An efficient trio-based mini-haplotyping method for genetic diagnosis of phenylketonuria

Objective: The phenylalanine hydroxylase (PAH) locus has high linkage disequilibrium. Haplotypes related to this locus may thus be considered sufficiently informative for genetic diagnosis and carrier screening using multi-allelic markers. In this study, we present an efficient method for haplotype analysis of PAH locus using multiplexing dyes. In addition, we explain how to resolve the dye shift challenge in multiplex short tandem repeat (STR) genotyping. Materials and Methods: One hundred family trios were included in this descriptive study. The forward primer of a tetra-nucleotide STR and the reverse primer of a variable number tandem repeat (VNTR) were labeled with three different non-overlapping dyes 5-carboxyfluorescein (FAM), 6-carboxy-N,N,N�,N�-tetramethylrhodamine (HEX) and 6-carboxy-N,N,N�,N�-tetramethylrhodamine (TAMRA). The polymerase chain reaction (PCR) products from each family trio were multiplexed for capillary electrophoresis and results were analyzed using Peak Scanner software. Results: Multiplexing trio products decreased the cost significantly. The TAMRA labeled products had a significant predictable shift (migrated at a slower electrophoretic rate) relative to the HEX and FAM labeled products. Through our methodology we achieve, the less inter-dye shift than intra-dye shift variance. Correcting the dye shift in the labeled products, according to the reference allele size, significantly decreased the inter-dye variability (P<0.001). Conclusion: Multiplexing trio products helps to detect and resolve the dye shift accurately in each family, which otherwise would result in diagnostic error. The dye system of FAM, HEX and TAMRA is more feasible and cheaper than other dye systems

Multi affected pedigree with congenital microcephaly: WES revealed PNKP gene mutation

Microcephaly is a rare neurological disorder, occurs in both isolated and syndromic forms. This classification could be confusing in rare disorders with variable phenotypic characteristics. However, identification of the causative gene through genetic study would allow determining the definite diagnosis. Here we reported a novel missense variant c.1133A>C (p.Lys378Thr) on the 13th exon of PNKP gene identified by whole exome sequencing (WES) in an Iranian multi-affected family with microcephaly, seizures and developmental delay (MCSZ) disorder. Data analysis suggested this variant as a pathogenic mutation which is co-segregate with the disease in the pedigree. PNKP gene mutation is consistent with the clinical features of the affected family members. Regarding both genetic findings and clinical examinations, the reported pedigree can be considered as another affected family with MCSZ syndrome, which has been reported about 10 cases worldwide. This study proves the application of WES for determining the final diagnosis in complicated neurodevelopmental disorders. © 2018 The Japanese Society of Child Neurolog

Electronic cigarettes cause alteration in cardiac structure and function in diet-induced obese mice.

In spite of the widespread use of electronic cigarettes, also known as e-cigarettes, and the proposed adverse cardiac effects of nicotine, the detrimental effects of e-cigarettes on the heart are not well known. This study examines the detrimental effects of e-cigarettes with nicotine at doses that yield circulating nicotine and cotinine in the ranges similar to the levels found in habitual smokers, and a high fat diet (HFD) on cardiac structure and function in a commonly used model of diet-induced obesity (DIO). C57BL/6J mice on an HFD were exposed to e-cigarette in the presence (2.4% nicotine) or absence (0% nicotine) of nicotine and saline aerosol for 12 weeks. Echocardiographic data demonstrated a decrease in left ventricular (LV) fractional shortening, LV ejection fraction, and velocity of circumferential fiber shortening (VCF) in mice treated with e-cigarette (2.4% nicotine) compared to e-cigarette (0% nicotine) or saline exposed mice. Cardiomyocytes (CMs) of mice treated with e-cigarette (2.4% nicotine) exhibited LV abnormalities, including lipid accumulation (ventricular steatosis), myofibrillar derangement and destruction, and mitochondrial hypertrophy, as revealed by transmission electron microscopy. The detrimental effects of e-cigarettes (2.4% nicotine) on cardiac structure and function was accompanied by increased oxidative stress, plasma free fatty acid levels, CM apoptosis, and inactivation of AMP-activated protein kinase and activation of its downstream target, acetyl-CoA-carboxylase. Our results indicate profound adverse effects of e-cigarettes (2.4% nicotine) on the heart in obese mice and raise questions about the safety of the nicotine e-cigarettes use