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Draft Genetic Test Review Hereditary
Hemochromatosis ANALYTIC VALIDITY Question
8: Is the test
qualitative or quantitative? Question 8:
Is the test qualitative or quantitative? The DNA test associated with HHC is qualitative (i.e., a mutation is reported as present or absent). Several mutations have been described, but when DNA analysis is proposed as a screening test for morbidity and mortality associated with iron overload in the setting of the general adult population, the only mutation of interest is C282Y. The genotype of interest is homozygosity for the C282Y mutation. Question 9: How often is the test positive when a mutation is present (analytic sensitivity)? Question 10: How often is the test negative when a mutation is not present (analytic specificity)?
Definitions Generically,
analytic specificity is the proportion of negative test results when
no detectable mutation is present.
Analytic specificity can also be expressed in terms of the
analytic false positive rate.
The false positive rate is the proportion of positive test
results when no detectable mutations are present (1-analytic
specificity). In keeping
with the specific definition of analytic sensitivity above, analytic
specificity is defined in this document as the proportion of individuals
that are not C282Y homozygotes who are correctly identified as not being
homozygous for C282Y. Optimal source(s) of data ·
mixing
of clinical and research laboratories and methodologies ·
relatively
few challenges ·
reporting
summary results in ways that do not allow a straightforward computation
of analytic sensitivity and specificity ·
challenges
that do not represent the ‘mix’ of genotypes expected in a screening
program (e.g., too few wild challenges and too many homozygotes). Future
analyses should be aimed at providing reliable, method-specific analytic
performance estimates. One
approach for collecting such data might include the following steps: ·
An
independent body (such as the College of American Pathologists, American
College of Medical Genetics, Food and Drug Administration or the Coriell
Institute of Medical Research, Camden, NJ) would develop a standard set
of samples, most of which would be randomly selected from the general
population. Included in the
standard set, however, would be additional, less common genotypes. ·
The
sample set would then be available for method validation.
Correct genotypes would be arrived at by consensus, or, if
disagreements emerged, by a reference method (e.g., sequencing).
The current validation practice of having a laboratory (or
manufacturer) run a series of samples with unknown genotype is
inadequate, since there is no ‘gold standard’ with which to compare.
For example, how would a laboratory running an unknown sample
determine whether a positive finding is a true, or a false, positive?
·
Ideally,
this blinded sample set would be available to manufacturers as part of
the pre-market approval process, with the understanding that multiple
laboratories using these commercial reagents would be asked by the
manufacturer to analyze portions of the sample set independently.
This initial assay validation process is distinct from assay
control samples that are discussed later (Question 13). Appropriate sample
size for determining analytic sensitivity and specificity has been
discussed in detail in an earlier ACCE review (Prenatal Cystic Fibrosis
Screening via Carrier Testing – Question 11 and 12).
In brief, a target sensitivity (or specificity) can be chosen,
along with an acceptable lower limit (assumed to be the lower limit of
the 95% confidence interval). Given
these targets, the number of necessary samples can be derived.
For example, if a laboratory chose a target specificity of 98%
and wanted to rule out a specificity of 90%, it would need to correctly
identify at least 49 of 50 known negative samples (estimated using the
binomial distribution). When
the estimates approach 100% and relatively tight confidence intervals
are sought, it may not be economically feasible for laboratories to
individually collect and analyze their data.
However, this level of confidence could be attained by a
consortium of laboratories using the same methodology, or by a
manufacturer that forms a consortium of laboratories using its reagents.
All of these suggested analyses could be done using a 2x2 table,
and all rates could be accompanied by 95% confidence intervals (CI). The ACMG/CAP external proficiency
testing scheme §
A
false negative result occurs when an individual who is homozygous for
C282Y has a test result that is not homozygous for C282Y (i.e.,
wild/wild or C282Y/wild) §
A
false positive result occurs when an individual who is not homozygous
for C282Y has a test result falsely indicating homozygosity for this
mutation. §
The
third type of error, wrong mutation, is not considered in this analysis,
since it is assumed that the test will only be directed at one mutation:
C282Y. A separate analysis of
analytic sensitivity and specificity for both the C282Y and H63D
mutations performed by chromosome can be found in Appendix 1 at the end
of this section. A listing
of other mutations in the HFE gene can be found in Appendix 2. Error rates for the ACMG/CAP external proficiency testing scheme Table 2-1 shows how each of the two types of error are
counted in the analyses of analytic performance.
Column 1 shows those individuals who are actually homozygous for
C282Y. The first entry in
that column contains those receiving positive test results (i.e., true
positives with a result of homozygous for C282Y).
Any other result in this column (rows 2 and 3) is considered a
false negative. Among true
heterozygotes (Column 2), the finding of homozygosity for C282Y would be
a false positive (first row). Any
other test result would be considered negative.
Column 3 shows the three possible test results in individuals
with no C282Y mutations. If
the aim of testing is to correctly identify individuals who are
homozygous for the C282Y mutation, the table could be collapsed
according to the darkened lines. Table
2-2 shows the results of the ACMG/CAP MGL survey for HFE
mutations in the format described in Table 2-1. That survey did include several challenges of the H63D
mutation. For this
analysis, the H63D mutations and corresponding results are ignored, but
the sample is still included. For
example, a compound heterozygote challenge of C282Y/H63D is viewed as a
C282Y heterozygote challenge. Overall,
20 of the 2,043 sample challenges were incorrectly genotyped for C282Y,
for an overall error rate of 1.0% (95 percent CI 0.6 to 1.5%).
As indicated earlier, the major goal of DNA screening for
hemochromatosis is to correctly identify C282Y homozygotes.
The “collapsed” table shows that 98.4% of the homozygous
genotypes were correctly identified (243/247, 95 percent CI 95.9 to
99.5%). In addition all but
four of the 1,796 negative (non-homozygous samples) were identified as
non-homozygotes (99.78%). The
error rate did not change appreciably over time, as shown in the summary
of challenges and errors displayed in Figure 2-1. Table 2-2. HFE Mutation
Testing: Results of the
ACMG/CAP Molecular Genetics Survey Figure 2-1. Summary of Errors Reported in the ACMG/CAP MGL Survey, with
Interpretation restricted to the C282Y mutation ·
In
1998, three samples were distributed to 67 laboratories (201 laboratory
challenges) No errors Error rate 0.0% (95 percent CI 0.0 to 1.8%) ·
In
1999, two samples were distributed to 78 laboratories (156 laboratory
challenges) One laboratory identified no C282Y mutations in a C282Y heterozygous sample One laboratory identified a C282Y mutation in a sample with no mutation present Error rate 1.3% (95 percent CI 0.2 to 4.6%) ·
In
2000-A, three samples were distributed to 81 laboratories (243
laboratory challenges) One laboratory identified a heterozygote as being homozygous for C282Y Error rate 0.4% (95 percent CI 0.1 to 2.3%) ·
In
2000-B, three samples were distributed to 90 laboratories (270
laboratory challenges) One identified a heterozygote when no C282Y mutations were present Two laboratories incorrectly identified a homozygote as having no mutations Error rate 1.1% (95 percent CI 0.2 to 3.2%) ·
In
2001-A, three samples were distributed to 100 laboratories (300
laboratory challenges) One identified a heterozygote when no C282Y mutations were present One identified a homozygote for C282Y when no C282Y mutations were present One laboratory identified no C282Y mutations in a C282Y heterozygous sample Error rate 1.0% (95 percent CI 0.2 to 2.9%) ·
In
2001-B, three samples were distributed to 90 laboratories (270
laboratory challenges) Two laboratories incorrectly identified a homozygote as being heterozygous Error rate 0.7% (95 percent CI 0.1to 2.7%) · In 2002-A, three samples were distributed to 103 laboratories (309 laboratory challenges) In two instances, a homozygote was reported when no C282Y mutations were present One identified a heterozygote when no C282Y mutations were present Two laboratories reported no C282Y mutations in a heterozygote One laboratory reported homozygosity for an individual heterozygous for C282Y Error rate 1.9% (95 percent CI 0.7 To 4.2%) · In 2002-B, three samples were distributed to 98 laboratories (294 laboratory challenges) One laboratory identified a heterozygote when non C282Y mutations were present Three laboratories reported no C282Y mutations in a heterozygote
Error rate 1.4% (95 percent CI 0.4 to 3.5%) Analytic sensitivity identifying C282Y homozygotes Only eight of the 20 errors identified in the proficiency
testing samples influence the analytic sensitivity of identifying C282Y
homozygotes (first column in Table 2-2).
Overall, the analytic sensitivity is 243/247, or 98.4% (95
percent CI 95.9 to 99.5%). These
confidence intervals could be considered pessimistic and optimistic
extremes of the analytic sensitivity.
Because of the relatively few challenges (and observed false
negatives), it is not possible to determine whether analytic sensitivity
varied over the four years. Analytic specificity for identifying non-C282Y homozygotes
The analytic specificity (computed using the second
and third columns in Table 2-2) is 1,191/1,193 or 99.8% (95 percent CI
99.4 to 99.9%). Genotyping
Errors and Method of Testing
According to the ACMG/CAP Participant Summary Reports, there was
no correlation between genotyping error and the laboratory method.
Errors were made by laboratories using restriction digestion and
ASO analysis. The majority
of laboratories (77% on the 2000-B survey) used PCR and restriction
digestion. Other methods of
analysis included ASO (9%), ARMS (8%), Light-cycler (3%), DNA sequencing
(2%), and other (1%). In
one survey (MGL 2001-A), seven errors were made that involved either
C282Y or H63D. The
Participant Summary Report notes that seven different laboratories made
these errors, and that six of those laboratories provided clinical test
results (only one was a research laboratory). Recognition
of a potential source of method-specific error As
part of the ACMG/CAP Survey Program, concern was raised regarding the
protocol validation of new laboratories, inexperienced in HFE
testing. Laboratories using
restriction analysis were encouraged to ensure that their assays contain
internal controls to validate enzyme restriction.
One other potential source for error is the use of the Feder
primers for C282Y analysis, due to the G5569A polymorphism in the
reverse primer. Laboratories
were cautioned that they should use alternate primers that do not
include this polymorphism and that decreased annealing temperatures of
50-55°C
would decrease the stringency of the PCR reaction and thus control for
non-amplification due to primer site polymorphisms.
In the MGL 2000-B ACMG/CAP Participant Summary Report,
participants reported that only 38/84 laboratories (45%) used the C282Y
Feder primers, while 58/82 laboratories (70%) still used the H63D Feder
primers. A more in-depth
discussion of this topic follows in the next two paragraphs. In
1999, Jeffrey et al. reported
that a previously described polymorphism, 5569A (Totaro et
al., 1997), was associated with misdiagnosis of 15 C282Y/5569A
heterozygotes as C282Y homozygotes.
Because this single base pair polymorphism is located in the
primer binding site for the C282Y wild type allele in exon 4, Jeffrey et
al. theorized that the Feder reverse primer might fail to anneal and
thus prevent amplification of the wild type allele.
Since only the mutant allele would then be amplified, this could
result in the appearance of a C282Y homozygote, and a false positive
result. Subsequently, two
other laboratories reported misclassification of C282Y heterozygotes as
homozygotes (Gomez et al.,
1999; Somerville et al.,
1999). Because this
polymorphism is relatively common (allele frequencies as high as 13%),
this report raised immediate concern about C282Y results in genotyping
studies worldwide and led some laboratories to re-analyze previous
results. The
ACMG/CAP Molecular Genetics Survey quickly responded that 67 U.S.
laboratories (many using the Feder primers) had correctly genotyped a
C282Y heterozygote sample that also carried the 5569A polymorphism (Noll
et al., 1999). Thorstensen
et al. (2000) also reported no
errors in genotyping in 433 patients tested using the Feder primers.
These authors suggested that the difference in performance might
relate to a change in a PCR reaction condition (i.e., primer annealing
temperature), and that most laboratories had used conditions that did
not affect result accuracy. The
European Haemochromatosis Consortium reported that some laboratories had
replaced the Feder reverse primer to remove the possibility of
misclassification, but that previous publications by member laboratories
had not been compromised. Therefore,
it appears that prevalence estimates of the C282Y mutation are unlikely
to have been overestimated. However,
clinical laboratories should avoid primers containing polymorphic sites
in which primer binding could affect test outcome. Other
polymorphisms The
DNA testing utilized for screening is aimed at identifying a specific
mutation (C282Y) that, when found in the homozygous state, can be the
cause of primary iron overload. The
test is designed to identify this mutation in any DNA sample, regardless
of the characteristics of the individual being tested (e.g., race or
ethnicity). Although the
prevalence of iron overload and the mix of mutations responsible for the
disorder may vary by race, the test should reliably identify the target
mutation. One exception to
this might occur if the presence and/or frequency of unknown
polymorphisms were found to vary by race/ethnicity (or some other
factor). In reality,
however, it would be difficult for laboratories to thoroughly examine
this possibility in all populations to which testing may be offered. Gap in Knowledge: Allele frequency by race/ethnicity. Variation in allele and polymorphism frequencies by race/ethnicity have been well described in the literature for some population groups, while others have much less information available. Laboratories should make efforts to report HFE allele frequencies as well as polymorphisms that could interfere with the analysis.References ACMG/CAP
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Updated on August 13, 2004