Takuya Naruto

RNU4-2 encodes U4 small nuclear RNA (snRNA), a non-coding RNA forming the spliceosome complex via the U4/U6 snRNA duplex. RNU4-2 heterozygous variants cause ReNU syndrome, which is characterised by intellectual disability, developmental delay, epilepsy, short stature and distinctive dysmorphic features. ReNU syndrome accounts for 0.4–0.5% of all cases of developmental delay, and RNU4-2 variants are located in the T-loop or stem III region of U4 snRNA, of which approximately 80% are the n.64_65insT variant in the T-loop. We identified four Japanese patients (4.3%) with novel and recurrent RNU4-2 variants from 93 individuals of developmental delay with negative results from exome sequencing. Genotype-phenotype correlations were observed in the present case series and a literature review. T-loop variants manifested severe developmental delay with more than 70% of cases being non-verbal. Stem III region variants resulted in milder developmental delay with fluent speech and nearly normal gross motor development milestones. In addition, we report a patient demonstrating intractable epilepsy with neurological regression harbouring a novel de novo heterozygous RNU4-2 variant (n.66A>G). This report expands the phenotypic spectrum of ReNU syndrome and suggests the presence of phenotypic variability related to variant location.

Chromatin remodeling is an important system controlling gene expression. CHD3, which is a causative gene of Snijders Blok-Campeau syndrome (SNIBCPS), is a member of the chromodomain helicase DNA-binding (CHD) family related to chromatin remodeling. SNIBCPS is characterized by developmental delay (DD), intellectual disability (ID), macrocephaly, and facial features including a prominent forehead and hypertelorism. Hypersociability/overfriendliness is a notable behavioral feature in patients. Here, we describe five SNIBCPS patients with CHD3 variants from four families, including a sibling pair caused by parental gonosomal mosaicism. We observed two distinct phenotypes in our patients in accordance with previous observations. Phenotype 1: macrocephaly, hypertelorism, overgrowth, DD, and ID; and Phenotype 2: microcephaly, growth retardation, DD, and ID. Phenotype 1 was consistent with the typical SNIBCPS phenotype, while Phenotype 2 was distinct. To understand further the features of the patients with SNIBCPS, we generated chd3-knockout (KO) zebrafish using CRISPR-Cas9 genome editing. No morphological changes were observed in chd3-KO zebrafish. However, behavioral tests showed that chd3-KO zebrafish had strong and sustained interest in others, and were less aggressive toward others, suggesting a recapitulation of the hypersociability/overfriendliness phenotype in patients with SNIBCPS. Metabolomic analysis using whole brains showed changes in metabolites processed by specific mitochondrial enzymes in chd3-KO zebrafish. The administration of metformin, which reportedly ameliorates mitochondrial dysfunction and behavioral abnormalities, attenuated the abnormal behavior of chd3-KO zebrafish. Our study helps delineate the phenotypes of patients with SNIBCPS, provides insights into a characteristic behavior of the disease, and suggests a potential treatment to improve the behavioral symptoms of patients.

FBXW7 (F-box and WD-repeat domain-containing 7) is a tumor suppressor gene, and its germline variants have been causally linked to Wilms tumors. Furthermore, germline variants of FBXW7 have also been implicated in a neurodevelopmental syndrome. However, little is known regarding the occurrence of Wilms tumor in patients with FBXW7-related neurodevelopmental syndrome. We identified a novel constitutional pathogenic variant of FBXW7 in a patient with intellectual disability, who also developed Wilms tumor. The variant was derived from his apparently normal mother, and was also detected in his sister who exhibited developmental delay. Furthermore, we detected a somatic nonsense variant on the paternal allele of FBXW7 in the tumor DNA. These results suggest that the development of Wilms tumor along with FBXW7-related neurodevelopmental syndrome follows the two-hit model, which needs to be validated to establish appropriate follow-up management and tumor surveillance.

Myeloid/natural killer (NK) cell precursor acute leukemia (MNKPL) has been described on the basis of its unique immunophenotype and clinical phenotype. However, there is no consensus on the characteristics for identifying this disease type because of its rarity and lack of defined distinctive molecular characteristics. In this study, multiomics analysis revealed that MNKPL is distinct from acute myeloid leukemia, T cell acute lymphoblastic leukemia, and mixed-phenotype acute leukemia (MPAL), and NOTCH1 and RUNX3 activation and BCL11B down-regulation are hallmarks of MNKPL. Although NK cells have been classically considered to be lymphoid lineage–derived, the results of our single-cell analysis using MNKPL cells suggest that NK cells and myeloid cells share common progenitor cells. Treatment outcomes for MNKPL are unsatisfactory, even when hematopoietic cell transplantation is performed. Multiomics analysis and in vitro drug sensitivity assays revealed increased sensitivity to l -asparaginase and reduced levels of asparagine synthetase (ASNS), supporting the clinically observed effectiveness of l -asparaginase.

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.

Dear Editor, We report a young Japanese female with suspected A20 haploinsufficiency (HA20) who was diagnosed after a recurrent myocardial infarction. She was treated with prednisolone (PSL) intermittently under a diagnosis of necrotizing lymphadenitis based on the recurrent fever and lymphadenopathy ( Fig. 1A). In her early 20s and 30s, she experienced two episodes of myocardial infarction with complete occlusion of the right coronary artery, requiring stenting intervention, without risk factors for arteriosclerosis or thromboembolism ( Fig. 1B). Her physical findings were normal except for diffuse hair loss without rash on the scalp. Most laboratory tests were within normal ranges ( Supplementary Table S1 , available at Rheumatology online), although her CRP level was temporarily elevated to 30 mg/l during fever. All examined disease-specific autoantibodies were negative except for low-titre anti-SSA antibody. However, the classification criteria for a diagnosis of SS were not met. Abdominal contrast CT revealed multiple infarctions and cortex atrophy in both kidneys ( Fig. 1C). Although renal or liver dysfunction was not reported in her history, abdominal angiography revealed multiple aneurysms and peripheral obstructions ( Fig. 1D and E), indicating subclinical vascular inflammation causing multiple occlusive events in the medium-sized arteries, and young-onset hypertension. Gastroscopy, colonoscopy, brain MRI, and PET-CT were conducted to detect further organ involvement, but no additional abnormal findings were identified.

Rubinstein-Taybi syndrome (RSTS) is characterized by dysmorphic facial features, broad thumbs, and intellectual disability. CREBBP or EP300 are causative genes. To elucidate the underlying genetic and genomic architecture related to the RSTS phenotype, we performed comprehensive genetic analysis targeting CREBBP and/or EP300 in 22 clinically diagnosed patients. During the 11-year study period, we used several analysis methods including high resolution melting, array-based comparative genomic hybridization, panel-based exome sequencing, whole exome sequencing, and whole genome sequencing (WGS). We identified the causative variants in 19 patients (86.3%), but they were variable and complex, so we must combine multiple analysis methods. Notably, we found genetic alterations in the non-coding regions of two patients (10.5%, 2/19): scattered deletions including a partial 5′-untranslated region of CREBBP in one patient (all coding exons were intact), and a deep 229-bp intronic deletion in another patient, resulting in a splicing error. Furthermore, we identified rare clinical findings: two patients with an EP300 variant showed abnormal development of the neural tube, and one patient with a CREBBP variant had anorectal atresia with a cloaca. Our findings expand the allelic heterogeneity of RSTS, underscore the utility of comprehensive genetic analysis, and suggest that WGS may be a practical diagnostic strategy.

Objective: Kawasaki disease (KD) is a systemic vasculitis in childhood that can lead to coronary artery lesions (CALs). Although early diagnosis and treatment is important for preventing KD patients from development of CALs, diagnosis depends on the clinical features of KD. We studied the usefulness of leucine-rich alpha-2-glycoprotein 1 (LRG1) and angiotensinogen (AGT), previously reported as KD-related proteins, for KD diagnosis and estimation of intravenous immunoglobulin (IVIG) efficacy.Methods: We undertook a prospective cohort study with patients having two or more KD symptoms in multiple centers in Japan, between July 2017 and February 2019.Results: Two hundred forty-two patients were included. In multivariable analysis, one unit increase in LRG1 was associated with higher odds of KD diagnosis (Odds ratio [OR] 1.02 [95% confidence interval (CI) 1.001–1.03]). Double-positivity for AGT (≥ 26 μg/mL) and LRG1 (≥ 123.5 μg/mL) was an independent biomarker for KD diagnosis in both the total cohort and the subgroup of patients with two to four KD symptoms (OR 5.01 [95% CI 1.86–13.50] and 3.71 [95% CI 1.23–11.16], respectively). There was no association between LRG1/AGT and IVIG efficacy.Conclusion: Double-positivity for LRG1 and AGT is an biomarker for KD diagnosis, especially useful in diagnosing incomplete KD from non-KD. Future studies with larger cohorts should seek to determine whether LRG1 and AGT are valuable as definitive data referred at the diagnosis of KD and for estimating the risk of CALs.

Autosomal recessive (AR) complete signal transducer and activator of transcription 1 (STAT1) deficiency is an extremely rare primary immunodeficiency that causes life-threatening mycobacterial and viral infections. Only seven patients from five unrelated families with this disorder have been so far reported. All causal STAT1 mutations reported are exonic and homozygous. We studied a patient with susceptibility to mycobacteria and virus infections, resulting in identification of AR complete STAT1 deficiency due to compound heterozygous mutations, both located in introns: c.128+2 T>G and c.542-8 A>G. Both mutations were the first intronic STAT1 mutations to cause AR complete STAT1 deficiency. Targeted RNA-seq documented the impairment of STAT1 mRNA expression and contributed to the identification of the intronic mutations. The patient’s cells showed a lack of STAT1 expression and phosphorylation, and severe impairment of the cellular response to IFN-γ and IFN-α. The case reflects the importance of accurate clinical diagnosis and precise evaluation, to include intronic mutations, in the comprehensive genomic study when the patient lacks molecular pathogenesis. In conclusion, AR complete STAT1 deficiency can be caused by compound heterozygous and intronic mutations. Targeted RNA-seq based systemic gene expression assay may help to increase diagnostic yield in inconclusive cases after comprehensive genomic study.