De novo variants in the non-coding spliceosomal snRNA gene RNU4-2 are a frequent cause of syndromic neurodevelopmental disorders.

Around 60% of individuals with neurodevelopmental disorders (NDD) remain undiagnosed after comprehensive genetic testing, primarily of protein-coding genes1. Increasingly, large genome-sequenced cohorts are improving our ability to discover new diagnoses in the non-coding genome. Here, we identify the non-coding RNA RNU4-2 as a novel syndromic NDD gene. RNU4-2 encodes the U4 small nuclear RNA (snRNA), which is a critical component of the U4/U6.U5 tri-snRNP complex of the major spliceosome2. We identify an 18 bp region of RNU4-2 mapping to two structural elements in the U4/U6 snRNA duplex (the T-loop and Stem III) that is severely depleted of variation in the general population, but in which we identify heterozygous variants in 119 individuals with NDD. The vast majority of individuals (77.3%) have the same highly recurrent single base-pair insertion (n.64_65insT). We estimate that variants in this region explain 0.41% of individuals with NDD. We demonstrate that RNU4-2 is highly expressed in the developing human brain, in contrast to its contiguous counterpart RNU4-1 and other U4 homologs, supporting RNU4-2's role as the primary U4 transcript in the brain. Overall, this work underscores the importance of non-coding genes in rare disorders. It will provide a diagnosis to thousands of individuals with NDD worldwide and pave the way for the development of effective treatments for these individuals.

Genetic counseling for congenital disorders of glycosylation (CDG).

Congenital disorders of glycosylation (CDGs) are a genetically and clinically diverse group of disorders that arise as a result of defects within glycosylation synthetic pathways. CDGs are caused by pathogenic variants in many different genes in the glycosylation network. With over 160 different CDG types currently identified and a vast range of severity and presentations existing within and across those types, the road to a CDG diagnosis is often lengthy and complicated. The perils of this arduous CDG diagnostic odyssey are fraught with various genetic counseling uncertainties: (1) confusion about family planning, (2) queries about inheritance, (3) managing treatment, and (4) dealing with the uncertainty of rare diseases. Thus, the role of the genetic counselor is paramount in helping affected individuals and their families navigate these genetic counseling complexities. Case examples of common genetic counseling difficulties for CDGs are outlined, providing clinical applications of what CDG presentations, diagnostic processes, and common difficulties look like. Information on the nomenclature, incidence, prevalence, diagnostic testing, treatment, and management of CDGs are also discussed to provide a comprehensive summary of CDGs for genetic counselors, and subsequently to affected individuals and their families.

Risks and benefits of anesthesia for combined pediatric procedures in the NIH undiagnosed diseases program.

PURPOSE: The NIH Undiagnosed Diseases Program (UDP) aims to provide diagnoses to patients who have previously received exhaustive evaluations yet remain undiagnosed. Patients undergo procedural anesthesia for deep phenotyping for analysis with genomic testing. METHODS: A retrospective chart review was performed to determine the safety and benefit of procedural anesthesia in pediatric patients in the UDP. Adverse perioperative events were classified as anesthesia-related complications or peri-procedural complications. The contribution of procedures performed under anesthesia to arriving at a diagnosis was also determined. RESULTS: From 2008 to 2020, 249 pediatric patients in the UDP underwent anesthesia for diagnostic procedures. The majority had a severe systemic disease (American Society for Anesthesiology status III, 79%) and/or a neurologic condition (91%). Perioperative events occurred in 45 patients; six of these were attributed to anesthesia. All patients recovered fully without sequelae. Nearly half of the 249 patients (49%) received a diagnosis, and almost all these diagnoses (88%) took advantage of information gleaned from procedures performed under anesthesia. CONCLUSIONS: The benefits of anesthesia involving multiple diagnostic procedures in a well-coordinated, multidisciplinary, research setting, such as in the pediatric UDP, outweigh the risks.

The contribution of mosaicism to genetic diseases and de novo pathogenic variants.

The contribution of mosaicism to diagnosed genetic disease and presumed de novo variants (DNV) is under investigated. We determined the contribution of mosaic genetic disease (MGD) and diagnosed parental mosaicism (PM) in parents of offspring with reported DNV (in the same variant) in the (1) Undiagnosed Diseases Network (UDN) (N = 1946) and (2) in 12,472 individuals electronic health records (EHR) who underwent genetic testing at an academic medical center. In the UDN, we found 4.51% of diagnosed probands had MGD, and 2.86% of parents of those with DNV exhibited PM. In the EHR, we found 6.03% and 2.99% and (of diagnosed probands) had MGD detected on chromosomal microarray and exome/genome sequencing, respectively. We found 2.34% (of those with a presumed pathogenic DNV) had a parent with PM for the variant. We detected mosaicism (regardless of pathogenicity) in 4.49% of genetic tests performed. We found a broad phenotypic spectrum of MGD with previously unknown phenotypic phenomena. MGD is highly heterogeneous and provides a significant contribution to genetic diseases. Further work is required to improve the diagnosis of MGD and investigate how PM contributes to DNV risk.

De novo variants in MRTFB have gain-of-function activity in Drosophila and are associated with a novel neurodevelopmental phenotype with dysmorphic features.

PURPOSE: Myocardin-related transcription factor B (MRTFB) is an important transcriptional regulator, which promotes the activity of an estimated 300 genes but is not known to underlie a Mendelian disorder. METHODS: Probands were identified through the efforts of the Undiagnosed Disease Network. Because the MRTFB protein is highly conserved between vertebrate and invertebrate model organisms, we generated a humanized Drosophila model expressing the human MRTFB protein in the same spatial and temporal pattern as the fly gene. Actin binding assays were used to validate the effect of the variants on MRTFB. RESULTS: Here, we report 2 pediatric probands with de novo variants in MRTFB (p.R104G and p.A91P) and mild dysmorphic features, intellectual disability, global developmental delays, speech apraxia, and impulse control issues. Expression of the variants within wing tissues of a fruit fly model resulted in changes in wing morphology. The MRTFBR104G and MRTFBA91P variants also display a decreased level of actin binding within critical RPEL domains, resulting in increased transcriptional activity and changes in the organization of the actin cytoskeleton. CONCLUSION: The MRTFBR104G and MRTFBA91P variants affect the regulation of the protein and underlie a novel neurodevelopmental disorder. Overall, our data suggest that these variants act as a gain of function.

Bi-allelic SNAPC4 variants dysregulate global alternative splicing and lead to neuroregression and progressive spastic paraparesis.

The vast majority of human genes encode multiple isoforms through alternative splicing, and the temporal and spatial regulation of those isoforms is critical for organismal development and function. The spliceosome, which regulates and executes splicing reactions, is primarily composed of small nuclear ribonucleoproteins (snRNPs) that consist of small nuclear RNAs (snRNAs) and protein subunits. snRNA gene transcription is initiated by the snRNA-activating protein complex (SNAPc). Here, we report ten individuals, from eight families, with bi-allelic, deleterious SNAPC4 variants. SNAPC4 encoded one of the five SNAPc subunits that is critical for DNA binding. Most affected individuals presented with delayed motor development and developmental regression after the first year of life, followed by progressive spasticity that led to gait alterations, paraparesis, and oromotor dysfunction. Most individuals had cerebral, cerebellar, or basal ganglia volume loss by brain MRI. In the available cells from affected individuals, SNAPC4 abundance was decreased compared to unaffected controls, suggesting that the bi-allelic variants affect SNAPC4 accumulation. The depletion of SNAPC4 levels in HeLa cell lines via genomic editing led to decreased snRNA expression and global dysregulation of alternative splicing. Analysis of available fibroblasts from affected individuals showed decreased snRNA expression and global dysregulation of alternative splicing compared to unaffected cells. Altogether, these data suggest that these bi-allelic SNAPC4 variants result in loss of function and underlie the neuroregression and progressive spasticity in these affected individuals.

MYH2-associated myopathy caused by a novel splice-site variant.

MYH2 encodes MyHCIIa, a myosin heavy chain found in fast type 2A fibers. Pathogenic variants in this gene have previously been implicated in dominant and recessive forms of myopathy. Three individuals reported here are part of a family in which four generations of individuals are affected by a slowly progressive, predominantly proximal myopathy in an autosomal dominant inheritance pattern. Affected individuals in this family lacked classic features of an MYH2-associated myopathy such as congenital contractures and ophthalmoplegia. A novel variant, MYH2 c.5673+1G>C, was detected in the proband and subsequently found to segregate with disease in five additional family members. Further studies demonstrated that this variant affects splicing, resulting in novel transcripts. These data and muscle biopsy findings in the proband, indicate that this family's MYH2 variant is causative of their myopathy, adding to our understanding of the clinical and molecular characteristics of the disease.

Bi-allelic ATG4D variants are associated with a neurodevelopmental disorder characterized by speech and motor impairment.

Autophagy regulates the degradation of damaged organelles and protein aggregates, and is critical for neuronal development, homeostasis, and maintenance, yet few neurodevelopmental disorders have been associated with pathogenic variants in genes encoding autophagy-related proteins. We report three individuals from two unrelated families with a neurodevelopmental disorder characterized by speech and motor impairment, and similar facial characteristics. Rare, conserved, bi-allelic variants were identified in ATG4D, encoding one of four ATG4 cysteine proteases important for autophagosome biogenesis, a hallmark of autophagy. Autophagosome biogenesis and induction of autophagy were intact in cells from affected individuals. However, studies evaluating the predominant substrate of ATG4D, GABARAPL1, demonstrated that three of the four ATG4D patient variants functionally impair ATG4D activity. GABARAPL1 is cleaved or "primed" by ATG4D and an in vitro GABARAPL1 priming assay revealed decreased priming activity for three of the four ATG4D variants. Furthermore, a rescue experiment performed in an ATG4 tetra knockout cell line, in which all four ATG4 isoforms were knocked out by gene editing, showed decreased GABARAPL1 priming activity for the two ATG4D missense variants located in the cysteine protease domain required for priming, suggesting that these variants impair the function of ATG4D. The clinical, bioinformatic, and functional data suggest that bi-allelic loss-of-function variants in ATG4D contribute to the pathogenesis of this syndromic neurodevelopmental disorder.

Continuing a search for a diagnosis: the impact of adolescence and family dynamics.

The "diagnostic odyssey" describes the process those with undiagnosed conditions undergo to identify a diagnosis. Throughout this process, families of children with undiagnosed conditions have multiple opportunities to decide whether to continue or stop their search for a diagnosis and accept the lack of a diagnostic label. Previous studies identified factors motivating a family to begin searching, but there is limited information about the decision-making process in a prolonged search and how the affected child impacts a family's decision. This study aimed to understand how families of children with undiagnosed diseases decide whether to continue to pursue a diagnosis after standard clinical testing has failed. Parents who applied to the Undiagnosed Disease Network (UDN) at the National Institutes of Health (NIH) were recruited to participate in semi-structured interviews. The 2015 Supportive Care Needs model by Pelenstov, which defines critical needs in families with rare/undiagnosed diseases, provided a framework for interview guide development and transcript analysis (Pelentsov et al in Disabil Health J 8(4):475-491, 2015. https://doi.org/10.1016/J.DHJO.2015.03.009 ). A deductive, iterative coding approach was used to identify common unifying themes. Fourteen parents from 13 families were interviewed. The average child's age was 11 years (range 3-18) and an average 63% of their life had been spent searching for a diagnosis. Our analysis found that alignment or misalignment of parent and child needs impact the trajectory of the diagnostic search. When needs and desires align, reevaluation of a decision to pursue a diagnosis is limited. However, when there is conflict between parent and child desires, there is reevaluation, and often a pause, in the search. This tension is exacerbated when children are adolescents and attempting to balance their dependence on parents for medical care with a natural desire for independence. Our results provide novel insights into the roles of adolescents in the diagnostic odyssey. The tension between desired and realistic developmental outcomes for parents and adolescents impacts if, and how, the search for a diagnosis progresses.

Genomic analysis, immunomodulation and deep phenotyping of patients with nodding syndrome.

The aetiology of nodding syndrome remains unclear, and comprehensive genotyping and phenotyping data from patients remain sparse. Our objectives were to characterize the phenotype of patients with nodding syndrome, investigate potential contributors to disease aetiology, and evaluate response to immunotherapy. This cohort study investigated members of a single-family unit from Lamwo District, Uganda. The participants for this study were selected by the Ugandan Ministry of Health as representative for nodding syndrome and with a conducive family structure for genomic analyses. Of the eight family members who participated in the study at the National Institutes of Health (NIH) Clinical Center, three had nodding syndrome. The three affected patients were extensively evaluated with metagenomic sequencing for infectious pathogens, exome sequencing, spinal fluid immune analyses, neurometabolic and toxicology testing, continuous electroencephalography and neuroimaging. Five unaffected family members underwent a subset of testing for comparison. A distinctive interictal pattern of sleep-activated bursts of generalized and multifocal epileptiform discharges and slowing was observed in two patients. Brain imaging showed two patients had mild generalized cerebral atrophy, and both patients and unaffected family members had excessive metal deposition in the basal ganglia. Trace metal biochemical evaluation was normal. CSF was non-inflammatory and one patient had CSF-restricted oligoclonal bands. Onchocerca volvulus-specific antibodies were present in all patients and skin snips were negative for active onchocerciasis. Metagenomic sequencing of serum and CSF revealed hepatitis B virus in the serum of one patient. Vitamin B6 metabolites were borderline low in all family members and CSF pyridoxine metabolites were normal. Mitochondrial DNA testing was normal. Exome sequencing did not identify potentially causal candidate gene variants. Nodding syndrome is characterized by a distinctive pattern of sleep-activated epileptiform activity. The associated growth stunting may be due to hypothalamic dysfunction. Extensive testing years after disease onset did not clarify a causal aetiology. A trial of immunomodulation (plasmapheresis in two patients and intravenous immunoglobulin in one patient) was given without short-term effect, but longer-term follow-up was not possible to fully assess any benefit of this intervention.

Complex effects on CaV2.1 channel gating caused by a CACNA1A variant associated with a severe neurodevelopmental disorder.

P/Q-type Ca2+ currents mediated by CaV2.1 channels are essential for active neurotransmitter release at neuromuscular junctions and many central synapses. Mutations in CACNA1A, the gene encoding the principal CaV2.1 α1A subunit, cause a broad spectrum of neurological disorders. Typically, gain-of-function (GOF) mutations are associated with migraine and epilepsy while loss-of-function (LOF) mutations are causative for episodic and congenital ataxias. However, a cluster of severe CaV2.1 channelopathies have overlapping presentations which suggests that channel dysfunction in these disorders cannot always be defined bimodally as GOF or LOF. In particular, the R1667P mutation causes focal seizures, generalized hypotonia, dysarthria, congenital ataxia and, in one case, cerebral edema leading ultimately to death. Here, we demonstrate that the R1667P mutation causes both channel GOF (hyperpolarizing voltage-dependence of activation, slowed deactivation) and LOF (slowed activation kinetics) when expressed heterologously in tsA-201 cells. We also observed a substantial reduction in Ca2+ current density in this heterologous system. These changes in channel gating and availability/expression manifested in diminished Ca2+ flux during action potential-like stimuli. However, the integrated Ca2+ fluxes were no different when normalized to tail current amplitude measured upon repolarization from the reversal potential. In summary, our findings indicate a complex functional effect of R1667P and support the idea that pathological missense mutations in CaV2.1 may not represent exclusively GOF or LOF.

Diagnosis and discovery: Insights from the NIH Undiagnosed Diseases Program.

Living with an undiagnosed medical condition places a tremendous burden on patients, their families, and their healthcare providers. The Undiagnosed Diseases Program (UDP) was established at the National Institutes of Health (NIH) in 2008 with the primary goals of providing a diagnosis for patients with mysterious conditions and advancing medical knowledge about rare and common diseases. The program reviews applications from referring clinicians for cases that are considered undiagnosed despite a thorough evaluation. Those that are accepted receive clinical evaluations involving deep phenotyping and genetic testing that includes exome and genomic sequencing. Selected candidate gene variants are evaluated by collaborators using functional assays. Since its inception, the UDP has received more than 4500 applications and has completed evaluations on nearly 1300 individuals. Here we present six cases that exemplify the discovery of novel disease mechanisms, the importance of deep phenotyping for rare diseases, and how genetic diagnoses have led to appropriate treatment. The creation of the Undiagnosed Diseases Network (UDN) in 2014 has substantially increased the number of patients evaluated and allowed for greater opportunities for data sharing. Expansion to the Undiagnosed Diseases Network International (UDNI) has the possibility to extend this reach even farther. Together, networks of undiagnosed diseases programs are powerful tools to advance our knowledge of pathophysiology, accelerate accurate diagnoses, and improve patient care for patients with rare conditions.

Progressive cerebellar atrophy in a patient with complex II and III deficiency and a novel deleterious variant in SDHA: A Counseling Conundrum.

BACKGROUND: Complex II is an essential component of the electron transport chain, linking it with the tricarboxylic acid cycle. Its four subunits are encoded in the nuclear genome, and deleterious variants in these genes, including SDHA (OMIM 600857), are associated with a wide range of symptoms including neurological disease, cardiomyopathy, and neoplasia (paraganglioma-pheochromocytomas (PGL/PCC), and gastrointestinal stromal tumors). Deleterious variants of SDHA are most frequently associated with Leigh and Leigh-like syndromes. METHODS AND RESULTS: Here, we describe a case of a 9-year-old boy with tremor, nystagmus, hypotonia, developmental delay, significant ataxia, and progressive cerebellar atrophy. He was found to have biallelic variants in SDHA, a known pathogenic variant (c.91C>T (p.R31*)), and a variant of unknown significance (c.454G>A (p.E152K)). Deficient activity of complexes II and III was detected in fibroblasts from the patient consistent with a diagnosis of a respiratory chain disorder. CONCLUSION: We, therefore, consider whether c.454G>A (p.E152K) is, indeed, a pathogenic variant, and what implications it has for family members who carry the same variant.

Prospective phenotyping of long-term survivors of generalized arterial calcification of infancy (GACI).

PURPOSE: Generalized arterial calcification of infancy (GACI), characterized by vascular calcifications that are often fatal shortly after birth, is usually caused by deficiency of ENPP1. A small fraction of GACI cases result from deficiency of ABCC6, a membrane transporter. The natural history of GACI survivors has not been established in a prospective fashion. METHODS: We performed deep phenotyping of 20 GACI survivors. RESULTS: Sixteen of 20 subjects presented with arterial calcifications, but only 5 had residual involvement at the time of evaluation. Individuals with ENPP1 deficiency either had hypophosphatemic rickets or were predicted to develop it by 14 years of age; 14/16 had elevated intact FGF23 levels (iFGF23). Blood phosphate levels correlated inversely with iFGF23. For ENPP1-deficient individuals, the lifetime risk of cervical spine fusion was 25%, that of hearing loss was 75%, and the main morbidity in adults was related to enthesis calcification. Four ENPP1-deficient individuals manifested classic skin or retinal findings of PXE. We estimated the minimal incidence of ENPP1 deficiency at ~1 in 200,000 pregnancies. CONCLUSION: GACI appears to be more common than previously thought, with an expanding spectrum of overlapping phenotypes. The relationships among decreased ENPP1, increased iFGF23, and rickets could inform future therapies.

An autosomal dominant neurological disorder caused by de novo variants in FAR1 resulting in uncontrolled synthesis of ether lipids.

PURPOSE: In this study we investigate the disease etiology in 12 patients with de novo variants in FAR1 all resulting in an amino acid change at position 480 (p.Arg480Cys/His/Leu). METHODS: Following next-generation sequencing and clinical phenotyping, functional characterization was performed in patients' fibroblasts using FAR1 enzyme analysis, FAR1 immunoblotting/immunofluorescence, and lipidomics. RESULTS: All patients had spastic paraparesis and bilateral congenital/juvenile cataracts, in most combined with speech and gross motor developmental delay and truncal hypotonia. FAR1 deficiency caused by biallelic variants results in defective ether lipid synthesis and plasmalogen deficiency. In contrast, patients' fibroblasts with the de novo FAR1 variants showed elevated plasmalogen levels. Further functional studies in fibroblasts showed that these variants cause a disruption of the plasmalogen-dependent feedback regulation of FAR1 protein levels leading to uncontrolled ether lipid production. CONCLUSION: Heterozygous de novo variants affecting the Arg480 residue of FAR1 lead to an autosomal dominant disorder with a different disease mechanism than that of recessive FAR1 deficiency and a diametrically opposed biochemical phenotype. Our findings show that for patients with spastic paraparesis and bilateral cataracts, FAR1 should be considered as a candidate gene and added to gene panels for hereditary spastic paraplegia, cerebral palsy, and juvenile cataracts.

DYRK1A pathogenic variants in two patients with syndromic intellectual disability and a review of the literature.

BACKGROUND: DYRK1A-Related Intellectual Disability Syndrome is a rare autosomal dominant condition characterized by intellectual disability, speech and language delays, microcephaly, facial dysmorphism, and feeding difficulties. Affected individuals represent simplex cases that result from de novo heterozygous pathogenic variants in DYRK1A (OMIM 614104), or chromosomal structural rearrangements involving the DYRK1A locus. Due to the rarity of DYRK1A-Related Intellectual Disability Syndrome, the spectrum of symptoms associated with this disease has not been completely defined. METHODS AND RESULTS: We present two unrelated cases of DYRK1A-Related Intellectual Disability Syndrome resulting from variants in DYRK1A. Both probands presented to the National Institutes of Health (NIH) with multiple dysmorphic facial features, primary microcephaly, absent or minimal speech, feeding difficulties, and cognitive impairment; features that have been previously reported in individuals with DYRK1A. During NIH evaluation, additional features of enlarged cerebral subarachnoid spaces, retinal vascular tortuosity, and bilateral anomalous large optic discs with increased cup-to-disc ratio were identified in the first proband and multiple ophthalmologic abnormalities and sensorineural hearing loss were identified in the second proband. CONCLUSION: We recommend that the workup of future of patients include a comprehensive eye exam. Early establishment of physical, occupational, and speech therapy may help in the management of ataxia, hypertonia, and speech impairments common in these patients.

The undiagnosed diseases program: Approach to diagnosis.

Undiagnosed and rare conditions are collectively common and affect millions of people worldwide. The NIH Undiagnosed Diseases Program (UDP) strives to achieve both a comprehensive diagnosis and a better understanding of the mechanisms of disease for many of these individuals. Through the careful review of records, a well-orchestrated inpatient evaluation, genomic sequencing and testing, and with the use of emerging strategies such as matchmaking programs, the UDP succeeds nearly 30 percent of the time for these highly selective cases. Although the UDP process is built on a unique set of resources, case examples demonstrate steps genetic professionals can take, in both clinical and research settings, to arrive at a diagnosis for their most challenging cases.

Kilquist syndrome: A novel syndromic hearing loss disorder caused by homozygous deletion of SLC12A2.

Syndromic sensorineural hearing loss is multigenic and associated with malformations of the ear and other organ systems. Herein we describe a child admitted to the NIH Undiagnosed Diseases Program with global developmental delay, sensorineural hearing loss, gastrointestinal abnormalities, and absent salivation. Next-generation sequencing revealed a uniparental isodisomy in chromosome 5, and a 22 kb homozygous deletion in SLC12A2, which encodes for sodium, potassium, and chloride transporter in the basolateral membrane of secretory epithelia. Functional studies using patient-derived fibroblasts showed truncated SLC12A2 transcripts and markedly reduced protein abundance when compared with control. Loss of Slc12a2 in mice has been shown to lead to deafness, abnormal neuronal growth and migration, severe gastrointestinal abnormalities, and absent salivation. Together with the described phenotype of the Slc12a2-knockout mouse model, our results suggest that the absence of functional SLC12A2 causes a new genetic syndrome and is crucial for the development of auditory, neurologic, and gastrointestinal tissues.

Cases from the Undiagnosed Diseases Network: The continued value of counseling skills in a new genomic era.

The "diagnostic odyssey" is well known and described in genetic counseling literature. Studies addressing the psychological, emotional, and financial costs of not having a diagnosis have shown how it permeates the lives of patients and families. The Undiagnosed Diseases Network aims to end this odyssey by providing diagnoses to individuals with undiagnosed conditions through multidisciplinary evaluations, whole exome and genome sequencing, and basic science research. It also provides an opportunity to learn from patients and families and to better understand their journeys and the impact of receiving a diagnosis. Seven cases are presented that outline challenges that come from working with chronically undiagnosed and newly diagnosed patients in a time when sequencing for clinical diagnosis is rapidly increasing. They illuminate the emotional journey of patients and families searching for a diagnosis and the mental health problems, financial distress, and chaos that can accompany not having answers. They also illustrate the surprising reactions patients and families can have to receiving a diagnosis, including anger, grief, and disappointment. While the lessons learned from these families are not novel, new strategies are presented for handling these challenges in undiagnosed and ultra-rare populations, groups that will increase with the rise of clinical sequencing.