ONT long-read WGS for variant discovery and orthogonal confirmation of short read WGS derived genetic variants in clinical genetic testing.

Technological advances in Next-Generation Sequencing dramatically increased clinical efficiency of genetic testing, allowing detection of a wide variety of variants, from single nucleotide events to large structural aberrations. Whole Genome Sequencing (WGS) has allowed exploration of areas of the genome that might not have been targeted by other approaches, such as intergenic regions. A single technique detecting all genetic variants at once is intended to expedite the diagnostic process while making it more comprehensive and efficient. Nevertheless, there are still several shortcomings that cannot be effectively addressed by short read sequencing, such as determination of the precise size of short tandem repeat (STR) expansions, phasing of potentially compound recessive variants, resolution of some structural variants and exact determination of their boundaries, etc. Therefore, in some cases variants can only be tentatively detected by short reads sequencing and require orthogonal confirmation, particularly for clinical reporting purposes. Moreover, certain regulatory authorities, for example, New York state CLIA, require orthogonal confirmation of every reportable variant. Such orthogonal confirmations often involve numerous different techniques, not necessarily available in the same laboratory and not always performed in an expedited manner, thus negating the advantages of "one-technique-for-all" approach, and making the process lengthy, prone to logistical and analytical faults, and financially inefficient. Fortunately, those weak spots of short read sequencing can be compensated by long read technology that have comparable or better detection of some types of variants while lacking the mentioned above limitations of short read sequencing. At Variantyx we have developed an integrated clinical genetic testing approach, augmenting short read WGS-based variant detection with Oxford Nanopore Technologies (ONT) long read sequencing, providing simultaneous orthogonal confirmation of all types of variants with the additional benefit of improved identification of exact size and position of the detected aberrations. The validation study of this augmented test has demonstrated that Oxford Nanopore Technologies sequencing can efficiently verify multiple types of reportable variants, thus ensuring highly reliable detection and a quick turnaround time for WGS-based clinical genetic testing.

The Science and Art of Clinical Genetic Variant Classification and Its Impact on Test Accuracy.

Clinical genetic variant classification science is a growing subspecialty of clinical genetics and genomics. The field's continued improvement is essential for the success of precision medicine in both germline (hereditary) and somatic (oncology) contexts. This review focuses on variant classification for DNA next-generation sequencing tests. We first summarize current limitations in variant discovery and definition, and then describe the current five- and four-tier classification systems outlined in dominant standards and guideline publications for germline and somatic tests, respectively. We then discuss measures of variant classification discordance and the field's bias for positive results, as well as considerations for panel size and population screening in the context of estimates of positive predictive value thatincorporate estimated variant classification imperfections. Finally, we share opinions on the current state of variant classification from some of the authors of the most widely used standards and guideline publications and from other domain experts.

Specifications of the ACMG/AMP standards and guidelines for mitochondrial DNA variant interpretation.

Mitochondrial DNA (mtDNA) variant pathogenicity interpretation has special considerations given unique features of the mtDNA genome, including maternal inheritance, variant heteroplasmy, threshold effect, absence of splicing, and contextual effects of haplogroups. Currently, there are insufficient standardized criteria for mtDNA variant assessment, which leads to inconsistencies in clinical variant pathogenicity reporting. An international working group of mtDNA experts was assembled within the Mitochondrial Disease Sequence Data Resource Consortium and obtained Expert Panel status from ClinGen. This group reviewed the 2015 American College of Medical Genetics and Association of Molecular Pathology standards and guidelines that are widely used for clinical interpretation of DNA sequence variants and provided further specifications for additional and specific guidance related to mtDNA variant classification. These Expert Panel consensus specifications allow for consistent consideration of the unique aspects of the mtDNA genome that directly influence variant assessment, including addressing mtDNA genome composition and structure, haplogroups and phylogeny, maternal inheritance, heteroplasmy, and functional analyses unique to mtDNA, as well as specifications for utilization of mtDNA genomic databases and computational algorithms.

Targeted gene sequencing in 6994 individuals with neurodevelopmental disorder with epilepsy.

PURPOSE: We aimed to gain insight into frequencies of genetic variants in genes implicated in neurodevelopmental disorder with epilepsy (NDD+E) by investigating large cohorts of patients in a diagnostic setting. METHODS: We analyzed variants in NDD+E using epilepsy gene panel sequencing performed between 2013 and 2017 by two large diagnostic companies. We compared variant frequencies in 6994 panels with another 8588 recently published panels as well as exome-wide de novo variants in 1942 individuals with NDD+E and 10,937 controls. RESULTS: Genes with highest frequencies of ultrarare variants in NDD+E comprised SCN1A, KCNQ2, SCN2A, CDKL5, SCN8A, and STXBP1, concordant with the two other epilepsy cohorts we investigated. In only 46% of the analyzed 262 dominant and X-linked panel genes ultrarare variants in patients were reported. Among genes with contradictory evidence of association with epilepsy, CACNB4, CLCN2, EFHC1, GABRD, MAGI2, and SRPX2 showed equal frequencies in cases and controls. CONCLUSION: We show that improvement of panel design increased diagnostic yield over time, but panels still display genes with low or no diagnostic yield. With our data, we hope to improve current diagnostic NDD+E panel design and provide a resource of ultrarare variants in individuals with NDD+E to the community.

KCTD7 deficiency defines a distinct neurodegenerative disorder with a conserved autophagy-lysosome defect.

OBJECTIVE: Several small case series identified KCTD7 mutations in patients with a rare autosomal recessive disorder designated progressive myoclonic epilepsy (EPM3) and neuronal ceroid lipofuscinosis (CLN14). Despite the name KCTD (potassium channel tetramerization domain), KCTD protein family members lack predicted channel domains. We sought to translate insight gained from yeast studies to uncover disease mechanisms associated with deficiencies in KCTD7 of unknown function. METHODS: Novel KCTD7 variants in new and published patients were assessed for disease causality using genetic analyses, cell-based functional assays of patient fibroblasts and knockout yeast, and electron microscopy of patient samples. RESULTS: Patients with KCTD7 mutations can exhibit movement disorders or developmental regression before seizure onset, and are distinguished from similar disorders by an earlier age of onset. Although most published KCTD7 patient variants were excluded from a genome sequence database of normal human variations, most newly identified patient variants are present in this database, potentially challenging disease causality. However, genetic analysis and impaired biochemical interactions with cullin 3 support a causal role for patient KCTD7 variants, suggesting deleterious alleles of KCTD7 and other rare disease variants may be underestimated. Both patient-derived fibroblasts and yeast lacking Whi2 with sequence similarity to KCTD7 have impaired autophagy consistent with brain pathology. INTERPRETATION: Biallelic KCTD7 mutations define a neurodegenerative disorder with lipofuscin and lipid droplet accumulation but without defining features of neuronal ceroid lipofuscinosis or lysosomal storage disorders. KCTD7 deficiency appears to cause an underlying autophagy-lysosome defect conserved in yeast, thereby assigning a biological role for KCTD7. Ann Neurol 2018;84:774-788.

Genetic testing including targeted gene panel in a diverse clinical population of children with autism spectrum disorder: Findings and implications.

BACKGROUND: Genetic testing of children with autism spectrum disorder (ASD) is now standard in the clinical setting, with American College of Medical Genetics and Genomics (ACMGG) guidelines recommending microarray for all children, fragile X testing for boys and additional gene sequencing, including PTEN and MECP2, in appropriate patients. Increasingly, testing utilizing high throughput sequencing, including gene panels and whole exome sequencing, are offered as well. METHODS: We performed genetic testing including microarray, fragile X testing and targeted gene panel, consistently sequencing 161 genes associated with ASD risk, in a clinical population of 100 well characterized children with ASD. Frequency of rare variants identified in individual genes was compared with that reported in the Exome Aggregation Consortium (ExAC) database. RESULTS: We did not diagnose any conditions with complete penetrance for ASD; however, copy number variants believed to contribute to ASD risk were identified in 12%. Eleven children were found to have likely pathogenic variants on gene panel, yet, after careful analysis, none was considered likely causative of disease. KIRREL3 variants were identified in 6.7% of children compared to 2% in ExAC, suggesting a potential role for KIRREL3 variants in ASD risk. Children with KIRREL3 variants more often had minor facial dysmorphism and intellectual disability. We also observed an increase in rare variants in TSC2. However, analysis of variant data from the Simons Simplex Collection indicated that rare variants in TSC2 occur more commonly in specific racial/ethnic groups, which are more prevalent in our population than in the ExAC database. CONCLUSION: The yield of genetic testing including microarray, fragile X (boys) and targeted gene panel was 12%. Gene panel did not increase diagnostic yield; however, we found an increase in rare variants in KIRREL3. Our findings reinforce the need for racial/ethnic diversity in large-scale genomic databases used to identify variants that contribute to disease risk.

Rare genetic variants in the endocannabinoid system genes CNR1 and DAGLA are associated with neurological phenotypes in humans.

Rare genetic variants in the core endocannabinoid system genes CNR1, CNR2, DAGLA, MGLL and FAAH were identified in molecular testing data from 6,032 patients with a broad spectrum of neurological disorders. The variants were evaluated for association with phenotypes similar to those observed in the orthologous gene knockouts in mice. Heterozygous rare coding variants in CNR1, which encodes the type 1 cannabinoid receptor (CB1), were found to be significantly associated with pain sensitivity (especially migraine), sleep and memory disorders-alone or in combination with anxiety-compared to a set of controls without such CNR1 variants. Similarly, heterozygous rare variants in DAGLA, which encodes diacylglycerol lipase alpha, were found to be significantly associated with seizures and neurodevelopmental disorders, including autism and abnormalities of brain morphology, compared to controls. Rare variants in MGLL, FAAH and CNR2 were not associated with any neurological phenotypes in the patients tested. Diacylglycerol lipase alpha synthesizes the endocannabinoid 2-AG in the brain, which interacts with CB1 receptors. The phenotypes associated with rare CNR1 variants are reminiscent of those implicated in the theory of clinical endocannabinoid deficiency syndrome. The severe phenotypes associated with rare DAGLA variants underscore the critical role of rapid 2-AG synthesis and the endocannabinoid system in regulating neurological function and development. Mapping of the variants to the 3D structure of the type 1 cannabinoid receptor, or primary structure of diacylglycerol lipase alpha, reveals clustering of variants in certain structural regions and is consistent with impacts to function.

TBC1D24 genotype-phenotype correlation: Epilepsies and other neurologic features.

OBJECTIVE: To evaluate the phenotypic spectrum associated with mutations in TBC1D24. METHODS: We acquired new clinical, EEG, and neuroimaging data of 11 previously unreported and 37 published patients. TBC1D24 mutations, identified through various sequencing methods, can be found online (http://lovd.nl/TBC1D24). RESULTS: Forty-eight patients were included (28 men, 20 women, average age 21 years) from 30 independent families. Eighteen patients (38%) had myoclonic epilepsies. The other patients carried diagnoses of focal (25%), multifocal (2%), generalized (4%), and unclassified epilepsy (6%), and early-onset epileptic encephalopathy (25%). Most patients had drug-resistant epilepsy. We detail EEG, neuroimaging, developmental, and cognitive features, treatment responsiveness, and physical examination. In silico evaluation revealed 7 different highly conserved motifs, with the most common pathogenic mutation located in the first. Neuronal outgrowth assays showed that some TBC1D24 mutations, associated with the most severe TBC1D24-associated disorders, are not necessarily the most disruptive to this gene function. CONCLUSIONS: TBC1D24-related epilepsy syndromes show marked phenotypic pleiotropy, with multisystem involvement and severity spectrum ranging from isolated deafness (not studied here), benign myoclonic epilepsy restricted to childhood with complete seizure control and normal intellect, to early-onset epileptic encephalopathy with severe developmental delay and early death. There is no distinct correlation with mutation type or location yet, but patterns are emerging. Given the phenotypic breadth observed, TBC1D24 mutation screening is indicated in a wide variety of epilepsies. A TBC1D24 consortium was formed to develop further research on this gene and its associated phenotypes.

Neural substrates of spontaneous narrative production in focal neurodegenerative disease.

Conversational storytelling integrates diverse cognitive and socio-emotional abilities that critically differ across neurodegenerative disease groups. Storytelling patterns may have diagnostic relevance and predict anatomic changes. The present study employed mixed methods discourse and quantitative analyses to delineate patterns of storytelling across focal neurodegenerative disease groups, and to clarify the neuroanatomical contributions to common storytelling characteristics. Transcripts of spontaneous social interactions of 46 participants (15 behavioral variant frontotemporal dementia (bvFTD), 7 semantic variant primary progressive aphasia (svPPA), 12 Alzheimer's disease (AD), and 12 healthy older normal controls (NC)) were analyzed for storytelling frequency and characteristics, and videos of the interactions were rated for patients' level of social attentiveness. Compared to controls, svPPAs told more stories and autobiographical stories, and perseverated on aspects of self during the interaction, whereas ADs told fewer autobiographical stories than NCs. svPPAs and bvFTDs were rated as less attentive to social cues. Aspects of storytelling were related to diverse cognitive and socio-emotional functions, and voxel-based anatomic analysis of structural magnetic resonance imaging revealed that temporal organization, narrative evaluations patterns, and social attentiveness correlated with atrophy corresponding to known intrinsic connectivity networks, including the default mode, limbic, salience, and stable task control networks. Differences in spontaneous storytelling among neurodegenerative groups elucidated diverse cognitive, socio-emotional, and neural contributions to narrative production, with implications for diagnostic screening and therapeutic intervention.

Hurt, tired and queasy: Specific variants in the ATPase domain of the TRAP1 mitochondrial chaperone are associated with common, chronic "functional" symptomatology including pain, fatigue and gastrointestinal dysmotility.

Functional disorders are common conditions with a substantial impact on a patients' wellbeing, and can be diagnostically elusive. There are bidirectional associations between functional disorders and mitochondrial dysfunction. In this study, provided clinical information and the exon sequence of the TRAP1 mitochondrial chaperone were retrospectively reviewed with a focus on the functional categories of chronic pain, fatigue and gastrointestinal dysmotility. Very-highly conserved TRAP1 variants were identified in 73 of 930 unrelated patients. Functional symptomatology is strongly associated with specific variants in the ATPase binding pocket. In particular, the combined presence of all three functional categories is strongly associated with p.Ile253Val (OR 7.5, P = 0.0001) and with two other interacting variants (OR 18, P = 0.0005). Considering a 1-2% combined variant prevalence and high odds ratios, these variants may be an important factor in the etiology of functional symptomatology.

Neural substrates of socioemotional self-awareness in neurodegenerative disease.

BACKGROUND: Neuroimaging studies examining neural substrates of impaired self-awareness in patients with neurodegenerative diseases have shown divergent results depending on the modality (cognitive, emotional, behavioral) of awareness. Evidence is accumulating to suggest that self-awareness arises from a combination of modality-specific and large-scale supramodal neural networks. METHODS: We investigated the structural substrates of patients' tendency to overestimate or underestimate their own capacity to demonstrate empathic concern for others. Subjects' level of empathic concern was measured using the Interpersonal Reactivity Index, and subject-informant discrepancy scores were used to predict regional atrophy pattern, using voxel-based morphometry analysis. Of the 102 subjects, 83 were patients with neurodegenerative diseases such as behavioral variant frontotemporal dementia (bvFTD) or semantic variant primary progressive aphasia (svPPA); the other 19 were healthy older adults. RESULTS: bvFTD and svPPA patients typically overestimated their level of empathic concern compared to controls, and overestimating one's empathic concern predicted damage to predominantly right-hemispheric anterior infero-lateral temporal regions, whereas underestimating one's empathic concern showed no neuroanatomical basis. CONCLUSIONS: These findings suggest that overestimation and underestimation of one's capacity for empathic concern cannot be interpreted as varying degrees of the same phenomenon, but may arise from different pathophysiological processes. Damage to anterior infero-lateral temporal regions has been associated with semantic self-knowledge, emotion processing, and social perspective taking; neuropsychological functions partly associated with empathic concern itself. These findings support the hypothesis that-at least in the socioemotional domain-neural substrates of self-awareness are partly modality-specific.

Clinically relevant variants - identifying, collecting, interpreting, and disseminating: the 2013 annual scientific meeting of the Human Genome Variation Society.

The dramatic advances in genetic sequencing technologies used in research laboratories are now entering the clinic, and applications of whole-genome and whole-exome sequencing to disease diagnosis, predisposition, and treatment will soon be commonplace. However, the standards and methods for identifying clinically relevant variants are currently being debated and defined. Multiple agencies worldwide have recognized that we have reached an exciting and critical transition point into the clinic, and many important issues are being discussed that impact how genetic variation data in the clinic will be interpreted and used. The 2013 annual scientific meeting of the Human Genome Variation Society (HGVS) had as its main theme the discovery, interpretation, and dissemination of clinically relevant DNA variants. The meeting featured the continuously developing technology of databasing genetic variation and computational tools for allelic variant discovery. Attention was given to curating and integrating these data with clinical findings, including approaches to distinguish between functional alleles underlying clinical phenotypes and benign sequence variants and making data sources interoperable and functional for clinical diagnostic utility, citing examples in specific diseases.

Mody-3: novel HNF1A mutation and the utility of glucagon-like peptide (GLP)-1 receptor agonist therapy.

OBJECTIVE: An estimated 1 to 2% of cases of diabetes mellitus have a monogenic basis; however, delayed diagnosis and misdiagnosis as type 1 and 2 diabetes are common. Correctly identifying the molecular basis of an individual's diabetes may significantly alter the management approach to both the patient and his or her relatives. We describe a case of mature onset diabetes of the young (MODY) with sufficient evidence to support the classification of a novel HNF1A (hepatocyte nuclear factor-1-α) mutation as a cause of MODY-3. METHODS: A 21-year-old Caucasian female presented to our office with a diagnosis of noninsulin-dependent diabetes mellitus (NIDDM) at age 10; glycemia was initially managed with oral antidiabetic (OAD) agents and insulin detemir. The patient reported a strong family history of early-onset NIDDM in both her mother and maternal grandmother, both of whom eventually required insulin therapy to control glycemia. The patient's medical and family history were highly suggestive of maturity-onset diabetes of the young (MODY), and genetic testing was performed. RESULTS: Genetic screening detected a mutation p. Arg200Trp in the HNF1A gene in the patient, her mother, and maternal grandmother, suggesting a diagnosis of MODY-3. This finding resulted in a change of antidiabetic therapy in all 3 patients, including the addition of once-daily liraglutide therapy, which helped improve their glycemic control. CONCLUSION: Our case report supports the classification of the p. Arg200Trp mutation as a cause of MODY-3. The findings also suggest that glucagon-like peptide-1 (GLP-1) receptor agonist therapy may be of value in managing glycemia in patients with MODY-3.

Infantile onset CMT2D/dSMA V in monozygotic twins due to a mutation in the anticodon-binding domain of GARS.

Mutations in the GARS gene cause Charcot-Marie-Tooth 2D and distal spinal muscular atrophy type V - allelic disorders characterized by predominantly distal upper extremity weakness and atrophy, typically beginning during the second decade of life. We report monozygotic twin girls with onset of weakness in infancy and a previously reported GARS mutation within the anticodon-binding domain. The severity and remarkable similarity in phenotypes of these girls and the reported case suggest that mutations within the anticodon-binding domain are more damaging to aminoacyl tRNA synthetase function than those within other domains of GARS.

Neuropsychological correlates of dominance, warmth, and extraversion in neurodegenerative disease.

INTRODUCTION: Changes in personality differ qualitatively and quantitatively among patients with different neurodegenerative diseases, likely due to divergent patterns of regional neurodegeneration. Regional damage to circuits underlying various cognitive and emotional functions have been associated with interpersonal traits like dominance, extraversion, and warmth in patients with neurodegenerative diseases, suggesting that personality may in part be mediated by these more basic neuropsychological functions. In this study, we hypothesized that different combinations of cognitive, neuropsychiatric, and emotional measures would predict different interpersonal traits in patients with neurodegenerative diseases. METHODS: A battery of cognitive, neuropsychiatric, and emotional measures was administered to 286 patients with various neurodegenerative diseases such as Alzheimer's disease, behavioral variant frontotemporal dementia, semantic dementia, and progressive supranuclear palsy, and informants described patients' dominance, extraversion, and warmth using the Interpersonal Adjective Scales (IAS) personality questionnaire. Regression modeling was performed to identify which neuropsychological factors uniquely predicted current personality, controlling for age, gender, and premorbid personality. RESULTS: Social dominance covaried with patients' capacity for cognitive control and verbal fluency. Conversely, warmth did not rely on these executive or verbal skills, but covaried primarily with patients' capacity for emotional responsiveness. Extraversion, representing a blend of dominance and warmth, demonstrated an intermediate degree of relationship to both executive/verbal and emotional functions. CONCLUSIONS: These findings suggest that different personality traits are partly subserved by specific cognitive and emotional functions in neurodegenerative disease patients. While this study was performed in the context of brain damage, the results raise the question of whether individual differences in these neuropsychological abilities may also underlie variability in normal personality.

Clinical characterization of bvFTD due to FUS neuropathology.

In 2009, inclusions containing the fused in sarcoma (FUS) protein were identified as a third major molecular class of pathology underlying the behavioral variant frontotemporal dementia (bvFTD) syndrome. Due to the low prevalence of FUS pathology, few clinical descriptions have been published and none provides information about specific social-emotional deficits despite evidence for severe behavioral manifestations in this disorder. We evaluated a patient with bvFTD due to FUS pathology using a comprehensive battery of cognitive and social- emotional tests. A structural MRI scan and genetic tests for tau, progranulin, and FUS mutations were also performed. The patient showed preserved general cognitive functioning and superior working memory, but severe deficits in emotion attribution, sensitivity to punishment, and diminished capacity for interpersonal warmth and empathy. The gray matter atrophy pattern corresponded to this focal deficit profile, with preservation of dorsolateral fronto-parietal regions associated with executive functioning but severe damage to right worse than left frontoinsula, temporal pole, subgenual anterior cingulate, medial orbitofrontal cortex, amygdala, and caudate. This patient demonstrates the striking focality associated with FUS neuropathology in patients with bvFTD.

Interpersonal traits change as a function of disease type and severity in degenerative brain diseases.

BACKGROUND: Different degenerative brain diseases result in distinct personality changes as a result of divergent patterns of brain damage; however, little is known about the natural history of these personality changes throughout the course of each disease. OBJECTIVE: To investigate how interpersonal traits change as a function of degenerative brain disease type and severity. METHODS: Using the Interpersonal Adjective Scales, informant ratings of retrospective premorbid and current scores for dominance, extraversion, warmth and ingenuousness were collected annually for 1 to 4 years on 188 patients (67 behavioural variant frontotemporal dementia (bvFTD), 40 semantic dementia (SemD), 81 Alzheimer's disease (AD)) and 65 older healthy controls. Using random coefficient models, interpersonal behaviour scores at very mild, mild or moderate-to-severe disease stages were compared within and between patient groups. RESULTS: Group-level changes from premorbid personality occurred as a function of disease type and severity, and were apparent even at a very mild disease stage (Clinical Dementia Rating=0.5) for all three diseases. Decreases in interpersonal traits were associated with emotional affiliation (ie, extraversion, warmth and ingenuousness) and more rigid interpersonal behaviour differentiated bvFTD and SemD patients from AD patients. CONCLUSIONS: Specific changes in affiliative interpersonal traits differentiate degenerative brain diseases even at a very mild disease stage, and patterns of personality change differ across bvFTD, SemD and AD with advancing disease. This study describes the typical progression of change of interpersonal traits in each disease, improving the ability of clinicians and caregivers to predict and plan for symptom progression.

Estimating the healthcare burden of osteomyelitis in Uganda.

Chronic osteomyelitis is a considerable healthcare burden in many developing countries, but this burden is poorly quantified. To estimate the clinical burden of osteomyelitis we systematically sampled the medical records of orthopaedic clinics at five hospitals in Uganda. To estimate the surgical burden of osteomyelitis we reviewed the diagnosis in 9354 operations conducted during a 1 year period at the same five hospitals. Of 1844 outpatients with a documented diagnosis sampled over 1 year, 187 (10%) had osteomyelitis. Only 20% of those with osteomyelitis were older than 20 years, whereas this age group accounted for 52% of patients with another orthopaedic diagnosis or no diagnosis (P<0.001). Osteomyelitis was diagnosed in 325 (3.5%) of the surgical operations; in 32% of these operations the patients were children aged between 10 and 14 years. The tibia was the bone most frequently involved (31%), and sequestrectomy was the most frequent surgical procedure (60%). These findings suggest that osteomyelitis disproportionately affects the young, and is a burden on both clinical and surgical services. To decrease this burden in populations with limited resources, improved diagnosis and more timely treatment of acute osteomyelitis is needed.