[Clinical phenotypic and genetic analysis of syndrome families with EYA1 gene variants].

Objective:To investigate the clinical phenotype of a family with branchio-oto syndrome （BOS） and to explore the genetic etiology of the syndrome in this family. Methods:Clinical data were collected from a child diagnosed with BOS and his family members. Genomic DNA was extracted from peripheral blood of the proband and his family members. Whole-exome sequencing was performed, and the mutation sites were verified and analyzed by Sanger sequencing. Results:The family consists of two generations with four members, three of whom exhibit the phenotype. Two members have hearing loss and bilateral preauricular fistulas and bilateral branchial cleft fistulas. One member has bilateral preauricular fistulas and bilateral branchial cleft fistulas. All of which were in line with the clinical diagnosis of gill ear syndrome, the inheritance mode of the family was autosomal dominant inheritance, genetic testing showed that all members of the family had c. 1744delC（p. L592Cfs*47） mutation in the EYA1 gene, while unaffected members have the wild-type allele at this locus. This mutation is a frameshift mutation, which results in the early appearance of the stop codon, and has not been reported so far. According to ACMG guidelines, the variant was preliminarily determined to be suspected pathogenic. Conclusion:The newly discovered EYA1c. 1744delC（p. L592Cfs*47） mutation in this family is the pathogenic mutant gene of the patients in this family, which further expands the mutation spectrum of EYA1 gene, gives us a new understanding of the disease, and provides an important reference for clinical diagnosis and genetic counseling.

Case report: Identification and clinical phenotypic analysis of novel mutation of the PPP1CB gene in NSLH2 syndrome.

Objective: To screen and analyze the genetic mutations in the PPP1CB gene in a patient with Noonan syndrome with loose anagen hair-2 (NSLH2) in Yunnan Province, China and explore the possible molecular pathogenesis. Methods: After obtaining informed consent, we collected the patient's medical history and carried out physical and laboratory examinations for the NSLH2 proband and the family members. Genomic DNA was extracted from the peripheral blood of all individuals. The coding regions including all pathogenic exons, parts of introns, and promoters of genes were sequenced by next-generation sequencing. Pathogenic mutations, which were detected in the probands and their parents, were verified by Sanger sequencing. Results: The clinical manifestations of NSLH2 included prominent forehead, yellowish hair, slightly wide eye distance, sparse eyebrows, bilateral auricle deformity, reduced muscle tension, and cardiac and visual abnormalities. The proband carried a c.371A>G mutation in exon 3 of PPP1CB, which is a missense mutation. This was a de novo mutation as the parents of the proband showed no mutation at this site. Conclusion: In this study, we identified a novel mutation of PPP1CB, which enriched the mutation spectrum of the PPP1CB gene and provided a basis for the diagnosis of NSLH2.

Hereditary hemorrhagic telangiectasia complicated by aortic sinus aneurysm caused by a pathogenic mutation in the ACVRL1 gene: a family survey / 中华皮肤科杂志

Objective:To investigate clinical and genetic characteristics of a family with hereditary hemorrhagic telangiectasia complicated by aortic sinus aneurysm, and to analyze causative genes.Methods:Clinical data and peripheral blood samples were collected from the proband and her relatives, and genomic DNA was extracted. Causative genes were screened by whole-exome sequencing, and then verified by Sanger sequencing.Results:A heterozygous mutation c.137G>A was identified at position 137 in exon 3 of the ACVRL1 gene in the proband, her daughter, grandson and granddaughter, which led to the substitution of cysteine by tyrosine at amino acid position 46 (p.C46Y) . The mutation was not found in any of the other 5 family members without clinical symptoms.Conclusion:A causative mutation c.137G>A (p.C46Y) in the ACVRL1 gene was identified in the family with hereditary hemorrhagic telangiectasia type 2 complicated by aortic sinus aneurysm, which had not been previously reported in Asian populations.

Mutation Profile of Aggressive Pheochromocytoma and Paraganglioma with Comparison of TCGA Data.

In pheochromocytoma and paraganglioma (PPGL), germline or somatic mutations in one of the known susceptibility genes are identified in up to 60% patients. However, the peculiar genetic events that drive the aggressive behavior including metastasis in PPGL are poorly understood. We performed targeted next-generation sequencing analysis to characterize the mutation profile in fifteen aggressive PPGL patients and compared accessible data of aggressive PPGLs from The Cancer Genome Atlas (TCGA) with findings of our cohort. A total of 115 germline and 34 somatic variants were identified with a median 0.58 per megabase tumor mutation burden in our cohort. The most frequent mutation was SDHB germline mutation (27%) and the second frequent mutations were somatic mutations for SETD2, NF1, and HRAS (13%, respectively). Patients were subtyped into three categories based on the kind of mutated genes: pseudohypoxia (n = 5), kinase (n = 5), and unknown (n = 5) group. In copy number variation analysis, deletion of chromosome arm 1p harboring SDHB gene was the most frequently observed. In our cohort, SDHB mutation and pseudohypoxia subtype were significantly associated with poor overall survival. In conclusion, subtyping of mutation profile can be helpful in aggressive PPGL patients with heterogeneous prognosis to make relevant follow-up plan and achieve proper treatment.

Assessment of the clinical application of detecting EGFR, KRAS, PIK3CA and BRAF mutations in patients with non-small cell lung cancer using next-generation sequencing.

BACKGROUND: Next-generation sequencing (NGS) has been widely applied in clinical research, while its application in routine clinical molecular testing requires careful validation. The aim of our study was to assess the clinical usefulness of the NextDaySeq Lung panel on Ion Torrent™ PGM in mutation detection of actionable genes in lung cancer. METHODS: The NextDaySeq assay was evaluated by blinded comparisons to Quantitative Real-Time PCR (qPCR) assays with 188 consecutive samples from Chinese patients with non-small cell lung cancer (NSCLC) to detect mutations in EGFR, KRAS, PIK3CA and BRAF. Discordant variants were further validated by Sanger sequencing and independent qPCR and NGS assays. RESULTS: Our results showed 93.3% concordance of reportable variants mutually covered in both NGS and qPCR assays, with a clinical sensitivity of 89.9%, specificity of 97.5%. Through the comparison, the NGS assays demonstrated its advantages in offering more clinical relevant information, such as detecting non-hotspot mutations and providing mutation allele frequencies (MAF) and accurate mutation sequences. The analytical sensitivity of NGS to detect mutations with low MAF needs further improvement. CONCLUSIONS: The NextDaySeq Lung panel exhibited good clinical performance, strongly supporting the implementation of the NGS assay in routine clinical use to facilitate therapeutic decision-making for lung cancer patients.

Hepatitis B virus (HBV) gene mutations related to entecavir-resistance in patients with chronic HBV infections / 中华临床感染病杂志

Objective To investigate hepatitis B virus (HBV) gene mutations related to entecavir (ETV)-resistance in patients with chronic HBV infections.Methods Serum samples were collected from 44 patients with chronic HBV infections and resistant to ETV treatment who were admitted in Ningbo No.2 Hospital during February 2010 and May 2014.The HBV polymerase regions were amplified by real-time fluorescent quantitative polymerase chain reaction (PCR) method,and the PCR products were analyzed with direct sequencing.SPSS 16.0 was used to assess the frequency of HBV polymerase gene mutations,and its relation to the viral genotype and clinical features.Results The most common HBV polymerase gene mutation was rtS202G/I (52.28％,23/44),followed by rtT184A/G/I/S (36.36％,16/44) and rtM250V/L (11.36％,5/44).Nine mutation patterns were detected,in which rtL180 + rtM204V + rtS202G/I (38.64％,17/44) and rtL180 +rtM204V + rtT184A/G/I/S (27.27％,12/44) were the most frequent ones.The difference in gene mutations between genotype B and C was of statistical significance (x2=12.294,P ＜0.01).Patients carrying rtT184A/G/I/S mutations were associated with worse liver function (x2 =14.499,P ＜ 0.01),and those carrying rtM250V/L mutations were associated with lower HBeAg positive rate (x2 =10.057,P ＜ 0.01).Conclusions rtL180M + rtM204V + rtS202G/I is the most common HBV polymerase gene mutation related to ETV resistance in patients with chronic HBV infections.Different gene mutations may be associated with HBV genotypes,severity of liver damages,and HBeAg positive rate.

ß-Thalassemia mutations in Western India: outcome of prenatal diagnosis in a hemoglobinopathies project.

Prenatal diagnosis (PND) is one of the most cost effective preventive methods, but it is available only in the large cities of India. Therefore, we initiated a program that offers PND and allows us to determine the prevalence of various mutations. Pregnant females (n = 111,426) were screened for hemoglobinopathies using complete blood count (CBC) and high performance liquid chromatography (HPLC). If the female had a hemoglobinopathy, her husband was then tested. If hemoglobinopathies were seen in both partners, a genetic mutation study was performed on the couple. Fetal samples were obtained by either chorionic villus sampling (CVS) in 70.6% or amniocentesis in 29.4%. The study included 282 couples. IVS-I-5 (G > C) was the most common mutation in all castes except in the Sindhis and Lohanas, where the 619 bp deletion was the most common. Prenatal testing was informative in 97.9% of the couples. A significant number of couples (41.0%) underwent PND during their first pregnancy. Seven patients with ß-thalassemia (ß-thal) trait had normal Hb A2 levels. The Hb A2 and Hb F values varied significantly (p < 0.0001 and 0.0082, respectively) among mutations associated with ß-thal. The IVS-I-5, 619 bp deletion, codons 41/42 (-CTTT), codons 8/9 (+G) and IVS-I-1 (G > T or G > A), were present in 81.0% of the couples tested. ß-Thalassemia mutation frequency varied among the different castes, underlining the need for evolving a testing strategy that considers the caste system. Targeting antenatal clinics could also prove to be a most cost effective way of preventing hemoglobinopathies.

Estudo da Beta-catenina em tumores adrenocorticais humanos / Study of beta-catenin in human adrenocortical tumors

Introdução: A incidência de tumores adrenocorticais em crianças é particularmente elevada nas regiões sudeste e sul do Brasil, correlacionandose com a ocorrência da mutação germinativa p.R337H do supressor tumoral p53, entretanto, o carcinoma adrenocortical é uma neoplasia endócrina maligna rara em todo o mundo com uma incidência aproximada de 0,5 - 2 casos por milhão por ano. Esta condição é uma doença heterogênea, apresentando frequentemente comportamento clínico agressivo e letal. A cascata de sinalização Wnt é uma via importante de transdução de sinal em cânceres humanos e tem sido implicada na tumorigênese adrenocortical. A atividade desta via de sinalização é dependente da quantidade de beta-catenina citoplasmática e nuclear. Mutações ativadoras no gene da beta-catenina (CTNNB1) foram relatadas em diversas neoplasias humanas. Estudos demonstraram que mutações no gene CTNNB1 são os defeitos genéticos mais frequentemente encontrados em adenomas e em carcinomas adrenocorticais. O estudo destas mutações demonstrou que as alterações no gene CTNNB1 localizam-se principalmente exon 3, que codifica a porção amino terminal da beta- catenina. Objetivos: determinar a ocorrência e a frequência das mutações somáticas no exon 3 do gene CTNNB1. Adicionalmente, determinar a imunorreatividade de beta-catenina e de p53 em tumores adrenocorticais benignos e malignos de crianças e adultos. Correlacionar os resultados da análise de mutações gênicas e os dados de imunorreatividade com as características hormonais, a mutação p.R337H do p53, o diagnóstico histológico e a evolução dos tumores adrenocorticais de crianças e adultos. Métodos: Neste estudo, a análise de imunohistoquímica para beta-catenina e p53 foi realizada em 103 tumores adrenocorticais benignos e malignos (40 crianças e 63 adultos), estando as amostras histológicas alocadas em micromatriz tecidual (TMA). A pesquisa de mutações no exon 3 do gene CTNNB1 foi determinada por seqüenciamento automático em 64 tumores...

Introduction: The incidence of adrenocortical tumors in children is particularly high in the southeastern and southern regions of Brazil, correlating with the occurrence of p.R337H p53 tumor suppressor germline mutation. However, adrenocortical carcinoma is a worldwide rare endocrine malignancy with an approximate incidence of 0.5 to 2 cases per million per year. This condition is a heterogeneous disease and is often lethal. The Wnt signaling pathway is an important signal transduction pathway in human cancers and has been implicated in adrenocortical tumorigenesis. The activity of this signaling pathway is dependent on the amount of nuclear and cytoplasmic beta-catenin. Activating mutations of ?-catenin (CTNNB1) gene have been reported in several human malignancies. Studies have shown that CTNNB1 mutations are the most common genetic defect found in adrenocortical adenomas and carcinomas. The study of these mutations demonstrated that the changes in CTNNB1 gene are mainly located in exon 3, which encodes the amino terminal portion of the beta- catenin. Objectives: to determine the occurrence and frequency of CTNNB1 somatic mutations and the abnormal beta-catenin and p53 accumulation in benign and malignant adrenocortical tumors in both children and adults. We also evaluated the correlation of the gene mutations analysis and immunohistochemistry data with the hormonal characteristics, the p.R337H germline mutation, the histological diagnosis and the prognosis of adrenocortical tumors in children and adults. Methods: In this study, immunohistochemistry for beta-catenin and p53 was performed in 103 benign and malignant (40 children and 63 adults) adrenocortical tumors. The histological samples were allocated in a tissue microarray (TMA). The study of the CTNNB1 gene was performed by direct sequencing of 64 adrenocortical tumors. Results: The beta-catenin abnormal accumulation was similar in benign and malignant adrenocortical tumors of children and adults (15...(au)

K-ras gene mutation status in 454 Chinese patients with colorectal cancer / 肿瘤

Objective: To analyze the mutation status of K-ras gene in CRC (colorectal cancer) tissues and plasma samples of CRC patients, ultimately to promote the targeted agents against EGFR (epidermal growth factor receptor) (such as cetuximab and panitumumab, etc.) used in personalized treatment for CRC patients. Methods: The sequence of K-ras gene at specific sites was investigated in 431 CRC tissues and 23 plasma samples collected from 454 CRC patients, respectively. To improve the detection sensitivity, a combinatory approach was chosen which included the COLD-PCR (coamplification at lower denaturation temperature PCR) method, followed by DNA sequencing. Chi-square test was employed to analyze K-ras mutation frequencies in various sample types or subgroups of CRC patients according to age, and the difference was considered statistically significant if P value was less than 0.05. Results: The overall K-ras mutation rate was 25.29% in 431 CRC tissue samples. The two major forms of K-ras mutation were Gl 2D (mutation of glycine to an aspartate residue at codon 12) and Gl 3D (mutation of glycine to an aspartate residue at codon 13), and their occurrence frequencies were 12.99% and 6.26%, respectively. Interestingly, the overall K-ras mutation rate was 21.74% in blood samples from additional 23 CRC patients, without a significant difference from the mutation rate in the tumor samples (P > 0.05). Moreover, the occurrence frequency of anyone form of K-ras mutation was 37.94% in CRC patients aged 60 and over, which was significantly higher than the detection rate (7.30%) of the patients under the age of 60 (P < 0.01). Conclusion: COLD-PCR amplification combined with DNA sequencing method can be used to detect the mutation status of K-ras gene, and plasma samples can be used insteadly if CRC tumor tissues are unavailable. In addition, for elderly patients aged of 60 and over, it is suggested that K-ras mutation status should be routinely detected before treatment. Copyright © 2013 by TUMOR.

Detection of epidermal growth factor receptor mutations in small specimens of non-small cell lung cancer by amplification refractory mutation system / 肿瘤

Objective: To explore the feasibility of the application of small specimens as alternatives of gross specimens to detect EGFR (epidermal growth factor receptor) mutation in NSCLC (non-small cell lung cancer) by ARMS (amplification refractory mutation system). Methods: Biopsy specimens from 181 cases of NSCLC were collected, in which 157 were small specimens (including specimens acquired through CT-guided percutaneous lung biopsy, endobronchial ultrasound-guided transbronchial needle aspiration, lymph node biopsy, bronchoscopic biopsy and aspiration of pleural effusion) and 24 were gross specimens. QIAGEN DNA extraction kit was used to extract DNAs from NSCLC specimens. Then AmoyDx EGFR Mutation Test Kit - a highly sensitive real-time PCR-based test was used to detect EGFR mutations. The detection rates of EGFR mutations in small specimens and gross specimens were compared by Chi-square test or Fisher's exact test. Results: The total EGFR mutation detection rate of all biopsy specimens was 39.8% (72/181). The detection rates of small specimens and gross specimens were 38.9% and 45.8%, respectively (P = 0.515). Furthermore, EGFR mutation frequencies were significantly higher in non-smokers (P = 0.033) and in patients with adenocarcinoma (P < 0.001). Conclusion: A relatively high detection rate of EGFR mutation in NSCLC small specimens can be obtained through ARMS. For patients with advanced NSCLC whose gross specimens cannot be easily obtained, the alternative small specimens can be used in clinical EGFR mutation detection. Copyright © 2012 by TUMOR.

Application of DNA microarray in the identification of Candida spp. and mutations of ERG11 gene resulting in fluconazole resistance / 中华皮肤科杂志

Objective To investigate the performance of DNA microarray in identifying 6 common Candida spp. and validating ERG11 mutations resulting in fluconazolc-resistance in Candida albicans. Methods Oligonucleotide probes were designed and synthesized targeting the species-specific sequence in the internal transcribed spacer 2 (ITS2) region of rDNA of Candida albicans, Candida tropicalis, Candida glabrata, Candida dubliniensis, Candida parapsilosis and Candida krusei, as well as 6 sequences embracing the following mutations respectively in ERG11 gene leading to fluconazole-resistance, i.c., T541C, A 1090G, C1361T, G1537A, G1547A, and T1559C, then arranged onto a chip. Twelve 50-base-pair oligonucleotides were artificially synthesized based on the above specific sequences, and utilized to hybridize with the DNA microarray. Thirty-lbur Candida strains, including 29 C. albicans, 1 Candida tropicalis, 1 Candida glabrata,1 Candida dubliniensis, 1 Candida parapsilosis and 1 Candida krusei, were detected with microarray. Genomic DNA was extracted from these tested strains and underwent multiple PCR for the amplification of ITS2 region and ERGI 1 gene. Sequencing was performed to analyze the sequence of ERG11 in 29 strains of C. albicans and the results were compared with those of DNA microarray hybridization. Results Multiple PCR successfully produced ITS2 fragment of 307-415 bp from all the 34 strains, as well as ERG11 fragment of 1712 bp from 29 C. albicans strains. DNA microarray hybridization offered the same results in species identification of the 34 strains with their given information, as well as in mutation detection of the 29 strains of C. albicans with ERG11 sequencing results. Also, the 6 synthesized oligonucleotides containing the muta- tions were identified precisely as T541C, A1090G, C1361T, G1537A, G1547A, and T1559C, and the 6 species specific oligonucleotieds were identified correctly as C. albicans, C. tropicalis, C. glabrata, C. dubliniensis, C. parapsilosis and C. krusei Both the sensitivity and the specificity of the microarray were 100%. Conclu- sion DNA microarray is a quite reliable method to identify Candida spp. and fluconazole resistance-associ- ated mutations in the ERG11 gene of C. albicans.

A Mutation in the Connexin30Gene in a Pedigree with Hidrotic Ectodermal Dysplasia / 中华皮肤科杂志

Objective To analyses the mutation of connexin30gene in a pedigree with hidrotic ec-todermal dysplasia(HED).Methods Blood samples were obtained from18affected and16normal individ-uals in this family.Mutation scanning was carried out by PCR and direct sequencing.Results A transition,at position on connexin30gene31(G→A),leading to a missense mutation(G11R),was detected in18patients,but was not found in16normal individuals in this HED family and in188unrelated,population-matched control individuals,which indicated that it did not represent common polymorphism.Conclusion A missense mutation(31G→A)in the connexin30gene has been determined in the pedigree with HED,which is probably one of the molecular bases of the pathogenesis of the disease.

Mutations in ED1 Gene of Two Pedigrees with X-linked Hypohidrotic Ectodermal Dysplasia / 中华皮肤科杂志

Objective To detect ED1 gene mutations in the families with X-linked hypohidrotic ec-todermal dysplasia (XLHED). Methods Blood samples were obtained from 2 pedigrees. All 8 exons and flanking intronic boundaries of ED1 gene were amplified with polymerase chain reaction technique and then directly sequenced. Results Two mutations were found in ED1 gene. One was splicing mutation (IVS8+5 del G), the other was missense mutation (A959G). None of the mutations was found in normal individuals of two XLHED families and in 188 unrelated, population-matched control individuals. Conclusion Out of the ED1 gene mutations identified in 2 Chinese XLHED families, IVS8+5del G is a novel mutation.

Study of mitochondrial DNA A1555G mutation among nonsyndromic hearing impairment in Chinese population / 中华检验医学杂志

Objective To study the prevalence of the mtDNA A1555G gene mutation in Chinese population with nonsyndromic hearing impairment.Methods PCR-RFLP,directional sequencing of PCR products were applied in 325 patients with nonsyndromic hearing impairment and 50 normal controls.Results The mutation rate of the mtDNA A1555G was 14.5% (47/325),28 of 47 cases were homozygosis,19 of 47 cases were heterozygosis.The same mutation was not detected in the control subjects.Conclusion The mutation rate of the mtDNA A1555G is relatively high in the Chinese NSHI patients,the mutation type includes both heterozygosis and homozygosis.

Founder effect of mutation of GJB2 on the patients of deafness in China / 解放军医学杂志

Objective To determine the frequency of GJB2 mutations in the China hearing loss population, and to screen the GJB2 gene in both hearing loss and normal populations. Methods 141 patients with hearing loss and 150 normal persons (control) underwent mutation screening of single coding exon of GJB2 with bidirectional sequencing to identify sequences alterations. Results Three polymorphisms were found: 79G→A, 109G→A, and 341A→G; and four pathologic mutations were identified: 235delC, 455A→G, 176-191del16 and 504insGCAA. Conclusion The 235delC mutation was found to be the significant cause of hearing loss in Chinese population.