MALDITOF Mass Spectrometry Detection of Mutations in CFTR

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MALDI-TOF Mass Spectrometry Detection of
Mutations in CFTR

• Caroline A. Martz
• DOE ERULF/ORSS Programs
• ORNL Life Sciences Division

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Outline of Presentation

• Background--cystic fibrosis, CFTR, MALDI-TOF mass
  spectrometry
• Overview of research project--amplification,
  hybridization, detection
• Future directions--optimization and
  miniaturization

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What is cystic fibrosis?

• autosomal recessive--CFTR gene
• affects 25,000 Americans (850 new diagnoses per
  year)

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Incidence of Cystic Fibrosis
Table modified from Genetic Testing for Cystic
Fibrosis. NIH Consensus Statement Online 1997
Apr 14-16 cited 2002 Dec 9 15(4) Table 1.
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What is cystic fibrosis?

• median age of survival in early 30s
• wide range of symptoms and severity

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Symptoms of Cystic Fibrosis

• Increased sweat chloride levels on skin
• meconium ileus
• chronic Pseudomonas aeruginosa lung infection
• decreased pulmonary function

• Asthma
• chronic pancreatitis
• congenital absence of the vas deferens (CBVAD) in
  males

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What is CFTR?

• Gene
• located on 7q
• consists of 24 exons

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Cystic Fibrosis Transmembrane Conductance
Regulator
MSD2
MSD1
EC
N
cytosol
NBD1
NBD2
C
R domain
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What is CFTR?

• Protein cystic fibrosis transmembrane
  conductance regulator
• acts as chloride channel
• regulates chloride-coupled bicarbonate transport
  and sodium transport (ENaC)

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How is cystic fibrosis explained on a molecular
level?

• Mutations and polymorphisms defects in
  protein processing/trafficking
• ?F508--67 of all CF mutations
• polymorphism in 5 splice site of exon 9 causes
  exon 9 skipping

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Why are new screening methods needed?

• Current methods expensive, inefficient, and
  often lacking sensitivity
• Advantages to improved screening
• early detection
• early nutritional and respiratory management
• longer time of survival

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Overview of Project
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What is MALDI-TOF Mass Spec?

• matrix-assisted laser desorption/ionization
• separation on the basis of mass and charge
• good for analysis of large biomolecules (proteins
  and oligonucleotides)

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What is MALDI-TOF Mass Spec?
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How will MALDI detect mutations?
Homozygous normal at 1282 locus
Heterozygous at 1282 locus
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Locus-specific PCR
Name Sequence 508-1B 5-CTCTTCTAGTTGGCATGCTTTGA
-3 508-2A 5-CCAACTATCTGAATCATGTGC-3 1282-1
5-GCAGAGTAATATGAATTTCTTGAGTAC-3 1282-2 5-GC
ACAGTGATAAAGGAAGTCTGCA-3

• Size
• Quality
• Quantity

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Hybridization
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Future Directions

• Optimization--hybridization and wash conditions
• Detection--switch from membranes to glass slides
• High-throughput--miniaturize for analysis of
  multiple mutations in multiple individuals

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Summary

• Cystic fibrosis is an autosomal recessive
  disorder caused by the defective
  processing/trafficking of the CFTR protein.
• MALDI-TOF mass spectrometry uses soft
  desorption and ionization to separate large
  biomolecules according to m/z.
• Hybridization of CFTR amplicons with mass-tagged
  probes is a feasible way to screen for CF-causing
  mutations.

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Acknowledgements

• DOE ERULF/ORSS programs
• Mentors Winston Chen, Lauri Sammartano, and Steve
  Allman
• Fellow students
• Oak Ridge National Laboratory and University of
  Tennessee Medical Center