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

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CFTR founder mutation causes protein trafficking defects in Chinese patients with cystic fibrosis

published_or_final_versio

CFTR: I1023R is a rare but recurrent disease-causing mutation found in Chinese patients with cystic fibrosis

Oral Free Paper SessionOutstanding Oral PresentationBackground: Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the gene CFTR. CF is a common in Caucasians, yet less than twenty Chinese patients with molecular confirmation have been reported. Our department is the only center that offers sweat test in Hong Kong, and from our data we estimate the incidence of CF as 1 in 300,000 in local Chinese. We report the findings of a comprehensive genetic analysis of 6 unrelated Chinese patients with the clinical suspicion of CF. Methods and results: Using NGS (next generation sequencing), Sanger sequencing and MLPA (multiplex ligationdependent probe amplification), we screened for single nucleotide variations and deletion/ duplications in all exons and three intronic hotspots of CFTR gene. Molecular diagnosis was confirmed in four unrelated patients. Importantly, three inherited the same missense mutation, I1023R (CFTR: NM_000492.3: c.T3068G), which was reported only in two Taiwanese siblings with CF but not in patients of other ethnicities. Patients with this recurrent mutation have typical CF features, including Pseudomonas aeuroginosa pneumonia, bronchiectasis and meconium ileus. It is not found in our in-house database of 200 exomes, or public databases like ESP6500 and 1000GP, indicating a very low allelic frequency. Using linkage and functional analysis, we showed that I1023R is likely a founder mutation in Hans Chinese and the mutant CFTR protein is potentially having a post-translational defect, resulting in reduced expression compared with the wild-type protein. Conclusion: We have summarized the diagnosis of all reported CF patients in the last twenty years. In addition to our genetic analysis of local patients, we propose that I1023R is a rare but recurrent, potential founder type disease-causing CFTR mutation in Chinese CF patients. Currently I1023R is not a screening target in the cystic fibrosis mutation panel (i.e. ACMG25) which includes the core mutations recommended by American College of Medical Genetics (ACMG). This finding has implications in the design of mutation panels, and analysis of NGS for molecular diagnosis of CF for Chinese patients

Frequent Inactivation of Axon Guidance Molecule RGMA in Human Colon Cancer Through Genetic and Epigenetic Mechanisms

Background & Aims: Repulsive guidance molecule member A (RGMA) is a glycosylphosphatidylinositol-anchored glycoprotein and axon guidance molecule that signals through its receptor, neogenin (NEO1), a homologue of the deleted-in-colorectal cancer (DCC) gene. RGMA also functions as a bone morphogenetic protein (BMP) coreceptor. We studied the potential roles of RGMA and NEO1 in colorectal cancer (CRC) pathogenesis. Methods: We analyzed expression of RGMA and NEO1, as well as their epigenetic and genetic changes, in a large series of CRC samples, normal colon tissues, adenomas, and cell lines. These studies were accompanied by in vitro functional assay. Results: RGMA and NEO1 expression were significantly down-regulated in most CRCs, adenomas, and cell lines. RGMA was frequently silenced by promoter methylation in CRCs (86.7%), adenomas (90.9%), and CRC cell lines (92.3%) but not in normal colon tissues; allelic imbalance of RGMA and NEO1 was observed in 40% and 49% of CRCs, respectively. In CRC samples, reduced RGMA levels were significantly associated with mismatch repair deficiency or mutations in KRAS or BRAF. Exposure to 5-aza-2′-deoxycytidine restored RGMA expression in CRC cell lines. Transfection of RGMA into CRC cells suppressed cell proliferation, migration, and invasion and also increased apoptosis in response to DNA-damaging agent. Conclusions: The frequent genetic and epigenetic inactivation of RGMA in CRCs and adenomas along with its in vitro function collectively support its role as a tumor suppressor in colon cells. These findings add to the expanding list of axon guidance molecules with disrupted function during colon carcinogenesis and create new opportunities for early detection and drug development. © 2009 AGA Institute.link_to_subscribed_fulltex

Monoallelic mutations in CC2D1A suggest a novel role in human heterotaxy and ciliary dysfunction

202109 bchyVersion of RecordPublishe

A specific DNA methylation signature associated with NSD1+/- mutations in Sotos syndrome reveals a significant genome-wide loss of DNA methylation (DNAm) targeting CGs in regulatory regions of key developmental genes

Sotos syndrome (SS) is characterized by somatic overgrowth and intellectual disability. Most SS cases (>75%) have mutations in NSD1 (nuclear receptor-binding SET domain protein 1). NSD1 binds near promoter elements and regulates transcription initiation and elongation via interactions with H3-K36Me and RNA polymerase II. To determine if NSD1 mutations impact stable epigenetic marks such as DNA methylation (DNAm), we compared DNAm in peripheral blood DNA from SS cases with NSD1 mutations (NSD1+/-; n=20) to controls (n=30) using the Illumina Infinium450methylation BeadChip. Differential DNAm analysis using non-parametric statistics (with correction for multiple testing) coupled with permutation analyses identified a surprisingly high number (n=2157) of differentially methylated (DM) CG sites (with >20% difference in DNAm) between SS and controls. These sites were distributed across the genome; 95% demonstrated loss of DNAm. Using unsupervised hierarchical clustering of the 2157 DM CG sites, all SS cases with NSD1 +/- clustered as a distinct group separate from controls. Moreover, DNAm at these sites clearly distinguished SS (NSD1+/-) from Weaver syndrome (EZH2+/-, n=5), another overgrowth syndrome which has considerable phenotypic overlap with SS. These results suggest that these DM CG sites constitute a DNAm signature that is specific for NSD1+/-. Also, the DNAm signature was successfully used to reclassify NSD1 variants of unknown significance (VUS) in six cases of SS into functionally damaging (n=1) and non-pathogenic (n=5) variants. The majority of these DM CGs mapped to enhancers and CpG island shores. Analysis of ChIP-seq data showed that NSD1+/- specific CG sites are associated with reduced H3K36me3 marks in both normal blood and embryonic stem cells. Also, Ingenuity analysis showed enrichment in neural and cellular development pathways (p<0.001). We then searched for binding motifs enriched in these NSD1+/- DNAm targets using MEME and JASPAR CORE database; SP1 was the most enriched with binding sites in 41% of the targets (NCOR=0.62). This is the first report of an NSD1+/- specific DNAm signature in SS and that loss-of-function mutations in NSD1 can deregulate the intricate transcriptional balance of key developmental genes. Further elucidation of this signature will significantly impact our understanding of the molecular pathophysiology of SS and identify the specific molecular targets for NSD1 that govern its action in early development.link_to_OA_fulltex

Identification of a CFTR founder mutations, c.3068T>G:p.I1023R, which causes protein trafficking defects in Chinese patients with cystic fibrosis

Poster Presentation: Group 1 - Genetics/Inborn Errors of Metabolism: no. G 3-

NSD1+/- DNA methylation (DNAm) signature: A novel functional diagnostic tool for Sotos syndrome

Session - 32. Molecular Insights into Mendelian Disorders: no. 130Sotos syndrome (SS) is characterized by somatic overgrowth and intellectual disability. Most SS cases have mutations in NSD1 (nuclear receptor-binding SET domain protein 1), a histone lysine methyltransferase. To determine if NSD1 mutations impact stable epigenetic marks such as DNAm, we compared DNAm in peripheral blood from SS cases with NSD1 mutations (NSD1+/-; n=20) to controls (n=53) using the Illumina Infinium450methylation BeadChip (450k array). Differential DNAm analysis using non-parametric statistics (with correction for multiple testing) identified a surprisingly high number of differentially methylated (DM) CG sites between SS and controls. The majority (99.3%) of these sites demonstrated loss of DNAm and were distributed across the genome. Using unsupervised hierarchical clustering of the significant DM sites, all SS cases with loss of function mutations in NSD1 clustered as a distinct group. The specificity of this signature was 100%; in comparison to DNAm profiles of 450k data from the GEO database for blood samples (n= 1200). The sensitivity of the NSD1+/- signature was tested in an independent replication cohort of 19 SS cases from Hong Kong with NSD1 loss of function mutations. The NSD1+/- signature demonstrated a sensitivity of 100%; highlighting its exceptional power in defining pathogenicity for mutations in NSD1. The classification of single nucleotide substitutions into benign or disease causing (damaging) variants represents an ongoing challenge in clinical diagnostics. To test the hypothesis that the NSD1+/- DNAm signature will inform the functional classification of NSD1 variants into benign or disease causing, we analyzed 16 cases with reported missense variants in NSD1. Using hierarchical clustering we classified these variants as pathogenic (n=9) or benign (n=7). Clinical re-assessment of 11/16 of these cases, for whom photos and adequate clinical information were available, was conducted by two experienced dysmorphologists (RW and DC) who were blinded to the methylation results. There was 100%; concordance between clinical impression and DNAm data. In comparison, 4/5 algorithms (SIFT, Polyphen-2, etc) were inconsistent in their prediction of pathogenicity. Our data suggest that the NSD1+/- DNAm signature reflects the functional effect of NSD1 variants on the methylome and can be used as a more robust predictor than existing algorithms for the functional classification of NSD1 variants in overgrowth disorders.link_to_OA_fulltex