The neuroaxonal dystrophies include INAD, NBIA and Schindler disease and share the distinctive pathologic feature of axonal degeneration with distended axons (spheroid bodies) throughout the central nervous system. INAD is characterized by progressive motor and sensory impairment, with spheroids also found in peripheral nerves1
online). NBIA comprises a clinically and genetically heterogeneous group of disorders with high basal ganglia iron and includes pantothenate kinase–associated neurodegeneration caused by mutations in PANK2
). Some individuals with INAD show high brain iron; hence, these phenotypes do overlap. To investigate the molecular bases of these two forms of neuroaxonal dystrophy, we undertook genome-wide linkage studies in 12 families with INAD and in a large consanguineous Pakistani family with NBIA (family 252). Using polymorphic microsatellite markers, we mapped a novel INAD1
locus to a 6.0-Mb region of 22q12.3–q13.2 (LOD score of 4.78 at D22S692
), with evidence of locus heterogeneity (Supplementary Methods
online). We also detected linkage to chromosome 22q12–q13 in family 252 with NBIA (LOD score 4.65), with a minimal region of linkage of 4.9 Mb between D22S426
containing the disease locus (Supplementary Fig. 1
and Supplementary Fig. 2
In view of the common regions of linkage, we predicted that the NBIA variant in family 252 and INAD would be allelic and proceeded to sequence candidate genes in DNA from family 252 and three INAD probands. After sequencing 70 of ~100 positional candidate genes, we detected mutations in PLA2G6
in all four kindreds ( and Supplementary Fig. 3
online). We detected a homozygous missense mutation (leading to the amino acid change L545T) in the proband of family 252. This mutation segregated with disease status in 15 affected and unaffected family members. We did not detect this change in 192 ethnically matched control chromosomes. We also detected PLA2G6
mutations in the three INAD probands. We did not detect any of these changes in 192 control chromosomes. We also identified PLA2G6
mutations in 28 additional probands with INAD and in the Karak syndrome family with high basal ganglia iron reported previously4
. We identified a total of 44 unique mutations (32 missense, five deletions leading to a frame-shift, three nonsense, two leading to amino acid deletions without a frameshift, one splice site and one large deletion) ( and ). Of the missense mutations, 85% occurred at amino acid positions that are conserved in vertebrates.
Phenotypically classified individuals and their mutations
Figure 1 Schematic showing iPLA2-VIA protein structure7 and mutation-derived changes. Variant 1 is shown with seven ankyrin repeats (blue), a proline-rich motif (purple box), a glycine-rich nucleotide binding motif (green diamond), a lipase motif (red with the (more ...)
We reviewed the clinical and brain magnetic resonance imaging (MRI) data from individuals with PLA2G6
mutations (Supplementary Fig. 4
online). Although brain MRI changes indicating high levels of iron have been reported only rarely in INAD5,6
, 8 of 20 kindreds (40%) with mutation-positive INAD showed high iron in the globus pallidus. This high frequency may be skewed by a bias in our collection toward those with high brain iron; nevertheless, INAD should clearly be included in the differential diagnosis of neurodegeneration with high brain iron. In contrast, we identified PLA2G6
mutations in only 4 of 24 additional individuals diagnosed with NBIA. PLA2G6
genotype correlates with phenotype only insofar as we found homozygous null mutations only in INAD and not in any individuals with a clinical diagnosis of NBIA.
Historically, diagnosis of INAD has required histopathological evidence of dystrophic axons. However, we found that the presence of spheroids on biopsy did not absolutely correlate with mutations in PLA2G6. Five individuals with clinical and pathological features of INAD were negative for PLA2G6 mutations. Incomplete detection of mutations could explain this; however, we were able to identify both mutated alleles in all but one individual with mutations. An alternative explanation is that INAD is genetically heterogeneous. Indeed, although PLA2G6 is the major disease locus, our linkage data support the existence of at least one additional INAD locus (data not shown).
Although spheroids are characteristic of INAD, three individuals with a clinical diagnosis of INAD but without spheroids were found to have mutations in PLA2G6
. Possible reasons for normal pathologic studies include biopsy at an age before pathological changes have developed or from a site where changes are not evident. Although individual 256 had only a single negative biopsy at age 2 years, individuals 16 and 172-2 (ref. 1
) had negative biopsies at 12 years of age. The latter’s sibling (172-1) was positive both for mutations in PLA2G6
and for spheroids (at age 11 years)1
. These observations indicate a role for PLA2G6
mutation detection in the diagnostic evaluation of all individuals suspected to have INAD, regardless of biopsy results. Indeed, we would argue that molecular testing should replace this more invasive procedure and that PLA2G6
mutation studies are indicated for individuals with a phenotype in the spectrum of NBIA as well.
-VI, a calcium-independent phospholipase. Phospholipase A2
enzymes catalyze the hydrolysis of glycerophospholipids at the sn
-2 position, generating a free fatty acid, usually arachidonic acid, and a lysophospholipid. The iPLA2
-VI protein is active as a tetramer, with proposed roles in phospholipid remodeling, arachidonic acid release, leukotriene and prostaglandin synthesis and apoptosis7
transcript variants encode multiple isoforms8
, and the two enzymatically active forms are predicted to be affected by all of the mutations reported here. A subset of mutations would also alter the shorter enzymatically inactive isoforms, which seem to act as dominant-negative inhibitors when incorporated into the tetramer7,8
. Because all of the mutations identified are likely to be inactivating, no additional effect is predicted for this subset, and indeed, no distinctive phenotype is recognized.
The calcium-independent PLA2
) enzymes are critical in cell membrane homeostasis. Levels of phosphatidylcholine, abundant in mammalian cell membranes and key in maintaining membrane integrity, are regulated by the opposing actions of CTP:cytidy-lylphosphocholine transferase and iPLA2
. Defects in iPLA2
-VIA could lead to a relative abundance of membrane phosphati-dylcholine and secondary structural abnormalities, which may underlie the axonal pathology observed in INAD and NBIA.
Although defective phospholipid metabolism has been suspected in neurodegeneration9,10
, our discovery clearly implicates phospholipase A2
dysfunction in neurodegeneration and more specifically in disease associated with brain iron dyshomeostasis. Impaired phospholipid metabolism has also been implicated in the pathogenesis of another form of NBIA, pantothenate kinase–associated neurodegeneration3
. Iron accumulates with age in brain regions that are targeted in Alzheimer disease and Parkinson disease, and iron is implicated in their pathogenesis11
. Hence, this new link between phospholipid defects and brain iron metabolism may generate insights into the pathogenesis of common neurodegenerative disorders and may spur ideas for new neuroprotective strategies.