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

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

Actionable pharmacogenetic variants in Hong Kong Chinese exome sequencing data and projected prescription impact in the Hong Kong population.

Preemptive pharmacogenetic testing has the potential to improve drug dosing by providing point-of-care patient genotype information. Nonetheless, its implementation in the Chinese population is limited by the lack of population-wide data. In this study, secondary analysis of exome sequencing data was conducted to study pharmacogenomics in 1116 Hong Kong Chinese. We aimed to identify the spectrum of actionable pharmacogenetic variants and rare, predicted deleterious variants that are potentially actionable in Hong Kong Chinese, and to estimate the proportion of dispensed drugs that may potentially benefit from genotype-guided prescription. The projected preemptive pharmacogenetic testing prescription impact was evaluated based on the patient prescription data of the public healthcare system in 2019, serving 7.5 million people. Twenty-nine actionable pharmacogenetic variants/ alleles were identified in our cohort. Nearly all (99.6%) subjects carried at least one actionable pharmacogenetic variant, whereas 93.5% of subjects harbored at least one rare deleterious pharmacogenetic variant. Based on the prescription data in 2019, 13.4% of the Hong Kong population was prescribed with drugs with pharmacogenetic clinical practice guideline recommendations. The total expenditure on actionable drugs was 33,520,000 USD, and it was estimated that 8,219,000 USD (24.5%) worth of drugs were prescribed to patients with an implicated actionable phenotype. Secondary use of exome sequencing data for pharmacogenetic analysis is feasible, and preemptive pharmacogenetic testing has the potential to support prescription decisions in the Hong Kong Chinese population

Use of clinical chromosomal microarray in Chinese patients with autism spectrum disorder—implications of a copy number variation involving DPP10

Abstract Background Array comparative genomic hybridization (aCGH) is recommended as a first-tier genetic test for children with autism spectrum disorder (ASD). However, interpretation of results can often be challenging partly due to the fact that copy number variants (CNVs) in non-European ASD patients are not well studied. To address this literature gap, we report the CNV findings in a cohort of Chinese children with ASD. Methods DNA samples were obtained from 258 Chinese ASD patients recruited from a child assessment center between January 2011 and August 2014. aCGH was performed using NimbleGen-CGX-135k or Agilent-CGX 60k oligonucleotide array. Results were classified based on existing guidelines and literature. Results Ten pathogenic CNVs and one likely pathogenic CNV were found in nine patients, with an overall diagnostic yield of 3.5%. A 138 kb duplication involving 3′ exons of DPP10 (arr[GRCh37] 2q14.1(116534689_116672358)x3), reported to be associated with ASD, was identified in one patient (0.39%). The same CNV was reported as variant of uncertain significance (VUS) in DECIPHER database. Multiple individuals of typical development carrying a similar duplication were identified among our ancestry-matched control with a frequency of 6/653 (0.92%) as well as from literature and genomic databases. Conclusions The DPP10 duplication is likely a benign CNV polymorphism enriched in Southern Chinese with a population frequency of ~1%. This highlights the importance of using ancestry-matched controls in interpretation of aCGH findings

Application of Prenatal Whole Exome Sequencing for Structural Congenital Anomalies—Experience from a Local Prenatal Diagnostic Laboratory

Fetal structural congenital abnormalities (SCAs) complicate 2–3% of all pregnancies. Whole-exome sequencing (WES) has been increasingly adopted prenatally when karyotyping and chromosomal microarray do not yield a diagnosis. This is a retrospective cohort study of 104 fetuses with SCAs identified on antenatal ultrasound in Hong Kong, where whole exome sequencing is performed. Molecular diagnosis was obtained in 25 of the 104 fetuses (24%). The highest diagnostic rate was found in fetuses with multiple SCAs (29.2%), particularly those with involvement of the cardiac and musculoskeletal systems. Variants of uncertain significance were detected in 8 out of the 104 fetuses (7.7%). Our study shows the utility of WES in the prenatal setting, and the extended use of the technology would be recommended in addition to conventional genetic workup

Genome-Wide DNA Methylation Analysis of Chinese Patients with Systemic Lupus Erythematosus Identified Hypomethylation in Genes Related to the Type I Interferon Pathway

<div><p>Background</p><p>Epigenetic variants have been shown in recent studies to be important contributors to the pathogenesis of systemic lupus erythematosus (SLE). Here, we report a 2-step study of discovery followed by replication to identify DNA methylation alterations associated with SLE in a Chinese population. Using a genome-wide DNA methylation microarray, the Illumina Infinium HumanMethylation450 BeadChip, we compared the methylation levels of CpG sites in DNA extracted from white blood cells from 12 female Chinese SLE patients and 10 healthy female controls.</p><p>Results</p><p>We identified 36 CpG sites with differential loss of DNA methylation and 8 CpG sites with differential gain of DNA methylation, representing 25 genes and 7 genes, respectively. Surprisingly, 42% of the hypomethylated CpG sites were located in CpG shores, which indicated the functional importance of the loss of DNA methylation. Microarray results were replicated in another cohort of 100 SLE patients and 100 healthy controls by performing bisulfite pyrosequencing of four hypomethylated genes, <i>MX1</i>, <i>IFI44L</i>, <i>NLRC5</i> and <i>PLSCR1</i>. In addition, loss of DNA methylation in these genes was associated with an increase in mRNA expression. Gene ontology analysis revealed that the hypomethylated genes identified in the microarray study were overrepresented in the type I interferon pathway, which has long been implicated in the pathogenesis of SLE.</p><p>Conclusion</p><p>Our epigenetic findings further support the importance of the type I interferon pathway in SLE pathogenesis. Moreover, we showed that the DNA methylation signatures of SLE can be defined in unfractionated white blood cells.</p></div

Co-expression analysis of differentially hypomethylated genes.

<p>The differentially hypomethylated genes found in the 450k microarray study were input into GeneMANIA for co-expression analysis, which revealed that they are related to type I interferon pathways, including “response to type I interferon” (FDR = 1.45e-34), “type I interferon-mediated signaling pathway” (FDR = 1.45e-34) and “cellular response to type I interferon” (FDR = 1.45e-34). Genes identified in these three co-expression pathways are overlapped with each other and are indicated in blue circles. Black circles indicate the differentially hypomethylated genes found in the 450k microarray, whereas circles with alternating black and blue stripes indicate hypomethylated genes that are overrepresented in type 1 interferon pathways.</p

Comparison of the methylation level of four hypomethylated genes between control and SLE patients.

<p>Bisulfite pyrosequencing was carried out on four of the genes that were found to be differentially methylated in the 450k microarray and in a larger cohort of 100 SLE patients and 100 healthy controls. These genes are <i>MX1</i>, <i>IFI44L</i>, <i>NLRC5</i> and <i>PLSCR1</i>. Bisulfite pyrosequencing of all four genes confirmed the microarray findings, showing that SLE patients have a significant loss of methylation when compared to healthy controls.</p