Draft Genetic Test Review

---

Hereditary Hemochromatosis
Clinical Validity

(260KB)

---

CLINICAL VALIDITY

Question 18: How often is the test positive when the disorder is present?
Question 19: How often is the test negative when the disorder is not present?
Question 20: Are there methods to resolve clinical false positive results in a timely manner?
Question 21: What is the prevalence of the disorder in this setting?
Question 22: Has the test been adequately validated on all populations to which it may be offered?
Question 23: What are the positive and negative predictive values?
Question 24: What are the genotype/phenotype relationships?
Question 25: What are the genetic, environmental or other modifiers?

---

CLINICAL VALIDITY

Question 20:  Are there methods to resolve false positive results in a timely manner?

Page 1 | Page 2 | Page 3

---

In the absence of confirmatory HFE testing, the analytic false positive rate for C282Y homozygosity is estimated to be 2 per 1,000 individuals tested in the general population (Question 10 – analytic specificity of 99.8, 95 percent CI 99.4 to 99.9%).  Confirmatory testing, on either the same or a second sample, might reduce this rate substantially, though definitive data are not currently available. Approximately 4 per 1000 non-Hispanic Caucasians are homozygous for C282Y, but only some of these will develop serious manifestations.  Figure 3-3 shows the relationship between analytic false positives and clinical false positives that might occur in the general population.   Among the 100,000 non-Hispanic Caucasians tested, 60 will be identified as homozygotes – 40 of these are true analytic positive results, and 20 are false positive analytic results.  The third row shows the impact of subsequent confirmatory testing.  The actual impact of confirmatory testing is unknown.  The table assumes that virtually all true positives will remain positive (40) but that 75 percent of the false positive test results will be identified and corrected (15).  The wide range of false positive results corrected (numbers in parentheses) is a direct consequence of the gap in knowledge about the impact of confirmatory testing.  Under these assumptions, 45 homozygotes are confirmed among the 10,000 individuals tested.  The fourth row shows the hypothetical outcome among these 45 individuals under the assumption that 10 percent of true positives will develop serious manifestations associated with hemochromatosis (penetrance).  According to the figure, most clinical false positives will be among true homozygotes who never develop disease, rather than due to individuals incorrectly classified as being homozygous for C282Y.

Gap in Knowledge:  The Extent to Which Confirmatory Testing Identifies False Positive Analytic Test Results.  It is not yet known to what extent analytic false positive test results will be correctable because: 1) confirmatory testing of homozygous test results is not routinely performed in all laboratories, 2) different types of confirmatory testing will have different rates of identifying errors (e.g., testing the same sample on the same methodology versus testing a new sample on a different methodology) and 3) the types of errors responsible for false positives are not known.

Figure 3-3  Diagram Showing the Causes and Frequencies of Clinical False Positive HFE Testing Results.

Subsequent Biochemical Testing.  Among individuals confirmed to be homozygous for the C282Y mutation after screening, most do not have current clinical manifestations (Asberg et al., 2001; Beutler et al., 2002).  However, biochemical testing might be undertaken to determine whether any of the individuals are iron loaded.  This can be accomplished by obtaining serum transferrin saturation and serum ferritin measurements.  Given what is known about the natural history of hemochromatosis, iron loading is a necessary prerequisite for the development of clinical manifestations (Burke et al., 1998).   Virtually all individuals with clinically diagnosed hemochromatosis have elevated transferrin saturation and serum ferritin measurements.  Such elevations are almost a prerequisite for establishing the clinical diagnosis, but this cannot be taken as evidence that all individuals with hemochromatosis have elevated values of these analytes.  Guidelines have been published providing reasonable cut-off levels (Witte et al., 1996).  For example, the transferrin saturation cut-off level is usually between 50 to 60 percent in males and lower in females.  The serum ferritin cut-off level is usually 300 or 400 ug/L or higher in males and lower in females.  However, given the variability of laboratory assays and methodologies, such cut-off levels often vary from study to study.

Results from Two Published Studies
The results from two cohort studies trials (Burt et al., 1998; McDonnell et al., 1999) are summarized in Table 3-8.  These provide further documentation of the usefulness of biochemical testing after genotyping.  In both studies, all individuals had both biochemical (at least transferrin saturation) and genetic testing performed.  The population was 79 percent female, and over 97 percent were non-Hispanic Caucasian.  The combined genotype frequency for C282Y homozygosity (44 per 10,000, 95 percent CI 22 to 78 per 10,000) is consistent with the consensus estimates of 41 per 10,000 (Table 3-4).  There were 11 C282Y homozygotes identified; 9 of the 11 (82%) had persistently elevated transferrin saturation test results.  Five of the nine also had elevated serum ferritin, all of whom had additional evidence of iron loading based on diagnostic test results (e.g., liver iron or mobilizable iron – the same definition used in Question 18).  C282Y homozygotes accounted for 35 percent of all persistently elevated transferrin saturation results (9/26), 46 percent of all elevated serum ferritin measurements (5/11) and 83 percent of iron loaded individuals (5/6).  No individual in the two studies was considered to have clinical evidence of hemochromatosis.  Among the six C282Y homozygotes who did not have evidence of iron loading, most were premenopausal women.  The two homozygotes with neither elevated transferrin saturation nor elevated serum ferritin measurements are the least likely to develop clinical manifestation in the near future.  On the other hand, the five individuals whose serum measurements both are elevated and whose tissue iron measurements indicate iron loading are at the highest risk of developing clinical manifestations.

Table 3-8.  Summary of Transferrin Saturation Screening Results and HFE Genotypes in Two Population-Based Studies Involving Non-Hispanic Caucasians

HFE Genotype	Number (rate per 10,000)	Elevated Transferrin Saturation N (%)1	Elevated Serum Ferritin N (%)2	Iron Overloadc N (%)3
282/282	11 ( 44)	9 (81.8)	5 (45.5)	5 (45.5)
282/63	54 ( 215)	3 ( 5.6)	0 ( 0.0)	0 ( 0.0)
63/63	74 ( 294)	2 ( 2.7)	1 ( 0.0)	1 ( 2.0)
282/wild	250 ( 994)	3 ( 1.2)	3 ( 1.2)	0 ( 0.0)
63/wild	587 (2335)	5 ( 0.9)	2 ( 0.3)	0 ( 0.0)
wild/wild	1538 (6118)	4 ( 0.3)	0 ( 0.0)	0 ( 0.0)
All	2514 (10,000)	26 ( 1.0)	11 ( 0.4)	6 ( 0.2)

¹  Burt et al., initial fasting transferrin saturation of 55% or more, repeat of 50.5% or more (97.5^th centile of nm/nm).  McDonnell et al., females >50%, males >60% on both fasting samples.

²  Burt et al., serum ferritin of > 160 ug/L (females) or > 300 ug/L (males).  McDonnell et al., > 95^th sex-adjusted centile (referred to as NHANES III ranges – found to be > 200 for females, > 400 for males).

³  At least 2 of the following 4 criteria: hepatic iron concentration >4,500 ug/g, hepatic iron index >2.0, 3-4+ stainable iron, removal of at least 4 grams of mobilizable iron

References

Asberg A, Hveem K, Thorstensen K, Ellekjter E, Kannelonning K, Fjosne U, Halvorsen TB, Smethurst HB, Sagen E, Bjerve KS.  2001  Screening for hemochromatosis: high prevalence and low morbidity in an unselected population of 65,238 persons. Scand J Gastroenterol 36:1108-15.

Beutler E, Felitti VJ, Koziol JA, Ho NJ, Gelbart T.  2002  Penetrance of 845G--> A (C282Y) HFE hereditary haemochromatosis mutation in the USA.  Lancet 359:211-8,

Burke W, Thomson E, Khoury MJ, McDonnell SM, Press N, Adams PC, Barton JC, Beutler E, Brittenham G, Buchanan A, Clayton EW, Cogswell ME, Meslin EM, Motulsky AG, Powell LW, Sigal E, Wilfond BS, Collins FS.  1998  Hereditary hemochromatosis: gene discovery and its implications for population-based screening.  JAMA 280:172-8.

Burt MJ, George PM, Upton JD, Collett JA, Frampton CM, Chapman TM, Walmsley TA, Chapman BA.  1998.  The significance of haemochromatosis gene mutations in the general population: implications for screening. Gut 43:830-6.

McDonald SM, Hover A, Gloe D, Ou CY, Cogswell ME, Grummer-Strawn L.  1999  Population-based screening for hemochromatosis using phenotype and DNA testing among employees of health maintenance organizations in Springfield, Missouri.  Am J Med 107:30-37.

Witte DL, Crosby WH, Edwards CQ, Fairbanks VF, Mitros FA.  1996.  Practice guideline development task force of the College of American Pathologists. Hereditary hemochromatosis.  Clin Chim Acta. 245:139-200.