Special Considerations in Custom Mutation Analysis

1
Special Considerations in CustomMutation Analysis

• Soma Das
• Department of Human Genetics
• The University of Chicago
• July 2006

2
Custom Mutation Analysis

• The process whereby mutations identified in a
  research setting can be relayed back to patients
  in a manner following CLIA regulated guidelines
  and used for clinical care and management of the
  patient and other family members
• This process was initiated and implemented in
  our laboratory in 1998

3
Custom Mutation Analysis
Genetic disease studied in research lab
Candidate gene analyzed
Mutations identified in patients (results relayed
in some instances)
Research lab pursues other interest
Family members request mutation testing/prenatal
testing
Research lab performs test
No test available
4
Custom Mutation Analysis
Genetic disease studied in research lab
Mutation identified in patient sample
Mutation confirmed in clinical lab on a new
sample from the patient
(e.g. X-linked myotubular myopathy)
Family members request mutation testing/prenatal
testing
Clinical lab performs testing
Eventually clinical laboratory can set up full
gene sequencing
5
Collaboration between Research and Clinical
Laboratories
Genetic disease studied in research lab
Patient sample received and DNA isolated in
clinical lab
DNA aliquot stored in clinical lab
DNA aliquot given to research lab
Mutation identified in sample
Mutation confirmed in clinical lab
(e.g. Lissencephaly)
Family members request mutation testing/prenatal
testing
Clinical lab performs testing
Eventually clinical laboratory can set up full
gene sequencing
6
Benefits of Custom Mutation Analysis
Once a disease mutation is identified in a
patient, in a research setting, genetic testing
for the patient and family members becomes simple

• Technically simple (single PCR and
  sequencing)
• Cost effective (200- 400)
• Can easily be performed by a clinical
  laboratory
• Allows genetic testing including prenatal
  diagnosis to be performed under
• CLIA regulations
• NOTE Negative results from a research lab
  cannot be confirmed

7
Custom Mutation Analysis - Considerations

• Nature of sequence variation and predicted
  effect on gene product and
• its function
• Supporting data from family studies, presence
  of the sequence variation
• in other affected individuals and absence of
  the sequence variation in
• unaffected individuals (if available)
• Whether or not the mutation can be detected by
  direct sequencing or
• other validated method (multiplex or dosage
  sensitive PCR)
• Utility and appropriateness of testing

8
Custom Mutation Analysis - Requirements

• From the patient or family
• Sample from a known mutation carrier obtained
  in a clinical
• setting for confirmation studies (new blood
  sample or sample
• stored in a CLIA/newborn screening
  laboratory)

• From the investigator
• Documentation of mutation
• PCR primer sequences and amplification
  conditions are helpful
• Evidence to support the pathogenicity of the
  sequence change

9
Confirmation of Familial Mutation

• It is important to confirm the presence of a
  familial mutation before testing other
• family members
• In some instances the patient is deceased and
  no new blood sample is available
• for mutation confirmation

Another family member who may have been studied
Obligate carrier - X-linked recessive disease
Parents - Autosomal recessive disease

• A DNA sample from the research lab is needed
  for use as a positive control,
• at the minimum, if nothing else is available
  
• - Critical to state in the report that research
  results could not be confirmed on a
• known mutation carrier

• Prenatal testing should not be performed if a
  familial mutation cannot be
• confirmed

10
Mutation Information

• Mutation type
• Mutation nomenclature
• PCR primers and amplification conditions
• Information supporting mutation to be
  disease-causing
• ? input from research laboratory
• ? information can be included in report

11
Validation of Custom Mutation Testing

• Custom mutation testing is not amenable to
  validation testing in the traditional
• sense
• This issue is similar to that of tests
  performed by full gene sequencing where
• testing is performed to identify the
  presence of any mutation in a gene - i.e.
• validation of every single mutation is not
  possible
• A validated technology needs to be used
• Self-validating assay - each mutation to be
  tested is confirmed in an individual
• known to carry the mutation or by the use of
  positive controls - i.e. sensitivity of
• assay is being tested for each mutation

12
Custom Mutation Analysis - when a mutation is
not confirmed

• Technical reasons
• - inability to amplify allele containing the
  mutation
• - e.g. due to SNP in PCR primer-binding site
• - use of different PCR primer set
• - test positive control
• Sample mix-up
• - test positive control

No further testing (particularly prenatal
testing) should be performed till clarified
13
The Role of the Genetic Counselor

• Utility and appropriateness of testing
• Coordination of testing
• ? custom mutation testing is usually initiated
  when patient is seen in clinic
• ? communication with research laboratory to get
  mutation information
• ? communication with clinical laboratory to set
  up testing
• ? may act as liason between research and
  clinical laboratories
• ? coordinating obtaining samples from relevant
  family members that may be
• needed for positive control purposes

14
Custom Mutation Testing - UC Experience
15
Custom Mutation Analysis - Prenatal case example
1630-1645del16 /-
1630-1645del16 /-
2004
2006
1630-1645del16 homozygous mutation ACADVL
gene Identified in research lab
1630-1645del16 /
1630-1645del16 /
Family with very long chain fatty acid
dehydrogenase deficiency (VLCAD)
16
Custom Mutation Analysis - Prenatal case example
T79A -ve
T79A -ve
T79A mutation ACTA1 gene Mutation identified in
research lab and confirmed in our lab
2001
2005
T79A -ve
T79A -ve
Family with nemaline myopathy
17
Custom Mutation Analysis - X-linked
Lymphoproliferative disease

reported carriers, not confirmed




XLP






SH2D1A del
18
GENETests
Search Options
Laboratory Directory . GeneReviews
Laboratory Directory GeneReviews
Disease
Gene
Locus
Protein
Feature
OMIM
Laboratory Directory
Services
Director
Location
Laboratory
19
GENETests
Search Options
Laboratory Directory
Laboratory Directory GeneReviews
Search for Services
Disease
Apoliproprotein E genotyping Custom Mutation
Analysis Clinical Confirmation of Mutations
Identified in Research Labs Custom Prenatal
Testing Specialized Cytogenetic Services
Comparative Genomic Hybridization (CGH)
FISH-interphase Genomic Microarray
Analysis Multicolor FISH (M-FISH)/Spectral
KaryotypingTM (SKYTM) Subtelomeric FISH
Screen Coronary Artery Disease Susceptibility
Testing for Risk Assessment Modification
Angiotensin I Converting Enzyme 1
Angiotensin II Receptor, Type 1
Angiotensin Integrin, Beta 3 DNA Banking
Gene
Locus
Protein
Testing
Research
Feature
OMIM
Laboratory Directory
Testing
Research
Services
Testing
Director
Location
Laboratory
20
Custom Mutation Analysis

• Cost of test
• First family member 400 - 500
• Subsequent family members 200 - 300
• Turn around time of test
• First family member 4 - 6 weeks
• Subsequent family members 2-3 weeks

21
Standards and Guidelines

• Guidelines have been developed for QC of
  sequencing-based and mutation
• scanning-based tests
• ? ACMG standards and guidelines for clinical
  molecular genetics
• http//www.acmg.net/Pages/ACMG_Activities/std
  s-2002/g.htm
• Standards and guidelines have recently been
  developed for ultra-rare disorders and
• approved by the ACMG
• ? http//www.acmg.net/Pages/ACMG_Activities/stds
  -2002/URD.htm