Creatine Deficiency Syndromes: Comparison of Screening Methods and Characterization of Four Novel Intronic Variants.

BACKGROUND: Cerebral creatine deficiency syndromes (CCDS) are disorders affecting creatine synthesis or transport. Several methods have been developed to measure creatine and guanidinoacetate (GAA) in different body fluids including methods based on gas chromatography-mass spectrometry (GC-MS) and High-pressure liquid chromatography mass spectrometry (HPLC-MS). The diagnosis of CCDS is then confirmed by sequencing of creatine biosynthesis genes guanidinoacetate methyltransferase (GAMT) and Arginine: glycine amidinotransferase (GATM) and creatine transporter gene solute carrier family 6 member 8 (SLC6A8) or by functional enzymatic assay. The aim of the current study was to find the most reliable and accurate screening method for CCDS by comparing methods using Nuclear Magnetic Resonance spectroscopy (NMR), GC-MS and HPLC-MS. Additionally, this study was performed to estimate the prevalence of CCDS in a cohort of Egyptian patients and potentially to discover novel variants. SUBJECTS AND METHODS: The study was conducted on 150 subjects with clinical signs and symptoms consistent with CCDS. Metabolic profiling of urine samples was performed using three techniques: 1) GC-MS 2) Ultra high-pressure (or performance) liquid chromatography - Tandem Mass Spectrometry (UHPLC- MS/MS) and 3) NMR. RESULTS: The linearity of peak areas for creatine and GAA by UHPLC-MS/MS and NMR covered and exceeded the ranges normally found in urine. The limit of quantification and the inter-day precision results for creatine and GAA were more robust by UHPLC-MS/MS than NMR. Ten cases were identified as being positive for CCDS by our analytical approaches and underwent next generation sequencing (NGS) for GAMT, GATM and SLC6A8 genes. NGS was performed and confirmed one patient with one likely Pathogenic variant in GAMT gene: (NC_000019.10:g.1401317C > G, NP_000147.1:p.Ala54Pro). Additionally, we describe four novel intronic variants in the GATM gene: c.1043-357del and c.1043-357_1043-356insT, and were predicted to activate cryptic acceptor site with potential alteration of splicing, c.979-227G > A was found to significantly alter the Exon Splice Enhancer (ESE) xon Splice Silencer (ESS) motifs ratio and c.1042 + 262del which was found to have no implications on splicing. CONCLUSIONS: Both UHPLC-MS/MS and NMR spectroscopy are comparable to GC-MS in screening for CCDS. Nonetheless, the UHPLC-MS/MS method had better performance than NMR spectroscopy. Additionally, Sequencing of the full length of GATM, GAMT, and SLC6A8 genes is needed to identify intronic variants that could cause CCDS via affecting splice sites.

Characterisation of COVID-19 Pandemic in Paediatric Age Group: A Systematic Review and Meta-Analysis.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is a pandemic first originated in Wuhan the capital of Hubei province, China in December 2019 and then spread globally. It is caused by SARS-CoV-2. Until 1st April 2020, the number of cases worldwide was recorded to be 823,626 with 40,598 deaths. Most of the reported cases were adults with few cases described in children and neonates. OBJECTIVES: We performed a systematic review and meta-analysis to analyse the disease characterisation in paediatric age group including the possibility of vertical transmission to the neonates. METHODS: Articles published up to 2nd April 2020 in PubMed and google Scholar were considered for this study. FINDINGS: The most frequently reported symptoms were cough 49% (95% CI: 42 - 55%) and fever 47% (95% CI: 41- 53%). Lymphopenia and increased Procalcitonin were recorded in (21%, 95% CI: 12 - 30%) and (28%, 95% CI: 18 - 37%) respectively. No sex difference for COVID-19 was found in paediatric age group (p = 0.7). Case fatality rate was 0%. Four out of 58 neonates (6.8%) born to COVID-19 confirmed mothers tested positive for the disease. CONCLUSION: The disease trajectory in Paediatric patients has good prognosis compared to adults. Intensive care unit and death are rare. Vertical transmission and virus shedding in breast milk are yet to be established.