Transcript Overview of Newborn Screening Molecular Assays

Overview of Newborn
Screening Molecular Assays
Susan M Tanksley, PhD
June 28, 2011
Outline
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Introduction to molecular testing for genetic
diseases
Brief history of molecular testing in NBS
When & why to use a molecular test
Availability of NBS molecular tests in
different states
Potential future applications
Genetic Variation in Humans
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Human genome is 99.9% identical across all
people
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~3 million nucleotide differences between 2 random
individuals
Mutation = Any change in the DNA sequence
Mutations are the source of differences between
individuals
Mutations can be....
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Helpful – Adaptability
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Neutral – ‘silent’ or polymorphic
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Color patterns for camouflage
Disease resistance
Useful as genetic markers
Identification, Forensics, Paternity
Gene mapping
Population studies
Harmful - Disease causing
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Sickle cell anemia
Phenylketonuria (PKU)
Cystic fibrosis
Genetic Disorders
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Caused by various types of mutations in genes
or chromosomes
Mutations may occur on
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An autosome (autosomal)
A sex chromosome (X-linked or Y-linked)
Multiple associated genes
Disease expression may be impacted by
environmental factors
Single Gene Disorders
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Caused by mutations in one gene
Generally follow Mendelian inheritance
patterns
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Dominant vs. Recessive
Expression may be impacted by genomic
imprinting or penetrance
Includes most inborn errors of metabolism
Classes of Single Gene Disorders
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Autosomal Dominant
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One copy of a mutated allele results in affected individual
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aka: AA or Aa
Heterozygotes and Homozygous Dominant Individuals are affected.
e.g. Achondroplasia, Huntington’s Disease
Autosomal Recessive
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Both alleles of the gene must be mutated to be affected
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aka: aa
Only Homozygous Recessive individuals are affected.
e.g. Sickle Cell Anemia, cystic fibrosis, galactosemia
Classes of Single Gene Disorders
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X-linked Recessive
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X-linked Dominant
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Males affected if X chromosome is defective
Females affected only if both X chromosomes are defective
e.g. Duchenne muscular dystrophy & Hemophilia
Individuals with 1 defective copy of X chromosome are
affected
e.g. Rett syndrome
Y-linked
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Individuals with a defective Y chromosome are affected
Rare
Complex/Multifactorial Disorders
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Associated with the effects of multiple genes
May be strongly impacted by environmental
factors (e.g. lifestyle)
Often cluster in families
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No clear-cut pattern of inheritance
Difficult to determine risk
e.g. heart disease, diabetes, obesity, cancer
Molecular Testing for Genetic Diseases
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Enabled by gene mapping to identify location of
genes on chromosomes AND ability to differentiate
between harmful and neutral mutations
Goal – identification of disease-causing mutations
for:
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Diagnosis – e.g. MCADD
Predictive testing – e.g. Huntington’s Disease, BRCA1
Carrier detection – e.g. Cystic Fibrosis
Prenatal screening – e.g. Trisomy 21
Preimplantation testing – e.g. Sickle Cell Anemia
Pharmacogenetics – e.g. PKU
Availability of Genetic Tests
GeneTESTS: Availability of Genetic Tests
599 Laboratories offering in-house
molecular genetic testing, specialized
cytogenetic testing, and biochemical
testing for inherited disorders
2334 Diseases
2072 Clinical Labs
262 Research Labs
As of 6/22/2011
Obstacles to Introduction of Genomic
Methods in Newborn Screening
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Volume/quality of specimen
Throughput (turn around time)
Cost ($$$) per sample
“Simple test” mentality
Public health infrastructure
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Equipment
Space
Trained personnel
Have test, no treatment
History of Molecular Testing in
Newborn Screening
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1994
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1998
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Wisconsin – CFTR mutation analysis for DF508
Washington – hemoglobin confirmatory testing (Hb
S, C, E by RFLP)
New England – 2 GALT mutations (Q & N) by
RFLP
1999
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New England – MCADD (985A>G) by RFLP
History of NBS Molecular Testing
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2005
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2006
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Wisconsin – MSUD (Y438N)
New York – Krabbe (3 polymorphisms & 5
mutations)
2008
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Wisconsin – SCID – TREC analysis
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1st use of molecular test as a primary full population screen
2010
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36 NBS programs in US use molecular testing for CF
Uses of Molecular Tests in NBS
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Primary Screening Test
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TREC analysis for detection of SCID
Second-Tier Test
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DNA test results provide supplemental
information to assist with diagnosis
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Often provided in separate report
b-globin and GALT mutation analysis
Genotypic information is required for
interpretation of the screen result
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Cystic fibrosis mutation analysis
When/Why Use a Molecular Test?
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To increase sensitivity without compromising
specificity
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Lower IRT cutoff to avoid missing CF cases
To increase specificity of a complex assay
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Allow differentiation of hemoglobinpathies &
thalassemias (e.g. Hb S/b-thalassemia)
Distinguish between patient & donor phenotypes
when patient was transfused
When/Why Use a Molecular Test?
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When the primary analyte is transient
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The primary analyte is present in the body for only a
limited time (e.g. VLCADD)
Analysis of a recollected specimen could result in a
false negative.
To speed diagnosis in order to avoid serious
medical consequences
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GALT enzyme activity is decreased by heat &
humidity, thus increasing false positive screens
Genotyping helps sort out the true positives for faster
diagnosis.
When/Why Use a Molecular Test?
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When there are significant founder mutations
in a population
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Due to high frequency (1 in 176 live births) of
MSUD in Mennonite population in WI, mutation
analysis for Y438N serves as primary screen for
MSUD for Mennonites.
CPT1a in Alaskan Innuit & Hutterite populations
When/Why Use a Molecular Test?
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When diagnostic testing is slow and/or
invasive
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Traditional confirmatory testing for VLCADD &
CPT1a involves skin biopsy (invasive to collect
and slow to grow)
When no other test exists for the analyte
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SCID, SMA
NBS Molecular Tests Available in US
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Primary Screen - SCID
Second-tier
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Hemoglobinopathies
Galactosemia
Cystic Fibrosis
MCAD and other FAOs
PKU and other aminoacidopathies
Krabbe
Potential Future Applications of
Molecular Testing in NBS
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Genome-wide association studies
Susceptibility Testing (heart disease, cancer,
obesity, diabetes)
Pharmacogenetics and NBS
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Drugs in clinical trials to treat specific CF causing
mutations (VX-770/G551D and VX-890/ DF508)
Ataluren (formerly PTC124) is an investigational drug
that reads through nonsense or STOP mutations
Conclusions
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NBS Molecular testing began in 1994 with second tier
CF DNA testing for DF508 mutation
Molecular tests are useful in NBS to:
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Increase sensitivity or specificity of a primary assay
Allow follow-up testing when the primary analyte is transient
Aid diagnostic process for disorders with serious consequences or
invasive follow-up tests
Screen founder populations with greater disease risk
Detect disorders for which no biochemical test exists
Wide availability of NBS molecular tests in the US
With expanding technologies, applications of NBS
molecular testing will continue to grow