 Can be syndromic or non-syndromic  Majority of cases are non-syndromic with no other features to assist in diagnosis

 Environmental and Genetic Factors  Pre and perinatal factors, infections, family histories, parental age, pesticides, drugs and chemicals

 Observed in all ethnic groups

 More than 600 genes described in literature  Most have not be replicated  Many individuals with autism are still unresolved – more genes/loci?

 Genetic testing is recommended for all children with ASD

 ~25-30% have an identified genetic syndrome or variant  Concordance in monozygotic twins  This means that ~70% have no mechanism identified as yet approaches 70%

 Comorbidity with ID, epilepsy, motor impairment, certain  Recurrence rates in siblings of children with dysmorphic features supports a likely underlying genetic ASD range from 5% to 20% etiology  Recurrence rate increases to 33% if a family  Future Goal: genetic characterization of etiology will has 2 children with ASD facilitate targeted treatments based on the underlying mechanism of the disease

Pediatr Clin N Am 2015;62:607-18 Nature 2012;485:242-5 Nature 2012;485:246-50

The Double Helix – April 1953

James Watson Francis Crick Rosalind Franklin

Chromosomes: 46, XX or 46, XY. Mitochondrial DNA 23 chromosomes from mother, 23 from father. Genes arranged on chromosomes which code for proteins (enzymes, transporters, etc). Maurice Wilkins X-ray diffraction photographs of DNA - 1951 2007  50 years later J. Craig Venter Decoded a full diploid genome – his and James Watson’s

It took 3 months and $300,000 each Currently it takes ~3 months and $5,000 to $7,000 It took 13 years and 3 billion dollars Goal is under $1000 for genome sequencing

Chromosome Technology Progress Fluorescent In Technology Resolution Sample Diagnosis Situ Hybridization Karyotype Whole Down syndrome (1970’s) Chromosome -FISH

Large Deletions or duplications (> 4 Mb) Looks for small specific Fluorescence in situ ~ 100 kb 22q11.2 syndrome Hybridization (FISH) VCFS sections of DNA (30-50 (1990’s) genes) that would be Tests a single locus at a time Need Prior knowledge of region missed by routine chromosome analysis Array CGH Flexible, only Submicroscopic (2000’s) limited by probe deletions/duplications Detects microdeletion spacing (> 1 kb) anywhere in the genome syndromes like: 22q11.2 deletion Can test whole-genome simultaneously 7q11 del Williams

Slide courtesy of Jennifer Mulle PhD

Fish for Williams syndrome 7q11.23 deletion 22q11.2 Region and FISH Incidence 1:10 to 15,000

Normal Deleted Array-based Comparative Genomic Hybridization

What is array CGH Patient DNA Genomic Comparative Genomic Hybridization? Clones

(aka - Chromosomal Microarray) NORMAL

Only detects What can it tell me? unbalanced rearrangements Control DNA

Pinkel et al., Nat Genet (1998), 20(2):207-11 Slide courtesy of Christa Martin, PhD

Array-based CGH Turner synd. (45,X) Normal (46,XX) 47,XXX

Patient DNA Genomic Clones

Loss: ratio < 0.8 Normal: ratio 0.8 - 1.2 Gain: ratio > 1.2 Control DNA

Diagnostic Yield for ID, ASD, DD and MCA:

Conventional Karyotype - 5%

3 Mb DGS Microarray – 20% positive for CNVs oligo probe region coverage on Yield for ASD alone – 10% positive for CNVs EmArray

FISH probe Recommendation: order a microarrays a first tier test for (~100 kb) used for 1. Intellectual Disability testing only 2. Multiple Congenital Anomalies covers this gene 3. Developmental Delays 4. Autism Spectrum Disorders

American Journal of Human Genetics 86, 749-764, May 13, 2010 Genetics in Medicine 15 (7) July 2013 DNA Testing DNA Primer

Transcription mRNA (messenger) tRNA (transfer) rRNA (ribosomal)

Translation

Protein Production

mRNA Gene – String of A’s, T’s Enzyme (U instead of T C’s and G’s Transporter etc. Single Stranded)

ATG GGG TTT TCT CCA CAC Met AUG GGG UUU UCU CCA CAC TAC CCC AAA AGA GGT GTG Gly Ser Pro Met Gly Phe Ser Pro His Phe His Codons Room for Normal Variation

Single base variants

Silent mutation: codes for same amino acid (AA) Conservative missense: codes for similar AA – protein works

Altered Biological Function = Disease Nonconservative missense: codes for different AA – protein may lose function Known Genes with Autism as a feature: Nonsense: STOP codon Autosomal Dominant – TSC1 and TSC2 Tuberous Sclerosis reduced or no protein made Autosomal Recessive – BCKDK - Branched Chain Ketoacid Dehydrogenase Kinase X-Linked – Fragile X syndrome Frame Shift: insertion or deletion shifts reading frame First Generation (Sanger Sequencing): • Single base variants

Other Mechanisms:

• Small Deletions Benefits: • Whole Gene Deletions - All nucleotides interrogated in a specific gene • Splice Mutations - Analyst reviews quality at every basepair - Gold standard • Chromosomal deletions • Rearrangements Limitations: • Epigenetic changes - Large deletions or duplications cannot be detected - Relatively high cost, labor intensive - Low through put - Limited automation in data review - Potentially complicated interpretation and reporting

Next Generation Sequencing - Second Generation (NextGeneration Sequencing): New sequencing Techniques

Sometime we know what gene to sequence Benefits: - Sophisticated bioinformatics Autism and Macrocephaly – sequence the PTEN - Highly automated tumor suppressor gene on chromosome 10q - Large amount of sequence

Limitations: - High cost for infrastructure Gene Panels - Sophisticated bioinformatics - Reagent cost - Complicated interpretation Utility of Panels: autism; cardiomyopathy; seizures etc and reporting Cost wise: panels are frequently close to the same cost as sequencing one single gene

Cardiomyopathy Panel Emory Autism Panel

• 63-gene cardiomyopathy NGS panel • 62 genes: • ADSL, AFF2, AP1S2, ARX, ATRX, BCKDK, BRAF, CACNA1C, CASK, CDKL5, CHD7, CHD8, CNTNAP2, CREBBP, CYP27A1, DHCR7, DMD, EHMT1, FGD1, FMR1, FOLR1, FOXG1, FOXP1, FOXP2, HPRT1, KDM5C, L1CAM, MAGEL2, MBD5, MECP2, MED12, MEF2C, MID1, NHS, NIPBL, NLGN3, NLGN4X, NR1I3, NRXN1, NSD1, OPHN1, PAFAH1B1, PCDH19, PHF6, PNKP, PQBP1, PTCHD1, PTEN, PTPN11, RAB39B, RAI1, RELN, SCN1A, SLC2A1, SLC9A6, SMARCB1, SMC1A, TCF4, UBE2A, UBE3A, VPS13B, ZEB2 • all except 3 of these genes are associated with known genetic syndromes or intellectual disability Venn diagram of the overlap of genes affected by hot zone de novo mutations across four neuropsychiatric disorders

Baker, Elizabeth and Jeste, Shafali: Pediatr Clin N Am 62 (2015) 607-618 Slide courtesy of: David B. Goldstein, Institute for Genomic Medicine, Columbia University

Exome sequencing The Next Test?

WES – Whole Exome Sequencing WGS – Whole Genome Sequencing

Next Generation Sequencing Panels vs Exome Genome vs. Exome

exon intron Panel Exome Cost $2,500-$3,200 $6,700 trios Turn-around 8-12 weeks 16 weeks time Analytical 99%; All coding exons of all genes on 92%; all exons of all genes are not sensitivity panel are analyzed; Del/Dup included covered; no del/dup Clinical All genes are associated with specific No specific phenotype needed; not all sensitivity phenotype of panel exons/genes covered Gene coverage Only genes included on panel are Captures exomes indiscriminately analyzed Parental testing Not required; parental follow up may be Recommended; can help with useful interpretation and classification Potential Mutations and VOUS identified in genes Mutations, VUS, and carrier status can Results associated with specific phenotype be identified in any gene, including adult onset, cancer and non-medically actionable genes