The hallmark of neuronopathic Gaucher disease (GD) is oculomotor abnormalities, but ophthalmological assessment is difficult in uncooperative patients. Chromatic pupillometry is a quantitative method to assess the pupillary light reflex (PLR) with minimal patient cooperation. Thus, we investigated whether chromatic pupillometry could be useful for neurological evaluations in GD. In our neuronopathic GD patients, red light-induced PLR was markedly impaired, whereas blue light-induced PLR was relatively spared. In addition, patients with non-neuronopathic GD showed no abnormalities. These novel findings show that chromatic pupillometry is a convenient method to detect neurological signs and monitor the course of disease in neuronopathic GD.

Cytochrome P450 oxidoreductase (POR) deficiency (PORD) is an autosomal recessive disorder caused by loss-of-function mutations in the POR (NM_000941.3) on chromosome 7q11.23. 1 POR serves as an electron donor enzyme to all microsomal cytochrome P450 enzymes. 1 A decreased POR function causes various symptoms, such as skeletal malformations, disorders of sex development, and adrenal insufficiency. 2,3 Skeletal malformations, such as joint contracture, radiohumeral synostosis, midface hypoplasia, and craniosynostosis, are distinctive clinical features of PORD, also known as Antley-Bixer syndrome, when associated with facial and cranial malformations and limb deformities. 1,3 The predicted cause of the malformations is impairment of cholesterol production and retinoic acid metabolism. 4 Scoliosis complications in patients with PORD have been reported, 2,3,5 but there are no reports of its severity or surgical intervention. Here, we describe two cases of PORD with severe scoliosis and surgery requirement.Patient 1 was a 7-year-old girl with a 46, XX karyotype. She was born at 40 weeks of gestation, weighed 2562 g, and measured 50 cm in length. She showed hypoplasia in the midline of the face, protrusion of the eyeball, clitoromegaly, labial fusion, radiohumeral synostosis, and finger joint contraction at birth. She was diagnosed with PORD based on POR genetic analysis, showing compound heterozygous mutations c.1370 G>A (p. R457H) and c.760 + 1 G>A. She was diagnosed with scoliosis at 6 months of age, and was fitted with an Osaka Medical College (OMC) brace at the age of 3 years, and then a trunk F I G U R E 1 X rays of the spine of the patient before and after surgery. A, Preoperative spine X-ray of patient 1. The Cobb angle is 85 . B, Post-operative X-ray of patient 1. C, Preoperative spine X-ray of patient 2. The cobb angle is 70 . D, Post-operative X-ray of patient 2

Pathogenic mitochondrial DNA heteroplasmy has mainly been assessed with bulk sequencing in individuals with mitochondrial disease. However, the distribution of heteroplasmy at the single-cell level in skin fibroblasts obtained from individuals, together with detailed clinical and biochemical information, remains to be investigated. We used the mitochondrial DNA single-cell assay for the transposase-accessible chromatin sequencing method. Skin fibroblasts were obtained from six individuals with mitochondrial disease and pathogenic m.3243A>G variants of differing severity. Different distributions of heteroplasmy at the single-cell level were identified in skin fibroblasts from all six individuals. Four individuals with different outcomes showed similar averaged heteroplasmy rates with normal mitochondrial respiratory chain enzyme activity, while the distribution of single-cell heteroplasmy patterns differed among the individuals. This study showed different heteroplasmy distribution patterns at the single-cell level in individuals with the m.3243A>G variant, who had a similar averaged heteroplasmy rates with normal mitochondrial respiratory chain enzyme activity. Whether such different heteroplasmy distribution patterns explain the different clinical outcomes should be assessed further in future studies. Measuring heteroplasmy of pathogenic mitochondrial DNA variants at the single-cell level could be important in individuals with mitochondrial disease.Atsuko Imai-Okazaki and Kazuhiro R. Nitta contributed equally to this work.

BackgroundEnoyl-CoA hydratase short-chain 1 (ECHS1) is an enzyme involved in the metabolism of branched chain amino acids and fatty acids. Mutations in theECHS1gene lead to mitochondrial short-chain enoyl-CoA hydratase 1 deficiency, resulting in the accumulation of intermediates of valine. This is one of the most common causative genes in mitochondrial diseases. While genetic analysis studies have diagnosed numerous cases withECHS1variants, the increasing number of variants of uncertain significance (VUS) in genetic diagnosis is a major problem.MethodsHere, we constructed an assay system to verify VUS function forECHS1gene. A high-throughput assay usingECHS1knockout cells was performed to index these phenotypes by expressing cDNAs containing VUS. In parallel with the VUS validation system, a genetic analysis of samples from patients with mitochondrial disease was performed. The effect on gene expression in cases was verified by RNA-seq and proteome analysis.ResultsThe functional validation of VUS identified novel variants causing loss ofECHS1function. The VUS validation system also revealed the effect of the VUS in the compound heterozygous state and provided a new methodology for variant interpretation. Moreover, we performed multiomics analysis and identified a synonymous substitution p.P163= that results in splicing abnormality. The multiomics analysis complemented the diagnosis of some cases that could not be diagnosed by the VUS validation system.ConclusionsIn summary, this study uncovered newECHS1cases based on VUS validation and omics analysis; these analyses are applicable to the functional evaluation of other genes associated with mitochondrial disease.

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