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1.  Sacral agenesis: a pilot whole exome sequencing and copy number study 
BMC Medical Genetics  2016;17:98.
Background
Caudal regression syndrome (CRS) or sacral agenesis is a rare congenital disorder characterized by a constellation of congenital caudal anomalies affecting the caudal spine and spinal cord, the hindgut, the urogenital system, and the lower limbs. CRS is a complex condition, attributed to an abnormal development of the caudal mesoderm, likely caused by the effect of interacting genetic and environmental factors. A well-known risk factor is maternal type 1 diabetes.
Method
Whole exome sequencing and copy number variation (CNV) analyses were conducted on 4 Caucasian trios to identify de novo and inherited rare mutations.
Results
In this pilot study, exome sequencing and copy number variation (CNV) analyses implicate a number of candidate genes, including SPTBN5, MORN1, ZNF330, CLTCL1 and PDZD2. De novo mutations were found in SPTBN5, MORN1 and ZNF330 and inherited predicted damaging mutations in PDZD2 (homozygous) and CLTCL1 (compound heterozygous). Importantly, predicted damaging mutations in PTEN (heterozygous), in its direct regulator GLTSCR2 (compound heterozygous) and in VANGL1 (heterozygous) were identified. These genes had previously been linked with the CRS phenotype.
Two CNV deletions, one de novo (chr3q13.13) and one homozygous (chr8p23.2), were detected in one of our CRS patients. These deletions overlapped with CNVs previously reported in patients with similar phenotype.
Conclusion
Despite the genetic diversity and the complexity of the phenotype, this pilot study identified genetic features common across CRS patients.
Electronic supplementary material
The online version of this article (doi:10.1186/s12881-016-0359-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s12881-016-0359-2
PMCID: PMC5178083  PMID: 28007035
Sacral agenesis; Caudal regression; Copy-number variation; Whole exome sequencing
2.  Genetic study of congenital bile-duct dilatation identifies de novo and inherited variants in functionally related genes 
BMC Medical Genomics  2016;9:75.
Background
Congenital dilatation of the bile-duct (CDD) is a rare, mostly sporadic, disorder that results in bile retention with severe associated complications. CDD affects mainly Asians. To our knowledge, no genetic study has ever been conducted.
Methods
We aim to identify genetic risk factors by a “trio-based” exome-sequencing approach, whereby 31 CDD probands and their unaffected parents were exome-sequenced. Seven-hundred controls from the local population were used to detect gene-sets significantly enriched with rare variants in CDD patients.
Results
Twenty-one predicted damaging de novo variants (DNVs; 4 protein truncating and 17 missense) were identified in several evolutionarily constrained genes (p < 0.01). Six genes carrying DNVs were associated with human developmental disorders involving epithelial, connective or bone morphologies (PXDN, RTEL1, ANKRD11, MAP2K1, CYLD, ACAN) and four linked with cholangio- and hepatocellular carcinomas (PIK3CA, TLN1 CYLD, MAP2K1). Importantly, CDD patients have an excess of DNVs in cancer-related genes (p < 0.025). Thirteen genes were recurrently mutated at different sites, forming compound heterozygotes or functionally related complexes within patients.
Conclusions
Our data supports a strong genetic basis for CDD and show that CDD is not only genetically heterogeneous but also non-monogenic, requiring mutations in more than one genes for the disease to develop. The data is consistent with the rarity and sporadic presentation of CDD.
Electronic supplementary material
The online version of this article (doi:10.1186/s12920-016-0236-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s12920-016-0236-z
PMCID: PMC5154011  PMID: 27955658
Choledochal cyst; Exome; De novo; Rare variants association
3.  Interplay between Schizophrenia Polygenic Risk Score and Childhood Adversity in First-Presentation Psychotic Disorder: A Pilot Study 
PLoS ONE  2016;11(9):e0163319.
A history of childhood adversity is associated with psychotic disorder, with an increase in risk according to number or severity of exposures. However, it is not known why only some exposed individuals go on to develop psychosis. One possibility is pre-existing genetic vulnerability. Research on gene-environment interaction in psychosis has primarily focused on candidate genes, although the genetic effects are now known to be polygenic. This pilot study investigated whether the effect of childhood adversity on psychosis is moderated by the polygenic risk score for schizophrenia (PRS). Data were utilised from the Genes and Psychosis (GAP) study set in South London, UK. The GAP sample comprises 285 first-presentation psychosis cases and 256 unaffected controls with information on childhood adversity. We studied only white subjects (80 cases and 110 controls) with PRS data, as the PRS has limited predictive ability in patients of African ancestry. The occurrence of childhood adversity was assessed with the Childhood Experience of Care and Abuse Questionnaire (CECA.Q) and the PRS was based on genome-wide meta-analysis results for schizophrenia from the Psychiatric Genomics Consortium. Higher schizophrenia PRS and childhood adversities each predicted psychosis status. Nevertheless, no evidence was found for interaction as departure from additivity, indicating that the effect of polygenic risk scores on psychosis was not increased in the presence of a history of childhood adversity. These findings are compatible with a multifactorial threshold model in which both genetic liability and exposure to environmental risk contribute independently to the etiology of psychosis.
doi:10.1371/journal.pone.0163319
PMCID: PMC5029892  PMID: 27648571
4.  A Mendelian randomization study of testosterone and cognition in men 
Scientific Reports  2016;6:21306.
Testosterone replacement for older men is increasingly common, with some observations suggesting a protective effect on cognitive function. We examined the association of endogenous testosterone with cognitive function among older men in a Mendelian randomization study using a separate-sample instrumental variable (SSIV) analysis estimator to minimize confounding and reverse causality. A genetic score predicting testosterone was developed in 289 young Chinese men from Hong Kong, based on selected testosterone-related single nucleotide polymorphisms (rs10046, rs1008805 and rs1256031). The association of genetically predicted testosterone with delayed 10-word recall score and Mini-Mental State Examination (MMSE) score was assessed at baseline and follow-up using generalized estimating equation among 4,212 older Chinese men from the Guangzhou Biobank Cohort Study. Predicted testosterone was not associated with delayed 10-word recall score (−0.02 per nmol/L testosterone, 95% confidence interval (CI) −0.06–0.02) or MMSE score (0.06, 95% CI −0.002–0.12). These estimates were similar after additional adjustment for age, education, smoking, use of alcohol, body mass index and the Framingham score. Our findings do not corroborate observed protective effects of testosterone on cognitive function among older men.
doi:10.1038/srep21306
PMCID: PMC4749999  PMID: 26864717
5.  Exome-wide association analysis reveals novel coding sequence variants associated with lipid traits in Chinese 
Nature Communications  2015;6:10206.
Blood lipids are important risk factors for coronary artery disease (CAD). Here we perform an exome-wide association study by genotyping 12,685 Chinese, using a custom Illumina HumanExome BeadChip, to identify additional loci influencing lipid levels. Single-variant association analysis on 65,671 single nucleotide polymorphisms reveals 19 loci associated with lipids at exome-wide significance (P<2.69 × 10−7), including three Asian-specific coding variants in known genes (CETP p.Asp459Gly, PCSK9 p.Arg93Cys and LDLR p.Arg257Trp). Furthermore, missense variants at two novel loci—PNPLA3 p.Ile148Met and PKD1L3 p.Thr429Ser—also influence levels of triglycerides and low-density lipoprotein cholesterol, respectively. Another novel gene, TEAD2, is found to be associated with high-density lipoprotein cholesterol through gene-based association analysis. Most of these newly identified coding variants show suggestive association (P<0.05) with CAD. These findings demonstrate that exome-wide genotyping on samples of non-European ancestry can identify additional population-specific possible causal variants, shedding light on novel lipid biology and CAD.
An important risk factor for coronary artery disease is the level of blood lipids. Here the authors conduct an exome-wide association study in Chinese cohorts and identify three novel loci associated with lipid levels as well as three Asian-specific variants in known loci.
doi:10.1038/ncomms10206
PMCID: PMC4703860  PMID: 26690388
6.  Exome sequencing reveals a high genetic heterogeneity on familial Hirschsprung disease 
Scientific Reports  2015;5:16473.
Hirschsprung disease (HSCR; OMIM 142623) is a developmental disorder characterized by aganglionosis along variable lengths of the distal gastrointestinal tract, which results in intestinal obstruction. Interactions among known HSCR genes and/or unknown disease susceptibility loci lead to variable severity of phenotype. Neither linkage nor genome-wide association studies have efficiently contributed to completely dissect the genetic pathways underlying this complex genetic disorder. We have performed whole exome sequencing of 16 HSCR patients from 8 unrelated families with SOLID platform. Variants shared by affected relatives were validated by Sanger sequencing. We searched for genes recurrently mutated across families. Only variations in the FAT3 gene were significantly enriched in five families. Within-family analysis identified compound heterozygotes for AHNAK and several genes (N = 23) with heterozygous variants that co-segregated with the phenotype. Network and pathway analyses facilitated the discovery of polygenic inheritance involving FAT3, HSCR known genes and their gene partners. Altogether, our approach has facilitated the detection of more than one damaging variant in biologically plausible genes that could jointly contribute to the phenotype. Our data may contribute to the understanding of the complex interactions that occur during enteric nervous system development and the etiopathology of familial HSCR.
doi:10.1038/srep16473
PMCID: PMC4642299  PMID: 26559152
7.  AB113. The first genetic study on congenital choledochal dilatation (CCD) implicates extracellular matrix proteins 
Annals of Translational Medicine  2015;3(Suppl 2):AB113.
Congenital choledochal dilatation (CCD) or paediatric choledochal cyst refers to the congenital dilatation of the choledochs (bile ducts) which leads to the obstruction of the ducts and bile retention. Symptoms include cholestatic jaundice, abdominal pain and liver enlargement complicated with cholangitis and pancreatitis. New-borns undergo surgery otherwise the liver could be permanently damaged. CCD is rare, mostly sporadic with variable population incidence, the highest being in Asia (1/1,000 in Asians; 1/150,000 in Caucasians). Its aetiology implicates congenital structural anomalies reflecting a failure in the hepatobiliary-pancreatic development. Thirty-one CCD trios were exome sequenced. Gene/pathway-set enrichment analyses grouped genes with at least one damaging allele into focal adhesion and extracellular matrix-receptor interaction pathways. Pathogenic mechanisms considered included de novo germ-line mutations and/or recessive inherited mutations in homozygosis, compound heterozygosis (CH) or as “di-genic/oligogenic” model of inheritance whereby variants in genes of related pathways coexist in a patient through parental inheritance. Fifteen gene members of those pathways were recurrently mutated and had variants at different sites (more than one damaging allele per gene). These alleles were in CH or co-existing with a mutated functional gene-partner in the same individual. Patients’ genetic profiling revealed CCD as not only genetically heterogeneous but with di/oligogenic inheritance. Yet, the relevant mutated genes are functionally convergent. Data are consistent with the sporadic presentation of CCD. Incidentally, the cholangiocarcinoma rate in Asians is also the highest world-wide. We are also aiming at finding possible links between these choledochal disorders and at explaining their high incidence in Asia.
doi:10.3978/j.issn.2305-5839.2015.AB113
PMCID: PMC4563397
Congenital choledochal dilatation (CCD); exome sequencing
8.  AB080. Genetic findings provide insight of biliary atresia patient complexity 
Annals of Translational Medicine  2015;3(Suppl 2):AB080.
Background
Biliary atresia (BA) is a rare complex disease with unknown etiology. Current treatment of BA is Kasai portoenterostomy but is ineffective. BA is now the most common cause of pediatric liver transplantation worldwide. Characterize the disease complexity and stratify patients for personalized medicine is necessary. Genetic variants underlie BA pathogenesis and yet comprehensive genotype-phenotype correlations are yet to be investigated.
Methods
We first reviewed the disease course of 89 isolated BA patients with long term follow up (median =17.2 years), whose blood DNA was genotyped on Affymetrix5.0. Copy number variants (CNVs) and single nucleotide polymorphisms (SNPs) were called. Meanwhile 23 BA patients’ liver DNA was submitted to exome sequencing for discovery of de novo mutations. After wise we narrowed the genotype callings down to BA unique mutations, i.e., CNVs, SNV/INDELs, and BA-associated genes through gene-based association test of SNPs, then genotype-phenotype correlations were interrogated. Last, interconnectivity among the candidate genes were examined, topology of the molecular network was then interrogated correlating to the BA clinical complexity.
Results
Clinical revision revealed that 41.57% isolated BAs had chronic extra-hepatic diseases, with high prevalence of autoimmune-atopic diseases (22.47%) and glucose-6-phosphate dehydrogenase deficiency (14.29% in males). In genotype data, we shortlisted 29 CNVs ≥100 kb private to BA and related sted 29 (I) de novo BA-CNVs, perturbing genes known to hepato-biliary diseases, associated with BA liver pathology; (II) three BA-CNVs encompassing genes known to immunity defects, correlated with comorbidities of those immune disorders in three carriers, and overall BA-CNVs intersected ‘immunologically-important’ genes (P=0.017). Biologically BA-CNVs are anchored to other BA candidate genes as interactions were observed between genes encompassed by BA-CNVs (N=102) and BA-associated genes tagged by SNPs (N=103) (empirical P=0.039). Additional SNV/INDELs associated with intrahepatic biliary anomalies were uncovered in exome-sequencing. All together the BA candidate genes converge into a molecular network with inflammatory regulators as the signalling hub, moreover, the network fell into multiple function modules, which coincides with the BA patients’ clinical profile.
Conclusions
Genetic variants underpin BA clinical manifestations. The BA-associated common and rare genetic converge in a molecular network, which support the plausible associations of BA with a host of ‘non-BA’ diseases, as supported by observation in patients comorbidities with non-BA disease. We propose this ‘diseasome’ network approach that integrates clinical/epidemiological data and BA genetic findings to decode the phenotypic complexity of this rare disease.
doi:10.3978/j.issn.2305-5839.2015.AB080
PMCID: PMC4563478
Rare complex disease; patient complexity; genetic screening; genotype-phenotype correlation; network
9.  Chinese family with diffuse oesophageal leiomyomatosis: a new COL4A5/COL4A6 deletion and a case of gonosomal mosaicism 
BMC Medical Genetics  2015;16:49.
Background
Diffuse oesophageal leiomyomatosis (DOL) is a rare disorder characterized by tumorous overgrowth of the muscular wall of the oesophagus. DOL is present in 5 % of Alport syndrome (AS) patients. AS is a rare hereditary disease that involves varying degrees of hearing impairment, ocular changes and progressive glomerulonephritis leading to renal failure. In DOL-AS patients, the genetic defect consists of a deletion involving the COL4A5 and COL4A6 genes on the X chromosome.
Case presentation
We report a two-generation family (4 individuals; parents and two children, one male and one female) with two members (mother and son) affected with oesophageal leiomyomatosis. Signs of potential renal failure, which characterizes AS, were only apparent in the index patient (son) 2 years and three months after the initial diagnosis of DOL. Blood DNA from the four family members were submitted to exome sequencing and array genotyping to perform a genome wide screening for disease causal single nucleotide (SN) and copy number (CN) variations. Analyses revealed a new 40kb deletion encompassing from intron 2 of COL4A5 to intron 1 of COL4A6 at Xq22.3. The breakpoints were also identified. Possible confounding pathogenic exonic variants in genes known to be involved in other extracellular matrices disorders were also shared by the two affected individuals. Meticulous analysis of the maternal DNA revealed a case of gonosomal mosaicism.
Conclusions
This is the first report of gonadosomal mosaicism associated to DOL-AS
Electronic supplementary material
The online version of this article (doi:10.1186/s12881-015-0189-7) contains supplementary material, which is available to authorized users.
doi:10.1186/s12881-015-0189-7
PMCID: PMC4557859  PMID: 26179878
Isolated diffuse oesophageal leiomyomatosis; Whole exome sequencing; Copy number variation; COL4A5; COL4A6; Gonosomal mosaicism
10.  Common Variants on Xq28 Conferring Risk of Schizophrenia in Han Chinese 
Schizophrenia Bulletin  2013;40(4):777-786.
Schizophrenia is a highly heritable, severe psychiatric disorder affecting approximately 1% of the world population. A substantial portion of heritability is still unexplained and the pathophysiology of schizophrenia remains to be elucidated. To identify more schizophrenia susceptibility loci, we performed a genome-wide association study (GWAS) on 498 patients with schizophrenia and 2025 controls from the Han Chinese population, and a follow-up study on 1027 cases and 1005 controls. In the follow-up study, we included 384 single nucleotide polymorphisms (SNPs) which were selected from the top hits in our GWAS (130 SNPs) and from previously implicated loci for schizophrenia based on the SZGene database, NHGRI GWAS Catalog, copy number variation studies, GWAS meta-analysis results from the international Psychiatric Genomics Consortium (PGC) and candidate genes from plausible biological pathways (254 SNPs).
Within the chromosomal region Xq28, SNP rs2269372 in RENBP achieved genome-wide significance with a combined P value of 3.98×10−8 (OR of allele A = 1.31). SNPs with suggestive P values were identified within 2 genes that have been previously implicated in schizophrenia, MECP2 (rs2734647, P combined = 8.78×10−7, OR = 1.28; rs2239464, P combined = 6.71×10−6, OR = 1.26) and ARHGAP4 (rs2269368, P combined = 4.74×10−7, OR = 1.25). In addition, the patient sample in our follow-up study showed a significantly greater burden for pre-defined risk alleles based on the SNPs selected than the controls. This indicates the existence of schizophrenia susceptibility loci among the SNPs we selected. This also further supports multigenic inheritance in schizophrenia. Our findings identified a new schizophrenia susceptibility locus on Xq28, which harbor the genes RENBP, MECP2, and ARHGAP4.
doi:10.1093/schbul/sbt104
PMCID: PMC4059435  PMID: 24043878
schizophrenia; genome-wide association study; Han Chinese; MECP2; ARHGAP4; RENBP
11.  Meta-analysis of two Chinese populations identifies an autoimmune disease risk allele in 22q11.21 as associated with systemic lupus erythematosus 
Introduction
Systemic lupus erythematosus (SLE) is a heterogeneous disease with a diverse spectrum of clinical symptoms, ranging from skin rash to end-organ damage. 22q11.21 has been identified as a susceptibility region for several autoimmune diseases, including SLE. However, detailed information for SLE association and the underlying functional mechanism(s) is still lacking.
Methods
Through meta-analysis of two genome-wide association studies (GWAS) on Han Chinese populations, comprising a total of 1,659 cases and 3,398 controls matched geographically, we closely examined the 22q11.21 region, especially on the reported single-nucleotide polymorphisms (SNPs) associated with different autoimmune diseases and their relationships. We further replicated the most significant associations of SNPs with SLE using 2,612 cases and 2,323 controls of Asian ancestry.
Results
All reported SNPs in the 22q11.21 region with different autoimmune diseases were examined using the two GWAS data and meta-analysis results, and supportive evidence of association with SLE was found (meta-analysis: P_meta ≤ 7.27E-05), which might require further investigation. SNP rs2298428 was identified as the most significant SNP associated with SLE in this region (P_meta =2.70E-09). It showed independent effects through both stepwise and conditional logistic regression, and there is no evidence of other independent association signals for SLE in this region. The association of rs2298428 was further replicated in three cohorts from Hong Kong, Anhui and Thailand comprising a total of 2,612 cases and 2,323 controls (joint analysis of GWAS and replication result: P_all =1.31E-11, odds ratio =1.23). SNP rs2298428 was shown to be an expression quantitative locus for UBE2L3 gene in different cell types, with the risk allele (T) being correlated with higher expression of UBE2L3. This is consistent with earlier reports on higher expression of UBE2L3 in patients with SLE.
Conclusions
Association with distinct autoimmune diseases highlights the significance of this region in autoreactive responses and potentially shared functional mechanisms in these diseases.
Electronic supplementary material
The online version of this article (doi:10.1186/s13075-015-0577-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s13075-015-0577-6
PMCID: PMC4404227  PMID: 25880549
12.  Cost effective assay choice for rare disease study designs 
High throughput assays tend to be expensive per subject. Often studies are limited not so much by the number of subjects available as by assay costs, making assay choice a critical issue. We have developed a framework for assay choice that maximises the number of true disease causing mechanisms ‘seen’, given limited resources. Although straightforward, some of the ramifications of our methodology run counter to received wisdom on study design. We illustrate our methodology with examples, and have built a website allowing calculation of quantities of interest to those designing rare disease studies.
doi:10.1186/s13023-015-0226-9
PMCID: PMC4334400  PMID: 25648394
WES; WGS; High-throughput assay; Rare disease; Study design
13.  Lumbar disc degeneration is linked to a carbohydrate sulfotransferase 3 variant 
The Journal of Clinical Investigation  2013;123(11):4909-4917.
Lumbar disc degeneration (LDD) is associated with both genetic and environmental factors and affects many people worldwide. A hallmark of LDD is loss of proteoglycan and water content in the nucleus pulposus of intervertebral discs. While some genetic determinants have been reported, the etiology of LDD is largely unknown. Here we report the findings from linkage and association studies on a total of 32,642 subjects consisting of 4,043 LDD cases and 28,599 control subjects. We identified carbohydrate sulfotransferase 3 (CHST3), an enzyme that catalyzes proteoglycan sulfation, as a susceptibility gene for LDD. The strongest genome-wide linkage peak encompassed CHST3 from a Southern Chinese family–based data set, while a genome-wide association was observed at rs4148941 in the gene in a meta-analysis using multiethnic population cohorts. rs4148941 lies within a potential microRNA-513a-5p (miR-513a-5p) binding site. Interaction between miR-513a-5p and mRNA transcribed from the susceptibility allele (A allele) of rs4148941 was enhanced in vitro compared with transcripts from other alleles. Additionally, expression of CHST3 mRNA was significantly reduced in the intervertebral disc cells of human subjects carrying the A allele of rs4148941. Together, our data provide new insights into the etiology of LDD, implicating an interplay between genetic risk factors and miRNA.
doi:10.1172/JCI69277
PMCID: PMC3809787  PMID: 24216480
14.  Gene Network Analysis of Candidate Loci for Human Anorectal Malformations 
PLoS ONE  2013;8(8):e69142.
Anorectal malformations (ARMs) are birth defects that require surgery and carry significant chronic morbidity. Our earlier genome-wide copy number variation (CNV) study had provided a wealth of candidate loci. To find out whether these candidate loci are related to important developmental pathways, we have performed an extensive literature search coupled with the currently available bioinformatics tools. This has allowed us to assign both genic and non-genic CNVs to interrelated pathways known to govern the development of the anorectal region. We have linked 11 candidate genes to the WNT signalling pathway and 17 genes to the cytoskeletal network. Interestingly, candidate genes with similar functions are disrupted by the same type of CNV. The gene network we discovered provides evidence that rare mutations in different interrelated genes may lead to similar phenotypes, accounting for genetic heterogeneity in ARMs. Classification of patients according to the affected pathway and lesion type should eventually improve the diagnosis and the identification of common genes/molecules as therapeutic targets.
doi:10.1371/journal.pone.0069142
PMCID: PMC3731316  PMID: 23936318
15.  Genetic and Environmental Contributions to General Cognitive Ability Through the First 16 Years of Life 
Developmental psychology  2004;40(5):805-812.
The genetic and environmental contributions to the development of general cognitive ability throughout the first 16 years of life were examined using sibling data from the Colorado Adoption Project. Correlations were analyzed along with structural equation models to characterize the genetic and environmental influences on longitudinal stability and instability. Intraclass correlations reflected both considerable genetic influence at each age and modest shared environmental influence within and across ages. Modeling results suggested that genetic factors mediated phenotypic stability throughout this entire period, whereas most age-to-age instability appeared to be due to nonshared environmental influences.
doi:10.1037/0012-1649.40.5.805
PMCID: PMC3710702  PMID: 15355167
16.  Genetic Analyses of a Three Generation Family Segregating Hirschsprung Disease and Iris Heterochromia 
PLoS ONE  2013;8(6):e66631.
We present the genetic analyses conducted on a three-generation family (14 individuals) with three members affected with isolated-Hirschsprung disease (HSCR) and one with HSCR and heterochromia iridum (syndromic-HSCR), a phenotype reminiscent of Waardenburg-Shah syndrome (WS4). WS4 is characterized by pigmentary abnormalities of the skin, eyes and/or hair, sensorineural deafness and HSCR. None of the members had sensorineural deafness. The family was screened for copy number variations (CNVs) using Illumina-HumanOmni2.5-Beadchip and for coding sequence mutations in WS4 genes (EDN3, EDNRB, or SOX10) and in the main HSCR gene (RET). Confocal microscopy and immunoblotting were used to assess the functional impact of the mutations. A heterozygous A/G transition in EDNRB was identified in 4 affected and 3 unaffected individuals. While in EDNRB isoforms 1 and 2 (cellular receptor) the transition results in the abolishment of translation initiation (M1V), in isoform 3 (only in the cytosol) the replacement occurs at Met91 (M91V) and is predicted benign. Another heterozygous transition (c.-248G/A; -predicted to affect translation efficiency-) in the 5′-untranslated region of EDN3 (EDNRB ligand) was detected in all affected individuals but not in healthy carriers of the EDNRB mutation. Also, a de novo CNVs encompassing DACH1 was identified in the patient with heterochromia iridum and HSCR
Since the EDNRB and EDN3 variants only coexist in affected individuals, HSCR could be due to the joint effect of mutations in genes of the same pathway. Iris heterochromia could be due to an independent genetic event and would account for the additional phenotype within the family.
doi:10.1371/journal.pone.0066631
PMCID: PMC3694150  PMID: 23840513
17.  Utility of the trnH–psbA Intergenic Spacer Region and Its Combinations as Plant DNA Barcodes: A Meta-Analysis 
PLoS ONE  2012;7(11):e48833.
Background
The trnH–psbA intergenic spacer region has been used in many DNA barcoding studies. However, a comprehensive evaluation with rigorous sequence preprocessing and statistical testing on the utility of trnH–psbA and its combinations as DNA barcodes is lacking.
Methodology/Principal Findings
Sequences were searched from GenBank for a meta-analysis on the usefulness of trnH–psbA and its combinations as DNA barcodes. After preprocessing, we constructed full and matching data sets that contained 17 983 trnH–psbA sequences and 2190 sets of trnH–psbA, matK, rbcL, and ITS2 sequences from the same sample, repectively. These datasets were used to analyze the ability of trnH–psbA and its combinations to discriminate species by the BLAST and BLAST+P methods. The Fisher's exact test was used to evaluate the significance of performance differences. For the full data set, the identification success rates of trnH–psbA exceeded 70% in 18 families and 12 genera, respectively. For the matching data set, the identification rates of trnH–psbA were significantly higher than those of the other loci in two families and four genera. Similarly, the identification rates of trnH–psbA+ITS2 were significantly higher than those of matK+rbcL in 18 families and 21 genera.
Conclusion/Significane
This study provides valuable information on the higher utility of trnH–psbA and its combinations. We found that trnH–psbA+ITS2 combination performs better or equally well compared with other combinations in most taxonomic groups investigated. This information will guide the optimal usage of trnH–psbA and its combinations for species identification.
doi:10.1371/journal.pone.0048833
PMCID: PMC3498263  PMID: 23155412
18.  Correction: A Genome-Wide Linkage and Association Scan Reveals Novel Loci for Hypertension and Blood Pressure Traits 
PLoS ONE  2012;7(6):10.1371/annotation/4415f88f-ab10-44dd-8ba9-1a57ade740c1.
doi:10.1371/annotation/4415f88f-ab10-44dd-8ba9-1a57ade740c1
PMCID: PMC3371059
20.  Homozygosity mapping on a single patient--identification of homozygous regions of recent common ancestry by using population data 
Human Mutation  2011;32(3):345-353.
Homozygosity mapping has played an important role in detecting recessive mutations using families of consanguineous marriages. However, detection of homozygous regions identity by descent (HBD) when family data is not available, or when relationship is hidden, is still a challenge. Making use of population data from high-density SNP genotyping may allow detection of regions HBD from recent common founders in singleton patients without genealogy information. We report a novel algorithm that detects such regions by estimating the population haplotype frequencies (HF) for an entire homozygous region. We also developed a simulation method to evaluate the probability of HBD for a homozygous region by examining the best regions in unaffected controls from the host population. The method can be applied to diseases of Mendelian inheritance and can be further extended to complex diseases to detect rare founder mutations using multiplex families or sporadic cases. Testing of the method on both real cases (singleton affected) and simulated data demonstrated its superb sensitivity and great resistance to genetic heterogeneity.
doi:10.1002/humu.21432
PMCID: PMC3357498  PMID: 21309031
homozygosity mapping; recessive mutation; founder mutation; runs of homozygosity; hidden relationship
21.  Genome-Wide Copy Number Analysis Uncovers a New HSCR Gene: NRG3 
PLoS Genetics  2012;8(5):e1002687.
Hirschsprung disease (HSCR) is a congenital disorder characterized by aganglionosis of the distal intestine. To assess the contribution of copy number variants (CNVs) to HSCR, we analysed the data generated from our previous genome-wide association study on HSCR patients, whereby we identified NRG1 as a new HSCR susceptibility locus. Analysis of 129 Chinese patients and 331 ethnically matched controls showed that HSCR patients have a greater burden of rare CNVs (p = 1.50×10−5), particularly for those encompassing genes (p = 5.00×10−6). Our study identified 246 rare-genic CNVs exclusive to patients. Among those, we detected a NRG3 deletion (p = 1.64×10−3). Subsequent follow-up (96 additional patients and 220 controls) on NRG3 revealed 9 deletions (combined p = 3.36×10−5) and 2 de novo duplications among patients and two deletions among controls. Importantly, NRG3 is a paralog of NRG1. Stratification of patients by presence/absence of HSCR–associated syndromes showed that while syndromic–HSCR patients carried significantly longer CNVs than the non-syndromic or controls (p = 1.50×10−5), non-syndromic patients were enriched in CNV number when compared to controls (p = 4.00×10−6) or the syndromic counterpart. Our results suggest a role for NRG3 in HSCR etiology and provide insights into the relative contribution of structural variants in both syndromic and non-syndromic HSCR. This would be the first genome-wide catalog of copy number variants identified in HSCR.
Author Summary
Copy number variations (CNVs) are significant genetic risk factors in disease pathogenesis and represent an important portion of missing heritability for some human diseases, making their discovery essential for the identification of genes and risk factors for a wide range of diseases, including Hirschsprung disease (HSCR, congenital colon aganglionosis). Since the discovery of the major HSCR gene, RET, a number of rare mutations have been reported in RET and other genes involved in the development of the enteric nervous system. However, these mutations contribute to only a small proportion of the disease susceptibility. Taking advantage of the recent technical and methodological advances, we have examined the contribution of CNVs to the disease. We have found that HSCR patients are enriched with CNVs encompassing genes. In particular, we found that deletions of NRG3, a paralog of the previously identified HSCR–susceptibility gene NRG1, were associated with the HSCR phenotype.
doi:10.1371/journal.pgen.1002687
PMCID: PMC3349728  PMID: 22589734
22.  Identification of IGF1, SLC4A4, WWOX, and SFMBT1 as Hypertension Susceptibility Genes in Han Chinese with a Genome-Wide Gene-Based Association Study 
PLoS ONE  2012;7(3):e32907.
Hypertension is a complex disorder with high prevalence rates all over the world. We conducted the first genome-wide gene-based association scan for hypertension in a Han Chinese population. By analyzing genome-wide single-nucleotide-polymorphism data of 400 matched pairs of young-onset hypertensive patients and normotensive controls genotyped with the Illumina HumanHap550-Duo BeadChip, 100 susceptibility genes for hypertension were identified and also validated with permutation tests. Seventeen of the 100 genes exhibited differential allelic and expression distributions between patient and control groups. These genes provided a good molecular signature for classifying hypertensive patients and normotensive controls. Among the 17 genes, IGF1, SLC4A4, WWOX, and SFMBT1 were not only identified by our gene-based association scan and gene expression analysis but were also replicated by a gene-based association analysis of the Hong Kong Hypertension Study. Moreover, cis-acting expression quantitative trait loci associated with the differentially expressed genes were found and linked to hypertension. IGF1, which encodes insulin-like growth factor 1, is associated with cardiovascular disorders, metabolic syndrome, decreased body weight/size, and changes of insulin levels in mice. SLC4A4, which encodes the electrogenic sodium bicarbonate cotransporter 1, is associated with decreased body weight/size and abnormal ion homeostasis in mice. WWOX, which encodes the WW domain-containing protein, is related to hypoglycemia and hyperphosphatemia. SFMBT1, which encodes the scm-like with four MBT domains protein 1, is a novel hypertension gene. GRB14, TMEM56 and KIAA1797 exhibited highly significant differential allelic and expressed distributions between hypertensive patients and normotensive controls. GRB14 was also found relevant to blood pressure in a previous genetic association study in East Asian populations. TMEM56 and KIAA1797 may be specific to Taiwanese populations, because they were not validated by the two replication studies. Identification of these genes enriches the collection of hypertension susceptibility genes, thereby shedding light on the etiology of hypertension in Han Chinese populations.
doi:10.1371/journal.pone.0032907
PMCID: PMC3315540  PMID: 22479346
24.  A Genome-Wide Linkage and Association Scan Reveals Novel Loci for Hypertension and Blood Pressure Traits 
PLoS ONE  2012;7(2):e31489.
Hypertension is caused by the interaction of environmental and genetic factors. The condition which is very common, with about 18% of the adult Hong Kong Chinese population and over 50% of older individuals affected, is responsible for considerable morbidity and mortality. To identify genes influencing hypertension and blood pressure, we conducted a combined linkage and association study using over 500,000 single nucleotide polymorphisms (SNPs) genotyped in 328 individuals comprising 111 hypertensive probands and their siblings. Using a family-based association test, we found an association with SNPs on chromosome 5q31.1 (rs6596140; P<9×10−8) for hypertension. One candidate gene, PDC, was replicated, with rs3817586 on 1q31.1 attaining P = 2.5×10−4 and 2.9×10−5 in the within-family tests for DBP and MAP, respectively. We also identified regions of significant linkage for systolic and diastolic blood pressure on chromosomes 2q22 and 5p13, respectively. Further family-based association analysis of the linkage peak on chromosome 5 yielded a significant association (rs1605685, P<7×10−5) for DBP. This is the first combined linkage and association study of hypertension and its related quantitative traits with Chinese ancestry. The associations reported here account for the action of common variants whereas the discovery of linkage regions may point to novel targets for rare variant screening.
doi:10.1371/journal.pone.0031489
PMCID: PMC3286457  PMID: 22384028
25.  RET Mutational Spectrum in Hirschsprung Disease: Evaluation of 601 Chinese Patients 
PLoS ONE  2011;6(12):e28986.
Rare (RVs) and common variants of the RET gene contribute to Hirschsprung disease (HSCR; congenital aganglionosis). While RET common variants are strongly associated with the commonest manifestation of the disease (males; short-segment aganglionosis; sporadic), rare coding sequence (CDS) variants are more frequently found in the lesser common and more severe forms of the disease (females; long/total colonic aganglionosis; familial).
Here we present the screening for RVs in the RET CDS and intron/exon boundaries of 601 Chinese HSCR patients, the largest number of patients ever reported. We identified 61 different heterozygous RVs (50 novel) distributed among 100 patients (16.64%). Those include 14 silent, 29 missense, 5 nonsense, 4 frame-shifts, and one in-frame amino-acid deletion in the CDS, two splice-site deletions, 4 nucleotide substitutions and a 22-bp deletion in the intron/exon boundaries and 1 single-nucleotide substitution in the 5′ untranslated region. Exonic variants were mainly clustered in RET the extracellular domain. RET RVs were more frequent among patients with the most severe phenotype (24% vs. 15% in short-HSCR). Phasing RVs with the RET HSCR-associated haplotype suggests that RVs do not underlie the undisputable association of RET common variants with HSCR. None of the variants were found in 250 Chinese controls.
doi:10.1371/journal.pone.0028986
PMCID: PMC3235168  PMID: 22174939

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