Post on 22-Jan-2020
General Nuts and Bolts of Genetics
Michael J. Gambello, MD, PhDSection Chief, Division of Medical Genetics
Children’s Healthcare of Atlanta
Primer in Genetics
Chromosomes: 46, XX or 46, XY.
23 chromosomes from mother, 23 from father.
Genes arranged on chromosomes which code for proteins(enzymes, transporters, collagens etc).
Mitochondrial DNA
Children’s Healthcare of AtlantaMaurice Wilkins
Rosalind FranklinJames Watson Francis Crick
The Double Helix – April 1953
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“ It has not escaped out notice thatThe specific pairing we have postulated Immediately suggests a possible copying mechanism for the genetic material.”
J.D. Watson and F.H.C. Crick
Replication Mechanism
AC
TG
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Chromosomes are DNA
Each Chromosome really OneMolecule of DNA wrapped aroundProteins - Histones
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The Genetic Code
Room for Normal Variation
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Transfer of Biological Information
Gene – String of A’s, T’sC’s and G’s
ATG GGG TTT TCT CCA CACTAC CCC AAA AGA GGT GTG
mRNA(U instead of TSingle Stranded)
AUG GGG UUU UCU CCA CAC
Codons
Met Gly Phe Ser Pro His
EnzymeTransporter etc.
Met
ProHis
Met
Gly
Phe
Ser
Pro
His
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Genes are Come in Pieces - Exons
Children’s Healthcare of AtlantaJournal of the American Academy of Nutrition and Dietetics 114(2):299-312 · February 2014, DOI: 10.1016/j.jand.2013.12.001 · Source: PubMed
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Genetic Disease
Mutations in DNA – Genetic Disease
Other Mechanisms:
• Small Gene Deletions• Whole Gene Deletions• Repeat Disorders• Whole Chromosome defects • Chromosomal deletions• Rearrangements• Epigenetic changes
• Single base variants
GENETIC TESTING TARGETSTHE LIEKLY MECHANISMS OFDISEASE
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The Age of Omics
• Suffix Ome = Collective study of Pools of Biological Molecules
– Genome: All the genes, and stuff in between
– Genomics: Study of all genes and their interactions
– Proteome: All the proteins
– Proteomics: Study of all proteins and their interactions
– Metabolome: All the metabolites, small molecules etc.
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Human Genome Project – First Genomic Project
• International Effort
• Completed 2003
• To understand the human genome.
• Determine the 3 billion A’s, T’s, G’s and C’s
• Map all our genes
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Knowledge from HGP
• Completed in 2003.
• Only ca. 1% of genome codes for protein –
• The EXOME (all the Exons )
• Genes are scattered non‐randomly across genome.
• Only 22,000 genes (much less than we thought).
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Genetic Testing
• Karyotype vs. Chromosome Microarray
• Repeat Disorders
• Next Generation Gene Panels
• Whole Exome vs Whole Genome Sequencing
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Whole Chromosome Aneuploidies
Rudolph’s Brief Atlas of the Newborn, 1998
G Banded Karyotypes
• Lymphocytes or amniocytes
• Only Used for Recognizable WHOLE chromosome syndrome (21, 13, 18, 47, XXY etc)
• Couples with recurrent miscarriage – looking for balanced rearrangements
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Translocation Down Syndrome
– Translocation Down syndrome
• ~ 4% cases due to 21 translocated to another chromosome
• 46,XYder(13;21)(q10;q10)+21
• MUST KARYOTYPE PARENTS!!
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Chromosome Microarray
• Can detect small deletions of duplications in the genome. Copy number variants = CNVs
• Using UCSC Genome Browser (Human Genome Project) we know exactly what genes are deleted or duplicated.
• Replaces traditional Karyotype – Much better resolution 5 Mb vs 30 kb
• Small pieces of entire genome placed on glass slide.
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Microarray
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Chromosome Microarray
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A consensus statement from the:International Standards for Cytogenomic Arrays (ISCA) Consortium
“Our recommendation based on current evidence is to offer CMA as the first‐tier genetic test, in place of G‐banded karyotype, for
patients with unexplained DD/ID, ASD, or MCA.”
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Microarray Results
Dynamic Mutation Diseases caused by Repeats:
SCA8SCA12
SCA17
…CCCCGCCCCGCG…EPM1
Adapted from Mirkin Nature 447, 932-40 (2007).
Fragile X Syndrome
http://medicineworld.org/images/blogs/6-2007/fragile-x-12701.gif
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FMR1 Molecular Genetics
Normal FMR1 Allele
(CGG)6-44
Transcription
ATGDNA
(CGG)30 AUG
mRNA
Translation
FMR1 PROTEIN
http://www.sueblimely.com/images/2008/fragilex-fmr1-protein.jpg
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Males with Fragile X Syndrome
(CGG) ATGDNA
≥ 200
CH3
Transcription FactorsCannot Bind
No mRNA Production
NO FMR1 ProteinProduction
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Phenotype NOT Consistent
http://www.nature.com/ejhg/journal/v11/n8/full/5200997a.html
So for any Boy with intellectual Disability order FMR1CGG Repeat Analysis
Single Gene Sanger Sequencing
mRNA AUG CUC CCC UUU UUC CUC UGAcDNA ATG CTC CCC TTT TTC CTC TGA
1
Hereditary Fructose Intolerance Patient
Homozygous mutation in ALDOBgenec.448G>C (p.A150P) Missense
c.448G>C (p.A149P)
A of ATG start is 1
protein
Alanine
ProlineCoding DNA
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Chromosome Microarray
• CANNOT DETECT
– Point mutations
– Repeat Disorders
– Very small intragenic deletions or duplications (e.g. Cystic Fibrosis c.1521‐1523 del CTT = p.Phe508del [aka deltaF508)
– Balanced Translocations
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Genomics in Medicine
• Based on Next Generation Sequencing Technology
– Gene Sequencing Panels
– Whole Exome vs Whole Genome Sequencing
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Gene Panels
• Can interrogate MANY genes with one test.
• Very useful with disorders that are genetically heterogeneous (MANY genes)
• Genetically heterogeneous diseases: Deafness, Epilepsy, Cardiomyopathy
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Whole Exome Sequencing
• Sequence MOST (92‐96%) EXONS (1% genome –protein coding regions
• Better coverage for known Mendelian disease genes
• Cost varies $4000‐6000
• Sequencing getting cheaper – analysis costly
• Diagnostic Yield 20‐30%
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WES and Undiagnosed Disease
• Performed mainly for patients with suspected genetic disease and without a clear clinical diagnosis or those having negative test results for genes known to be associated with the disorder.
• Many examples of novel disease discovery or atypical manifestation of known disease.
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Gene for Kabuki syndrome
•WES of 10 unrelated individuals
•Genotype Phenotype stratification
•Identified mutations in MLL2
•Clinical testing for Kabuki syndrome nowavailable
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New Disease Genes Every month!!
• Study of autosomal recessive osteogenesis imperfecta in Arabia reveals a novel locus defined by TMEM38Bmutation.
• Mutations in ATP1A3 Cause Alternating Hemiplegia of Childhood.
• Two novel CCDC88Cmutations confirm the role of DAPLE in autosomal recessive congenital hydrocephalus.
• Whole‐Exome Capture and Sequencing Identifies HEATR2Mutation as a Cause of Primary Ciliary Dyskinesia.
• CSF1Rmutations identified in three families with autosomal dominantly inherited leukoencephalopathy.
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Successes with Gene Panels or WES
CASE 1
• 15 yo and 22 yo siblings with RP and epiplepsy. Eye disorders panel. Diagnoses with Neuronal Ceroid Lipofuscinosis CLN8
CASE 2
• 19 yo with PKU but learning and behavior deficits not consistent with PKU. WES identified mutation EHMT1 causing Kleefstra syndrome
CASE 3
• 21 yo with Learning disabilities, dysmorphic, short stature, brachydactyly. WES identified SMAD4mutation causing Myhresyndrome. Risk for pericardial effusion (once with no FU). Now followed closely by cardiology.
0 – 5 incidental finding per exome:- Carrier status of autosomal recessive conditions- Adult-onset conditions
0 – 2 pharmacogenetic findings per exome
Most common variants associated with:HemochromatosisG6PD deficiency
Incidental Findings: Exome Sequencing
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Incidental Findings
• Consent process very important.
• Patients can opt out of receiving information
• Important to have Clinical Genetics Involved inpatient or outpatient
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Genomics – Where are we Going?• Whole Genome Sequencing??? – RESEARCH ONLY NOW
• Personalized Medicine
– Primary Care • DNA vs Family history
• Predictive Health
• Targeted Cancer Therapies
• Pharmacogenomics
– Warfarin and CYP2C9 and VKORC1 genotypes
– CHALLENGE: Demonstrate Clinical Utility
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Newborn Screening and Genomics?
• NBS based mainly on analyte analysis. MS/MS
• WES or WGS for NBS????
• Several Studies underway
• Many issues that need to be addressed.
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NBS and WES or WGS
• The significance of many variants unknown.
– GENOTYPE vs. PHENOTYPE
• Cost still too prohibitive.
• Exon capture not 100%.
• What do you do with Adult onset diseases?
• Do you report carrier status?
• Do you only report some information in newborn period, and other information after age 18?
• Ethical , Legal and Social issues.
ResourcesGene Reviews https://www.ncbi.nlm.nih.gov/books/NBK1116/
OMIM – On Line Mendelian Inheritance in Manhttps://www.omim.org
Unique The Rare Chromosome Disorder Support Grouphttp:/www.rarechromo.org/html/home.asp
Newborn Screeninghttp://dph.Georgia.gov/nbs-providers
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• Genomic Medicine is here and progressing full speed ahead.
• There is still a huge amount of work to do to understand our genome and its relationship to health and disease.
• Goal – Improved health for all.
• Increasing Number of Nuts and Bolts!!!!