title: Targeted massively parallel sequencing of autism spectrum disorder-associated genes in a case control cohort reveals rare loss-of-function risk variants.
abstract: BACKGROUND
  Autism spectrum disorder (ASD) is highly heritable, yet genome-wide association studies (GWAS), copy number variation screens, and candidate gene association studies have found no single factor accounting for a large percentage of genetic risk. ASD trio exome sequencing studies have revealed genes with recurrent de novo loss-of-function variants as strong risk factors, but there are relatively few recurrently affected genes while as many as 1000 genes are predicted to play a role. As such, it is critical to identify the remaining rare and low-frequency variants contributing to ASD.
  
  
  METHODS
  We have utilized an approach of prioritization of genes by GWAS and follow-up with massively parallel sequencing in a case-control cohort. Using a previously reported ASD noise reduction GWAS analyses, we prioritized 837 RefSeq genes for custom targeting and sequencing. We sequenced the coding regions of those genes in 2071 ASD cases and 904 controls of European white ancestry. We applied comprehensive annotation to identify single variants which could confer ASD risk and also gene-based association analysis to identify sets of rare variants associated with ASD.
  
  
  RESULTS
  We identified a significant over-representation of rare loss-of-function variants in genes previously associated with ASD, including a de novo premature stop variant in the well-established ASD candidate gene RBFOX1. Furthermore, ASD cases were more likely to have two damaging missense variants in candidate genes than controls. Finally, gene-based rare variant association implicates genes functioning in excitatory neurotransmission and neurite outgrowth and guidance pathways including CACNAD2, KCNH7, and NRXN1.
  
  
  CONCLUSIONS
  We find suggestive evidence that rare variants in synaptic genes are associated with ASD and that loss-of-function mutations in ASD candidate genes are a major risk factor, and we implicate damaging mutations in glutamate signaling receptors and neuronal adhesion and guidance molecules. Furthermore, the role of de novo mutations in ASD remains to be fully investigated as we identified the first reported protein-truncating variant in RBFOX1 in ASD. Overall, this work, combined with others in the field, suggests a convergence of genes and molecular pathways underlying ASD etiology.
authors.affiliation: John P. Hussman Institute for Human Genomics, Miller School of Medicine, University of Miami, Miami, FL 33136 USA.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Griswold
authors.affiliation: John P. Hussman Institute for Human Genomics, Miller School of Medicine, University of Miami, Miami, FL 33136 USA.
authors.forename: Nicole D
authors.initials: ND
authors.lastname: Dueker
authors.affiliation: John P. Hussman Institute for Human Genomics, Miller School of Medicine, University of Miami, Miami, FL 33136 USA.
authors.forename: Derek
authors.initials: D
authors.lastname: Van Booven
authors.affiliation: John P. Hussman Institute for Human Genomics, Miller School of Medicine, University of Miami, Miami, FL 33136 USA.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Rantus
authors.affiliation: John P. Hussman Institute for Human Genomics, Miller School of Medicine, University of Miami, Miami, FL 33136 USA.
authors.forename: James M
authors.initials: JM
authors.lastname: Jaworski
authors.affiliation: John P. Hussman Institute for Human Genomics, Miller School of Medicine, University of Miami, Miami, FL 33136 USA.
authors.forename: Susan H
authors.initials: SH
authors.lastname: Slifer
authors.affiliation: John P. Hussman Institute for Human Genomics, Miller School of Medicine, University of Miami, Miami, FL 33136 USA.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Schmidt
authors.affiliation: John P. Hussman Institute for Human Genomics, Miller School of Medicine, University of Miami, Miami, FL 33136 USA.
authors.forename: William
authors.initials: W
authors.lastname: Hulme
authors.affiliation: John P. Hussman Institute for Human Genomics, Miller School of Medicine, University of Miami, Miami, FL 33136 USA.
authors.forename: Ioanna
authors.initials: I
authors.lastname: Konidari
authors.affiliation: John P. Hussman Institute for Human Genomics, Miller School of Medicine, University of Miami, Miami, FL 33136 USA.
authors.forename: Patrice L
authors.initials: PL
authors.lastname: Whitehead
authors.affiliation: John P. Hussman Institute for Human Genomics, Miller School of Medicine, University of Miami, Miami, FL 33136 USA ; Dr. John T. Macdonald Foundation Department of Human Genetics, Miller School of Medicine, University of Miami, Miami, FL 33136 USA.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Cuccaro
authors.affiliation: John P. Hussman Institute for Human Genomics, Miller School of Medicine, University of Miami, Miami, FL 33136 USA ; Dr. John T. Macdonald Foundation Department of Human Genetics, Miller School of Medicine, University of Miami, Miami, FL 33136 USA.
authors.forename: Eden R
authors.initials: ER
authors.lastname: Martin
authors.affiliation: Department of Epidemiology and Biostatistics, Case Western Reserve University, Cleveland, OH 44106 USA.
authors.forename: Jonathan L
authors.initials: JL
authors.lastname: Haines
authors.affiliation: John P. Hussman Institute for Human Genomics, Miller School of Medicine, University of Miami, Miami, FL 33136 USA ; Dr. John T. Macdonald Foundation Department of Human Genetics, Miller School of Medicine, University of Miami, Miami, FL 33136 USA.
authors.forename: John R
authors.initials: JR
authors.lastname: Gilbert
authors.affiliation: Hussman Institute for Autism, Baltimore, MD 21201 USA.
authors.forename: John P
authors.initials: JP
authors.lastname: Hussman
authors.affiliation: John P. Hussman Institute for Human Genomics, Miller School of Medicine, University of Miami, Miami, FL 33136 USA ; Dr. John T. Macdonald Foundation Department of Human Genetics, Miller School of Medicine, University of Miami, Miami, FL 33136 USA.
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Pericak-Vance