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Introduction and objectives: Omenn syndrome (OS) is a very rare type of severe combined immunodeficiencies manifested with erythroderma, eosinophilia, hepatosplenomegaly, lymphadenopathy, and elevated level of serum IgE. OS is inherited with an autosomal recessive mode of inheritance. Germline mutations in the human RAG1 gene cause OS. Materials and methods: In this study, we investigated a 2-month-old boy with cough, mild anaemia, pneumonia, immunodeficiency, repeated infection, feeding difficulties, hepatomegaly, growth retardation, and heart failure. Parents of the proband were phenotypically normal. Results: Karyotype analysis and chromosomal microarray analysis found no chromosomal struc-tural abnormalities (46, XY) and no pathogenic copy number variations (CNVs) in the proband. Whole-exome sequencing identified a novel homozygous single nucleotide deletion (c.2662delC) in exon 2 of the RAG1 gene in the proband. Sanger sequencing confirmed that both the proband parents were carrying this variant in a heterozygous state. This variant was not identified in two elder sisters and one elder brother of the proband and in the 100 ethnically matched normal healthy individuals. This novel homozygous deletion (c.2662delC) leads to the frameshift, which finally results in the formation of the truncated protein (p.Leu888Phefs*3) V(D)J recombination-activating protein 1 with 890 amino acids compared with the wildtype V(D)J recombination-activating protein 1 of 1043 amino acids. Hence, it is a loss-of-function variant. Conclusions: Our present study expands the mutational spectrum of the RAG1 gene associated with OS. We also strongly suggested the importance of whole-exome sequencing for the genetic screening of patients with OS.

Isolated sulfite oxidase deficiency (ISOD) is a rare type of life-threatening neurometabolic disorders characterized by neonatal intractable seizures and severe developmental delay with an autosomal recessive mode of inheritance. Germline mutation in SUOX gene causes ISOD. Till date, only 32 mutations of SUOX gene have been identified and reported to be associated with ISOD.Here, we investigated a 5-days old Chinese female child, presented with intermittent tremor or seizures of limbs, neonatal encephalopathy, subarachnoid cyst and haemorrhage, dysplasia of corpus callosum, neonatal convulsion, hyperlactatemia, severe metabolic acidosis, hyperglycemia, and hyperkalemia.Whole exome sequencing identified a novel homozygous transition (c.1227G A) in exon 6 of the SUOX gene in the proband. This novel homozygous variant leads to the formation of a truncated sulfite oxidase (p.Trp409*) of 408 amino acids. This variant causes partial loss of the dimerization domain of sulfite oxidase. Hence, it is a loss-of-function variant. Proband's father and mother is carrying this novel variant in a heterozygous state. This variant was not found in 200 ethnically matched normal healthy control individuals.Our study not only expanded the mutational spectrum of SUOX gene associated with ISOD, but also strongly suggested the significance of whole exome sequencing for identifying candidate genes and novel disease-causing variants.

Omenn syndrome (OS) is a very rare type of severe combined immunodeficiencies manifested with erythroderma, eosinophilia, hepatosplenomegaly, lymph-adenopathy, and elevated level of serum IgE. OS is inherited with an autosomal recessive mode of inheritance. Germline mutations in the humanIn this study, we investigated a 2-month-old boy with cough, mild anaemia, pneumonia, immunodeficiency, repeated infection, feeding difficulties, hepatomegaly, growth retardation, and heart failure. Parents of the proband were phenotypically normal.Karyotype analysis and chromosomal microarray analysis found no chromosomal structural abnormalities (46, XY) and no pathogenic copy number variations (CNVs) in the proband. Whole-exome sequencing identified a novel homozygous single nucleotide deletion (c.2662delC) in exon 2 of theOur present study expands the mutational spectrum of the

Background: Isolated sulfite oxidase deficiency (ISOD) is the rarest types of life-threatening neurometabolic disorders characterized by neonatal intractable seizures and severe developmental delay with an autosomal recessive mode of inheritance. ISOD is extremely rare and till date only 32 mutations have been identified and reported worldwide. Germline mutation in SUOX gene causes ISOD. Methods: Here, we investigated a 5-days old Chinese female child, presented with intermittent tremor or seizures of limbs, neonatal encephalopathy, subarachnoid cyst and haemorrhage, dysplasia of corpus callosum, neonatal convulsion, respiratory failure, cardiac failure, hyperlactatemia, severe metabolic acidosis, hyperglycemia, hyperkalemia, moderate anemia, atrioventricular block and complete right bundle branch block. Results: Whole exome sequencing identified a novel homozygous transition (c.1227G>A) in exon 6 of the SUOX gene in the proband. This novel homozygous variant leads to the formation of a truncated sulfite oxidase (p.Trp409*) of 408 amino acids. Hence, it is a loss-of-function variant. Proband’s father and mother is carrying this novel variant in a heterozygous state. This variant was not identified in 200 ethnically matched normal healthy control individuals. Conclusions: Our study not only expand the mutational spectrum of SUOX gene associated ISOD, but also strongly suggested the application of whole exome sequencing for identifying candidate genes and novel disease-causing mutations.

Summary Tumor multiregion sequencing reveals intratumor heterogeneity (ITH) and clonal evolution playing a key role in tumor progression and metastases. Large-scale high-depth multiregional sequencing of colorectal cancer, comparative analysis among patients with right-sided colon cancer (RCC), left-sided colon cancer (LCC), and rectal cancer (RC), as well as the study of lymph node metastasis (LN) with extranodal tumor deposits (ENTDs) from evolutionary perspective remain weakly explored. Here, we recruited 68 patients with RCC (18), LCC (20), and RC (30). We performed high-depth whole-exome sequencing of 206 tumor regions including 176 primary tumors, 19 LN, and 11 ENTD samples. Our results showed ITH with a Darwinian pattern of evolution and the evolution pattern of LCC and RC was more complex and divergent than RCC. Genetic and evolutionary evidences found that both LN and ENTD originated from different clones. Moreover, ENTD was a distinct entity from LN and evolved later., Graphical abstract, Highlights • Clonal evolution of colorectal tumors followed Darwinian pattern of evolution • Intratumor heterogeneity in patients with right-sided and left-sided colon and rectal cancer • Evolution of left-sided colon cancer and rectal cancer was more complex and divergent • Lymph node metastasis and extranodal tumor deposits were of polyclonal in origin, Evolutionary biology; Cancer systems biology; Genomics

BackgroundGermline variants of ten keratin genes (K1,K2,K5,K6A,K6B,K9,K10,K14,K16, andK17) have been reported for causing different types of genodermatoses with an autosomal dominant mode of inheritance. Among all the variants of these ten keratin genes, most of them are missense variants. Unlike pathogenic and likely pathogenic variants, understanding the clinical importance of novel missense variants or variants of uncertain significance (VUS) is the biggest challenge for clinicians or medical geneticists. Functional characterization is the only way to understand the clinical association of novel missense variants or VUS but it is time consuming, costly, and depends on the availability of patient’s samples. Existing databases report the pathogenic variants of the keratin genes, but never emphasize the systematic effects of these variants on keratin protein structure and genotype-phenotype correlation.ResultsTo address this need, we developed a comprehensive database KVarPredDB, which contains information of all ten keratin genes associated with genodermatoses. We integrated and curated 400 reported pathogenic missense variants as well as 4629 missense VUS. KVarPredDB predicts the pathogenicity of novel missense variants as well as to understand the severity of disease phenotype, based on four criteria; firstly, the difference in physico-chemical properties between the wild type and substituted amino acids; secondly, the loss of inter/intra-chain interactions; thirdly, evolutionary conservation of the wild type amino acids and lastly, the effect of the substituted amino acids in the heptad repeat. Molecular docking simulations based on resolved crystal structures were adopted to predict stability changes and get the binding energy to compare the wild type protein with the mutated one. We use this basic information to determine the structural and functional impact of novel missense variants on the keratin coiled-coil heterodimer. KVarPredDB was built under the integrative web application development framework SSM (SpringBoot, Spring MVC, MyBatis) and implemented in Java, Bootstrap, React-mutation-mapper, MySQL, Tomcat. The website can be accessed throughhttp://bioinfo.zju.edu.cn/KVarPredDB. The genomic variants and analysis results are freely available under the Creative Commons license.ConclusionsKVarPredDB provides an intuitive and user-friendly interface with computational analytical investigation for each missense variant of the keratin genes associated with genodermatoses.

Background The correlation between long-term treatment outcomes with genotypes in congenital hypogonadotropic hypogonadism (CHH) males is rarely reported. Aim To investigate the correlations among genotypes, phenotypes, and treatment outcomes for CHH male patients. Methods Whole exome sequencing was performed for 73 Chinese CHH males from one academic center. Patients self-selected one of the 4 treatments: pulsatile Gonadorelin pump (PGP), cyclical gonadotropins therapy (CGT), human menopausal gonadotropin monotherapy, or testosterone replacement treatment. Clinical assessments were performed every 3 months for 3 years. Outcomes The pathogenicity of variants was determined. Baseline clinical features, spermatogenesis outcomes were analysed. RESULTS 62 variants were identified in 51 patients (69.9%), 17 of which were novel. Among these mutations, variants on FGFR1, PROKR2, CHD7, ANOS1 and NSMF gene were 16.1%, 16.1%, 11.3%, 8.1% and 8.1% respectively. 11 patients followed the oligogenic pattern (21.6%). All CHD7 patients had hearing impairment or structural deformities of external/inner ear, and were diagnosed as CHARGE syndrome. 24.7% of CHH patients manifested with ear/hearing anomalies. KS patients had higher rates of cryptorchidism history and ear/hearing anomalies than normosmic CHH subjects. Male patients with PROKR2 mutations showed relatively better testicular development, less dental deformity when compared with FGFR1 mutations. About 30% normosmic patients defined by simple olfactory assessment showed olfactory nerve center (ONC) dysplasia under nasal sinus MRI examination. Among the CHH males treated with CGT or PGP, 70.2% reached spermatogenesis within 3 years of treatment. Clinical Implications No direct correlation was observed between certain responsible genes and spermatogenic outcomes. When CHH patients were identified with CHD7 variants, ear/hearing evaluation should be carefully performed. The precise assessment of ONC development was advised for normosmic CHH subjects. Strengths & Limitations This study provided informative long-term treatment data of CHH male patients screened with whole exome sequencing. The limitations included small number of subgroups with multifaceted gene variants, clinical heterogeneity, and uncontrolled sperm-inducing treatment method. The seventeen novel mutations worth experimental validation in the future. CONCLUSION The clinical severity is partially related with specific gene variants, and detailed individualized data and outcomes were provided. Ear/hearing anomalies were closely connected with CHD7 variants, and were common problems for CHH patients. Simple olfactory assessment underestimated the true olfactory deficit.

BackgroundIsolated sulfite oxidase deficiency (ISOD) is the rarest types of life-threatening neurometabolic disorders characterized by neonatal intractable seizures and severe developmental delay with an autosomal recessive mode of inheritance. ISOD is extremely rare and only 29 mutations have been identified and reported worldwide. Germline mutation in SUOX gene causes ISOD. MethodsHere, we investigated a 5-days old Chinese girl, presented with intermittent tremor or seizures of limbs, neonatal encephalopathy, subarachnoid cyst and haemorrhage, dysplasia of corpus callosum, neonatal convulsion, respiratory failure, cardiac failure, hyperlactatemia, severe metabolic acidosis, hyperglycemia, hyperkalemia, moderate anemia, atrioventricular block and complete right bundle branch block. Karyotype and chromosomal microarray analysis were performed and identified no chromosomal abnormalities in the proband. Whole exome sequencing was performed for the proband. Sanger sequencing was performed for the proband and her family members. ResultsWhole exome sequencing identified a novel heterozygous deletion (c.1406_1421delCCTGGCAGGTGGCTAA) and a previously reported heterozygous substitution (c.1200C>G) in exon 6 of the SUOX gene in the proband. The novel heterozygous deletion leads to frameshift (p.Thr469Serfs*20) which results into the formation of a truncated sulfite oxidase of 488 amino acids. The substitution leads to a premature stop codon (p.Tyr400*) followed by the formation of a truncated sulfite oxidase of 399 amino acids. Hence, both the variants are loss-of-function variants. The proband’s father and mother is carrying the substitution and deletion in a heterozygous state respectively. These two variants were not identified in the elder brother of the proband as well as in the 100 healthy individuals. ConclusionHere, we reported the first variant of SUOX gene associated ISOD in Chinese population. Our study not only expand the mutational spectrum of SUOX gene associated ISOD, but also strongly suggested the application of whole exome sequencing for identifying the novel disease-causing mutation in candidate genes.

BackgroundTumor multi-region sequencing reveals intratumor heterogeneity (ITH) and clonal evolution which play a key role in progression and metastases of the tumor. However, large-scale high depths multiregional sequencing of colorectal cancer (CRC) has not been well studied. In addition, the comparative analysis among right-sided colon cancer (RCC), left-sided colon cancer (LCC) and rectal cancer (RC) patients as well as the study of lymph node metastasis (LN) with extranodal tumor deposits (ENTD) from evolutionary perspective remain unknown.ResultsIn this prospective study, we recruited different stages of 68 CRC patients with RCC (18), LCC (20) and RC (30). We performed high-depth whole exome sequencing (WES) of 206 tumor regions including 176 primary tumors, 19 LN and 11 ENTD samples. Our results showed ITH with a Darwinian pattern of evolution. We identified that the evolution pattern of LCC and RC was more complex and divergent than RCC, suggesting the evolutionary diversity in the initiation and progression of LCC and RC. Genetic and evolutionary evidences found that both LN and ENTD were of polyclonal in origin. Moreover, ENTD was a distinct entity from LN and evolved later.ConclusionsIn conclusion, our study showed the Darwinian pattern of evolution with differences in clonal evolution between RCC with LCC and RC.

LS is a colorectal cancer (CRC) predisposing syndrome with an autosomal dominant mode of inheritance Lynch et al...

Idiopathic ventricular tachycardia (IVT) is the major cause of sudden cardiac death. Patients with IVT were usually manifested without structural heart disease. In this present study, we performed family-based whole genome sequencing (WGS) and Sanger sequencing for a 5-year-old Chinese boy with IVT and all the unaffected family members in order to identify the candidate gene and disease-causing mutation underlying the disease phenotype. Results showed that a novel heterozygous single-nucleotide duplication (c.128dup) and a novel heterozygous missense (c.3328A > G) variant in ABCA5 gene were identified in the proband. The single-nucleotide duplication (c.128dupT), inherited from his father and patrilineal grandfather, leads to a frameshift which results into the formation of a truncated ABCA5 protein of 50 (p.Leu43Phefs*8) amino acids. Hence, it is a loss-of-function mutation. The missense (c.3328A > G) variant, inherited from his mother, leads to the replacement of isoleucine by valine at the position of 1110 (p.Ile1110Val) of the ABCA5 protein. Multiple sequence alignment showed that p.Ile1110 is evolutionarily conserved among several species indicating both the structural and functional significance of the p.Ile1110 residue in the wild-type ABCA5 protein. Quantitative RT-PCR showed that the ABCA5 mRNA expression levels were decreased in the proband. These two novel variants of ABCA5 gene were co-segregated well among all the members of this family. Our present study also strongly supports the importance of using family-based whole genome sequencing for identifying novel candidate genes associated with IVT.

Hypertriglyceridaemia is a very rare disorder caused by the mutations of LPL gene, with an autosomal recessive mode of inheritance. Here, we identified two unrelated Chinese patients manifested with severe hypertriglyceridaemia and acute pancreatitis. The clinical symptoms of proband 1 are more severe than proband 2. Whole exome sequencing and Sanger sequencing were performed. Functional analysis of the identified mutations has been done. Whole exome sequencing identified two pairs of variants in LPL gene in the proband 1 (c.162C>A and c.1322+1G>A) and proband 2 (c.835C>G and c.1322+1G>A). The substitution (c.162C>A) leads to the formation of a truncated (p.Cys54*) LPL protein. The substitution (c.835C>G) leads to the replacement of leucine to valine (p.Leu279Val). The splice donor site mutation (c.1322+1G>A) leads to the formation of alternative transcripts with the loss of 134 bp in exon 8 of the LPL gene. The proband 1 and his younger son also harbouring a heterozygous variant (c.553G>T; p.Gly185Cys) in APOA5 gene. The relative expression level of the mutated LPL mRNA (c.162C>A, c.835C>G and c.1322+1G>A) showed significant differences compared to wild-type LPL mRNA, suggesting that all these three mutations affect the transcription of LPL mRNA. These three mutations (c.162C>A, c.835C>G and c.1322+1G>A) showed noticeably decreased LPL activity in cell culture medium but not in cell lysates. Here, we identified three mutations in LPL gene which causes severe hypertriglyceridaemia with acute pancreatitis in Chinese patients. We also described the significance of whole exome sequencing for identifying the candidate gene and disease-causing mutation in patients with severe hypertriglyceridaemia and acute pancreatitis.

Muscular dystrophy‐dystroglycanopathy (limb‐girdle), type C, 9 (MDDGC9) is the rarest type of autosomal recessive muscular dystrophies. MDDGC9 is manifested with an early onset in childhood. Patients with MDDGC9 usually identified with defective glycosylation of DAG1, hence it is known as “dystroglycanopathies”. Here, we report a Chinese pedigree presented with mild MDDGC9. The proband is a 64 years old Chinese man. In this family, both the proband and proband's younger brother have been suffering from mild and late onset MDDGC9. Muscle biopsy showed that the left deltoid muscle with an advanced stage of dystrophic change. Immunohistochemistry staining of dystrophin, α‐sarcoglycan, β‐sarcoglycan and dysferlin are normal. Molecular genetic analysis of the proband has been done with whole exome sequencing. A homozygous novel missense mutation (c.2326C>T; p.R776C) in the exon 3 of the DAG1 gene has been identified in the proband. Sanger sequencing revealed that this missense mutation is co‐segregated well among the affected and unaffected (carrier) family members. This mutation is not detected in 200 normal healthy control individuals. This novel homozygous missense mutation (c.2326C>T) causes substitution of arginine by cystine at the position of 776 (p.R776C) which is evolutionarily highly conserved. Immunoblotting studies revealed that a significant reduction of α‐dystroglycan expression in the muscle tissue. The novelty of our study is that it is a first report of DAG1 associated muscular dystrophy‐dystroglycanopathy (limb‐girdle), type C, 9 (MDDGC9) with mild and late age of onset. In Chinese population this is the first report of DAG1 associated MDDGC9.

Lynch syndrome (LS) is one of the most common familial forms of colorectal cancer predisposing syndrome with an autosomal dominant mode of inheritance. LS is caused by the germline mutations in DNA mismatch repair (MMR) genes including MSH2, MLH1, MSH6 and PMS2. Clinically, LS is characterized by high incidence of early-onset colorectal cancer as well as endometrial, small intestinal and urinary tract cancers, usually occur in the third to fourth decade of the life. Here we describe a five generation Chinese family with LS clinically diagnosed according to the Amsterdam II criteria. Immuno-histochemical staining of MSH2 and MSH6 shows only foci nuclear positive on the surface of the tumor with strong expression of MLH1 and PMS2 with diffuse immunoreactivity. In order to dig into the molecular basis of this LS pedigree, we collected the proband's blood sample, extracted the genomic DNA and applied the genetic screening. As a result, we identified a novel heterozygous deletion in MSH2 gene by targeted next generation sequencing, which is also proved to be co-segregated among other affected family members by following validation. To our knowledge, this novel heterozygous deletion (c.1676_1679 delTAAA) in MSH2 gene causes frameshift mutation (p.Asn560Lysfs*29) and leads to the formation of a truncated MSH2 protein which is confirmed to be a deleterious mutation according to the variant interpretation guidelines of American College of Medical Genetics and Genomics (ACMG). Identification of novel DNA mismatch repair (MMR) gene mutations can definitely benefit to the clinical diagnosis and management.

// Hui Huang 1, * , Ying Wang 2, 3, * , Huishuang Chen 1, * , Yanhua Chen 1, 4 , Jing Wu 1 , Pei-Wen Chiang 5 , Ning Fan 2 , Yan Su 1 , Jianlian Deng 1 , Dongna Chen 1 , Yang Li 1 , Xinxin Zhang 1, 6 , Mengxin Zhang 7 , Shengran Liang 1 , Santasree Banerjee 1 , Ming Qi 1, 8, 9 and Xuyang Liu 2, 10 1 BGI-Shenzhen, Shenzhen, China 2 Shenzhen Key Laboratory of Ophthalmology, Shenzhen Eye Hospital, Jinan University, Shenzhen, China 3 Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Optoelectronic Engineering, Shenzhen University, Shenzhen, China 4 School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, China 5 Casey Eye Institute Molecular Diagnostic Laboratory, Portland, Oregon, USA 6 BGI Education Center, University of Chinese Academy of Sciences, Shenzhen, China 7 Department of Applied Biology with Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong 8 School of Basic Medical Sciences, Zhejiang University, Hangzhou, China 9 Functional Genomics Center, Department of Pathology & Laboratory Medicine, University of Rochester Medical Center, West Henrietta, New York, USA 10 School of Ophthalmology & Optometry, Shenzhen University, Shenzhen, China * These authors have contributed equally to this work Correspondence to: Xuyang Liu, email: xliu1213@126.com Ming Qi, email: qiming@genomics.cn Keywords: targeted next generation sequencing, gene panel, novel mutation, retinitis pigmentosa, Leber's congenital amaurosis Received: October 26, 2016 Accepted: March 15, 2017 Published: April 12, 2017 ABSTRACT As the most common inherited retinal degenerations, retinitis pigmentosa (RP) is clinically and genetically heterogeneous. Some of the RP genes are also associated with other retinal diseases, such as LCA (Leber's congenital amaurosis) and CORD (cone-rod dystrophy). Here, in our molecular diagnosis of 99 Chinese RP patients using targeted gene capture sequencing, three probands were found to carry mutations of RPGRIP1 , which was known to be associated with pathogenesis of LCA and CORD. By further clinical analysis, two probands were confirmed to be RP patients and one was confirmed to be LCA patient. These novel mutations were co-segregated with the disease phenotype in their families. Our result not only expands the mutational spectrum of the RPGRIP1 gene but also gives supports to clinical diagnosis and molecular treatment of RP patients.

// Dan Wang 1, * , Shengyun Liang 2, * , Zhao Zhang 3, * , Guoru Zhao 2 , Yuan Hu 4 , Shengran Liang 5 , Xipeng Zhang 3 , Santasree Banerjee 6, * 1 Department of Pathology, Tianjin Medical University General Hospital, Tianjin 300052, China 2 Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China 3 Department of Colorectal Surgery, Tianjin Union Medical Center, Tianjin 300121, China 4 Department of Hematology, Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510006, China 5 School of Life Science and Biopharmaceuticals, Guangdong Pharmaceutical University, Guangzhou 510006, China 6 Department of Cell Biology and Medical Genetics, School of Medicine, Zhejiang University, Hangzhou 310000, China * These authors contributed equally to this work Correspondence to: Santasree Banerjee, email: santasree.banerjee@yahoo.com Xipeng Zhang, email: zxp2266@163.com Keywords: familial adenomatous polyposis, APC gene, splice acceptor site mutation, targeted next-generation sequencing, colorectal cancer Received: September 06, 2016 Accepted: January 27, 2017 Published: February 20, 2017 ABSTRACT Familial adenomatous polyposis (FAP) is an autosomal dominant precancerous condition, clinically characterized by the presence of multiple colorectal adenomas or polyps. Patients with FAP has a high risk of developing colorectal cancer (CRC) from these colorectal adenomatous polyps by the mean age of diagnosis at 40 years. Germline mutations of the APC gene cause familial adenomatous polyposis (FAP). Colectomy has recommended for the FAP patients with significant polyposis. Here, we present a clinical molecular study of a four generation Chinese family with FAP. Clinical diagnosis of FAP has been done according to the phenotype, family history and medical records. Patient’s blood samples were collected and genomic DNA was extracted. In order to identify the pathogenic mutation underlying the disease phenotype targeted next-generation sequencing and confirmatory sanger sequencing has undertaken. Targeted next generation sequencing identified a novel heterozygous splice-acceptor site mutation [c.1744-1G>A] in intron 14 of APC gene, which is co-segregated with the FAP phenotypes in the proband and amongst all the affected family members. This mutation is not present in unaffected family members and in normal healthy controls of same ethnic origin. According to the LOVD database for Chinese colorectal cancer patients, in Chinese population, 60% of the previously reported APC gene mutations causes FAP, are missense mutations. This novel splice-acceptor site mutation causing FAP in this Chinese family expands the germline mutation spectrum of the APC gene in the Chinese population.

// Santasree Banerjee 1,* , Dongzhu Lei 2,* Shengran Liang 1,* , Li Yang 3,* , Saijun Liu 1 , Zhu Wei 4 and Jian Ping Tang 4,* 1 BGI-Shenzhen, Shenzhen, China 2 Center of Prenatal Diagnosis, ChenZhou No.1 peoples hospital, Hunan, China 3 Biological therapy center, The Third Affiliated Hospital, Sun-Yet-San University, Guangzhou, China 4 Department of dermatology, Hunan Children’s Hospital, Hunan, China * These authors have contributed equally to the manuscript Correspondence to: Jian Ping Tang, email: // Keywords : neurofibromatosis type 1, next generation sequencing, novel mutation, NF1 gene, mutational screening Received : July 10, 2016 Accepted : December 06, 2016 Published : December 14, 2016 Abstract Neurofibromatosis type 1 (NF1) is an autosomal dominant, multi-system, neurocutaneous disorder, manifested with neurofibromas and Cafe´-au-lait spots. Germline mutations in NF1 gene are associated with Neurofibromatosis type 1. NF1 gene encodes neurofibromin, a RAS-specific GTPase activating protein. In our study, we present a clinical molecular study of four Chinese probands with NF1 from four unrelated families, showing extreme phenotypic variation with rare phenotype. In family 1, the proband is a 16 months old girl with multiple cafe-au-lait spots throughout her whole body. In family 2, the proband is a 6 months old girl with several cafe-au-lait spots mostly in her trunk and in lower limbs. In family 3, the proband is a 4 months old boy with several cafe-au-lait spots, tibial pseudarthrosis, and chronic iron deficiency anemia. In family 4, the proband is a 14 years old boy with multiple cafe-au-lait spots of variable sizes. Targeted exome capture based next generation sequencing and Sanger sequencing identified a novel mutation and three previously reported mutations in these four probands. These four mutations in NF1 gene were causing disease phenotypes in these four probands and was absent in unaffected family members and in healthy controls. According to the variant interpretation guideline of American College of Medical Genetics and Genomics (ACMG), these four mutations, are classified as “likely pathogenic”. Our result expands the mutational spectrum of the NF1 gene associated with neurofibromatosis type1.

Background Recently, increasing innovations improved the accuracy of next generation sequencing (NGS) data. However, the validation of all NGS variants increased the cost and turn‐around time of clinical diagnosis, and therefore limited the further development of clinical applications. We aimed to comprehensively assess the necessity of validating NGS variants. Methods Validation data of 7,601 NGS variants involving 1,045 genes were collected from 5,190 clinical samples and sequenced by one of five targeted capture panels and two NGS chemistries, respectively. These genes and variants were widely distributed in 24 human chromosomes and mitochondrial genome. Variants validation was firstly processed by Sanger sequencing. If validation results were unavailable or inconsistent with NGS calls, another validation test would be performed by mass spectrometry genotyping. Results A total of 6,939 high quality NGS variants with ≥35 × depth coverage and ≥35% heterozygous ratio were 100% confirmed by a secondary methodology. 5,775 heterozygous variants were separated from 760 homozygous variants and 404 hemizygous variants by 80% heterozygous ratio. A total of 1.5% (98/6,939) of NGS variants were validated by mass spectrometry genotyping. Conclusion Considering of the above comprehensive assessment, a new variant with high quality from a well‐validated capture‐based NGS workflow can be reported directly without validation.

Meckel syndrome (MKS) is a pre‐ or perinatal multisystemic ciliopathic lethal disorder with an autosomal recessive mode of inheritance. Meckel syndrome is usually manifested with meningo‐occipital encephalocele, polycystic kidney dysplasia, postaxial polydactyly and hepatobiliary ductal plate malformation. Germline variants in CEP290 cause MKS4. In this study, we investigated a 35‐years‐old Chinese female who was 17+1 weeks pregnant. She had a history of adverse pregnancy of having foetus with multiple malformations. We performed ultrasonography and identified the foetus with occipital meningoencephalocele and enlarged cystic dysplastic kidneys. So, she decided to terminate her pregnancy and further genetic molecular analysis was performed. We identified the aborted foetus without postaxial polydactyly. Histological examination of foetal kidney showed cysts in kidney and thinning of the renal cortex with glomerular atrophy. Whole exome sequencing identified a novel homozygous variant (c.2144T>G; p.L715*) in exon 21 of the CEP290 in the foetus. Sanger sequencing confirmed that both the parents of the foetus were carrying this variant in a heterozygous state. This variant was not identified in two elder sisters of the foetus as well as in the 100 healthy individuals. Western blot analysis showed that this variant leads to the formation of truncated CEP290 protein with the molecular weight of 84 KD compared with the wild‐type CEP290 protein of 290 KD. Hence, it is a loss‐of‐function variant. We also found that the mutant cilium appears longer in length than the wild‐type cilium. Our present study reported the first variant of CEP290 associated with MKS4 in Chinese population.

Hereditary hearing impairment is one of the major and common birth defects in Chinese population. Non-syndromic sensorineural hearing loss (NSHL) is the most common types of hereditary hearing impairment. Genotypically and phenotypically NSHL is extremely heterogenous and follow either autosomal dominant or autosomal recessive or X-linked mode of inheritance. Presently, 127 genes have been identified to be associated with both syndromic and (NSHL). Here, we studied a Chinese family with moderate and profound hearing impairment. The proband is a 30-year old Chinese man. The proband was born with normal hearing and at the age of 5-years, the proband was first noticed with hearing impairment. Gradually and progressively the proband was presented with loss of hearing in his both right and left ears at the age of 30 years. The clinical symptoms, age of onset or progression to loss of hearing was similar in both the proband and his younger brother. The proband’s parents are phenotypically normal and non-consanguineous. Clinical diagnosis of the proband and his younger brother has been done by classical pure tone audiogram (PTA). Computed Tomography (CT) found no abnormality in bilateral external ear, middle ear and inner ear. Targeted next generation sequencing was performed with a panel of 127 genes reported to be associated with hereditary hearing impairment. A novel homozygous single nucleotide deletion (c.427delT) in exon 4 of ILDR1 gene has been identified in proband and in his younger brother. Sanger sequencing confirmed that proband’s father and mother are carrying this mutation in a heterozygous manner. This mutation has not been identified in 100 normal healthy control individuals. This mutation (c.427delT) causes frameshift (p.Tyr143Ilefs∗19) which leads to the formation of a truncated ILDR1 protein of 162 amino acids instead of the wild type ILDR1 protein of 546 amino acids. ILDR1 associated hereditary hearing impairment is very rare and this is the first report of identifying a loss-of-function mutation in ILDR1 gene associated with hereditary hearing impairment in Chinese population. Our present study also emphasized the significance of rapid, accurate and cost-effective screening for the patient with hereditary hearing impairment by targeted next generation sequencing.

Rigid spine muscular dystrophy 1 (RSMD1) is a neuromuscular disorder, manifested with poor axial muscle strength, scoliosis and neck weakness, and a variable degree of spinal rigidity with an early ventilatory insufficiency which can lead to death by respiratory failure. Mutations of SEPN1 gene are associated with autosomal recessive RSMD1. Here, we present a clinical molecular study of a Chinese proband with RSMD1. The proband is a 17 years old male, showing difficulty in feeding, delayed motor response, problem in running with frequent fall down, early onset respiratory insufficiency, general muscle weakness and rigid cervical spine. Muscle biopsy identified increased variability of fiber size with atrophic muscle cells consistent with non-specific myopathic changes. Proband's elder brother presented with same phenotype as the proband and died at the age of 15 years due to acute respiratory failure. Proband's father and mother are phenotypically normal. Targeted exome capture based next generation sequencing and Sanger sequencing identified that the proband was a compound heterozygote with two novel mutations in SEPN1 gene; a novel missense mutation (c.1384T>C; p.Sec462Arg) and a novel nonsense mutation (c.1525C>T; p.Gln509Ter), inherited from his father and mother respectively. These two mutations are co-segregated with the disease phenotypes in the proband and was absent in normal healthy controls. Our present study expands the mutational spectrum of the SEPN1 associated RSMD1.

// Santasree Banerjee 1,* , Huishuang Chen 1,* , Hui Huang 1,* , Jing Wu 1 , Zhiyun Yang 2 , Weiping Deng 3 , Dongna Chen 1 , Jianlian Deng 1 , Yan Su 1 , Yang Li 1 , Chao Wu 6 , Ye Wang 4 , Hao Zeng 4 , Yiming Wang 5,1 and Xunhua Li 6 1 BGI-Shenzhen, Shenzhen, China 2 Department of Medical Imaging, 1st affiliated hospital, Sun Yat-sen University, Guangzhou, PR China 3 Department of Dermatology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, PR China 4 Department of Medical Genetics, Center for Genome Research, Sun Yat-sen University, Guangzhou, PR China 5 Xinhua College, Sun Yat-sen university, Guangzhou, PR China 6 Department of Neurology, 1st affiliated hospital, Sun Yat-sen University, Guangzhou, PR China * These authors have contributed equally to this work Correspondence to: Xunhua Li, email: // Yiming Wang, email: // Keywords : microcephaly 2, WDR62 mutation, compound heterozygosity, novel mutations, exome sequencing, Pathology Section Received : June 25, 2016 Accepted : November 01, 2016 Published : November 10, 2016 Abstract Microcephaly (MCPH) is a developmental disorder characterized by reduced brain size and intellectual disability. A proportion of microcephaly is caused by defects in a single gene. Microcephaly 2 (MCPH2) is one of the most frequent subtypes of MCPH.WD repeat-containing protein 62 gene ( WDR62 ) is the most frequently mutated gene in MCPH2 patients. Phenotypes involving dermatological changes in MCPH2 have not been reported. We have identified and investigated a 5-year-old Chinese girl with markedly reduced brain size (86% of normal size), intellectual disability and psychomotor developmental delay. The patient also exhibited spattered blisters and reduced hair density on her head, anisochromasia with reticular hyperpigmentation and hypopigmentation on the trunk, which she has had since the age of 4 and had been found by her parents. Histological examination of a skin biopsy revealed acanthosis, hyperkeratosis and necrotic keratinocytes. Whole exome and Sanger sequencing identified two novel missense mutations, c.28G>T and c.189G>T, in the WDR62 gene. Both the mutations non-synonymously affect evolutionarily conserved amino acids and are predicted to be disease causing. We report the first case of MCPH2 that also presented with marked dermatological changes. Our findings expand the mutational and phenotypical spectra of MCPH2 and are valuable in the mutation-based pre- and post-natal screening and genetic diagnosis for MCPH2.

Germline mutations of the APC gene are associated with an autosomal dominant precancerous condition, termed familial adenomatous polyposis (FAP). FAP is clinically manifested by the presence of multiple colorectal adenomas or polyps. Gradually, these colorectal adenomas or polyps inevitably result in colorectal cancer by the third-to fourth decade of life. Surgical interventions or total proctocolectomy is the best possible treatment for FAP. Here, we present a clinical molecular study of a five generation Chinese family with FAP. Diagnosis of FAP was made on the basis of clinical manifestations, family history and medical (colonoscopy and histopathology) records. Blood samples were collected and genomic DNA was extracted. Genetic screening of the APC gene was performed by targeted next-generation sequencing and quantitative real-time PCR. Targeted next generation sequencing identified a novel heterozygous large deletion [exon5-exon16; c.423_8532del] of APC gene, which segregated with the FAP phenotypes in the proband and in all the affected family members. Unaffected family members and normal controls did not carry this deletion. In the Chinese population, most of the previously reported APC gene mutations are missense mutations. This is the first report describing the largest deletion of the APC gene in the Chinese population associated with FAP.

// Santasree Banerjee 1,2 , Qian Wu 1 , Yuyi Ying 1 , Yanni Li 1 , Matsuyuki Shirota 3 , Dante Neculai 1 and Chen Li 1 1 Department of Cell Biology and Medical Genetics, School of Medicine, Zhejiang University, Hangzhou, China 2 BGI-Shenzhen, Shenzhen, China 3 Department of Applied Information Sciences, Graduate School of Information Sciences, Tohoku University, Sendai, Japan Correspondence to: Chen Li, email: // Keywords : genodermatoses, missense mutations, coiled-coil heterodimer, in silico analysis, Pathology Section Received : March 30, 2016 Accepted : May 17, 2016 Published : July 13, 2016 Abstract The K1 and K10 associated genodermatoses are characterized by clinical symptoms of mild to severe redness, blistering and hypertrophy of the skin. In this paper, we set out to computationally investigate the structural and functional effects of missense mutations on the 2B domain of K1/K10 heterodimer and its consequences in disease phenotype. We modeled the structure of the K1/K10 heterodimer based on crystal structures for the human homolog K5/K14 heterodimer, and identified that the missense mutations exert their effects on stability and assembly competence of the heterodimer by altering physico-chemical properties, interatomic interactions, and inter-residue atomic contacts. Comparative structural analysis between all the missense mutations and SNPs showed that the location and physico-chemical properties of the substituted amino acid are significantly correlated with phenotypic variations. In particular, we find evidence that a particular SNP ( K10 , p.E443K) is a pathogenic nsSNP which disrupts formation of the hydrophobic core and destabilizes the heterodimer through the loss of interatomic interactions. Our study is the first comprehensive report analyzing the mutations located on 2B domain of K1/K10 heterodimeric coiled-coil complex.

Lynch syndrome is a genetically and clinically heterogeneous disorder; it is caused by a germline mutation in DNA mismatch repair (MMR) genes. Individuals with a heterozygous mutation in MLH1 have an increased risk for developing colorectal cancer. Here we described a 5-generation Chinese Lynch syndrome family with different severity and onset age. A novel heterozygous germline mutation (c.3G>T, p.Met1Ile) inMLH1gene was discovered by next generation sequencing. Our study also revealed by qPCR that the MLH1 mRNA expression in peripheral blood of patients in this family was remarkably lower than that of the unaffected carriers and non-carriers. The research results indicated that the mRNA expression level may provide predictive suggestions of treatment and management for carriers with the initiation codon mutation ofMLH1in this family. Further studies are undertaken in this family as well as other families with Lynch syndrome to interrogate the exact reasons affecting the MLH1 mRNA expression level and whether mRNA expression in peripheral blood could be a significant factor for early diagnosis and surveillance of Lynch syndrome.

Hereditary breast cancer is an autosomal dominant syndrome caused by germ-line mutations in the human breast cancer genes, BRCA1 and BRCA2. Mutations in either BRCA1 or BRCA2 are the major causes of familial and early-onset breast cancer. The present study investigated a 33-year-old Chinese female patient with breast cancer using targeted next generation sequencing. A novel heterozygous deletion-insertion was also identified in the BRCA1 gene, c.311_312delinsAGGTTTGCA, which causes the formation of a truncated BRCA1 protein of 109 amino acids instead of a wild-type BRCA1 protein of 1,863 amino acids. These results could potentially expand the mutational spectra of BRCA1-associated breast cancer. In addition, these findings may be valuable for the mutation-based screening and genetic diagnosis of breast cancer.

Hereditary hearing impairment is one of the major and common birth defects in Chinese population. Non-syndromic sensorineural hearing loss (NSHL) is the most common types of hereditary hearing impairment. Genotypically and phenotypically NSHL is extremely heterogenous and follow either autosomal dominant or autosomal recessive or X-linked mode of inheritance. Presently, 127 genes have been identified to be associated with both syndromic and (NSHL). Here, we studied a Chinese family with moderate and profound hearing impairment. The proband is a 30-year old Chinese man. The proband was born with normal hearing and at the age of 5-years, the proband was first noticed with hearing impairment. Gradually and progressively the proband was presented with loss of hearing in his both right and left ears at the age of 30 years. The clinical symptoms, age of onset or progression to loss of hearing was similar in both the proband and his younger brother. The proband’s parents are phenotypically normal and non-consanguineous. Clinical diagnosis of the proband and his younger brother has been done by classical pure tone audiogram (PTA). Computed Tomography (CT) found no abnormality in bilateral external ear, middle ear and inner ear. Targeted next generation sequencing was performed with a panel of 127 genes reported to be associated with hereditary hearing impairment. A novel homozygous single nucleotide deletion (c.427delT) in exon 4 of ILDR1 gene has been identified in proband and in his younger brother. Sanger sequencing confirmed that proband’s father and mother are carrying this mutation in a heterozygous manner. This mutation has not been identified in 100 normal healthy control individuals. This mutation (c.427delT) causes frameshift (p.Tyr143Ilefs∗19) which leads to the formation of a truncated ILDR1 protein of 162 amino acids instead of the wild type ILDR1 protein of 546 amino acids. ILDR1 associated hereditary hearing impairment is very rare and this is the first report of identifying a loss-of-function mutation in ILDR1 gene associated with hereditary hearing impairment in Chinese population. Our present study also emphasized the significance of rapid, accurate and cost-effective screening for the patient with hereditary hearing impairment by targeted next generation sequencing.

Acute intermittent porphyria (AIP) is a rare hereditary metabolic disease with an autosomal dominant mode of inheritance. Germline mutations of HMBS gene causes AIP. Mutation of HMBS gene results into the partial deficiency of the heme biosynthetic enzyme hydroxymethylbilane synthase. AIP is clinically manifested with abdominal pain, vomiting, and neurological complaints. Additionally, an extreme phenotypic heterogeneity has been reported in AIP patients with mutations in HMBS gene. Here, we investigated a Chinese patient with AIP. The proband is a 28-year-old Chinese male manifested with severe stomach ache, constipation, nausea and depression. Proband's father and mother is normal. Proband's blood sample was collected and genomic DNA was extracted. Whole exome sequencing and Sanger sequencing identified a heterozygous novel single nucleotide deletion (c.809delC) in exon 12 of HMBS gene in the proband. This mutation leads to frameshift followed by formation of a truncated (p.Ala270Valfs∗2) HMBS protein with 272 amino acids comparing with the wild type HMBS protein of 361 amino acids. This mutation has not been found in proband's unaffected parents as well as in 100 healthy normal control. According to the variant interpretation guidelines of American College of Medical Genetics and Genomics (ACMG), this variant is classified as "likely pathogenic" variant. Our findings expand the mutational spectra of HMBS gene related AIP which are significant for screening and genetic diagnosis for AIP.

Purpose: Primary ciliary dyskinesia (PCD) is a rare genetic disorder manifested with recurrent infections of respiratory tract and infertility. Mutations in more than 20 genes including the Coiled-Coil Domain Containing 40 (CCDC40) gene are associated with PCD. A Chinese proband with a clinical diagnosis of PCD was analyzed for mutations in these genes to identify the genetic basis of the disease in the family. The proband showed altered mucociliary clearance of the airways, various degree of hyperemia and edema of the mucous membrane, left/right body asymmetry, infertility and ultrastructural abnormality of cilia in both sperm and bronchioles. Methods: The DNA from the proband was analyzed for genetic variation in a subset of genes known to cause PCD using targeted next generation sequencing in order to understand the molecular and genetic basis of the PCD in present family. The result of targeted next generation sequencing has been validated by Sanger sequencing and q-PCR. Results: Targeted next-generation sequencing identified two novel mutations (c.1259delA and EX17_20 deletion) in CCDC40 gene that causes abnormal CCDC40 mRNA expression. These two novel variants cause disorganization of axoneme filaments, which resulted in reduction of sperm motility and phenotypic diversity in ultrastructure of cilia in the proband. Conclusion: These findings highlight the significance of the mutations in CCDC40 as novel candidates for genetic testing in PCD patients as well as the key role of ICSI treatment for the families affected by this ciliary dysmotility. Our findings showed that our work enriched the performance of cilia ultrastructure which were not previously reported in PCD patients.

Diffuse palmoplantar keratoderma (DPPK) is an autosomal-dominant genodermatosis characterized by restricted, uniform hyperkeratosis on the palm and sole epidermis. DPPK is normally associated with dominant-negative mutations in the keratin-encoding gene, KRT1. We report a heterozygous novel point mutation in the exon 6 splice donor site of KRT1 (c.1254G>C) by next-generation sequencing, resulting in the formation of two alternative transcripts, which segregates with DPPK in a four-generation Chinese family. This results in both the complete loss of exon 6 and the simultaneous utilization of a novel in-frame splice site 54 bases downstream of the mutation with the subsequent deletion of 42 amino acids and the insertion of 18 amino acids into the protein's 2B domain. This is the first report of a novel splice donor site mutation with aberrant splicing and the formation of two alternative transcripts causing DPPK. This study also demonstrates the value of next-generation sequencing in the identification of novel disease-causing mutations.

Neurofibromatosis type 1 (NF1) is a dysregulated neurocutaneous disorder, characterized by neurofibromas and café-au-lait spots. NF1 is caused by mutations in the NF1 gene, encoding neurofibromin. Here, we present a clinical molecular study of a three-generation Chinese family with NF1. The proband was a male patient who showed café-au-lait spots and multiple subcutaneous neurofibromas over the whole body, but his siblings only had regional lesions. The man's daughter presented with severe headache and vomiting. Neurological examination revealed an intracranial space occupying lesion. Surgery was undertaken and the histopathological examination showed a grade I-II astrocytoma. Next-Generation sequencing (Illumina HiSeq2500 Analyzers; Illumina, San Diego, CA, USA) and Sanger sequencing (ABI PRISM 3730 automated sequencer; Applied Biosystems, Foster City, CA, USA) identified the c.227delA mutation in the NF1 gene in the man. The mutation is co-segregated with the disease phenotypes among the affected members of this family and was absent in 100 healthy controls. This novel mutation results in a frameshift (p.Asn78IlefsX7) as well as truncation of neurofibromin by formation of a premature stop codon. Our results not only extended the mutational and phenotypic spectra of the gene and the disease, but also highlight the importance of the other genetic or environmental factors in the development and severity of the disease.

Familial adenomatous polyposis (FAP) is an autosomal dominant precancerous condition which is associated with germline mutations of the APC gene. Clinically, FAP is characterized by the development of multiple colorectal adenomas or polyps which finally result in colorectal cancer by the 40 years age of the patient, if no surgical interventions have been undertaken. In this study, we present a clinical molecular study of a four generation Chinese family with FAP. Diagnosis of FAP was made on the basis of clinical manifestations, family history and medical (colonoscopy and histopathology) records. Genetic screening of the proband and all affected family members were performed by targeted next-generation sequencing and confirmatory Sanger sequencing. Targeted next generation sequencing identified a germline novel heterozygous single nucleotide deletion [c.3418delC; p.Pro1140Leufs*25] in exon18 of APC gene, which segregated with the FAP phenotypes in the proband and in all the affected family members whereas absent in unaffected family members as well as in normal healthy controls of same ethnic origin. Our present study expands the mutational spectrum of APC gene and provides evidence to understand the function of APC gene in FAP.

Autosomal dominant adult-onset demyelinating leukodystrophy (ADLD) is a very rare neurological disorder featured with late onset, slowly progressive central nervous system demyelination. Duplication or over expression of the lamin B1 (LMNB1) gene causes ADLD. In this study, we undertook a comprehensive clinical evaluation and genetic detection for a Chinese family with ADLD. The proband is a 52-year old man manifested with autonomic abnormalities, pyramidal tract dysfunction. MRI brain scan identified bilateral symmetric white matter (WM) hyper-intensities in periventricular and semi-oval WM, cerebral peduncles and middle cerebellar peduncles. The proband has a positive autosomal dominant family history with similar clinical manifestations with a trend of genetic anticipation. In order to understand the genetic cause of the disease in this family, target exome capture based next generation sequencing has been done, but no causative variants or possibly pathogenic variants has been identified. However, Multiplex ligand-dependent probe amplification (MLPA) showed whole duplication of LMNB1 gene which is co-segregated with the disease phenotype in this family. This is the first genetically confirmed LMNB1 associated ADLD pedigree from China.

Background Inherited eye diseases are major causes of vision loss in both children and adults. Inherited eye diseases are characterized by clinical variability and pronounced genetic heterogeneity. Genetic testing may provide an accurate diagnosis for ophthalmic genetic disorders and allow gene therapy for specific diseases. Methods A targeted gene capture panel was designed to capture exons of 283 inherited eye disease genes including 58 known causative retinitis pigmentosa (RP) genes. 180 samples were tested with this panel, 68 were previously tested by Sanger sequencing. Systematic evaluation of our method and comprehensive molecular diagnosis were carried on 99 RP patients. Results 96.85% targeted regions were covered by at least 20 folds, the accuracy of variants detection was 99.994%. In 4 of the 68 samples previously tested by Sanger sequencing, mutations of other diseases not consisting with the clinical diagnosis were detected by next-generation sequencing (NGS) not Sanger. Among the 99 RP patients, 64 (64.6%) were detected with pathogenic mutations, while in 3 patients, it was inconsistent between molecular diagnosis and their initial clinical diagnosis. After revisiting, one patient’s clinical diagnosis was reclassified. In addition, 3 patients were found carrying large deletions. Conclusions We have systematically evaluated our method and compared it with Sanger sequencing, and have identified a large number of novel mutations in a cohort of 99 RP patients. The results showed a sufficient accuracy of our method and suggested the importance of molecular diagnosis in clinical diagnosis.

Phelan-McDermid syndrome is a neurodevelopmental disorder caused by the terminal deletion of chromosome 22 (22q13) followed by the loss of function of the SHANK3 gene. Various terminal deletions of chromosome 22q13 are associated with Phelan-McDermid with a spectrum of phenotypic severity. Here, we have done a clinical molecular study of a Chinese proband with Phelan-McDermid syndrome. Both the proband and her younger brother are associated with this syndrome while their parents are phenotypically normal. We used a karyotype in order to detect the genotype of the proband and her younger brother. We have also used whole genome low-coverage paired-end next generation sequencing to determine whether the parent is the carrier of translocation with terminal 22q13 deletions. We found that both proband and her younger brother are comprises of a novel deletion of 22q13.31q13.33, harboring genes were associated with several clinical phenotype such as severity of speech delay, neonatal hypotonia, delayed in age of walking, male genital anomalies, dysplastic toenails, large and fleshy hands, macrocephaly, short stature, facial asymmetry, and atypical reflexes. Probands and her younger brother inherited this translocation from their mother whereas their father is genotypically normal. In conclusion, our present study expands the deletion spectrum and report a novel deletion associated with Phelan-McDermid syndrome.

The Human Variome Project (HVP) is an international consortium of clinicians, geneticists, and researchers from over 30 countries, aiming to facilitate the establishment and maintenance of standards, systems, and infrastructure for the worldwide collection and sharing of all genetic variations effecting human disease. The HVP-China Node will build new and supplement existing databases of genetic diseases. As the first effort, we have created a novel variant database of BRCA1 and BRCA2, mismatch repair genes (MMR), and APC genes for breast cancer, Lynch syndrome, and familial adenomatous polyposis (FAP), respectively, in the Chinese population using the Leiden Open Variation Database (LOVD) format. We searched PubMed and some Chinese search engines to collect all the variants of these genes in the Chinese population that have already been detected and reported. There are some differences in the gene variants between the Chinese population and that of other ethnicities. The database is available online at http://www.genomed.org/LOVD/. Our database will appear to users who survey other LOVD databases (e.g., by Google search, or by NCBI GeneTests search). Remote submissions are accepted, and the information is updated monthly.

Epidermolysis bullosa simplex (EBS) is a genodermatosis caused by mutations in keratins 5 and 14 (K5 and K14), which leads to fragility of basal keratinocytes and eventually epidermal cytolysis and blistering. Depending upon the severity of symptoms, EBS is classified into three major subtypes. In order of increasing severity these classes are EBS, localized (EBS-loc), EBS, other generalized (EBS, gen-nonDM), and EBS, Dowling-Meara (EBS-DM). We have searched and assembled 36 previously reported point mutations located on the 2B domain of K5/K14 in order to investigate the effects of point mutations. By performing a comprehensive in silico analysis we determine the underlying relationship between the mutation and its phenotypic effects. Our result showed that all pathogenic point mutations exert their dominant negative effect on the K5/K14 coiled-coil heterodimer complex by altering interchain interaction, leading to the changes in stability and assembly competence of the heterodimer complex. The physico-chemical properties of substituted amino acid and location of the mutation are also deeply correlated with disease severity. In addition, we found a SNP previously reported as non-pathogenic (K14 p.M338R) that likely affects the stability of the dimer structure due to the loss of interchain interaction and steric clashes. Overall, our finding demonstrates the significance of in silico characterization of EBS severity and would allow for accurate genetic counseling and prenatal diagnosis.