The identification of predisposing genes has been a daunting endeavor for genetically complex neuro-psychiatric disorders such as ASD. This is in part due to extensive genetic heterogeneity that reduces statistical power for linkage and association studies. Hypothesizing that analyzing a quantitative trait that represents a single feature of ASD may reduce heterogeneity, we analyzed NVC to reveal potential contributory genes. Our earlier publication10
established that NVC can be measured using items from the ADI-R,11
a tool used extensively by researchers for the diagnosis of ASD. In those analyses, we established the necessary criteria that NVC is familial, varies among the affected and correlates with ASD. We also conducted analyses to identify QTL likely to harbor NVC genes, and among the five detected, a chromosome-1 locus was detected.
The work reported here builds sequentially on the findings of that previous study. The first analyses were conducted under the assumption that replication is an essential criterion for gene identification in complex disorders such as ASD. It requires a sufficiently large sample ascertained from an independent panel of families with a comparable diagnosis and should be based on the same quantitative trait measured with the same instrument. Those replication criteria were met by the chromosome-1 QTL, and given our confidence that this QTL harbors gene(s) contributing to deficits in NVC and the availability of GWAS data in this sample, we then conducted a targeted-association study. Those analyses operated under the principle that QTL identify the chromosome regions most likely to harbor trait genes with larger effects, which are easier to detect. One caveat, however, is that allelic heterogeneity in multiple-risk genes within a QTL will allow the region to be flagged by linkage, but may not provide sufficient effect sizes for gene detection with follow-up association studies. To capitalize on the likely heterogeneity within associated genes, we conducted association analyses of haplotype blocks rather than individual SNPs. Blocks divide the QTL into a smaller number of regions that require a less stringent correction for multiple testing than tests of individual SNPs. Causal variants within associated haplotype blocks are likely to reside on different individual haplotypes that can also be tested for association with NVC. To provide support for the associated genes, an additional analysis of their haplotype blocks was conducted in an independent study sample.
Two genes were flagged in these analyses, although they did not exhibit equivalent degrees of support. The first, KCND3, had the strongest association in a single block in the AGRE sample. Support in another block among 22 in KCND3 tested in the AGP sample was somewhat reassuring, but not overwhelming. On the other hand, the NGF gene showed association in three of seven blocks in the original sample and in an adjacent block in the AGP sample. These association signals are consistent with the model of multiple NGF variants contributing to variation in this trait, although the number of independent signals cannot be clearly discerned without sequencing for ‘causal variants’. The results do not preclude an important role for KCND3, as the presence of a replicated QTL reflects multiple signals that are likely to include the effects of more than one gene. Siblings with similar NVC scores are more likely to share such variants, consistent with the strong linkage signal observed and the weaker association findings at any one block of the NGF gene. Here, although the replication in NGF did not involve the same block or SNP, it does replicate at the gene level, with evidence for association in two cohorts in essentially the same region of the gene. These two adjacent haplotype blocks fall within an intronic region with low mammalian conservation, between exons 2 and 3 of this approximately 50 kb gene, so neither are likely to harbor the true functional variant.
is the member of the neurotrophin family of genes and is characterized by its fundamental role in regulating nerve-cell growth, survival and differentiation during early brain development and in the adult both in the peripheral and the central nervous systems. NGF
is expressed in the cerebral cortex, hippocampus and olfactory bulb in the central nervous systems,33
although its levels vary considerably by region. The majority of neuroscience research has focused on the neuroprotective role of NGF
in neurodegenerative disease, especially as a potential therapy in Alzheimer Disease.34
Prior to this study, very little research has linked NGF
to ASD. Riikonen and Vanhala35
showed that NGF
levels were normal in children with infantile autism and low to negligible in children with Rett syndrome, however, they did not examine these levels during earlier or later time points. Nelson et al36
examined and compared archived newborn blood samples in children that developed ASD (n
= 69), mental retardation without ASD (n
= 60), cerebral palsy (n
= 63) and control children (n
= 54). They measured NGF, brain-derived neurotrophic factor, neurotrophin 3 (NT3) and neurotrophins 4/5 (NT4/5), finding that children with ASD and mental retardation without ASD showed higher levels of brain-derived neurotrophic factor, and NT4/5, but not NGF, when compared with control children. In a potentially related neurodevelopmental disorder, Schizophrenia, Parikh et al37
measured plasma NGF levels in 24 medicated first-episode psychotic patients and in 24 chronic medicated schizophrenia patients by measuring NGF levels. These investigators found that NGF levels were decreased in both groups when compared with the normal group, but NGF levels were significantly higher in chronic patients who were treated with antipsychotic medicine as compared with the first-episode psychosis patients.
With regards to previously published genetic evidence supporting a role for NGF
in ASD, none of the published GWAS or studies of structural variation have identified clear pathogenic variants in NGF
in patients with ASD. However, a hypothesis-driven candidate-gene association study focusing on a variety of neuronal signaling pathways did identify evidence for association in the gene NTRK1
which is the canonical receptor for NGF
. Remarkably, NTRK1
was one of only 2 out of approximately 60 genes that survived significance thresholds in the two cohorts investigated in that study. Combined with the current study, these data suggest the involvement of the NGF
signaling pathway in ASD pathogenesis.
It should also be noted that although NGF
is well known for its neurotrophic activity, it has a significant role in immune modulation.39–41
Additionally, its receptor NTRK1
is expressed by several classes of immune cells.42
Given the emerging evidence for the role of immune or inflammatory activation in ASD,43
and the observation of an increase in autoimmune disorders in parents of autistic children,44
it is tempting to speculate a potential neuroimmune mechanism. Nevertheless, these results suggest that further in-depth sequence analysis of the NGF
gene is warranted, so as to clarify its potential role in ASD.