Sex- and age-dependent association of SLC11A1 polymorphisms with tuberculosis in Chinese: a case control study

BACKGROUND: Host genetic factors are important determinants in tuberculosis (TB). The SLC11A1 (or NRAMP1) gene has been studied extensively for genetic association with TB, but with inconsistent findings. In addition, no study has yet looked into the effect of sex and age on the relationship between SLC11A1 polymorphisms and TB. METHODS: A case-control study was conducted. In total, 278 pulmonary TB patients and 282 sex- and age-matched controls without TB were recruited. All subjects were ethnic Chinese. On the basis of linkage disequilibrium pattern, three genetic markers from SLC11A1 and one from the nearby IL8RB locus were selected and examined for association with TB susceptibility. These markers were genotyped using single strand conformation polymorphism analysis or fragment analysis of amplified products. RESULTS: Statistically significant differences in allele (P = 0.0165, OR = 1.51) and genotype (P = 0.0163, OR = 1.59) frequencies of the linked markers SLC6a/b (classically called D543N and 3'UTR) of the SLC11A1 locus were found between patients and controls. With stratification by sex, positive associations were identified in the female group for both allele (P = 0.0049, OR = 2.54) and genotype (P = 0.0075, OR = 2.74) frequencies. With stratification by age, positive associations were demonstrated in the young age group (age ≤65 years) for both allele (P = 0.0047, OR = 2.52) and genotype (P = 0.0031, OR = 2.92) frequencies. All positive findings remained significant even after correction for multiple comparisons. No significant differences were noted in either the male group or the older age group. No significant differences were found for the other markers (one SLC11A1 marker and one IL8RB marker) either. CONCLUSION: This study confirmed the association between SLC11A1 and TB susceptibility and demonstrated for the first time that the association was restricted to females and the young age group

The use of genome-wide DNA methylation microarray to study both the common and rare diseases

DNA methylation plays many important roles in human physiology such as imprinting and X chromosome inactivation (XCI), and therefore disruption in DNA methylation can lead to disease development. The objective of this study is to study the role of DNA methylation in both the common and rare disease, using Systemic Lupus Erythematosus (SLE) and chromosome X translocation as the example respectively. The genome-wide DNA methylation analysis was studied by Illumina HumanMethylation450 BeadChip, which is the microarray that allows the detection of more than 480,000 CpG sites across 99% of reference sequence genes. Numerous studies have shown that hypomethylation occurred in specific genes of SLE patients, and genome-wide DNA methylation analysis has never been performed in Chinese before. Since DNA methylation can be ethnicity-specific, we aim to identify the Chinese-specific DNA methylation pattern in SLE patients. Microarray results showed that differential DNA methylation changes were loci-specific, where 36 CpG sites showed loss of DNA methylation while 8 of them showed gain of it, representing 24 genes and 7 genes respectively. In order to replicate the microarray findings, bisulfite pyrosequencing was performed in an additional cohort of 100 patients and 100 controls on four hypomethylated genes, which were selected based on their relevance with immunity. Bisulfite pyrosequencing confirmed the microarray result that hypomethylation occurred in SLE, and were associated with increased in the corresponding gene’s mRNA expressions. Gene ontology analysis revealed that hypomethylated genes identified in the microarray study were overrepresented in type I interferon pathway, where type I interferon has long been implicated in SLE pathogenesis. Therefore this study also support the importance of type I interferon in SLE from the epigenetic point of view. X;autosome translocation is a rare condition and the autosome with chromosome X translocated on can be inactivated by XCI, but DNA methylation change is rarely used to investigate the spread of XCI. In this study, we aim to identify genes subjected to XCI in X;15 translocation using the DNA methylation microarray. Results of microarray showed that 586 CpG sites spanning the long arm of chromosome 15 had DNA methylation gain of more than 20%. Since genes subjected to XCI are known to have gain of DNA methylation in their corresponding CpG-island promoters, the analysis was then focused on CpG sites in these regions, and a total of 75 sites representing 24 genes were hypermethylated. Nearly all of these CpG sites are located in region proximal to the breakpoint, from 15q11.2 to 15q21.3 accounting for 35Mb, suggesting that XCI was spread to the proximal region of 15q. Gain of DNA methylation, especially in the CpG-island promoter, can result in functional inactivation of genes, and therefore could explain the worsen phenotype of the patient. In conclusion, we successfully demonstrated the use of genome-wide DNA methylation microarray in different diseases, allowing the identification of genes or pathways important in diseases and opened the door for further investigation of the effect of these differentially methylated genes on disease.published_or_final_versionPaediatrics and Adolescent MedicineMasterMaster of Philosoph

Paternal uniparental disomy of chromosome 19 in a pair of monochorionic diamniotic twins with dysmorphic features and developmental delay

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Potentials and challenges of launching the pilot phase of Hong Kong Genome Project

Genomic medicine and precision medicine initiatives have taken centre stage in scientific, clinical, as well as health economics and utility research on the global scene for the past decade. It is clear the important role genomic advancement has played in enhancing diagnostic rate, streamlining personalised treatment, and improving efficacy of the overall clinical management of undiagnosed, rare, and common diseases for humankind. The Hong Kong Genome Institute (HKGI) was established in May 2020 within the Food and Health Bureau, Hong Kong Special Administrative Region, to integrate genomic medicine into mainstream healthcare. The main goals of setting up HKGI are to (1) improve the diagnostic rate and future care for individuals affected by undiagnosed diseases and hereditary cancers using whole genome sequencing; (2) advance research in genomic science; (3) nurture talents in genomic medicine; and (4) enhance public genomic literacy and overall engagement through the launching of the Hong Kong Genome Project (HKGP). In this paper, we review the current landscape and specific challenges encountered during the construction of the infrastructure and implementation of the pilot phase of HKGP. Through reviewing what has been achieved and established to date, and the potentials and prospects that have emerged in the process, this paper will provide insights into planning the main phase of HKGP, and considerations for our international counterparts when building similar projects

Severe acute respiratory syndrome coronavirus ORF3a protein activates the NLRP3 inflammasome by promoting TRAF3-dependent ubiquitination of ASC

Severe acute respiratory syndrome coronavirus (SARS-CoV) is capable of inducing a storm of proinflammatory cytokines. In this study, we show that the SARS-CoV open reading frame 3a (ORF3a) accessory protein activates the NLRP3 inflammasome by promoting TNF receptor-associated factor 3 (TRAF3)-mediated ubiquitination of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). SARS-CoV and its ORF3a protein were found to be potent activators of pro-IL-1beta gene transcription and protein maturation, the 2 signals required for activation of the NLRP3 inflammasome.This work was supported by the Hong Kong Health and Medical Research Fund (13121032, 14130822, and HKM-15- M01), Hong Kong Research Grants Council (T11-707/15-R, C7011-15R, and 17124415), Government of Spain (BIO2013- 42869-R), U.S. National Institutes of Health (NIH) National Institute of Allergy and Infectious Diseases (2P01AI060699- 06A1), and European Commission (Innovative Medicines Initiative Grant Agreement 115760). C.C.-R. received a fellowship from Fundacion La Caixa

Identification of mutations in the PI3K-AKT-mTOR signalling pathway in patients with macrocephaly and developmental delay and/or autism

Abstract Background Macrocephaly, which is defined as a head circumference greater than or equal to + 2 standard deviations, is a feature commonly observed in children with developmental delay and/or autism spectrum disorder. Although PTEN is a well-known gene identified in patients with this syndromic presentation, other genes in the PI3K-AKT-mTOR signalling pathway have also recently been suggested to have important roles. The aim of this study is to characterise the mutation spectrum of this group of patients. Methods We performed whole-exome sequencing of 21 patients with macrocephaly and developmental delay/autism spectrum disorder. Sources of genomic DNA included blood, buccal mucosa and saliva. Germline mutations were validated by Sanger sequencing, whereas somatic mutations were validated by droplet digital PCR. Results We identified ten pathogenic/likely pathogenic mutations in PTEN (n = 4), PIK3CA (n = 3), MTOR (n = 1) and PPP2R5D (n = 2) in ten patients. An additional PTEN mutation, which was classified as variant of unknown significance, was identified in a patient with a pathogenic PTEN mutation, making him harbour bi-allelic germline PTEN mutations. Two patients harboured somatic PIK3CA mutations, and the level of somatic mosaicism in blood DNA was low. Patients who tested positive for mutations in the PI3K-AKT-mTOR pathway had a lower developmental quotient than the rest of the cohort (DQ = 62.8 vs. 76.1, p = 0.021). Their dysmorphic features were non-specific, except for macrocephaly. Among the ten patients with identified mutations, brain magnetic resonance imaging was performed in nine, all of whom showed megalencephaly. Conclusion We identified mutations in the PI3K-AKT-mTOR signalling pathway in nearly half of our patients with macrocephaly and developmental delay/autism spectrum disorder. These patients have subtle dysmorphic features and mild developmental issues. Clinically, patients with germline mutations are difficult to distinguish from patients with somatic mutations, and therefore, sequencing of buccal or saliva DNA is important to identify somatic mosaicism. Given the high diagnostic yield and the management implications, we suggest implementing comprehensive genetic testing in the PI3K-AKT-mTOR pathway in the clinical evaluation of patients with macrocephaly and developmental delay and/or autism spectrum disorder

Cell lineage-specific genome-wide DNA methylation analysis of patients with paediatric-onset systemic lupus erythematosus

Patients with paediatric-onset systemic lupus erythematosus (SLE) often present with more severe clinical courses than adult-onset patients. Although genome-wide DNA methylation (DNAm) profiling has been performed in adult-onset SLE patients, parallel data on paediatric-onset SLE are not available. Therefore, we undertook a genome-wide DNAm study in paediatric-onset SLE patients across multiple blood cell lineages. The DNAm profiles of four purified immune cell lineages (CD4 + T cells, CD8 + T cells, B cells and neutrophils) and whole blood were compared in 16 Chinese patients with paediatric-onset SLE and 13 healthy controls using the Illumina HumanMethylationEPIC BeadChip. Comparison of DNAm in whole blood and within each independent cell lineage identified a consistent pattern of loss of DNAm at 21 CpG sites overlapping 15 genes, which represented a robust, disease-specific DNAm signature for paediatric-onset SLE in our cohort. In addition, cell lineage-specific changes, involving both loss and gain of DNAm, were observed in both novel genes and genes with well-described roles in SLE pathogenesis. This study also highlights the importance of studying DNAm changes in different immune cell lineages rather than only whole blood, since cell type-specific DNAm changes facilitated the elucidation of the cell type-specific molecular pathophysiology of SLE

Diagnostic value of whole‐exome sequencing in Chinese pediatric‐onset neuromuscular patients

Abstract Background Neuromuscular disorders (NMDs) comprise a group of heterogeneous genetic diseases with a broad spectrum of overlapping the clinical presentations that makes diagnosis challenging. Notably, the recent introduction of whole‐exome sequencing (WES) is introducing rapid changes on the genetic diagnosis of NMDs. We aimed to investigate the diagnostic value of WES for pediatric‐onset NMDs. Methods We applied integrated diagnostic approach and performed WES in 50 Chinese subjects (30 males, 20 females) with undiagnosed pediatric‐onset NMDs despite previous specific tests. The patients were categorized in four subgroups according to phenotyping and investigation findings. Variants on NMDs gene list and open exome analysis for those with initial negative findings were identified. Results WES identified causative variants in ACTA1 (n = 2), POMT1, COL6A1 (n = 2), MTMR2, LMNA, SELENON, DNM2, TGFB1, MPZ, IGHMBP2, and LAMA2 in 13 patients. Two subjects have variants of uncertain significance (VUSs) in TTN and SCN11A, unlikely to be pathogenic due to incompatible phenotypes. The mean interval time from symptom onset to genetic diagnosis was 10.4 years (range from 1 month to 33 years). The overall diagnostic yield of WES in our cohort was 26%. Open exome analysis was necessary to identify the pathogenic variant in TGFB1 that caused skeletal dysplasia with neuromuscular presentation. Conclusion Our study shows a clear role of WES in the pathway of integrated diagnostic approach to shorten the diagnostic odyssey in patients with rare NMDs