Overview of Newborn Screening Molecular Assays

Susan M Tanksley, PhDJune 28, 2011

Outline 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

Human genome is 99.9% identical across all people ~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.... Helpful – Adaptability

Color patterns for camouflage Disease resistance

Neutral – ‘silent’ or polymorphic Useful as genetic markers Identification, Forensics, Paternity Gene mapping Population studies

Harmful - Disease causing Sickle cell anemia Phenylketonuria (PKU) Cystic fibrosis

Genetic Disorders Caused by various types of mutations in

genes or chromosomes Mutations may occur on

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 Caused by mutations in one gene Generally follow Mendelian inheritance

patterns Dominant vs. Recessive Expression may be impacted by genomic

imprinting or penetrance Includes most inborn errors of metabolism

Classes of Single Gene Disorders Autosomal Dominant

One copy of a mutated allele results in affected individual aka: AA or Aa Heterozygotes and Homozygous Dominant Individuals are affected. e.g. Achondroplasia, Huntington’s Disease

Autosomal Recessive Both alleles of the gene must be mutated to be affected

aka: aa Only Homozygous Recessive individuals are affected. e.g. Sickle Cell Anemia, cystic fibrosis, galactosemia

Classes of Single Gene Disorders X-linked Recessive

Males affected if X chromosome is defective Females affected only if both X chromosomes are defective e.g. Duchenne muscular dystrophy & Hemophilia

X-linked Dominant Individuals with 1 defective copy of X chromosome are affected e.g. Rett syndrome

Y-linked Individuals with a defective Y chromosome are affected Rare

Complex/Multifactorial Disorders Associated with the effects of multiple genes May be strongly impacted by environmental

factors (e.g. lifestyle) Often cluster in families

No clear-cut pattern of inheritance Difficult to determine risk

e.g. heart disease, diabetes, obesity, cancer

Molecular Testing for Genetic Diseases 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: 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

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

GeneTESTS: Availability of Genetic Tests

As of 6/22/2011

Obstacles to Introduction of Genomic Methods in Newborn Screening Volume/quality of specimen Throughput (turn around time) Cost ($$$) per sample “Simple test” mentality Public health infrastructure

Equipment Space Trained personnel

Have test, no treatment

History of Molecular Testing in Newborn Screening 1994

Wisconsin – CFTR mutation analysis for F508 Washington – hemoglobin confirmatory testing (Hb S,

C, E by RFLP) 1998

New England – 2 GALT mutations (Q & N) by RFLP 1999

New England – MCADD (985A>G) by RFLP

History of NBS Molecular Testing 2005

Wisconsin – MSUD (Y438N) 2006

New York – Krabbe (3 polymorphisms & 5 mutations) 2008

Wisconsin – SCID – TREC analysis 1st use of molecular test as a primary full population screen

2010 36 NBS programs in US use molecular testing for CF

Uses of Molecular Tests in NBS Primary Screening Test

TREC analysis for detection of SCID Second-Tier Test

DNA test results provide supplemental information to assist with diagnosis Often provided in separate report -globin and GALT mutation analysis

Genotypic information is required for interpretation of the screen result Cystic fibrosis mutation analysis

When/Why Use a Molecular Test? To increase sensitivity without compromising

specificity Lower IRT cutoff to avoid missing CF cases

To increase specificity of a complex assay Allow differentiation of hemoglobinpathies &

thalassemias (e.g. Hb S/-thalassemia) Distinguish between patient & donor phenotypes

when patient was transfused

When/Why Use a Molecular Test? When the primary analyte is transient

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 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? When there are significant founder mutations

in a population 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? When diagnostic testing is slow and/or

invasive Traditional confirmatory testing for VLCADD &

CPT1a involves skin biopsy (invasive to collect and slow to grow)

When no other test exists for the analyte SCID, SMA

NBS Molecular Tests Available in US Primary Screen - SCID Second-tier

Hemoglobinopathies Galactosemia Cystic Fibrosis MCAD and other FAOs PKU and other aminoacidopathies Krabbe

Potential Future Applications of Molecular Testing in NBS Genome-wide association studies Susceptibility Testing (heart disease, cancer,

obesity, diabetes) Pharmacogenetics and NBS

Drugs in clinical trials to treat specific CF causing mutations (VX-770/G551D and VX-890/F508)

Ataluren (formerly PTC124) is an investigational drug that reads through nonsense or STOP mutations

Conclusions NBS Molecular testing began in 1994 with second tier CF DNA

testing for F508 mutation Molecular tests are useful in NBS to:

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