Harnessing Next-Generation Sequencing as a Timely and Accurate Second-Tier Screening Test for Newborn Screening of Inborn Errors of Metabolism.

In this study, we evaluated the implementation of a second-tier genetic screening test using an amplicon-based next-generation sequencing (NGS) panel in our laboratory during the period of 1 September 2021 to 31 August 2022 for the newborn screening (NBS) of six conditions for inborn errors of metabolism: citrullinemia type II (MIM #605814), systemic primary carnitine deficiency (MIM #212140), glutaric acidemia type I (MIM #231670), beta-ketothiolase deficiency (#203750), holocarboxylase synthetase deficiency (MIM #253270) and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency (MIM # 246450). The custom-designed NGS panel can detect sequence variants in the relevant genes and also specifically screen for the presence of the hotspot variant IVS16ins3kb of SLC25A13 by the copy number variant calling algorithm. Genetic second-tier tests were performed for 1.8% of a total of 22,883 NBS samples. The false positive rate for these six conditions after the NGS second-tier test was only 0.017%, and two cases of citrullinemia type II would have been missed as false negatives if only biochemical first-tier testing was performed. The confirmed true positive cases were citrullinemia type II (n = 2) and systemic primary carnitine deficiency (n = 1). The false positives were later confirmed to be carrier of citrullinemia type II (n = 2), carrier of glutaric acidemia type I (n = 1) and carrier of systemic primary carnitine deficiency (n = 1). There were no false negatives reported. The incorporation of a second-tier genetic screening test by NGS greatly enhanced our program's performance with 5-working days turn-around time maintained as before. In addition, early genetic information is available at the time of recall to facilitate better clinical management and genetic counseling.

Phylogenetic and Structural Significance of Dihydrofolate Synthase (folC) Mutations in Drug-Resistant Mycobacterium tuberculosis.

The aim of this study is to investigate the mutation pattern of the folC gene in drug-resistant Mycobacterium tuberculosis (MTB) clinical isolates of global and Hong Kong cohorts. The public sequence read archives of 1,124 MTB genomes from three independent studies were retrieved and folC mutations existing solely in drug-resistant MTB strains were identified. A phylogenetic tree was constructed to analyze the segregation of mutation-related amino acid residues in the FolC structure. These mutation sites were further supported by direct Sanger sequencing of the folC gene among 254 clinical MTB isolates in a Hong Kong cohort. Homology modeling of wild-type and mutated FolC was performed, and the predicted structures were docked with hydroxydihydropteroate, the metabolic derivative of para-aminosalicylic acid (PAS), to evaluate the resultant binding affinity changes. Combining the results of three previous cohorts and our cohort, E40, I43, S150, and E153 are the most frequently affected amino acid residues in resistant isolates. Based on the distribution of mutations in the genome-based phylogenetic tree, lineage-specific mutation patterns were observed. Regarding the segregation of affected amino acid residues, the four most frequently affected residues are all in close proximity of the binding pocket for the PAS derivative. Molecular modeling results showed that mutations at E40, I43, and S150 can alter the structure of FolC putative binding pocket, causing the PAS derivative to bind outside of the now deformed pocket. This might ablate the interaction between the protein and the PAS derivative. To conclude, this study is the first comprehensive mutation pattern and bioinformatics analysis of the folC gene in MTB drug-resistant isolates. The distribution of mutations in phylogenetic lineages and protein structure is reported, analyzed, and discussed.

The FHL2 regulation in the transcriptional circuitry of human cancers.

The Four-and-a-half LIM (FHL)-only protein is a subfamily of protein members under the LIM-only protein family. These proteins are identified by their characteristic four and a half cysteinerich LIM homeodomain. Five members have been categorized into the FHL subfamily, which are FHL1, FHL2, FHL3, FHL4 and activator of CREM in testis (ACT) in human. FHL2 is amongst the most examined members within the family. Fhl2, the gene that code for the protein, is transcriptionally regulated by diverse types of transcription factors, for example, p53, serum response factor (SRF), and specificity protein 1 (Sp1). The expression of FHL2 is found in different tissues and organs and has been reported as a critical participant influencing the wide types of cancer such as breast cancer, gastrointestinal (GI) cancers, liver cancer and prostate cancer. The expression profile of FHL2 appeared to have a significant functional role in the carcinogenesis of these cancers which are mediated by different types of transcription factor including both tumor suppressors and inducers. In this review, we will first describe the molecular network governing FHL2 expression, which focus on the transcription factors conveying FHL2-initiated responses. In the second part, FHL2-linked cancers and the underlying molecular machinery will be discussed. Factors other than transcriptional regulation which may involve the cancer progression such as mutations of fhl2 and posttranslational modifications of the protein will also be mentioned.

The involvement of DNA methylation and histone modification on the epigenetic regulation of embryonic stem cells and induced pluripotent stem cells.

Stem cell research has been developing rapidly in diverse areas such as the fields of genetics and molecular biology over the past decades. Genomic studies on both embryonic stem cells (ESCs) and terminally-differentiated cells illustrated that factors apart from their hereditary information disparity are associated with gene expression patterns of ESCs. Therefore, current research is trying to explore the effects of epigenetic processes in stem cell physiology and phenotypic changes. In-depth analyses of the molecular mechanisms underpinning such epigenetic-mediated functions have also been conducted. These findings suggest the importance of understanding the epigenetic influences in stem cell activities. Accordingly this review will describe the regulatory machineries of stem cells development targeting the two epigenetic processes: (1) DNA methylation and (2) histones modification. In addition, up-to-date findings concerning the functional roles of these processes in stem cells homeostasis will be covered.