In this study, we identified mutations in the phospholipase A2 group VI gene (PLA2G6) in the majority of patients clinically diagnosed with INAD and in a smaller fraction of patients with idiopathic NBIA. This latter condition is thought to encompass several different disorders, accounting for the lower frequency of mutations observed in this group. We found that genotype correlates with phenotype: mutations predicted to lead to absent protein were associated with the INAD profile of early onset and rapid progression, whereas compound heterozygous missense mutations correlated with the less severe phenotype of NBIA, consistent with residual function in the mutant protein ().
The term “infantile neuroaxonal dystrophy” is well established in the literature, as with other disorders historically described based on their clinical and pathologic features. Rather than abandon this terminology in the face of the PLA2G6 gene discovery, we instead propose a functional classification of mutation-positive patients to reflect the shared molecular etiology of their disease. Children presenting with psychomotor regression in the first year of life and loss of ambulation within 5 years should be designated as having “classic INAD.” When disease onset is later and progression slower, the term “atypical neuroaxonal dystrophy” should be used. For patients with brain iron on imaging but without a defined genetic basis for their disease, we propose continuing to use the term “idiopathic NBIA.” As more people are discovered to have mutations in PLA2G6, we anticipate that the phenotypic spectrum of atypical neuroaxonal dystrophy will probably expand but that this classification will remain useful.
Prior to the discovery of the causal gene, a diagnosis of INAD was confirmed by demonstrating axonal spheroids in a peripheral nerve biopsy. In our study, we found that some patients with mutations lacked spheroids and some without mutations had spheroids (), indicating incomplete detection using either pathologic or molecular methods. We were unable to identify a mutation in nearly one-fifth of patients with a strong clinical profile of INAD, including five patients with peripheral spheroids. We presume these patients to have undetected PLA2G6 mutations, indicating an 87% detection rate using current molecular and pathologic methods in combination. With the development of methods to identify large deletions, duplications, and insertions, we predict that the PLA2G6 mutation detection rate will exceed 95% by molecular methods alone, as has been achieved for other rare autosomal recessive disorders. Such assays are being developed for clinical use, and preliminary studies in patients with strong clinical and radiographic features for INAD suggest that up to 25% of alleles negative by sequencing contain large deletions. When INAD is suspected, we recommend that genetic testing precede tissue biopsy, which would be unnecessary when PLA2G6 mutations are identified by DNA analysis. In rare cases, biopsy of peripheral tissue (conjunctiva, rectal mucosa, skin, or sural nerve) may still be useful in the diagnostic evaluation of mutation-negative patients.
The spectrum of clinical features associated with mutations in PLA2G6 is broader than previously described, and genotype correlates with phenotype. We found the clinical profile of classic INAD to be a fairly uniform one of early hypotonia and tetraparesis that progresses rapidly to complete psychomotor retardation. We observed optic atrophy, nystagmus, and strabismus in most patients. We found abnormalities on various electrophysiologic measures, including a decrease in nerve conduction velocity, denervation on EMG, and fast rhythms on EEG. In contrast, we found atypical neuroaxonal dystrophy to be clinically heterogeneous, with patients showing later onset disease and slower progression, with variable ataxia, spasticity, and neurobehavioral abnormalities.
The imaging findings in classic infantile and atypical neuroaxonal dystrophy will guide diagnostic molecular testing. We observed cerebellar atrophy in nearly all PLA2G6
mutation-positive patients, and globus pallidus iron accumulation in half, evident as low signal intensity on T2-weighted MRI ().2
Since cerebellar atrophy is not typically found in other disorders associated with high brain iron, this combination of features is strongly predictive of a PLA2G6
Thus, if neuroimaging shows cerebellar atrophy with or without evidence of iron accumulation in a patient with psychomotor regression, molecular testing for a PLA2G6
mutation is indicated.
Figure 2 Pattern on brain MRI
The pathology associated with mutations in PLA2G6
suggests a shared pathogenesis with both Parkinson disease and Alzheimer disease. In this study, we saw not only the expected peripheral and central spheroids, but also α-synuclein-positive Lewy bodies, dystrophic neurites, and neurofibrillary tangles in the brain tissue of a 23-year-old patient with atypical neuroaxonal dystrophy. Interestingly, although synuclein-positive spheroids have been observed in classic early onset cases with death before age 10,13
Lewy bodies and neurofibrillary tangles have not been reported in association with this phenotype. This suggests that in order to form these structures either exposure to the neuropathologic process must be prolonged, or the brain must reach a certain stage of maturation. Our patient is among the youngest with any disease in whom Lewy bodies and neurofibrillary tangles have been described.
The pathologic and imaging features shared by disorders of distinct molecular etiology may lend further insights into pathogenesis. The similar appearance of spheroids and iron in classic and atypical neuroaxonal dystrophy and in PKAN, another inherited neurodegenerative disorder with brain iron accumulation, suggests that defects in the respective mutant proteins lead to a common mechanism of iron dyshomeostasis in basal ganglia. The PLA2G6 protein is critical for cellular membrane phospholipid homeostasis and remodeling,7
and defects in this protein may cause accumulation of membranes, organelles, and protein in distal axons that represent spheroids. The protein that is defective in PKAN normally regulates the biosynthesis of coenzyme A, which is critical to fatty acid metabolism. Both proteins associate with mitochondria and play a role in lipid metabolism, which when perturbed may alter the regulation of iron transport and utilization. By identifying genes that underlie neurodegenerative disorders with high brain iron, we have improved diagnostic testing and gained insights into pathogenesis. This new knowledge may guide the development of therapeutic strategies not only for these rare disorders but also for pathogenically related more common illnesses such as Parkinson disease and Alzheimer disease.