Target gene panel method versus whole-exome sequencing in detection of idiopathic hypogonadotropic hypogonadism in males / 中华男科学杂志

Objective@#To compare the efficiency of the target gene panel method and whole-exome sequencing (WES) in detecting idiopathic hypogonadotropic hypogonadism (IHH), and select a more suitable gene detection method.@*METHODS@#We selected 24 genes closely related to the molecular pathogenesis of IHH to make up the gene panel, detected the mutation sites in 73 patients with IHH using the panel method, and verified the results of sequencing with the Sanger method. Using the key words "idiopathic hypogonadotropic hypogonadism", we searched databases for relevant literature, calculated the positive rate of IHH detected by WES and compared it with that detected with the panel method.@*RESULTS@#Of the 73 cases of IHH detected with the panel method, 7 were found with pathogenic mutations, including 2 cases of FGFR1, 2 cases of CHD7, 2 cases of KISS1R, and 1 case of NR5A1 mutation. Sanger sequencing showed that the positive rate of the panel method was 9.7%. Of the 1 336 articles retrieved, 5 met the inclusion criteria and were included, in which WES revealed a positive rate of about 30%.@*CONCLUSIONS@#For detection of the diseases with clear mutated genes, the panel method is relatively inexpensive and has a high sequencing depth, while for detection of the diseases with complicated genetic patterns and unclear mutated genes, WES is more efficient. Further studies are needed for choice of the two methods for different purpose of detection./.

Clinical Validity of Next-Generation Sequencing Multi-Gene Panel Testing for Detecting Pathogenic Variants in Patients With Hereditary Breast-Ovarian Cancer Syndrome

BACKGROUND: Hereditary breast and ovarian cancer syndrome (HBOC) is caused by pathogenic variants in BRCA and other cancer-related genes. We analyzed variants in BRCA gene and other cancer-related genes in HBOC patients to evaluate the clinical validity of next-generation sequencing (NGS) multi-gene panel testing. METHODS: The BRCA1/2 NGS testing was conducted for 262 HBOC patients. Multiplex ligation-dependent probe amplification and direct Sanger sequencing were performed for confirmation. Multi-gene panel testing was conducted for 120 patients who did not possess BRCA1/2 pathogenic variants but met the National Comprehensive Cancer Network criteria. RESULTS: Pathogenic variants in BRCA1/2 were detected in 30 HBOC patients (11.5%). Additionally, four out of the 120 patients possessed pathogenic variants by multi-gene panel testing (3.3%): MSH2 (c.256G>T, p.Glu86*), PMS2 (c.1687C>T, p.Arg563*), CHEK2 (c.546C>A, p.Tyr182*), and PALB2 (c.3351-1G>C). All the four patients had a family history of cancer. CONCLUSIONS: Multi-gene panel testing could be a significant screening tool for HBOC patients, especially for those with a family history of cancer.

Genetic Mutation Profiles in Korean Patients with Inherited Retinal Diseases

BACKGROUND: Because of genetically and phenotypically heterogenous features, identification of causative genes for inherited retinal diseases (IRD) is essential for diagnosis and treatment in coming gene therapy era. To date, there are no large-scale data of the genes responsible for IRD in Korea. The aim of this study was to identify the distribution of genetic defects in IRD patients in Korea. METHODS: Medical records and DNA samples from 86 clinically diagnosed IRD patients were consecutively collected between July 2011 and May 2015. We applied the next-generation sequencing strategy (gene panel) for screening 204 known pathogenic genes associated with IRD. RESULTS: Molecular diagnoses were made in 38/86 (44.2%) IRD patients: 18/44 (40.9%) retinitis pigmentosa (RP), 8/22 (36.4%) cone dystrophy, 6/7 (85.7%) Stargardt disease, 1/1 (100%) Best disease, 1/1 (100%) Bardet-Biedl syndrome, 1/1 (100%) congenital stationary night blindness, 1/1 (100%) choroideremia, and 2/8 (25%) other macular dystrophies. ABCA4 was the most common causative gene associated with IRD and was responsible for causing Stargardt disease (n = 6), RP (n = 1), and cone dystrophy (n = 1). In particular, mutations in EYS were found in 4 of 14 autosomal recessive RP (29%). All cases of Stargardt disease had a mutation in the ABCA4 gene with an autosomal recessive trait. CONCLUSION: This study provided the distribution of genetic mutations responsible for causing IRD in the Korean patients. This data will serve as a reference for future genetic screening and treatment for Korean IRD patients.

A New Integrated Newborn Screening Workflow Can Provide a Shortcut to Differential Diagnosis and Confirmation of Inherited Metabolic Diseases

PURPOSE: We developed a new workflow design which included results from both biochemical and targeted gene sequencing analysis interpreted comprehensively. We then conducted a pilot study to evaluate the benefit of this new approach in newborn screening (NBS) and demonstrated the efficiency of this workflow in detecting causative genetic variants. MATERIALS AND METHODS: Ten patients in Group 1 were diagnosed clinically using biochemical assays only, and 10 newborns in Group 2 were diagnosed with suspected inherited metabolic disease (IMD) in NBS. We applied NewbornDiscovery (SD Genomics), an integrated workflow design that encompasses analyte-phenotype-gene, single nucleotide variant/small insertion and deletion/copy number variation analyses along with clinical interpretation of genetic variants related to each participant's condition. RESULTS: A molecular genetic diagnosis was established in 95% (19/20) of individuals. In Group 1, 13 and 7 of 20 alleles were classified as pathogenic and likely pathogenic, respectively. In Group 2, 11 and 6 of 17 alleles with identified causative variants were pathogenic and likely pathogenic, respectively. There were no variants of uncertain significance. For each individual, the NewbornDiscovery and biochemical analysis results reached 100% concordance, since the single newborn testing negative for causative genetic variant in Group 2 showed a benign clinical course. CONCLUSION: This integrated diagnostic workflow resulted in a high yield. This approach not only enabled early confirmation of specific IMD, but also detected conditions not included in the current NBS.

Analysis method based on the gene-panel sequencing data / 上海交通大学学报（医学版）

Objective· To establish an integrative method for the gene-panel sequencing data to automatically complete quality control, detection of gene mutation and visualization. Methods · Integrate several methods, e.g. FastQC, preprocessing and information of sequences (Prinseq) to develop an R package that can be used to visualize and control the quality of the raw sequencing reads and final mutations result. The sequencing reads mapped against to the reference genome using Burrows-Wheeler Alignment Tool (BWA)/Torrent Mapping Alignment Program (TMAP). Lofreq, Varscan2, the Genome Analysis Toolkit (GATK) and Torrent Variant Caller (TVC) were used to detect gene mutation and get the variant call format (VCF) format file. Annotate the gene mutation sites using Annovar. Results · Thirty-six cases of acute myeloid leukemia sequencing from Ion Torrent Personal Genome Machine (PGM) platform were passed by this analysis tool.Ten mutation sites of 2 demo data were found in DNMT3A,TET2,JAK2,PHF6,ASXL1,NPM1 and CEBPA which were validated by sanger sequencing. Conclusion · The analysis method that integrated and developed several tools for gene-panel sequencing data analysis can accomplish the gene-panel sequencing data analysis effectively. Besides, it can reduce the false positive ratio and improve the sensitivity of gene mutation detection that provides support for the analysis of gene-panel sequencing data.

Diagnóstico molecular de enfermedades genéticas: del diagnóstico genético al diagnóstico genómico con la secuenciación masiva / Molecular diagnosis of genetic diseases: from genetic to genomic diagnosis using next generation sequencing

En la actualidad se conocen 8.000 enfermedades genéticas monogénicas. La mayoría de ellas son heterogéneas, por lo que el diagnóstico molecular por técnicas convencionales de secuenciación suele ser largo y costoso debido al gran número de genes implicados. El tiempo estimado para el diagnóstico molecular se encuentra entre 1 y 10 años, y este retraso impide que los pacientes reciban medidas terapéuticas y de rehabilitación específicas, que sus familiares entren en programas preventivos y que reciban asesoramiento genético. La secuenciación masiva está cambiando el modelo de diagnóstico molecular de los afectos, sin embargo, los médicos y profesionales de la salud se enfrentan al dilema de la selección del método más eficiente, con el menor coste sanitario y con la mayor precisión de sus resultados. El objetivo de este trabajo es revisar la tecnología de secuenciación masiva y definir las ventajas y los problemas en su utilización.

Currently 8000 monogenic genetic diseases are known. Most of them are heterogeneous, so their molecular diagnosis by conventional sequencing techniques is labour intensive and time consuming due to the large number of genes involved. The estimated time is between 1 and 10 years for molecular diagnosis and this delay prevents patients from receiving therapy and rehabilitation measures, and their families from entering prevention programs and being given genetic counselling. Next generation sequencing (NGS) is changing the model of molecular diagnosis of patients; however, doctors and health professionals are faced with the dilemma of choosing the most efficient method, with lower health care costs and the most accurate results. The aim of this paper is to review the NGS technology and define the advantages and problems in the use of this technology.