NAD(P)HX dehydratase protein-truncating mutations are associated with neurodevelopmental disorder exacerbated by acute illness

The identification of mutations in NAXD in six cases from the six families reported by Van Bergen et al. (2019) was the first-ever report of the association of the NAXD gene with a mitochondrial disorder. The affected individuals presented with fever-induced, severe multisystem diseases with repeated episodes of skin lesions, and died within the first decade of life (Van Bergen et al., 2019). The article described the compelling evidence of the pathogenicity of NAXD variants. Later, Zhou et al. (2020) described a patient with NAXD novel biallelic mutations with a phenotype like that in the previous article (Zhou et al., 2020). Herein, we describe another case with NAXD biallelic protein-truncating mutations, one of which is novel (c.44delG; p.Arg15Glnfs*3) and the other previously reported (c.51_54delAGAA; p.Ala20Phefs*9), in a Japanese patient with a neurodevelopmental disorder, autism spectrum disorder (ASD) and a muscular-dystrophy-like myopathy. Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form (NADP) play essential roles in cellular metabolism. NAD (reduced form, NADH) is involved in a series of catabolic reactions and mitochondrial energy production, whereas NADP (reduced form, NADPH) is a crucial component of numerous biosynthetic processes and cellular antioxidant protection systems (Agledal et al., 2010). The hydrated form of NADH (called NADHX) and NADPH (NADPHX) also spontaneously arises enzymatically, in acidic conditions, or at elevated temperature under stress, and further degrades irreversibly to cyclic NAD(P)HX (Rafter et al., 1954; Marbaix et al., 2014). These damaged cofactors inhibit several dehydrogenases (Prabhakar et al., 1998) and the accumulated toxic products inhibit critical metabolism. Therefore, functioning metabolite repair systems are crucial for the detoxification of toxic metabolic side-products, to ensure precise metabolism (Linster et al., 2013; Van Schaftingen et al., 2013). The nicotinamide nucleotide repair system consists of the NAXD [NAD(P)HX dehydratase, MIM#615910] and NAXE [NAD(P)HX epimerase, MIM#608862] enzymes. Disrupted function in the nicotinamide repair system can cause human diseases, notably fever-induced neurological deterioration (Spiegel et al., 2016; Van Bergen et al., 2019). In this study, we report the case of a 7-year-old male who experienced rapid deterioration after gastroenteritis, and died suddenly. The boy was born at term without remarkable complications. He was the second child of healthy, unrelated parents. At birth, his weight was 2800 g. His 11-year-old elder brother was healthy, and the paternal uncle died due to an acute myocardial infarction at the age of 28. The patient was developmentally well until the age of 1, and then he was admitted to the hospital due to bronchitis, hypotonia and hyperCKaemia. During his hospital stay, biochemical analyses revealed mild anaemia (Hb: 10.7 g/dl), elevated liver enzymes [alanine transaminase (ALT): 204 IU/l, ASL: 279 IU/l, lactate dehydrogenase (LDH): 1501 IU/l, alkaline phosphatase (ALP): 569 IU/l], hyperCKemia (CK: 9376 IU/l), and erythrocyte sedimentation rate (ESR) of 129 mm/h. Detailed virology and toxicology screening found no viral or parasitic infections, but Hemophilus influenzae and Corynebacterium species were evident. A muscle MRI excluded the diagnosis of myositis. Persistent elevation of serum enzymes (AST, ALT, ALP, LDH and CK) and muscle weakness led physicians to consider a diagnosis of muscular dystrophy. Gene panel analysis for muscular dystrophy could not identify any causative mutation. At 4 years of age, his neurodevelopmental growth was considered unsatisfactory and he exhibited poor communication skills, clumsiness and repetitive behaviour. Because of his limited interactions, his neurodevelopment was evaluated using the Pervasive Developmental Disorders Autism Society Japan Rating Scale (PARS-TR) (Ito et al., 2012). The PARS-TR is a comprehensive and widely used Japanese scale for the diagnosis and evaluation of the severity of ASD symptoms, with the help of a family member of the subject. The evaluation of the symptoms is based on structured items about behavioural problems or symptoms of ASD. A diagnosis of ASD is suspected when the PARS-TR score is ≥9 points in a preschooler: the patient scored 10 points. He was also assessed on another standardized developmental assessment scale for Japanese infants, named the Kyoto Scale of Psychological Development 2001 (KSPD) (Ikuzawa et al., 2001). His KSPD score was within the normal range, but the physician observed the patient’s poor communication skills. The Wechsler Intelligence Scale for Children version IV (Wechsler, 2003) was used for the intellectual assessment of the patient, and he had an average score on the full-scale IQ assessment. The patient was admitted to the hospital with gastroenteritis at 7 years and 3 months of age. He experienced frequent vomiting. Laboratory investigations upon admission revealed: a blood urea nitrogen to creatinine (BUN/Cre) ratio of 37.5:1 (normal range 10:1 to 20:1); AST, 220 IU/l; ALT, 220 IU/l; LDH, 1000 IU/l (normal range for children: 60–170); CK, 5346 IU/l; lactate, 125 mg/dl (normal range: <18 mg/dl); and glucose, 230 mg/dl (normal range: 70–100). The patient suffered from intravascular dehydration, lactic acidosis (pH, 7.15; pCO2, 36 torr; pO2, 37 torr; HCO3−⁠, 12 mmol/l; and base excess, 15 mmol/l), hyperglycaemia and acute liver injury. After correction of the dehydration, he was able to take food orally, and the maintenance dose of fluid was continued. Early the following morning, he suddenly deteriorated and immediate resuscitation failed to save his life. An autopsy imaging CT scan and autopsy were performed with his parents’ consent, to determine the cause of death. CT revealed the thickening of the myocardial wall, no apparent lesions in the skull or airways, and gross hypertrophic cardiomyopathy. Acute inflammation of the digestive tract (oesophagus, stomach, small intestine and large intestine), acute myocarditis, acute tubular necrosis and congestion of various organs (liver, both lungs and spleen) were observed macroscopically and histologically. Whole-exome sequencing of the patient’s blood DNA identified biallelic variants in the NAXD gene [NM_001242882.1: c.44delG (p.Arg15Glnfs*3); c.51_54delAGAA (p.Ala20Phefs*9)]. We could not identify any other candidate variants in the mitochondrial genome. Segregation analysis showed that his parents are heterozygous for the two variants (Fig. 1A and B). Homozygous deletion of the c.51_54delAGAA variant in a patient with early-onset progressive encephalopathy with brain oedema and/or leukoencephalopathy-2 was reported by Van Bergen et al. (2019). The novel variant c.44delG has not been reported in the dbSNP (https://www.ncbi.nlm.nih.gov), gnomAD (Lek et al., 2016) or 3.5KJPN (Nagasaki et al., 2015) databases. Conservation of amino acid sequences among the reported transcripts and predicted mitochondrial (mtNAXD) and cytosolic (cNAXD) isoforms are shown in Fig. 1C. Both variants are localized in the mitochondria-targeted isoform (mtNAXD, NP_001229811.1) (Fig. 1C). NAXD variants information and NAXD protein levels. (A) Pedigree of the patient’s family showing NAXD variants. Sanger sequencing of the patient with NAXD variants showing the biallelic inheritance pattern. (B) Amino acid (AA) alignments of four different NAXD isoforms showing the AA sequence position of the variants. Alignments were constructed with ClustalW2 (http://www.ebi.ac.uk/clustalw2/). The upper red bar indicates the sequence conservation and the ‘Consensus’ bars indicate the reference sequences. mtNAXD = mitochondria-targeted NAXD; cNAXD = cytosolic NAXD; NP_060680.2 (NM_018210.3); NP_001229810.1 (NM_001242881.1); NP_001229811.1 (NM_001242882.1); and NP_001229812.1 (NM_001242883.1). (C) Western blot immunoblotting of cytoplasmic (Cyto) and mitochondrial (Mito) extracts of V5-tagged wild-type mtNAXD (mtNAXD-WT) and mutated mtNAXD (mtNAXD-Arg15Glnfs*3; mtNAXD-Ala20Phefs*9) showing loss-of-protein expression due to mutations compared with wild-type NAXD. The plasmids were constructed based on NM_001242882.1 transcript. HSP60 and ACTB were used as loading controls. Anti-NAXD antibody (ab82820, Abcam) and anti-V5 antibody (R960-25, Invitrogen) were used. The predicted band size of the NAXD protein is 37 kDa. We constructed plasmids to overexpress mutated NAXD in the HEK293FT cell line to assess the effects of both variants. The plasmids encoding mtNAXD (NM_001242882.1) wild-type, mtNAXD-delG and mtNAXD-delAGAA cDNA, with a C-terminal V5-tag, were generated in the pCS-CA-MCS vector systems. Transient overexpression (72 h post-transfection) of the constructs in the HEK293FT cell line revealed loss-of-protein function due to NAXD variants (Fig. 1D). We also performed OXPHOS enzymology from autopsy-derived cardiomyocytes using a Cary 300 Bio (Varian) and observed complex I deficiency (CI: 19.6%). Although previous reports have not confirmed the effect of NAXD variants on protein expression, our report has demonstrated it. At least two identified variants (localized on the N-terminal side of the mtNAXD isoform) cause loss-of-protein expression or a change in the translation-initiation site, due to frameshift deletions. Thus, the previously reported variant and the newly identified variant both affect the presence of NAXD protein in mitochondria. We compare the reported NAXD cases in Table 1. Clinical features of the patients with NAXD mutations C1: c.839+1G>T, c.922C>T C2: c.187G>A, c.948_949insTT C3: c.51_54delAGAA C4: c.308C>T C5: c.54_57delAAGA C6: c.331C>T and c.776T>G C1: c.839+1G>T, c.922C>T C2: c.187G>A, c.948_949insTT C3: c.51_54delAGAA C4: c.308C>T C5: c.54_57delAAGA C6: c.331C>T and c.776T>G AR = autosomal recessive; C1‒C6 = Cases 1‒6; CI = complex I; CIV = complex IV; CII+CIII = complexes II and III; HSV = herpes simplex virus; RSV = respiratory syncytial virus. Clinical features of the patients with NAXD mutations C1: c.839+1G>T, c.922C>T C2: c.187G>A, c.948_949insTT C3: c.51_54delAGAA C4: c.308C>T C5: c.54_57delAAGA C6: c.331C>T and c.776T>G C1: c.839+1G>T, c.922C>T C2: c.187G>A, c.948_949insTT C3: c.51_54delAGAA C4: c.308C>T C5: c.54_57delAAGA C6: c.331C>T and c.776T>G AR = autosomal recessive; C1‒C6 = Cases 1‒6; CI = complex I; CIV = complex IV; CII+CIII = complexes II and III; HSV = herpes simplex virus; RSV = respiratory syncytial virus. ASD is a complex neurodevelopmental disorder characterized by impaired social interaction and communication skills, impaired language skills, limited interests and repetitive behaviours (Nygren et al., 2012). Although the pathophysiology of ASD is incomplete, various genetic, neurological, metabolic and immunological factors are known to be involved in the disease process. Isolated ASD features, without other neurological symptoms, can be associated with mitochondrial DNA depletion and m.3243A>G mtDNA mutation (Pons et al., 2004). Among the causes of ASD, oxidative stress, reduced antioxidant activity, mitochondrial dysfunction and impaired energy metabolism (NAD+, NADH, ATP, pyruvate and lactate) are notable (Essa et al., 2013). Vitamins and minerals are crucial cofactors for the production of many neurotransmitters and their precursors; the levels of NADH and NADPH, vitamins, minerals and amino acids in the plasma of children with ASD are significantly lower than normal (Adams et al., 2011). More specifically, pyridoxine (vitamin B6) and magnesium have been shown to be involved in serotonin, dopamine and norepinephrine synthesis, whereas ascorbic acid and inositol exert effects on the dopaminergic pathway and serotonin levels, respectively (Gogou and Kolios, 2017). NADPH indirectly functions as an antioxidant factor which reduces the oxidized glutathione to reduced glutathione and directly scavenges toxic free radicals (Kirsch and De Groot, 2001). Glutathione is an antioxidant, and a reduced level of total glutathione, increased ratio of oxidized to reduced glutathione and increased level of oxidized glutathione have been observed in ASD (Ghanizadeh et al., 2012). ASD is also associated with non-specific gastrointestinal symptoms, intestinal mucosal abnormalities and microbiome synthesis, and gut-brain-based processes may contribute to the pathophysiology in ASD (Fulceri et al., 2016). ASD is a complex neurodevelopmental disorder and the neurobiology of ASD is associated with oxidative stress. In this study, we describe an additional case of NAXD biallelic mutations, with ASD, which was exacerbated by acute gastroenteritis. NAXD mutations are potential causes of damage to the brain since the mutations impair metabolism, cause accumulation of toxic products and increase oxidative stress. The data are available from the corresponding author. We thank the patient’s parents for their support. We thank Dr Hiroyuki Miyoshi of Keio University and RIKEN BioResource Centre for the pCS-CA-MCS plasmid (https://dnaconda.riken.jp/search/RDB_clone/RDB05/RDB05963.html). We thank the Laboratory of Molecular and Biochemical Research, Research Support Center, Juntendo University Graduate School of Medicine, for technical assistance. This work was carried out at the Intractable Disease Research Center, Juntendo University Graduate School of Medicine. We thank Claire Barnes, PhD, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript. The work was supported by grants from the ‘Practical Research Project for Rare/Intractable Diseases’ [grant numbers 19ek0109273, 18ek0109177] and the ‘Program for an Integrated Database of Clinical and Genomic Information’ [grant numbers 19kk0205014, 18kk0205002] from the Japan Agency for Medical Research and Development (AMED) (http://www.amed.go.jp/en/). The authors report no competing interests.