Dandy-Walker malformation (DWM) is a rare congenital abnormality of the posterior fossa and the cerebellum and has an incidence of 1 in 10 000 to 30 000 births. Although DWM can present in isolation, it is often associated with other central nervous system (CNS) abnormalities or extra-CNS anomalies (DWM+). A molecular cause is not identified in the majority of individuals with DWM+. This is due, in part, to uncertainty regarding optimal testing strategies and an incomplete understanding of the genetic causes of DWM+. In this study, we analyzed clinical exome sequencing (cES) data from 91 individuals with DWM+ to determine its diagnostic efficacy. A definitive or probable diagnosis was made for 32 individuals, yielding a diagnostic rate of 35.2% (32/91). Commercially available brain malformation panels would have detected only 24.2% (8/33) to 54.5% (18/33) of the diagnoses made by cES. We then used data from our cohort, published cases, and mouse models to identify nine phenotypic expansions involving DWM. Our results suggest that cES should be considered for all individuals with DWM+ for whom a molecular diagnosis has not been established and that additional testing to identify a genetic cause of DWM is likely not warranted in individuals with genetic syndromes caused by variants in ANKRD11, C2CD3, COL4A1, KMT2D, KRAS, OPHN1, SHOC2, SMARCB1, and WDR73.

PURPOSE:To define the phenotypic spectrum and genotype-phenotype correlations associated with pathogenic RERE variants and inform clinical management and genetic counseling for neurodevelopmental disorder with or without anomalies of the brain, eye, or heart (NEDBEH). METHODS:We assembled a cohort of 54 individuals with heterozygous pathogenic, likely pathogenic, and variants of uncertain significance in RERE, including 30 previously unreported cases. Individuals were classified into 5 subcohorts based on variant type and location: loss-of-function, missense variants inside and outside a specific histidine-rich region (HRR), and HRR in-frame deletions and duplications. Phenotypic features were analyzed and compared across groups. Protein modeling was performed to assess potential structural effects. RESULTS:Developmental delay, intellectual disability, and/or autism spectrum disorder were prevalent across all groups. Loss-of-function variants are associated with fewer multisystem anomalies than missense variants and are more likely to be inherited from a mildly symptomatic or asymptomatic parent. In contrast, HRR-associated missense variants and in-frame HRR duplications were associated with more multisystem phenotypes and usually arise de novo. HRR missense variants were structurally stabilizing, suggesting a gain-of-function or dominant-negative mechanism. CONCLUSION:These findings expand the clinical spectrum of RERE-related disorders, refine genotype-phenotype correlations, and support variant-specific approaches to management and genetic counseling.

WD and tetratricopeptide repeats protein 1 (WDTC1) encodes a component of the cullin-RING E3 ligase complexes that mediate polyubiquitination of specific target proteins for degradation and has been shown to regulate lipid storage in studies performed in Drosophila, mice, and humans. WDTC1 is expressed in a wide variety of tissues and organs including the brain and is predicted to be loss-of-function intolerant. To determine the phenotypes associated with WDTC1 haploinsufficiency, we identified seven individuals, six of whom have not been previously reported, who are heterozygous for loss-of-function or putatively damaging missense variants in WDTC1. None of these individuals were reported to be obese, but all had neurodevelopmental phenotypes that included developmental delay and intellectual disability. Seizures were also recurrently reported. One loss-of-function variant was inherited from an affected mother, and one missense variant was inherited from an unaffected father. Our findings suggest that WDTC1 haploinsufficiency causes a neurodevelopmental syndrome characterized by variable developmental delay, intellectual disability, and seizures. The identification of additional patients with loss-of-function variants will be needed to identify recurrent patterns of less common phenotypes, determine with greater certainty if obesity is or is not a feature associated with this disorder, and confirm that this disorder is incompletely penetrant.

PURPOSE:RAPGEF2 encodes a guanine nucleotide exchange factor (GEF) that activates small GTPases and has not been linked to a Mendelian disorder. RAPGEF2 is highly intolerant to loss-of-function variants. We report 5 de novo heterozygous variants in RAPGEF2 in unrelated individuals with developmental delay, attention deficit hyperactivity disorder, epilepsy, dysmorphic features, or other manifestations. We used a Drosophila model to assess the functional impact of the identified human variants. METHODS:We generated a Kozak-GAL4 null allele of the Drosophila ortholog of RAPGEF2, PDZ-GEF, and used the allele to determine the gene expression pattern as well as the loss-of-function phenotypes. We expressed the reference and variant RAPGEF2 in PDZ-GEF mutant background to conduct "humanization" studies. RESULTS:Our experiments show that PDZ-GEF is expressed in the central nervous system. Loss of PDZ-GEF leads to severe locomotion defects, aberrant microtubular stability in motor neuron axons, and synaptic overgrowth at neuromuscular junctions in third instar larvae. Mutant animals are lethal at various developmental stages. Importantly, the neurodevelopmental phenotypes can be rescued by expression of the human RAPGEF2 reference cDNA but not by any of the variants. CONCLUSION:Our findings provide functional evidence that the tested RAPGEF2 variants are loss-of-function alleles and that the RAPGEF2 variants are associated with a neurodevelopmental disorder.

Background: Germline heterozygous variants in the ETS transcription factor, ERG , cause “ERG deficiency syndrome”, characterised by bone marrow failure (BMF), haematological malignancy and primary lymphoedema predisposition. Given the recent discovery of ERG deficiency syndrome and the limited number of reported cases, the full phenotypic spectrum of the disorder remains to be defined. Methods: Through international collaborations, we ascertained germline ERG variants in individuals with aortic/mitral valve abnormalities and/or aortic aneurysm. We conducted explorative genotype/phenotype analysis of population and disease databases, including in-depth analysis of the UK Biobank, to identify additional variants. To assess both variant-level evidence demonstrating a damaging effect on gene function (DNA binding, transactivation) and experimental evidence supporting the role of the gene in related phenotypic features, we performed functional assays and characterised ERG expression during a critical timepoint of murine aortic valve development. Results: We report 10 heterozygous ERG variants (7 likely pathogenic/pathogenic) in 11 patients (including one family) with aortic/mitral valve abnormalities and aortic aneurysm (6 co-segregate with a BMF or lymphoedema), before 50 years of age, broadening the phenotypic landscape of ERG deficiency syndrome. Providing a possible molecular explanation for aortic valve defects, ERG is present at high levels in aortic valves at a critical point of murine aortic valve development. Explorative genotype/phenotype analysis of population and disease databases identified 3 ERG variants, demonstrating that rare pathogenic variants are hidden within such cohorts. Seven variants are predicted to cause premature protein termination and all three missense variants disrupted transactivation and/or DNA-binding in vitro . Further in-depth analysis of the UK Biobank identified two predicted pathogenic ERG missense variants in ostensibly asymptomatic individuals obscured by somatic genetic rescue, a competitive, ERG-deficiency-driven stem cell phenomenon. Conclusion: ERG deficiency syndrome encompasses a broader clinical phenotype than previously recognised, including cardiovascular manifestations; aortic and mitral valve abnormalities and aortic aneurysms. Identification of patients with hidden germline ERG variants and the expansion of clinical features of ERG deficiency will improve genetic diagnosis and direct clinical management to individuals and families.

Haploinsufficiency of SKI, PRDM16, RERE, PAX7, and GRHL3 have been implicated in the development of orofacial clefting (OFC) associated with chromosome 1p36 deletions based on human and/or mouse data. Haploinsufficiency of SPEN, a 1p36 gene that encodes a transcriptional repressor, causes Radio-Tartaglia syndrome, a neurodevelopmental syndrome in which high/narrow palates are common, and OFC is occasionally observed. We show that Spen-null embryos have abnormal palatal shelf elevation and extension leading to the development of cleft palate. Mesenchymal cell proliferation in the medial halves of the palatal shelves of Spen-null embryos at E13.5 is significantly reduced. This contributes to the delay of palatal shelf elevation. Tissue specific ablation of Spen in the cranial neural crest cells results in delayed palatal development. This pattern of abnormal palatal development mimics the pattern described in RERE-deficient mice. We show that Rere and Spen are expressed in same cell types, that Rere and Spen interact genetically in the development of the palate, that Spen expression is reduced in the palates of RERE-deficient embryos at E14.5, and that the rate of OFC in individuals with 1p36 deletions involving both RERE and SPEN is higher than those of individuals with RERE or SPEN haploinsufficiency. Our results suggest that SPEN is required for normal mammalian palatal development, that RERE and SPEN interact in a common pathway during palatal development, and that haploinsufficiency of RERE and SPEN are likely to contribute to the development of OFC in individuals with 1p36 deletions.

SEMA6A is a transmembrane protein that plays a role in axon guidance and cell migration. Sema6a null mice have cerebral anatomical defects and altered social interactions and working memory. However, the phenotypes associated with loss of SEMA6A function have not been clearly defined in humans. Here we describe 11 individuals who are heterozygous for putatively damaging variants affecting SEMA6A. All of these individuals (100%) had neurodevelopmental phenotypes that included developmental delay, intellectual disability, and/or autism spectrum disorder. Abnormal behaviors were seen in 73% with oppositional defiant disorder being diagnosed in 27% and acting out, overeating, and tantrums each being described in 18% of individuals. Disorders of attention were documented in 45%. Among the six individuals who had a brain MRI, 50% had at least one abnormal finding. Of the eight SEMA6A variants with known inheritance, five were inherited. Taken together, our data suggest that loss of SEMA6A function may be associated with an increased risk of neurodevelopmental phenotypes, abnormal behaviors, disorders of attention, and brain anomalies. Additional studies will be needed to determine if SEMA6A haploinsufficiency is best characterized as an autosomal dominant disorder with incomplete penetrance or as a risk factor for these phenotypes.

PURPOSE:The current diagnostic rate for patients with suspected Mendelian genetic disorders is low, despite exome/genome sequencing being the standard of care. One reason for this low diagnostic rate is that traditional exome/genome sequencing analysis methods struggle to detect RNA splicing aberrations. Causative variants often involve splicing changes, with numerous splice-altering variants being responsible for known Mendelian disorders. Therefore, it is crucial to develop reliable tools to detect, quantify, prioritize, and visualize RNA splicing aberrations from patient RNA sequencing data. METHODS:We developed Modeling Alternative Junction Inclusion Quantification for Clinical Applications (MAJIQ-CLIN), a method to identify RNA splicing aberrations in patients' RNA sequencing data compared with a cohort of control samples. MAJIQ-CLIN can efficiently process large datasets, avoiding reprocessing when new data are added, while effectively detecting local splicing variations with deviations in a given patient, termed outlier local splicing variation, or unique to the patient, termed private local splicing variation. RESULTS:We performed a systematic evaluation of the accuracy of tools for detecting patients' RNA splicing aberrations from RNA sequence using synthetic data across several aberration types and transcript inclusion levels. Then, we used several real datasets to assess MAJIQ-CLINs ability to identify solved test cases and control for the effect of confounders such as batches. We showed that MAJIQ-CLIN compares favorably to existing tools in both accuracy and efficiency. We also used MAJIQ-CLIN to investigate several unsolved patient cases from the Undiagnosed Diseases Network. CONCLUSION:MAJIQ-CLIN offers an efficient, accurate, and user-friendly tool to aid in diagnosing Mendelian disease-causing variants from RNA sequence data.

Children in Health Professional Shortage Areas (HPSAs), including the US border regions, experience delays in the diagnosis of hereditary hearing loss (HL), driven primarily by limited access to specialized genetic services. Although approximately 60% of congenital HL has a genetic etiology, many affected children in these communities remain undiagnosed, delaying timely intervention. Project GIVE is an NIH-funded virtual genomics program that expands access to genome sequencing (GS) for children with undiagnosed multisystemic conditions along the Texas-Mexico border in the Rio Grande Valley (RGV) and El Paso regions of Texas, USA. Children (0-18 years) with suspected rare diseases were referred by regional healthcare professionals through a virtual portal, Consultagene, and underwent comprehensive virtual clinical genetics evaluation and trio GS. Among 23 Hispanic/Latino children evaluated for HL, 16 (~70%) received a molecular diagnosis. Of these, 56% had changes to medical management. Our findings demonstrate that high diagnostic yield for pediatric HL can be achieved in under-resourced populations when genomic testing barriers are addressed. As targeted therapies for hereditary HL emerge, broader implementation of comprehensive genetic testing in HPSAs is crucial to ensure timely medical interventions.

WNT4 is a secreted protein that plays a critical role in the regulation of cell fate and embryogenesis. Biallelic variants in WNT4 have been linked to SERKAL syndrome, an autosomal recessive disorder characterized by 46,XX sex reversal and dysgenesis of the kidneys, adrenals, and lungs. SERKAL syndrome has only been described in a single consanguineous kindred with four affected fetuses. Additional features seen in a subset of affected fetuses included ventricular septal defect (VSD), congenital diaphragmatic hernia (CDH), and orofacial clefting (OFC). To determine if these additional features were likely to be caused by WNT4 deficiency, we used machine learning to compare WNT4 to genes known to cause VSD, CDH, and OFC. When compared to all RefSeq genes, WNT4's rank annotation scores for these congenital anomalies were 94%, 99%, and 98.5%, respectively, indicating a high level of similarity. We subsequently identified a second consanguineous family with SERKAL syndrome in which an affected fetus had CDH and an affected child had OFC. We then demonstrated that a subset of Wnt4 null embryos have perimembranous VSDs, anterior and posterior sac CDH, and soft palate clefts. These findings suggest that WNT4 deficiency can cause VSD, CDH, and palatal anomalies in mice and humans with SERKAL syndrome. These studies also suggest that our machine learning approach can be used as a candidate gene prioritization tool, and that targeted mouse phenotyping can serve as a means of confirming the roles of candidate genes in mammalian development.

LONP1 encodes a mitochondrial protease essential for protein quality control and metabolism. Variants in LONP1 are associated with a diverse and expanding spectrum of disorders, including Cerebral, Ocular, Dental, Auricular, and Skeletal anomalies syndrome (CODAS), congenital diaphragmatic hernia (CDH), and neurodevelopmental disorders (NDD), with some individuals exhibiting features of mitochondrial encephalopathy. We report 16 novel LONP1 variants identified in 16 individuals (11 with NDD, 5 with CDH), further expanding the clinical spectrum. Structural mapping of disease-associated missense variants revealed phenotype-specific clustering, with CODAS variants enriched in the proteolytic chamber and NDD variants more broadly distributed. CODAS is caused by biallelic variants and CDH by monoallelic variants, both of which are predicted to act through loss-of-function mechanisms. Both monoallelic and biallelic variants are associated with LONP1-related NDD, suggesting complex mechanisms such as dominant-negative effects. Our findings broaden the phenotypic and genetic spectrum of LONP1-associated disorders and highlight the essential role of LONP1 in mitochondrial function and development.

DDX3X dysfunction causes an X-linked multisystem disorder with high penetrance and variable expressivity. The phenotypic spectrum spans from learning disability without somatic involvement to profound intellectual disability with severe impairments in the central nervous system and other organs. A few multicenter studies and single case reports have previously highlighted some common phenotypic patterns but were unable to delineate correlations between underlying DDX3X variants and phenotypic findings.From the second largest patient cohort published to date, we analysed clinical, psychometric and diagnostic findings of 52 female and 7 male individuals, harbouring de novo and inherited DDX3X variants. These female patients revealed previously unknown quantitative correlations between variant type and localization and specific phenotypic findings (growth features, epilepsy, brain anomalies, dysmorphisms, motor-focused neurological findings). Moreover, by analysing the in silico folding and RNA binding capability of mutant DDX3X monomers, we were able to delineate novel correlations between DDX3X monomer misfolding grade and phenotypic severity.

POLG2 encodes an accessory subunit in DNA polymerase gamma that is required for mitochondrial DNA synthesis. Monoallelic pathogenic variants in POLG2 are associated primarily with progressive external ophthalmoplegia with mitochondrial DNA deletions, autosomal dominant type 4 (PEOA4, MIM #610131). We report a rare case of severe infantile hepatocerebral syndrome associated with biallelic variants in POLG2. The proband, a 5-week-old female infant, presented with seizures and acute liver failure. Extensive metabolic workup, including untargeted metabolomics analysis and elevated plasma growth differentiation factor 15, was suggestive of mitochondrial dysfunction. Rapid trio genome sequencing identified compound heterozygous variants, a likely pathogenic variant and a variant of uncertain significance in POLG2. This case expands the clinical phenotype associated with POLG2-related mitochondrial disease to include a severe hepatocerebral syndrome manifesting in early childhood. This case underscores the utility of integrated genomic and metabolomic analyses in diagnosing rare and complex mitochondrial disorders. These findings also emphasize the importance of considering POLG2-related mitochondrial disease in the differential diagnosis of infants presenting with liver failure and neurological symptoms and enhance our understanding of the phenotypic spectrum associated with this disorder.

Congenital Anomalies of Kidney and Urinary Tract (CAKUT) can occur in isolation or in conjunction with one or more non-CAKUT associated congenital anomalies or neurodevelopmental disorders (CAKUT+). A molecular cause is not identified in most individuals with CAKUT+. This is due, in part, to uncertainty regarding the efficacy of genetic testing and an incomplete understanding of the genes that cause CAKUT+. Here, we use data from 515 individuals with CAKUT+ (n = 500) or isolated CAKUT (n = 15) to determine the efficacy of clinical exome sequencing (cES) and to identify new phenotype expansions that involve CAKUT. We determined that cES established a molecular diagnosis in 27.4% (141/515) of individuals in this cohort. No statistically significant difference in efficacy was seen with regards to age, sex, CAKUT phenotype, or associated organ system abnormality. Only 3.5% (5/144) to 14.6% (21/144) of the individual diagnoses made in our cohort could have been identified using one of four clinically available CAKUT gene panels. We then used a machine-learning approach to confirm that PHIP is a CAKUT gene and to implicate ADNP and SETD5 genes associated with an increased risk of CAKUT. These findings lead us to conclude that cES should be considered in individuals with CAKUT+ for whom a molecular diagnosis has not been identified, that cES has the potential to identify many diagnoses in individuals with CAKUT+ that would be missed using a CAKUT gene panel, and that individuals with ADNP-, PHIP-, and SETD5-related disorders may present with CAKUT phenotypes.

Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart defect (CHD). TOF may present in isolation or in conjunction with one or more non-cardiac congenital anomalies or neurodevelopmental disorders (TOF+). Uncertainty regarding the efficacy of various genetic testing strategies, and an incomplete understanding of the genetic causes of TOF+, may lead to hesitancy in recommending genetic testing, particularly, clinical exome sequencing (cES). Here, we analyzed cES data from 131 individuals with TOF+. A definitive or probable diagnosis was made for 31 individuals, yielding a diagnostic rate of 23.6% (31/131). One individual received three diagnoses. Commercially available CHD panels would have detected only 27.3% (9/33) to 63.6% (21/33) of the diagnoses made by cES. We then used a machine learning approach to identify four genes for which there is sufficient evidence to support a phenotypic expansion including TOF: DVL3, MED13L, PUF60, and MEIS2. Since chromosomal microarray analysis (CMA) has been reported to have a diagnostic efficacy of 10-20% in individuals with TOF, we conclude that cES should be considered for all individuals with TOF+ for whom a molecular diagnosis has not been established by CMA. We also conclude that TOF represents a low penetrance phenotype associated with genetic syndromes caused by pathogenic variants in DVL3, MED13L, PUF60, and MEIS2.

The current diagnostic rate for patients with suspected Mendelian genetic disorders is only 25 to 58%, even though whole exome sequencing (WES) is part of the standard of care. One reason for the low diagnostic rate is that traditional WES analysis methods struggle to detect RNA splicing aberrations. It is estimated that 15-50% of human pathogenic variants alter splicing, with numerous splice-altering variants being causal for known Mendelian disorders. Developing reliable diagnostic tools to detect, quantify, prioritize, and visualize RNA splicing aberrations from patient RNA sequencing is therefore crucial. We present MAJIQ-CLIN, a method to address this need to augment clinical diagnostic using RNA-Seq and compare it to existing tools. We include the first systematic evaluation of the accuracy of such tools using synthetic data across several aberration types and transcript inclusion levels; we also evaluate accuracy on several datasets of biologically validated solved test cases. We show that MAJIQ-CLIN compares favorably to existing tools in both accuracy and efficiency, then use MAJIQ-CLIN to investigate several unsolved patient cases from the Undiagnosed Diseases Network.

Childhood -onset essential hypertension (COEH) is an uncommon form of hypertension that manifests in childhood or adolescence and, in the United States, disproportionately affects children of African ancestry. The etiology of COEH is unknown, but its childhood onset, low prevalence, high heritability, and skewed ancestral demography suggest the potential to identify rare genetic variation segregating in a Mendelian manner among affected individuals and thereby implicate genes important to disease pathogenesis. However, no COEH genes have been reported to date. Here, we identify recessive segregation of rare and putatively damaging missense variation in the spectrin domain of spectrin repeat containing nuclear envelope protein 1 ( SYNE1 ), a cardiovascular candidate gene, in 3 of 16 families with early -onset COEH without an antecedent family history. By leveraging exome sequence data from an additional 48 COEH families, 1,700 in-house trios, and publicly available data sets, we demonstrate that compound heterozygous SYNE1 variation in these COEH individuals occurred more often than expected by chance and that this class of biallelic rare variation was significantly enriched among individuals of African genetic ancestry. Using in vitro shRNA knockdown of SYNE1 , we show that reduced SYNE1 expression resulted in a substantial decrease in the elasticity of smooth muscle vascular cells that could be rescued by pharmacological inhibition of the downstream RhoA/Rho-associated protein kinase pathway. These results provide insights into the molecular genetics and underlying pathophysiology of COEH and suggest a role for precision therapeutics in the future.

Fine-Lubinsky syndrome is a rare clinically defined syndrome sometimes referred to as brachycephaly, deafness, cataract, microstomia, and impaired intellectual development syndrome. Here we provide a clinical and molecular update for a sibling pair diagnosed with Fine-Lubinsky syndrome. An extensive genetic work-up, including chromosomal microarray analysis and quad exome sequencing, was nondiagnostic. However, a research reanalysis of their exome sequencing data revealed that both were homozygous for an intronic c.749+39G>A [NM_001383.6] variant in DPH1. RNAseq analysis performed on RNA from fibroblasts revealed significantly reduced expression of DPH1 transcripts suggestive of abnormal splicing followed by nonsense mediated mRNA decay. Since the phenotypes of this sibling pair were consistent with those associated with the inheritance of biallelic pathogenic variants in DPH1, they were given a diagnosis of developmental delay with short stature, dysmorphic facial features, and sparse hair 1 (DEDSSH1). This leads us to recommend that all individuals with a clinical diagnosis of Fine-Lubinsky syndrome be screened for variants in DPH1. The clinical histories of this sibling pair emphasize that hearing loss associated with DEDSSH1 may remit over time and that individuals with DEDSSH1 should be monitored for the development of cardiomyopathy. This case also demonstrates the clinical utility of RNAseq as a means of functionally validating the effects of intronic variants that may affect splicing.

FOXP1 encodes a transcription factor involved in tissue regulation and cell-type-specific functions. Haploinsufficiency of FOXP1 is associated with a neurodevelopmental disorder: autosomal dominant mental retardation with language impairment with or without autistic features. More recently, heterozygous FOXP1 variants have also been shown to cause a variety of structural birth defects including central nervous system (CNS) anomalies, congenital heart defects, congenital anomalies of the kidney and urinary tract, cryptorchidism, and hypospadias. In this report, we present a previously unpublished case of an individual with congenital diaphragmatic hernia (CDH) who carries an approximately 3.8 Mb deletion. Based on this deletion, and deletions previously reported in two other individuals with CDH, we define a CDH critical region on chromosome 3p13 that includes FOXP1 and four other protein-coding genes. We also provide detailed clinical descriptions of two previously reported individuals with CDH who carry de novo, pathogenic variants in FOXP1 that are predicted to trigger nonsense-mediated mRNA decay. A subset of individuals with putatively deleterious FOXP4 variants has also been shown to develop CDH. Since FOXP proteins function as homo- or heterodimers and the homologs of FOXP1 and FOXP4 are expressed at the same time points in the embryonic mouse diaphragm, they may function together as a dimer, or in parallel as homodimers, to regulate gene expression during diaphragm development. Not all individuals with heterozygous, loss-of-function changes in FOXP1 develop CDH. Hence, we conclude that FOXP1 acts as a susceptibility factor that contributes to the development of CDH in conjunction with other genetic, epigenetic, environmental, and/or stochastic factors.