Background: Next-generation sequencing-based genetic testing represents a great opportunity to identify hereditary predispositions to specific pathological conditions and to promptly implement health surveillance or therapeutic protocols in case of disease. The term secondary finding refers to the active search for causative variants in genes associated with medically actionable conditions. Methods: We evaluated 59 medically actionable ACMG genes using a targeted in silico analysis of clinical exome sequencing performed in 383 consecutive individuals referred to our Medical Genetics Unit. A three-tier classification system of SFs for assessing their clinical impact and supporting a decision-making process for reporting was established. Results: We identified SFs with high/moderate evidence of pathogenicity in 7.0% (27/383) of analyzed subjects. Among these, 12/27 (44.4%) were carriers of a high-risk recessive disease allele. The most represented disease domains were cancer predisposition (33.3%), cardiac disorders (16.7%), and familial hypercholesterolemia (12.5%). Conclusion: Although still debated, ensuring during NGS-based genetic testing an opportunistic screening might be valuable for personal and familial early management and surveillance of medically actionable disorders, the individual\'s reproductive choices, and the prevalence assessment of underestimated hereditary genetic diseases.

Massive parallel sequencing technology has become the predominant technique for genetic diagnostics and research. Many genetic laboratories have wrestled with the challenges of setting up genetic testing workflows based on a completely new technology. The learning curve we went through as a laboratory was accompanied by growing pains while we gained new knowledge and expertise. Here we discuss some important mistakes that have been made in our laboratory through 10 years of clinical exome sequencing but that have given us important new insights on how to adapt our working methods. We provide these examples and the lessons that we learned to help other laboratories avoid to make the same mistakes.

We describe a 2 year old boy with two previously undescribed frameshift mutations in the interferon (IFN)α/β receptor 2 (IFNAR2) gene presenting with hemophagocytic lymphohistiocytosis (HLH) following measles-mumps-rubella vaccination. Functional analyses show the absence of response to type I IFN in the patient\'s cells, as revealed by the lack of phosphorylation of STAT1 and the lack of induction of interferon-stimulated genes upon ex vivo stimulation with IFNα. HLH has been reported in patients with inborn errors of type I IFN-mediated immune responses following vaccination with live-attenuated viruses. The relation between HLH and defective type I IFN-mediated responses is unclear. We show that in patient\'s natural killer (NK) cells stimulated with IFNα the expected increase in degranulation and inhibition of IFNγ production were affected. These data support a role for NK cell function dysregulation and lack of inhibition of IFNγ production as contributors to the development of HLH in patients with impaired type I IFN signaling.

Establishing a genetic diagnosis for individuals with intellectual disability (ID) benefits patients and their families as it may inform the prognosis, lead to appropriate therapy, and facilitate access to medical and supportive services. Exome sequencing has been successfully applied in a diagnostic setting, but most clinical exome referrals are pediatric patients, with many adults with ID lacking a comprehensive genetic evaluation.
Our unique recruitment strategy involved partnering with service and education providers for individuals with ID. We performed exome sequencing and analysis, and clinical variant interpretation for each recruited family.
All five families enrolled in the study opted-in for the return of genetic results. In three out of five families exome sequencing analysis identified pathogenic or likely pathogenic variants in KANSL1, TUSC3, and MED13L genes. Families discussed the results and any potential medical follow-up in an appointment with a board certified clinical geneticist.
Our study suggests high yield of exome sequencing as a diagnostic tool in adult patients with ID who have not undergone comprehensive sequencing-based genetic testing. Research studies including an option of return of results through a genetic clinic could help minimize the disparity in exome diagnostic testing between pediatric and adult patients with ID.

Autoinflammatory diseases comprise a wide range of syndromes caused by dysregulation of the innate immune response. They are difficult to diagnose due to their phenotypic heterogeneity and variable expressivity. Thus, the genetic origin of the disease remains undetermined for an important proportion of patients. We aim to identify causal genetic variants in patients with suspected autoinflammatory disease and to test the advantages and limitations of the clinical exome gene panels for molecular diagnosis. Twenty-two unrelated patients with clinical features of autoinflammatory diseases were analyzed using clinical exome sequencing (~4800 genes), followed by bioinformatic analyses to detect likely pathogenic variants. By integrating genetic and clinical information, we found a likely causative heterozygous genetic variant in NFKBIA (p.D31N) in a North-African patient with a clinical picture resembling the deficiency of interleukin-1 receptor antagonist, and a heterozygous variant in DNASE2 (p.G322D) in a Spanish patient with a suspected lupus-like monogenic disorder. We also found variants likely to increase the susceptibility to autoinflammatory diseases in three additional Spanish patients: one with an initial diagnosis of juvenile idiopathic arthritis who carries two heterozygous UNC13D variants (p.R727Q and p.A59T), and two with early-onset inflammatory bowel disease harbouring NOD2 variants (p.L221R and p.A728V respectively). Our results show a similar proportion of molecular diagnosis to other studies using whole exome or targeted resequencing in primary immunodeficiencies. Thus, despite its main limitation of not including all candidate genes, clinical exome targeted sequencing can be an appropriate approach to detect likely causative variants in autoinflammatory diseases.

Background Dilated cardiomyopathy may be heritable but shows extensive genetic heterogeneity. The utility of whole exome sequencing as a first-line genetic test for patients with dilated cardiomyopathy in a contemporary \"real-world\" setting has not been specifically established. Using whole exome sequencing with rigorous, evidence-based variant interpretation, we aimed to identify the prevalence of a molecular diagnosis in patients with dilated cardiomyopathy in a clinical setting. Methods and Results Whole exome sequencing was performed in eligible patients (n=83) with idiopathic or familial dilated cardiomyopathy. Variants were prioritized for curation in up to 247 genes and classified using American College of Medical Genetics and Genomics-based criteria. Ten (12%) had a pathogenic or likely pathogenic variant. Eight (10%) participants had truncating TTN variants classified as variants of uncertain significance. Five (6%) participants had variants of unknown significance according to strict American College of Medical Genetics and Genomics criteria but classified as either pathogenic or likely pathogenic by other clinical laboratories. Pathogenic or likely pathogenic variants were found in 8 genes (all within tier 1 genes), 2 (20%) of which are not included in a standard commercially available dilated cardiomyopathy panel. Using our bioinformatics pipeline, there was an average of 0.74 variants of uncertain significance per case with ≈0.75 person-hours needed to interpret each of these variants. Conclusions Whole exome sequencing is an effective diagnostic tool for patients with dilated cardiomyopathy. With stringent classification using American College of Medical Genetics and Genomics criteria, the rate of detection of pathogenic variants is lower than previous reports. Efforts to improve adherence to these guidelines will be important to prevent erroneous misclassification of nonpathogenic variants in dilated cardiomyopathy genetic testing and inappropriate cascade screening.

The exponential knowledge on the genetic etiology and the trend towards genetically-specific therapies for previously untreatable disorders, requires neurologists to be familiar with the strengths and weaknesses of Next-Generation Sequencing (NGS). Our aim was to assess the diagnostic yield of NGS studies in clinical practice in our setting. We performed a retrospective, cross-sectional, 18 months long study, from a single Portuguese center, of consecutive neurological patients for whom a NGS study was requested. A diagnosis rate (DR) of 33.2% was achieved for a total of 190 patients (89 children). It was higher for muscle diseases (DR 61.1%). In 20%, an inconclusive molecular diagnosis was obtained. The rate of incidental findings (IF) was 5.3%. We found better DR for clinical exome (52.6%, p < 0.05) although only 14% of patients were characterized using this approach. The performance of gene panels for muscle diseases was better but not statistically significant (DR 56.3% vs. 31.7% overall, p > 0.05). The reduced number of patients in several phenotypic groups limits the interpretation of specific diagnostic yields. The better yield of gene panels for muscle diseases suggests that gene panels may be a more cost-effective first-line test in well-defined phenotypes. For heterogeneous phenotypes and overall, WES-based virtual panels or clinical exome should be favored. We present daily practice evidence that, with the constraints of our health system, for one third of the patients with neurological disorders of undetermined etiology a definitive diagnosis can be reached with NGS.

During mouse embryonic development the protein kinase domain containing, cytoplasmic (Pkdcc) gene, also known as Vlk, is expressed in several tissues including the ventral midbrain, with particularly strong expression in branchial arches and limb buds. Homozygous Pkdcc knockout mice have dysmorphic features and shortened long bones as the most obvious morphological abnormalities. The human PKDCC gene has currently not been associated with any disorders.
To use clinical diagnostic exome sequencing (DES) for providing genetic diagnoses to two apparently unrelated patients with similar skeletal abnormalities comprising rhizomelic shortening of limbs and dysmorphic features.
Patient-parents trio DES was carried out and the identified candidate variants were confirmed by Sanger sequencing.
Each patient had a homozygous gene disrupting variant in PKDCC considered to explain the skeletal phenotypes shared by both. The first patient was homozygous for the nonsense variant p.(Tyr217*) (NM_1 38 370 c.651C>A) expected to result in nonsense-mediated decay of the mutant transcripts, whereas the second patient was homozygous for the splice donor variant c.639+1G>T predicted to abolish the donor splice site by three in silico splice prediction algorithms.
Biallelic gene disrupting variants in PKDCC in humans, just like in mice, cause dysmorphic features and rhizomelic shortening of limbs.

Reanalysis of exome sequencing data when results are negative may yield additional diagnoses. We sought to estimate the contribution of clinical geneticists to the interpretation of sequencing data of their patients.
The cohort included 84 probands attending a tertiary genetics institute (2015-2018) with a nondiagnostic result on clinical exome sequencing performed in one of five external laboratories. The raw data were uploaded to the Emedgene bioinformatics and interpretation platform for reanalysis by a team of two clinical geneticists, the geneticist directly involved in the patient\'s care, and a bioinformatician.
In ten probands (11.9%), a new definitive diagnosis was reached based on genes that were known to be associated with the phenotype at the time the original report was issued. The main reasons for a negative exome result were incorrect interpretation of the clinical context and absence of OMIM entry. Pathogenic variants in genes with previously unknown gene-disease associations were discovered to be causative in three probands. In total, new diagnoses were established in 13/84 individuals (15.5%).
Direct access to complete clinical data and shortening of time to including gene-phenotype associations in databases can assist the analytics team and reduce the need for additional unnecessary tests.

In this study, we report three cases of nonketotic hyperglycinemia (NKHG) diagnosed biochemically and molecularly. Clinical exome analysis in two families revealed two novel mutations in the aminomethyltransferase (AMT) gene, that is, c.14_15insT (p.Ser6LysfsTer22) and c.259-2A > T, both of them adversely affecting the protein. This is the first report of AMT gene mutations in NKHG from India. Prenatal diagnosis in the first family showed an unaffected fetus in the third pregnancy. The role of AMT protein is pivotal for the synthesis of 5,10-methylene tetrahydrofolate, the first metabolite in one-carbon metabolism that regulates DNA synthesis, repair, and methylation.