Novel Mutation in A patient with Canavan Disease Osama Zaki1, Heba S. El Abd1, Shaimaa A. Mohamed1, Navaneeth Krish2, Hatem Zayed*3 1. Medical Genetics Unit, Pediatric Department, Faculty of Medicine, Ain-Shams University, Cairo 11665, Egypt, 2. Heart Science Centre, National Heart and Lung Institute, Imperial College London, Harefield, UK, 3. *College of Health and Sciences, Biomedical Program, Qatar University, Doha Qatar,
[email protected]
Canavan disease (CD) is a rare fatal childhood neurological autosomal recessive genetic disease caused by mutations in the ASPA gene, which lead to catalytic deficiency of the ASPA enzyme that catalyzes the deacetylation of NAA. It is a severe progressive leukodystrophy characterized by spongiform degeneration of the white matter of the brain. CD occurs frequently among Ashkenazi Jewish population, however it has been reported in many other ethnic groups with significantly lower frequency. Here, we report on a 2 year-old Egyptian child with severe CD who harbors a novel homozygous missense variant (c.91G > T, p.V31F) in the ASPA gene. The clinical, radiological, structural, and molecular genetic profiles are reviewed in details.
Canavan disease (CD; MIM#271900) is a rare autosomal recessive neurodegenerative disease caused by deficiency of the aspartoacylase enzyme (ASPA; NP_000040.1), due to genetic mutations in the ASPA gene (refseq NM_000049.2). ASPA enzyme is a functional homodimer and the key for myelin synthesis, which catalyzes the hydrolysis of Nacetyl-L-aspartate (NAA) into aspartate and acetate in the human brain. Patients with deficient ASPA enzyme have abnormal elevation of NAA in the brain. This can be detected using magnetic resonance spectroscopy (MRS) before increasing its concentration in the urine, which is suitable for early diagnosis of CD [1]. Clinical symptoms are not manifested at the time of birth; however the clinical triad of hypotonia, macrocephaly, and head lag, are initial diagnostic manifestations for CD in early childhood. CD patients have characteristic swelling, spongiform degeneration of the white matter of the brain, dysmyelination, and intramyelinic oedema with consequent impairment of psychomotor development in the first months of life, which is characterized by cognitive delay, ataxia, and irritability. Other symptoms include preserved social interactions, difficulties in visual tracking, sucking, and progressive macrocephaly. Disease progression is marked by seizures, atrophy of the optic nerve, intellectual disability, spastic tetraparesis, and early death [2]. The spongy degeneration of the white matter in CD patients can be diagnosed using magnetic resonance imaging (MRI) that typically shows signal abnormalities of the white matter and the basal ganglia (Michel and Given 2006). Here we present an Egyptian patient with CD, that can be attributed to a novel homozygous variant c.91G > T (p.V31F) in the ASAP gene.
Fig. 2. Magnetic Resonance Spectroscopy measuring the NAA levels (j,k) at TE 31 ms (j) showing elevation of N-acetyle aspartate as well as glutamate and myoinisitol. MRS at TE 144 ms (k) showing elevation of N-acetyle aspartate (NAA/creatine = 3.19).
In this study, we report the identification of a novel homozygous missense variant (c.91G > T, p.V31F) in the ASPA gene in an Egyptian patient with CD. This variant was not seen in 200 healthy controls, not reported in the exome variant database (http://evs.gs.washington.edu/EVS/), nor in the 1000 genome database, in silico analysis using PolyPhen-2, SIFT, Mutation Taster, and SNPs&GO predicts this variant to be pathogenic with high probability scores (data not shown). Valine 31 was found to be conserved among different species (Fig. 3b), this was tested using the protein of each organism and multiple sequencing alignment was done using Clustal Omega multiple sequence alignment tool (http://www.ebi.ac.uk/Tools/msa/clustalo/).
Fig. 3. Molecular analysis of the novel mutation. a The electropherogram shows a homozygous G > T transversion at position 91 of the ASPA gene of the patient (case), indicated with an arrow, compared to a healthy control, resulting in the substitution of Valine 31 residue by a Phenylalanine residue (p.V31F). b Amino acid sequence alignment with different species, the conserved V31 is shown in bold, the sequence was retrieved from the NCBI web site, and multiple sequence alignment was performed using Clustal Omega (http://www.ebi.ac.uk/Tools/ msa/clustalo/).
Fig. 1. Neuroradiology of the patient. Axial T2WI (a, b, c) shows diffuse. signal abnormality of cerebral and cerebellar hemispheres. Dorsal brain stem (short arrow), middle cerebellar peduncles (long arrow) are involved, the globus pallidus (g) and posterior limb of internal capsule (arrowhead) are also abnormal.DWI (d,e,f) and ADC (g,h,i) shows areas of diffusion restriction which appear to have occipito-frontal gradient (*). Facilitated diffusion is seen in the middle cerebellar peduncles (white arrow).
Fig. 5. Structural consequences of the p.V31F variant. a) and b) Superimposed structures of both Apo and p.V31F shown with their mutational impact on the dimer, where in B the variant induced changes near the mutational spot are highlighted in yellow (Apo) and red (p.V31F). c) The key residues that are in the vicinity of V31 are displayed in yellow sticks. d) For the clarity, the comparison of Apo and mutant p.V31F dimers are shown only with the most affected region, helix α1,α2 and sheet β2. e) The changes in the backbone of the Zn2+ binding coordinates that are close to the mutation spot are shown in sticks. In the figure, Zn2+ is represented as sphere.
Fig. 4. Structural stability of the systems. a) Structural deviation between the systems during the MD simulations b) Average fluctuations of the residues and c) Average number of hydrogen bonds of the Apo and p.V31F forms in the course of MD simulations
The structural analysis provides significant insight to the functional relevance, to compare the different structures we superposed the representative structures (most frequently occurring and energetically stable from the MD simulations) of the Apo and p.V31F. The overall deviation of 2.4 Å between the structures (Fig. 5a) indicates that the variant affects the entire structure and it might result in a different conformation than the functional Apo form. In particular, the impact was initiated in the middle of the helix α1, where the variant is located and this effect was transferred to the helix α2 and the sheet β2 via the loops on both sides of the helix (Fig. 5b). The vicinity of V31 is critical as it contributes for coordinating Zn2+ (H21 and E24) and for substrate binding (R63) [3] (Fig. 5c-d). A significant structural change was noticed on the backbone of the key residues, H21, E24 and R63, near the catalytic site (Fig. 5e). These findings demonstrate the impact of the p.V31F variant and its consequence to the catalytic site, which might lead to modification in the normal function of the protein.
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