SUPPLEMENTARY NOTE

Listed below are the Gene Scoring Criteria (GSC v1.0) we developed to assess support for 196 ASD candidate genes; these criteria can also be found at https://gene.sfari.org/autdb/GS_Classification.do Each category description also includes an example, with the exception of category 1, as no genes met those criteria at the time of submission.

Gene scores will change over time in response to new data and community-driven efforts to develop scoring criteria; this is a key strength to AutDB 2.0. Viewable score histories, together with a coupling of gene scores to the specific GSC version used to generate them, will ensure clarity in the face of such changes.

Category S (Syndromic)

S - Monogenic (syndromic) disease genes are included here if mutations are associated with a substantial degree of increased risk (e.g. 10x or >5-10%). If there is independent evidence implicating a gene in idiopathic ASD, it will be listed as #S (e.g. 2S, 3S, etc.). If there is no such independent evidence, the gene will be listed simply as ‘S’

Example: FMR1 (https://gene.sfari.org/autdb/GeneDetail/FMR1#GS) is assigned to category S. It has been consistently shown that more than 20% of individuals with FMR1 mutations causing fragile X syndrome also meet criteria for ASD ¹.

Category 1 (High confidence)

Genome-wide significance plus replication specific to a unique gene

1.1 - Recurrent and convincing mutations accompanied by a rigorous statistical comparison with the mutation frequency in controls, and independent replication. Full sequencing of a comparable number of cases and controls is required. Convincing is defined as ‘likely to be functional’, or showing perfect segregation of mutations and phenotype in a large pedigree.

1.2 - For results from association studies, those that reach genome-wide significance, uniquely implicating a single gene, and are independently replicated or reach genome-wide significance via meta-analysis of all current association studies. For genome-wide significant variants in an intergenic region, a nearby flanking gene would be included if it’s the only gene in strong LD with the intergenic variant, or if the variant if also associated with altered expression of a particular flanking gene (or another line of strong evidence implicating this gene).

At the time of submission, no genes met these criteria.

Category 2 (Strong candidate)

Genome-wide significance without replication, or very consistent replication + function deemed equivalent to genome-wide significance

2.1 - Rare mutations that are recurrent and convincing accompanied by a rigorous statistical comparison with the mutation frequency in controls. Independent replication is not required. Full sequencing of a comparable number of cases and controls is required. Convincing is defined as ‘likely to be functional’, or showing perfect segregation of mutations and phenotype in a large pedigree.

2.2 - For results from association studies, those that reach genome-wide significance, uniquely implicating a single gene, but with no independent replication. For genome-wide significant variants in an intergenic region, a nearby flanking gene would be included if it’s the only gene in strong LD with the intergenic variant, or if the variant if also associated with altered expression of a particular flanking gene (or another line of strong evidence implicating this gene).

2.3 - Consistently replicated association of the same allele, falling short of genome wide significance, but is accompanied by evidence that the risk variant has a relevant functional effect in humans. ‘Consistently replicated’ means replication of the same variant in each follow-up association study that is appropriately powered and involves a population of the same ancestry, or inconsistent but overall significant by meta-analysis. In this regard, ‘gene-based’ tests of association are acceptable as evidence of an association if performed in the original study, but would not be considered as evidence simply to ‘rescue’ a subsequent study that finds a variant association that is different from the associated variant in the original study.

Example: CNTN4 (https://gene.sfari.org/autdb/GeneDetail/CNTN4#GS) is assigned to category 2 (criterion 2.1). Inherited (maternal and paternal) deletions were observed in 7/~2000 unrelated cases and 0/~2500 controls, 1/7 unrelated individuals confirmed by qPCR (2 sibs actually confirmed). Statistical support for effect at this gene (4.7 x 10-4) is similar to that observed for either 15q11-13 or NRXN1, but still nominal given number of tests performed. Inherited duplications may also be enriched in cases with 7 unrelated probands carrying such events versus only 1 control (p = 0.0078) ². Paternally inherited deletions observed in 2/3 autistic siblings from screen of 80 probands, paternally inherited duplication seen in a third unrelated child with autism, no similar event in > 750 controls including those from NIMH normal control initiative, resulting in nominal significance ³. A translocation was also identified in an individual with autism (follow-up comment confirms diagnosis) ⁴.

Category 3 (Suggestive evidence)

Several independent lines of nominal evidence pointing to the same gene

3.1 - Genes that meet at least two of criteria 4.1, 4.2, and 4.3, or meet one of those criteria and at least one line of category 4 accessory evidence.

3.2 - Genes that meet category 2.3 criteria, but without accompanying functional evidence.

Example: OXTR (https://gene.sfari.org/autdb/GeneDetail/OXTR#GS) is assigned to category 3 (criterion 4.3 plus accessory #1). A number of studies have focused on the genetic association of the OXTR gene with autism, including negative association. Cohorts and populations that have shown positive associations include AGRE, Chinese Han, Caucasian, Japanese, Irish, Portuguese, US and the United Kingdom ^5-8. In addition, association has been found with social cognition ⁹. One study also found a rare deletion in the OXTR gene in an autistic family and potential transcriptional misregulation through DNA methylation ¹⁰.

Category 4 (Minimal evidence)

Nominal evidence with neither genome-wide significance nor consistent replication

4.1 - Any gene in an ASD-associated multi-genic CNV (including syndromic) for which there is no other independent evidence.

4.2 - Genes proximal to genome-wide significant intergenic variants that don’t meet category 1 or 2 criteria.

4.3 - Any significant, convincing, but unreplicated association study. Also, multiple but inconsistent reports of association that is not overall significant by meta-analysis.

4.4 - Genes with a series of two or more putative mutations identified (e.g. non-synonymous substitutions, single-gene deletion, duplication, disruption by translocation) for which there is not rigorous statistical comparison with controls.

Accessory evidence to raise a gene from category 4 to category 3:

• Altered expression or function in ASD cases vs. controls in any tissue, whether as a function of genotype or not.

• Strong evidence for involvement in a related phenotype or disorder (specifically including ADHD, ASD-related endophenotypes (e.g. language impairment), bipolar disorder, epilepsy, intellectual disability, schizophrenia.

• Genes shown to be associated with ASD risk via genetic epistasis with another gene.

Example: CENTG2 (https://gene.sfari.org/autdb/GeneDetail/CENTG2#GS) is assigned to category 4 (criterion 4.4). There is a single publication showing several rare mutations in autism families, as well as some contradictory linkage findings ¹¹.

Category 5 (Hypothesized but untested)

Reason to consider the gene for further study but lack of evidence to date

5.1 - Genes for which the only evidence comes from studies of model organisms, without statistical/genetic support in human studies.

5.2 Genes in a region of linkage with no unique evidence for that gene vs. others nearby.

5.3 Genes shown to functionally interact with category 1-3 ASD candidate genes.

Example: DLX1 (https://gene.sfari.org/autdb/GeneDetail/DLX1#GS) is assigned to category 5 (criterion 5.2). DLX1 is in a linkage region for ASD ¹².

Category 6 (Evidence does not support a role in ASD)

Evidence against association

6.1 The weight of the evidence argues against a role in autism.

Example: FABP7 (https://gene.sfari.org/autdb/GeneDetail/FABP7#GS) is assigned to category 6. Maekawa et al. examined the genes FABP3, 5, and 7 in 285 ASD probands by resequencing, and obtained no support for involvement of FABP7 ¹³.

Supplementary References:

1. Rogers, S.J., Wehner, D.E. & Hagerman, R. The behavioral phenotype in fragile X: symptoms of autism in very young children with fragile X syndrome, idiopathic autism, and other developmental disorders. J Dev Behav Pediatr 22, 409-17 (2001).

2. Glessner, J.T. et al. Autism genome-wide copy number variation reveals ubiquitin and neuronal genes. Nature 459, 569-73 (2009).

3. Roohi, J. et al. Disruption of contactin 4 in three subjects with autism spectrum disorder. J Med Genet 46, 176-82 (2009).

4. Fernandez, T. et al. Disruption of contactin 4 (CNTN4) results in developmental delay and other features of 3p deletion syndrome. Am J Hum Genet 74, 1286-93 (2004).

5. Wu, S. et al. Positive association of the oxytocin receptor gene (OXTR) with autism in the Chinese Han population. Biol Psychiatry 58, 74-7 (2005).

6. Jacob, S. et al. Association of the oxytocin receptor gene (OXTR) in Caucasian children and adolescents with autism. Neurosci Lett 417, 6-9 (2007).

7. Lerer, E. et al. Association between the oxytocin receptor (OXTR) gene and autism: relationship to Vineland Adaptive Behavior Scales and cognition. Mol Psychiatry (2007).

8. Yrigollen, C.M. et al. Genes Controlling Affiliative Behavior as Candidate Genes for Autism. Biol Psychiatry (2008).

9. Park, J. et al. Evidence that genetic variation in the oxytocin receptor (OXTR) gene influences social cognition in ADHD. Prog Neuropsychopharmacol Biol Psychiatry 34, 697-702 (2010).

10. Gregory, S.G. et al. Genomic and epigenetic evidence for oxytocin receptor deficiency in autism. BMC Med 7, 62 (2009).

11. Wassink, T.H. et al. Evaluation of the chromosome 2q37.3 gene CENTG2 as an autism susceptibility gene. Am J Med Genet B Neuropsychiatr Genet 136, 36-44 (2005).

12. Bacchelli, E. et al. Screening of nine candidate genes for autism on chromosome 2q reveals rare nonsynonymous variants in the cAMP-GEFII gene. Molecular Psychiatry 8, 916-24 (2003).

13. Maekawa, M. et al. Polymorphism screening of brain-expressed FABP7, 5 and 3 genes and association studies in autism and schizophrenia in Japanese subjects. J Hum Genet 55, 127-30 (2010).