Draft Genetic Test Review
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Hereditary
Hemochromatosis
Analytic Validity
(119KB)
ANALYTIC VALIDITY
Question 8: Is the test qualitative or quantitative?
Question 9: How often is a 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)?
Question 11: Is an internal QC program defined and externally monitored?
Question 12: Have repeated measurements been made on specimens?
Question 13. What is the within- and between-laboratory precision?
Question 14: If appropriate, how is confirmatory testing performed?
Question 15: What range of patient specimens has been tested?
Question 16: How often does the test fail to give a useable result?
Question 17: How similar are results obtained in multiple laboratories using the same, or different, technology?
Appendix 1. Data used to calculate analytic sensitivity and specificity
Analytic sensitivity and specificity by genotype
Tables 2-3 through 2-7 summarize the HFE-related external proficiency testing results obtained by ACMG/CAP for the years 1998 through 2002. Samples with known genotypes have been distributed to participants since 1998. For orientation, the first column of Table 2-4 contains the distribution label (98 MGL-16 indicates the 16th DNA sample distributed as part of the Molecular Genetics Laboratory survey in 1998). The second column contains the number of participating laboratories, and the third column lists the consensus genotype of the sample. The number of laboratories reporting specific genotypes is then provided, along with a tabulation of their ‘correct' and ‘incorrect' responses. The last two columns provide an adjusted interpretation by taking into account that some laboratories do not test for the H63D mutation. The last column also shows the type of error and is listed by chromosome. Some analyses use errors listed by genotype instead. For example, identifying a C282Y homozygote as having no identifiable mutations is considered two errors when counting by chromosome, but only one error when counting by genotype. The main analysis, which ignores H63D mutations and is performed by genotype, is shown at the end of each year's results and is labeled ‘282 performance measures'. Table 2-9 summarizes results from all five years.
Table 2-3. Computations for the 1998 ACMG/CAP Proficiency Testing Surveys
| 98 MGL-16 |
67 |
C282Y/C282Y |
|
|
|
|
| |
58 |
C282Y/C282Y |
116 |
0 |
116 |
0 |
| |
9* |
C282Y/C282Y |
18 |
0 |
18 |
0 |
| 98 MGL-17 |
67 |
C282Y/H63D |
|
|
|
|
| |
58 |
C282Y/H63D |
116 |
0 |
116 |
0 |
| |
9* |
C282Y/N |
9 |
9 |
18 |
0 |
| 98 MGL-18 |
67 |
C282Y/N |
|
|
|
|
| |
58 |
C282Y/N |
116 |
0 |
116 |
0 |
| |
9* |
C282Y/N |
18 |
0 |
18 |
0 |
| Totals 1998 |
|
402 alleles |
393 |
9 |
402 |
0 |
| |
|
|
|
|
|
|
282 Performance Measures For Homozygosity |
|
Genotype challenges |
Errors |
Rate (%) |
Sensitivity |
|
67 |
0 |
100 |
Specificity |
|
67+67=134 |
0 |
100 |
* These laboratories do not test for the H63D mutation
Table 2-4. Computations for the 1999 ACMG/CAP Proficiency Testing Survey
| 99 MGL-17 |
78 |
C282Y/H63D |
|
|
|
|
| |
70 |
C282Y/H63D |
140 |
0 |
140 |
0 |
|
7* |
C282Y/N |
7 |
7 |
14 |
0 |
| |
1 |
H63D/N |
1 |
1 |
1 |
1 (fn 282) |
| 99 MGL-18 |
78 |
N/N |
|
|
|
|
| |
69 |
N/N |
138 |
0 |
138 |
0 |
|
7* |
N/N |
14 |
0 |
14 |
0 |
| |
1 |
H63D/N |
1 |
1 |
1 |
1 (fp 63) |
| |
1 |
C282Y/N |
1 |
1 |
1 |
1 (fp 282) |
| Totals 1998 |
|
312 alleles |
302 |
10 |
309 |
3 |
| |
|
|
|
|
|
|
282 Performance Measures For Homozygosity |
|
Genotype challenges |
Errors |
Rate (%) |
Sensitivity |
|
0 |
0 |
- |
Specificity |
|
78+78=156 |
0 |
100 |
* These laboratories do not test for the H63D mutation
Table 2-5. Computations for the 2000 ACMG/CAP Proficiency Testing Survey
| 00 MGL-04 |
81 |
N/N |
|
|
|
|
| |
72 |
N/N |
144 |
0 |
144 |
0 |
| |
9* |
N/N |
18 |
0 |
18 |
0 |
| 00 MGL-05 |
81 |
N/N |
|
|
|
|
| |
72 |
N/N |
144 |
0 |
144 |
0 |
| |
9* |
N/N |
18 |
0 |
18 |
0 |
| 00 MGL-06 |
81 |
282/N |
|
|
|
|
| |
72 |
282/N |
144 |
0 |
144 |
0 |
| |
8* |
282/N |
16 |
0 |
16 |
0 |
| |
1* |
282/282 |
1 |
1 |
1 |
1 (fp 282) |
| 00 MGL-16 |
90 |
63/N |
|
|
|
|
| |
79 |
63/N |
158 |
0 |
158 |
0 |
| |
8* |
N/N |
8 |
8 |
16 |
0 |
| |
2 |
N/N |
2 |
2 |
2 |
2 (fn 63) |
| |
1 |
63/282 |
1 |
1 |
1 |
1 (fp 282) |
| 00 MGL-17 |
90 |
N/N |
|
|
|
|
| |
82 |
N/N |
164 |
0 |
164 |
0 |
| |
8* |
N/N |
16 |
0 |
16 |
0 |
| 00 MGL-18 |
90 |
282/282 |
|
|
|
|
| |
80 |
282/282 |
160 |
0 |
160 |
0 |
| |
8* |
282/282 |
16 |
0 |
16 |
0 |
| |
2 |
N/N |
0 |
4 |
0 |
4 (fn 282) |
| Totals 2000 |
|
1,026 alleles |
1,010 |
16 |
1,008 |
8 |
| |
|
|
|
|
|
|
282 Performance Measures For Homozygosity |
|
Genotype challenges |
Errors |
Rate (%) |
Sensitivity |
|
90 |
2 |
97.8 |
Specificity |
|
81+81+81+90+90=423 |
1 |
99.8 |
* These laboratories do not test for the H63D mutation
Table 2-6. Computations for the 2001 ACMG/CAP Proficiency Testing Survey
| 01 MGL-04 |
100 |
N/N |
|
|
|
|
| |
90 |
N/N |
180 |
0 |
180 |
0 |
| |
8* |
N/N |
16 |
0 |
16 |
0 |
| |
1 |
282/63 |
0 |
2 |
0 |
2 (fp 282, fp 63) |
| |
1 |
63/N |
1 |
1 |
1 |
1 (fp 63) |
| 01 MGL-05 |
100 |
63/63 |
|
|
|
|
| |
88 |
63/63 |
176 |
0 |
176 |
0 |
| |
8* |
N/N |
0 |
16 |
16 |
0 |
| |
2 |
N/N |
0 |
4 |
0 |
4 (fn 63) |
| |
1 |
63/N |
1 |
1 |
1 |
1 (fn 63) |
| |
1 |
282/282 |
0 |
2 |
0 |
2 (fp 282) |
| 01 MGL-06 |
100 |
282/63 |
|
|
|
|
| |
91 |
282/63 |
182 |
0 |
182 |
0 |
| |
8* |
282/N |
8 |
8 |
16 |
0 |
| |
1 |
63/N |
1 |
1 |
1 |
1 (fn 282) |
| 01 MGL-16 |
90 |
N/N |
|
|
|
|
| |
84 |
N/N |
168 |
0 |
168 |
0 |
| |
5* |
N/N |
10 |
0 |
10 |
0 |
| |
1 |
63/N |
1 |
1 |
1 |
1 (fp 63) |
| 01 MGL-17 |
90 |
N/N |
|
|
|
|
| |
85 |
N/N |
170 |
0 |
170 |
0 |
| |
5* |
N/N |
10 |
0 |
10 |
0 |
| 01 MGL-18 |
90 |
282/282 |
|
|
|
|
| |
83 |
282/282 |
166 |
0 |
166 |
0 |
| |
5* |
282/282 |
10 |
0 |
10 |
0 |
| |
2 |
282/N |
2 |
2 |
2 |
2 (fn 282) |
| Totals 01 |
|
1140 alleles |
1,102 |
38 |
1,126 |
14 |
| |
|
|
|
|
|
|
282 Performance Measures For Homozygosity |
|
Genotype challenges |
Errors |
Rate (%) |
Sensitivity |
|
90 |
2 |
97.8 |
Specificity |
|
100+100+100+90+90=480 |
1 |
99.8 |
* These laboratories do not test for the H63D mutation
Table 2-7. Computations for the 2002 ACMG/CAP Proficiency Testing Survey
| 02 MGL-04 |
1041 |
N/N |
|
|
|
|
| |
94 |
N/N |
188 |
0 |
188 |
0 |
| |
7* |
N/N |
14 |
0 |
14 |
0 |
| |
1 |
282/N |
1 |
1 |
1 |
1 (fp 282) |
| |
1 |
282/282 |
0 |
2 |
0 |
2 (2fp 282) |
| |
1 |
63/N |
1 |
1 |
1 |
1 (fp 63) |
| 02 MGL-05 |
1031 |
N/N |
|
|
|
|
| |
94 |
N/N |
188 |
0 |
188 |
0 |
| |
7* |
N/N |
14 |
0 |
14 |
0 |
| |
1 |
63/N |
1 |
1 |
1 |
1 (fp 63) |
| |
1 |
282/282 |
0 |
2 |
0 |
2 (fp 282) |
| 02 MGL-06 |
1031 |
282/N |
|
|
|
|
| |
93 |
282/N |
186 |
0 |
186 |
0 |
| |
6* |
282/N |
12 |
0 |
12 |
0 |
| |
1* |
N/N |
1 |
1 |
1 |
1 (fn 282) |
| |
1 |
N/N |
1 |
1 |
22 |
0 |
| |
1 |
282/282 |
1 |
1 |
22 |
0 |
| |
1 |
63/N |
1 |
1 |
1 |
1 (wm) |
| 02 MGL-10 |
98 |
63/N |
|
|
|
|
| |
89 |
63/N |
178 |
0 |
178 |
0 |
| |
7* |
N/N |
7 |
7 |
14 |
0 |
| |
1 |
N/N |
1 |
1 |
1 |
1 (fn 63) |
| |
1 |
282/63 |
1 |
1 |
1 |
1 (fp 282) |
| 02 MGL-11 |
98 |
N/N |
|
|
|
|
| |
91 |
N/N |
182 |
0 |
182 |
0 |
| |
6* |
N/N |
12 |
0 |
12 |
0 |
| |
1 |
63/N |
1 |
1 |
1 |
1 (fp 63) |
| 02 MGL-12 |
97 |
282/63 |
|
|
|
|
| |
86 |
282/63 |
172 |
0 |
172 |
0 |
| |
6* |
282/N |
6 |
6 |
12 |
0 |
| |
2 |
282/N |
2 |
2 |
2 |
2 (fn 63) |
| |
2 |
63/N |
2 |
2 |
2 |
2 (fn 282) |
| |
1 |
N/N |
0 |
2 |
0 |
2 (fn 282 fn 63) |
| Totals 02 |
|
1,206 alleles |
1,102 |
38 |
1,188 |
18 |
| |
|
|
|
|
|
|
282 Performance Measures For Homozygosity |
|
Genotype challenges |
Errors |
Rate (%) |
Sensitivity |
|
0 |
0 |
|
Specificity |
|
104+103+103+98+98+97=603 |
2 |
99.7 |
* These laboratories do not test for the H63D mutation
1 Assumes the laboratory reporting incorrectly was actually testing for both C282Y and H63D
2 Does not count a reporting error (each allele reported as a genotype)
Table 2-8. Computations for the ACMG/CAP Proficiency Testing Surveys for the Combined Years 1998-2002
| Totals 98-02 |
|
4,086 alleles |
3,980 |
106 |
4,043 |
43 |
| |
|
|
|
|
|
|
282 Performance Measures For Homozygosity |
|
Genotype challenges |
Errors |
Rate (%) |
Sensitivity |
|
67+0+90+90+0=247 |
4 |
98.4 |
Specificity |
|
134+156+423+480+603=1,796 |
4 |
99.8 |
Analytic sensitivity and specificity for the C282Y mutation by chromosome
Table 2-9 shows the raw data used in the analysis of analytic sensitivity and specificity for the C282Y mutation, using data from the ACMG/CAP Survey. This analysis differs from that shown in Tables 2-3 through 2-8, in that each chromosome is analyzed separately. For example, there is no explicit analysis of the rate at which laboratories incorrectly identify homozygous C282Y individuals. Rather, the analytic sensitivity is a measure of how often a chromosome with a C282Y mutation is correctly identified as being positive. Analytic specificity is a measure of how often a chromosome without a C282Y mutation is correctly identified as being negative. Overall, analytic sensitivity by chromosome is 97.8% (95 percent CI 96.3 to 98.8%) and analytic specificity is 99.6% (95 percent CI 99.3 to 99.8%). The analytic performance when estimated by chromosome is less good than the analysis by genotype, because all errors are counted.
Table 2-9. Computation of Analytic Sensitivity and Specificity by Chromosome for the C282Y Mutation Using Data from the ACMG/CAP Molecular Genetics Laboratory External Proficiency Testing Surveys
98 MGL-16 |
67/67 |
67/67 |
- |
- |
98 MGL-17 |
67/67 |
- |
67/67 |
- |
98 MGL-18 |
67/67 |
- |
67/67 |
- |
99 MGL-17 |
77/78 (1 fn) |
- |
78/78 |
- |
99 MGL-18 |
- |
- |
77/78 (1fp) |
78/78 |
00 MGL-04 |
- |
- |
81/81 |
81/81 |
00 MGL-05 |
- |
- |
81/81 |
81/81 |
00 MGL-06 |
81/81 |
- |
80/81 (1 fp) |
- |
00 MGL-16 |
|
- |
89/89 (1fp) |
90/90 |
00 MGL-17 |
|
- |
90/90 |
90/90 |
00 MGL-18 |
88/90 (2 fn) |
88/90 (2 fn) |
|
|
01 MGL-04 |
- |
- |
100/100 |
99/100 (1 fp) |
01 MGL-05 |
|
|
99/10 (1 fp) |
99/100 (1 fp) |
01 MGL-06 |
99/100 (1 fn) |
- |
100/100 |
- |
01 MGL-16 |
- |
- |
90/90 |
90/90 |
01 MGL-17 |
- |
- |
90/90 |
90/90 |
01 MGL-18 |
90/90 |
88/90 (2 fn) |
|
|
02 MGL-04 |
|
|
102/103 (1 fp) |
101/103 (2 fp) |
02 MGL-05 |
|
|
102/103 (1 fp) |
102/103 (1 fp) |
02 MGL-06 |
101/103 (2 fn) |
|
102/103 (1 fp) |
|
02 MGL-10 |
|
|
97/98 (1 fp) |
98/98 |
02 MGL-11 |
|
|
98/98 |
98/98 |
02 MGL-12 |
95/98 (3 fn) |
|
98/98 |
|
All |
583/596 (13 fn)
97.8% (95% CI 96.3 – 98.8%) |
3123/3136 (13 fp)
99.6% (95% CI 99.3 – 99.8%) |
Analytic sensitivity and specificity for the H63D mutation by chromosome
Table 2-10 shows the raw data used in the analysis of analytic sensitivity and specificity for the H63D mutation using data from the Molecular Genetics Laboratory Survey sponsored by ACMG/CAP. In this analysis, each chromosome is analyzed separately. The analytic sensitivity is a measure of how often a chromosome with an H63D mutation is correctly identified as being positive. Analytic specificity is a measure of how often a chromosome without an H63D mutation is correctly identified as being negative. Overall, analytic sensitivity is 98.4% (95 percent CI 97.1 to 99.2%) and analytic specificity is 99.7% (95 percent CI 99.5 to 99.9%).
Table 2-10. Computation of Analytic Sensitivity and Specificity by Chromosome for the H63D Mutation Using Data from the ACMG/CAP Molecular Genetics Laboratory External Proficiency Testing Surveys for the Years 1998-2002
98 MGL-16 |
- |
- |
58/58 |
58/58 |
98 MGL-17 |
58/58 |
- |
58/58 |
- |
98 MGL-18 |
- |
- |
58/58 |
58/58 |
99 MGL-17 |
71/71 |
- |
71/71 |
- |
99 MGL-18 |
|
|
71/71 |
70/71 (1 fp) |
00 MGL-04 |
- |
- |
72/72 |
72/72 |
00 MGL-05 |
- |
- |
72/72 |
72/72 |
00 MGL-06 |
- |
- |
72/72 |
72/72 |
00 MGL-16 |
80/82 (2 fn) |
- |
82/82 |
- |
00 MGL-17 |
- |
- |
82/82 |
82/82 |
00 MGL-18 |
- |
- |
82/82 |
82/82 |
01 MGL-04 |
|
|
92/92 |
90/92 (2 fp) |
01 MGL-05 |
90/92 (2 fn) |
89/92 (3 fn) |
- |
- |
01 MGL-06 |
92/92 |
- |
92/92 |
- |
01 MGL-16 |
- |
- |
85/85 |
84/85 (1fp) |
01 MGL-17 |
- |
- |
85/85 |
85/85 |
01 MGL-18 |
- |
- |
85/85 |
85/85 |
02 MGL-04 |
|
|
96/97 (1 fp) |
97/97 |
02 MGL-05 |
|
|
96/97 (1 fp) |
97/97 |
02 MGL-06 |
|
|
96/97 (1 fp) |
97/97 |
02 MGL-10 |
90/91 (1 fn) |
|
91/91 |
|
02 MGL-11 |
|
|
90/91 (1 fp) |
91/91 |
02 MGL-12 |
88/91 (3 fn) |
|
91/91 |
|
All |
658/669 (11 fn)
98.4% (95% CI 97.1 – 99.2%) |
3069/3077 (8 fp)
99.7% (95% CI 99.5 – 99.9%) |
Appendix 2.
Mutations in the HFE gene The two most common mutations are C282Y and H63D, and the majority of clinical laboratories test for both of these mutations, since at least some samples are submitted primarily for diagnostic testing. Recently, the S65C variant has been described and tentatively linked to a mild form of iron overload, although this variant is not associated with increased transferrin saturation in voluntary blood donors (Arya et al., 1999) and has not been included in the ACMG/CAP proficiency testing program. Other mutations have been identified in the HFE gene (Table 2-11), but are less frequent and/or of low penetrance. Based on the extensive published literature of more than 12,000 individuals genotyped (including more than 2,000 C282Y homozygotes), it is clear that the two common mutations, C282Y and H63D, are very rarely observed in the ‘cis' phase (both mutations on the same chromosome). Two patients, apparently unrelated and of different ethnic backgrounds, have been described who are homozygous for the C282Y mutation and heterozygous for the H63D mutation (Thorstensen et al., 2000; Best et al., 2001). This finding of C282Y and H63D in the ‘cis' phase is likely to represent two independent recombination events and probably occurs at a frequency much less than 1 in 1,000 (Best et al., 2001).
Table 2-11. HFE Gene Mutations Reported in the Literature
|
|
|
|
C282Y (845G→A) |
4 |
~7 |
Feder et al., 1996 |
H63D (187C?G) |
2 |
~20 |
Feder et al., 1996 |
S65C (193A?T) |
2 |
~2 |
Henz et al., 1997 |
I105T (314T?C) |
2 |
V Low/Private |
Barton et al., 1999 |
G93R (277G?C) |
2 |
V Low/Private |
Barton et al., 1999 |
Q127H (381A?C) |
3 |
V Low/Private |
De Villiers et al., 1999 |
V53M (157A?G) |
2 |
V Low/Private |
De Villiers et al., 1999 |
V59M (175G?A) |
2 |
V Low/Private |
De Villiers et al., 1999 |
E168Q (502G?C) |
3 |
Private |
Oberkanins et al., 2000 |
V212V (636G?C) |
4 |
??? |
Bradbury et al., 1999 |
V272L (814G?T) |
4 |
Private |
Worwood et al., 1999 |
E277K (829G?A) |
4 |
Private |
Bradbury et al., 1999 |
R330M (989G?T) |
5 |
V Low/Private |
De Villiers et al., 1999 |
|
|
|
|
E168X (502G?T) |
3 |
Rare |
Piperno et al., 2000 |
W169X (506G?A) |
3 |
Rare |
Piperno et al., 2000 |
E74X (211C?T) |
2 |
Private |
Beutler et al., 2002 |
|
|
|
|
V68delT (203delT) |
2 |
Private |
Liechti-Gallati et al., 1999 |
P160delC (478delC) |
3 |
Private |
Pointon et al., 2000 |
|
|
|
|
IVS3+1G?T |
Intron 3 |
Private |
Wallace et al., 1999 |
|