Truncating variants of the sterol recognition region of SHH cause hypertelorism phenotype rather than hypotelorism-holoprosencephaly.

Sonic hedgehog signaling molecule (SHH) is a key molecule in the cilia-mediated signaling pathway and a critical morphogen in embryogenesis. The association between loss-of-function variants of SHH and holoprosencephaly is well established. In mice experiments, reduced or increased signaling of SHH have been shown to be associated with narrowing or excessive expansion of the facial midline, respectively. Herein, we report two unrelated patients with de novo truncating variants of SHH presenting with hypertelorism rather than hypotelorism. The first patient was a 13-year-old girl. Her facial features included hypertelorism, strabismus, telecanthus, malocclusion, frontal bossing, and wide widow's peak. She had borderline developmental delay and agenesis of the corpus callosum. She had a nonsense variant of SHH: Chr7(GRCh38):g.155802987C > T, NM_000193.4:c.1302G > A, p.(Trp434*). The second patient was a 25-year-old girl. Her facial features included hypertelorism and wide widow's peak. She had developmental delay and agenesis of the corpus callosum. She had a frameshift variant of SHH: Chr7(GRCh38):g.155803072_155803074delCGGinsT, NM_000193.4:c.1215_1217delCCGinsA, p.(Asp405Glufs*92). The hypertelorism phenotype contrasts sharply with the prototypical hypotelorism-holoprosencephaly phenotype associated with loss-of-function of SHH. We concluded that a subset of truncating variants of SHH could be associated with hypertelorism rather than hypotelorism.

Gene-specific somatic epigenetic mosaicism of FDFT1 underlies a non-hereditary localized form of porokeratosis.

Porokeratosis is a clonal keratinization disorder characterized by solitary, linearly arranged, or generally distributed multiple skin lesions. Previous studies showed that genetic alterations in MVK, PMVK, MVD, or FDPS-genes in the mevalonate pathway-cause hereditary porokeratosis, with skin lesions harboring germline and lesion-specific somatic variants on opposite alleles. Here, we identified non-hereditary porokeratosis associated with epigenetic silencing of FDFT1, another gene in the mevalonate pathway. Skin lesions of the generalized form had germline and lesion-specific somatic variants on opposite alleles in FDFT1, representing FDFT1-associated hereditary porokeratosis identified in this study. Conversely, lesions of the solitary or linearly arranged localized form had somatic bi-allelic promoter hypermethylation or mono-allelic promoter hypermethylation with somatic genetic alterations on opposite alleles in FDFT1, indicating non-hereditary porokeratosis. FDFT1 localization was uniformly diminished within the lesions, and lesion-derived keratinocytes showed cholesterol dependence for cell growth and altered expression of genes related to cell-cycle and epidermal development, confirming that lesions form by clonal expansion of FDFT1-deficient keratinocytes. In some individuals with the localized form, gene-specific promoter hypermethylation of FDFT1 was detected in morphologically normal epidermis adjacent to methylation-related lesions but not distal to these lesions, suggesting that asymptomatic somatic epigenetic mosaicism of FDFT1 predisposes certain skin areas to the disease. Finally, consistent with its genetic etiology, topical statin treatment ameliorated lesions in FDFT1-deficient porokeratosis. In conclusion, we identified bi-allelic genetic and/or epigenetic alterations of FDFT1 as a cause of porokeratosis and shed light on the pathogenesis of skin mosaicism involving clonal expansion of epigenetically altered cells.

Gut microbiota of one-and-a-half-year-old food-allergic and healthy children.

BACKGROUND: Intestinal bacteria may play a role in the development of food allergies. This study aimed to analyze and compare the gut microbiota of food-allergic children with that of healthy children of the same age. METHODS: Stool samples were collected from one-and-a-half-year-old food-allergic (FA group, n = 29) and healthy controls (HC group, n = 19). A questionnaire was provided to examine the children's birth, dietary, medical, and social histories. The gut microbiota was profiled by 16S rRNA sequencing. Differences in taxonomic composition were assessed using linear discriminant analysis effect size (LEfSe), and microbial functional profiles were predicted with Tax4Fun2. RESULTS: No significant difference in the alpha diversity index between the two groups; however, a negative correlation was observed between the Shannon diversity index and the relative abundance of Bacteroides. A significant difference was observed in beta diversity (permutational multivariate analysis of variance) in the bacterial composition between the FA and HC groups (P < 0.05). The FA group had a higher abundance of Escherichia and Anaeromassilibacillus and a lower abundance of Bacteroides, Oscillibacter, Ruminococcus, Hungateiclostridium and Anaerotaenia than the HC group (LEfSe: linear discriminant analysis score >2). The FA group showed a predicted increase in the expression levels of genes associated with intestinal pathogenicity compared with that in the HC group. CONCLUSIONS: The gut microbiota of food-allergic children has a higher abundance of bacteria involved in intestinal inflammation and a lower abundance of bacteria involved in immune tolerance than that of healthy children. This dysbiosis may also be associated with food allergies.

Successful skipping of abnormal pseudoexon by antisense oligonucleotides in vitro for a patient with beta-propeller protein-associated neurodegeneration.

Pathogenic variants in WDR45 on chromosome Xp11 cause neurodegenerative disorder beta-propeller protein-associated neurodegeneration (BPAN). Currently, there is no effective therapy for BPAN. Here we report a 17-year-old female patient with BPAN and show that antisense oligonucleotide (ASO) was effective in vitro. The patient had developmental delay and later showed extrapyramidal signs since the age of 15 years. MRI findings showed iron deposition in the globus pallidus and substantia nigra on T2 MRI. Whole genome sequencing and RNA sequencing revealed generation of pseudoexon due to inclusion of intronic sequences triggered by an intronic variant that is remote from the exon-intron junction: WDR45 (OMIM #300526) chrX(GRCh37):g.48935143G > C, (NM_007075.4:c.235 + 159C > G). We recapitulated the exonization of intron sequences by a mini-gene assay and further sought antisense oligonucleotide that induce pseudoexon skipping using our recently developed, a dual fluorescent splicing reporter system that encodes two fluorescent proteins, mCherry, a transfection marker designed to facilitate evaluation of exon skipping and split eGFP, a splicing reaction marker. The results showed that the 24-base ASO was the strongest inducer of pseudoexon skipping. Our data presented here have provided supportive evidence for in vivo preclinical studies.

Identification of a novel splice-site WWOX variant with paternal uniparental isodisomy in a patient with infantile epileptic encephalopathy.

WOREE syndrome is an early infantile epileptic encephalopathy characterized by drug-resistant seizures and severe psychomotor developmental delays. We report a case of a WWOX splice-site mutation with uniparental isodisomy. A 1-year and 7-month-old girl presented with nystagmus and epileptic seizures from early infancy, with no fixation or pursuit of vision. Physical examination revealed small deformities, such as swelling of both cheeks, folded fingers, rocking feet, and scoliosis. Brain imaging revealed slight hypoplasia of the cerebrum. Electroencephalogram showed focal paroxysmal discharges during the interictal phase of seizures. Vitamin B6 and zonisamide were administered for early infantile epileptic encephalopathy; however, the seizures were not relieved. Despite altering the type and dosage of antiepileptic drugs and ACTH therapy, the seizures were intractable. Whole-exome analysis revealed the homozygosity of WWOX(NM_016373.4):c.516+1G>A. The WWOX mRNA sequencing using peripheral blood RNA confirmed that exon 5 was homozygously deleted. Based on these results, the patient was diagnosed with WOREE syndrome at 5 months. The WWOX variant found in this study is novel and has never been reported before. WOREE syndrome being extremely rare, further case series and analyses of its pathophysiology are warranted.

SALL4 deletion and kidney and cardiac defects associated with VACTERL association.

Congenital anomalies of the kidney and urinary tract (CAKUT) can be a part of the VACTERL association, which represents the non-random combination of the following congenital anomalies: vertebral anomalies, anal anomalies, cardiac anomalies, tracheal-esophageal anomalies, kidney anomalies, and limb anomalies. VACTERL association is generally considered to be a non-genetic condition. Exceptions include a patient with a heterozygous nonsense SALL4 variant and anal stenosis, tetralogy of Fallot, sacro-vertebral fusion, and radial and thumb anomalies. SALL4 encodes a transcription factor that plays a critical role in kidney morphogenesis. Here, we report a patient with VACTERL association and a heterozygous 128-kb deletion spanning SALL4 who presented with renal hypoplasia, radial and atrio-septal defects, and patent ductus arteriosus. The present report of SALL4 deletion, in addition to a previously reported patient with VACTERL association phenotype and SALL4 nonsense mutation, further supports the notion that SALL4 haploinsufficiency can lead to VACTERL association.

X -linked inheritance of primary ciliary dyskinesia and retinitis pigmentosa due to RPGR variant: A case report and literature review.

Bronchiectasis is a chronic respiratory condition characterized by irreversible bronchial dilation, often caused by infection or inflammation. It can be associated with primary ciliary dyskinesia (PCD), a hereditary disorder affecting cilia function in various organs and flagella. PCD's genetic heterogeneity leads to varying disease severity. PCD may be more prevalent in Asia, but its diagnosis is often delayed in Japan. This study reviewed a case of PCD and retinitis pigmentosa (RP) with the relevant literature. The patient had a persistent cough, sputum, and diffuse bronchiectasis. He was diagnosed with a combination of PCD and RP, with the presence of an X-linked retinitis pigmentosa GTPase regulator (RPGR) variant confirmed through electron microscopy, retinal scan, and genetic testing. Although co-occurrence of bronchiectasis and RP is rare, PCD should be considered in cases of persistent wet cough in childhood or unidentified bronchiectasis aetiology. Ophthalmologists should consider concomitant PCD in RP patients.

[Initiative on Rare and Undiagnosed Diseases: Achievements and Future Prospects].

The Initiative on Rare and Undiagnosed Diseases (IRUD) was a project for patients who remain undiagnosed after undergoing various clinical tests. This project includes both the clinical aspect of diagnosing rare diseases and the research aspect of identifying new diseases. Since 2015, the diagnosis rate for undiagnosed patients has been 40-50%. From a clinical perspective, this project demonstrated how genome analysis has become indispensable in rare diseases, especially in pediatrics. From a research standpoint, over 30 diseases have been identified through this project. This project was coordinated with a similar undiagnosed disease program from outside the country, and we strongly believe that much success has been achieved. Future challenges include examining which approaches (e.g., long-read genome analysis, transcriptome analysis, and methylation analysis) would be effective for undiagnosed patients after exome analysis, and developing therapeutic drugs for rare diseases. Future challenges include addressing the approximately 50% of patients whose undiagnosed diseases cannot be diagnosed even after exome analysis and developing therapeutic agents for the rare diseases that have been diagnosed.

BMP2 is a potential causative gene for isolated dextrocardia situs solitus.

BMP2 (bone morphogenic protein-2) is a member of the TGF-ß superfamily and has essential roles in the development of multiple organs, including osteogenesis. Because of its crucial role in organ and skeletal development, Bmp2 null mice is fetal lethal. The recent report has characterized multiple patients with BMP2 haploinsufficiency, describing individuals with BMP2 sequence variants and deletions associated with short stature without endocrinological abnormalities, a recognizable craniofacial gestalt, skeletal anomalies, and congenital heart disease. However, due to a small number of reported patients with BMP2 haploinsufficiency, the genotype and phenotype correlations are not fully understood. We experienced a family of BMP2 haploinsufficiency with a novel frameshift variant NM_001200.4: c.231dup (p.Tyr78Leufs*38) which was predicted to be "pathogenic" by the American College of Genetics and Genomics (ACGM) criteria. In addition to short stature, impaired hearing ability and minor skeletal deformities, the proband exhibited isolated dextrocardia situs solitus without cardiac anomalies and abnormal locations of other visceral organs. Our study would shed light on the crucial role of BMP2 in determining the cardiac axis, and further studies are needed to assemble more cases to elucidate BMP2 role in human heart development.

Café-au-lait Spots and Cleft Palate: Not a Chance Association.

The recognition of syndromic forms of cleft palate is important for condition-specific management. Here, we report a patient with cleft palate, congenital heart disease, intellectual disability, and café-au-lait spots who had a deletion of chromosome 15q14. The identification of the precise breakpoints using a Nanopore-based long-read sequencer showed that the deletion spanned MEIS2 and SPRED1 loci. Cleft palate and café-au-lait spots can be ascribed to MEIS2 and SPRED1, respectively. Patients with cleft palate and café-au-lait spots should be encouraged to undergo a detailed genomic evaluation, including screening for a 15q14 deletion, to enable appropriate anticipatory medico-surgical management and genetic counseling.

Parkinsonism in spinocerebellar ataxia with axonal neuropathy caused by adult-onset COA7 variants: a case report.

BACKGROUND: Individuals with variants of cytochrome c oxidase assembly factor 7 (COA7), a mitochondrial functional-related gene, exhibit symptoms of spinocerebellar ataxia with axonal neuropathy before the age of 20. However, COA7 variants with parkinsonism or adult-onset type cases have not been described. CASE PRESENTATION: We report the case of a patient who developed cerebellar symptoms and slowly progressive sensory and motor neuropathy in the extremities, similar to Charcot-Marie-Tooth disease, at age 30, followed by parkinsonism at age 58. Exome analysis revealed COA7 missense mutation in homozygotes (NM_023077.2:c.17A > G, NP_075565.2: p.Asp6Gly). Dopamine transporter single-photon emission computed tomography using a 123I-Ioflupane revealed clear hypo-accumulation in the bilateral striatum. However, 123I-metaiodobenzylguanidine myocardial scintigraphy showed normal sympathetic nerve function. Levodopa administration improved parkinsonism in this patient. CONCLUSIONS: COA7 gene variants may have caused parkinsonism in this case because mitochondrial function-related genes, such as parkin and PINK1, are known causative genes in some familial Parkinson's diseases.

Oculofaciocardiodental syndrome caused by a novel BCOR variant.

Oculofaciocardiodental syndrome is caused by variants in the BCL6 corepressor (BCOR) gene. We identified a novel heterozygous frameshift variant, NM_001123385.2(BCOR):c.2326del, that arose de novo in a Japanese girl with characteristic facial features, congenital heart disease, bilateral syndactyly of toes 2 and 3, congenital cataracts, dental abnormalities, and mild intellectual disability. Reports of BCOR variants are rare, and further case accumulation is warranted.

Heterozygous loss-of-function DHX9 variants are associated with neurodevelopmental disorders: Human genetic and experimental evidences.

DExH-box helicases are involved in unwinding of RNA and DNA. Among the 16 DExH-box genes, monoallelic variants of DHX16, DHX30, DHX34, and DHX37 are known to be associated with neurodevelopmental disorders. In particular, DHX30 is well established as a causative gene for neurodevelopmental disorders. Germline variants of DHX9, the closest homolog of DHX30, have not been reported until now as being associated with congenital disorders in humans, except that one de novo heterozygous variant, p.(Arg1052Gln) of the gene was identified during comprehensive screening in a patient with autism; unfortunately, the phenotypic details of this individual are unknown. Herein, we report a patients with a heterozygous de novo missense variant, p.(Gly414Arg) of DHX9 who presented with a short stature, intellectual disability, and ventricular non-compaction cardiomyopathy. The variant was located in the glycine codon of the ATP-binding site, G-C-G-K-T. To assess the pathogenicity of these variants, we generated transgenic Drosophila lines expressing human wild-type and mutant DHX9 proteins: 1) the mutant proteins showed aberrant localization both in the nucleus and the cytoplasm; 2) ectopic expression of wild-type protein in the visual system led to the rough eye phenotype, whereas expression of the mutant proteins had minimal effect; 3) overexpression of the wild-type protein in the retina led to a reduction in axonal numbers, whereas expression of the mutant proteins had a less pronounced effect. Furthermore, in a gene-editing experiment of Dhx9 G416 to R416, corresponding to p.(Gly414Arg) in humans, heterozygous mice showed a reduced body size, reduced emotionality, and cardiac conduction abnormality. In conclusion, we established that heterozygosity for a loss-of-function variant of DHX9 can lead to a new neurodevelopmental disorder.

Precise definition of the breakpoints of an apparently balanced translocation between chromosome 3q26 and chromosome 7q36: Role of KMT2C disruption.

When a de novo balanced reciprocal translocation is identified in the patient, the cause of phenotype of the patient can be explained by detecting the breakpoints of the genes. Here, we report a 3-year-old patient with developmental delay, autism spectrum disorder, and distinctive facial features who had an apparently balanced translocation between chromosome 3q26 and chromosome 7q36. Nanopore long-read sequencing revealed that balanced translocation disrupted the KMT2C gene, the haploinsufficiency of which leads to Kleefstra syndrome 2 characterized by delayed psychomotor development, variable intellectual disability and mild dysmorphism. Nanopore long-read sequencing was shown to be useful in elucidating the exact genetic etiology of patients with nonspecific clinical findings.

Ketogenic diet in action: Metabolic profiling of pyruvate dehydrogenase deficiency.

The pyruvate dehydrogenase complex serves as the main connection between cytosolic glycolysis and the tricarboxylic acid cycle within mitochondria. An infant with pyruvate dehydrogenase complex deficiency was treated with vitamin B1 supplementation and a ketogenic diet. These dietary modifications resolved the renal tubular reabsorption, central apnea, and transfusion-dependent anemia. A concurrent metabolome analysis demonstrated the resolution of the amino aciduria and an increased total amount of substrates in the tricarboxylic acid cycle, reflecting the improved mitochondrial energetics. Glutamate was first detected in the cerebrospinal fluid, accompanied by a clinical improvement, after the ketogenic ratio was increased to 3:1; thus, glutamate levels in cerebrospinal fluid may represent a biomarker for neuronal recovery. Metabolomic analyses of body fluids are useful for monitoring therapeutic effects in infants with inborn errors of carbohydrate metabolism.

Involvement of mTOR pathway in neurodegeneration in NSF-related developmental and epileptic encephalopathy.

Membrane fusion is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. During neurotransmitter exocytosis, SNARE proteins on a synaptic vesicle and the target membrane form a complex, resulting in neurotransmitter release. N-ethylmaleimide-sensitive factor (NSF), a homohexameric ATPase, disassembles the complex, allowing individual SNARE proteins to be recycled. Recently, the association between pathogenic NSF variants and developmental and epileptic encephalopathy (DEE) was reported; however, the molecular pathomechanism of NSF-related DEE remains unclear. Here, three patients with de novo heterozygous NSF variants were presented, of which two were associated with DEE and one with a very mild phenotype. One of the DEE patients also had hypocalcemia from parathyroid hormone deficiency and neuromuscular junction impairment. Using PC12 cells, a neurosecretion model, we show that NSF with DEE-associated variants impaired the recycling of vesicular membrane proteins and vesicle enlargement in response to exocytotic stimulation. In addition, DEE-associated variants caused neurodegenerative change and defective autophagy through overactivation of the mammalian/mechanistic target of rapamycin (mTOR) pathway. Treatment with rapamycin, an mTOR inhibitor or overexpression of wild-type NSF ameliorated these phenotypes. Furthermore, neurons differentiated from patient-derived induced pluripotent stem cells showed neurite degeneration, which was also alleviated by rapamycin treatment or gene correction using genome editing. Protein structure analysis of NSF revealed that DEE-associated variants might disrupt the transmission of the conformational change of NSF monomers and consequently halt the rotation of ATP hydrolysis, indicating a dominant negative mechanism. In conclusion, this study elucidates the pathomechanism underlying NSF-related DEE and identifies a potential therapeutic approach.