The 7q11.23 Protein DNAJC30 Interacts with ATP Synthase and Links Mitochondria to Brain Development.

Despite the known causality of copy-number variations (CNVs) to human neurodevelopmental disorders, the mechanisms behind each gene's contribution to the constellation of neural phenotypes remain elusive. Here, we investigated the 7q11.23 CNV, whose hemideletion causes Williams syndrome (WS), and uncovered that mitochondrial dysfunction participates in WS pathogenesis. Dysfunction is facilitated in part by the 7q11.23 protein DNAJC30, which interacts with mitochondrial ATP-synthase machinery. Removal of Dnajc30 in mice resulted in hypofunctional mitochondria, diminished morphological features of neocortical pyramidal neurons, and altered behaviors reminiscent of WS. The mitochondrial features are consistent with our observations of decreased integrity of oxidative phosphorylation supercomplexes and ATP-synthase dimers in WS. Thus, we identify DNAJC30 as an auxiliary component of ATP-synthase machinery and reveal mitochondrial maladies as underlying certain defects in brain development and function associated with WS.

The detection of a strong episignature for Chung-Jansen syndrome, partially overlapping with Börjeson-Forssman-Lehmann and White-Kernohan syndromes.

Chung-Jansen syndrome is a neurodevelopmental disorder characterized by intellectual disability, behavioral problems, obesity and dysmorphic features. It is caused by pathogenic variants in the PHIP gene that encodes for the Pleckstrin homology domain-interacting protein, which is part of an epigenetic modifier protein complex. Therefore, we hypothesized that PHIP haploinsufficiency may impact genome-wide DNA methylation (DNAm). We assessed the DNAm profiles of affected individuals with pathogenic and likely pathogenic PHIP variants with Infinium Methylation EPIC arrays and report a specific and sensitive DNAm episignature biomarker for Chung-Jansen syndrome. In addition, we observed similarities between the methylation profile of Chung-Jansen syndrome and that of functionally related and clinically partially overlapping genetic disorders, White-Kernohan syndrome (caused by variants in DDB1 gene) and Börjeson-Forssman-Lehmann syndrome (caused by variants in PHF6 gene). Based on these observations we also proceeded to develop a common episignature biomarker for these disorders. These newly defined episignatures can be used as part of a multiclass episignature classifier for screening of affected individuals with rare disorders and interpretation of genetic variants of unknown clinical significance, and provide further insights into the common molecular pathophysiology of the clinically-related Chung-Jansen, Börjeson-Forssman-Lehmann and White-Kernohan syndromes.

Identification of DNA methylation episignature for the intellectual developmental disorder, autosomal dominant 21 syndrome, caused by variants in the CTCF gene.

PURPOSE: The main objective of this study was to assess clinical features and genome-wide DNA methylation profiles in individuals affected by intellectual developmental disorder, autosomal dominant 21 (IDD21) syndrome, caused by variants in the CCCTC-binding factor (CTCF) gene. METHODS: DNA samples were extracted from peripheral blood of 16 individuals with clinical features and genetic findings consistent with IDD21. DNA methylation analysis was performed using the Illumina Infinium Methylation EPIC Bead Chip microarrays. The methylation levels were fitted in a multivariate linear regression model to identify the differentially methylated probes. A binary support vector machine classification model was constructed to differentiate IDD21 samples from controls. RESULTS: We identified a highly specific, reproducible, and sensitive episignature associated with CTCF variants. Six variants of uncertain significance were tested, of which 2 mapped to the IDD21 episignature and clustered alongside IDD21 cases in both heatmap and multidimensional scaling plots. Comparison of the genomic DNA methylation profile of IDD21 with that of 56 other neurodevelopmental disorders provided insights into the underlying molecular pathophysiology of this disorder. CONCLUSION: The robust and specific CTCF/IDD21 episignature expands the growing list of neurodevelopmental disorders with distinct DNA methylation profiles, which can be applied as supporting evidence in variant classification.

Congenital heart defects in CTNNB1 syndrome: Raising clinical awareness.

CTNNB1 [OMIM *116806] encodes ß-catenin, an integral part of the cadherin/catenin complex, which functions as effector of Wnt signaling. CTNNB1 is highly expressed in brain as well as in other tissues, including heart. Heterozygous CTNNB1 pathogenic variations are associated with a neurodevelopmental disorder characterized by spastic diplegia and visual defects (NEDSDV) [OMIM #615075], featuring psychomotor delay, intellectual disability, behavioral disturbances, movement disorders, visual defects and subtle facial and somatic features. We report on a new series of 19 NEDSDV patients (mean age 10.3 years), nine of whom bearing novel CTNNB1 variants. Notably, five patients showed congenital heart anomalies including absent pulmonary valve with intact ventricular septum, atrioventricular canal with hypoplastic aortic arch, tetralogy of Fallot, and mitral valve prolapse. We focused on the cardiac phenotype characterizing such cases and reviewed the congenital heart defects in previously reported NEDSDV patients. While congenital heart defects had occasionally been reported so far, the present findings configure a higher rate of cardiac anomalies, suggesting dedicated heart examination to NEDSDV clinical management.

Novel biallelic variants expand the phenotype of NAA20-related syndrome.

NAA20 is the catalytic subunit of the NatB complex, which is responsible for N-terminal acetylation of approximately 20% of the human proteome. Recently, pathogenic biallelic variants in NAA20 were associated with a novel neurodevelopmental disorder in five individuals with limited clinical information. We report two sisters harboring compound heterozygous variant (c.100C>T (p.Gln34Ter) and c.11T>C p.(Leu4Pro)) in the NAA20 gene, identified by exome sequencing. In vitro studies showed that the missense variant p.Leu4Pro resulted in a reduction of NAA20 catalytic activity due to weak coupling with the NatB auxiliary subunit. In addition, unpublished data of the previous families were reported, outlining the core phenotype of the NAA20-related disorder mostly characterized by cognitive impairment, microcephaly, ataxia, brain malformations, dysmorphism and variable occurrence of cardiac defect and epilepsy. Remarkably, our two patients featured epilepsy onset in adolescence suggesting this may be a part of syndrome evolution. Functional studies are needed to better understand the complexity of NAA20 variants pathogenesis as well as of other genes linked to N-terminal acetylation.

DNA Methylation in the Fields of Prenatal Diagnosis and Early Detection of Cancers.

The central objective of the metamorphosis of discovery science into biomedical applications is to serve the purpose of patients and curtail the global disease burden. The journey from the discovery of DNA methylation (DNAm) as a biological process to its emergence as a diagnostic tool is one of the finest examples of such metamorphosis and has taken nearly a century. Particularly in the last decade, the application of DNA methylation studies in the clinic has been standardized more than ever before, with great potential to diagnose a multitude of diseases that are associated with a burgeoning number of genes with this epigenetic alteration. Fetal DNAm detection is becoming useful for noninvasive prenatal testing, whereas, in very preterm infants, DNAm is also shown to be a potential biological indicator of prenatal risk factors. In the context of cancer, liquid biopsy-based DNA-methylation profiling is offering valuable epigenetic biomarkers for noninvasive early-stage diagnosis. In this review, we focus on the applications of DNA methylation in prenatal diagnosis for delivering timely therapy before or after birth and in detecting early-stage cancers for better clinical outcomes. Furthermore, we also provide an up-to-date commercial landscape of DNAm biomarkers for cancer detection and screening of cancers of unknown origin.

DNA methylation episignature testing improves molecular diagnosis of Mendelian chromatinopathies.

PURPOSE: Chromatinopathies include more than 50 disorders caused by disease-causing variants of various components of chromatin structure and function. Many of these disorders exhibit unique genome-wide DNA methylation profiles, known as episignatures. In this study, the methylation profile of a large cohort of individuals with chromatinopathies was analyzed for episignature detection. METHODS: DNA methylation data was generated on extracted blood samples from 129 affected individuals with the Illumina Infinium EPIC arrays and analyzed using an established bioinformatic pipeline. RESULTS: The DNA methylation profiles matched and confirmed the sequence findings in both the discovery and validation cohorts. Twenty-five affected individuals carrying a variant of uncertain significance, did not show a methylation profile matching any of the known episignatures. Three additional variant of uncertain significance cases with an identified KDM6A variant were re-classified as likely pathogenic (n = 2) or re-assigned as Wolf-Hirschhorn syndrome (n = 1). Thirty of the 33 Next Generation Sequencing negative cases did not match a defined episignature while three matched Kabuki syndrome, Rubinstein-Taybi syndrome and BAFopathy respectively. CONCLUSION: With the expanding clinical utility of the EpiSign assay, DNA methylation analysis should be considered part of the testing cascade for individuals presenting with clinical features of Mendelian chromatinopathy disorders.

Genome-wide DNA methylation profiling and exome sequencing resolved a long-time misdiagnosed case.

The search for aetiology of Mendelian disorders is traditionally based on the observation of clinical phenotypes and molecular screening of associated genes. However, a disease-specific diagnosis can be challenging. In this study we detail how the combinatorial genomic and epigenomic assessment allowed to find the underlying molecular event of a clinical case that remained misdiagnosed for years. The individual was referred as affected by an atypical form of Kabuki syndrome with a variant of uncertain significance in the KMT2D gene. However, significant inconsistencies with this diagnosis emerged upon familial segregation of the variant and after the clinical re-evaluation. Therefore, we applied an epigenomic strategy by studying the DNA methylation profile which resulted not consistent with the Kabuki syndrome episignature or with any other disorder-specific episignature described so far, providing strong evidence that the Kabuki syndrome diagnosis does not apply. This result led us to further investigate for epigenetic machinery diseases by using a multigene panel for chromatinopathies. Since this analysis yielded negative results, we applied a whole exome sequencing and identified a de novo pathogenic variant in the CTNNB1 gene associated to NEDSDV syndrome, a neurodevelopmental disorder characterized by intellectual disability and craniofacial anomalies. Based on molecular results and the updated clinical features, we confirmed the NEDSDV diagnosis. Our findings show that the combination of genomic and epigenomics strategies, along with a deeper analysis of clinical phenotype, may provide a significant improvement in the diagnostic protocols for rare genetic disorders and help resolve long-time misdiagnosed and unsolved case.

PIGN encephalopathy: Characterizing the epileptology.

OBJECTIVE: Epilepsy is common in patients with PIGN diseases due to biallelic variants; however, limited epilepsy phenotyping data have been reported. We describe the epileptology of PIGN encephalopathy. METHODS: We recruited patients with epilepsy due to biallelic PIGN variants and obtained clinical data regarding age at seizure onset/offset and semiology, development, medical history, examination, electroencephalogram, neuroimaging, and treatment. Seizure and epilepsy types were classified. RESULTS: Twenty six patients (13 female) from 26 families were identified, with mean age 7 years (range = 1 month to 21 years; three deceased). Abnormal development at seizure onset was present in 25 of 26. Developmental outcome was most frequently profound (14/26) or severe (11/26). Patients presented with focal motor (12/26), unknown onset motor (5/26), focal impaired awareness (1/26), absence (2/26), myoclonic (2/26), myoclonic-atonic (1/26), and generalized tonic-clonic (2/26) seizures. Twenty of 26 were classified as developmental and epileptic encephalopathy (DEE): 55% (11/20) focal DEE, 30% (6/20) generalized DEE, and 15% (3/20) combined DEE. Six had intellectual disability and epilepsy (ID+E): two generalized and four focal epilepsy. Mean age at seizure onset was 13 months (birth to 10 years), with a lower mean onset in DEE (7 months) compared with ID+E (33 months). Patients with DEE had drug-resistant epilepsy, compared to 4/6 ID+E patients, who were seizure-free. Hyperkinetic movement disorder occurred in 13 of 26 patients. Twenty-seven of 34 variants were novel. Variants were truncating (n = 7), intronic and predicted to affect splicing (n = 7), and missense or inframe indels (n = 20, of which 11 were predicted to affect splicing). Seven variants were recurrent, including p.Leu311Trp in 10 unrelated patients, nine with generalized seizures, accounting for nine of the 11 patients in this cohort with generalized seizures. SIGNIFICANCE: PIGN encephalopathy is a complex autosomal recessive disorder associated with a wide spectrum of epilepsy phenotypes, typically with substantial profound to severe developmental impairment.

Clinical heterogeneity of Kabuki syndrome in a cohort of Italian patients and review of the literature.

Kabuki syndrome (KS) is a well-recognized disorder characterized by postnatal growth deficiency, dysmorphic facial features, skeletal anomalies, and intellectual disability. The syndrome is caused by KMT2D gene mutations or less frequently KDM6A gene mutations or deletions. We report a systematic evaluation of KS patients from Campania region of Italy; data were also compared with literature ones. We collected data of 15 subjects (8 males and 7 females with age range 10-26 years; mean age 16.9 years) with confirmed diagnosis of KS, representing the entire cohort of patients from Campania Region. Each patient performed biochemical testing and instrumental investigation. Neuro-intellectual development, cranio-facial dysmorphisms, and multisystem involvement data were collected retrospectively. For each category, type of defects and frequency of the anomalies were analyzed. Our observation shows that KS patients from Campania region have some particular and previously underscored, neurological and immunological findings. We found high prevalence of EEG's abnormalities (43%) and MRI brain abnormalities (60%). Microcephaly resulted more common in our series (33%), if compared with major cohorts described in literature. Biochemical features of immunodeficiency and autoimmune diseases including thyroid autoimmunity, polyserositis, and vitiligo were observed with high prevalence (54.5%). Low immunoglobulins levels were a frequent finding. Lymphocyte class investigation showed significantly reduced CD8 levels in one patient.Conclusions: These data confirm great heterogeneity of clinical manifestations in KS and suggest to introduce further clinical diagnostic criteria in order to perform a correct and precocious diagnosis. What is Known • Kabuki syndrome is characterized by growth deficiency, dysmorphic facial features, skeletal anomalies, and intellectual disability • Immune dysfunction is a common finding but autoimmune diseases are rarely seen • Neurological features are common What is New • Some particular facial features could help gestalt diagnosis (hypertelorism, broad nasal bridge, micrognathia, tooth agenesis, cutaneous haemangiomas and strabismus) • Higher prevalence of autoimmune disorders than previously reported • Particular neurological features are present in this cohort (EEG and MRI brain abnormalities).

Clinical Utility of a Unique Genome-Wide DNA Methylation Signature for KMT2A-Related Syndrome.

Wiedemann-Steiner syndrome (WDSTS) is a Mendelian syndromic intellectual disability (ID) condition associated with hypertrichosis cubiti, short stature, and characteristic facies caused by pathogenic variants in the KMT2A gene. Clinical features can be inconclusive in mild and unusual WDSTS presentations with variable ID (mild to severe), facies (typical or not) and other associated malformations (bone, cerebral, renal, cardiac and ophthalmological anomalies). Interpretation and classification of rare KMT2A variants can be challenging. A genome-wide DNA methylation episignature for KMT2A-related syndrome could allow functional classification of variants and provide insights into the pathophysiology of WDSTS. Therefore, we assessed genome-wide DNA methylation profiles in a cohort of 60 patients with clinical diagnosis for WDSTS or Kabuki and identified a unique highly sensitive and specific DNA methylation episignature as a molecular biomarker of WDSTS. WDSTS episignature enabled classification of variants of uncertain significance in the KMT2A gene as well as confirmation of diagnosis in patients with clinical presentation of WDSTS without known genetic variants. The changes in the methylation profile resulting from KMT2A mutations involve global reduction in methylation in various genes, including homeobox gene promoters. These findings provide novel insights into the molecular etiology of WDSTS and explain the broad phenotypic spectrum of the disease.

Customised next-generation sequencing multigene panel to screen a large cohort of individuals with chromatin-related disorder.

BACKGROUND: The regulation of the chromatin state by epigenetic mechanisms plays a central role in gene expression, cell function, and maintenance of cell identity. Hereditary disorders of chromatin regulation are a group of conditions caused by abnormalities of the various components of the epigenetic machinery, namely writers, erasers, readers, and chromatin remodelers. Although neurological dysfunction is almost ubiquitous in these disorders, the constellation of additional features characterizing many of these genes and the emerging clinical overlap among them indicate the existence of a community of syndromes. The introduction of high-throughput next generation sequencing (NGS) methods for testing multiple genes simultaneously is a logical step for the implementation of diagnostics of these disorders. METHODS: We screened a heterogeneous cohort of 263 index patients by an NGS-targeted panel, containing 68 genes associated with more than 40 OMIM entries affecting chromatin function. RESULTS: This strategy allowed us to identify clinically relevant variants in 87 patients (32%), including 30 for which an alternative clinical diagnosis was proposed after sequencing analysis and clinical re-evaluation. CONCLUSION: Our findings indicate that this approach is effective not only in disorders with locus heterogeneity, but also in order to anticipate unexpected misdiagnoses due to clinical overlap among cognate disorders. Finally, this work highlights the utility of a prompt diagnosis in such a clinically and genetically heterogeneous group of disorders that we propose to group under the umbrella term of chromatinopathies.

Identification of a DNA Methylation Episignature in the 22q11.2 Deletion Syndrome.

The 22q11.2 deletion syndrome (22q11.2DS) is the most common genomic disorder in humans and is the result of a recurrent 1.5 to 2.5 Mb deletion, encompassing approximately 20-40 genes, respectively. The clinical presentation of the typical deletion includes: Velocardiofacial, Di George, Opitz G/BBB and Conotruncalanomaly face syndromes. Atypical deletions (proximal, distal or nested) are rare and characterized mainly by normal phenotype or mild intellectual disability and variable clinical features. The pathogenetic mechanisms underlying this disorder are not completely understood. Because the 22q11.2 region harbours genes coding for transcriptional factors and chromatin remodelers, in this study, we performed analysis of genome-wide DNA methylation of peripheral blood from 49 patients with 22q11.2DS using the Illumina Infinium Methylation EPIC bead chip arrays. This cohort comprises 43 typical, 2 proximal and 4 distal deletions. We demonstrated the evidence of a unique and highly specific episignature in all typical and proximal 22q11.2DS. The sensitivity and specificity of this signature was further confirmed by comparing it to over 1500 patients with other neurodevelopmental disorders with known episignatures. Mapping the 22q11.2DS DNA methylation episignature provides both novel insights into the molecular pathogenesis of this disorder and an effective tool in the molecular diagnosis of 22q11.2DS.

Dissecting KMT2D missense mutations in Kabuki syndrome patients.

Kabuki syndrome is a rare autosomal dominant condition characterized by facial features, various organs malformations, postnatal growth deficiency and intellectual disability. The discovery of frequent germline mutations in the histone methyltransferase KMT2D and the demethylase KDM6A revealed a causative role for histone modifiers in this disease. However, the role of missense mutations has remained unexplored. Here, we expanded the mutation spectrum of KMT2D and KDM6A in KS by identifying 37 new KMT2D sequence variants. Moreover, we functionally dissected 14 KMT2D missense variants, by investigating their impact on the protein enzymatic activity and the binding to members of the WRAD complex. We demonstrate impaired H3K4 methyltransferase activity in 9 of the 14 mutant alleles and show that this reduced activity is due in part to disruption of protein complex formation. These findings have relevant implications for diagnostic and counseling purposes in this disease.

Effect of diazoxide on Friedreich ataxia models.

Friedreich ataxia (FRDA) is an inherited recessive disorder caused by a deficiency in the mitochondrial protein frataxin. There is currently no effective treatment for FRDA available, especially for neurological deficits. In this study, we tested diazoxide, a drug commonly used as vasodilator in the treatment of acute hypertension, on cellular and animal models of FRDA. We first showed that diazoxide increases frataxin protein levels in FRDA lymphoblastoid cell lines, via the mammalian target of rapamycin (mTOR) pathway. We then explored the potential therapeutic effect of diazoxide in frataxin-deficient transgenic YG8sR mice and we found that prolonged oral administration of 3 mpk/d diazoxide was found to be safe, but produced variable effects concerning efficacy. YG8sR mice showed improved beam walk coordination abilities and footprint stride patterns, but a generally reduced locomotor activity. Moreover, they showed significantly increased frataxin expression, improved aconitase activity, and decreased protein oxidation in cerebellum and brain mitochondrial tissue extracts. Further studies are needed before this drug should be considered for FRDA clinical trials.

Correction: A restricted spectrum of missense KMT2D variants cause a multiple malformations disorder distinct from Kabuki syndrome.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A restricted spectrum of missense KMT2D variants cause a multiple malformations disorder distinct from Kabuki syndrome.

PURPOSE: To investigate if specific exon 38 or 39 KMT2D missense variants (MVs) cause a condition distinct from Kabuki syndrome type 1 (KS1). METHODS: Multiple individuals, with MVs in exons 38 or 39 of KMT2D that encode a highly conserved region of 54 amino acids flanked by Val3527 and Lys3583, were identified and phenotyped. Functional tests were performed to study their pathogenicity and understand the disease mechanism. RESULTS: The consistent clinical features of the affected individuals, from seven unrelated families, included choanal atresia, athelia or hypoplastic nipples, branchial sinus abnormalities, neck pits, lacrimal duct anomalies, hearing loss, external ear malformations, and thyroid abnormalities. None of the individuals had intellectual disability. The frequency of clinical features, objective software-based facial analysis metrics, and genome-wide peripheral blood DNA methylation patterns in these patients were significantly different from that of KS1. Circular dichroism spectroscopy indicated that these MVs perturb KMT2D secondary structure through an increased disordered to ɑ-helical transition. CONCLUSION: KMT2D MVs located in a specific region spanning exons 38 and 39 and affecting highly conserved residues cause a novel multiple malformations syndrome distinct from KS1. Unlike KMT2D haploinsufficiency in KS1, these MVs likely result in disease through a dominant negative mechanism.

Kabuki syndrome: international consensus diagnostic criteria.

BACKGROUND: Kabuki syndrome (KS) is a clinically recognisable syndrome in which 70% of patients have a pathogenic variant in KMT2D or KDM6A. Understanding the function of these genes opens the door to targeted therapies. The purpose of this report is to propose diagnostic criteria for KS, particularly when molecular genetic testing is equivocal. METHODS: An international group of experts created consensus diagnostic criteria for KS. Systematic PubMed searches returned 70 peer-reviewed publications in which at least one individual with molecularly confirmed KS was reported. The clinical features of individuals with known mutations were reviewed. RESULTS: The authors propose that a definitive diagnosis can be made in an individual of any age with a history of infantile hypotonia, developmental delay and/or intellectual disability, and one or both of the following major criteria: (1) a pathogenic or likely pathogenic variant in KMT2D or KDM6A; and (2) typical dysmorphic features (defined below) at some point of life. Typical dysmorphic features include long palpebral fissures with eversion of the lateral third of the lower eyelid and two or more of the following: (1) arched and broad eyebrows with the lateral third displaying notching or sparseness; (2) short columella with depressed nasal tip; (3) large, prominent or cupped ears; and (4) persistent fingertip pads. Further criteria for a probable and possible diagnosis, including a table of suggestive clinical features, are presented. CONCLUSION: As targeted therapies for KS are being developed, it is important to be able to make the correct diagnosis, either with or without molecular genetic confirmation.

TRIM50 regulates Beclin 1 proautophagic activity.

Autophagy is a catabolic process needed for maintaining cell viability and homeostasis in response to numerous stress conditions. Emerging evidence indicates that the ubiquitin system has a major role in this process. TRIMs, an E3 ligase protein family, contribute to selective autophagy acting as receptors and regulators of the autophagy proteins recognizing endogenous or exogenous targets through intermediary autophagic tags, such as ubiquitin. Here we report that TRIM50 fosters the initiation phase of starvation-induced autophagy and associates with Beclin1, a central component of autophagy initiation complex. We show that TRIM50, via the RING domain, ubiquitinates Beclin 1 in a K63-dependent manner enhancing its binding with ULK1 and autophagy activity. Finally, we found that the Lys-372 residue of TRIM50, critical for its own acetylation, is necessary for its E3 ligase activity that governs Beclin1 ubiquitination. Our study expands the roles of TRIMs in regulating selective autophagy, revealing an acetylation-ubiquitination dependent control for autophagy modulation.

GNB5 Mutations Cause an Autosomal-Recessive Multisystem Syndrome with Sinus Bradycardia and Cognitive Disability.

GNB5 encodes the G protein ß subunit 5 and is involved in inhibitory G protein signaling. Here, we report mutations in GNB5 that are associated with heart-rate disturbance, eye disease, intellectual disability, gastric problems, hypotonia, and seizures in nine individuals from six families. We observed an association between the nature of the variants and clinical severity; individuals with loss-of-function alleles had more severe symptoms, including substantial developmental delay, speech defects, severe hypotonia, pathological gastro-esophageal reflux, retinal disease, and sinus-node dysfunction, whereas related heterozygotes harboring missense variants presented with a clinically milder phenotype. Zebrafish gnb5 knockouts recapitulated the phenotypic spectrum of affected individuals, including cardiac, neurological, and ophthalmological abnormalities, supporting a direct role of GNB5 in the control of heart rate, hypotonia, and vision.