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Rare diseases are estimated to affect 3.5%–5.9% of the population worldwide and are difficult to diagnose. Genome analysis is useful for diagnosis. However, since some variants, especially missense variants, are also difficult to interpret, tools to accurately predict the effect of missense variants are very important and needed. Here we developed a method, “VarMeter”, to predict whether a missense variant is damaging based on Gibbs free energy and solvent-accessible surface area calculated from the AlphaFold 3D protein model. We applied this method to the whole-exome sequencing data of 900 individuals with rare or undiagnosed disease in our in-house database, and identified four who were hemizygous for missense variants of arylsulfatase L (ARSL; known as the genetic cause of chondrodysplasia punctata 1, CPDX1). Two individuals had a novel Ser89 to Asn (Ser89Asn) or Arg469 to Trp (Arg469Trp) substitution, respectively predicted as “damaging” or “benign”; the other two had an Arg111 to His (Arg111His) or Gly117 to Arg (Gly117Arg) substitution, respectively predicted as “damaging” or “possibly damaging” and previously reported in patients showing clinical manifestations of CDPX1. Expression and analysis of the missense variant proteins showed that the predicted pathogenic variants (Ser89Asn, Arg111His, and Gly117Arg) had complete loss of sulfatase activity and reduced protease resistance due to destabilization of protein structure, while the predicted benign variant (Arg469Trp) had activity and protease resistance comparable to those of wild-type ARSL. The individual with the novel pathogenic Ser89Asn variant exhibited characteristics of CDPX1, while the individual with the benign Arg469Trp variant exhibited no such characteristics. These findings demonstrate that VarMeter may be used to predict the deleteriousness of variants found in genome sequencing data and thereby support disease diagnosis.

NAA10 is an enzyme involved in the N-terminal acetylation of proteins. NAA10-related syndrome is caused by a pathogenic variant of NAA10 on X chromosome, resulting in several phenotypes, including mental retardation, hypotonia, growth retardation, and various external malformations, with varying degrees of severity. With regard to cardiac diseases, hypertrophic cardiomyopathy is a possible complication. Some mutations are also associated with long QT syndrome. Herein, we describe the case of a 7-year-old boy with a novel NAA10 mutation who experienced cardiopulmonary arrest possibly due to long QT syndrome and was implanted with a subcutaneous implantable cardioverter defibrillator. Résumé: La NAA10 est une enzyme qui intervient dans l’acétylation N-terminale des protéines. Le syndrome lié au gène NAA10 est causé par un variant pathogène du NAA10 sur le chromosome X qui entraîne plusieurs phénotypes, comme une déficience intellectuelle, une hypotonie, un retard de croissance ou différentes malformations externes, et ce, à divers degrés de sévérité. En ce qui concerne les maladies cardiaques, une cardiomyopathie hypertrophique est une complication possible. Certaines mutations sont également associées au syndrome du QT long. Nous décrivons ici le cas d’un garçon âgé de sept ans qui présente une nouvelle mutation du gène NAA10 et qui a fait un arrêt cardiorespiratoire, possiblement en raison d’un syndrome du QT long. L’enfant a reçu un défibrillateur cardiaque implantable sous-cutané.

Abstract Background Although infantile hepatic hemangioma and hepatic mesenchymal hamartoma are relatively common in benign pediatric liver tumors, coexistence of the two tumors is rare. Placental mesenchymal dysplasia is also a rare disorder. We report the case of a baby girl born after a pregnancy complicated by placental mesenchymal dysplasia, who developed both infantile hepatic hemangioma and hepatic mesenchymal hamartoma. Case presentation The patient was born at 32 weeks and 5 days of gestation for impending placental abruption, weighing 1450 g. Liver tumors, composed of both hypervascular solid and large cystic lesions, were detected after birth and markedly increased to create abdominal distention within 9 months. Diagnostic imaging suspected the coexistence of infantile hepatic hemangioma and cystic hepatic mesenchymal hamartoma. Following propranolol therapy for infantile hepatic hemangioma and needle puncture of a large cyst, the cystic lesions and adjacent hypervascular lesions were partially resected via laparotomy. Pathological findings confirmed the coexistence of hepatic mesenchymal hamartoma and infantile hepatic hemangioma, which had no association with androgenetic/biparental mosaicism. The postoperative course was uneventful, and the tumor had not regrown after 3 years. Conclusions Although the coexistence of infantile hepatic hemangioma and hepatic mesenchymal hamartoma associated with placental mesenchymal dysplasia is extremely rare, the pathological and pathogenetic similarities between these disorders suggest that they could have derived from similar embryologic origins rather than being a mere coincidence. Further follow-up is required, with careful attention to the potential for malignant hepatic mesenchymal hamartoma transformation.

Background and purposeHereditary spastic paraplegias (HSPs) are a set of heterogeneous neurodegenerative disorders characterized by bilateral lower limb spasticity. They may present from infancy onwards at any time. Although next-generation sequencing has allowed the identification of many causative genes, little is known about which genes are specifically associated with pediatric-onset variants.MethodsThis study retrospectively evaluated the genetic analyses, family history clinical courses, magnetic resonance imaging (MRI) findings, and electrophysiologic findings of patients diagnosed with HSP in childhood at a tertiary pediatric hospital in Japan. Genetic analyses were performed using direct sequencing, disease-associated panels, and whole-exome sequencing.ResultsOf the 37 patients included, 14 had a family history of HSP and 23 had a sporadic form of the disease. In 20 patients, HSP was the pure type, whereas the remaining 17 patients had complex types of HSP. Genetic data were available for 11 of the pure-type patients and 16 of those with complex types. Of these, genetic diagnoses were possible in 5 (45%) of the pure-type and 13 (81%) of the complex-type patients. SPAST variants were found in five children, KIF1A variants in four, ALS2 variants in three, SACS and L1CAM variants in two each, and an ATL1 variant in one. One child had a 10p15.3p13 duplication. Four patients with pure-type HSPs had SPAST variants and one had an ALT1 variant. The KIF1A, ALS2, SACS, and L1CAM variants and the 10p15.3p13 duplication were seen in children with complex-type HSPs, with just one complex-type patient having a SPAST variant. The identification of brain abnormalities on MRI was significantly more common among children with complex-type (11 [69%] of 16) than pure-type HSPs (one [5%] of 19) (p

Abstract Spondyloepiphyseal dysplasia congenita (SEDC) is a multisystemic skeletal disorder caused by pathogenic variants in COL2A1. Here, we report the genotype-phenotype correlations in five Japanese patients with SEDC based on their clinical and radiological findings. All five patients had novel missense variants resulting in glycine substitutions (G474V, G543E, G567S, G594R, and G1170R). Genetic testing is important for early intervention for the extraskeletal complications of SEDC. Spondyloepiphyseal dysplasia congenita (SEDC) (OMIM#183900) is an autosomal dominant chondrodysplasia characterized by disproportionate short stature, abnormal epiphyses, flattened vertebral bodies (skeletal abnormalities), and extraskeletal features, including myopia, retinal degeneration with retinal detachment, and cleft palate. SEDC is caused by a heterozygous variant in the collagen II alpha 1 (COL2A1) gene.

Little information is available on age-related electrocardiographic changes in patients with Noonan syndrome. This single-center study evaluated the electrocardiograms of patients with Noonan syndrome. We divided the patients (n = 112; electrocardiograms, 256) into four groups according to age: G1 (1 month–1 year), G2 (1–6 years), G3 (6–12 years), and G4 (>12 years). Typical Noonan syndrome-related electrocardiographic features such as left-axis deviation, abnormal Q wave, wide QRS complex, and small R wave in precordial leads were detected. A high percentage of QRS axis abnormalities was found in all groups. Significant differences in right-axis deviation (RAD) were noted among the groups: 56.5% of G1 patients showed RAD compared with 33.3% of G2, 21.1% of G3, and 19.2% of G4 patients. The small R was also significantly different among the groups: 32.6% of G1 patients showed a small R wave compared with 14.9% of G2, 8.5% of G3, and 15.4% of G4 patients. Of the 53 patients with Noonan syndrome aged 1 month to 2 years, 18 had T-positive V1 with a higher prevalence of pulmonary stenosis and cardiac interventions. QRS axis abnormalities, small R in V6, and T-positive V1 could help diagnose Noonan syndrome in infants or young children.

Various genetic and environmental factors are associated with developmental disorders (DDs). It has been suggested that interaction between genetic and environmental factors (G × E) is involved in the etiology of DDs. There are two major approaches to analyze the interaction: genome-wide and candidate gene-based approaches. In this mini-review, we demonstrate how these approaches can be applied to reveal the G × E related to DDs focusing on zebrafish and mouse models. We also discuss novel approaches to analyze the G × E associated with DDs.

Abstract Background Human 15q11–13 is responsible for Prader-Willi syndrome (PWS) and Angelman syndrome (AS) and includes several imprinted genes together with bipartite elements named AS-IC (imprinting center) and PWS-IC. These concertedly confer allele specificity on 15q11–13. Here, we report DNA methylation status of 15q11–13 and other autosomal imprinted differentially methylated regions (iDMRs) in cases with various deletions within the PWS/AS-responsible region. Methods We performed array-based methylation analysis and examined the methylation status of CpG sites in 15q11–13 and in 71 iDMRs in six cases with various microdeletions, eight cases with conventional deletions within 15q11–13, and healthy controls. Results We detected 89 CpGs in 15q11–13 showing significant methylation changes in our cases. Of them, 14 CpGs in the SNORD116s cluster presented slight hypomethylation in the PWS cases and hypermethylation in the AS cases. No iDMRs at regions other than 15q11–13 showed abnormal methylation. Conclusions We identified CpG sites and regions in which methylation status is regulated by AS-IC and PWS-IC. This result indicated that each IC had unique functions and coordinately regulated the DNA methylation of respective alleles. In addition, only aberrant methylation at iDMRs in 15q11–13 leads to the development of the phenotypes in our cases.

Abstract Background A weakness of exome analysis lies in inability to characterize aberrant splicing other than those involving consensus donor‐acceptor sequence. To overcome this limitation, we developed a novel analytic method SAVNet that combines transcriptome and exome analysis which enabled the successful detection of carriers of splicing variants in the disease‐causing genes of autosomal recessive disorders within a normal cohort. However, the clinical utility of the SAVNet analysis in delineating splicing defects in patients without a diagnosis has yet to be documented. Method We performed SAVNet analysis using the integrated analysis of exome and transcriptome analysis from the peripheral blood of the patient. The patient is an undiagnosed Japanese female patient with submucous cleft palate, scaphocephaly and intellectual disability with no words at 8 years of age. Dysmorphic features included a long face, a short palpebral fissure, thick lips with an open month, premaxillary hypoplasia, a depressed nasal bridge, and satyr ears. Result A SAVNet analysis showed that a heterozygous intronic variant located at the −10 position of exon 5 of the HNRNPK gene on chromosome 9 created a new splice acceptor sequence “ag” and led to the incorporation of 9 intronic nucleotides into the coding sequence. The mutant protein would have three extra amino acid residues, Leu‐Leu‐Gln, inserted within the critical KH domain. The patient was diagnosed as having recently delineated Au–Kline syndrome, which is characterized by cleft palate, craniosynostosis, and intellectual disability. Conclusion The successful molecular diagnosis of the presently reported patient illustrates the diagnostic utility of the SAVNet analysis as an innovative way of implementing an integrated exome‐transcriptome analysis in clinical settings.

Intellectual disability (ID) is a developmental disorder that includes both intellectual and adaptive functioning deficits in conceptual, social, and practical domains. Although evidence-based interventions for patients have long been desired, their progress has been hindered due to various determinants. One of these determinants is the complexity of the origins of ID. The ceramide transport protein (CERT) encoded by CERT1 mediates inter-organelle trafficking of ceramide for the synthesis of intracellular sphingomyelin. Utilizing whole exome sequencing analysis, we identified a novel CERT variant, which substitutes a serine at position 135 (S135) for a proline in a patient with severe ID. Biochemical analysis showed that S135 is essential for hyperphosphorylation of a serine-repeat motif of CERT, which is required for down-regulation of CERT activity. Amino acid replacements of S135 abnormally activated CERT and induced an intracellular punctate distribution pattern of this protein. These results identified specific ID-associated CERT1 mutations that induced gain-of-function effects on CERT activity. These findings provide a possible molecular basis for not only new diagnostics but also a conceivable pharmaceutical intervention for ID disorders caused by gain-of-function mutations in CERT1.

Christianson syndrome (CS) is an X-linked intellectual disorder caused by mutations in the SLC9A6 gene. Clinical features of CS include an inability to speak, truncal ataxia, postnatal microcephaly, hyperkinesis, and epilepsy. Almost all patients with CS develop drug-resistant epilepsy—its most serious complication. We report two cases of CS with drug-resistant epilpesy associated with the Lennox–Gastaut syndrome (LGS). One patient experienced generalized tonic seizures since 9 months of age with cognitive regression, which evolved to include atonic seizures at the age of 7 years. Electroencephalography (EEG) showed generalized slow spike–wave complexes and generalized paroxysmal fast activity. Seizures remained drug-resistant despite multiple anti-seizure drugs. The second patient experienced generalized tonic seizures since the age of 17 months and arrested development. EEG showed generalized slow spike–wave complexes, with frequent atonic seizures since the age of 6 years. Electrical status epilepticus during slow-wave sleep (ESES) developed at the age of 7 years. Our cases illustrate that CS may cause LGS in addition to other developmental and epileptic encephalopathies of the neonatal and infantile period. We suggest that generalized tonic or tonic–clonic seizures and generalized slow spike–wave complexes in interictal EEG be included as potential electroclinical features of epilepsy in CS. Keywords: Epilepsy, Christianson syndrome, SLC9A6, Lennox–Gastaut syndrome, Electrical status epilepticus in slow-wave sleep

Background: Glycogen storage disease type IV (GSD IV), caused by GBE1 mutations, has a quite wide phenotypic variation. While the classic hepatic form and the perinatal/neonatal neuromuscular forms result in early mortality, milder manifestations include non-progressive form (NP-GSD IV) and adult polyglucosan body disease (APBD). Thus far, only one clinical case of a patient with compound heterozygous mutations has been reported for the molecular analysis of NP-GSD IV. This study aimed to elucidate the molecular basis in a NP-GSD IV patient via protein expression analysis and to obtain a clearer genotype-phenotype relationship in GSD IV. Case presentation: A Japanese boy presented hepatosplenomegaly at 2 years of age. Developmental delay, neurological symptoms, and cardiac dysfunction were not apparent. Observation of hepatocytes with periodic acid-Schiff-positive materials resistant to diastase, coupled with resolution of hepatosplenomegaly at 8 years of age, yielded a diagnosis of NP-GSD IV. Glycogen branching enzyme activity was decreased in erythrocytes. At 13 years of age, he developed epilepsy, which was successfully controlled by carbamazepine. Molecular analysis: In this study, we identified compound heterozygous GBE1 mutations (p.Gln46Pro and p.Glu609Lys). The branching activities of the mutant proteins expressed using E. coli were examined in a reaction with starch. The result showed that both mutants had approximately 50% activity of the wild type protein. Conclusion: This is the second clinical report of a NP-GSD IV patient with a definite molecular elucidation. Based on the clinical and genotypic overlapping between NP-GSD IV and APBD, we suggest both are in a continuum. Keywords: Epilepsy, Functional analysis, Glycogen storage disease type IV, Glycogenosis, glycogen branching enzyme 1, GSD

Abstract Background Congenital disorders of glycosylation (CDGs) are genetic diseases caused by pathogenic variants of genes involved in protein or lipid glycosylation. De novo variants in the SLC35A2 gene, which encodes a UDP‐galactose transporter, are responsible for CDGs with an X‐linked dominant manner. Common symptoms related to SLC35A2 variants include epilepsy, psychomotor developmental delay, hypotonia, abnormal facial and skeletal features, and various magnetic resonance imaging (MRI) findings. Methods Whole‐exome sequencing was performed on the patient's DNA, and candidate variants were confirmed by Sanger sequencing. cDNA analysis was performed to assess the effect of the splice site variant using peripheral leukocytes. The X‐chromosome inactivation pattern was studied using the human androgen receptor assay. Results We identified a de novo splice site variant in SLC35A2 (NM_005660.2: c.274+1G>A) in a female patient who showed severe developmental delay, spastic paraplegia, mild cerebral atrophy, and delayed myelination on MRI, but no seizures. The variant led to an aberrant splicing resulting in an in‐frame 33‐bp insertion, which caused an 11‐amino acid insertion in the presumptive cytoplasmic loop. X‐inactivation pattern was random. Partial loss of galactose and sialic acid of the N‐linked glycans of serum transferrin was observed. Conclusion This case would expand the phenotypic spectrum of SLC35A2‐related disorders to delayed myelination with spasticity and no seizures.

CHARGE syndrome is caused by heterozygous mutations in the chromatin remodeler, CHD7, and is characterized by a set of malformations that, on clinical grounds, were historically postulated to arise from defects in neural crest formation during embryogenesis. To better delineate neural crest defects in CHARGE syndrome, we generated induced pluripotent stem cells (iPSCs) from two patients with typical syndrome manifestations, and characterized neural crest cells differentiated in vitro from these iPSCs (iPSC-NCCs). We found that expression of genes associated with cell migration was altered in CHARGE iPSC-NCCs compared to control iPSC-NCCs. Consistently, CHARGE iPSC-NCCs showed defective delamination, migration and motility in vitro, and their transplantation in ovo revealed overall defective migratory activity in the chick embryo. These results support the historical inference that CHARGE syndrome patients exhibit defects in neural crest migration, and provide the first successful application of patient-derived iPSCs in modeling craniofacial disorders.