NAD(P)HX dehydratase (NAXD) deficiency due to a novel biallelic missense variant and review of literature

Abstract

Encephalopathy, progressive, early-onset, with brain edema and/or leukoencephalopathy, 2 (PEBEL2; MIM# 618321), caused by biallelic pathogenic variants in the NAD(P)HX dehydratase (NAXD) is a rare metabolite repair disorder. It is characterized by progressive neurological deterioration usually associated with a febrile illness. The other common findings include skin lesions, elevated serum or cerebrospinal fluid lactate levels, and brain neuroimaging abnormalities. Currently, variants in NAXD have been reported in eight unrelated individuals including six truncating and six missense variants. We report on an additional individual with characteristic findings of PEBEL2, and an additional finding of sparse scalp hair. A novel missense variant c.301G>A, p.(Ala101Thr) in a homozygous state was identified through exome sequencing. This study adds to the phenotypic and mutational spectrum of PEBEL2. We review the existing phenotypic and genotypic information for the individuals with this neurometabolic condition.

Introduction

Metabolite repair system plays essential role in the efficient functioning of metabolic networks. NAD(P)HX dehydratase (NAXD; MIM*615910) gene encodes for one of the key enzymes in the nicotinamide nucleotide repair system (Marbaix et al., 2011). Encephalopathy, progressive, early-onset, with brain edema and/or leukoencephalopathy, 2 (PEBEL2; MIM# 618321) resulting in NAXD deficiency is the third known disorder of metabolite repair in addition to encephalopathy, progressive, early-onset, with brain edema and/or leukoencephalopathy, 1 (PEBEL1; MIM# 617186) and L-2-hydroxyglutaricaciduria (MIM# 236792) (Kremer et al., 2016; Van Schaftingen et al., 2009). It is an autosomal recessive disorder and results in a rapidly progressive neurological deterioration associated with a febrile illness (Van Bergen et al., 2019). The molecular basis of this disorder is recently linked to the pathogenic variants in the NAXD gene (Van Bergen et al., 2019). To date, including the present individual, a total of nine unrelated individuals with PEBEL2 have been reported (Van Bergen et al., 2019; Zhou et al., 2019).

Here we describe in detail the findings of an individual with PEBEL2 harboring a novel homozygous missense variant identified through exome sequencing (ES). Further, we review the phenotypic and genotypic findings of eight additional individuals of PEBEL2.

Clinical report

We ascertained a 2-years-and-9-months female first-born of third-degree consanguineous healthy parents (Fig. 1.A). She was born at term by emergency cesarean section due to meconium-stained liquor. Her birth weight was 2.8 kg (−1.1 SD) and the perinatal period was uneventful. She was healthy until 1 year of age when she was first admitted for fever, lower respiratory tract infection, vomiting, and acute gastroenteritis. It was followed by seizures, manifesting as involuntary movements of all four limbs with perioral twitching. It was diagnosed as febrile seizures and with supportive treatment her condition improved. She was readmitted at the age of 1 year 7 months for fever and acute gastroenteritis for 2 days followed by generalized tonic-clonic seizures. She was developmentally normal and spoke around ten words before this episode of illness but later showed regression of speech and could not speak. At 1 year 7 months, her electroencephalography (EEG) showed mild to moderate generalized disturbance of electrical function suggestive of mild to moderate encephalopathy. Her echocardiography and brain magnetic resonance imaging (MRI) were apparently normal. She had raised C-reactive protein (CRP >350 mg/L, normal range 0–10 mg/L), mild anemia (Hb: 10.5 gm/dL), and neutropenia (Neutrophil count: 10.1%, normal range 42–74%; absolute neutrophil count: 0.61×10³/μL, normal range 1.5–8.0×10³/μL). She received supportive treatment and gradually recovered. Subsequently, she had 4–5 hospitalizations till the age of 2 years 9 months for seizures. These episodes of seizures were triggered by febrile illness such as respiratory infection or acute gastroenteritis. Her brain MRI at 2 years 6 months was apparently normal (Fig. 1.C1).

Figure 1:

(A) Pedigree of the family; (B) Clinical photographs of the proband: sparse scalp hair and erythematous and eroded plaques with crusting on neck and groin; (C) Serial brain MRI of the proband showing: (C1) normal findings at 2 years 6 months, and (C2) mild cerebral atrophy at 2 years 9 months; (D) IGV of proband showing sequence variant c.301G>A in NAXD in homozygous state and chromatograms showing c.301G>A variant in homozygous state in the proband and heterozygous state in her parents

She was referred for genetic evaluation to us at the age of 2-years-and-9-months with complaints of recurrent episodes of seizures. At this age she could walk, run, scribble, draw vertical lines, and wave bye-bye but could only speak bisyllables. On examination at the age of 2-years-and-9-months, her occipitofrontal circumference was 45 cm (−2.4 SD), height was 81 cm (−3 SD), and weight was 9.3 kg (−3 SD). She had sparse scalp hair, pallor, and skin lesions in the form of erythematous and eroded plaques with crusting predominantly in the flexural creases of neck, axilla and the groin region (Fig. 1.B). Her detailed neurological and systemic examination were unremarkable. She did not have focal neurological deficits or any neurocutaneous markers. MRI brain at this age revealed mildly dilated ventricles and prominent sub-arachnoid spaces suggestive of mild cerebral atrophy (Fig. 1.C2). Baseline investigations showed raised CRP (>350 mg/L), mild anemia (Hb: 10.6 gm/dL), and neutropenia (Neutrophil count: 8%; absolute neutrophil count: 0.30×10³/μL). Cell count, glucose, protein, and lactate of the cerebrospinal fluid (CSF) were normal. Hepatic and renal function, serum electrolytes, blood glucose, lactate, ammonia, thyroid studies, antinuclear antibody profile, and lupus studies were in normal range. She was clinically diagnosed with epileptic encephalopathy and managed on antibiotics, antipyretics, and antiepileptics. In view of skin lesions and gastroenteritis, biotin (10 mg/day) and multivitamin complex were added. She responded to the treatment and was discharged on oral antiepileptics and biotin. Skin lesions also gradually improved.

At the age of 3 years, she was readmitted following a five days history of fever, lower respiratory tract infection, seizures and severe encephalopathy. Blood investigations showed microcytic anemia (Hb: 7.7 gm/dL), neutropenia (Neutrophil count: 4.8%; absolute neutrophil count: 0.16 × 10³/μL), mildly elevated serum lactate (24.2 mg/dL, normal range 4.5–19.8 mg/dL), and high CRP (260.6 mg/dL). Chest radiograph was suggestive of bronchopneumonia. She did not respond to the supportive treatment and succumbed during this episode of illness.

Methods

The EDTA blood samples were collected from the proband and her parents after obtaining written informed consent from her parents.

Molecular testing

Singleton ES (Illumina, Inc., San Diego, California, USA) was performed for the proband as described earlier (Girisha et al., 2019). The ES raw data was obtained and the called variants were analyzed based on variant prioritization and in-house filtering strategy. Variants with frequency of less than 1% in population database gnomAD and in-house database of 1167 exomes were included. Intergenic, intronic and synonymous variants were excluded. Exonic and splice site variants in genes with known OMIM phenotype were then prioritized based on its concordance to the observed phenotype. Validation of the variant identified by ES and segregation analysis was done by Sanger sequencing.

In silico protein modelling and analysis

Structures of wild-type and mutant p.(Ala101Thr) human NAXD protein (UniProt accession: Q8IW45) was predicted using the online web-server SWISS-MODEL (https://swissmodel.expasy.org/). The resultant protein model was loaded on PyMol (The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC) and the in silico mutagenesis was performed to obtain the mutant protein. This was followed by analysing the alteration in the polar contacts of the residue of interest (Ala101) with surrounding amino acids as compared to the mutant type (Thr101). Multiple sequence alignment was performed using Clustal Omega. The structural effect of the missense substitution Ala101Thr was predicted using HOPE (http://www.cmbi.ru.nl/hope/) and I-Mutant (http://folding.biofold.org/imutant/imutant2.0.html).

Results

Molecular testing

On analysis of the ES data, a novel missense variant c.301G>A, p.(Ala101Thr) was observed in exon 4 of NAXD (NM_001242882.2) in a homozygous state in the proband. The variant was not observed in a homozygous state in population database gnomAD and our in-house data of 1167 exomes. However, it was present in the heterozygous state in 4/250640 individuals (allele frequency 0.0000159) in gnomAD database. Multiple in-silico analysis tools (MutationTaster, SIFT, PROVEAN, LRT, and FATHMM_MKL) consistently predict the variant as damaging to NAXD protein function. On segregation analysis, the unaffected parents were found to be carriers for this variant (Fig. 1.D).

In silico protein modelling and analysis

In silico protein modelling of this variant followed by mutagenesis and comparative analysis of the wild type and mutant protein resulted in gain of two polar contacts with the neighboring residues, Gly74 and Leu103 (Fig. 2.B and C). In silico protein structure analysis tool HOPE predicted the effects caused by differences between the wild type and mutant amino acids with respect to domain, size and hydrophobicity. The variant was found to be located in a YjeF C-terminal of the main domain that predicts the abolition of its function. The mutant residue was found to be larger in size and less hydrophobic as compared to the wild type residue. I-mutant predicted reduced structural stability of the protein due to the effect of mutant residue Thr101 (DDG −1.10).

Figure 2:

In silico protein modelling followed by mutagenesis: (A) Amino acid conservation using Clustal Omega indicating Ala101 as a highly conserved residue. An asterisk (*) represents fully conserved residue, colon (:) represents residues with strongly similar properties, and period (.) represents residues with weakly similar properties; (B) Predicted polar contacts of wild type Ala101 residue with Ala96 and Arg268; (C) Mutagenesis showing two additional polar contacts of mutant Thr101 residue with Leu103 and Gly74 in addition to Ala96 and Arg268 of the wild type residue; (D) Graphical illustration of pathogenic sequence variants identified in individuals with PEBEL2 including the present proband.

Discussion

NAD(P)HX epimerase (NAXE, MIM* 608862) and NAD(P)HX dehydratase (NAXD; MIM*615910) are the two key enzymes involved in the nicotinamide nucleotide repair system. Pathogenic variants in NAXE and NAXD can result in fever-induced neurological deterioration. The characteristic clinical features of NAXD deficiency such as neurological deterioration following febrile illness, skin lesions, movement abnormalities, and early death overlap with NAXE deficiency with additional features such as hypotonia, and nystagmus observed in later (Kremer et al., 2016; Van Bergen et al., 2019).

The human NAXD (formerly CARKD; MIM*615910) gene was recently described to be associated with a severe multisystem neurodegenerative disorder causing early death in life (Van Bergen et al., 2019). Van Bergen et al. reported six unrelated individuals with pathogenic variants in the NAXD causing febrile illness-induced neurodegeneration and early death (Van Bergen et al., 2019). A similar phenotype was reported in a Chinese and a Japanese individual with biallelic pathogenic variants in the NAXD gene (Zhou et al., 2019; Borna et al., 2020). We report on an additional individual of Indian origin who presented with a period of normal development followed by episodic neurological deterioration following episodes of infectious illnesses, as seen in previously reported eight individuals (Table 1). All nine individuals had an identified trigger event like acute respiratory illness, gastroenteritis, or vomiting (Van Bergen et al., 2019; Zhou et al., 2019; Borna et al., 2020). Seizures were associated with febrile illness in seven individuals, including the proband in the study. Developmental delay before the onset of febrile illness was reported in two individuals, which was followed by regression of milestones after the febrile illness. In six individuals, including the present proband, developmental regression was noted following episodic illnesses. Five of the eight reported individuals had varied skin lesions (Van Bergen et al., 2019; Zhou et al., 2019). The present individual also had skin lesions similar to those observed previously in other affected individuals. These lesions gradually improved with the subsidence of illness. The present individual also had sparse scalp hair which was not observed in previously reported individuals. Three of the eight reported individuals had cardiac presentations and two had myopathy which were not seen in the present proband. Gait and movement abnormalities like ataxia, dystonia, athetosis, and choreiform movements were observed in five reported individuals but not seen in the present individual. The ophthalmic evaluation in three reported individuals revealed ophthalmoplegia in two and bilateral cataracts in one individual. However, the present individual had normal ophthalmic findings.

Table 1:

Clinical and molecular findings in individuals with variants in NAXD gene

	Present study	Van Bergen et al., 2019						Zhou et al., 2019	Borna et al., 2020	Summary
Individuals	III-3	Individual 1: II-2	Individual 2: II-1	Individual 3: II-2	Individual 4: II-1	Individual 5: II-3	Individual 6: II-2	II-1	II-2	9
Gender	Female	Male	Female	Female	Male	Female	Female	Male	Male	Males: 4 Females: 5
Consanguinity	+	-	-	-	-	-	-	-	-	1/9
Age at presentation	1 year	3 years 7 months	1 year 2 months	10 months	3 months	2 years 6 months	8 months	2 years 10 months	1 year	3 months to 3 years 7 months
Age at death	3 years	4 years 6 months	3 years 10 months	1 year	4 months	3 years	1 year 10 months	Alive	7 years	4 months to 7 years
Episodes of fever or illness prior to deterioration	+	+	+	+	+	+	+	+	+	9/9
Development delay before onset	-	+	-	-	-	-	+	-	-	2/9
Developmental regression (age)	-	+ (3 years 7 months)	+ (1 year 2 months)	-	+ (3 months)	+ (2 years 6 months)	+ (1 year 7 months)	+ (3 years 10 months)	+ (4 years)	7/9
Seizures	+	+	-	+	+	+	+	+	-	7/9
Skin lesions	+	+	+	-	+	-	+	+	-	6/9
Respiratory infection	+	+	-	-	+	+	-	+	+	6/9
Gastroenteritis	+	+	+	+	-	-	+	-	+	6/9
Vomiting	+	-	-	+	-	+	-	+	-	4/9
Cardiac involvement	-	-	-	-	Acute myocarditis	Dilated cardiomyopathy	-	-	Acute myocarditis	3/9
Myopathy	-	-	-	-	-	+	-	-	+	2/9
Gait and movement abnormality	-	Ataxia, choreiform movements, dystonia	Ataxia, dystonia	-	-	Ataxia	Impaired coordinati on	Blinking of eyelids, head shaking, athetosis, dystonia	-	5/9
Ophthalmic abnormality	Absent	Ophthalmoplegia	Ophthalmoplegia	NA	Bilateral cataracts	NA	NA	NA	NA	3/4
Hematological abnormality	Mild anemia, neutropenia	Pancytopenia, neutropenia	Pancytopenia, neutropenia	Mild anemia	Pancytopenia, neutropenia	Mild anemia	NA	Absent	Mild anemia	7/8
Investigations
Elevated serum or CSF lactate	+	-	-	+	-	+	NA	-	+	4/8
C-reactive protein (mg/L)	High (>350)	High (225)	NA	NA	High (133)	High (21)	High (NA)	NA	NA	5/5
EEG abnormality	Generalized electrical disturbances	Bilateral epileptiform activity	NA	NA	Hypsarrhythmia	NA	Normal	Slow waves in bilateral occipital regions	NA	4/5
Brain MRI and MRS abnormalities
Cerebral atrophy	+	+	-	NA	+	+	-	+	NA	5/7
White matter signal abnormalities	-	+	-	NA	-	+	+	-	NA	3/7
Thin corpus callosum	-	+	-	NA	-	-	-	-	NA	1/7
Basal ganglia changes	-	-	+	NA	+	-	+	+	NA	4/7
Cerebellar changes	-	-	-	NA	-	-	-	+	NA	1/7
MR spectroscopy	NA	NA	NA	Normal	Lactate peak in the deep grey nuclei and in the cerebrum	NA	NA	NA	NA	1/2
Genetic testing
Sequence variants in NAXD (NM_001242882.2)	c.301G>A	c.839+1G>T/c.922C>T	c.187G>A/ c.948_949insTT	c.51_54delAGAA	c.308C>T	c.54_57delAAGA	c.331C>T/c.776T>G	c.101_102d elTA/c.318C>G	c.44delG/c.51_54delAGAA
HGVS Protein	p.(Ala101Thr)	p.(?)/ p.(Arg308Cys)	p.(Gly63S er)/ p.(Ala317Leufs*64)	p.(Ala20Phefs*9)	p.(Pro103L eu)	p.(Ala20Ph efs*9)	p.(Leu111Phe)/ p.(Leu259Arg)	(p.Thr35Ph efs*63)/ (p.Ile106Met)	(p.Arg15Gl nfs*3)/ (p.Ala20Ph efs*9)
Location	Exon 4	Intron 9/ exon 10	Exon 2/ exon 10	Exon 2	Exon 4	Exon 2	Exon 4/ exon 9	Exon 2/ exon 4	Exon 1/ Exon 2
Type of variant	Missense	Splicing/ missense	Missense/ frameshift	Frameshif t	Missense	Frameshift	Missense/ missense	Frameshift/ missense	Frameshift	Frameshift: 5 Missense: 7 Splicing: 1
Zygosity	Homozygous	Compound heterozygous	Compound heterozygous	Homozygous	Homozygous	Homozygous	Compound heterozygous	Compound heterozygo us	Compound heterozygo us	Homozygous: 4
										Compound heterozygous: 5

(+): observed; (−): not observed; NA: data not available; EEG: Electroencephalography; MRI: Magnetic resonance imaging; MRS: Magnetic resonance spectroscopy; HGVS: Human Genome Variation Society

Mild anemia, neutropenia, and pancytopenia were variably observed in all individuals, including the present proband. C-reactive protein and serum lactate levels were increased in the present proband as found in earlier reported individuals. The observed brain MRI findings include cerebral atrophy, white matter signal abnormalities, thin corpus callosum, basal ganglia changes, and oedema of the pons, medulla, and cerebellum (Van Bergen et al., 2019; Zhou et al., 2019). The present individual had normal imaging findings earlier and mild cerebral atrophy later in the course of the illness. One individual was reported with a lactate peak in the deep grey nuclei and cerebrum on brain magnetic resonance spectroscopy. Detailed clinical features of all the reported individuals, including the present proband, are reviewed in Table 1.

Exome sequencing revealed a novel homozygous missense variant c.301G>A p.(Ala101Thr) in NAXD in the proband. According to the American College of Medical Genetics and Genomics (ACMG) sequence variants interpretation guidelines, this variant is classified as a variant of uncertain significance (PM2 and PP3; Richards et al., 2015) and submitted to Leiden Open Variation Database. (LOVD; Variant ID: 00314930). This variant occurs on highly conserved residues (GERP++RS – 5.37) (Fig. 2.A). In silico protein modelling of the wild type residue (Ala101) was predicted to have two polar contacts with Ala96 and Arg268 (Fig. 2.B). This interaction was predicted to be intact in the mutant (Thr101), however two additional polar contacts were established with Gly74 and Leu103 (Fig. 2.C). Further, as a consequence of the missense variant Thr101, a decrease in the structural stability of the protein was predicted using I-Mutant. Of note, the mutant protein was predicted to lose hydrophobic interactions in the core of the protein or on the surface owing to its decreased hydrophobic nature as compared to wild type residue. Moreover, as the mutant residue is larger in size, it might lead to collision and altered protein stability, ultimately disturbing NAXD protein function.

NAXD gene encodes for an ATP-dependent NAD(P)HX dehydratase, one of the critical enzymes in the nicotinamide nucleotide repair system. This enzyme is targeted to several subcellular compartments, including the mitochondrion, and has a ubiquitous distribution across all tissues (Marbaix et al., 2014). However, as the brain has a constant demand for energy supply generated through mitochondrion, it is predicted to be more vulnerable to the deficient or impaired NAXD enzyme (Van Bergen et al., 2019). NAD(P)H are essential cofactors in either anabolic or catabolic cellular reactions and play a significant role in mitochondrial energy production. These are prone to hydration either by GAPDH enzyme or stressful conditions like acidic pH and increased temperature ((Rafter et al., 1954; Yoshida and Dave, 1975). Accumulation of these NAD(P)HX (hydrated forms of NAD(P)H) derivatives are found to inhibit several key mitochondrial dehydrogenases (Yoshida and Dave, 1975). These hydrated cofactors are suggested to be toxic to cells, thereby making detoxification with the help of metabolite repair system very critical. NAXD plays an important role in converting the (S)-epimer of NAD(P)HX cofactor back into their active form NAD(P)H (Marbaix et al., 2011). Pathogenic variants in NAXD gene are known to cause fever-induced neurological deterioration and skin lesions leading to PEBEL2 disorder (Van Bergen et al., 2019).

Previous studies have reported five frameshift variants, six missense, and one splicing variant in NAXD (Fig. 2.D; Table 1; Van Bergen et al., 2019; Zhou et al., 2019; Borna et al., 2020) to cause this neurometabolic condition. The frameshift and the splicing variants affect the NAXD protein and is predicted to cause loss of the protein function. Whereas, the missense variants in NAXD lead to a partial loss of enzyme activity and result in a significant decrease in thermostability, especially at higher temperatures (Van Bergen et al., 2019). This suggests loss of function as an underlying disease mechanism. Van Bergen et al. (2019) illustrated decreased expression of complex I and IV subunits and reduced respiratory chain activity in patient-derived fibroblasts. This provided evidence on the mitochondrial function being compromised in NAXD deficient individuals (Van Bergen et al., 2019).

To conclude, we report an additional individual with a novel homozygous missense variant in NAXD gene. The onset of neurological deterioration following febrile illness, skin and possibly the hair findings appear to be significant clues for clinical diagnosis of this condition.