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The Journal of Applied Laboratory... Jan 2024Pharmacogenetics or pharmacogenomics (PGx) is the study of the role of inherited or acquired sequence change in drug response. With the rapid evolution of molecular...
BACKGROUND
Pharmacogenetics or pharmacogenomics (PGx) is the study of the role of inherited or acquired sequence change in drug response. With the rapid evolution of molecular techniques, bioinformatic tools, and increased throughput of functional genomic studies, the discovery of PGx associations and clinical implementation of PGx test results have now moved beyond a handful variants in single pharmacogenes and multi-gene panels that interrogate a few pharmacogenes to whole-exome and whole-genome scales. Although some laboratories have adopted next-generation sequencing (NGS) as a testing platform for PGx and other molecular tests, most clinical laboratories that offer PGx tests still use targeted genotyping approaches.
CONTENT
This article discusses primarily the technical considerations for clinical laboratories to develop NGS-based PGx tests including whole-genome and whole-exome sequencing analyses and highlights the challenges and opportunities in test design, content selection, bioinformatic pipeline for PGx allele and diplotype assignment, rare variant classification, reporting, and briefly touches a few additional areas that are important for successful clinical implementation of PGx results.
SUMMARY
The accelerated speed of technology development associated with continuous cost reduction and enhanced ability to interrogate complex genome regions makes it inevitable for most, if not all, clinical laboratories to transition PGx testing to an NGS-based platform in the near future. It is important for laboratories and relevant professional societies to recognize both the potential and limitations of NGS-based PGx profiling, and to work together to develop a standard and consistent practice to maximize the variant or allele detection rate and utility of PGx testing.
Topics: Humans; Pharmacogenetics; Alleles; Computational Biology; High-Throughput Nucleotide Sequencing
PubMed: 38167765
DOI: 10.1093/jalm/jfad097 -
International Journal of Molecular... Sep 2023Imprinted genes play diverse roles in mammalian development, homeostasis, and disease. Most imprinted chromosomal domains express one or more long non-coding RNAs... (Review)
Review
Imprinted genes play diverse roles in mammalian development, homeostasis, and disease. Most imprinted chromosomal domains express one or more long non-coding RNAs (lncRNAs). Several of these lncRNAs are strictly nuclear and their mono-allelic expression controls in the expression of protein-coding genes, often developmentally regulated. Some imprinted lncRNAs act in as well, controlling target gene expression elsewhere in the genome. The regulation of imprinted gene expression-including that of imprinted lncRNAs-is susceptible to stochastic and environmentally triggered epigenetic changes in the early embryo. These aberrant changes persist during subsequent development and have long-term phenotypic consequences. This review focuses on the expression and the - and -regulatory roles of imprinted lncRNAs and describes human disease syndromes associated with their perturbed expression.
Topics: Humans; Animals; RNA, Long Noncoding; Alleles; Embryo, Mammalian; Epigenesis, Genetic; Homeostasis; Mammals
PubMed: 37686455
DOI: 10.3390/ijms241713647 -
Nature Communications Oct 2023Differential allele-specific expression (ASE) is a powerful tool to study context-specific cis-regulation of gene expression. Such effects can reflect the interaction...
Differential allele-specific expression (ASE) is a powerful tool to study context-specific cis-regulation of gene expression. Such effects can reflect the interaction between genetic or epigenetic factors and a measured context or condition. Single-cell RNA sequencing (scRNA-seq) allows the measurement of ASE at individual-cell resolution, but there is a lack of statistical methods to analyze such data. We present Differential Allelic Expression using Single-Cell data (DAESC), a powerful method for differential ASE analysis using scRNA-seq from multiple individuals, with statistical behavior confirmed through simulation. DAESC accounts for non-independence between cells from the same individual and incorporates implicit haplotype phasing. Application to data from 105 induced pluripotent stem cell (iPSC) lines identifies 657 genes dynamically regulated during endoderm differentiation, with enrichment for changes in chromatin state. Application to a type-2 diabetes dataset identifies several differentially regulated genes between patients and controls in pancreatic endocrine cells. DAESC is a powerful method for single-cell ASE analysis and can uncover novel insights on gene regulation.
Topics: Humans; Alleles; Gene Expression Regulation; Cell Differentiation; Computer Simulation; Diabetes Mellitus, Type 2; Single-Cell Analysis; Sequence Analysis, RNA; Gene Expression Profiling
PubMed: 37813843
DOI: 10.1038/s41467-023-42016-9 -
American Journal of Human Genetics Aug 2023Hyperferritinemia is a frequent finding in several conditions, both genetic and acquired. We previously studied eleven healthy subjects from eight different families...
Hyperferritinemia is a frequent finding in several conditions, both genetic and acquired. We previously studied eleven healthy subjects from eight different families presenting with unexplained hyperferritinemia. Their findings suggested the existence of an autosomal-recessive disorder. We carried out whole-exome sequencing to detect the genetic cause of hyperferritinemia. Immunohistochemistry and flow cytometry assays were performed on liver biopsies and monocyte-macrophages to confirm the pathogenic role of the identified candidate variants. Through a combined approach of whole-exome sequencing and homozygosity mapping, we found bi-allelic STAB1 variants in ten subjects from seven families. STAB1 encodes the multifunctional scavenger receptor stabilin-1. Immunohistochemistry and flow cytometry analyses showed absent or markedly reduced stabilin-1 in liver samples, monocytes, and monocyte-derived macrophages. Our findings show a strong association between otherwise unexplained hyperferritinemia and bi-allelic STAB1 mutations suggesting the existence of another genetic cause of hyperferritinemia without iron overload and an unexpected function of stabilin-1 in ferritin metabolism.
Topics: Humans; Hyperferritinemia; Iron Overload; Ferritins; Macrophages; Alleles
PubMed: 37490907
DOI: 10.1016/j.ajhg.2023.07.004 -
Allelic effects on uromodulin aggregates drive autosomal dominant tubulointerstitial kidney disease.EMBO Molecular Medicine Dec 2023Missense mutations in the uromodulin (UMOD) gene cause autosomal dominant tubulointerstitial kidney disease (ADTKD), one of the most common monogenic kidney diseases....
Missense mutations in the uromodulin (UMOD) gene cause autosomal dominant tubulointerstitial kidney disease (ADTKD), one of the most common monogenic kidney diseases. The unknown impact of the allelic and gene dosage effects and fate of mutant uromodulin leaves open the gap between postulated gain-of-function mutations, end-organ damage and disease progression in ADTKD. Based on two prevalent missense UMOD mutations with divergent disease progression, we generated Umod and Umod knock-in mice that showed strong allelic and gene dosage effects on uromodulin aggregates and activation of ER stress and unfolded protein and immune responses, leading to variable kidney damage. Deletion of the wild-type Umod allele in heterozygous Umod mice increased the formation of uromodulin aggregates and ER stress. Studies in kidney tubular cells confirmed differences in uromodulin aggregates, with activation of mutation-specific quality control and clearance mechanisms. Enhancement of autophagy by starvation and mTORC1 inhibition decreased uromodulin aggregates. These studies substantiate the role of toxic aggregates as driving progression of ADTKD-UMOD, relevant for therapeutic strategies to improve clearance of mutant uromodulin.
Topics: Animals; Mice; Alleles; Disease Progression; Kidney; Kidney Diseases; Mutation; Uromodulin
PubMed: 37885358
DOI: 10.15252/emmm.202318242 -
Methods in Molecular Biology (Clifton,... 2024The diagnosis of alpha-1-antitrypsin (A1AT) deficiency is established by quantitation of protein concentration in serum (immunoassay) followed by determination of...
The diagnosis of alpha-1-antitrypsin (A1AT) deficiency is established by quantitation of protein concentration in serum (immunoassay) followed by determination of specific allelic variants by phenotyping (isoelectric focusing (IEF) gel electrophoresis) and/or allele-specific genotyping. Various phenotyping and genotyping methodologies are available, and each has their own advantages and disadvantages. As an alternative, mass spectrometry is emerging as a powerful tool in the identification and quantitation of proteins and peptides. The method described here, referred to as proteotyping, is a proteomic method using trypsin digestion and tandem mass spectrometry that detects the most common deficiency alleles, S and Z, associated with A1AT deficiency.This qualitative mass spectrometry method is based on the principle that the S and Z mutations lead to amino acid changes which result in a change in the mass of the A1AT protein. When the A1AT protein is proteolytically digested, multiple peptides are generated, two of which include the sites of the S and Z mutations, respectively. Peptides generated from wild-type A1AT (M alleles) differ in sequence and mass from peptides generated from the S and Z alleles at these two specific locations. The mass difference allows for differentiation of S and Z peptides, representing the deficiency alleles, from non-S and non-Z peptides, representing the wild-type alleles (M). Interpretation of the peptide patterns in conjunction with A1AT quantitation by immunoassay allows for an accurate assessment for the presence of deficiency alleles in the majority of patients.
Topics: Humans; Liquid Chromatography-Mass Spectrometry; Chromatography, Liquid; Proteomics; Tandem Mass Spectrometry; Alleles
PubMed: 38108970
DOI: 10.1007/978-1-0716-3605-3_9 -
Gene Dec 2023Autism spectrum disorder (ASD) is neurodevelopmental disorder characterized by stereotyped behavior and deficits in communication and social interactions. To date,... (Meta-Analysis)
Meta-Analysis Review
BACKGROUND
Autism spectrum disorder (ASD) is neurodevelopmental disorder characterized by stereotyped behavior and deficits in communication and social interactions. To date, numerous studies have investigated the associations between genetic variants and ASD risk. However, the results of these published studies lack a clear consensus. In the present study, we performed a systematic review on the association between genetic variants and ASD risk. Meanwhile, we conducted a meta-analysis on available data to identify the association between the single nucleotide polymorphisms (SNPs) of candidate genes and ASD risk.
METHODS
We systematically searched public databases including English and Chinese from their inception to August 1, 2022. Two independent reviewers extracted data and assessed study quality. Odds ratio and 95 % confidence interval were used as effect indexes to evaluate the association between the SNPs of candidate genes and the risk of ASD. Heterogeneity was explored through subgroup, sensitivity, and meta-regression analyses. Publication bias was assessed by using Egger's and Begg's tests for funnel plot asymmetry. In addition, TSA analysis were performed to confirm the study findings.
RESULTS
We summarized 84 SNPs of 32 candidate genes from 81 articles included in the study. Subsequently, we analyzed 16 SNPs of eight genes by calculating pooled ORs, and identified eight significant SNPs of contactin associated protein 2 (CNTNAP2), methylentetrahydrofolate reductase (MTHFR), oxytocin receptor (OXTR), and vitamin D receptor (VDR). Results showed that seven SNPs, including the CNTNAP2 rs2710102 (homozygote, heterozygote, dominant and allelic models) and rs7794745 (heterozygote and dominant models), MTHFR C677T (homozygote, heterozygote, dominant, recessive and allelic models) and A1298C (dominant and allelic models), OXTR rs2254298 (homozygote and recessive models), VDR rs731236 (homozygote, dominant, recessive and allelic models) and rs2228570 (homozygote and recessive models), were showed to be correlated with an increased ASD risk. By contrast, the VDR rs7975232 was correlated with a decreased the risk of ASD under the homozygote and allelic models.
CONCLUSION
Our study summarized research evidence on the genetic variants of ASD and provides a broad and detailed overview of ASD risk genes. The C677T and A1298C polymorphisms of MTHFR, rs2710102 and rs7794745 polymorphisms of CNTNAP2, rs2254298 polymorphism of OXTR, and rs731236 and rs2228570 polymorphisms of VDR were genetic risk factors. The rs7975232 polymorphism of VDR was a genetic protective factor for ASD. Our study provides novel clues to clinicians and healthcare decision-makers to predict ASD susceptibility.
Topics: Humans; Autism Spectrum Disorder; Genetic Predisposition to Disease; Polymorphism, Single Nucleotide; Alleles; Heterozygote; Methylenetetrahydrofolate Reductase (NADPH2)
PubMed: 37598788
DOI: 10.1016/j.gene.2023.147723 -
European Journal of Human Genetics :... Sep 2023DNA methylation classifiers ("episignatures") help to determine the pathogenicity of variants of uncertain significance (VUS). However, their sensitivity is limited due...
DNA methylation classifiers ("episignatures") help to determine the pathogenicity of variants of uncertain significance (VUS). However, their sensitivity is limited due to their training on unambiguous cases with strong-effect variants so that the classification of variants with reduced effect size or in mosaic state may fail. Moreover, episignature evaluation of mosaics as a function of their degree of mosaicism has not been developed so far. We improved episignatures with respect to three categories. Applying (i) minimum-redundancy-maximum-relevance feature selection we reduced their length by up to one order of magnitude without loss of accuracy. Performing (ii) repeated re-training of a support vector machine classifier by step-wise inclusion of cases in the training set that reached probability scores larger than 0.5, we increased the sensitivity of the episignature-classifiers by 30%. In the newly diagnosed patients we confirmed the association between DNA methylation aberration and age at onset of KMT2B-deficient dystonia. Moreover, we found evidence for allelic series, including KMT2B-variants with moderate effects and comparatively mild phenotypes such as late-onset focal dystonia. Retrained classifiers also can detect mosaics that previously remained below the 0.5-threshold, as we showed for KMT2D-associated Kabuki syndrome. Conversely, episignature-classifiers are able to revoke erroneous exome calls of mosaicism, as we demonstrated by (iii) comparing presumed mosaic cases with a distribution of artificial in silico-mosaics that represented all the possible variation in degree of mosaicism, variant read sampling and methylation analysis.
Topics: Humans; Phenotype; DNA Methylation; Abnormalities, Multiple; Alleles; Mosaicism
PubMed: 37365401
DOI: 10.1038/s41431-023-01406-9 -
Nature Protocols Sep 2023A lack of generic and effective genetic manipulation methods for Pseudomonas has restricted fundamental research and utilization of this genus for biotechnology... (Review)
Review
A lack of generic and effective genetic manipulation methods for Pseudomonas has restricted fundamental research and utilization of this genus for biotechnology applications. Phage-encoded homologous recombination (PEHR) is an efficient tool for bacterial genome engineering. This PEHR system is based on a lambda Red-like operon (BAS) from Pseudomonas aeruginosa phage Ab31 and a Rac bacteriophage RecET-like operon (Rec-TE) from P. syringae pv. syringae B728a and also contains exogenous elements, including the RecBCD inhibitor (Redγ or Pluγ) or single-stranded DNA-binding protein (SSB), that were added to enhance the PEHR recombineering efficiency. To solve the problem of false positives in Pseudomonas editing with the PEHR system, the processive enzyme Cas3 with a minimal Type I-C Cascade-based system was combined with PEHR. This protocol describes the utilization of a Pseudomonas-specific PEHR-Cas3 system that was designed to universally and proficiently modify the genomes of Pseudomonas species. The pipeline uses standardized cassettes combined with the concerted use of SacB counterselection and Cre site-specific recombinase for markerless or seamless genome modification, in association with vectors that possess the selectively replicating template R6K to minimize recombineering background. Compared with the traditional allelic exchange editing method, the PEHR-Cas3 system does not need to construct suicide plasmids carrying long homologous arms, thus simplifying the experimental procedure and shortening the traceless editing period. Compared with general editing systems based on phage recombinases, the PEHR-Cas3 system can effectively improve the screening efficiency of mutants using the cutting ability of Cas3 protein. The entire procedure requires ~12 days.
Topics: Humans; Bacteriophages; Alleles; Homologous Recombination; Pseudomonas; CRISPR-Associated Proteins
PubMed: 37626246
DOI: 10.1038/s41596-023-00856-1 -
American Journal of Transplantation :... Sep 2023Technological advances in the field of histocompatibility have allowed us to define anti-human leukocyte antigen (HLA) antibody specificity at the allelic level....
Technological advances in the field of histocompatibility have allowed us to define anti-human leukocyte antigen (HLA) antibody specificity at the allelic level. However, how allele-specific antibodies affect organ allocation is poorly studied. We examined allelic specificities of class I HLA antibodies in 6726 consecutive serum samples from 2953 transplant candidates and evaluated their impact on the corresponding crossmatch and organ allocation. Out of 17 class I HLA antigens represented by >1 allele in the LABScreen single antigen bead assay, 12 had potential allele-specific reactivity. Taking advantage of our unbiased cohort of deceased donor-candidate testing (123,135 complement-dependent cytotoxicity crossmatches between 2014 and 2017), we estimated that the presence of allele-specific antibody detected using a single antigen bead assay (median fluorescence intensity, >3000) against only the rare allele was a poor predictor of a positive complement-dependent cytotoxicity crossmatch, with a positive predictive value of 0% to 7%, compared with 52.5% in allele-concordant class I HLA antibodies against A or B locus antigens. Further, we confirmed allele-specific reactivity using flow crossmatch in 3 scenarios: A11:01/A11:02, A68:01/A68:02, and B44:02/B44:03. Our results suggest that allele-specific antibodies may unnecessarily exclude transplant candidates (up to 10%) from organ offers by overcalling unacceptable antigens; incorporation of selective reactivity pattern in allocation may promote precision matching and more equitable allocation.
Topics: Humans; Isoantibodies; Alleles; Histocompatibility Testing; Histocompatibility Antigens Class I; HLA Antigens; Antigens
PubMed: 37257653
DOI: 10.1016/j.ajt.2023.05.021