General Nuts and Bolts of Genetics - Pediatric...

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


“ 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


Chromosomes are DNA

Each Chromosome really OneMolecule of DNA wrapped aroundProteins - Histones


The Genetic Code

Room for Normal Variation


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


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


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


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.


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


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).


Genetic Testing

• Karyotype vs. Chromosome Microarray

• Repeat Disorders

• Next Generation Gene Panels

• Whole Exome vs Whole Genome Sequencing


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


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!!


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.


Microarray




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.”


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


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


Males with Fragile X Syndrome

(CGG) ATGDNA

≥ 200

CH3

Transcription FactorsCannot Bind

No mRNA Production

NO FMR1 ProteinProduction


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



• 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


Genomics in Medicine

• Based on Next Generation Sequencing Technology

– Gene Sequencing Panels

– Whole Exome vs Whole Genome Sequencing


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


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%


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.


Gene for Kabuki syndrome

•WES of 10 unrelated individuals

•Genotype Phenotype stratification

•Identified mutations in MLL2

•Clinical testing for Kabuki syndrome nowavailable


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.


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


Incidental Findings

• Consent process very important.

• Patients can opt out of receiving information

• Important to have Clinical Genetics Involved inpatient or outpatient


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


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.


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


• 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!!!!

General Nuts and Bolts of Genetics - Pediatric...

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