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 Francis Crick James Watson Rosalind Franklin 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 J. Craig Venter 50 years later 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 I t took 1 3 years and 3 billion dollars Goal is under $1000 for genome sequencing Chromosome Technology Progress Fluorescent In Situ Hybridization Technology Resolution Sample Diagnosis Karyotype Whole Down syndrome - FISH (1970’s) Chromosome Large Deletions or duplications (> 4 Mb) Looks for small specific Fluorescence in situ ~ 100 kb 22q11.2 syndrome sections of DNA (30-50 Hybridization (FISH) VCFS genes) that would be (1990’s) Tests a single locus at a time missed by routine Need Prior knowledge of region chromosome analysis Array CGH Flexible, only Submicroscopic Detects microdeletion (2000’s) limited by probe deletions/duplications spacing (> 1 kb) anywhere in the syndromes like: genome 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% Microarray – 20% positive for CNVs 3 Mb DGS oligo probe region Yield for ASD alone – 10% positive for CNVs coverage on EmArray Recommendation: order a microarrays a first tier test for FISH probe (~100 kb) used for 1 . Intellectual Disability testing only 2. Multiple Congenital Anomalies covers this 3. Developmental Delays gene 4. Autism Spectrum Disorders American Journal of Human Genetics 86, 749-764, May 13, 2010 Genetics in Medicine 15 (7) July 2013
DNA Primer DNA Testing Transcription mRNA (messenger) tRNA (transfer) rRNA (ribosomal) Translation Protein Production mRNA Enzyme Gene – String of A’s, T’s (U instead of T Transporter etc. C’s and G’s Single Stranded) ATG GGG TTT TCT CCA CAC Met AUG GGG UUU UCU CCA CAC TAC CCC AAA AGA GGT GTG Gly Ser Pro Phe Met Gly Phe Ser Pro His 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 Nonconservative missense: Altered Biological Function = Disease 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: - All nucleotides interrogated in a specific gene • Whole Gene Deletions - Analyst reviews quality at every basepair • Splice Mutations - 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: Autism and Macrocephaly – sequence the PTEN - Sophisticated bioinformatics - Highly automated tumor suppressor gene on chromosome 10q - Large amount of sequence Limitations: Gene Panels - High cost for infrastructure - Sophisticated bioinformatics - Reagent cost Utility of Panels: autism ; cardiomyopathy; seizures etc - Complicated interpretation 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
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