Biallelic variants in LARS1 induce steatosis in developing zebrafish liver via enhanced autophagy.

BACKGROUND: Biallelic pathogenic variants of LARS1 cause infantile liver failure syndrome type 1 (ILFS1), which is characterized by acute hepatic failure with steatosis in infants. LARS functions as a protein associated with mTORC1 and plays a crucial role in amino acid-triggered mTORC1 activation and regulation of autophagy. A previous study demonstrated that larsb-knockout zebrafish exhibit conditions resembling ILFS. However, a comprehensive analysis of larsb-knockout zebrafish has not yet been performed because of early mortality. METHODS: We generated a long-term viable zebrafish model carrying a LARS1 variant identified in an ILFS1 patient (larsb-I451F zebrafish) and analyzed the pathogenesis of the affected liver of ILFS1. RESULTS: Hepatic dysfunction is most prominent in ILFS1 patients during infancy; correspondingly, the larsb-I451F zebrafish manifested hepatic anomalies during developmental stages. The larsb-I451F zebrafish demonstrates augmented lipid accumulation within the liver during autophagy activation. Inhibition of DGAT1, which converts fatty acids to triacylglycerols, improved lipid droplets in the liver of larsb-I451F zebrafish. Notably, treatment with an autophagy inhibitor ameliorated hepatic lipid accumulation in this model. CONCLUSIONS: Our findings suggested that enhanced autophagy caused by biallelic LARS1 variants contributes to ILFS1-associated hepatic dysfunction. Furthermore, the larsb-I451F zebrafish model, which has a prolonged survival rate compared with the larsb-knockout model, highlights its potential utility as a tool for investigating the pathophysiology of ILFS1-associated liver dysfunction.

Biallelic loss-of-function variants of EZH1 cause a novel developmental disorder with central precocious puberty.

Pathogenic variants of polycomb repressive complex-2 (PRC2) subunits are associated with overgrowth syndromes and neurological diseases. EZH2 is a major component of PRC2 and mediates the methylation of H3K27 trimethylation (H3K27me3). Germline variants of EZH2 have been identified as a cause of Weaver syndrome (WS), an overgrowth/intellectual disability (OGID) syndrome characterized by overgrowth, macrocephaly, accelerated bone age, intellectual disability (ID), and characteristic facial features. Germline variants of SUZ12 and EED, other components of PRC2, have also been reported in the WS or Weaver-like syndrome. EZH1 is a homolog of EZH2 that interchangeably associates with SUZ12 and EED. Recently, pathogenic variants of EZH1 have been reported in individuals with dominant and recessive neurodevelopmental disorders. We herein present sisters with biallelic loss-of-function variants of EZH1. They showed developmental delay, ID, and central precocious puberty, but not the features of WS or other OGID syndromes.

Diagnostic delay of MYH9-related disorder in Japan.

MYH9-related disorder (MYH9-RD) is characterized by congenital macrothrombocytopenia and granulocyte inclusion bodies. MYH9-RD is often misdiagnosed as chronic immune thrombocytopenia. In this study, we investigated age at definitive diagnosis and indicative thrombocytopenia in 41 patients with MYH9-RD from the congenital thrombocytopenia registry in Japan. Our cohort comprises 54.8% adults over 18 years at confirmed diagnosis. We found a significant difference (p < 0.0001) between the median age at definitive diagnosis of 25.0 years and for indicative thrombocytopenia it was 9.0 years. Our findings strongly suggest diagnostic delay of MYH9-RD in Japan. Our registry system will continue to contribute to this issue.

Functional analysis of RRAS2 pathogenic variants with a Noonan-like phenotype.

Introduction: RRAS2, a member of the R-Ras subfamily of Ras-like low-molecular-weight GTPases, is considered to regulate cell proliferation and differentiation via the RAS/MAPK signaling pathway. Seven RRAS2 pathogenic variants have been reported in patients with Noonan syndrome; however, few functional analyses have been conducted. Herein, we report two patients who presented with a Noonan-like phenotype with recurrent and novel RRAS2 pathogenic variants (p.Gly23Val and p.Gly24Glu, respectively) and the results of their functional analysis. Materials and methods: Wild-type (WT) and mutant RRAS2 genes were transiently expressed in Human Embryonic Kidney293 cells. Expression of RRAS2 and phosphorylation of ERK1/2 were confirmed by Western blotting, and the RAS signaling pathway activity was measured using a reporter assay system with the serum response element-luciferase construct. WT and p.Gly23Val RRAS2 were expressed in Drosophila eye using the glass multiple reporter-Gal4 driver. Mutant mRNA microinjection into zebrafish embryos was performed, and the embryo jaws were observed. Results: No obvious differences in the expression of proteins WT, p.Gly23Val, and p.Gly24Glu were observed. The luciferase reporter assay showed that the activity of p.Gly23Val was 2.45 ± 0.95-fold higher than WT, and p.Gly24Glu was 3.06 ± 1.35-fold higher than WT. For transgenic flies, the p.Gly23Val expression resulted in no adults flies emerging, indicating lethality. For mutant mRNA-injected zebrafish embryos, an oval shape and delayed jaw development were observed compared with WT mRNA-injected embryos. These indicated hyperactivity of the RAS signaling pathway. Discussion: Recurrent and novel RRAS2 variants that we reported showed increased in vitro or in vivo RAS signaling pathway activity because of gain-of-function RRAS2 variants. Clinical features are similar to those previously reported, suggesting that RRAS2 gain-of-function variants cause this disease in patients.

Bernard-Soulier syndrome caused by a novel GP1BB variant and 22q11.2 deletion.

Bernard-Soulier syndrome (BSS) is caused by defects in GP1BA, GP1BB, or GP9 genes. Patients with 22q11.2 deletion syndrome (22q11.2DS) are obligate carriers of BSS because GP1BB resides on chromosome 22q11.2. A 15-month-old girl without bleeding symptoms had giant platelets and thrombocytopenia. Physical findings and macrothrombocytopenia suggested 22q11.2DS, which was confirmed by fluorescence in situ hybridization. Flow cytometry showed decreased GPIbα on the platelets. Gene panel testing revealed a novel variant in GP1BB, p.(Val169_Leu172del). These findings confirmed that the patient had BSS. This case suggests that any patient with 22q11.2DS and macrothrombocytopenia should be further tested for BSS.

Renal coloboma syndrome/dominant optic atrophy with severe retinal atrophy and de novo digenic mutations in PAX2 and OPA1.

Renal coloboma syndrome (RCS) and dominant optic atrophy are mainly caused by heterozygous mutations in PAX2 and OPA1, respectively. We describe a patient with digenic mutations in PAX2 and OPA1. A female infant was born without perinatal abnormalities. Magnetic resonance imaging at 4 months of age showed bilateral microphthalmia and optic nerve hypoplasia. Appropriate body size was present at 2 years of age, and mental development was favorable. Color fundus photography revealed severe retinal atrophy in both eyes. Electroretinography showed slight responses in the right eye, but no responses in the left eye, suggesting a high risk of blindness. Urinalysis results were normal, creatinine-based estimated glomerular filtration rate was 63.5 mL/min/1.73 m2, and ultrasonography showed bilateral hypoplastic kidneys. Whole exome sequencing revealed de novo frameshift mutations in PAX2 and OPA1. Both variants were classified as pathogenic (PVS1, PS2, PM2) based on the guidelines from the American College of Medical Genetics and Genomics (ACMG). Genetic testing for ocular diseases should be considered for patients with suspected RCS and a high risk of total blindness.

Case Report: An Adult Case of Poretti-Boltshauser Syndrome Diagnosed by Medical Checkup.

This report describes an adult case of Poretti-Boltshauser syndrome (PTBHS) and with novel variants of LAMA1. A 65-year-old Japanese woman with cerebellar malformation identified during a medical checkup was referred to our hospital. Subsequently, neurological examination, brain imaging, and genetic investigation via whole-exome sequencing were performed. The patient presented with mild cerebellar ataxia and intellectual disability. Magnetic resonance imaging revealed cerebellar dysplasia and cysts and an absence of molar tooth sign. Genetic analysis revealed a novel homozygous variant of c.1711_1712del in LAMA1 (NM_005559.4). Most cases with PTBHS are reported in pediatric patients; however, our patient expressed a mild phenotype and was undiagnosed until her 60 s. These findings suggest that PTBHS should be considered in not only pediatric cerebellar dysplasia but also adult cerebellar ataxia with mild presentation.

Clinical details of individuals with Rauch-Steindl syndrome due to NSD2 truncating variants.

BACKGROUND: Rauch-Steindl syndrome (RAUST) is a very rare genetic syndrome caused by a pathogenic variant in NSD2 on chromosome 4p16.3. Although NSD2 was previously thought to be the major gene in Wolf-Hirschhorn syndrome (WHS), a contiguous gene syndrome of chromosome 4p16.3 deletion, RAUST has been found to present different facial and clinical features from WHS. In this study, we report the details of two newly diagnosed individuals with RAUST in order to better understand the molecular and clinical features of RAUST. METHODS: Whole-genome sequencing was performed on two individuals with psychomotor delay and growth failure. Detailed clinical evaluation of growth parameters, craniofacial features, electroencephalogram (EEG), magnetic resonance imaging of the brain, and developmental assessment were performed. RESULTS: Both individuals had de novo truncating variants in NSD2. One had a novel variant (c.2470C>T, p.Arg824*), and the other had a recurrent variant (c.4028del, p.Pro1343Glnfs*49). Both exhibited characteristic RAUST facial features, growth failure, and mild psychomotor delay. A novel finding of RAUST was seen in individual 2, a Chiari malformation type 1, and both showed delayed bone age. They lacked common WHS features such as congenital heart defects, cleft lip/palate, and seizures (EEG with abnormal findings). CONCLUSION: We present a novel variant and clinical presentations of RAUST, expand the molecular and clinical diversity of RAUST, and improve our understanding of this rare syndrome, which is distinct from WHS. Further researches are needed on more RAUST cases and on functional analysis of NSD2.

A novel DLG4 variant causes DLG4-related synaptopathy with intellectual regression.

DLG4-related synaptopathy is a neurodevelopmental disorder caused by a DLG4 variant. We identified a novel de novo heterozygous frameshift variant, NM_001321075.3(DLG4):c.554_563del, in a Japanese girl. Intellectual regression without motor delay was observed at 2 years of age, and she was diagnosed with autism spectrum disorder and attention-deficit/hyperactivity disorder. Recognizing the possibility of DLG4-related synaptopathy in patients with intellectual regression is important for ensuring an accurate diagnosis.

Frontal deficits and atrophy in a patient with familial encephalopathy with neuroserpin inclusion bodies detected by single-case voxel-based morphometry: a case report.

BACKGROUND: Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is a rare genetic disorder characterized by progressive cognitive decline and myoclonic epilepsy, caused by pathogenic variants of SERPINI1. We reported a case of genetically confirmed FENIB with de novo H338R mutation in the SERPINI1, in which frontal deficits including inattention and disinhibition, and relevant atrophy in the vmPFC on brain MRI were observed in the early stage of the disease. CASE PRESENTATION: A 23-year-old Japanese man presented with progressive inattention and disinhibition over 4 years followed by myoclonic epilepsy. The whole-genome sequencing and filtering analysis showed de novo heterozygous H338R mutation in the SERPINI1, confirming the diagnosis of FENIB. Single-case voxel-based morphometry using brain magnetic resonance imaging obtained at the initial visit revealed focal gray matter volume loss in the ventromedial prefrontal cortices, which is presumed to be associated with inattention and disinhibition. CONCLUSION: Frontal deficits including inattention and disinhibition can be the presenting symptoms of patients with FENIB. Single-case voxel-based morphometry may be useful for detecting regional atrophy of the frontal lobe in FENIB. Detecting these abnormalities in the early stage of disease may be key findings for differentiating FENIB from other causes of progressive myoclonic epilepsy.

Exome-wide benchmark of difficult-to-sequence regions using short-read next-generation DNA sequencing.

Next-generation DNA sequencing (NGS) in short-read mode has recently been used for genetic testing in various clinical settings. NGS data accuracy is crucial in clinical settings, and several reports regarding quality control of NGS data, primarily focusing on establishing NGS sequence read accuracy, have been published thus far. Variant calling is another critical source of NGS errors that remains unexplored at the single-nucleotide level despite its established significance. In this study, we used a machine-learning-based method to establish an exome-wide benchmark of difficult-to-sequence regions at the nucleotide-residue resolution using 10 genome sequence features based on real-world NGS data accumulated in The Genome Aggregation Database (gnomAD) of the human reference genome sequence (GRCh38/hg38). The newly acquired metric, designated the 'UNMET score,' along with additional lines of structural information from the human genome, allowed us to assess the sequencing challenges within the exonic region of interest using conventional short-read NGS. Thus, the UNMET score could provide a basis for addressing potential sequential errors in protein-coding exons of the human reference genome sequence GRCh38/hg38 in clinical sequencing.

Exploring the genetic diversity of the Japanese population: Insights from a large-scale whole genome sequencing analysis.

The Japanese archipelago is a terminal location for human migration, and the contemporary Japanese people represent a unique population whose genomic diversity has been shaped by multiple migrations from Eurasia. We analyzed the genomic characteristics that define the genetic makeup of the modern Japanese population from a population genetics perspective from the genomic data of 9,287 samples obtained by high-coverage whole-genome sequencing (WGS) by the National Center Biobank Network. The dataset comprised populations from the Ryukyu Islands and other parts of the Japanese archipelago (Hondo). The Hondo population underwent two episodes of population decline during the Jomon period, corresponding to the Late Neolithic, and the Edo period, corresponding to the Early Modern era, while the Ryukyu population experienced a population decline during the shell midden period of the Late Neolithic in this region. Haplotype analysis suggested increased allele frequencies for genes related to alcohol and fatty acid metabolism, which were reported as loci that had experienced positive natural selection. Two genes related to alcohol metabolism were found to be 12,500 years out of phase with the time when they began to increase in the allele frequency; this finding indicates that the genomic diversity of Japanese people has been shaped by events closely related to agriculture and food production.

Predicting the pathogenicity of missense variants based on protein instability to support diagnosis of patients with novel variants of ARSL.

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.

A novel HECW2 variant in an infant with congenital long QT syndrome.

Pathogenic variants of HECW2 have been reported in cases of neurodevelopmental disorder with hypotonia, seizures, and absent language (NDHSAL; OMIM #617268). A novel HECW2 variant (NM_001348768.2:c.4343 T > C,p.Leu1448Ser) was identified in an NDHSAL infant with severe cardiac comorbidities. The patient presented with fetal tachyarrhythmia and hydrops and was postnatally diagnosed with long QT syndrome. This study provides evidence that HECW2 pathogenic variants can cause long QT syndrome along with neurodevelopmental disorders.

Measurement of immature platelet fraction is useful in the differential diagnosis of MYH9 disorders.

INTRODUCTION: Although the presence of large and giant platelets is important in screening for MYH9 disorders, platelet morphology evaluation is dependent on operator subjectivity. Immature platelet fraction (IPF%) is widely used in clinical practice because of its rapidity and reproducibility; however, IPF% has been rarely analyzed in MYH9 disorders. Therefore, our study aimed to clarify the usefulness of IPF% in the differential diagnosis of MYH9 disorders. METHODS: We assessed 24 patients with MYH9 disorders, 10 with chronic immune thrombocytopenia (cITP), 14 with myelodysplastic syndromes (MDS) with thrombocytopenia (<100 × 109 /L), and 20 healthy volunteers. Platelet-related data, including IPF% and platelet morphology (diameter, surface area, and staining), were retrospectively analyzed. RESULTS: Median IPF% in MYH9 disorders, 48.7%, was significantly higher than in all other groups (cITP: 13.4%, MDS: 9.4%, controls: 2.6%). IPF% in MYH9 disorders was significantly negatively correlated with platelet count and significantly positively correlated with the diameter and surface area of platelets, but a correlation was not found between IPF% and platelet staining. The area under the curve of IPF% for the differential diagnosis of MYH9 disorders was 0.987 (95% CI: 0.969-1.000), with a sensitivity of 95.8% and specificity of 93.2% when the cutoff value of IPF% was 24.3%. CONCLUSION: Our study strongly suggests that IPF% is useful in the differential diagnosis between MYH9 disorders and other types of thrombocytopenia.

Clinical characteristics of muscle cramps in hereditary angiopathy with nephropathy, aneurysms, and muscle cramps syndrome associated with a novel COL4A1 pathogenic variant: A family case study.

BACKGROUND: Muscle cramps are a common problem characterized by a sudden, painful, and involuntary contraction of a muscle or muscle group. Most muscle cramps develop in the calf muscles, particularly in situations of prolonged exercise; however, some may be related to underlying systemic conditions such as the hereditary angiopathy with nephropathy, aneurysms, and muscle cramps (HANAC) syndrome. Muscle cramps appear to be the initial symptom of the HANAC syndrome; however, the clinical characteristics of these muscle cramps have rarely been described in detail. CASE PRESENTATION: We report a familial case of autosomal-dominant muscle cramps in four members of a Japanese family spanning across three generations. The muscle cramps were recognized as systemic symptoms of the HANAC syndrome associated with a novel COL4A1 pathogenic variant, NM_001845:c.1538G > A, p.(Gly513Asp). The four affected individuals indicated that the first episodes of the muscle cramps occurred in early childhood. In addition, they reported that the muscle cramps are characterized by an abrupt onset of severe pain without muscle contraction. The painful recurrent attacks occurred spontaneously in various muscles throughout the body, but rarely in the calf muscle. The muscle pain lasts for several minutes, cannot be ameliorated by stretching the affected muscle, and leaves a feeling of discomfort that lasts for 24-48 h. The serum creatine kinase levels of the patients were persistently elevated; however, their electromyography results did not reveal any specific abnormalities. CONCLUSIONS: Recognition of the clinical characteristics of the muscle cramps in the HANAC syndrome may facilitate early diagnosis of the syndrome and enable proper treatment of the patients, improve their long-term outcomes, and facilitate the design and adaption of appropriate genetic counseling.

A gain-of-function mutation in microRNA 142 is sufficient to cause the development of T-cell leukemia in mice.

MicroRNAs (miRNAs) play a crucial role in regulating gene expression. MicroRNA expression levels fluctuate, and point mutations and methylation occur in cancer cells; however, to date, there have been no reports of carcinogenic point mutations in miRNAs. MicroRNA 142 (miR-142) is frequently mutated in patients with follicular lymphoma, diffuse large B-cell lymphoma, chronic lymphocytic leukemia (CLL), and acute myeloid leukemia/myelodysplastic syndrome (AML/MDS). To understand the role of miR-142 mutation in blood cancers, the CRISPR-Cas9 system was utilized to successfully generate miR-142-55A>G mutant knock-in (Ki) mice, simulating the most frequent mutation in patients with miR-142 mutated AML/MDS. Bone marrow cells from miR-142 mutant heterozygous Ki mice were transplanted, and we found that the miR-142 mutant/wild-type cells were sufficient for the development of CD8+ T-cell leukemia in mice post-transplantation. RNA-sequencing analysis in hematopoietic stem/progenitor cells and CD8+ T-cells revealed that miR-142-Ki/+ cells had increased expression of the mTORC1 activator, a potential target of wild-type miR-142-3p. Notably, the expression of genes involved in apoptosis, differentiation, and the inhibition of the Akt-mTOR pathway was suppressed in miR-142-55A>G heterozygous cells, indicating that these genes are repressed by the mutant miR-142-3p. Thus, in addition to the loss of function due to the halving of wild-type miR-142-3p alleles, mutated miR-142-3p gained the function to suppress the expression of distinct target genes, sufficient to cause leukemogenesis in mice.

Biallelic CC2D2A variants, SNV and LINE-1 insertion simultaneously identified in siblings using long-read whole-genome sequencing and haplotype phasing.

Joubert syndrome (JBTS) is characterized by a magnetic resonance imaging appearance called 'molar tooth sign', neonatal breathing dysregulation and hypotonia, and developmental delay. Whole-exome analysis based on short-read sequencing has often contributed to the identification of causative single-nucleotide variants in patients clinically diagnosed with JBTS. However, ~10% of them are still undiagnosed even though a single possible pathogenic variant has been identified. We report a successful identification of biallelic variants using long-read whole-genome sequencing and haplotype phasing analysis in a family with two Japanese siblings having morphological brain abnormalities. The affected siblings had a novel nonsynonymous variant (CC2D2A:NM_001080522.2:c.4454A>G:p.(Tyr1485Cys)) and an exonic insertion of Long INterspercsed Element-1 (LINE-1). The allelicity of these variants was clearly proven without the data of parents. Finally, our survey of in-house genome sequencing data indicates that there are rare carriers of CC2D2A related diseases, who harbour the exonic LINE-1 insertion in the CC2D2A gene.

GRIA3 p.Met661Thr variant in a female with developmental epileptic encephalopathy.

The X-linked human glutamate receptor subunit 3 (GRIA3) gene (MIM *305915, Xq25) encodes ionotropic α amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptor subunit 3, which mediates postsynaptic neurotransmission. Variants in this gene can cause a variety of neurological disorders, primarily reported in male patients. Here, we report a female patient with developmental and epileptic encephalopathy who carries the novel de novo GRIA3 variant NM_007325.5: c.1982T > C: p.Met661Thr.