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.

Rapid exome sequencing as the first-tier investigation for diagnosis of acutely and severely ill children and adults in Thailand.

The use of rapid DNA sequencing technology in severely ill children in developed countries can accurately identify diagnoses and positively impact patient outcomes. This study sought to evaluate the outcome of Thai children and adults with unknown etiologies of critical illnesses with the deployment of rapid whole exome sequencing (rWES) in Thailand. We recruited 54 unrelated patients from 11 hospitals throughout Thailand. The median age was 3 months (range, 2 days-55 years) including 47 children and 7 adults with 52% males. The median time from obtaining blood samples to issuing the rWES report was 12 days (range, 5-27 days). A molecular diagnosis was established in 25 patients (46%), resulting in a change in clinical management for 24 patients (44%) resulting in improved clinical outcomes in 16 patients (30%). Four out of seven adult patients (57%) received the molecular diagnosis which led to a change in management. The 25 diagnoses comprised 23 different diseases. Of the 34 identified variants, 15 had never been previously reported. This study suggests that use of rWES as a first-tier investigation tool can provide tremendous benefits in critically ill patients with unknown etiology across age groups in Thailand.

The Thai reference exome (T-REx) variant database.

To maximize the potential of genomics in medicine, it is essential to establish databases of genomic variants for ethno-geographic groups that can be used for filtering and prioritizing candidate pathogenic variants. Populations with non-European ancestry are poorly represented among current genomic variant databases. Here, we report the first high-density survey of genomic variants for the Thai population, the Thai Reference Exome (T-REx) variant database. T-REx comprises exome sequencing data of 1092 unrelated Thai individuals. The targeted exome regions common among four capture platforms cover 30.04 Mbp on autosomes and chromosome X. 345 681 short variants (18.27% of which are novel) and 34 907 copy number variations were found. Principal component analysis on 38 469 single nucleotide variants present worldwide showed that the Thai population is most genetically similar to East and Southeast Asian populations. Moreover, unsupervised clustering revealed six Thai subpopulations consistent with the evidence of gene flow from neighboring populations. The prevalence of common pathogenic variants in T-REx was investigated in detail, which revealed subpopulation-specific patterns, in particular variants associated with erythrocyte disorders such as the HbE variant in HBB and the Viangchan variant in G6PD. T-REx serves as a pivotal addition to the current databases for genomic medicine.

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.

De novo genome assembly and transcriptome sequencing in foot and mantle tissues of Megaustenia siamensis reveals components of adhesive substances.

The semislug Megaustenia siamensis, commonly found in Thailand, is notable for its exceptional capacity to produce biological adhesives, enabling it to adhere to tree leaves even during heavy rainfall. In this study, we generated the first reference genome for M. siamensis using a combination of three sequencing technologies: Illumina's short-read, Pac-Bio's HIFI long-read, and Hi-C. The assembled genome size was 2593 billion base pairs (bp), containing 34,882 protein-coding genes. Our analysis revealed positive selection in pathways associated with the ubiquitin-proteasome system. Furthermore, RNA sequencing of foot and mantle tissues unveiled the primary constituents of the adhesive, including lectin-like proteins (C-lectin, H-lectin, and C1q) and matrilin-like proteins (VWA and EGF). Additionally, antimicrobial peptides were identified. The comprehensive M. siamensis genome and tissue-specific transcriptomic data provided here offer valuable resources for understanding its biology and exploring potential medical applications.

A novel BAG5 variant impairs the ER stress response pathway, causing dilated cardiomyopathy and arrhythmia.

Pathogenic BAG5 variants recently linked to dilated cardiomyopathy (DCM) prompt further investigation into phenotypic, mutational, and pathomechanistic aspects. We explored the clinical and molecular characteristics of DCM associated with BAG5 variants, uncovering the consistently severe manifestations of the disease and its impact on the endoplasmic reticulum (ER) stress response. The analysis involved three siblings affected by DCM and arrhythmia, along with their four unaffected siblings, their unaffected father, and their mother who exhibited arrhythmia. The parents were consanguineous. Exome and Sanger sequencing identified a novel BAG5 variant, c.444_445delGA (p.Lys149AsnfsTer6), homozygous in affected siblings and heterozygous in parents and unaffected siblings. We generated heterozygous and homozygous Bag5 point mutant knock-in (KI) mice and evaluated cardiac pathophysiology under stress conditions, including tunicamycin (TN) administration. Bag5-/- mice displayed no abnormalities up to 12 months old and showed no anomalies during an exercise stress test. However, following TN injection, Bag5-/- mice exhibited significantly reduced left ventricular fractional shortening (LVFS) and ejection fraction (LVEF). Their cardiac tissues exhibited a notable increase in apoptotic cells, despite non-distinctive changes in CHOP and GRP78 levels. Interestingly, only Bag5 KI male mice demonstrated arrhythmia, which was more pronounced in Bag5-/- than in Bag5+/-males. Here, our study reveals a novel BAG5 mutation causing DCM by impairing the ER stress response, with observed sex-specific arrhythmia differences.

Comprehensive genome assembly reveals genetic diversity and carcass consumption insights in critically endangered Asian king vultures.

The Asian king vulture (AKV), a vital forest scavenger, is facing globally critical endangerment. This study aimed to construct a reference genome to unveil the mechanisms underlying its scavenger abilities and to assess the genetic relatedness of the captive population in Thailand. A reference genome of a female AKV was assembled from sequencing reads obtained from both PacBio long-read and MGI short-read sequencing platforms. Comparative genomics with New World vultures (NWVs) and other birds in the Family Accipitridae revealed unique gene families in AKV associated with retroviral genome integration and feather keratin, contrasting with NWVs' genes related to olfactory reception. Expanded gene families in AKV were linked to inflammatory response, iron regulation and spermatogenesis. Positively selected genes included those associated with anti-apoptosis, immune response and muscle cell development, shedding light on adaptations for carcass consumption and high-altitude soaring. Using restriction site-associated DNA sequencing (RADseq)-based genome-wide single nucleotide polymorphisms (SNPs), genetic relatedness and inbreeding status of five captive AKVs were determined, revealing high genomic inbreeding in two females. In conclusion, the AKV reference genome was established, providing insights into its unique characteristics. Additionally, the potential of RADseq-based genome-wide SNPs for selecting AKV breeders was demonstrated.

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.

A LILRB1 variant with a decreased ability to phosphorylate SHP-1 leads to autoimmune diseases.

Inborn errors of immunity are known to cause not only immunodeficiencies and allergies but also autoimmunity. Leukocyte immunoglobulin-like receptor B1 (LILRB1) is a receptor on leukocytes playing a role in regulating immune responses. No phenotypes have been reported to be caused by germline mutations in LILRB1. We aimed to identify the causative variant in a three-generation family with nine members suffering from one of the three autoimmune diseases-Graves' disease, Hashimoto's thyroiditis, or systemic lupus erythematosus. Whole-genome linkage study revealed a locus on chromosome 19q13.4 with the maximum LOD score of 2.71. Whole-exome sequencing identified a heterozygous missense variant, c.479G > A (p. G160E) in LILRB1, located within the chromosomal-linked region, in all nine affected members. The variant has never been previously reported. Jurkat cells transfected with the mutant LILRB1, compared with those with the wild-type LILRB1, showed decreased phosphorylation of both LILRB1 and its downstream protein, SHP-1. Flow cytometry was used to study immunophenotype and revealed that LILRB1 was significantly lower on the surface of activated regulatory T lymphocytes (Treg) cells of patients. Single-cell RNA sequencing showed substantially increased M1-like monocytes in peripheral blood mononuclear cells of affected individuals. This study, for the first time, implicates LILRB1 as a new disease gene for autoimmunity.