Human complete NFAT1 deficiency causes a triad of joint contractures, osteochondromas, and B-cell malignancy.

The discovery of humans with monogenic disorders has a rich history of generating new insights into biology. Here we report the first human identified with complete deficiency of nuclear factor of activated T cells 1 (NFAT1). NFAT1, encoded by NFATC2, mediates calcium-calcineurin signals that drive cell activation, proliferation, and survival. The patient is homozygous for a damaging germline NFATC2 variant (c.2023_2026delTACC; p.Tyr675Thrfs∗18) and presented with joint contractures, osteochondromas, and recurrent B-cell lymphoma. Absence of NFAT1 protein in chondrocytes caused enrichment in prosurvival and inflammatory genes. Systematic single-cell-omic analyses in PBMCs revealed an environment that promotes lymphomagenesis with accumulation of naïve B cells (enriched for oncogenic signatures MYC and JAK1), exhausted CD4+ T cells, impaired T follicular helper cells, and aberrant CD8+ T cells. This work highlights the pleiotropic role of human NFAT1, will empower the diagnosis of additional patients with NFAT1 deficiency, and further defines the detrimental effects associated with long-term use of calcineurin inhibitors.

Lethal respiratory course and additional features expand the phenotypic spectrum of PIEZO2-related distal arthrogryposis type 5.

Distal arthrogryposes (DA) are a group of conditions presenting with multiple congenital contractures in the distal joints. The 10 types of DA are distinguished by different extra-articular manifestations. Heterozygous gain-of-function variants in PIEZO2 are known to cause a spectrum of DA conditions including DA type 3, DA type 5, and possibly Marden Walker syndrome, which are usually distinguished by the presence of cleft palate (DA3), ptosis and restriction in eye movements (DA5), and specific facial abnormalities and central nervous system involvement, respectively. We report on a boy with a recurrent de novo heterozygous PIEZO2 variant in exon 20 (NM_022068.3: c.2994G > A, p.(Met998Ile); NM_001378183.1: c.3069G > A, p.(Met1023Ile)), who presented at birth with DA and later developed respiratory insufficiency. His phenotype broadly fits the PIEZO2 phenotypic spectrum and potentially extends it with novel phenotypic features of pretibial linear vertical crease, immobile skin, immobile tongue, and lipid myopathy.

NOTCH1 loss of the TAD and PEST domain: An antimorph?

The Notch proteins play key roles in cell fate determination during development. Germline pathogenic variants in NOTCH1 predispose to a spectrum of cardiovascular malformations including Adams-Oliver syndrome and a wide variety of isolated complex and simple congenital heart defects. The intracellular C-terminus of the single-pass transmembrane receptor encoded by NOTCH1 contains a transcriptional activating domain (TAD) required for target gene activation and a PEST domain (a sequence rich in proline, glutamic acid, serine, and threonine), regulating protein stability and turnover. We present a patient with a novel variant encoding a truncated NOTCH1 protein without the TAD and PEST domain (NM_017617.4: c.[6626_6629del];[=], p.(Tyr2209CysfsTer38)) and extensive cardiovascular abnormalities consistent with a NOTCH1-mediated mechanism. This variant fails to promote transcription of target genes as assessed by luciferase reporter assay. Given the roles of the TAD and PEST domains in NOTCH1 function and regulation, we hypothesize that loss of both the TAD and the PEST domain results in a stable, loss-of-function protein that acts as an antimorph through competition with wild-type NOTCH1.

Linkage analysis identifies an isolated strabismus locus at 14q12 overlapping with FOXG1 syndrome region.

Strabismus is a common condition, affecting 1%-4% of individuals. Isolated strabismus has been studied in families with Mendelian inheritance patterns. Despite the identification of multiple loci via linkage analyses, no specific genes have been identified from these studies. The current study is based on a seven-generation family with isolated strabismus inherited in an autosomal dominant manner. A total of 13 individuals from a common ancestor have been included for linkage analysis. Among these, nine are affected and four are unaffected. A single linkage signal has been identified at an 8.5 Mb region of chromosome 14q12 with a multipoint LOD (logarithm of the odds) score of 4.69. Disruption of this locus is known to cause FOXG1 syndrome (or congenital Rett syndrome; OMIM #613454 and *164874), in which 84% of affected individuals present with strabismus. With the incorporation of next-generation sequencing and in-depth bioinformatic analyses, a 4 bp non-coding deletion was prioritised as the top candidate for the observed strabismus phenotype. The deletion is predicted to disrupt regulation of FOXG1, which encodes a transcription factor of the Forkhead family. Suggestive of an autoregulation effect, the disrupted sequence matches the consensus FOXG1 and Forkhead family transcription factor binding site and has been observed in previous ChIP-seq studies to be bound by Foxg1 in early mouse brain development. Future study of this specific deletion may shed light on the regulation of FOXG1 expression and may enhance our understanding of the mechanisms contributing to strabismus and FOXG1 syndrome.

Impact of variation in practice in the prenatal reporting of variants of uncertain significance by commercial laboratories: Need for greater adherence to published guidelines.

OBJECTIVE: To evaluate the impact of implementing commercial whole exome sequencing (WES) and targeted gene panel testing in pregnancies with fetal anomalies. METHODS: A retrospective chart review of 124 patients with sequencing performed by commercial laboratories. RESULTS: The diagnostic yield of WES and panel testing was 21.5% and 26%, respectively, based on likely pathogenic (LP) or pathogenic (P) variants. Forty-two percent of exomes and 32% of panels analysed had one or more variants of uncertain significance (VUS) reported. A multidisciplinary in-depth review of the fetal phenotype, disease phenotype, variant data, and, in some patients, additional prenatal or postnatal investigations increased the diagnostic yield by 5% for exome analysis and 6% for panel analysis. CONCLUSIONS: The diagnostic yield of WES and panel testing combined was 23% based on LP and P variants. Although the reporting of VUS contributed to a 5% increase in diagnostic yield for WES and 6% for panels, the large number of VUS reported by commercial laboratories has significant resource implications. Our results support the need for greater adherence to the recommendations on the prenatal reporting of VUS and the importance of a multidisciplinary approach that brings together clinical and laboratory expertise in prenatal genetics and genomics.

Pathologic Skull Fracture in a Near-Term Neonate with Arthrochalasia Type Ehlers-Danlos Syndrome: A Case Report.

Background: Arthrochalasia type Ehlers-Danlos Syndrome (EDS) is a connective tissue disease characterized by severe generalized joint hypermobility, congenital bilateral hip dislocations, and recurrent joint subluxations and dislocations. Only one study has reported bone fragility resulting in fractures. The genetic abnormality underlying this disorder is a variant in the COL1A1 gene causing entire or partial loss of exon 6, resulting in defective type 1 collagen synthesis. Case Report: We report a female infant born at 35 weeks of gestation presenting with pathologic skull fracture following vaginal delivery. Genetic testing revealed a pathogenic variant in the COL1A1 gene (c.472-1G > C), consistent with arthrochalasia type EDS, reported previously. Conclusion: This report adds pathologic fractures to the phenotypic breadth of this type of EDS and reinforces the importance of including the condition on the differential diagnosis when early onset non-accidental injury or trauma is being considered.

New cases that expand the genotypic and phenotypic spectrum of Congenital NAD Deficiency Disorder.

Nicotinamide adenine dinucleotide (NAD) is an essential coenzyme involved in over 400 cellular reactions. During embryogenesis, mammals synthesize NAD de novo from dietary l -tryptophan via the kynurenine pathway. Biallelic, inactivating variants in three genes encoding enzymes of this biosynthesis pathway (KYNU, HAAO, and NADSYN1) disrupt NAD synthesis and have been identified in patients with multiple malformations of the heart, kidney, vertebrae, and limbs; these patients have Congenital NAD Deficiency Disorder HAAO and four families with biallelic variants in KYNU. These patients present similarly with multiple malformations of the heart, kidney, vertebrae, and limbs, of variable severity. We show that each variant identified in these patients results in loss-of-function, revealed by a significant reduction in NAD levels via yeast genetic complementation assays. For the first time, missense mutations are identified as a cause of malformation and shown to disrupt enzyme function. These missense and frameshift variants cause moderate to severe NAD deficiency in yeast, analogous to insufficient synthesized NAD in patients. We hereby expand the genotypic and corresponding phenotypic spectrum of Congenital NAD Deficiency Disorder.

PIGG variant pathogenicity assessment reveals characteristic features within 19 families.

PURPOSE: Phosphatidylinositol Glycan Anchor Biosynthesis, class G (PIGG) is an ethanolamine phosphate transferase catalyzing the modification of glycosylphosphatidylinositol (GPI). GPI serves as an anchor on the cell membrane for surface proteins called GPI-anchored proteins (GPI-APs). Pathogenic variants in genes involved in the biosynthesis of GPI cause inherited GPI deficiency (IGD), which still needs to be further characterized. METHODS: We describe 22 individuals from 19 unrelated families with biallelic variants in PIGG. We analyzed GPI-AP surface levels on granulocytes and fibroblasts for three and two individuals, respectively. We demonstrated enzymatic activity defects for PIGG variants in vitro in a PIGG/PIGO double knockout system. RESULTS: Phenotypic analysis of reported individuals reveals shared PIGG deficiency-associated features. All tested GPI-APs were unchanged on granulocytes whereas CD73 level in fibroblasts was decreased. In addition to classic IGD symptoms such as hypotonia, intellectual disability/developmental delay (ID/DD), and seizures, individuals with PIGG variants of null or severely decreased activity showed cerebellar atrophy, various neurological manifestations, and mitochondrial dysfunction, a feature increasingly recognized in IGDs. Individuals with mildly decreased activity showed autism spectrum disorder. CONCLUSION: This in vitro system is a useful method to validate the pathogenicity of variants in PIGG and to study PIGG physiological functions.

Further delineation of the clinical spectrum of KAT6B disorders and allelic series of pathogenic variants.

PURPOSE: Genitopatellar syndrome and Say-Barber-Biesecker-Young-Simpson syndrome are caused by variants in the KAT6B gene and are part of a broad clinical spectrum called KAT6B disorders, whose variable expressivity is increasingly being recognized. METHODS: We herein present the phenotypes of 32 previously unreported individuals with a molecularly confirmed diagnosis of a KAT6B disorder, report 24 new pathogenic KAT6B variants, and review phenotypic information available on all published individuals with this condition. We also suggest a classification of clinical subtypes within the KAT6B disorder spectrum. RESULTS: We demonstrate that cerebral anomalies, optic nerve hypoplasia, neurobehavioral difficulties, and distal limb anomalies other than long thumbs and great toes, such as polydactyly, are more frequently observed than initially reported. Intestinal malrotation and its serious consequences can be present in affected individuals. Additionally, we identified four children with Pierre Robin sequence, four individuals who had increased nuchal translucency/cystic hygroma prenatally, and two fetuses with severe renal anomalies leading to renal failure. We also report an individual in which a pathogenic variant was inherited from a mildly affected parent. CONCLUSION: Our work provides a comprehensive review and expansion of the genotypic and phenotypic spectrum of KAT6B disorders that will assist clinicians in the assessment, counseling, and management of affected individuals.

Heterozygous Loss-of-Function Mutations in DLL4 Cause Adams-Oliver Syndrome.

Adams-Oliver syndrome (AOS) is a rare developmental disorder characterized by the presence of aplasia cutis congenita (ACC) of the scalp vertex and terminal limb-reduction defects. Cardiovascular anomalies are also frequently observed. Mutations in five genes have been identified as a cause for AOS prior to this report. Mutations in EOGT and DOCK6 cause autosomal-recessive AOS, whereas mutations in ARHGAP31, RBPJ, and NOTCH1 lead to autosomal-dominant AOS. Because RBPJ, NOTCH1, and EOGT are involved in NOTCH signaling, we hypothesized that mutations in other genes involved in this pathway might also be implicated in AOS pathogenesis. Using a candidate-gene-based approach, we prioritized DLL4, a critical NOTCH ligand, due to its essential role in vascular development in the context of cardiovascular features in AOS-affected individuals. Targeted resequencing of the DLL4 gene with a custom enrichment panel in 89 independent families resulted in the identification of seven mutations. A defect in DLL4 was also detected in two families via whole-exome or genome sequencing. In total, nine heterozygous mutations in DLL4 were identified, including two nonsense and seven missense variants, the latter encompassing four mutations that replace or create cysteine residues, which are most likely critical for maintaining structural integrity of the protein. Affected individuals with DLL4 mutations present with variable clinical expression with no emerging genotype-phenotype correlations. Our findings demonstrate that DLL4 mutations are an additional cause of autosomal-dominant AOS or isolated ACC and provide further evidence for a key role of NOTCH signaling in the etiology of this disorder.

Mutations in NOTCH1 cause Adams-Oliver syndrome.

Notch signaling determines and reinforces cell fate in bilaterally symmetric multicellular eukaryotes. Despite the involvement of Notch in many key developmental systems, human mutations in Notch signaling components have mainly been described in disorders with vascular and bone effects. Here, we report five heterozygous NOTCH1 variants in unrelated individuals with Adams-Oliver syndrome (AOS), a rare disease with major features of aplasia cutis of the scalp and terminal transverse limb defects. Using whole-genome sequencing in a cohort of 11 families lacking mutations in the four genes with known roles in AOS pathology (ARHGAP31, RBPJ, DOCK6, and EOGT), we found a heterozygous de novo 85 kb deletion spanning the NOTCH1 5' region and three coding variants (c.1285T>C [p.Cys429Arg], c.4487G>A [p.Cys1496Tyr], and c.5965G>A [p.Asp1989Asn]), two of which are de novo, in four unrelated probands. In a fifth family, we identified a heterozygous canonical splice-site variant (c.743-1 G>T) in an affected father and daughter. These variants were not present in 5,077 in-house control genomes or in public databases. In keeping with the prominent developmental role described for Notch1 in mouse vasculature, we observed cardiac and multiple vascular defects in four of the five families. We propose that the limb and scalp defects might also be due to a vasculopathy in NOTCH1-related AOS. Our results suggest that mutations in NOTCH1 are the most common cause of AOS and add to a growing list of human diseases that have a vascular and/or bony component and are caused by alterations in the Notch signaling pathway.

Diagnosis of Van den Ende-Gupta syndrome: Approach to the Marden-Walker-like spectrum of disorders.

Marden-Walker syndrome is challenging to diagnose, as there is significant overlap with other multi-system congenital contracture syndromes including Beals congenital contractural arachnodactyly, D4ST1-Deficient Ehlers-Danlos syndrome (adducted thumb-clubfoot syndrome), Schwartz-Jampel syndrome, Freeman-Sheldon syndrome, Cerebro-oculo-facio-skeletal syndrome, and Van den Ende-Gupta syndrome. We discuss this differential diagnosis in the context of a boy from a consanguineous union with Van den Ende-Gupta syndrome, a diagnosis initially confused by the atypical presence of intellectual disability. SNP microarray and whole exome sequencing identified a homozygous frameshift mutation (p.L870V) in SCARF2 and predicted damaging mutations in several genes, most notably DGCR2 (p.P75L) and NCAM2 (p.S147G), both possible candidates for this child's intellectual disability. We review distinguishing features for each Marden-Walker-like syndrome and propose a clinical algorithm for diagnosis among this spectrum of disorders. © 2016 Wiley Periodicals, Inc.

Paternal uniparental disomy 11p15.5 in the pancreatic nodule of an infant with Costello syndrome: Shared mechanism for hyperinsulinemic hypoglycemia in neonates with Costello and Beckwith-Wiedemann syndrome and somatic loss of heterozygosity in Costello syndrome driving clonal expansion.

Costello syndrome (CS) entails a cancer predisposition and is caused by activating HRAS mutations, typically arising de novo in the paternal germline. Hypoglycemia is common in CS neonates. A previously reported individual with the rare HRAS p.Gln22Lys had hyperinsulinemic hypoglycemia. Autopsy showed a discrete pancreatic nodule. The morphologic and immunohistochemistry findings, including loss of p57(Kip2) protein, were identical to a focal lesion of congenital hyperinsulinism, however, no KCNJ11 or ABCC8 mutation was identified and germline derived DNA showed no alternation of the maternal or paternal 11p15 alleles. Here we report paternal uniparental disomy (pUPD) within the lesion, similar to the pUPD11p15.5 in Beckwith-Wiedemann syndrome (BWS). The similar extent of the pUPD suggests a similar mechanism driving hyperinsulinemia in both conditions. After coincidental somatic LOH and pUPD, the growth promoting effects of the paternally derived HRAS mutation, in combination with the increased function of the adjacent paternally expressed IGF2, may together result in clonal expansion. Although this somatic LOH within pancreatic tissue resulted in hyperinsulinism, similar LOH in mesenchymal cells may drive embryonal rhabdomyosarcoma (ERMS). Interestingly, biallelic IGF2 expression has been linked to rhabdomyosarcoma tumorigenesis and pUPD11 occurred in all 8 ERMS samples from CS individuals. Somatic KRAS and HRAS mutations occur with comparable frequency in isolated malignancies. Yet, the malignancy risk in CS is notably higher than in Noonan syndrome with a KRAS mutation. It is conceivable that HRAS co-localization with IGF2 and the combined effect of pUPD 11p15.5 on both genes contributes to the oncogenic potential.

A genome-wide copy number association study of osteoporotic fractures points to the 6p25.1 locus.

BACKGROUND: Osteoporosis is a systemic skeletal disease characterised by reduced bone mineral density and increased susceptibility to fracture; these traits are highly heritable. Both common and rare copy number variants (CNVs) potentially affect the function of genes and may influence disease risk. AIM: To identify CNVs associated with osteoporotic bone fracture risk. METHOD: We performed a genome-wide CNV association study in 5178 individuals from a prospective cohort in the Netherlands, including 809 osteoporotic fracture cases, and performed in silico lookups and de novo genotyping to replicate in several independent studies. RESULTS: A rare (population prevalence 0.14%, 95% CI 0.03% to 0.24%) 210 kb deletion located on chromosome 6p25.1 was associated with the risk of fracture (OR 32.58, 95% CI 3.95 to 1488.89; p = 8.69 × 10(-5)). We performed an in silico meta-analysis in four studies with CNV microarray data and the association with fracture risk was replicated (OR 3.11, 95% CI 1.01 to 8.22; p = 0.02). The prevalence of this deletion showed geographic diversity, being absent in additional samples from Australia, Canada, Poland, Iceland, Denmark, and Sweden, but present in the Netherlands (0.34%), Spain (0.33%), USA (0.23%), England (0.15%), Scotland (0.10%), and Ireland (0.06%), with insufficient evidence for association with fracture risk. CONCLUSIONS: These results suggest that deletions in the 6p25.1 locus may predispose to higher risk of fracture in a subset of populations of European origin; larger and geographically restricted studies will be needed to confirm this regional association. This is a first step towards the evaluation of the role of rare CNVs in osteoporosis.

Prenatal and postnatal findings in serpentine fibula polycystic kidney syndrome and a review of the NOTCH2 spectrum disorders.

Serpentine fibula polycystic kidney syndrome (SFPKS; OMIM600330) is a rare skeletal dysplasia with a characteristic phenotype that includes polycystic kidneys, S-shaped fibulas, and abnormal craniofacial features. SFPKS shares features with Alagille (AGS; OMIM) and Hajdu-Cheney (HCS; OMIM10250) syndromes. All three syndromes result from mutations in the gene that encodes NOTCH2, one of the receptors involved in Notch signaling. Notch signaling is a major developmental signaling pathway, as well as a key regulator of numerous cellular processes. In this report, we present the prenatal ultrasound and postnatal findings in a 23-week fetus with severe manifestations of SPKS and heterozygosity for a de novo mutation in exon 34 of NOTCH2. These findings expand the phenotypic spectrum of NOTCH2 mutations and demonstrate the findings in the prenatal period.

A cryptic familial rearrangement of 11p15.5, involving both imprinting centers, in a family with a history of short stature.

Silver-Russell syndrome (SRS) is a heterogeneous disorder characterized by intrauterine and postnatal growth retardation, dysmorphic facial features and body asymmetry. Both hypomethylation of the telomeric imprinting control region 1 (ICR1) at 11p15.5 and maternal duplication of 11p15.5 have been implicated in the etiology of this disorder. Here we report the origin and segregation of the first reported between-arm intrachromosomal insertion of 11p15.5 that encompasses both ICR1 and ICR2 in a multigenerational family with a history of short stature. One (or any odd number) crossover within the centromeric segment during meiosis would produce recombinant chromosomes; one with a duplication of the inserted segment and the other a deletion. In this 4-generation family, there were six instances of transmission of the recombinant chromosome with duplication of the11p15.5 segment, which leads to a SRS phenotype when maternally inherited and a Beckwith-Wiedemann phenotype when paternally transmitted. The size of the duplicated region is ~1.9 Mb as determined by microarray analysis. This study provides further evidence that maternally inherited duplications of 11p15.5 result in a SRS phenotype that includes short stature and other variable features. The methylation status of the extra copy of the duplicated region of 11p15.5 ultimately predicts the resulting phenotype. Thus, the different phenotype based on parental mode of transmission is of importance in the genetic counseling of these patients.

Prevalence of ocular anomalies is increased in women with polycystic ovary syndrome-exploration of association with PAX6 genotype.

BACKGROUND: Polycystic Ovarian Syndrome (PCOS), the most common reproductive endocrine disorder affecting premenopausal women, is frequently associated with central obesity and pancreatic ß-cell dysfunction. Aniridia, a rare congenital eye disorder with haploinsufficiency of the PAX6 gene, was observed to co-occur with PCOS in a proband. This study investigates eye health and PAX6 genotypes of women with PCOS and controls. MATERIALS AND METHODS: This is a cross-sectional study of 203 premenopausal women (100 healthy controls, 103 with PCOS) conducted at an academic medical center in Vancouver, Canada. Ophthalmological exams and detailed medical histories were obtained from each participant. DNA extracted from saliva was Sanger-sequenced for the exons, intron-exon boundaries, and untranslated regions of PAX6. RESULTS: Women with PCOS had eye abnormalities, including abnormalities of the anterior segment, optic nerve, and retina, that were not observed in controls (p = 0.0002). Myopia prevalence was similar in both groups. Dry eye syndrome, by history, was markedly more prevalent in women with PCOS (22.3%) than controls (5%), p = 0.004. PAX6 genotype did not significantly differ between the two groups, nor was it associated with the greater prevalence of eye anomalies observed in women with PCOS. CONCLUSION: This is the first study to systematically perform an ophthalmological examination in women with PCOS, who were found to have a higher prevalence of potentially serious eye health problems compared with controls. These data suggest that ophthalmological-metabolic-genetic connections in women with PCOS require further investigation. Confirmation of these data and increased attention to eye health in women with PCOS appears warranted.

Comprehensive human amniotic fluid metagenomics supports the sterile womb hypothesis.

As metagenomic approaches for detecting infectious agents have improved, each tissue that was once thought to be sterile has been found to harbor a variety of microorganisms. Controversy still exists over the status of amniotic fluid, which is part of an immunologically privileged zone that is required to prevent maternal immune system rejection of the fetus. Due to this privilege, the exclusion of microbes has been proposed to be mandatory, leading to the sterile womb hypothesis. Since nucleic acid yields from amniotic fluid are very low, contaminating nucleic acid found in water, reagents and the laboratory environment frequently confound attempts to address this hypothesis. Here we present metagenomic criteria for microorganism detection and a metagenomic method able to be performed with small volumes of starting material, while controlling for exogenous contamination, to circumvent these and other pitfalls. We use this method to show that human mid-gestational amniotic fluid has no detectable virome or microbiome, supporting the sterile womb hypothesis.

The practice of genomic medicine: A delineation of the process and its governing principles.

Genomic medicine, an emerging medical discipline, applies the principles of evolution, developmental biology, functional genomics, and structural genomics within clinical care. Enabling widespread adoption and integration of genomic medicine into clinical practice is key to achieving precision medicine. We delineate a biological framework defining diagnostic utility of genomic testing and map the process of genomic medicine to inform integration into clinical practice. This process leverages collaboration and collective cognition of patients, principal care providers, clinical genomic specialists, laboratory geneticists, and payers. We detail considerations for referral, triage, patient intake, phenotyping, testing eligibility, variant analysis and interpretation, counseling, and management within the utilitarian limitations of health care systems. To reduce barriers for clinician engagement in genomic medicine, we provide several decision-making frameworks and tools and describe the implementation of the proposed workflow in a prototyped electronic platform that facilitates genomic care. Finally, we discuss a vision for the future of genomic medicine and comment on areas for continued efforts.

Genome-wide sequencing and the clinical diagnosis of genetic disease: The CAUSES study.

Genome-wide sequencing (GWS) is a standard of care for diagnosis of suspected genetic disorders, but the proportion of patients found to have pathogenic or likely pathogenic variants ranges from less than 30% to more than 60% in reported studies. It has been suggested that the diagnostic rate can be improved by interpreting genomic variants in the context of each affected individual's full clinical picture and by regular follow-up and reinterpretation of GWS laboratory results. Trio exome sequencing was performed in 415 families and trio genome sequencing in 85 families in the CAUSES study. The variants observed were interpreted by a multidisciplinary team including laboratory geneticists, bioinformaticians, clinical geneticists, genetic counselors, pediatric subspecialists, and the referring physician, and independently by a clinical laboratory using standard American College of Medical Genetics and Genomics (ACMG) criteria. Individuals were followed for an average of 5.1 years after testing, with clinical reassessment and reinterpretation of the GWS results as necessary. The multidisciplinary team established a diagnosis of genetic disease in 43.0% of the families at the time of initial GWS interpretation, and longitudinal follow-up and reinterpretation of GWS results produced new diagnoses in 17.2% of families whose initial GWS interpretation was uninformative or uncertain. Reinterpretation also resulted in rescinding a diagnosis in four families (1.9%). Of the families studied, 33.6% had ACMG pathogenic or likely pathogenic variants related to the clinical indication. Close collaboration among clinical geneticists, genetic counselors, laboratory geneticists, bioinformaticians, and individuals' primary physicians, with ongoing follow-up, reanalysis, and reinterpretation over time, can improve the clinical value of GWS.