Background. In Syria, CML patients are started on tyrosine kinase inhibitors (TKIs) and monitored until complete molecular response is achieved. BCR-ABL mRNA transcript type is not routinely identified, contrary to the recommendations. In this study we aimed to identify the frequency of different BCR-ABL transcripts in Syrian CML patients and highlight their significance on monitoring and treatment protocols. Methods. CML patients positive for BCR-ABL transcripts by quantitative RT-PCR were enrolled. BCR-ABL transcript types were investigated using a home-made PCR method that was adapted from published protocols and optimized. The transcript types were then confirmed using a commercially available research kit. Results. Twenty-four transcripts were found in 21 patients. The most common was b2a2, followed by b3a2, b3a3, and e1a3 present solely in 12 (57.1%), 3 (14.3%), 2 (9.5%), and 1 (4.8%), respectively. Three samples (14.3%) contained dual transcripts. While b3a2 transcript was apparently associated with warning molecular response to imatinib treatment, b2a2, b3a3, and e1a3 transcripts collectively proved otherwise (P = 0.047). Conclusion. It might be advisable to identify the BCR-ABL transcript type in CML patients at diagnosis, using an empirically verified method, in order to link the detected transcript with the clinical findings, possible resistance to treatment, and appropriate monitoring methods.

Screening of GJB6 gene large deletions among Syrians with congenital hearing impairment.

BACKGROUND: Research into the genetics of congenital hearing impairment in the Syrian population, where cases are noticeably encountered, is still in its infancy. AIMS: Our goal was to estimate the frequencies of the del(GJB6-D13S1830) and del(GJB6-D13S1854) mutations in a group of Syrians with autosomal recessive nonsyndromic hearing loss (ARNSHL). METHODS: Forty-one unrelated Syrian probands, already screened for exon 2, GJB2 gene mutations, were reanalyzed for del(GJB6-D13S1830) and del(GJB6-D13S1854) mutations by polymerase chain reaction. RESULTS: The del(GJB6-D13S1830) mutation was only found in homozygosity in 1 of 41 probands (2.43%), while the del(GJB6-D13S1854) mutation was not detected in any of the enrolled patients. Coexistence of GJB2 and GJB6 mutations was not encountered in any case. CONCLUSIONS: Our study reports the first case in Syria with the del(GJB6-D13S1830) mutation. This mutation might be considered in the diagnosis and genetic counseling of inherited hearing impairment in the Syrian population.

Prevalence of "anti-HBc alone" among Syrian blood donors.

INTRODUCTION: We aimed to evaluate the prevalence of "anti-HBc alone" among Syrian blood donors, highlighting the possibility of representing occult HBV infection. METHODOLOGY: Sera of 3,896 healthy blood donors were tested for both HBsAg and anti-HBc. HBsAg-negative, anti-HBc-positive samples were further tested for the antibodies to HBsAg (anti-HBs), and "anti-HBc alone" sera were tested for HBV DNA. RESULTS: Of 3,830 HBsAg-negative donors, 63 were "anti-HBc alone" donors, five of whom were HBV DNA positive. CONCLUSIONS: Greater consideration should be given to the "anti-HBc alone" serological profile in blood screening, premarital testing, organ transplantation tests, and other HBV transmission-related procedures in Syria.

Rethinking therapeutic decisions for hepatitis B infection in Syria: insights into molecular monitoring.

INTRODUCTION: Hepatitis B virus patients are usually treated in Syria with alpha interferon and nucleos(t)ide analogues. Genotypic viral factors causing inadequate response or relapse following initial response are not routinely investigated. This study aimed to explore and discuss local therapeutic decisions from a molecular perspective. METHODOLOGY: Fifty patients with hepatitis B from Syria were tested for HBV genotyping and drug-resistance mutations by DNA sequencing. RESULTS: All patients had genotype D, which is characterized by relatively low response to interferon-based therapy. Drug-resistant viral mutant variants were detected in one fifth of the enrolled patients, and distributed similarly in both nucleos(t)ide analogues-naïve and -treated patients. However, nucleos(t)ide analogues-based therapy was associated with the existence of more mutations and hence increased resistance. CONCLUSIONS: Investigating HBV genotypes and drug-resistance mutations to support treatment decisions is critically needed for efficient therapy and patients' survival.

Comparative evaluation of published cytomegalovirus primers for rapid real-time PCR: which are the most sensitive?

Standardization of human cytomegalovirus (CMV) PCR is highly recommended. As primer design is essential for PCR sensitivity, this study evaluated all published CMV primer pairs to identify the most sensitive for single-round real-time PCR. PubMed (1993-2004) was searched for original papers aimed at CMV PCR. Fifty-seven papers were identified revealing 82 different primer pairs. Of these, 17 primer sets were selected for empirical study, as they were either used in real-time PCR or were evaluated comparatively by conventional PCR. After optimizing the PCR conditions, these primer sets were evaluated by real-time PCR using a SYBR Green format. Analytical sensitivities were assessed by testing the reference standard CMV strain AD169. A blast search was performed to identify mismatches with published sequences. Additionally, 60 clinical samples were tested with the three primer sets showing highest analytical sensitivity and the best match to all CMV strains. Three primer sets located in the glycoprotein B (UL55) gene region were found to be the most sensitive using strain AD169. However, two of these showed a considerable number of mismatches with clinical isolates in a blast search. Instead, two other pairs from the lower matrix phosphoprotein (UL83) gene and DNA polymerase (UL54) gene showed reasonable sensitivity and no mismatches with clinical isolates. These three pairs were further tested with clinical samples, which indicated that the two primer sets from UL55 and UL54 were the most sensitive. Interestingly, the analytical sensitivity of the PCR was inversely correlated with the size of the PCR product. In conclusion, these two primer pairs are recommended for a standardized, highly sensitive, real-time PCR.

Optimization of human cytomegalovirus LightCycler real-time PCR.

BACKGROUND: Real-time PCR has been widely considered as a powerful tool for the evaluation of Human Cytomegalovirus (CMV) DNA kinetics. Successful PCR relies on optimization, which is an extremely demanding procedure. Nevertheless, certain values could be optimal for most primers in use. METHODOLOGY: Seventeen CMV primer sets recommended in the literature were selected for optimization in terms of MgCl2 and primers concentrations as well as annealing temperature using the LightCycler instrument and SYBR Green I detection format. Optimal values were considered as those showing the lowest crossing point (Cp), the highest fluorescence intensity, the steepest sigmoid curve slope, and the absence of non-specific PCR products. RESULTS: Optimal values for most studied primers were found to be 3 mM for MgCl2 concentration, 0.5 microM and 0.6 microM for primers concentration, and 55 degrees C for annealing temperature. CONCLUSION: Adopting the resulting values for CMV-specific primers generally used in single-target real-time PCR assays with the same thermal cycler may guarantee their efficient performance minimizing cost and time needed for optimization.