High diagnosis rate for nonimmune hydrops fetalis with prenatal clinical exome from the Hydrops-Yielding Diagnostic Results of Prenatal Sequencing (HYDROPS) Study

PurposeNonimmune hydrops fetalis (NIHF) presents as life-threatening fluid collections in multiple fetal compartments and can be caused by both genetic and non-genetic etiologies. We explored incremental diagnostic yield of testing with prenatal exome sequencing (ES) for NIHF following a negative standard NIHF workup.MethodsParticipants enrolled into the Hydrops-Yielding Diagnostic Results of Prenatal Sequencing (HYDROPS) study met a strict definition of NIHF and had negative standard-of-care workup. Clinical trio ES from fetal samples and parental blood was performed at a CLIA-certified reference laboratory with clinical reports returned by geneticists and genetic counselors. Negative exomes were reanalyzed with information from subsequent ultrasounds and records.ResultsTwenty-two fetal exomes reported 11 (50%) diagnostic results and five possible diagnoses (22.7%). Diagnosed cases comprised seven de novodominant disorders, three recessive disorders, and one inherited dominant disorder including four Noonan syndromes (PTPN11, RAF1, RIT1, and RRAS2), three musculoskeletal disorders (RYR1, AMER1, and BICD2), two metabolic disorders (sialidosis and multiple sulfatase deficiency), one Kabuki syndrome, and one congenital anemia (KLF1).ConclusionThe etiology of NIHF predicts postnatal prognosis and recurrence risk in future pregnancies. ES provides high incremental diagnostic yield for NIHF after standard-of-care testing and should be considered in the workup.     

Unexpected diagnosis of myotonic dystrophy type 2 repeat expansion by genome sequencing

Several neurological disorders, such as myotonic dystrophy are caused by expansions of short tandem repeats (STRs) which can be difficult to detect by molecular tools. Methodological advances have made repeat expansion (RE) detection with whole genome sequencing (WGS) feasible. We recruited a multi-generational family (family A) ascertained for genetic studies of autism spectrum disorder. WGS was performed on seven children from four nuclear families from family A and analyzed for REs of STRs known to cause neurological disorders. We detected an expansion of a heterozygous intronic CCTG STR in CNBP in two siblings. This STR causes myotonic dystrophy type 2 (DM2). The expansion did not segregate with the ASD phenotype. Repeat-primed PCR showed that the DM2 CCTG motif was expanded above the pathogenic threshold in both children and their mother. On subsequent examination, the mother had mild features of DM2. We show that screening of STRs in WGS datasets has diagnostic utility, both in the clinical and research domain, with potential management and genetic counseling implications.Subject terms: Movement disorders, Medical genomics     

Systematic reanalysis of clinical exome data yields additional diagnoses: implications for providers

PurposeClinical exome sequencing is nondiagnostic for about 75% of patients evaluated for a possible Mendelian disorder. We examined the ability of systematic reevaluation of exome data to establish additional diagnoses.MethodsThe exome and phenotypic data of 40 individuals with previously nondiagnostic clinical exomes were reanalyzed with current software and literature.ResultsA definitive diagnosis was identified for 4 of 40 participants (10%). In these cases the causative variant is de novo and in a relevant autosomal-dominant disease gene. The literature to tie the causative genes to the participants’ phenotypes was weak, nonexistent, or not readily located at the time of the initial clinical exome reports. At the time of diagnosis by reanalysis, the supporting literature was 1 to 3 years old.ConclusionApproximately 250 gene–disease and 9,200 variant–disease associations are reported annually. This increase in information necessitates regular reevaluation of nondiagnostic exomes. To be practical, systematic reanalysis requires further automation and more up-to-date variant databases. To maximize the diagnostic yield of exome sequencing, providers should periodically request reanalysis of nondiagnostic exomes. Accordingly, policies regarding reanalysis should be weighed in combination with factors such as cost and turnaround time when selecting a clinical exome laboratory.     

RNA sequencing solved the most common but unrecognized NEB pathogenic variant in Japanese nemaline myopathy

PurposeThe diagnostic rate for Mendelian diseases by exome sequencing (ES) is typically 20–40%. The low rate is partly because ES misses deep-intronic or synonymous variants leading to aberrant splicing. In this study, we aimed to apply RNA sequencing (RNA-seq) to efficiently detect the aberrant splicings and their related variants.MethodsAberrant splicing in biopsied muscles from six nemaline myopathy (NM) cases unresolved by ES were analyzed with RNA-seq. Variants related to detected aberrant splicing events were analyzed with Sanger sequencing. Detected variants were screened in NM patients unresolved by ES.ResultsWe identified a novel deep-intronic NEB pathogenic variant, c.1569+339A>G in one case, and another novel synonymous NEB pathogenic variant, c.24684G>C (p.Ser8228Ser) in three cases. The c.24684G>C variant was observed to be the most frequent among all NEB pathogenic variants in normal Japanese populations with a frequency of 1 in 178 (20 alleles in 3552 individuals), but was previously unrecognized. Expanded screening of the variant identified it in a further four previously unsolved nemaline myopathy cases.ConclusionThese results indicated that RNA-seq may be able to solve a large proportion of previously undiagnosed muscle diseases.     

Mobile element insertion detection in 89,874 clinical exomes

PurposeExome sequencing (ES) is increasingly used for the diagnosis of rare genetic disease. However, some pathogenic sequence variants within the exome go undetected due to the technical difficulty of identifying them. Mobile element insertions (MEIs) are a known cause of genetic disease in humans but have been historically difficult to detect via ES and similar targeted sequencing methods.MethodsWe developed and applied a novel MEI detection method prospectively to samples received for clinical ES beginning in November 2017. Positive MEI findings were confirmed by an orthogonal method and reported back to the ordering provider. In this study, we examined 89,874 samples from 38,871 cases.ResultsDiagnostic MEIs were present in 0.03% (95% binomial test confidence interval: 0.02–0.06%) of all cases and account for 0.15% (95% binomial test confidence interval: 0.08–0.25%) of cases with a molecular diagnosis. One diagnostic MEI was a novel founder event. Most patients with pathogenic MEIs had prior genetic testing, three of whom had previous negative DNA sequencing analysis of the diagnostic gene.ConclusionMEI detection from ES is a valuable diagnostic tool, reveals molecular findings that may be undetected by other sequencing assays, and increases diagnostic yield by 0.15%.     

Putting genome-wide sequencing in neonates into perspective

PurposeSeveral studies have reported diagnostic yields up to 57% for rapid exome or genome sequencing (rES/GS) as a single test in neonatal intensive care unit (NICU) patients, but the additional yield of rES/GS compared with other available diagnostic options still remains unquantified in this population.MethodsWe retrospectively evaluated all genetic NICU consultations in a 2-year period.ResultsIn 132 retrospectively evaluated NICU consultations 27 of 32 diagnoses (84.4%) were made using standard genetic workup. Most diagnoses (65.6%) were made within 16 days. Diagnostic ES yield was 5/29 (17.2%). Genetic diagnoses had a direct effect on clinical management in 90.6% (29/32) of patients.ConclusionsOur study shows that exome sequencing has a place in NICU diagnostics, but given the associated costs and the high yield of alternative diagnostic strategies, we recommend to first perform clinical genetic consultation.     

Exome sequencing is a useful diagnostic tool for complicated forms of hereditary spastic paraplegia

Hereditary spastic paraplegias constitute a heterogeneous group of neurodegenerative diseases encompassing pure and complicated forms, for which at least 52 loci and 31 causative genes have been identified. Although mutations in the SPAST gene explain approximately 40% of the pure autosomal dominant forms, molecular diagnosis can be challenging for the sporadic and recessive forms, which are often complicated and clinically overlap with a broad number of movement disorders. The validity of exome sequencing as a routine diagnostic approach in the movement disorder clinic needs to be assessed. The main goal of this study was to explore the usefulness of an exome analysis for the diagnosis of a complicated form of spastic paraplegia. Whole‐exome sequencing was performed in two Spanish siblings with a neurodegenerative syndrome including upper and lower motor neuron, ocular and cerebellar signs. Exome sequencing revealed that both patients carry a novel homozygous nonsense mutation in exon 15 of the SPG11 gene (c.2678G>A; p.W893X), which was not found in 584 Spanish control chromosomes. After many years of follow‐up and multiple time‐consuming genetic testing, we were able to diagnose these patients by making use of whole‐exome sequencing, showing that this is a cost‐efficient diagnostic tool for the movement disorder specialist.     

Meta-analysis and multidisciplinary consensus statement: exome sequencing is a first-tier clinical diagnostic test for individuals with neurodevelopmental disorders

PurposeFor neurodevelopmental disorders (NDDs), etiological evaluation can be a diagnostic odyssey involving numerous genetic tests, underscoring the need to develop a streamlined algorithm maximizing molecular diagnostic yield for this clinical indication. Our objective was to compare the yield of exome sequencing (ES) with that of chromosomal microarray (CMA), the current first-tier test for NDDs.MethodsWe performed a PubMed scoping review and meta-analysis investigating the diagnostic yield of ES for NDDs as the basis of a consensus development conference. We defined NDD as global developmental delay, intellectual disability, and/or autism spectrum disorder. The consensus development conference included input from genetics professionals, pediatric neurologists, and developmental behavioral pediatricians.ResultsAfter applying strict inclusion/exclusion criteria, we identified 30 articles with data on molecular diagnostic yield in individuals with isolated NDD, or NDD plus associated conditions (such as Rett-like features). Yield of ES was 36% overall, 31% for isolated NDD, and 53% for the NDD plus associated conditions. ES yield for NDDs is markedly greater than previous studies of CMA(15–20%).ConclusionOur review demonstrates that ES consistently outperforms CMA for evaluation of unexplained NDDs. We propose a diagnostic algorithm placing ES at the beginning of the evaluation of unexplained NDDs.     

Parents’ perceptions of personal utility of exome sequencing results

PurposeClinical genome or exome sequencing (GS/ES) provides a diagnosis for many individuals with suspected genetic disorders, but also yields negative or uncertain results for the majority. This study examines how parents of a child with an undiagnosed condition attribute personal utility to all types of ES results.MethodsReturn of 31 exome sequencing results was observed during clinic sessions, followed by semistructured interviews with parents one month later. Observations and interviews were recorded and transcribed. Data display matrices were used for content analysis and systematic comparisons of parents’ perceptions of utility.ResultsES results could not provide all the answers to parents’ questions, especially in cases of clinically uninformative results, but parents nonetheless attributed utility to the knowledge gained. Parents across all results categories used the genomic information to rule out possible causes, end or postpone the diagnostic odyssey, and shift focus to treatment and management of symptoms.ConclusionThis study suggests that parents value even uninformative ES results while expressing hope for future discoveries. As pediatric genetics moves toward GS/ES as a first-tier test, how parents perceive the personal utility of negative or uncertain results is an important topic for genetic counseling and further research.     

TANGO2: expanding the clinical phenotype and spectrum of pathogenic variants

PurposeTANGO2-related disorders were first described in 2016 and prior to this publication, only 15 individuals with TANGO2-related disorder were described in the literature. Primary features include metabolic crisis with rhabdomyolysis, encephalopathy, intellectual disability, seizures, and cardiac arrhythmias. We assess whether genotype and phenotype of TANGO2-related disorder has expanded since the initial discovery and determine the efficacy of exome sequencing (ES) as a diagnostic tool for detecting variants.MethodsWe present a series of 14 individuals from 11 unrelated families with complex medical and developmental histories, in whom ES or microarray identified compound heterozygous or homozygous variants in TANGO2.ResultsThe initial presentation of patients with TANGO2-related disorders can be variable, including primarily neurological presentations. We expand the phenotype and genotype for TANGO2, highlighting the variability of the disorder.ConclusionTANGO2-related disorders can have a more diverse clinical presentation than previously anticipated. We illustrate the utility of routine ES data reanalysis whereby discovery of novel disease genes can lead to a diagnosis in previously unsolved cases and the need for additional copy-number variation analysis when ES is performed.     

Accurate detection of clinically relevant uniparental disomy from exome sequencing data

PurposeUniparental disomy (UPD) is the rare occurrence of two homologous chromosomes originating from the same parent and is typically identified by marker analysis or single-nucleotide polymorphism (SNP)-based microarrays. UPDs may lead to disease due to imprinting effects, underlying homozygous pathogenic variants, or low-level mosaic aneuploidies. In this study we detected clinically relevant UPD events in both trio and single exome sequencing (ES) data.MethodsUPD was detected by applying a method based on Mendelian inheritance errors to a cohort of 4912 ES trios (all UPD types) and by using median absolute deviation–scaled regions of homozygosity to a cohort of 29,723 single ES samples (isodisomy only).ResultsAs positive controls, we accurately identified three mixed UPD, three isodisomy, as well as two segmental UPD events that were all previously reported by SNP-based microarrays. In addition, we identified three segmental UPD and 11 isodisomy events. This resulted in a novel diagnosis based on imprinting for one patient, and adjusted genetic counseling for another patient.ConclusionUPD can easily be identified using both single and trio ES and may be clinically relevant to patients. UPD analysis should become routine in clinical ES, because it increases the diagnostic yield and could affect genetic counseling.     

An approach to integrating exome sequencing for fetal structural anomalies into clinical practice

PurposeWe investigated the diagnostic and clinical performance of trio exome sequencing (ES) in parent–fetus trios where the fetus had sonographic abnormalities but normal karyotype, microarray and, in some cases, normal gene-specific sequencing.MethodsES was performed from DNA of 102 anomalous fetuses and from peripheral blood from their parents. Parents provided consent for the return of diagnostic results in the fetus, medically actionable findings in the parents, and identification as carrier couple for significant autosomal recessive conditions.ResultsIn 21/102 (20.6%) fetuses, ES provided a positive-definitive or positive-probable diagnosis. In 10/102 (9.8%), ES provided an inconclusive-possible result. At least 2/102 (2.0%) had a repeat pregnancy during the study period and used the information from the study for prenatal diagnosis in the next pregnancy. Six of 204 (2.9%) parents received medically actionable results that affected their own health and 3/102 (2.9%) of couples received results that they were carriers for the same autosomal recessive condition.ConclusionES has diagnostic utility in a select population of fetuses where a genetic diagnosis was highly suspected. Challenges related to genetics literacy, variant interpretation, and various types of diagnostic results affecting both fetal and parental health must be addressed by highly tailored pre- and post-test genetic counseling.     

The Exome Clinic and the role of medical genetics expertise in the interpretation of exome sequencing results

PurposeEvaluation of the clinician’s role in the optimal interpretation of clinical exome sequencing (ES) results.MethodsRetrospective chart review of the first 155 patients who underwent clinical ES in our Exome Clinic and direct interaction with the ordering geneticist to evaluate the process of interpretation of results.ResultsThe most common primary indication was neurodevelopmental problems (~66%), followed by multiple congenital anomalies (~10%). Based on sequencing data, the overall diagnostic yield was 36%. After assessment by the medical geneticist, incorporation of detailed phenotypic and molecular data, and utilization of additional diagnostic modalities, the final diagnostic yield increased to 43%. Seven patients in our cohort were included in initial case series that described novel genetic syndromes, and 23% of patients were involved in subsequent research studies directly related to their results or involved in efforts to move beyond clinical ES for diagnosis. Clinical management was directly altered due to the ES findings in 12% of definitively diagnosed cases.ConclusionsOur results emphasize the usefulness of ES, demonstrate the significant role of the medical geneticist in the diagnostic process of patients undergoing ES, and illustrate the benefits of postanalytical diagnostic work-up in solving the “diagnostic odyssey.”Genet Med advance online publication 02 March 2017     

Clinical and genetic spectrum of a large cohort of children with epilepsy in China

PurposeGenetic diagnosis for children suffering from epilepsy has important implications for treatment, prognosis, and development of precision medicine strategies.MethodsWe performed exome sequencing (ES) or targeted sequencing on 733 children with epilepsy onset within the first year of life. We subgrouped our patients based on the onset age of seizure into neonatal and before 1 year (1–12 months), to compare the clinical and genetic features.ResultsThe subgroups with different onset age of seizure showed different pathogenic variant spectrum, and the 1-year age group was more likely to have developmental delays than the neonate group (p = 0.000614). The diagnostic rate was 26.7% for targeted sequencing using a 2742-gene panel, and 42% for ES. We identified 12 genes, which covered 48.7% of diagnostic cases. Our data revealed that 41.9% of patients in the neonate group and 49.7% patients in the 1-year group had treatment options based on molecular diagnosis.ConclusionThe 12 most commonly implicated genes in this cohort and the genes with treatment options should be considered as part of the essential panel for early diagnosis of epilepsy onset, if large medical exome analyses or ES are not feasible as first-tier analysis. Genetic results are beginning to improve therapy by antiepileptic medication selections and precision medicine approaches.     

Phenotype based prediction of exome sequencing outcome using machine learning for neurodevelopmental disorders

PurposeAlthough the introduction of exome sequencing (ES) has led to the diagnosis of a significant portion of patients with neurodevelopmental disorders (NDDs), the diagnostic yield in actual clinical practice has remained stable at approximately 30%. We hypothesized that improving the selection of patients to test on the basis of their phenotypic presentation will increase diagnostic yield and therefore reduce unnecessary genetic testing.MethodsWe tested 4 machine learning methods and developed PredWES from these: a statistical model predicting the probability of a positive ES result solely on the basis of the phenotype of the patient.ResultsWe first trained the tool on 1663 patients with NDDs and subsequently showed that diagnostic ES on the top 10% of patients with the highest probability of a positive ES result would provide a diagnostic yield of 56%, leading to a notable 114% increase. Inspection of our model revealed that for patients with NDDs, comorbid abnormal (lower) muscle tone and microcephaly positively correlated with a conclusive ES diagnosis, whereas autism was negatively associated with a molecular diagnosis.ConclusionIn conclusion, PredWES allows prioritizing patients with NDDs eligible for diagnostic ES on the basis of their phenotypic presentation to increase the diagnostic yield, making a more efficient use of health care resources.     

Discordant results between conventional newborn screening and genomic sequencing in the BabySeq Project

PurposeNewborn screening (NBS) is performed to identify neonates at risk for actionable, severe, early-onset disorders, many of which are genetic. The BabySeq Project randomized neonates to receive conventional NBS or NBS plus exome sequencing (ES) capable of detecting sequence variants that may also diagnose monogenic disease or indicate genetic disease risk. We therefore evaluated how ES and conventional NBS results differ in this population.MethodsWe compared results of NBS (including hearing screens) and ES for 159 infants in the BabySeq Project. Infants were considered “NBS positive” if any abnormal result was found indicating disease risk and “ES positive” if ES identified a monogenic disease risk or a genetic diagnosis.ResultsMost infants (132/159, 84%) were NBS and ES negative. Only one infant was positive for the same disorder by both modalities. Nine infants were NBS positive/ES negative, though seven of these were subsequently determined to be false positives. Fifteen infants were ES positive/NBS negative, all of which represented risk of genetic conditions that are not included in NBS programs. No genetic explanation was identified for eight infants referred on the hearing screen.ConclusionThese differences highlight the complementarity of information that may be gleaned from NBS and ES in the newborn period.     

Exome sequencing compared with standard genetic tests for critically ill infants with suspected genetic conditions

PurposeAs exome sequencing (ES) is increasingly used as a diagnostic tool, we aimed to compare ES with status quo genetic diagnostic workup for infants with suspected genetic disorders in terms of identifying diagnoses, survival, and cost of care.MethodsWe studied newborns and infants admitted to intensive care with a suspected genetic etiology within the first year of life at a US quaternary-referral children’s hospital over 5 years. In this propensity-matched cohort study using electronic medical record data, we compared patients who received ES as part of a diagnostic workup (ES cohort, n = 368) with clinically similar patients who did not receive ES (No-ES cohort, n = 368).ResultsDiagnostic yield (27.4% ES, 25.8% No-ES; p = 0.62) and 1-year survival (80.2% ES, 84.8% No-ES; p = 0.10) were no different between cohorts. ES cohort patients had higher cost of admission, diagnostic investigation, and genetic testing (all p < 0.01).ConclusionES did not differ from status quo genetic testing collectively in terms of diagnostic yield or patient survival; however, it had high yield as a single test, led to complementary classes of diagnoses, and was associated with higher costs. Further work is needed to define the most efficient use of diagnostic ES for critically ill newborns and infants.     

Comprehensive use of extended exome analysis improves diagnostic yield in rare disease: a retrospective survey in 1,059 cases

PurposeWe sought to determine the analytical sensitivity of several extended exome variation analysis approaches in terms of their contribution to diagnostic yield and their clinical feasibility.MethodsWe retrospectively analyzed the results of genetic testing in 1,059 distinct cases referred for exome sequencing to our institution. In these, we routinely employed extended exome analysis approaches in addition to basic variant analysis, including (i) copy-number variation (CNV) detection, (ii) nonconsensus splice defect detection, (ii) genomic breakpoint detection, (iv) homozygosity mapping, and (v) mitochondrial variant analysis.ResultsExtended exome analysis approaches assisted in identification of causative genetic variant in 44 cases, which represented a 4.2% increase in diagnostic yield. The greatest contribution was associated with CNV analysis (1.8%) and splice variant prediction (1.2%), and the remaining approaches contributed an additional 1.2%. Analysis of workload has shown that on average nine additional variants per case had to be interpreted in the extended analysis.ConclusionWe show that extended exome analysis approaches improve the diagnostic yield of heterogeneous genetic disorders and result in considerable increase of diagnostic yield of exome sequencing with a minor increase of interpretative workload.     

Cost-effectiveness frameworks for comparing genome and exome sequencing versus conventional diagnostic pathways: A scoping review and recommended methods

PurposeMethodological challenges have limited economic evaluations of genome sequencing (GS) and exome sequencing (ES). Our objective was to develop conceptual frameworks for model-based cost-effectiveness analyses (CEAs) of diagnostic GS/ES.MethodsWe conducted a scoping review of economic analyses to develop and iterate with experts a set of conceptual CEA frameworks for GS/ES for prenatal testing, early diagnosis in pediatrics, diagnosis of delayed-onset disorders in pediatrics, genetic testing in cancer, screening of newborns, and general population screening.ResultsReflecting on 57 studies meeting inclusion criteria, we recommend the following considerations for each clinical scenario. For prenatal testing, performing comparative analyses of costs of ES strategies and postpartum care, as well as genetic diagnoses and pregnancy outcomes. For early diagnosis in pediatrics, modeling quality-adjusted life years (QALYs) and costs over ≥20 years for rapid turnaround GS/ES. For hereditary cancer syndrome testing, modeling cumulative costs and QALYs for the individual tested and first/second/third-degree relatives. For tumor profiling, not restricting to treatment uptake or response and including QALYs and costs of downstream outcomes. For screening, modeling lifetime costs and QALYs and considering consequences of low penetrance and GS/ES reanalysis.ConclusionOur frameworks can guide the design of model-based CEAs and ultimately foster robust evidence for the economic value of GS/ES.