Novel insertion mutation in the PLA2G6 gene in an Iranian family with infantile neuroaxonal dystrophy

Abstract Background Infantile neuroaxonal dystrophy is an autosomal recessive neurological disorder. Individuals with infantile neuroaxonal dystrophy experience progressive loss of vision, mental skills and muscular control, and other variable clinical signs. Pathogenic variants in the PLA2G6 gene, encoding phospholipase A2, are recognized to be the fundamental reason for infantile neuroaxonal dystrophy. This study aimed to detect pathogenic variant in a consanguine Iranian family with infantile neuroaxonal dystrophy. Methods The mutation screening was done by whole exome sequencing followed by direct Sanger sequencing. Results We identified a homozygous insertion mutation, NM_003560: c.1548_1549insCG (p.G517Rfs*29) in exon 10 of PLA2G6 in the patient. The parents were heterozygous for variant. Conclusions Because of the clinical heterogeneity and rarity of infantile neuroaxonal dystrophy, whole exome sequencing is critical to confirm the diagnosis and is an excellent tool for INAD management.


PLA2G6-associated neurodegeneration (PLAN) is a heterogeneous
class of autosomal recessive neurodegenerative conditions that categorized into four subtypes, based on onset age, containing infantile neuroaxonal dystrophy (INAD), autosomal recessive early-onset parkinsonism, dystonia-parkinsonism, and atypical neuroaxonal dystrophy (ANAD). 1,2 Mutation in PLA2G6 is the causative gene for PLAN. 3 Infantile neuroaxonal dystrophy (INAD) is a very rare inherited disorder with autosomal recessive pattern that affects the nervous system. 4 Its precise incidence is unknown. Subjects with INAD usually do not have any symptoms at birth and symptoms typically present between ages six months and three years. 4,5 Most patients with INAD show a progressive disorder with psychomotor regression or delay and mental deterioration, hypotonia, cerebellar ataxia, hyperreflexia, spastic tetraplegia, and visual impairment including strabismus and nystagmus. Hearing loss and seizures may occur in some affected children. 6 In many affected patients, death usually occurs by the age of ten years. 4 Homozygote mutations in the PLA2G6 gene have been recognized as the most common cause of INAD. 3,[7][8][9] The PLA2G6 gene is located on of the chromosome 22q13.1 and contains of 17 exons.
The protein encoded by this gene called a phospholipase A2 (Group VI) that involved the release of fatty acids from phospholipids. 3 Phospholipase A2 is expressed in several tissues such as the brain, spinal cord, kidney, lung, pancreas, and gut (www.genec ards.org).
Phospholipid metabolism is essential for various body processes, including helping to maintain the integrity of the cell membrane. 10 Few cases of INAD patients have been reported in the worldwide, especially in Iran. 11 The aims of the study were mutational analysis from a consanguineous family coming from Iran, whose proband showed progressive hypotonia and motor neuron defect.

| Family recruitment and ethical statement
One pediatric subject with progressive hypotonia and motor neuron defect, who was referred to the pediatric and genetics Clinic of Beski Hospital, was included in the current study. The parents provided their written informed consent to participate in this study,

| Whole exome sequencing
Genomic DNA from 6ml peripheral blood was extracted from all participants using the salting out method. The quality of DNA extraction is checked using 1% agarose gel electrophoresis. One μg of gDNA from patients was sheared, and exome capture was done using Sure Select Human All Exon V7r2. The enriched libraries were sequenced by NovaSeq 6000 platform with the coverage of target region about 100%. The sequencing data alignment, variant calling, annotation, variant prioritization, and prediction were performed as mentioned previously. 12
The PCR was performed in a total volume of 50 μl containing 20 μl of PCR Master mix, 1 μl of each forward and reverse primers (10 Pmol), 26 μl of ddH2O and 2 μl DNA. Thermal cycling conditions were primary denaturation step at 95°C for 4ʹ, then 34 cycles of denaturation step at 94°C for 28ʺ, annealing step 59°C for 25ʺ, and extension step at 72°C for 26ʺ, and a final extension step at 72°C for 6ʹ. The PCR products for direct Sanger sequencing were done on an automated ABI PRISM 3130XL (Applied Biosystems,). Then, the mutation was investigated in available family members for disease segregation analyses. Lastly, the sequencing results were aligned with a reference sequence in NCBI using the Chromas software.

| Clinical description
The proband (IV-3), a 2.5-year-old female, originating from Turkmen ethnicity from a consanguineous marriage who has shown signs of progressive hypotonia since 17 months of age (Figure 1). At the moment, she has signs such as developmental regression, destruction in the anterior horn neurons, nystagmus, feeding difficulty, speech delay, swallowing problems, and inability to walk. Brain MRI showed brief brainstorm involvement. Also, EMG-NCS revealed evidence of anterior horn cell involvement. Her elder sibling deceased at the age of 7 years with related symptoms.

| Mutational analysis
We identified a novel pathogenic insertion variation NM_003560: c.1548_1549insCG (p.G517Rfs*29) in our patient (IV-3) ( Figure 2B). Although our study has a limitation regarding lack of functional studies and the frequency of this novel PLA2G6 mutation await future examinations, we believe that the recognition of genetic defects related to INAD will eventually shed light on the underlying pathological mechanisms and help improve more effective management plans for INAD subjects in the future.

K E Y WO R DS
gene, homozygous, infantile neuroaxonal dystrophy, mutation, PLA2G6 F I G U R E 2 (A) The mutation status of PLA2G6 (c.1548_1549insCG) was validated by the Sanger sequencing, (B) the parents (III-5 & III-6) were in the heterozygous state, and the patient (IV-3) was homozygous for (c.1548_1549insCG)