Unraveling metagenomics through long-read sequencing: a comprehensive review.

The study of microbial communities has undergone significant advancements, starting from the initial use of 16S rRNA sequencing to the adoption of shotgun metagenomics. However, a new era has emerged with the advent of long-read sequencing (LRS), which offers substantial improvements over its predecessor, short-read sequencing (SRS). LRS produces reads that are several kilobases long, enabling researchers to obtain more complete and contiguous genomic information, characterize structural variations, and study epigenetic modifications. The current leaders in LRS technologies are Pacific Biotechnologies (PacBio) and Oxford Nanopore Technologies (ONT), each offering a distinct set of advantages. This review covers the workflow of long-read metagenomics sequencing, including sample preparation (sample collection, sample extraction, and library preparation), sequencing, processing (quality control, assembly, and binning), and analysis (taxonomic annotation and functional annotation). Each section provides a concise outline of the key concept of the methodology, presenting the original concept as well as how it is challenged or modified in the context of LRS. Additionally, the section introduces a range of tools that are compatible with LRS and can be utilized to execute the LRS process. This review aims to present the workflow of metagenomics, highlight the transformative impact of LRS, and provide researchers with a selection of tools suitable for this task.

Pentanucleotide Repeat Insertions in RAI1 Cause Benign Adult Familial Myoclonic Epilepsy Type 8.

BACKGROUND: Benign adult familial myoclonic epilepsy (BAFME) is an autosomal dominant disorder characterized by cortical tremors and seizures. Six types of BAFME, all caused by pentanucleotide repeat expansions in different genes, have been reported. However, several other BAFME cases remain with no molecular diagnosis. OBJECTIVES: We aim to characterize clinical features and identify the mutation causing BAFME in a large Malian family with 10 affected members. METHODS: Long-read whole genome sequencing, repeat-primed polymerase chain reaction and RNA studies were performed. RESULTS: We identified TTTTA repeat expansions and TTTCA repeat insertions in intron 4 of the RAI1 gene that co-segregated with disease status in this family. TTTCA repeats were absent in 200 Malian controls. In the affected individuals, we found a read with only nine TTTCA repeat units and somatic instability. The RAI1 repeat expansions cause the only BAFME type in which the disease-causing repeats are in a gene associated with a monogenic disorder in the haploinsufficiency state (ie, Smith-Magenis syndrome [SMS]). Nevertheless, none of the Malian patients exhibited symptoms related to SMS. Moreover, leukocyte RNA levels of RAI1 in six Malian BAFME patients were no different from controls. CONCLUSIONS: These findings establish a new type of BAFME, BAFME8, in an African family and suggest that haploinsufficiency is unlikely to be the main pathomechanism of BAFME. © 2023 International Parkinson and Movement Disorder Society. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Host genetic factors of COVID-19 susceptibility and disease severity in a Thai population.

Host genetic factors have been shown to play a role in SARs-CoV-2 infection in diverse populations. However, the genetic landscape differs among various ethnicities; therefore, we explored the host genetic factors associated with COVID-19 disease susceptivity and disease severity in a Thai population. We recruited and genotyped 212 unrelated COVID-19 Thai patients and 36 controls using AxiomTM Human Genotyping SARs-COV-2 array, including 847,384 single nucleotide polymorphisms related to SARs-COV-2 pathogenesis, immune response, and related comorbidity No SNPs passed the genome-wide significance threshold of p value <1 × 10-8. However, with a threshold of p value <1 × 10-5, a locus on chromosome 5q32 was found to have a suggestive association with COVID-19 disease susceptibility (p value 6.9 × 10-6; Q-Q plot λ = 0.805, odds ratio 0.02). Notably, IL17B is a gene located in this linkage disequilibrium block and is previously shown to play a part in inflammation and pneumonia. Additionally, a suggestive locus on chromosome 12q22, harboring EEA1 and LOC643339, was associated with COVID-19 disease severity (p value 1.3 × 10-6 - 4.4 × 10-6, Q-Q plot λ = 0.997, odds ratio 0.28-0.31). EEA1 is involved in viral entry into cells, while LOC643339 is a long non-coding RNA. In summary, our study suggested loci on chromosomes 5q32 and 12q22 to be linked to COVID-19 disease susceptibility and disease severity, respectively. The small sample size of this study may lessen the likelihood that the association found is real, but it could still be true. Further study with a larger cohort is required to confirm these findings.

Identification of a Negative Regulator for Salt Tolerance at Seedling Stage via a Genome-Wide Association Study of Thai Rice Populations.

Salt stress is a major limiting factor in crop production and yield in many regions of the world. The objective of this study was to identify the genes responsible for salt tolerance in Thai rice populations. We performed a genome-wide association study with growth traits, relative water content, and cell membrane stability at the seedling stage, and predicted 25 putative genes. Eleven of them were located within previously reported salt-tolerant QTLs (ST-QTLs). OsCRN, located outside the ST-QTLs, was selected for gene characterization using the Arabidopsis mutant line with T-DNA insertion in the orthologous gene. Mutations in the AtCRN gene led to the enhancement of salt tolerance by increasing the ability to maintain photosynthetic pigment content and relative water content, while the complemented lines with ectopic expression of OsCRN showed more susceptibility to salt stress detected by photosynthesis performance. Moreover, the salt-tolerant rice varieties showed lower expression of this gene than the susceptible rice varieties under salt stress conditions. The study concludes that by acting as a negative regulator, OsCRN plays an important role in salt tolerance in rice.

An integrative analysis of genome-wide methylation and expression in ameloblastoma: A pilot study.

OBJECTIVE: DNA methylation regulates the expression of various genes involved in tumorigenesis. Ameloblastoma is a benign odontogenic jaw tumor. It is locally aggressive with a high level of recurrence. A delay in treatment can lead to severe facial disfigurement. To the best of our knowledge, this is the first integrated analysis of DNA methylation and gene expression in ameloblastoma with the aim to identify genes that may be regulated by DNA methylation. MATERIALS AND METHODS: We used an Infinium MethylationEPIC array to measure genome-wide methylation and the Illumina HiSeq platform to obtain gene expression data in ameloblastoma tissues from five patients and dental follicles from three healthy subjects. An integration analysis was performed using City of Hope CpG Island Analysis Pipeline software. RESULTS: We identified 25,255 differentially methylated CpG sites and 17 differentially methylated CpG islands; six of the islands were negatively correlated with the expression of BAIAP2, DUSP6, FGFR2, FOXF2, NID2, and PAK6. Pyrosequencing and immunostaining techniques were further used to validate FGFR2, NID2, and PAK6. CONCLUSIONS: This analysis identifies a group of novel genes that may be regulated by DNA methylation and will possibly lead to new insights into the pathology and invasion mechanism of ameloblastoma.

TTTCA repeat insertions in an intron of YEATS2 in benign adult familial myoclonic epilepsy type 4.

Epilepsy is a common neurological disorder and identification of its causes is important for a better understanding of its pathogenesis. We previously studied a Thai family with a type of epilepsy, benign adult familial myoclonic epilepsy type 4 (BAFME4), and localized its gene to chromosome 3q26.32-q28. Here, we used single-molecule real-time sequencing and found expansions of TTTTA and insertions of TTTCA repeats in intron 1 of YEATS2 in one affected member of the family. Of all the available members in the family-comprising 13 affected and eight unaffected-repeat-primed PCR and long-range PCR revealed the co-segregation of the TTTCA repeat insertions with the TTTTA repeat expansions and the disease status. For 1116 Thai control subjects, none were found to harbour the TTTCA repeats while four had the TTTTA repeat expansions. Therefore, our findings suggest that BAFME4 is caused by the insertions of the intronic TTTCA repeats in YEATS2. Interestingly, all four types of BAFMEs for which underlying genes have been found (BAFMEs 1, 4, 6 and 7) are caused by the same molecular pathology, suggesting that the insertions of non-coding TTTCA repeats are involved in their pathogenesis.

Genome-wide association study for salinity tolerance at the flowering stage in a panel of rice accessions from Thailand.

BACKGROUND: Salt stress, a major plant environmental stress, is a critical constraint for rice productivity. Dissecting the genetic loci controlling salt tolerance in rice for improving productivity, especially at the flowering stage, remains challenging. Here, we conducted a genome-wide association study (GWAS) of salt tolerance based on exome sequencing of the Thai rice accessions. RESULTS: Photosynthetic parameters and cell membrane stability under salt stress at the flowering stage; and yield-related traits of 104 Thai rice (Oryza sativa L.) accessions belonging to the indica subspecies were evaluated. The rice accessions were subjected to exome sequencing, resulting in 112,565 single nucleotide polymorphisms (SNPs) called with a minor allele frequency of at least 5%. LD decay analysis of the panel indicates that the average LD for SNPs at 20 kb distance from each other was 0.34 (r2), which decayed to its half value (~ 0.17) at around 80 kb. By GWAS performed using mixed linear model, two hundred loci containing 448 SNPs on exons were identified based on the salt susceptibility index of the net photosynthetic rate at day 6 after salt stress; and the number of panicles, filled grains and unfilled grains per plant. One hundred and forty six genes, which accounted for 73% of the identified loci, co-localized with the previously reported salt quantitative trait loci (QTLs). The top four regions that contained a high number of significant SNPs were found on chromosome 8, 12, 1 and 2. While many are novel, their annotation is consistent with potential involvement in plant salt tolerance and in related agronomic traits. These significant SNPs greatly help narrow down the region within these QTLs where the likely underlying candidate genes can be identified. CONCLUSIONS: Insight into the contribution of potential genes controlling salt tolerance from this GWAS provides further understanding of salt tolerance mechanisms of rice at the flowering stage, which can help improve yield productivity under salinity via gene cloning and genomic selection.

Reduction in replication-independent endogenous DNA double-strand breaks promotes genomic instability during chronological aging in yeast.

The mechanism that causes genomic instability in nondividing aging cells is unknown. Our previous study of mutant yeast suggested that 2 types of replication-independent endogenous DNA double-strand breaks (RIND-EDSBs) exist and that they play opposing roles. The first type, known as physiologic RIND-EDSBs, were ubiquitous in the G0 phase of both yeast and human cells in certain genomic locations and may act as epigenetic markers. Low RIND-EDSB levels were found in mutants that lacked chromatin-condensing proteins, such as the high-mobility group box (HMGB) proteins and Sir2. The second type is referred to as pathologic RIND-EDSBs. High pathological RIND-EDSB levels were found in DSB repair mutants. Under normal physiologic conditions, these excess RIND-EDSBs are repaired in much the same way as DNA lesions. Here, chronological aging in yeast reduced physiological RIND-EDSBs and cell viability. A strong correlation was observed between the reduction in RIND-EDSBs and viability in aging yeast cells ( r = 0.94, P < 0.0001). We used galactose-inducible HO endonuclease (HO) and nhp6a∆, an HMGB protein mutant, to evaluate the consequences of reduced physiological RIND-EDSB levels. The HO-induced cells exhibited a sustained reduction in RIND-EDSBs at various levels for several days. Interestingly, we found that lower physiologic RIND-EDSB levels resulted in decreased cell viability ( r = 0.69, P < 0.0001). Treatment with caffeine, a DSB repair inhibitor, increased pathological RIND-EDSBs, which were distinguished from physiologic RIND-EDSBs by their lack of sequences prior to DSB in untreated cells [odds ratio (OR) ≤1]. Caffeine treatment in both the HO-induced and nhp6a∆ cells markedly increased OR ≤1 breaks. Therefore, physiological RIND-EDSBs play an epigenetic role in preventing pathological RIND-EDSBs, a type of DNA damage. In summary, the reduction of physiological RIND-EDSB level is a genomic instability mechanism in chronologically aging cells.-Thongsroy, J., Patchsung, M., Pongpanich, M., Settayanon, S., Mutirangura, A. Reduction in replication-independent endogenous DNA double-strand breaks promotes genomic instability during chronological aging in yeast.

Two novel compound heterozygous BMP1 mutations in a patient with osteogenesis imperfecta: a case report.

BACKGROUND: Osteogenesis imperfecta (OI) is a collagen-related bone dysplasia leading to a susceptibility to fractures. OI can be caused by mutations in several genes including BMP1. It encodes two isoforms, bone morphogenetic protein 1 (BMP1) and mammalian tolloid (mTLD); both have proteolytic activity to remove the C-propeptide from procollagen. CASE PRESENTATION: We report a Thai OI patient who had his first fracture at the age of three months. Using next generation sequencing, we successfully identified two novel compound heterozygous BMP1 mutations. One mutation, c.796_797delTT (p.Phe266Argfs*25) affects both BMP1 and mTLD isoforms, while the other, c.2108-2A > G, affects only the BMP1 isoform. Preservation of the mTLD may explain the relatively less severe clinical phenotype in this patient. Intravenous bisphosphonate was given from the age of 8 months to 5 years. He was free from fractures for 9 months before discontinuation. CONCLUSION: This case expands the mutation spectrum of BMP1, strengthens the correlation between genotype and phenotype, and supports the benefits of bisphosphonate in OI patients with BMP1 mutations.

Short stature, platyspondyly, hip dysplasia, and retinal detachment: an atypical type II collagenopathy caused by a novel mutation in the C-propeptide region of COL2A1: a case report.

BACKGROUND: Heterozygous mutations in COL2A1 create a spectrum of clinical entities called type II collagenopathies that range from in utero lethal to relatively mild conditions which become apparent only during adulthood. We aimed to characterize the clinical, radiological, and molecular features of a family with an atypical type II collagenopathy. CASE PRESENTATION: A family with three affected males in three generations was described. Prominent clinical findings included short stature with platyspondyly, flat midface and Pierre Robin sequence, severe dysplasia of the proximal femora, and severe retinopathy that could lead to blindness. By whole exome sequencing, a novel heterozygous deletion, c.4161_4165del, in COL2A1 was identified. The phenotype is atypical for those described for mutations in the C-propeptide region of COL2A1. CONCLUSIONS: We have described an atypical type II collagenopathy caused by a novel out-of-frame deletion in the C-propeptide region of COL2A1. Of all the reported truncating mutations in the C-propeptide region that result in short-stature type II collagenopathies, this mutation is the farthest from the C-terminal of COL2A1.

Upstream mononucleotide A-repeats play a cis-regulatory role in mammals through the DICER1 and Ago proteins.

A-repeats are the simplest form of tandem repeats and are found ubiquitously throughout genomes. These mononucleotide repeats have been widely believed to be non-functional 'junk' DNA. However, studies in yeasts suggest that A-repeats play crucial biological functions, and their role in humans remains largely unknown. Here, we showed a non-random pattern of distribution of sense A- and T-repeats within 20 kb around transcription start sites (TSSs) in the human genome. Different distributions of these repeats are observed upstream and downstream of TSSs. Sense A-repeats are enriched upstream, whereas sense T-repeats are enriched downstream of TSSs. This enrichment directly correlates with repeat size. Genes with different functions contain different lengths of repeats. In humans, tissue-specific genes are enriched for short repeats of <10 bp, whereas housekeeping genes are enriched for long repeats of ≥10 bp. We demonstrated that DICER1 and Argonaute proteins are required for the cis-regulatory role of A-repeats. Moreover, in the presence of a synthetic polymer that mimics an A-repeat, protein binding to A-repeats was blocked, resulting in a dramatic change in the expression of genes containing upstream A-repeats. Our findings suggest a length-dependent cis-regulatory function of A-repeats and that Argonaute proteins serve as trans-acting factors, binding to A-repeats.

Characteristics of replication-independent endogenous double-strand breaks in Saccharomyces cerevisiae.

BACKGROUND: Replication-independent endogenous double-strand breaks (RIND-EDSBs) occur in both humans and yeast in the absence of inductive agents and DNA replication. In human cells, RIND-EDSBs are hypermethylated, preferentially retained in the heterochromatin and unbound by γ-H2AX. In single gene deletion yeast strains, the RIND-EDSB levels are altered; the number of RIND-EDSBs is higher in strains with deletions of histone deacetylase, endonucleases, topoisomerase, or DNA repair regulators, but lower in strains with deletions of the high-mobility group box proteins or Sir2. In summary, RIND-EDSBs are different from pathologic DSBs in terms of their causes and consequences. In this study, we identified the nucleotide sequences surrounding RIND-EDSBs and investigated the features of these sequences as well as their break locations. RESULTS: In recent work, we detected RIND-EDSBs using ligation mediated PCR. In this study, we sequenced RIND-EDSB PCR products of resting state Saccharomyces cerevisiae using next-generation sequencing to analyze RIND-EDSB sequences. We found that the break locations are scattered across a number of chromosomes. The number of breaks correlated with the size of the chromosomes. Most importantly, the break occurrences had sequence pattern specificity. Specifically, the majority of the breaks occurred immediately after the sequence "ACGT" (P = 2.2E-156). Because the "ACGT" sequence does not occur primarily in the yeast genome, this specificity of the "ACGT" sequence cannot be attributed to chance. CONCLUSIONS: RIND-EDSBs occur non-randomly; that is, they are produced and retained by specific mechanisms. Because these particular mechanisms regulate their generation and they possess potentially specific functions, RIND-EDSBs could be epigenetic marks.

Studying gene and gene-environment effects of uncommon and common variants on continuous traits: a marker-set approach using gene-trait similarity regression.

Genomic association analyses of complex traits demand statistical tools that are capable of detecting small effects of common and rare variants and modeling complex interaction effects and yet are computationally feasible. In this work, we introduce a similarity-based regression method for assessing the main genetic and interaction effects of a group of markers on quantitative traits. The method uses genetic similarity to aggregate information from multiple polymorphic sites and integrates adaptive weights that depend on allele frequencies to accomodate common and uncommon variants. Collapsing information at the similarity level instead of the genotype level avoids canceling signals that have the opposite etiological effects and is applicable to any class of genetic variants without the need for dichotomizing the allele types. To assess gene-trait associations, we regress trait similarities for pairs of unrelated individuals on their genetic similarities and assess association by using a score test whose limiting distribution is derived in this work. The proposed regression framework allows for covariates, has the capacity to model both main and interaction effects, can be applied to a mixture of different polymorphism types, and is computationally efficient. These features make it an ideal tool for evaluating associations between phenotype and marker sets defined by linkage disequilibrium (LD) blocks, genes, or pathways in whole-genome analysis.

Long-read Nanopore sequencing identified D4Z4 contractions in patients with facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is a genetic muscle disorder caused by abnormal expression of the DUX4 protein, commonly resulting from a contraction of D4Z4 repeat units with the presence of a polyadenylation (polyA) signal. More than 10 units of the D4Z4 repeat, with a length of 3.3 kb per unit, are typically required to silence DUX4 expression. Consequently, molecular diagnosis of FSHD is challenging. We used Oxford Nanopore technology to perform whole-genome sequencing of seven unrelated patients with FSHD, their six unaffected parents, and 10 unaffected controls. All seven patients were successfully identified to harbor one to five D4Z4 repeat units and the polyA signal, whereas none of the 16 unaffected individuals met the molecular diagnostic criteria. Our newly developed method provides a straightforward and powerful molecular diagnostic tool for FSHD.

Genome-Wide Association Study of Starch Properties in Local Thai Rice.

Rice (Oryza sativa L.) is the main source of energy for humans and a staple food of high cultural significance for much of the world's population. Rice with highly resistant starch (RS) is beneficial for health and can reduce the risk of disease, especially type II diabetes. The identification of loci affecting starch properties will facilitate breeding of high-quality and health-supportive rice. A genome-wide association study (GWAS) of 230 rice cultivars was used to identify candidate loci affecting starch properties. The apparent amylose content (AAC) among rice cultivars ranged from 7.04 to 33.06%, and the AAC was positively correlated with RS (R2 = 0.94) and negatively correlated with rapidly available glucose (RAG) (R2 = -0.73). Three loci responsible for starch properties were detected on chromosomes 1, 6, and 11. On chromosome 6, the most significant SNP corresponded to LOC_Os06g04200 which encodes granule-bound starch synthase I (GBSSI) or starch synthase. Two novel loci associated with starch traits were LOC_Os01g65810 and LOC_Os11g01580, which encode an unknown protein and a sodium/calcium exchanger, respectively. The markers associated with GBSSI and LOC_Os11g01580 were tested in two independent sets of rice populations to confirm their effect on starch properties. The identification of genes associated with starch traits will further the understanding of the molecular mechanisms affecting starch in rice and may be useful in the selection of rice varieties with improved starch.

HLA-B*46:01:01:01 and HLA-DRB1*09:01:02:01 are associated with anti-rHuEPO-induced pure red cell aplasia.

Treatment of anemia in patients with chronic kidney disease (CKD) with recombinant human erythropoietin (rHuEPO) can be disrupted by a severe complication, anti-rHuEPO-induced pure red cell aplasia (PRCA). Specific HLA genotypes may have played a role in the high incidence of PRCA in Thai patients (1.7/1,000 patient years vs. 0.03/10,000 patient years in Caucasians). We conducted a case-control study in 157 CKD patients with anti-rHuEPO-induced PRCA and 56 controls. The HLA typing was determined by sequencing using a highly accurate multiplex single-molecule, real-time, long-read sequencing platform. Four analytical models were deployed: Model 1 (additive: accounts for the number of alleles), Model 2 (dominant: accounts for only the presence or absence of alleles), Model 3 (adjusted additive with rHuEPO types) and Model 4 (adjusted dominant with rHuEPO types). HLA-B*46:01:01:01 and DRB1*09:01:02:01 were found to be independent risk markers for anti-rHuEPO-induced PRCA in all models [OR (95%CI), p-values for B*46:01:01:01: 4.58 (1.55-13.51), 0.006; 4.63 (1.56-13.75), 0.006; 5.72 (1.67-19.67), 0.006; and 5.81 (1.68-20.09), 0.005; for DRB1*09:01:02:01: 3.99 (1.28-12.49), 0.017, 4.50 (1.32-15.40), 0.016, 3.42 (1.09-10.74), 0.035, and 3.75 (1.08-13.07), 0.038, in Models 1-4, respectively. HLA-B*46:01:01:01 and DRB1*09:01:02:01 are susceptible alleles for anti-rHuEPO-induced PRCA. These findings support the role of HLA genotyping in helping to monitor patients receiving rHuEPO treatment.

Salt stress responses and SNP-based phylogenetic analysis of Thai rice cultivars.

Genetic diversity is important for developing salt-tolerant rice (Oryza sativa L.) cultivars. Certain Thai rice accessions display salt tolerance at the adult or reproductive stage, but their response to salinity at the seedling stage is unknown. In this study, a total of 10 rice cultivars/line, including eight Thai cultivars and standard salt-tolerant cultivar and susceptible line, were screened using a hydroponic system to identify salt-tolerant genotypes at the seedling stage. Different morphophysiological indicators were used to classify tolerant and susceptible genotypes. Phylogenetic analyses were performed to determine the evolutionary relationships between the cultivars. Results showed that 'Lai Mahk', 'Jao Khao', 'Luang Pratahn', and 'Ma Gawk' exhibited salt stress tolerance comparable with the standard salt-tolerance check 'Pokkali'. Whole-exome single-nucleotide polymorphism (SNP)-based phylogenetic analysis showed that the Thai rice cultivars were monophyletic and distantly related to Pokkali and IR29. Lai Mahk and Luang Pratahn were found closely related when using the whole-exome SNPs for the analysis. This is also the case for the analysis of SNPs from 164 salt-tolerance genes and transcription regulatory genes. The tolerant cultivars shared the same haplotype for 16 genes. Overall, the findings of this study identified four salt-stress-tolerant Thai rice cultivars, which could be used in rice breeding programs for salinity tolerance.

Genotypic and phenotypic landscapes of 51 pharmacogenes derived from whole-genome sequencing in a Thai population.

Differences in drug responses in individuals are partly due to genetic variations in pharmacogenes, which differ among populations. Here, genome sequencing of 171 unrelated Thai individuals from all regions of Thailand was used to call star alleles of 51 pharmacogenes by Stargazer, determine allele and genotype frequencies, predict phenotype and compare high-impact variant frequencies between Thai and other populations. Three control genes, EGFR, VDR, and RYR1, were used, giving consistent results. Every individual had at least three genes with variant or altered phenotype. Forty of the 51 pharmacogenes had at least one individual with variant or altered phenotype. Moreover, thirteen genes had at least 25% of individuals with variant or altered phenotype including SLCO1B3 (97.08%), CYP3A5 (88.3%), CYP2C19 (60.82%), CYP2A6 (60.2%), SULT1A1 (56.14%), G6PD (54.39%), CYP4B1 (50.00%), CYP2D6 (48.65%), CYP2F1 (46.41%), NAT2 (40.35%), SLCO2B1 (28.95%), UGT1A1 (28.07%), and SLCO1B1 (26.79%). Allele frequencies of high impact variants from our samples were most similar to East Asian. Remarkably, we identified twenty predicted high impact variants which have not previously been reported. Our results provide information that contributes to the implementation of pharmacogenetic testing in Thailand and other Southeast Asian countries, bringing a step closer to personalized medicine.

Long-read Amplicon Sequencing of the CYP21A2 in 48 Thai Patients With Steroid 21-Hydroxylase Deficiency.

CONTEXT: Congenital adrenal hyperplasia is most commonly caused by 21-hydroxylase deficiency (21-OHD), an autosomal recessive disorder resulting from biallelic pathogenic variants (PVs) in CYP21A2. With a highly homologous pseudogene and various types of single nucleotide and complex structural variants, identification of PVs in CYP21A2 has been challenging. OBJECTIVE: To leverage long-read next-generation sequencing combined with locus-specific polymerase chain reaction (PCR) to detect PVs in CYP21A2 and to determine its diagnostic yield in patients with 21-OHD. METHODS: Forty-eight Thai patients with 21-OHD comprising 38 sporadic cases and 5 pairs of siblings were enrolled. Two previously described locus-specific PCR methods were performed. Amplicons were subject to long-read sequencing. RESULTS: Ninety-six PVs in CYP21A2 in the 48 patients were successfully identified. The combined techniques were able to detect 26 structural chimeric variants (27%; 26/96) in 22 patients with 18 having monoallelic and 4 having biallelic chimeras. The remaining PVs were pseudogene-derived mutations (63%; 60/96), entire gene deletions (2%; 2/96), missense variants (3%; 3/96), a splice-site variant (2%; 2/96), frameshift variants (2%; 2/96), and a nonsense variant (1%; 1/96). Notably, a splice-site variant, IVS7 + 1G > T, which was identified in a pair of siblings, has not previously been reported. CONCLUSIONS: Our approach exploiting locus-specific PCR and long-read DNA sequencing has a 100% diagnostic yield for our cohort of 48 patients with 21-OHD.

The roles of HMGB1-produced DNA gaps in DNA protection and aging biomarker reversal.

The endogenous DNA damage triggering an aging progression in the elderly is prevented in the youth, probably by naturally occurring DNA gaps. Decreased DNA gaps are found during chronological aging in yeast. So we named the gaps "Youth-DNA-GAPs." The gaps are hidden by histone deacetylation to prevent DNA break response and were also reduced in cells lacking either the high-mobility group box (HMGB) or the NAD-dependent histone deacetylase, SIR2. A reduction in DNA gaps results in shearing DNA strands and decreasing cell viability. Here, we show the roles of DNA gaps in genomic stability and aging prevention in mammals. The number of Youth-DNA-GAPs were low in senescent cells, two aging rat models, and the elderly. Box A domain of HMGB1 acts as molecular scissors in producing DNA gaps. Increased gaps consolidated DNA durability, leading to DNA protection and improved aging features in senescent cells and two aging rat models similar to those of young organisms. Like the naturally occurring Youth-DNA-GAPs, Box A-produced DNA gaps avoided DNA double-strand break response by histone deacetylation and SIRT1, a Sir2 homolog. In conclusion, Youth-DNA-GAPs are a biomarker determining the DNA aging stage (young/old). Box A-produced DNA gaps ultimately reverse aging features. Therefore, DNA gap formation is a potential strategy to monitor and treat aging-associated diseases.