In the last decade, an incredible improvement has been made in elucidating the genetic bases of cardiomyopathies. Here we report the impact of either the European Society of Cardiology (ESC) guidelines or the use of whole exome sequencing (WES) in terms of a number of variants of uncertain significance (VUS) and missed diagnoses in a series of 260 patients affected by inherited cardiac disorders. Samples were analyzed using a targeted gene panel of 128 cardiac-related genes and/or WES in a subset of patients, with a three-tier approach. Analyzing (i) only a subset of genes related to the clinical presentation, strictly following the ESC guidelines, 20.77% positive test were assessed. The incremental diagnostic rate for (ii) the whole gene panel, and (iii) the WES was 4.71% and 11.67%, respectively. The diverse analytical approaches increased the number of VUSs and incidental findings. Indeed, the use of WES highlights that there is a small percentage of syndromic conditions that standard analysis would not have detected. Moreover, the use of targeted sequencing coupled with \"narrow\" analytical approach prevents the detection of variants in actionable genes that could allow for preventive treatment. Our data suggest that genetic testing might aid clinicians in the diagnosis of inheritable cardiac disorders.

OBJECTIVE: This document addresses the clinical application of next-generation sequencing (NGS) technologies for prenatal genetic diagnosis and aims to establish clinical practice recommendations in Spain to ensure uniformity in implementing these technologies into prenatal care.
METHODS: A joint committee of expert obstetricians and geneticists was created to review the existing literature on fetal NGS for genetic diagnosis and to make recommendations for Spanish healthcare professionals.
RESULTS: This guideline summarises technical aspects of NGS technologies, clinical indications in prenatal setting, considerations regarding findings to be reported, genetic counselling considerations as well as data storage and protection policies.
CONCLUSIONS: This document provides updated recommendations for the use of NGS diagnostic tests in prenatal diagnosis. These recommendations should be periodically reviewed as our knowledge of the clinical utility of NGS technologies, applied during pregnancy, may advance.

Objective: To compare the differences between the variation interpretation standards and guidelines issued by the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) in 2015 (The 2015ACMG/AMP guideline) and the Deafness Specialist Group of the Clinical Genome Resource (ClinGen) in 2018 for hereditary hearing loss (Healing loss, HL) issued the expert specification of the variation interpretation guide (The 2018 HL-EP guideline) in evaluating the pathogenicity of OTOF gene variation in patients with auditory neuropathy. Methods: Thirty-eight auditory neuropathy patients with OTOF gene variant were selected as the study subjects (23 males and 15 females, aged 0.3-25.9 years). Using whole-genome sequencing, whole exome sequencing or target region sequencing (Panel) combined with Sanger sequencing, 38 cases were found to carry more than two OTOF mutation sites. A total of 59 candidate variants were independently interpreted based on the 2015 ACMG/AMP guideline and 2018 HL-EP guideline. Compared with the judgment results in 2015 ACMG/AMP guideline, the variants interpreted as lower pathogenic classifications in the 2018 HL-EP guideline were defined as downgraded variants, and the variants regarded as higher pathogenic classifications were defined as upgraded variants. Statistical analysis was conducted using SPSS 20.0. Results: The concordance rate of variant classification between the guidelines was 72.9%(43/59). The 13.6%(8/59) of variants were upgraded and 13.6% (8/59) of variants downgraded in the classifications of the 2018 HL-EP guideline. A couple of rules saw significant differences between the guidelines (PVS1, PM3, PP2, PP3 and PP5). The distribution of pathogenicity of splicing mutation was statistically different (P=0.013). Conclusions: The 2018 HL-EP guideline is inconsistent with the 2015 ACMG/AMP guideline, when judging the pathogenicity of OTOF gene variants in patients with auditory neuropathy. Through the deletion and refinement of evidence and the breaking of solidification thinking, the 2018 HL-EP guideline makes the pathogenicity grading more traceable and improves the credibility.
目的： 对比2015年美国医学遗传学与基因组学学会（American College of Medical Genetics and Genomics，ACMG）和分子病理学会（Association for Molecular Pathology，AMP）发布的变异解读标准与指南（本文中简称2015 ACMG/AMP指南）与2018年临床基因组资源中心（Clinical Genome Resource，ClinGen）耳聋专病小组针对遗传性听力损失（hearing loss，HL）发布的变异解读指南专家规范（本文中简称2018HL专病指南）在评估听神经病患者OTOF基因变异致病性中的异同。 方法： 以38例OTOF基因变异听神经病患者作为研究对象（男23例、女15例，年龄范围0.3~25.9岁），经全基因组重测序、全外显子组测序或目标区域靶向（Panel）测序结合一代Sanger测序验证，38例听神经病患者均检出携带两个以上OTOF变异位点，共计59个候选位点，分别使用2015ACMG/AMP指南以及2018HL专病指南对其致病性进行判断。与2015年指南判断结果相比，2018年指南判断的致病性等级更强定义为升级，更弱定义为降级。采用SPSS 20.0软件进行统计学分析。 结果： 2015 ACMG/AMP指南和2018 HL专病指南的变异分类一致率为72.9%（43/59）。致病性升级变异位点占13.6%（8/59），致病性降级变异位点占13.6%（8/59）。两指南致病性判定不一致主要集中在PVS1、PM3、PP2、PP3以及PP5等级证据的应用上。剪接变异、错义变异、框内插入/缺失以及同义变异致病性分布发生改变，其中剪接变异改变差异具有统计学意义（P=0.013）。 结论： 针对听神经病患者OTOF基因变异进行致病性判断时，2018HL专病指南与2015ACMG/AMP指南存在不一致，2018HL专病指南对证据进行删减及进一步细分，打破常规对于变异类型的固化思维，使得致病性分级更有迹可循，提高可信度。.

Exome sequencing is a first-tier diagnostic test for individuals with neurodevelopmental disorders, including intellectual disability/developmental delay and autism spectrum disorder; however, this recommendation does not include cerebral palsy.
To evaluate if the diagnostic yield of exome or genome sequencing in cerebral palsy is similar to that of other neurodevelopmental disorders.
The study team searched PubMed for studies published between 2013 and 2022 using cerebral palsy and genetic testing terms. Data were analyzed during March 2022.
Studies performing exome or genome sequencing in at least 10 participants with cerebral palsy were included. Studies with fewer than 10 individuals and studies reporting variants detected by other genetic tests were excluded. Consensus review was performed. The initial search identified 148 studies, of which 13 met inclusion criteria.
Data were extracted by 2 investigators and pooled using a random-effects meta-analysis. Incidence rates with corresponding 95% CIs and prediction intervals were calculated. Publication bias was evaluated by the Egger test. Variability between included studies was assessed via heterogeneity tests using the I2 statistic.
The primary outcome was the pooled diagnostic yield (rate of pathogenic/likely pathogenic variants) across studies. Subgroup analyses were performed based on population age and on the use of exclusion criteria for patient selection.
Thirteen studies were included consisting of 2612 individuals with cerebral palsy. The overall diagnostic yield was 31.1% (95% CI, 24.2%-38.6%; I2 = 91%). The yield was higher in pediatric populations (34.8%; 95% CI, 28.3%-41.5%) than adult populations (26.9%; 95% CI, 1.2%-68.8%) and higher among studies that used exclusion criteria for patient selection (42.1%; 95% CI, 36.0%-48.2%) than those that did not (20.7%; 95% CI, 12.3%-30.5%).
In this systematic review and meta-analysis, the genetic diagnostic yield in cerebral palsy was similar to that of other neurodevelopmental disorders for which exome sequencing is recommended as standard of care. Data from this meta-analysis provide evidence to support the inclusion of cerebral palsy in the current recommendation of exome sequencing in the diagnostic evaluation of individuals with neurodevelopmental disorders.

The American College of Medical Genetics and Genomics (ACMG)-recommended five variant classification categories (pathogenic, likely pathogenic, uncertain significance, likely benign, and benign) have been widely used in medical genetics. However, these guidelines are fundamentally constrained in practice owing to their focus upon Mendelian disease genes and their dichotomous classification of variants as being either causal or not. Herein, we attempt to expand the ACMG guidelines into a general variant classification framework that takes into account not only the continuum of clinical phenotypes, but also the continuum of the variants\' genetic effects, and the different pathological roles of the implicated genes.
As a disease model, we employed chronic pancreatitis (CP), which manifests clinically as a spectrum from monogenic to multifactorial. Bearing in mind that any general conceptual proposal should be based upon sound data, we focused our analysis on the four most extensively studied CP genes, PRSS1, CFTR, SPINK1 and CTRC. Based upon several cross-gene and cross-variant comparisons, we first assigned the different genes to two distinct categories in terms of disease causation: CP-causing (PRSS1 and SPINK1) and CP-predisposing (CFTR and CTRC). We then employed two new classificatory categories, \"predisposing\" and \"likely predisposing\", to replace ACMG\'s \"pathogenic\" and \"likely pathogenic\" categories in the context of CP-predisposing genes, thereby classifying all pathologically relevant variants in these genes as \"predisposing\". In the case of CP-causing genes, the two new classificatory categories served to extend the five ACMG categories whilst two thresholds (allele frequency and functional) were introduced to discriminate \"pathogenic\" from \"predisposing\" variants.
Employing CP as a disease model, we expand ACMG guidelines into a five-category classification system (predisposing, likely predisposing, uncertain significance, likely benign, and benign) and a seven-category classification system (pathogenic, likely pathogenic, predisposing, likely predisposing, uncertain significance, likely benign, and benign) in the context of disease-predisposing and disease-causing genes, respectively. Taken together, the two systems constitute a general variant classification framework that, in principle, should span the entire spectrum of variants in any disease-related gene. The maximal compliance of our five-category and seven-category classification systems with the ACMG guidelines ought to facilitate their practical application.

Prenatal exome sequencing (ES) is increasingly used for prenatal diagnosis because emerging data indicate it has incremental diagnostic benefit in pregnancies with fetal anomalies without identified genetic abnormalities by karyotyping and chromosomal microarray analysis. The aim of this study was to evaluate the medical community\'s attitude toward the clinical utility and use of exome sequencing for prenatal diagnosis and to address differences in attitudes and responses by type of practitioner, level of training, and years passed since last full-time training. We analyzed the answers of 109 trainees and professionals in the fields of genetic counseling, laboratory science, and medicine to an online survey addressing these topics. Multiple-choice questions asked participants about their awareness of prenatal ES and what genetic test they would choose to order in certain scenarios. Likert-scale questions assessed participants\' opinions of statements asserting when prenatal ES should be used for diagnostic testing. Attitude toward the use of prenatal ES statistically differed (p < 0.05) by type of participant and level of training. Practicing genetic counselors and physicians were more selective in their recommendations for prenatal ES than laboratory scientists. Genetic counseling students and practicing genetic counselors felt similarly about indications for the use of prenatal ES, whereas medical students were more liberal in their recommendations for prenatal ES than practicing physicians. This study shows a lack of consensus among the medical community regarding the clinical utility and indications for prenatal ES.

Infants with hypotonia can present with a variety of potentially severe clinical signs and symptoms and often require invasive testing and multiple procedures. The wide range of clinical presentations and potential etiologies leaves diagnosis and prognosis uncertain, underscoring the need for rapid elucidation of the underlying genetic cause of disease.
The clinical application of exome sequencing or genome sequencing has dramatically improved the timely yield of diagnostic testing for neonatal hypotonia, with diagnostic rates of greater than 50% in academic neonatal intensive care units (NICUs) across Australia, Canada, the UK, and the US, which compose the International Precision Child Health Partnership (IPCHiP). A total of 74% (17 of 23) of patients had a change in clinical care in response to genetic diagnosis, including 2 patients who received targeted therapy. This narrative review discusses the common causes of neonatal hypotonia, the relative benefits and limitations of available testing modalities used in NICUs, and hypotonia management recommendations.
This narrative review summarizes the causes of neonatal hypotonia and the benefits of prompt genetic diagnosis, including improved prognostication and identification of targeted treatments which can improve the short-term and long-term outcomes. Institutional resources can vary among different NICUs; as a result, consideration should be given to rule out a small number of relatively unique conditions for which rapid targeted genetic testing is available. Nevertheless, the consensus recommendation is to use rapid genome or exome sequencing as a first-line testing option for NICU patients with unexplained hypotonia. As part of the IPCHiP, this diagnostic experience will be collected in a central database with the goal of advancing knowledge of neonatal hypotonia and improving evidence-based practice.

OBJECTIVE: Next Generation Sequencing (NGS) is increasingly used for the diagnosis of rare genetic disorders. The aim of this study is to review the different approaches for economic evaluations of Next Generation Sequencing (NGS) in pediatric care used to date, to identify all costs, effects, and time horizons taken into account.
METHODS: A systematic literature review was conducted to identify published economic evaluations of NGS applications in pediatric diagnostics, i.e. exome sequencing (ES) and/or genome sequencing (GS). Information regarding methodological approach, costs, effects, and time horizon was abstracted from these publications.
RESULTS: Twenty-eight economic evaluations of ES/GS within pediatrics were identified. Costs included were mainly restricted to direct in-hospital healthcare costs and varied widely in inclusion of sort of costs and time-horizon. Nineteen studies included diagnostic yield and eight studies included cost-effectiveness as outcome measures. Studies varied greatly in terms of included sort of costs data, effects, and time horizon.
CONCLUSIONS: Large differences in inclusion of cost and effect parameters were identified between studies. Validity of outcomes can therefore be questioned, which hinders valid comparison and widespread generalization of conclusions. In addition to current health economic guidance, specific guidance for evaluations in pediatric care is therefore necessary to improve the validity of outcomes and furthermore facilitate comparable decision-making for implementing novel NGS-based diagnostic modalities in pediatric genetics and beyond.

A novel approach is developed to address the challenge of annotating with phenotypic effects those exome variants for which relevant empirical data are lacking or minimal. The predictive annotation method is implemented as a stacked ensemble of supervised base-learners, including distributed random forest and gradient boosting machines. Ensemble models were trained and cross-validated on evidence-based categorical variant effect annotations from the ClinVar database, and were applied to 84 million non-synonymous single nucleotide variants (SNVs). The consensus model combined 39 functional mutation impacts, cross-species conservation score, and gene indispensability score. The indispensability score, accounting for differences in variant pathogenicities including in essential and mutation-tolerant genes, considerably improved the predictions. The consensus combination is consistent with as many input scores as possible while minimizing false predictions. The input scores are ranked based on their ability to predict effects. The score rankings and categorical phenotypic variant effect predictions are aimed for direct use in clinical and biological applications to prioritize human exome variants and mutations.

Whole-exome sequencing (WES), one of the next-generation sequencing (NGS), has become a powerful tool to identify exonic variants. Investigating causality of the sequence variants in human disease becomes an important part in NGS for the research and clinical applications. Recently, important guidelines on them have been published and will keep on updating. In our study, two Chinese families, with the clinical diagnosis of \"Epilepsy\", which presented with seizures, psychomotor retardation, hypotonia and etc. features, were sequenced by Trio-WES (including the proband and the unaffected parents), and a standard interpretation of the identified variants was performed referring to the recently updated guidelines. Finally, we identified three novel mutations (c.71 C > T, p.P24L; c.1387-1389delGAG, p.E463-; c.134 G > A, p.W45*; NM_000026) in ADSL in the two Chinese families, and confirmed them as the causal variants to the disease-Adenylosuccinate Lyase Deficiency. Previous reported specific therapy was also introduced to the patients after our refined molecular diagnosis, however, the effect was very limited success. In summary, our study demonstrated the power and advantages of WES in exploring the etiology of human disease. Using the constantly updated guidelines to conduct the WES study and to interpret the sequence variants are a necessary strategy to make the molecular diagnosis and to guide the individualized treatment of human disease.