A Diagnosis for All Rare Genetic Diseases: The Horizon and the Next Frontiers.

The introduction of exome sequencing in the clinic has sparked tremendous optimism for the future of rare disease diagnosis, and there is exciting opportunity to further leverage these advances. To provide diagnostic clarity to all of these patients, however, there is a critical need for the field to develop and implement strategies to understand the mechanisms underlying all rare diseases and translate these to clinical care.

What's out there for parents? A systematic review of online information about prenatal microarray and exome sequencing.

OBJECTIVE: To identify what online patient information (presented in English) is available to parents about prenatal microarray (CMA) and exome sequencing (ES), and evaluate its content, quality, and readability. METHOD: Systematic searches (Google and Bing) were conducted, and websites were categorised according to their purpose. Websites categorised as patient information were included if they were: in English, directed at patients, or were a text, video, or online version of an information leaflet. Author-developed content checklists, the DISCERN Genetics tool, and readability tests (the Flesch Reading Ease Score, the Gunning Fog Index, and the Simple Measure of Gobbledygook Index) were then used to assess those sources of patient information. RESULTS: Of the 665 websites screened, 18 met the criteria. A further 8 sources were found through a targeted search of professional organisations, resulting in 26 sources available for further evaluation. In general, this was found to be low in quality, omitted details recommended by national or international guidance, and was written at a level too advanced for average readers. CONCLUSION: Improvements should be made to the content, quality, and readability of online information so that it both reinforces and complements the discussions between parents and clinicians about testing options during pregnancy.

The Third Revolution in Sequencing Technology.

Forty years ago the advent of Sanger sequencing was revolutionary as it allowed complete genome sequences to be deciphered for the first time. A second revolution came when next-generation sequencing (NGS) technologies appeared, which made genome sequencing much cheaper and faster. However, NGS methods have several drawbacks and pitfalls, most notably their short reads. Recently, third-generation/long-read methods appeared, which can produce genome assemblies of unprecedented quality. Moreover, these technologies can directly detect epigenetic modifications on native DNA and allow whole-transcript sequencing without the need for assembly. This marks the third revolution in sequencing technology. Here we review and compare the various long-read methods. We discuss their applications and their respective strengths and weaknesses and provide future perspectives.

Clinical utility of exome sequencing in infantile heart failure.

PURPOSE: Pediatric cardiomyopathy is rare, has a broad differential diagnosis, results in high morbidity and mortality, and has suboptimal diagnostic yield using next-generation sequencing panels. Exome sequencing has reported diagnostic yields ranging from 30% to 57% for neonates in intensive care units. We aimed to characterize the clinical utility of exome sequencing in infantile heart failure. METHODS: Infants diagnosed with acute heart failure prior to 1 year old over a period of 34 months at a large tertiary children's hospital were recruited. Demographic and diagnostic information was obtained from medical records. Fifteen eligible patients were enrolled. RESULTS: Dilated cardiomyopathy was the predominant cardiac diagnosis, seen in 60% of patients. A molecular diagnosis was identified in 66.7% of patients (10/15). Of those diagnoses, 70% would not have been detected using multigene next-generation sequencing panels focused on cardiomyopathy or arrhythmia disease genes. Genetic testing changed medical decision-making in 53% of all cases and 80% of positive cases, and was especially beneficial when testing was expedited. CONCLUSION: Given the broad differential diagnosis and critical status of infants with heart failure, rapid exome sequencing provides timely diagnoses, changes medical management, and should be the first-tier molecular test.

Copy number alterations involving 59 ACMG-recommended secondary findings genes.

In clinical exome/genome sequencing, the American College of Medical Genetics and Genomics (ACMG) recommends reporting of secondary findings unrelated to a patient's phenotype when pathogenic single-nucleotide variants (SNVs) are observed in one of 59 genes associated with a life-threatening, medically actionable condition. Little is known about the incidence and sensitivity of chromosomal microarray analysis (CMA) for detection of pathogenic copy number variants (CNVs) comprising medically-actionable genes. Clinical CMA has been performed on 8865 individuals referred for molecular cytogenetic testing. We retrospectively reviewed the CMA results to identify patients with CNVs comprising genes included in the 59-ACMG list of secondary findings. We evaluated the clinical significance of these CNVs in respect to pathogenicity, phenotypic manifestations, and heritability. We identified 23 patients (0.26%) with relevant CNV either deletions comprising the entire gene or intragenic alterations involving one or more secondary findings genes. A number of patients and/or their family members with pathogenic CNVs manifest or expected to develop an anticipated clinical phenotype and would benefit from preventive management similar to the patients with pathogenic SNVs. To improve patients' care standardization should apply to reporting of both sequencing and CNVs obtained via clinical genome-wide analysis, including chromosomal microarray and exome/genome sequencing.

Evolution of a prenatal genetic clinic-A 10-year cohort study.

OBJECTIVE: To (a) evaluate the proportion of women where a unifying genetic diagnosis was obtained following assessment of an observed pattern of fetal anomalies and (b) assess trends in genetic testing in a joint fetal-medicine genetic clinic. METHOD: Retrospective cohort study of all women attending the clinic. Outcomes included (a) indication for referral, (b) genetic test performed and (c) diagnoses obtained. RESULTS: From 2008 to 2019, 256 patients were referred and reviewed, of which 23% (n = 59) were consanguineous. The main indication for referral was the observed pattern of fetal anomalies. Over 10 years, the number of patients reviewed increased from 11 to 35 per annum. A unifying genetic diagnosis was obtained in 43.2% (n = 79/183), the majority of which were diagnosed prenatally (50.6% [n = 40/79]). The main investigation(s) that was the ultimate diagnostic test was targeted gene panel sequencing 34.2% (n = 27/79), with this and exome sequencing becoming the dominant genetic test by 2019. Pregnancies reviewed due to an abnormal karyotype or microarray decreased as an indication for referral during the study period (21.6% [n = 16/74] 2008-2012 vs 16.5% [n = 30/182] in 2012-2019). CONCLUSION: A prenatal genetic clinic with a structured multi-disciplinary team approach may be successful in obtaining a unifying prenatal genetic diagnosis.

Case for genome sequencing in infants and children with rare, undiagnosed or genetic diseases.

Up to 350 million people worldwide suffer from a rare disease, and while the individual diseases are rare, in aggregate they represent a substantial challenge to global health systems. The majority of rare disorders are genetic in origin, with children under the age of five disproportionately affected. As these conditions are difficult to identify clinically, genetic and genomic testing have become the backbone of diagnostic testing in this population. In the last 10 years, next-generation sequencing technologies have enabled testing of multiple disease genes simultaneously, ranging from targeted gene panels to exome sequencing (ES) and genome sequencing (GS). GS is quickly becoming a practical first-tier test, as cost decreases and performance improves. A growing number of studies demonstrate that GS can detect an unparalleled range of pathogenic abnormalities in a single laboratory workflow. GS has the potential to deliver unbiased, rapid and accurate molecular diagnoses to patients across diverse clinical indications and complex presentations. In this paper, we discuss clinical indications for testing and historical testing paradigms. Evidence supporting GS as a diagnostic tool is supported by superior genomic coverage, types of pathogenic variants detected, simpler laboratory workflow enabling shorter turnaround times, diagnostic and reanalysis yield, and impact on healthcare.

Clinical Application of Genome and Exome Sequencing as a Diagnostic Tool for Pediatric Patients: a Scoping Review of the Literature.

PURPOSE: Availability of clinical genomic sequencing (CGS) has generated questions about the value of genome and exome sequencing as a diagnostic tool. Analysis of reported CGS application can inform uptake and direct further research. This scoping literature review aims to synthesize evidence on the clinical and economic impact of CGS. METHODS: PubMed, Embase, and Cochrane were searched for peer-reviewed articles published between 2009 and 2017 on diagnostic CGS for infant and pediatric patients. Articles were classified according to sample size and whether economic evaluation was a primary research objective. Data on patient characteristics, clinical setting, and outcomes were extracted and narratively synthesized. RESULTS: Of 171 included articles, 131 were case reports, 40 were aggregate analyses, and 4 had a primary economic evaluation aim. Diagnostic yield was the only consistently reported outcome. Median diagnostic yield in aggregate analyses was 33.2% but varied by broad clinical categories and test type. CONCLUSION: Reported CGS use has rapidly increased and spans diverse clinical settings and patient phenotypes. Economic evaluations support the cost-saving potential of diagnostic CGS. Multidisciplinary implementation research, including more robust outcome measurement and economic evaluation, is needed to demonstrate clinical utility and cost-effectiveness of CGS.

Misattributed parentage as an unanticipated finding during exome/genome sequencing: current clinical laboratory practices and an opportunity for standardization.

PURPOSE: Clinical laboratories performing exome or genome sequencing (ES/GS) are familiar with the challenges associated with proper consenting for and reporting of medically actionable secondary findings based on recommendations from the American College of Medical Genetics and Genomics (ACMG). Misattributed parentage is another type of unanticipated finding a laboratory may encounter during family-based ES/GS; however, there are currently no professional recommendations related to the proper consenting for and reporting of misattributed parentage encountered during ES/GS. METHODS: We surveyed 10 clinical laboratories offering family-based ES/GS regarding their consent language, discovery, and reporting of misattributed parentage. RESULTS: Many laboratories have already developed their own practices/policies for these issues, which do not necessarily agree with those from other labs. CONCLUSION: There are several other possibilities besides true misattributed parentage that could result in similar laboratory findings, and laboratories often feel they lack sufficient information to make formal conclusions on a report regarding the true genetic relatedness of the submitted samples. However, understanding the genetic relatedness (or lack thereof) of the samples submitted for family-based ES/GS has medical relevance. Therefore, professional recommendations for the appropriate handling of suspected misattributed parentage encountered during ES/GS are needed to help standardize current clinical laboratory practices.

Pandora's pregnancy: NIPT, CMA, and genome sequencing-A new era for prenatal genetic testing.

OBJECTIVES: We delineate in this article a shift from the "traditional" technologies of karyotyping in PND to the current phase of advanced genetic technologies including noninvasive prenatal testing (NIPT), chromosomal microarray analysis (CMA), and whole-exome sequencing (WES) with their higher detection rate and related abundance of uncertain data. METHODS: Conceptual analysis based on seminal works that shaped the socioethical discourse surrounding the experiences of parents as well as professionals with prenatal diagnosis in the last 30 years. RESULTS: We consider the implications of this new era of PND for patients and health professionals by drawing on previous studies documenting how probability and uncertainty affect informed consent/choice, health risks communication, customer satisfaction and decision making, and parent-child bonding. CONCLUSIONS: We argue that these changes move us beyond the idioms and realities of the tentative pregnancy and moral pioneering, to uncertainty, probability-based counseling, and moral/translational gambling. We conclude by discussing what is needed to maintain hope in the era of Pandora's pregnancy.

Genome sequencing in the clinic: the past, present, and future of genomic medicine.

Genomic sequencing has undergone massive expansion in the past 10 yr, from a rarely used research tool into an approach that has broad applications in a clinical setting. From rare disease to cancer, genomics is transforming our knowledge of biology. The transition from targeted gene sequencing, to whole exome sequencing, to whole genome sequencing has only been made possible due to rapid advancements in technologies and informatics that have plummeted the cost per base of DNA sequencing and analysis. The tools of genomics have resolved the etiology of disease for previously undiagnosable conditions, identified cancer driver gene variants, and have impacted the understanding of pathophysiology for many diseases. However, this expansion of use has also highlighted research's current voids in knowledge. The lack of precise animal models for gene-to-function association, lack of tools for analysis of genomic structural changes, skew in populations used for genetic studies, publication biases, and the "Unknown Proteome" all contribute to voids needing filled for genomics to work in a fast-paced clinical setting. The future will hold the tools to fill in these voids, with new data sets and the continual development of new technologies allowing for expansion of genomic medicine, ushering in the days to come for precision medicine. In this review we highlight these and other points in hopes of advancing and guiding precision medicine into the future for optimal success.

Importance of complete phenotyping in prenatal whole exome sequencing.

Whole exome sequencing (WES) is an emerging technique in prenatal diagnosis. In this retrospective study, we examined diagnostic utility and limitations of WES in prenatal cases with structural birth defects. DNA from 20 trios (fetal and parental), with normal karyotype and microarray findings, underwent WES and variant interpretation at a reference laboratory. The WES results were later re-evaluated in our academic center utilizing prenatal and postnatal phenotyping. Initial analysis using only prenatal ultrasound findings revealed no pathogenic or likely pathogenic variants in 20 pregnancies with structural birth defects. Re-analysis of WES variants and combination of prenatal and postnatal phenotyping yielded pathogenic variants in at least 20% of cases including PORCN gene in a fetus with split-hand/foot malformation, as well as variants of uncertain significance in NEB and NOTCH1 in fetuses with postnatal muscle weakness and Adams-Oliver syndrome, respectively. Furthermore, Sanger sequencing in a patient with holoprosencephaly, elucidated by postnatal MRI, revealed a pathogenic 47-base pairs deletion in ZIC2 which was missed by prenatal WES. This study suggests that incomplete prenatal phenotyping and lack of prenatal ultrasound-genotype databases are the limiting factors for current interpretation of WES data in prenatal diagnosis. Development of prenatal phenotype-genotype databases would significantly help WES interpretation in this setting. Patients who underwent prenatal clinical WES may benefit from the re-analysis based on detailed postnatal findings.

Meeting the challenges of implementing rapid genomic testing in acute pediatric care.

PURPOSE: The purpose of the study was to implement and prospectively evaluate the outcomes of a rapid genomic diagnosis program at two pediatric tertiary centers. METHODS: Rapid singleton whole-exome sequencing (rWES) was performed in acutely unwell pediatric patients with suspected monogenic disorders. Laboratory and clinical barriers to implementation were addressed through continuous multidisciplinary review of process parameters. Diagnostic and clinical utility and cost-effectiveness of rWES were assessed. RESULTS: Of 40 enrolled patients, 21 (52.5%) received a diagnosis, with median time to report of 16 days (range 9-109 days). A result was provided during the first hospital admission in 28 of 36 inpatients (78%). Clinical management changed in 12 of the 21 diagnosed patients (57%), including the provision of lifesaving treatment, avoidance of invasive biopsies, and palliative care guidance. The cost per diagnosis was AU$13,388 (US$10,453). Additional cost savings from avoidance of planned tests and procedures and reduced length of stay are estimated to be around AU$543,178 (US$424,101). The clear relative advantage of rWES, joint clinical and laboratory leadership, and the creation of a multidisciplinary "rapid team" were key to successful implementation. CONCLUSION: Rapid genomic testing in acute pediatrics is not only feasible but also cost-effective, and has high diagnostic and clinical utility. It requires a whole-of-system approach for successful implementation.

Whole-exome sequencing reanalysis at 12 months boosts diagnosis and is cost-effective when applied early in Mendelian disorders.

PURPOSE: Whole-exome sequencing (WES) has revolutionized Mendelian diagnostics, however, there is no consensus on the timing of data review in undiagnosed individuals and only preliminary data on the cost-effectiveness of this technology. We aimed to assess the utility of WES data reanalysis for diagnosis in Mendelian disorders and to analyze the cost-effectiveness of this technology compared with a traditional diagnostic pathway. METHODS: WES was applied to a cohort of 54 patients from 37 families with a variety of Mendelian disorders to identify the genetic etiology. Reanalysis was performed after 12 months with an improved WES diagnostic pipeline. A comparison was made between costs of a modeled WES pathway and a traditional diagnostic pathway in a cohort with intellectual disability (ID). RESULTS: Reanalysis of WES data at 12 months improved diagnostic success from 30 to 41% due to interim publication of disease genes, expanded phenotype data from referrer, and an improved bioinformatics pipeline. Cost analysis on the ID cohort showed average cost savings of US$586 (AU$782) for each additional diagnosis. CONCLUSION: Early application of WES in Mendelian disorders is cost-effective and reanalysis of an undiagnosed individual at a 12-month time point increases total diagnoses by 11%.

Novel missense mutation in PTPN22 in a Chinese pedigree with Hashimoto's thyroiditis.

BACKGROUND: Hashimoto's thyroiditis is a complex autoimmune thyroid disease, the onset of which is associated with environmental exposures and specific susceptibility genes. Its incidence in females is higher than its incidence in males. Thus far, although some susceptibility loci have been elaborated, including PTPN22, FOXP3, and CD25, the aetiology and pathogenesis of Hashimoto's thyroiditis remains unclear. METHODS: Four affected members from a Chinese family with Hashimoto's thyroiditis were selected for whole-exome sequencing. Missense, nonsense, frameshift, or splicing-site variants shared by all affected members were identified after frequency filtering against public and internal exome databases. Segregation analysis was performed by Sanger sequencing among all members with available DNA. RESULTS: We identified a missense mutation in PTPN22 (NM_015967.5; c. 77A > G; p.Asn26Ser) using whole-exome sequencing. PTPN22 is a known susceptibility gene associated with increased risks of multiple autoimmune diseases. Cosegregation analysis confirmed that all patients in this family, all of whom were female, carried the mutation. All public and private databases showed that the missense mutation was extremely rare. CONCLUSIONS: We found a missense mutation in PTPN22 in a Chinese HT pedigree using whole-exome sequencing. Our study, for the first time, linked a rare variant of PTPN22 to Hashimoto's thyroiditis, providing further evidence of the disease-causing or susceptibility role of PTPN22 in autoimmune thyroid disease. Functional studies regarding the effects of this variant on thyroid autoimmunity and thyroid function are warranted.

Stakeholder views on secondary findings in whole-genome and whole-exome sequencing: a systematic review of quantitative and qualitative studies.

PURPOSE: As whole-exome sequencing (WES) and whole-genome sequencing (WGS) move into routine clinical practice, it is timely to review data that might inform the debate regarding secondary findings (SF) and the development of policies that maximize participant benefit. METHODS: We systematically searched for qualitative and quantitative studies that explored stakeholder views on SF in WES/WGS. Framework analysis was undertaken to identify major themes. RESULTS: Forty-four articles reporting the views of 11,566 stakeholders were included. Stakeholders were broadly supportive of returning "actionable" findings, but definitions of actionability varied. Stakeholder views on SF disclosure exist along a spectrum: potential WES/WGS recipients' views were largely influenced by a sense of rights, whereas views of genomics professionals were informed by a sense of professional responsibility. Experience with genetic illness and testing resulted in greater caution about SF, suggesting that truly informed decisions require an understanding of the implications and limitations of WES/WGS and possible findings. CONCLUSION: This review suggests that bidirectional interaction during consent might best facilitate informed decision making about SF and that dynamic forms of consent, allowing for changing preferences, should be considered. Research exploring views from wider perspectives and from recipients who have received SF is critical if evidence-based policies are to be achieved.Genet Med 19 3, 283-293.

Emerging Monogenic Complex Hyperkinetic Disorders.

PURPOSE OF REVIEW: Hyperkinetic movement disorders can manifest alone or as part of complex phenotypes. In the era of next-generation sequencing (NGS), the list of monogenic complex movement disorders is rapidly growing. This review will explore the main features of these newly identified conditions. RECENT FINDINGS: Mutations in ADCY5 and PDE10A have been identified as important causes of childhood-onset dyskinesias and KMT2B mutations as one of the most frequent causes of complex dystonia in children. The delineation of the phenotypic spectrum associated with mutations in ATP1A3, FOXG1, GNAO1, GRIN1, FRRS1L, and TBC1D24 is revealing an expanding genetic overlap between epileptic encephalopathies, developmental delay/intellectual disability, and hyperkinetic movement disorders,. Thanks to NGS, the etiology of several complex hyperkinetic movement disorders has been elucidated. Importantly, NGS is changing the way clinicians diagnose these complex conditions. Shared molecular pathways, involved in early stages of brain development and normal synaptic transmission, underlie basal ganglia dysfunction, epilepsy, and other neurodevelopmental disorders.

New generation genetic testing entering the clinic.

New generation sequencing (NGS) genetic testing is a powerful diagnostic tool and is increasingly used in the clinical workup of patients, especially in unusual presentations or where a positive family history suggests heritable disease. This review addresses the NGS technologies Targeted sequencing (TS), Whole exome sequencing (WES), Whole genome sequencing (WGS), and the use of gene panels or gene lists for clinical diagnostic purposes. These methods primarily assess nucleotide sequence but can also detect copy number variants and many tandem repeat expansions, greatly simplifying diagnostic algorithms for movement disorders. Studies evaluating the efficacy of NGS in diagnosing movement disorders have reported a diagnostic yield of up to 10.1% for familial and 15.7% for early-onset PD, 11.7-37.5% for dystonia, 12.1-61.8% for ataxia/spastic paraplegia and 11.3-28% for combined movement disorders. Patient selection and stringency in the interpretation of the detected variants and genotypes affect diagnostic yield. Careful comparison of the patient's or family's disease features with the previously reported phenotype associated with the same variant or gene can avoid false-positive diagnoses, although some genes are implicated in various phenotypes. Moving from TS to WES and WGS increases the number of patients correctly diagnosed, but for many patients, a genetic cause cannot be identified today. However, new genetically defined entities are discovered at rapid pace, and genetic databases and our knowledge of genotype-phenotype correlations expand steadily. We discuss the need for clear communication of genetic results and suggest a list of aspects to consider when reporting neurogenetic disorders using NGS testing.

Molecular subtyping in pancreatic neuroendocrine neoplasms: New insights into clinical, pathological unmet needs and challenges.

Pancreatic neuroendocrine neoplasms (PanNENs) contain two primary subtypes with distinct molecular features and associated clinical outcomes: well-differentiated neuroendocrine tumors (NETs) and poorly differentiated neuroendocrine carcinomas (NECs). PanNENs are a group of clinically heterogeneous tumors, whose diagnosis is based on tumor morphologic features and proliferation indices. However, these standards incompletely meet clinical needs by failing to adequately assess the likelihood of tumor recurrence and the potential for therapeutic response. We therefore focused on discussing molecular advances that facilitate the understanding of heterogeneity and exploration of reliable recurrence/treatment predictors. Taking advantage of high-throughput technologies, emerging methods of molecular subtyping in PanNETs include classifications based on co-existing multi-gene mutations, a large-scale loss of heterozygosity or copy number variation, and islet cell type-specific signatures. PanNEC molecular updates were discussed as well. This review aims to help the field classify PanNEN molecular subtypes, gain insights to aid in the solving of clinical, pathological unmet needs, and detect challenges and concerns of genetically-driven trials.

New insights from unbiased panel and whole-exome sequencing in a large Chinese cohort with disorders of sex development.

CONTEXT: Diagnosis of non-chromosomal type disorders of sex development (DSD) has long been challenging. There is still no research on overview of a large Chinese DSD cohort. OBJECTIVE: To determine the etiologic diagnosis through unbiased large-scale panel sequencing and whole-exome sequencing (WES) within a large Chinese DSD cohort. DESIGN: Patients were recruited according to the inclusion criteria of DSD. The applied panel contains 2742 known disease-causing genes, including all known diagnostic genes for DSD. METHODS: Targeted panel sequencing (TPS) was performed, and identified candidate variants were verified. Variant pathogenicities were evaluated according to established guidelines. WES was performed for randomly selected negative samples. RESULTS: This study included 125 patients. Seventy-five variants were identified by TPS and 31 variants were reported for the first time. Pathogenic and likely pathogenic variants accounted for 38.7 and 30.7%, respectively. On the basis of clinical certainty, etiologic diagnostic rates of 46.9 and 10.3% were obtained for 46,XY and 46,XX DSD patients, respectively. We reported novel candidate genes (BMPR1B, GNAS, GHR) and regions of copy number variants outside the expected DSD genotype-phenotype correlation and determined a founder mutation (SRD5A2 p.R227Q) in patients with 5α-reductase deficiency. Further WES in randomly selected negative samples identified only one among 14 negative samples as a variant of uncertain significance, indicating that WES did not improve the diagnostic rate. CONCLUSIONS: This is the first report of the applying unbiased TPS in a large Chinese cohort of patients with 46,XY and 46,XX DSD. Our findings expand the gene, mutation and phenotype spectra of the rare types of DSD in the Chinese population and provide new insight into the current understanding of the etiologies of DSD.