TMEM151A Variants Cause Paroxysmal Kinesigenic Dyskinesia: A Large-Sample Study.

BACKGROUND: Paroxysmal kinesigenic dyskinesia (PKD) is the most common type of paroxysmal dyskinesias. Only one-third of PKD patients are attributed to proline-rich transmembrane protein 2 (PRRT2) mutations. OBJECTIVE: We aimed to explore the potential causative gene for PKD. METHODS: A cohort of 196 PRRT2-negative PKD probands were enrolled for whole-exome sequencing (WES). Gene Ranking, Identification and Prediction Tool, a method of case-control analysis, was applied to identify the candidate genes. Another 325 PRRT2-negative PKD probands were subsequently screened with Sanger sequencing. RESULTS: Transmembrane Protein 151 (TMEM151A) variants were mainly clustered in PKD patients compared with the control groups. 24 heterozygous variants were detected in 25 of 521 probands (frequency = 4.80%), including 18 missense and 6 nonsense mutations. In 29 patients with TMEM151A variants, the ratio of male to female was 2.63:1 and the mean age of onset was 12.93 ± 3.15 years. Compared with PRRT2 mutation carriers, TMEM151A-related PKD were more common in sporadic PKD patients with pure phenotype. There was no significant difference in types of attack and treatment outcome between TMEM151A-positive and PRRT2-positive groups. CONCLUSIONS: We consolidated mutations in TMEM151A causing PKD with the aid of case-control analysis of a large-scale WES data, which broadens the genotypic spectrum of PKD. TMEM151A-related PKD were more common in sporadic cases and tended to present as pure phenotype with a late onset. Extensive functional studies are needed to enhance our understanding of the pathogenesis of TMEM151A-related PKD. © 2021 International Parkinson and Movement Disorder Society.

Large-area flexible MWCNT/PDMS pressure sensor for ergonomic design with aid of deep learning learning.

The achievement of well-performing pressure sensors with low pressure detection, high sensitivity, large-scale integration, and effective analysis of the subsequent data remains a major challenge in the development of flexible piezoresistive sensors. In this study, a simple and extendable sensor preparation strategy was proposed to fabricate flexible sensors on the basis of multiwalled carbon nanotube/polydimethylsiloxane (MWCNT/PDMS) composites. A dispersant of tetrahydrofuran (THF) was added to solve the agglomeration of MWCNTs in PDMS, and the resistance of the obtained MWCNT/PDMS conductive unit with 7.5 wt.% MWCNTs were as low as 180 Ω/hemisphere. Sensitivity (0.004 kPa-1), excellent response stability, fast response time (36 ms), and excellent electromechanical properties were demonstrated within the pressure range from 0 to 100 kPa. A large-area flexible sensor with 8 × 10 pixels was successfully adopted to detect the pressure distribution on the human back and to verify its applicability. Combining the sensor array with deep learning, inclination of human sitting was easily recognized with high accuracy, indicating that the combined technology can be used to guide ergonomic design.

[A consensus on the standardization of the next generation sequencing process for the diagnosis of genetic diseases (1) - Procedures prior to genetic testing].

Pre-testing preparation is the basis and starting point of genetic testing. The process includes collection of clinical information, formulation of testing scheme, genetic counseling before testing, and completion of informed consent and testing authorization. To effectively identify genetic diseases in clinics can greatly improve the diagnostic rate of next generation sequencing (NGS), thereby reducing medical cost and improving clinical efficacy. The analysis of NGS results relies, to a large extent, on the understanding of genotype-phenotype correlations, therefore it is particularly important to collect and evaluate clinical phenotypes and describe them in uniform standard terms. Different types of genetic diseases or mutations may require specific testing techniques, which can yield twice the result with half the effort. Pre-testing genetic counseling can help patients and their families to understand the significance of relevant genetic testing, formulate individualized testing strategies, and lay a foundation for follow-up.

[A consensus on the standardization of the next generation sequencing process for the diagnosis of genetic diseases (3) - Data analysis].

Bioinformatic analysis and variant classification are the key components of high-throughput sequencing-based genetic diagnostic approach. This consensus is part of the effort to develop a standardized process for next generation sequencing (NGS)-based test for germline mutations underlying Mendelian disorders in China. The flow-chart, common software, key parameters of bioinformatics pipeline for data processing, annotation, storage and variant classification are reviewed, which is aimed to help improving and maintaining a high-quality process and obtaining consistent outcomes for NGS-based molecular diagnosis.

[A consensus on the standardization of the next generation sequencing process for the diagnosis of genetic diseases (4) - Report interpretation and genetic counseling].

Clinical genetic testing results are compiled into a standardized report by genetic specialists and provided to clinicians and patients (Should the patient be intellectually disabled or under 18, the report will be provided to his/her parents or legal guardians). The content of genetic testing report should conform to relevant guidelines, industry standards and consensus. The decisions of clinicians will be made based on the report and clinical indications. Genetic counselors should provide post-test counseling to clinicians and patients or their authorized family members. A mechanism of follow-up visit after the genetic testing should be established with informed consent. Data should be shared by clinical institutions and genome sequencing institutions. As findings upon follow-up visit can help with further evaluation of the results, genome sequencing institutions should regularly re-analyze historical and follow-up data, and the updated results should be shared with clinical institutions. All activities involving reporting, genetic counselling, follow-up visiting, and re-analyzing should follow the relevant guidelines and regulations.

[An overview of tools for post-analysis of high-throughput sequencing data in clinical study].

With the advance of high-throughout sequencing technology and its extensive application in clinical diagnosis, analysis of sequencing data has become an important part of clinical diagnosis. To date, the development and establishment of various software and databases have made it convenient to extract useful information from massive amounts of high-throughput sequencing data. However, it is still a challenge for correlating the clinical-genetic diagnosis based on the above-mentioned sequence data with the screened DNA variations and disease phenotypes. Further validation of the proposed pathogenesis with the discovered molecular defects are required. Here a comprehensive review is provided for the strategies of sequencing data analysis, commonly used phenotype-genotype correlation tools, and functional analysis and verification methods for the genetic diagnosis.

[Discussion on the standard of clinical genetic testing report and the consensus of gene testing industry].

The widespread application of next generation sequencing (NGS) in clinical settings has enabled testing, diagnosis, treatment and prevention of genetic diseases. However, many issues have arisen in the meanwhile. One of the most pressing issues is the lack of standards for reporting genetic test results across different service providers. The First Forum on Standards and Specifications for Clinical Genetic Testing was held to address the issue in Shenzhen, China, on October 28, 2017. Participants, including geneticists, clinicians, and representatives of genetic testing service providers, discussed problems of clinical genetic testing services across in China and shared opinions on principles, challenges, and standards for reporting clinical genetic test results. Here we summarize expert opinions presented at the seminar and report the consensus, which will serve as a basis for the development of standards and guidelines for reporting of clinical genetic testing results, in order to promote the standardization and regulation of genetic testing services in China.

Alteration of gene expression profiling including GPR174 and GNG2 is associated with vasovagal syncope.

Vasovagal syncope (VVS) causes accidental harm for susceptible patients. However, pathophysiology of this disorder remains largely unknown. In an effort to understanding of molecular mechanism for VVS, genome-wide gene expression profiling analyses were performed on VVS patients at syncope state. A total of 66 Type 1 VVS child patients and the same number healthy controls were enrolled in this study. Peripheral blood RNAs were isolated from all subjects, of which 10 RNA samples were randomly selected from each groups for gene expression profile analysis using Gene ST 1.0 arrays (Affymetrix). The results revealed that 103 genes were differently expressed between the patients and controls. Significantly, two G-proteins related genes, GPR174 and GNG2 that have not been related to VVS were among the differently expressed genes. The microarray results were confirmed by qRT-PCR in all the tested individuals. Ingenuity pathway analysis and gene ontology annotation study showed that the differently expressed genes are associated with stress response and apoptosis, suggesting that the alteration of some gene expression including G-proteins related genes is associated with VVS. This study provides new insight into the molecular mechanism of VVS and would be helpful to further identify new molecular biomarkers for the disease.

mvPPT: A Highly Efficient and Sensitive Pathogenicity Prediction Tool for Missense Variants.

Next-generation sequencing technologies both boost the discovery of variants in the human genome and exacerbate the challenges of pathogenic variant identification. In this study, we developed Pathogenicity Prediction Tool for missense variants (mvPPT), a highly sensitive and accurate missense variant classifier based on gradient boosting. mvPPT adopts high-confidence training sets with a wide spectrum of variant profiles, and extracts three categories of features, including scores from existing prediction tools, frequencies (allele frequencies, amino acid frequencies, and genotype frequencies), and genomic context. Compared with established predictors, mvPPT achieves superior performance in all test sets, regardless of data source. In addition, our study also provides guidance for training set and feature selection strategies, as well as reveals highly relevant features, which may further provide biological insights into variant pathogenicity. mvPPT is freely available at http://www.mvppt.club/.

A Novel Heterozygous Missense Variant in Parathyroid Hormone 1 is Related to the Occurrence of Developmental Dysplasia of the Hip.

Introduction: Developmental dysplasia of the hip (DDH) is one of the most common diseases in the pediatric orthopedics, with an incidence of 1-5%. Genetic factors are the bases of the pathogenesis of DDH, but the pathogenic variants and pathogenesis of DDH are still unknown. There are no key accurate diagnostic or prognostic molecular markers for DDH. The purpose of our study was to screen for genetic variant associated with DDH and explore its pathogenesis. Materials and Methods: The genetic variation of DDH was tested by variant NGS-based exome analyses, verified by the Sanger sequencing. Results: A four-generation family in which DDH was present in three generations was recruited. A novel heterozygous missense variant c.629C>T (p.(Ala210Val)) in exon 7/8 of the parathyroid hormone 1 receptor (PTH1R) gene was identified through screening of two affected and one unaffected family members. The candidate variant was validated in all available family members with all three affected members being positive for the PTH1R variant. Conclusion: Our results are highly supportive of PTH1R as a novel candidate gene for DDH and demonstrated that the combination of pedigree information and next-generation sequencing is an effective method for identifying pathogenic variants associated with DDH.

Establishment of an improved high-efficiency thermal asymmetric interlaced PCR for identification of genomic integration sites mediated by phiC31 integrase.

Streptomyces phage phiC31 integrase is widely used to mediate the integration of exogenous genes into host genomes for gene therapy and genomic modification, as it autonomously performs efficient, unidirectional, site-specific integration into pseudo attP sites of the host genome. Although pseudo attP sites are rarely found within exons, it is necessary to map their precise locations to avoid the risk of insertion mutagenesis. High-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR) is a technique that has been developed to recover genomic sequences that flank insertion tags. We have found, however, that this technique is poorly efficient, as it amplifies many non-specific targets and frequently does not generate sufficient product for downstream analysis. Therefore, we have modified the hiTAIL-PCR procedure and re-designed the random primers. As a result, both the amount and specificity of the reaction product were enhanced for each integration site. Restriction analysis of known sequences within the integrated vector, which co-amplified with the flanking genomic sequences, validated 90% of these bands for sequencing. In contrast, only 30% of the bands produced by previous hiTAIL-PCR could be validated. Compared with the original hiTAIL-PCR, our improved hiTAIL-PCR procedure identified phiC31 integration sites more accurately and efficiently.

Textile-Based Mechanical Sensors: A Review.

Innovations related to textiles-based sensors have drawn great interest due to their outstanding merits of flexibility, comfort, low cost, and wearability. Textile-based sensors are often tied to certain parts of the human body to collect mechanical, physical, and chemical stimuli to identify and record human health and exercise. Until now, much research and review work has been carried out to summarize and promote the development of textile-based sensors. As a feature, we focus on textile-based mechanical sensors (TMSs), especially on their advantages and the way they achieve performance optimizations in this review. We first adopt a novel approach to introduce different kinds of TMSs by combining sensing mechanisms, textile structure, and novel fabricating strategies for implementing TMSs and focusing on critical performance criteria such as sensitivity, response range, response time, and stability. Next, we summarize their great advantages over other flexible sensors, and their potential applications in health monitoring, motion recognition, and human-machine interaction. Finally, we present the challenges and prospects to provide meaningful guidelines and directions for future research. The TMSs play an important role in promoting the development of the emerging Internet of Things, which can make health monitoring and everyday objects connect more smartly, conveniently, and comfortably efficiently in a wearable way in the coming years.

A multiplex ligation­dependent probe amplification­based next­generation sequencing approach for the detection of copy number variations in the human genome.

The aim of the present study was to describe a multiplex ligation­dependent probe amplification (MLPA)­based next­generation sequencing (NGS) assay that exhibited a significantly higher efficiency in detecting copy number variations (CNVs) and known single­nucleotide variants, compared with traditional MLPA. MLPA polymerase chain reaction products were used to construct a library with indexed adapters, which was subsequently tested on an NGS platform, and the resulting data were analyzed by a series of analytical software. The reads from each probe reflected genetic variations in the target regions, and fragment differentiation was based on the specific base composition of the sequences, rather than fragment length, which was determined by capillary electrophoresis. The results of this approach were not only consistent with the MLPA results following capillary electrophoresis, but also coincided with the CNV results from the single­nucleotide polymorphism array chip. This method allowed high­throughput screening for the number of fragments and samples by integrating additional indices for detection. Furthermore, this technology precisely and accurately performed large­scale detection and quantification of DNA variations, thereby serving as an effective and sensitive method for diagnosing genetic disorders caused by CNVs and known single­nucleotide variations. Notably, MLPA­NGS circumvents the problems associated with the inaccuracies of NGS in CNV detection due to the use of target sequence capture.

A recurrent mutation in bone morphogenetic proteins-2-inducible kinase gene is associated with developmental dysplasia of the hip.

Developmental dysplasia of the hip (DDH) is a complex disorder of the hip joint affecting 1-5‰ of newborns. While genetic influence on DDH has been long known, DDH has not been ascribed to any specific genetic event. The present study reported on variants contributing to DDH susceptibility in a family with four individuals affected across three generations. Whole-exome sequencing was performed in three affected and two unaffected individuals of a pedigree with DDH. Candidate variants were confirmed by Sanger sequencing and then validated in available family members and 37 sporadic DDH patients. Two novel heterozygous, inframe mutations causing multi-nucleotide substitution polymorphisms (c.1432_1440delCAGCAGCAG corresponding with p.Gln478_480del and c.1440_1441insCAG corresponding with p.Gln480ins) in exon 11 of chromosome 4 in bone morphogenetic proteins-2-inducible kinase (BMP2K) were identified; these were found in members of the pedigree affected by DDH and in the unaffected grandmother of the proband, who was deemed to be the carrier of potential mutations, but not in the unaffected normal control saunt of the proband. These two variants shared the same genomic coordinate but with different types of mutation in BMP2K. BMP2K is known to be associated with bone and cartridge development and heterozygous mutations were found to be present in 4/4 (100%) of the affected family members, 4/15 (26.7%) of the unaffected family members and 0/7 (0%) of the unaffected unrelated family members. Genotyping of 37 unrelated, sporadic DDH patients showed that three cases were positive for the BMP2K c.1432_1440delCAGCAGCAG variants (8.12%). These findings provided strong evidence for the role of BMP2K variants in causing DDH and demonstrated that the combination of pedigree information and next-generation sequencing is an effective method for identifying pathogenic sites associated with DDH.

A gain-of-function ACTC1 3'UTR mutation that introduces a miR-139-5p target site may be associated with a dominant familial atrial septal defect.

The ostium secundum atrial septal defect (ASDII) is the most common type of congenital heart disease and is characterized by a left to right shunting of oxygenated blood caused by incomplete closure of the septum secundum. We identified a familial form of isolated ASDII that affects four individuals in a family of five and shows autosomal dominant inheritance. By whole genome sequencing, we discovered a new mutation (c.*1784T > C) in the 3'-untranslated region (3'UTR) of ACTC1, which encodes the predominant actin in the embryonic heart. Further analysis demonstrated that the c.*1784T > C mutation results in a new target site for miRNA-139-5p, a microRNA that is involved in cell migration, invasion, and proliferation. Functional analysis demonstrated that the c.*1784T > C mutation specifically downregulates gene expression in a luciferase assay. Additionally, miR-139-5p mimic causes further decrease, whereas miR-139-5p inhibitor can dramatically rescue the decline in gene expression caused by this mutation. These findings suggest that the familial ASDII may be a result of an ACTC1 3'UTR gain-of-function mutation caused by the introduction of a new miR-139-5p target site. Our results provide the first evidence of a pathogenic mutation in the ACTC1 3'UTR that may be associated with familial isolated ASDII.

A profile of native integration sites used by φC31 integrase in the bovine genome.

The Streptomyces phage φC31 integrase can efficiently target attB-bearing transgenes to endogenous pseudo attP sites within mammalian genomes. To better understand the activity of φC31 integrase in the bovine genome, DNA sequences of 44 integration events were analyzed, and 32 pseudo attP sites were identified. The majority of these sites share a sequence motif that contains inverted repeats and has similarities to wild-type attP site. Genomic DNA flanking these sites typically contained repetitive sequence elements, such as short and long interspersed repetitive elements. These sequence features indicate that DNA sequence recognition plays an important role in guiding φC31-mediated site-specific integration. In addition, BF27 integration hotspot sites were identified in the bovine genome, which accounted for 13.6% of all isolated integration events and mapped to an intron of the deleted in liver cancer 1 (DLC1) gene. Also we found that the pseudo attP sites in the bovine genome had other features in common with those in the human genome. This study represents the first time that the sequence features of pseudo attP sites in the bovine genome were analyzed. We conclude that this site-specific integrase system has great potential for applied modifications of the bovine genome.

Application of high-resolution melting for genotyping bovine mitochondrial DNA.

Recent studies have demonstrated that mitochondrial DNA (mtDNA) haplotype has a significant impact on the efficiency of bovine somatic cell nuclear transfer. Conventional methods for detecting mtDNA variations and haplotypes, such as restriction fragment length polymorphism (RFLP), temporal temperature gradient gel electrophoresis, dHPLC and sequencing, are labor intensive or expensive and have low sensitivity. High-resolution melting (HRM) analysis is a new technique for mutation detection and has the advantages of speed, cost, and accuracy. Here, we describe the genotyping of bovine mtDNA using HRM analysis. DNA samples containing mtDNA were extracted from 75 Holstein cows and subjected to rapid-cycle (<20 min) PCR of small amplicons (<120 bp) using specific primer sets. Capillaries containing the PCR products were then subjected to HRM analysis; data were acquired in 2 min and analyzed using the instrument's software. Five common bovine mtDNA single nucleotide polymorphisms were identified: 9602 G>A, 169 A>G, 166A>G with 173A>G, and 363C>G. These results agree with both sequencing and RFLP analysis. In addition, a very small amount of heteroplasmic variants (<5%) was sufficiently to be distinguished by HRM analysis that would be very useful to differentiate heteroplasmy vs. homoplasmy. HRM analysis thus provides a new approach to genotyping bovine mtDNA sequence variations and has many advantages over other methods, including speed of analysis, cost, and accuracy. We believe this will be a valuable technique for determining the efficiency of nuclear transfer in cloned embryos and for studying maternal effects on nuclear-cytoplasm interactions.