Compound Heterozygous Variants Research Articles

Case Report: Novel ADA2 variants cause atypical adenosine deaminase 2 deficiency

Case presentationA girl aged 2 years and 5 months presented to the hospital with chief complaints of intermittent fever and weakness of the left limb for more than 1 month. The child had transient urticaria appearing on her face for 5 days. The inflammatory biomarkers were significantly increased. Brain MRI showed multiple ischemic lesions in the brain’s small vessels. The patient exhibited significant systemic inflammation and multiple vasculitis. Whole-exome sequencing showed c.1358A>G p. (Tyr453Cys) and c.1082-7T>A compound heterozygous variants in the adenosine deaminase 2 (ADA2) gene, of which the c.1082-7T>A variant has not been reported yet in previous literature. Peripheral blood mRNA reverse transcription-Sanger sequencing confirmed that this variant affected mRNA splicing, resulting in a frameshift with premature stop codon c.1083_1103del p. (Leu362Glnfs*45). Peripheral blood test suggested a significant decrease in ADA2 activity. Eventually, the patient was diagnosed with deficiency of adenosine deaminase 2 (DADA2). Her condition improved after treatment with etanercept. She had no more fevers, and no hemiplegia attacks were observed during the 3 years of follow-up.ConclusionFever and hemiplegia were the main manifestations in this patient, without typical rashes. DADA2 was finally confirmed by enzymology and genetic testing, and we believe this is the first reported case of the c.1082-7T>A intronic variant in DADA2, and the RNA studies conducted in this case have been pivotal in assessing its pathogenicity.

Pyridoxal phosphate binding protein (PLPBP) deficiency mimicking opsoclonus‐myoclonus‐ataxia syndrome

AbstractIntroductionGenetic and metabolic conditions can mimic diagnoses such as hypoxic‐ischemic encephalopathy, meningoencephalitis, epilepsy, and opsoclonus‐myoclonus‐ataxia syndrome (OMAS). Without a high index of suspicion and proper testing, diagnoses can be missed, and treatment delayed.MethodsA 3‐year‐old girl with a history of neonatal seizures and previous nondiagnostic epilepsy gene panel presented with seizures, behavioral changes, and discrete episodes of myoclonus, tremors, and abnormal eye movements following a viral illness.Objective and InterpretationInitial evaluation was concerning for OMAS, though metabolic causes remained on the differential. Metabolic testing revealed elevated glycine and glutamine, suggestive of a possible inborn error of metabolism. Whole exome sequencing demonstrated compound heterozygous variants in the PLPBP gene associated with pyridoxine‐dependent epilepsy (PDE), consistent with her clinical presentation and leading to her diagnosis of PLPBP deficiency.DiscussionClinicians should gauge the indications, advantages, and limitations of targeted sequencing panels versus whole exome sequencing. Continued evaluation is recommended in patients with a history of neonatal and infantile epilepsy, especially if they present with episodic crises related to viral illness even if prior genetic and metabolic investigations have been nondiagnostic. This report also highlights the clinical overlap between PLPBP deficiency and OMAS, and the differences in pathophysiology, treatment pathways, and implications.

Case Report: Prenatal diagnosis of novel compound heterozygous variants in WDR35 gene causing short-rib thoracic dysplasia 7 with or without polydactyly

BackgroundWhole exome sequencing (WES) technology has been increasingly used for the etiological diagnosis of fetuses with ultrasound anomalies. In this article, we report a novel deletion compound combined with a causative variant in WDR35 gene leading to short-rib thoracic dysplasia 7 (SRTD7) with or without polydactyly using WES.MethodsThis study involved a Chinese fetus with clinical features of skeletal dysplasia on ultrasound imaging, in whom chromosome abnormalities and copy number variants (CNVs) were detected by chromosomal microarray analysis (CMA), and sequence variants were detected by WES. The obtained results were further verified by Sanger sequencing or real time quantitative PCR (qPCR).ResultsNo chromosomal abnormality or CNVs were identified in the fetus by CMA. However, WES result revealed a 14.38-kb large novel deletion compound covering exon 7 to exon 12 combined with a missense variant NM_001006657.2:c.932G>T(p.W311l) in WDR35. Both variants were thought of as pathogenic, which was further confirmed by Sanger sequencing and qPCR. In addition, two compound heterozygous variants NM_015102.5:c.[1196A>G(p.E399G)];[1972C>T(p.R658*)] in NPHP4 gene were also identified in the fetus, which may be partially responsible for fetal kidney hyperechogenicity and oligohydramnios.ConclusionThis is the first study reporting a novel deletion compound combined with a causative missense variant in WDR35 leading to SRTD7. This finding may broaden the spectrum of variants of WDR35 gene and provide a valuable reference for clinical counseling of related abnormalities in pregnancies.

Clinical follow-up of 2 families with glomerulopathy caused by COQ8B gene variants and literature review

BackgroundPrimary coenzyme Q10 (CoQ10) deficiency is an autosomal recessive genetic disease caused by mitochondrial dysfunction. Variants in Coenzyme Q8B (COQ8B) can cause primary CoQ10 deficiency. COQ8B-related glomerulopathy is a recently recognized glomerular disease that most often presents as steroid-resistant nephrotic syndrome (SRNS) in childhood. The disease often progresses to kidney failure and the renal histopathology is most commonly focal segmental glomerulosclerosis (FSGS).MethodsFour SRNS cases (2 females and 2 males) from 2 unrelated families who were followed clinically for nearly 3 years. Clinical exome testing and analyses were performed by MyGenostics Laboratory in China to evaluate unexplained proteinuria given the strong family history of glomerular disease and histologic evidence of SRNS. Pathogenic variants were identified in COQ8B in the exome studies and confirmed by direct sequencing.ResultsClinical exome sequencing revealed biallelic variants of the COQ8B gene in 2 families. In the Family 1, the oldest of three affected siblings died of renal failure at 11 years of age. Based on the results of genetic testing which identified a homozygous variant of COQ8B, the other two affected siblings with mild proteinuria and normal renal function were treated with CoQ10 oral supplementation at an early stage. Coenzyme Q10 treatment was effective in reducing proteinuria levels in both patients from Family 1 over the first 6 months and the two patients still have low-level proteinuria and normal renal function at nearly three years. In Family 2, clinical exome sequencing revealed a compoundheterozygous variants of COQ8B in a patient with biopsy- proven FSGS. His disease was unresponsive to prior treatment with glucocorticoids and cyclosporine. Oral CoQ10 was initiated based on his genetic diagnosis and was it was effective in reducing proteinuria over the first 5 months months of therapy. However after 1 year, his disease progressed tokidney failure. Kidney transplantation was performed at 5 years of age and his condition has been stable without rejection and no recurrence of disease.ConclusionsCOQ8B gene variant-related glomerulopathy often presents as SRNS without obvious extrarenal manifestations. The histopathology is mainly FSGS and follows an autosomal recessive mode of inheritance. Some patients may benefit from early coenzyme Q10 supplementation. For patients whose disease progresses to kidney failure, kidney transplantation can be an effective treatment. For children with unexplained proteinuria and abnormal renal function, genetic testing should be performed early in the course of disease to guide therapy where possible and improve prognosis.

The impact and clinical implication of variants in the start codon of HBA gene on the phenotype of thalassemia

To analyze the correlation between variants in the start codon of the α-globin gene and phenotypes of thalassemia, so as to provide a basis for the diagnosis and prevention of α-thalassemia. A retrospective study was conducted on 7 patients diagnosed by Yangjiang People's Hospital and Guangzhou Hybribio Co. Ltd., from June 2019 to October 2022. Routine blood tests and hemoglobin electrophoresis were carried out. Potential variants were identified through polymerase chain reaction (PCR) combined with Reverse dot blotting (RDB), Gap-PCR, and Sanger sequencing. This study has been approved by the Medical Ethics Committee of People's Hospital of Yangjiang (Ethics No: 20240001). For the 7 patients, results of blood routine test of one case was unknown, and that of another was normal. The remaining 5 cases had presented with microcytic hypochromic anemia. The results of hemoglobin electrophoresis showed that one case had normal Hb A and slightly lower Hb A2, whilst another had significantly decreased Hb A and Hb A2, in addition with the appearance of a Hb H band. The content of Hb Bart's in four neonates was ≥ 0.4%. The remaining one case had no result. Genetic testing has identified 4 rare start codon mutations, namely HBA2: c.2delT, HBA2: c.1A>G, HBA2: c.1A>T, and HBA1: c.2T>C. Among these, Patient 1 had harbored compound heterozygous variants of HBA2: c.427T>C (Hb CS) and HBA2: c.2delT. Patient 4 had harbored compound heterozygous variants of HBA2: c.1A>G and Southeast Asian type deletion. Heterozygotes with HBA start codon variants usually present as silent or mild thalassemia, and the symptoms of anemia may deteriorate when combined with other α-thalassemia variant. The HBA2: c.1A>T start codon variant was unreported previously in China. The detection of start codon variants has helped to clarify the causes of anemia, genetic counseling, and guidance for reproduction.

Bone marrow transplantation reverses metabolic alterations in multiple sulfatase deficiency: a case series

BackgroundMultiple sulfatase deficiency (MSD) is an exceptionally rare neurodegenerative disorder due to the absence or deficiency of 17 known cellular sulfatases. The activation of all these cellular sulfatases is dependent on the presence of the formylglycine-generating enzyme, which is encoded by the SUMF1 gene. Disease-causing homozygous or compound heterozygous variants in SUMF1 result in MSD. Other than symptomatic treatment, no curative therapy exists as of yet for MSD. Eight out of these 17 sulfatases are primarily localized in the lysosome.MethodsTwo siblings with attenuated MSD underwent hematopoietic cell transplantation (HCT), evaluating the possibility of lysosomal enzymatic cross-correction from the donor cells.ResultsThere is evidence of correction of currently available biomarkers within 3 months post-HCT. Untargeted metabolomics also shows continued correction of multiple biochemical abnormalities in the post-HCT period. Furthermore, this article also presents the neuropsychological outcomes of these children as well as the results of untargeted metabolomics analysis in this condition.ConclusionsThese data suggest biochemical benefits post-transplant along with slowing of disease progression. Long-term follow-up is necessary to fully evaluate the therapeutic benefit of HCT in MSD.

Identification of novel CDH23 heterozygous variants causing autosomal recessive nonsyndromic hearing loss.

Hearing loss adversely impacts language development, acquisition, and the social and cognitive maturation of affected children. The hearing loss etiology mainly includes genetic factors and environmental factors, of which the former account for about 50-60%. This study aimed to investigate the genetic basis of autosomal recessive non-syndromic hearing loss (NSHL) by identifying and characterizing novel variants in the CDH23 gene. Furthermore, it seeks to determine the pathogenic potential of the noncanonical splice site variant c.2398-6G > A. Comprehensive clinical evaluation and whole-exome sequencing (WES) were performed on the girl. The WES analysis revealed two novel variants in the CDH23 gene, associated with nonsyndromic deafness 12 (DFNB12). To further explore the pathogenicity of these variants, functional studies involving in vivo splicing analysis were performed on the novel noncanonical splice site variant, c.2398-6G > A, which was initially classified as a variant of uncertain significance (VUS). Whole-exome sequencing of the patient identified two compound heterozygous variants in CDH23: c.2398-6G > A, a noncanonical splice site variant, and c.6068C > A (p. Ser2023Ter), a nonsense mutation. In vitro splicing assays demonstrated that c.2398-6G > A caused aberrant splicing, leading to a frameshift (p. Val800Alafs*6) and the production of a truncated protein, as confirmed by structural protein analysis. The study revealed novel mutations as likely pathogenic, linking both variants to autosomal recessive NSHL. Our analyses revealed novel compound heterozygous mutations in CDH23 associated with autosomal recessive NSHL, thereby expanding the mutational landscape of CDH23-related hearing loss and increasing knowledge about the CDH23 splice site variants.

Transcriptome-wide outlier approach identifies individuals with minor spliceopathies.

RNA-sequencing has improved the diagnostic yield of individuals with rare diseases. Current analyses predominantly focus on identifying outliers in single genes that can be attributed to cis-acting variants within or near that gene. This approach overlooks causal variants with trans-acting effects on splicing transcriptome-wide, such as variants impacting spliceosome function. We present a transcriptomics-first method to diagnose individuals with rare diseases by examining transcriptome-wide patterns of splicing outliers. Using splicing outlier detection methods - FRASER and FRASER2 - we identified splicing outliers from whole blood for 390 individuals from the Genomics Research to Elucidate the Genetics of Rare Diseases (GREGoR) and Undiagnosed Diseases Network (UDN) consortia. We examined all samples for excess intron retention events in minor intron containing genes. Minor introns, which make up about 0.5% of all introns in the human genome, are removed by small nuclear RNAs (snRNAs) in the minor spliceosome. This approach identified five cases with excess intron retention events in minor intron containing genes, all of which were found to harbor rare, biallelic variants in the minor spliceosome snRNAs. Four had rare, compound heterozygous variants in RNU4ATAC. These results led to the reclassification of four variants. Additionally, one case had rare, highly conserved, compound heterozygous variants in RNU6ATAC that may disrupt the formation of the catalytic spliceosome, suggesting a novel disease-gene candidate. These results demonstrate that examining RNA-sequencing data for known transcriptome-wide signatures can increase the diagnostic yield of individuals with rare diseases, provide variant-to-functional interpretation of spliceopathies, and potentially uncover novel disease genes.

Recessive but damaging alleles of muscle-specific ribosomal protein gene RPL3L drive neonatal dilated cardiomyopathy.

The heart employs a specialized ribosome in its muscle cells to translate genetic information into proteins, a fundamental adaptation with an elusive physiological role. Its significance is underscored by the discovery of neonatal patients suffering from often fatal heart failure caused by rare compound heterozygous variants in RPL3L, a muscle-specific ribosomal protein that replaces the ubiquitous RPL3 in cardiac ribosomes. RPL3L -linked heart failure represents the only known human disease arising from mutations in tissue-specific ribosomes, yet the underlying pathogenetic mechanisms remain poorly understood despite an increasing number of reported cases. While the autosomal recessive inheritance pattern suggests a loss-of-function mechanism, Rpl3l -knockout mice display only mild phenotypes, attributed to up-regulation of the ubiquitous Rpl3. Interestingly, living human knockouts of RPL3L have been identified. Here, we report two new cases of RPL3L -linked severe neonatal heart failure and uncover an unusual pathogenetic mechanism through integrated analyses of population genetic data, patient cardiac tissue, and isogenic cells expressing RPL3L variants. Our findings demonstrate that patient hearts lack sufficient RPL3 compensation. Moreover, contrary to a simple loss-of-function mechanism often associated with autosomal recessive diseases, RPL3L -linked disease is driven by a combination of gain-of-toxicity and loss-of-function. Most patients carry a recurrent toxic missense variant alongside a non-recurrent loss-of-function variant. The non-recurrent variants trigger partial compensation of RPL3 similar to Rpl3l -knockout mice. In contrast, the recurrent missense variants exhibit increased affinity for the RPL3/RPL3L chaperone GRWD1 and 60S biogenesis factors, sequester 28S rRNA in the nucleus, disrupt ribosome biogenesis, and trigger severe cellular toxicity that extends beyond the loss of ribosomes. These findings elucidate the pathogenetic mechanisms underlying muscle-specific ribosome dysfunction in neonatal heart failure, providing critical insights for genetic screening and therapeutic development. Our findings also suggest that gain-of-toxicity mechanisms may be more widespread in autosomal recessive diseases, especially for those involving genes with paralogs.

Whole exome sequencing identifies ABHD14A and MRNIP as novel candidate genes for developmental language disorder

Developmental language disorder (DLD) is a neurodevelopmental disorder involving impaired language abilities. Its genetic etiology is heterogeneous, involving rare variations in multiple susceptibility loci. However, family-based studies on gene mutations are scarce. We performed whole-exome sequencing (WES) of a first-time-described Tunisian-family with DLD. Analyses of segregation patterns with stringent filtering of the exome data identified disease-causing compound heterozygous variants. In the MRNIP gene, two variants were detected including a synonymous low-frequency variant c.345G > C and a nonsense rare variant c.112G > A predicted pathogenic. In the ABHD14A gene, four variants were identified including a rare missense variant c.689T > G and three splice-site variants c.70-8C > T, c.282-25A > T and c.282-10G > C with low-frequency MAF < 5%. Complementary analyses showed that these variants are predicted pathogenic and the missense variant Leu230Arg significantly affects the stability and structure modelling of the ABHD14A protein. Biological functions and interconnections analyses predicted the potential roles of ABHD14A and MRNIP in neuronal development pathways. These results suggest ABHD14A and MRNIP, as putative candidate genes for DLD susceptibility. Our findings reveal the involvement of novel candidate genes in the genetic etiology of DLD and explore the potential future utility of WES in the diagnosis of such complex disorders.

DYNC2H1 splicing variants causing severe prenatal short-rib polydactyly syndrome and postnatal orofaciodigital syndrome.

The DYNC2H1 gene has been associated with short-rib polydactyly syndrome (SRPS), among other skeletal ciliopathies. Two cases are presented of distinctive phenotypes resulting from splicing variants in DYNC2H1. The first is a 14-week-old fetus with enlarged nuchal translucency, oral hamartoma, malformed uvula, bifid epiglottis, short ribs, micromelia, long bone agenesis, polysyndactyly, heart defect, pancreatic cysts, multicystic dysplastic kidney, megabladder and trident acetabulum. A ciliopathies NGS panel revealed two compound heterozygous variants in DYNC2H1: c.7840-18T>G r.7841_7964del p.Gly2614Aspfs*5 and c.11070G>A r.11044_11116del p.Ile3682Aspfs*2. Both variants were initially classified as variants of uncertain significance but were reclassified as likely pathogenic after PCR-based RNA testing. The second is an 11-year-old overweight male with multiple accessory oral frenula, median cleft lip and alveolar ridge, polysyndactyly, brachydactyly, normal rib length, and hypogonadism. Exome sequencing revealed two compound heterozygous variants in DYNC2H1: c.6315del p.(Thr2106Glnfs*7), classified as likely pathogenic, and c.3303-16A>G p.(?), classified as a variant of uncertain significance. PCR-based RNA testing suggested that c.3303-16A>G induces an in-frame deletion: r.3303_3458del p.Asp1102_Arg1153del, although the normal transcript is still produced. These results are consistent with both SRPS type I/III in the first case and orofaciodigital syndrome in the second, an unprecedented description. This work thus improves the clinical and molecular knowledge of the phenotypes associated with splicing variants in the DYNC2H1 gene.

Bernard-Soulier syndrome case caused by novel compound heterozygous variants in the GP1BA gene: Case report

Bernard-Soulier syndrome case caused by novel compound heterozygous variants in the GP1BA gene: Case report

Biallelic TXNDC15 variants associated with Joubert syndrome-related molar tooth sign and forebrain malformation.

TXNDC15 encodes thioredoxin domain-containing protein 15, a protein disulfide isomerase that plays a role in ciliogenesis. Biallelic TXNDC15 variants have been reported in six individuals of Meckel syndrome (MKS) with perinatal lethal phenotypes, but have not been reported in patients with Joubert syndrome (JS). Here, we describe a 1-year-old female patient with compound heterozygous TXNDC15 variants demonstrating cerebellar vermis hypoplasia with the molar tooth sign, mild holoprosencephaly, and cortical abnormalities. She had severe developmental delay and epilepsy. Her clinical features were similar to those of JS, but distinctive forebrain abnormalities were also noted including mild holoprosencephaly and cortical abnormalities, which have been reported in a severe form of ciliopathy. Biallelic TXNDC15 variants manifest as overlapping phenotypes of JS and MKS, including the molar tooth sign, cortical dysgenesis, and mild holoprosencephaly. This report supports the hypothesis that JS and MKS are spectrum ciliopathy disorders with overlapping causative genes and hypomorphic TXNDC15 variants might contribute to JS.

Interpreting Variants of Uncertain Significance in PCD: Abnormal Splicing Caused by a Missense Variant of DNAAF3.

Primary ciliary dyskinesia (PCD) is a rare autosomal recessive disorder characterized by dysfunction of motile cilia. While approximately 50 genes have been identified, around 25% of PCD patients remain genetically unexplained; elucidating the pathogenicity of specific variants remains a challenge. Whole exome sequencing (WES) and Sanger sequencing were conducted to identify potential pathogenic variants of PCD. Minigene assays were performed to evaluate the pathogenicity of variants. Transmission electron microscopy (TEM) and high-speed video analysis (HSVA) were conducted to analyze the function of cilia in respiratory epithelial cells. We identified two variants of DNAAF3: c.557G>A, p.G186E in exon 5, and c.1364G>A, p.G455D at the terminal nucleotide of exon 10 in a 16-year-old male patient. Through a minigene assay, we demonstrated that the c.1364G>A variant led to a four-nucleotide skipping. The cilia in epithelial ciliary cells of the proband were almost immotile. The absence of outer dynein arms and inner dynein arms was also observed. Our study identified two compound heterozygous variants of DNAAF3, a pathogenic gene for PCD, and proved that a novel missense variant c.1364G>A affects splicing. Our findings not only expanded the spectrum of mutations in the DNAAF3 gene but also highlighted the importance of investigating variants of uncertain significance (VUS) for comprehensive genetic diagnoses.

A Possible Phenotype-to-Genotype Association of Novel Single-Nucleotide Variants in the Coding Exons of the ZNF469 Gene to Arterial Aneurysmal and Dissection Diseases.

After reporting the first known clinical case associating compound heterozygous single-nucleotide variants in Exon 2 of ZNF469 to aortic aneurysmal and iliac dissection, we began prospective surveillance in our vascular genetic practice for similar cases. Herein, we present nine (9) subjects from a total cohort of 135 with arterial aneurysms or dissections who revealed single-nucleotide variants in ZNF469 with no other alterations in a panel of 35 genes associated with aneurysmal and dissection disorders. Five out of nine (5/9) single-nucleotide variants were in Exon 1, and four out of nine (4/9) mutations were in Exon 2, both of which are principal coding exons for this gene. Eight out of nine (8/9) were ACMG variants of unknown significance (VUSs), and one out of nine (1/9) was an ACMG pathogenic mutation previously associated to brittle cornea syndrome (BCS). Of our nine subjects, four (44.4%) experienced clinically significant vascular dissection, and four (44.4%) had a family history of one or more first-degree relatives with aneurysmal or dissection diseases. This novel genetic case series significantly strengthens our initial discovery of ZNF469's potential association with arterial aneurysmal/dissection diseases through the study of this cohort of unrelated patients.

Unravelling variants in Farber disease: diagnostic and prenatal challenges in atypical presentations

Abstract Farber disease (FD; OMIM #228000), also known as Farber's lipogranulomatosis, is a rare lysosomal storage disease caused by acid ceramidase deficiency. Clinically, FD is typically identified by a triad of symptoms: subcutaneous nodules, joint pain, and voice hoarseness. However, diagnosing mild or attenuated variants can be difficult, as some symptoms may be absent or overlooked. In such cases, genetic testing is essential for confirming the diagnosis and enabling prenatal diagnosis. Case presentation A couple presented with a history of losing two children at 1.5 years of age, both of whom exhibited severe developmental delay, hypotonia, weak cry, and progressive skin lesions. Neither child underwent genetic testing, and no DNA samples were preserved. Subsequent parental carrier screening identified a heterozygous variant of uncertain significance (VUS) in the ASAH1 (N-Acylsphingosine Amidohydrolase 1) (OMIM #613468) gene in both parents. Exome sequencing was later performed on the couple’s third affected child, revealing compound heterozygous variants in the ASAH1 gene, consistent with those found in the parents. This case presented with atypical manifestations of FD, necessitating comprehensive investigations, including laboratory tests, radiological assessments, genetic analysis, and histopathological examination of skin biopsy samples. These findings led to the identification of likely pathogenic mutations in ASAH1. Based on these results, prenatal diagnosis was successfully conducted in the couple's fourth pregnancy. Conclusion This case illustrates the challenges of diagnosing FD when atypical presentations and genetic variants of uncertain significance are involved. The absence of classic symptoms like subcutaneous nodules and the presence of an ASAH1 gene variant complicated the diagnosis and delayed options for prenatal diagnosis in future pregnancies. It underscores the critical role of genetic testing and the need to reclassify VUS through detailed genotype–phenotype correlations to enhance diagnostic accuracy and support timely interventions.

Severe congenital liver disease caused by FOCAD gene compound heterozygous variation in a child

Severe congenital liver disease caused by FOCAD gene compound heterozygous variation in a child

Use of patient-derived cell models for characterization of compound heterozygous hypomorphic C2CD3 variants in a patient with isolated nephronophthisis.

Primary ciliopathies are a heterogeneous group of rare disorders predominantly caused by autosomal-recessive genetic variants that disrupt non-motile ciliary function. They often manifest as a syndromic phenotype, frequently involving the kidney. Biallelic pathogenic variants in C2CD3 disrupt ciliogenesis and Sonic Hedgehog (SHH) signaling, resulting in a severe ciliopathy (Orofaciodigital syndrome XIV, OMIM 615948). We present compound heterozygous missense variants in C2CD3 that partially disrupt ciliary function in a patient with isolated renal disease. Exome sequencing identified biallelic C2CD3 missense variants (p.Pro168Leu; p.Thr2079Met). Patient-derived fibroblasts and urinary renal epithelial cells (URECs), and human RPE-1 C2CD3 knockout (KO) cell-lines were used for in vitro studies. Cilia length was significantly shorter in patient-derived fibroblasts compared to an unaffected sibling (2.309 vs. 2.850μm, P < 0.0001), while URECs showed significantly shortened cilia (2.068 vs. 2.807μm, P < 0.0001) and a 40.8% reduction in ciliation (P < 0.001). The latter was not observed in fibroblasts, suggesting a kidney-specific effect. SHH signaling was dysregulated in patient cells as expression of GLI3 activator protein and GLI1 mRNA was significantly reduced. C2CD3 localization to the basal body was significantly reduced in patient URECs. Finally, rescue experiments in C2CD3 KO RPE-1 cells corroborated these findings by demonstrating a reduced capacity to restore ciliogenesis for each variant. Biallelic hypomorphic missense variants in C2CD3 may contribute to an isolated nephronophthisis phenotype with impaired ciliogenesis and SHH signaling. Our findings underscore the importance of functional testing to characterize candidate gene-disease relationships in patients with nephropathy of unknown etiology.

Variants in EP400, encoding a chromatin remodeler, cause epilepsy with neurodevelopmental disorders.

EP400 encodes a core catalytic ATPase subunit of ATP-dependent chromatin remodeling complexes. The gene-disease association of EP400 is undetermined. In this study, we performed trio-based whole-exome sequencing in a cohort of 402 families with epilepsy and neurodevelopmental disorders (NDDs) and identified compound heterozygous EP400 variants in six unrelated individuals. Six additional EP400 individuals were recruited via the match platform of China, including two de novo heterozygous and four compound heterozygous variants. The individual with a heterozygous de novo frameshift variant presented with NDDs, while the others exhibited epilepsy and NDDs, explained by the damaged genetic dependence quantity. EP400 presented significantly higher excesses of variants in the individuals. Clustering analysis revealed that the majority paralogs of EP400 were associated with NDDs/epilepsy and co-expressed highly with EP400. Analysis of the spatiotemporal expression indicated that EP400 is highly expressed in the developing brain and cells during differentiation, indicating its vital role in neurodevelopment; EP400 is predominantly expressed in inhibitory neurons in the early stage but in excitatory neurons in the mature stage. The development-dependent expression pattern of neuron specificity explained the favorable outcome of epilepsy. Knockdown of EP400 ortholog in Drosophila caused significantly increased susceptibility to seizures and abnormal neuronal firing. The ep400 crispant zebrafish exhibited brain developmental abnormalities, poorer adaptability, lower response to stimulation, epileptic discharges, abnormal cellular apoptosis, and increased susceptibility to seizures. Transcriptome analysis showed that ep400 deficiency caused expressional dysregulation of 84 epilepsy/NDD-associated genes, including 11 highly dose-sensitive genes. This study identified EP400 as a causative gene of epilepsy/NDDs.

Combined in silico/in vitro approaches for identifying modulators of the activity of the p.Tyr110Cys Carnitine O-Acetyltransferase (CRAT) variant associated to an early onset case of Leigh syndrome.

Carnitine O-acetyltransferase (CRAT) is a crucial enzyme involved in mitochondrial energy metabolism. Alterations in CRAT activity have emerged as significant contributors to the pathogenesis of Leigh syndrome and related mitochondrial disorders. In this study we employed an integrated approach combining in silico docking analysis and virtual screening of chemical libraries with subsequent in vitro validation to identify small molecule modulators of the activity of the wild type (WT) CRAT and the p.Tyr110Cys (Y110C) variant associated to an early onset case of Leigh syndrome. Through 3D molecular modeling, docking simulations, and virtual screening of chemical libraries, potential CRAT modulators were prioritized based on their predicted binding affinities and interactions with the 3D models of the WT-CRAT and of the p.Tyr110Cys-CRAT mutant. The performed in silico analyses were validated through in vitro assays on the purified recombinant CRAT proteins and cell-lysates from control fibroblasts and the fibroblasts of a patient with genetic diagnosis of CRAT-deficiency, carrying the compound heterozygous missense variants in the CRAT gene, namely p.Tyr110Cys and p.Val569Met. Based on the above screening by applying the indicated filtering strategy and mentioned criteria, 3 commercially available approved drugs (also known for their possible interactions with mitochondria) namely glimepiride, artemisinin and dorzolamide, as well as suramin (already known for its ability to interact with mitochondrial proteins) were tested in in vitro assays. We found that suramin (1-1000 μM) dose-dependently inhibited the activity of both WT-CRAT and p.Tyr110Cys_CRAT variant. Artemisinin (0.1-200μM) dose-dependently stimulated the activity ofthe recombinant p.Tyr110Cys CRAT mutant, whereas glimepiride and dorzolamide did not change the activity of these proteins towards acetyl-CoA. This study demonstrates the effectiveness of this combined approach in identifying novel compounds for modulating CRAT enzyme activity, providing valuable insights for potential therapeutic interventions targeting CRAT-related disorders.