Biallelic CRELD1 variants cause a multisystem syndrome, including neurodevelopmental phenotypes, cardiac dysrhythmias, and frequent infections.

PURPOSE: We sought to delineate a multisystem disorder caused by recessive cysteine-rich with epidermal growth factor-like domains 1 (CRELD1) gene variants. METHODS: The impact of CRELD1 variants was characterized through an international collaboration utilizing next-generation DNA sequencing, gene knockdown, and protein overexpression in Xenopus tropicalis, and in vitro analysis of patient immune cells. RESULTS: Biallelic variants in CRELD1 were found in 18 participants from 14 families. Affected individuals displayed an array of phenotypes involving developmental delay, early-onset epilepsy, and hypotonia, with about half demonstrating cardiac arrhythmias and some experiencing recurrent infections. Most harbored a frameshift in trans with a missense allele, with 1 recurrent variant, p.(Cys192Tyr), identified in 10 families. X tropicalis tadpoles with creld1 knockdown displayed developmental defects along with increased susceptibility to induced seizures compared with controls. Additionally, human CRELD1 harboring missense variants from affected individuals had reduced protein function, indicated by a diminished ability to induce craniofacial defects when overexpressed in X tropicalis. Finally, baseline analyses of peripheral blood mononuclear cells showed similar proportions of immune cell subtypes in patients compared with healthy donors. CONCLUSION: This patient cohort, combined with experimental data, provide evidence of a multisystem clinical syndrome mediated by recessive variants in CRELD1.

Disruption of NEUROD2 causes a neurodevelopmental syndrome with autistic features via cell-autonomous defects in forebrain glutamatergic neurons.

While the transcription factor NEUROD2 has recently been associated with epilepsy, its precise role during nervous system development remains unclear. Using a multi-scale approach, we set out to understand how Neurod2 deletion affects the development of the cerebral cortex in mice. In Neurod2 KO embryos, cortical projection neurons over-migrated, thereby altering the final size and position of layers. In juvenile and adults, spine density and turnover were dysregulated in apical but not basal compartments in layer 5 neurons. Patch-clamp recordings in layer 5 neurons of juvenile mice revealed increased intrinsic excitability. Bulk RNA sequencing showed dysregulated expression of many genes associated with neuronal excitability and synaptic function, whose human orthologs were strongly associated with autism spectrum disorders (ASD). At the behavior level, Neurod2 KO mice displayed social interaction deficits, stereotypies, hyperactivity, and occasionally spontaneous seizures. Mice heterozygous for Neurod2 had similar defects, indicating that Neurod2 is haploinsufficient. Finally, specific deletion of Neurod2 in forebrain excitatory neurons recapitulated cellular and behavioral phenotypes found in constitutive KO mice, revealing the region-specific contribution of dysfunctional Neurod2 in symptoms. Informed by these neurobehavioral features in mouse mutants, we identified eleven patients from eight families with a neurodevelopmental disorder including intellectual disability and ASD associated with NEUROD2 pathogenic mutations. Our findings demonstrate crucial roles for Neurod2 in neocortical development, whose alterations can cause neurodevelopmental disorders including intellectual disability and ASD.

A retrospective cohort analysis of the Yale pediatric genomics discovery program.

The Pediatric Genomics Discovery Program (PGDP) at Yale uses next-generation sequencing (NGS) and translational research to evaluate complex patients with a wide range of phenotypes suspected to have rare genetic diseases. We conducted a retrospective cohort analysis of 356 PGDP probands evaluated between June 2015 and July 2020, querying our database for participant demographics, clinical characteristics, NGS results, and diagnostic and research findings. The three most common phenotypes among the entire studied cohort (n = 356) were immune system abnormalities (n = 105, 29%), syndromic or multisystem disease (n = 103, 29%), and cardiovascular system abnormalities (n = 62, 17%). Of 216 patients with final classifications, 77 (36%) received new diagnoses and 139 (64%) were undiagnosed; the remaining 140 patients were still actively being investigated. Monogenetic diagnoses were found in 67 (89%); the largest group had variants in known disease genes but with new contributions such as novel variants (n = 31, 40%) or expanded phenotypes (n = 14, 18%). Finally, five PGDP diagnoses (8%) were suggestive of novel gene-to-phenotype relationships. A broad range of patients can benefit from single subject studies combining NGS and functional molecular analyses. All pediatric providers should consider further genetics evaluations for patients lacking precise molecular diagnoses.

DLG5 variants are associated with multiple congenital anomalies including ciliopathy phenotypes.

BACKGROUND: Cilia are dynamic cellular extensions that generate and sense signals to orchestrate proper development and tissue homeostasis. They rely on the underlying polarisation of cells to participate in signalling. Cilia dysfunction is a well-known cause of several diseases that affect multiple organ systems including the kidneys, brain, heart, respiratory tract, skeleton and retina. METHODS: Among individuals from four unrelated families, we identified variants in discs large 5 (DLG5) that manifested in a variety of pathologies. In our proband, we also examined patient tissues. We depleted dlg5 in Xenopus tropicalis frog embryos to generate a loss-of-function model. Finally, we tested the pathogenicity of DLG5 patient variants through rescue experiments in the frog model. RESULTS: Patients with variants of DLG5 were found to have a variety of phenotypes including cystic kidneys, nephrotic syndrome, hydrocephalus, limb abnormalities, congenital heart disease and craniofacial malformations. We also observed a loss of cilia in cystic kidney tissue of our proband. Knockdown of dlg5 in Xenopus embryos recapitulated many of these phenotypes and resulted in a loss of cilia in multiple tissues. Unlike introduction of wildtype DLG5 in frog embryos depleted of dlg5, introduction of DLG5 patient variants was largely ineffective in restoring proper ciliation and tissue morphology in the kidney and brain suggesting that the variants were indeed detrimental to function. CONCLUSION: These findings in both patient tissues and Xenopus shed light on how mutations in DLG5 may lead to tissue-specific manifestations of disease. DLG5 is essential for cilia and many of the patient phenotypes are in the ciliopathy spectrum.

Functional testing for variant prioritization in a family with long QT syndrome.

Next-generation sequencing platforms are being increasingly applied in clinical genetic settings for evaluation of families with suspected heritable disease. These platforms potentially improve the diagnostic yield beyond that of disease-specific targeted gene panels, but also increase the number of rare or novel genetic variants that may confound precise diagnostics. Here, we describe a functional testing approach used to interpret the results of whole exome sequencing (WES) in a family presenting with syncope and sudden death. One individual had a prolonged QT interval on electrocardiogram (ECG) and carried a diagnosis of long QT syndrome (LQTS), but a second individual did not meet criteria for LQTS. Filtering WES results for uncommon variants with arrhythmia association identified four for further analyses. In silico analyses indicated that two of these variants, KCNH2 p.(Cys555Arg) and KCNQ1 p.(Arg293Cys), were likely to be causal in this family's LQTS. We subsequently performed functional characterization of these variants in a heterologous expression system. The expression of KCNQ1-Arg293Cys did not show a deleterious phenotype but KCNH2-Cys555Arg demonstrated a loss-of-function phenotype that was partially dominant. Our stepwise approach identified a precise genetic etiology in this family, which resulted in the establishment of a LQTS diagnosis in the second individual as well as an additional asymptomatic family member, enabling personalized clinical management. Given its ability to aid in the diagnosis, the application of functional characterization should be considered as a value adjunct to in silico analyses of WES.

Expansion of NEUROD2 phenotypes to include developmental delay without seizures.

De novo heterozygous variants in the brain-specific transcription factor Neuronal Differentiation Factor 2 (NEUROD2) have been recently associated with early-onset epileptic encephalopathy and developmental delay. Here, we report an adolescent with developmental delay without seizures who was found to have a novel de novo heterozygous NEUROD2 missense variant, p.(Leu163Pro). Functional testing using an in vivo assay of neuronal differentiation in Xenopus laevis tadpoles demonstrated that the patient variant of NEUROD2 displays minimal protein activity, strongly suggesting a loss of function effect. In contrast, a second rare NEUROD2 variant, p.(Ala235Thr), identified in an adolescent with developmental delay but lacking parental studies for inheritance, showed normal in vivo NEUROD2 activity. We thus provide clinical, genetic, and functional evidence that NEUROD2 variants can lead to developmental delay without accompanying early-onset seizures, and demonstrate how functional testing can complement genetic data when determining variant pathogenicity.

The latest FADS: Functional analysis of GLDN patient variants and classification of GLDN-associated AMC as a type of viable fetal akinesia deformation sequence.

Recessive variants in the GLDN gene, which encodes the gliomedin protein and is involved in nervous system development, have recently been associated with Arthrogryposis Multiplex Congenita (AMC), a heterogenous condition characterized by congenital contractures of more than one joint. Two cohorts of patients with GLDN-associated AMC have previously been described, evolving the understanding of the condition from lethal to survivable with the provision of significant neonatal support. Here, we describe one additional patient currently living with the syndrome, having one novel variant, p.Leu365Phe, for which we provide functional data supporting its pathogenicity. We additionally provide experimental data for four other previously reported variants lacking functional evidence, including p.Arg393Lys, the second variant present in our patient. We discuss unique and defining clinical features, adding calcium-related findings which appear to be recurrent in the GLDN cohort. Finally, we compare all previously reported patients and draw new conclusions about scope of illness, with emphasis on the finding of pulmonary hypoplasia, suggesting that AMC secondary to GLDN variants may be best fitted under the umbrella of fetal akinesia deformation sequence (FADS).

DYNC1H1-related disorders: A description of four new unrelated patients and a comprehensive review of previously reported variants.

Heterozygous variants in the DYNC1H1 gene have been associated chiefly with intellectual disability (ID), malformations in cortical development (MCD), spinal muscular atrophy (SMA), and Charcot-Marie-Tooth axonal type 20 (CMT), with fewer reports describing other intersecting phenotypes. To better characterize the variable syndromes associated with DYNC1H1, we undertook a detailed analysis of reported patients in the medical literature through June 30, 2019. In sum we identified 200 patients from 143 families harboring 103 different DYNC1H1 variants, and added reports for four unrelated patients identified at our center, three with novel variants. The most common features associated with DYNC1H1 were neuromuscular (NM) disease (largely associated with variants in the stem domain), ID with MCD (largely associated with variants in the motor domain), or a combination of these phenotypes. Despite these trends, exceptions are noted throughout. Overall, DYNC1H1 is associated with variable neurodevelopmental and/or neuromuscular phenotypes that overlap. To avoid confusion DYNC1H1 disorders may be best categorized at this time by more general descriptions rather than phenotype-specific nomenclature such as SMA or CMT. We therefore propose the terms: DYNC1H1-related NM disorder, DYNC1H1-related CNS disorder, and DYNC1H1-related combined disorder. Our single center's experience may be evidence that disease-causing variants in this gene are more prevalent than currently recognized.

De novo pathogenic variants in neuronal differentiation factor 2 (NEUROD2) cause a form of early infantile epileptic encephalopathy.

BACKGROUND: Early infantile epileptic encephalopathies are severe disorders consisting of early-onset refractory seizures accompanied often by significant developmental delay. The increasing availability of next-generation sequencing has facilitated the recognition of single gene mutations as an underlying aetiology of some forms of early infantile epileptic encephalopathies. OBJECTIVES: This study was designed to identify candidate genes as a potential cause of early infantile epileptic encephalopathy, and then to provide genetic and functional evidence supporting patient variants as causative. METHODS: We used whole exome sequencing to identify candidate genes. To model the disease and assess the functional effects of patient variants on candidate protein function, we used in vivo CRISPR/Cas9-mediated genome editing and protein overexpression in frog tadpoles. RESULTS: We identified novel de novo variants in neuronal differentiation factor 2 (NEUROD2) in two unrelated children with early infantile epileptic encephalopathy. Depleting neurod2 with CRISPR/Cas9-mediated genome editing induced spontaneous seizures in tadpoles, mimicking the patients' condition. Overexpression of wild-type NEUROD2 induced ectopic neurons in tadpoles; however, patient variants were markedly less effective, suggesting that both variants are dysfunctional and likely pathogenic. CONCLUSION: This study provides clinical and functional support for NEUROD2 variants as a cause of early infantile epileptic encephalopathy, the first evidence of human disease caused by NEUROD2 variants.

A novel SAMD9 mutation causing MIRAGE syndrome: An expansion and review of phenotype, dysmorphology, and natural history.

Germline gain-of-function variants in SAMD9 have been associated with a high risk of mortality and a newly recognized constellation of symptoms described by the acronym MIRAGE: Myelodysplasia, Infection, Restriction of growth, Adrenal insufficiency, Genital phenotypes, and Enteropathy. Here, we describe two additional patients currently living with the syndrome, including one patient with a novel de novo variant for which we provide functional data supporting its pathogenicity. We discuss features of dysmorphology, contrasting with previously described patients as well as drawing attention to additional clinical features, dysautonomia and hearing loss that have not previously been reported. We detail both patients' courses following diagnosis, with attention to treatment plans and recommended specialist care. Our patients are the oldest known with arginine-substituting amino acid variants, and we conclude that early diagnosis and multidisciplinary management may positively impact outcomes for this vulnerable group of patients.

A metabolic signature for NADSYN1-dependent congenital NAD deficiency disorder.

Nicotinamide adenine dinucleotide (NAD) is essential for embryonic development. To date, biallelic loss-of-function variants in 3 genes encoding nonredundant enzymes of the NAD de novo synthesis pathway - KYNU, HAAO, and NADSYN1 - have been identified in humans with congenital malformations defined as congenital NAD deficiency disorder (CNDD). Here, we identified 13 further individuals with biallelic NADSYN1 variants predicted to be damaging, and phenotypes ranging from multiple severe malformations to the complete absence of malformation. Enzymatic assessment of variant deleteriousness in vitro revealed protein domain-specific perturbation, complemented by protein structure modeling in silico. We reproduced NADSYN1-dependent CNDD in mice and assessed various maternal NAD precursor supplementation strategies to prevent adverse pregnancy outcomes. While for Nadsyn1+/- mothers, any B3 vitamer was suitable to raise NAD, preventing embryo loss and malformation, Nadsyn1-/- mothers required supplementation with amidated NAD precursors (nicotinamide or nicotinamide mononucleotide) bypassing their metabolic block. The circulatory NAD metabolome in mice and humans before and after NAD precursor supplementation revealed a consistent metabolic signature with utility for patient identification. Our data collectively improve clinical diagnostics of NADSYN1-dependent CNDD, provide guidance for the therapeutic prevention of CNDD, and suggest an ongoing need to maintain NAD levels via amidated NAD precursor supplementation after birth.

Provider- and System-Level Barriers and Facilitators to Colonoscopy and Multi-Target Stool DNA for Colorectal Cancer Screening in Rural/Remote Alaska Native Communities.

The Alaska Tribal Health System is working to increase colorectal cancer (CRC) screening among Alaska Native people, who experience the highest CRC rates in the world. This study examined CRC screening provider- and system-level barriers and facilitators from the perspective of healthcare providers serving Alaska Native people in rural/remote communities. A total of 28 provider (physicians, advanced practice, and Community Health Aides/Practitioners) interviews were held from 1 February to 30 November 2021. Colonoscopy provider-level barrier themes included time, competing priorities, and staffing, while system-level barriers included travel costs, weather, and the COVID-19 pandemic. Multi-target stool DNA (mt-sDNA) barrier themes included test viability and unfamiliarity, and previous stool tests experiences. For both tests, limited medical record reminders was a major barrier. Facilitator themes for both tests included community outreach, cultural competency and patient navigation, and clinic/system improvements. In-depth interviews with tribal health providers showed that adding mt-sDNA testing may help address system-level colonoscopy barriers such as waitlists and travel costs, but other barriers remain. Further research is needed into patient barriers and facilitators, as well as the effectiveness of integrating mt-sDNA into a geographically dispersed tribal health system to reduce cancer disparities and build equity in CRC prevention among Alaska Native people.

TBX5 variant with the novel phenotype of mixed­type total anomalous pulmonary venous return in Holt­Oram Syndrome and variable intrafamilial heart defects.

Variants in T­box transcription factor 5 (TBX5) can result in a wide phenotypic spectrum, specifically in the heart and the limbs. TBX5 has been implicated in causing non­syndromic cardiac defects and Holt­Oram syndrome (HOS). The present study investigated the underlying molecular etiology of a family with heterogeneous heart defects. The proband had mixed­type total anomalous pulmonary venous return (mixed­type TAPVR), whereas her mother had an atrial septal defect. Genetic testing through trio­based whole­exome sequencing was used to reveal the molecular etiology. A nonsense variant was identified in TBX5 (c.577G>T; p.Gly193*) initially showing co­segregation with a presumably non­syndromic presentation of congenital heart disease. Subsequent genetic investigations and more complete phenotyping led to the correct diagnosis of HOS, documenting the novel association of mixed­type TAPVR with HOS. Finally, protein modeling of the mutant TBX5 protein that harbored this pathogenic nonsense variant (p.Gly193*) revealed a substantial drop in the quantity of non­covalent bonds. The decrease in the number of non­covalent bonds suggested that the resultant mutant dimer was less stable compared with the wild­type protein, consequently affecting the protein's ability to bind DNA. The present findings extended the phenotypic cardiac defects associated with HOS; to the best of our knowledge, this is the first association of mixed­type TAPVR with TBX5. Prior to the current analysis, the molecular association of TAPVR with HOS had never been documented; hence, this is the first genetic investigation to report the association between TAPVR and HOS. Furthermore, it was demonstrated that the null­variants reported in the T­box domain of TBX5 were associated with a wide range of cardiac and/or skeletal anomalies on both the inter­and intrafamilial levels. In conclusion, genetic testing was highlighted as a potentially powerful approach in the prognostication of the proper diagnosis.

A novel variant in MAP3K7 associated with an expanded cardiospondylocarpofacial syndrome phenotype.

The transforming growth factor-ß-activated kinase 1 (TAK1) encoded by mitogen-activated protein kinase kinase kinase 7 (MAP3K7) is widely expressed and participates in multiple molecular and cellular processes, including growth, differentiation, inflammation, and apoptosis. Pathogenic variants in MAP3K7 have recently been associated with two disorders: cardiospondylocarpofacial syndrome (CSCFS) and frontometaphyseal dysplasia 2 (FMD2). To date, all small in-frame deletions and splice variants in MAP3K7 have been associated with CSCFS, whereas missense variants have been reported in both CSCFS and FMD2. Here, we present a patient with a novel heterozygous likely pathogenic variant, c.125_127del, p.(Val42del), in MAP3K7, only the sixth variant associated with CSCFS to be described in the literature. Although this patient has a phenotype that is most consistent with that of CSCFS, including valvular heart disease, short stature, fusions of the spine and bones of the hands and feet, and certain facial dysmorphisms, he interestingly has some features reported previously in FMD2 but not CSCFS. These include flexion contractures of the elbow and widely spaced first and second toes, highlighting new points of overlap between these two syndromes. We additionally point out features in the patient presented here that are rare but recurrent among CSCFS patients previously reported in the literature, as well as a new distinctive cutaneous finding not previously reported.

Identification of a novel MYOC variant in a Hispanic family with early-onset primary open-angle glaucoma with elevated intraocular pressure.

Primary open-angle glaucoma (POAG) is the leading cause of irreversible blindness worldwide. Most cases are multifactorial in etiology, but some are associated with variants in the myocilin gene, MYOC Here, we report the identification of a novel MYOC variant, c.1153G>A, in a 24-yr-old female patient with a personal and family history of juvenile/early-onset POAG. Further genetic testing within her family demonstrated that this variant segregates with the POAG phenotype in an autosomal dominant pattern. Identification of this MYOC variant in multiple affected relatives provides evidence for its pathogenicity, supporting previous findings linking MYOC mutations, in particular in the third exon's olfactomedin domain, to juvenile-onset POAG. This case also emphasizes the potential value of genetic testing in families with histories of eye disorders.

Two siblings with a novel nonsense variant provide further delineation of the spectrum of recessive KLHL7 diseases.

Mutations in Kelch-like family member 7 (KLHL7) have recently been described as a cause of a constellation of clinical findings with descriptions of both a Crisponi syndrome (CS)/cold-induced sweating syndrome type 1 (CISS1)-like, as well as a Bohring-Opitz syndrome (BOS)-like presentation. Here we report two siblings of Guatelmalan descent with a novel homozygous nonsense mutation (p.Arg326*) in KLHL7. These children have multiple dysmorphic features and developmental delay. Interestingly, their clinical traits inconsistently overlap both the CS/CISS1-like and BOS-like phenotypes, and the siblings also have subtle differences from each other, suggesting that clinicians need to be aware of the degree of variability in the presentations of these patients. Still, there is enough in common between patients with recessive KLHL7 mutations to define a novel multisystem disease that features various neurodevelopmental, musculoskeletal, dysmorphic, and other unique components. This report adds to the clinical features and disease-associated variants of the newly-recognized spectrum of KLHL7 mutations, and offers a new description, PERCHING, for the resulting syndrome.

Siblings with lethal primary pulmonary hypoplasia and compound heterozygous variants in the AARS2 gene: further delineation of the phenotypic spectrum.

Variants in the mitochondrial alanyl-tRNA synthetase 2 gene AARS2 (OMIM 612035) are associated with infantile mitochondrial cardiomyopathy or later-onset leukoencephalopathy with premature ovarian insufficiency. Here, we report two newborn siblings who died soon after birth with primary pulmonary hypoplasia without evidence of cardiomyopathy. Whole-exome sequencing detected the same compound heterozygous AARS2 variants in both siblings (c.1774C>T, p.Arg592Trp and c.647dup, p.Cys218Leufs*6) that have previously been associated with infantile mitochondrial cardiomyopathy. Segregation analysis in the family confirmed carrier status of the parents and an unaffected sibling. To our knowledge, this is the first report of primary pulmonary hypoplasia in the absence of cardiomyopathy associated with recessive AARS2 variants and further defines the phenotypic spectrum associated with this gene.

Familial Dilated Cardiomyopathy Associated With a Novel Combination of Compound Heterozygous TNNC1 Variants.

Familial dilated cardiomyopathy (DCM), clinically characterized by enlargement and dysfunction of one or both ventricles of the heart, can be caused by variants in sarcomeric genes including TNNC1 (encoding cardiac troponin C, cTnC). Here, we report the case of two siblings with severe, early onset DCM who were found to have compound heterozygous variants in TNNC1: p.Asp145Glu (D145E) and p.Asp132Asn (D132N), which were inherited from the parents. We began our investigation with CRISPR/Cas9 knockout of TNNC1 in Xenopus tropicalis, which resulted in a cardiac phenotype in tadpoles consistent with DCM. Despite multiple maneuvers, we were unable to rescue the tadpole hearts with either human cTnC wild-type or patient variants to investigate the cardiomyopathy phenotype in vivo. We therefore utilized porcine permeabilized cardiac muscle preparations (CMPs) reconstituted with either wild-type or patient variant forms of cTnC to examine effects of the patient variants on contractile function. Incorporation of 50% WT/50% D145E into CMPs increased Ca2+ sensitivity of isometric force, consistent with prior studies. In contrast, incorporation of 50% WT/50% D132N, which had not been previously reported, decreased Ca2+ sensitivity of isometric force. CMPs reconstituted 50-50% with both variants mirrored WT in regard to myofilament Ca2+ responsiveness. Sinusoidal stiffness (SS) (0.2% peak-to-peak) and the kinetics of tension redevelopment (k TR) at saturating Ca2+ were similar to WT for all preparations. Modeling of Ca2+-dependence of k TR support the observation from Ca2+ responsiveness of steady-state isometric force, that the effects on each mutant (50% WT/50% mutant) were greater than the combination of the two mutants (50% D132N/50% D145E). Further studies are needed to ascertain the mechanism(s) of these variants.