Interpretation of Test Results

Mutation analyses have only three possible results, detection of no, one, or two mutations. However, reporting of CF results is complex because: (a) detection in a targeted mutation assay is less than 100% and (b) there are many interpretations for each of the possible results, depending on the indication for the test and the ethnicity and family history of the consultand.

For example, the finding of one mutation in an asymptomatic patient demonstrates the consultand to be a carrier but has a very different interpretation in a diagnostic context. For a symptomatic patient, detection of one mutation could mean that the patient is indeed a CF carrier; however, this patient may also be a compound heterozygote for one identified mutation and a second unidentified mutation that is either private or rare.

Most laboratories offer CF tests using a single orderable test code. Prenatal tests are easily identified based on the fetal sample type (amniocytes or chorionic villus biopsies), but it is not easy to distinguish carrier from diagnostic test orders, since both use whole blood specimens. This issue can be addressed by offering clients three separate test codes for each of the three test indications: carrier, prenatal, or diagnostic testing.

The finding of two mutations in a diagnostic test confirms a diagnosis of CF. However, genotype-phenotype relationships are not well established enough that prognostic statements can be made for the many different possible homozygous and compound heterozygous genotypes. The interpretation of one or no detected mutations for a symptomatic patient must include a recommendation for diagnostic sweat electrolyte analysis.

Since the late 1990s, more than 95% of all CF tests are for carrier screening. Although the finding of one mutation in this context is straightforward, the majority of these analyses are negative, with no mutations detected. In the setting of a negative test result, the physician must be informed that the revised carrier risk, while reduced from the prior risk, is a nonzero number because mutation detection is incomplete. As shown in Table 10-3, prior and revised carrier risks both are dependent on patient ethnicity and family history. Prior carrier risks for patients without a family history of CF are calculated from the ethnic frequency of CF, which is highest among whites (1 in 25), lower in Hispanics (1 in 46), and blacks (1 in 62), and lowest among Asians (1 in 90).

Prior carrier risks for patients with a positive family history are based on pedigree analysis. Meaningful risks vary from a high of 2 in 3 for an individual with an affected sibling to a low of 1 in 16 for a patient with an affected second cousin. The prior risk for a consultand with a more distant affected relative is small relative to the population risk.

Revised risks also are based on the frequency of the tested mutations in the patient's ethnic group. The data in Table 10-3 illustrate that the frequency of mutation detec-

Table 10-3. Prior and Residual CF Risks for Patients with a Neg

ative Carrier Test

A. White, Ashkenazi Jewish: 97% Detection

Affected Relative

Prior Risk

Revised Risk

No affected relative

1 in 25

1 in 801

Sibling

2 in 3

1 in 18

Niece/nephew/half-sibling

1 in 2

1 in 34

Aunt/uncle

1 in 3

1 in 68

First cousin

1 in 4

1 in 101

First cousin, once removed

1 in 8

1 in 234

Second cousin

1 in 16

1 in 494

B. White, European: 90% Detection

Affected Relative

Prior Risk

Revised Risk

No affected relative

1 in 25

1 in 241

Sibling

2 in 3

1 in 6

Niece/nephew/half-sibling

1 in 2

1 in 11

Aunt/uncle

1 in 3

1 in 21

First cousin

1 in 4

1 in 31

First cousin, once removed

1 in 8

1 in 71

Second cousin

1 in 16

1 in 151

C. Hispanic: 57% Detection

Affected Relative

Prior Risk

Revised Risk

No affected relative

1 in 46

1 in 110

Sibling

2 in 3

1 in 2.2

Niece/nephew/half-sibling

1 in 2

1 in 3.3

Aunt/uncle

1 in 3

1 in 5.7

First cousin

1 in 4

1 in 8

First cousin, once removed

1 in 8

1 in 17

Second cousin

1 in 16

1 in 36

D. Black: 69% Detection

Affected Relative

Prior Risk

Revised Risk

No affected relative

1 in 62

1 in 198

Sibling

2 in 3

1 in 2.6

Niece/nephew/half-sibling

1 in 2

1 in 4.2

Aunt/uncle

1 in 3

1 in 7.5

First cousin

1 in 4

1 in 11

First cousin, once removed

1 in 8

1 in 23

Second cousin

1 in 16

1 in 50

tion using the ACMG/ACOG panel ranges from 97% for individuals of Ashkenazi Jewish descent to 57% for His-panics. Residual risks are not given for Asian patients because these risks are not known. Risk revision is even more complicated for individuals of mixed heritage.

Thus, responsible reporting for CF mutation test interpretation includes risk assessment based on the indication for the test, patient ethnicity, and family history. Often, samples are referred for testing without patient information and, since this information is not an interpretative requirement for routine clinical tests, clients must be educated to provide it. A two-tiered approach for gathering preanalytic information can be used. First, a specialized requisition that solicits patient information can be used for test ordering. When only partial or no information is received on the requisition, a follow-up questionnaire can be faxed to the client. Gathering patient information by telephone is not recommended since such calls interrupt workflow in the client's office and also because the information may not be readily available to the recipient of the call. Using this system,sufficient information is obtained to provide a patient-specific report for more than 85% of all samples referred for carrier screening. Ethnicity and family history are self-reported by patients, and inaccurate information can be transmitted to the laboratory. It is not unusual for a client to state that the patient is white, omit the information that the patient is of Ashkenazi Jewish descent, and simultaneously order several carrier tests for Jewish recessive diseases. In such an instance, follow-up with the client is required to clarify whether the patient is or is not Jewish, and whether the patient's partner is Jewish.

For negative carrier screens, the report can be tailored to the patient's specific ethnicity and family history when this information is available (see Table 10-3), or a comprehensive report covering all possibilities is written when the information is not provided. To emphasize to the ordering physician the impact of patient information on the interpretation, the report also contains the ethnic-family tables shown in Table 10-3.

The published ACMG/ACOG recommendations1 include well-written model laboratory reports for detection of no or one mutation in a carrier context for individuals with a negative personal and family history. These model reports are also included in the educational materials that have been distributed to the members of ACOG and ACMG. They contain a field for patient ethnicity, but the interpretation is not patient specific. In the model mutationnegative report, the prior and revised carrier risks for all ethnicities are presented in the interpretation field in a tabular format. However, in the author's opinion, it is incumbent upon the laboratory to include specific interpretive information about the patient in negative test reports.

Laboratories can anticipate the most common reporting formats and prepare standard template reports. In practice, as many as 50 templates may be needed, which can then be modified to the patient's specific clinical situation with a minimum number of keystrokes. This approach accommodates high test volumes while maintaining reporting standards. A sample template report for the most common report, a negative test for a patient with a negative family history, is shown in Figure 10-1. Each report contains a patient-specific risk and a statement that the carrier risk of the consultand is reduced but that the risk for having a child affected by CF is also a function of the partner's carrier status. In this way, the nonzero value of the revised risk for a negative test is emphasized as well as the interplay of the genotypes of both parents. Clients have the choice of first testing only one member of the couple, most often a pregnant woman presenting for prenatal care, or testing both members of a couple simultaneously.

Although the ACMG/ACOG mutation panel is designed for carrier screening for individuals without a family history of CF, individuals with an affected relative

CYSTIC FIBROSIS GENOTYPR® TEST CODE 5356: CARRIER STUDY

SOUCE:

Blood

INDICATION:

Carrier screening, negative history

ETHNICITY:

ETHNICITYa

PRIOR CARRIER RISK:

REVISED CARRIER RISK:

Negative for the mutationns analyzed RRISKc

INTERPRETATION: Molecular analysis revealed that this patient is negative for common mutations that comprise PERCENT1%dof cystic fibrosis (CF) mutations in his/her ethnic group. lt is our understanding that this patient has a negative personal and family history of CF.

INTERPRETATION: Molecular analysis revealed that this patient is negative for common mutations that comprise PERCENT1%dof cystic fibrosis (CF) mutations in his/her ethnic group. lt is our understanding that this patient has a negative personal and family history of CF.

The revised risk that this patient is a carrir, based on stated ethnicity, negative family and personal history of CF, and thi negative mutation analtsis.is 1 in RRISKb (0.PECENT2%e ). The risk that this patient will have a child with CF is decreased by this result. This risk, however, is also dependent on the carrier status of his/her partner.

METHOD:

The CF Transmembrane Conductance Regulator Gene was tested for the presence of 25 mutations that are recommended by the American College of Human Genetics for CF camier screening (G85E, R117H, I148T, 621(+1)G>T, 711(+1)G>T, 1078delT, R334W, R347P, A455E, Delta 1507, Delta F508,1717(-1)G>A, G542X, G551D, R553X. R560T, 1898(+1)G>A, 2184delA, 2789(+5)G>A, 3120+(1)G>A, R1162X, 3659delC, 3849(+10kb)C>T, W1282X and N1303K) using the polymerase chain reactions and allele-specific oligonucleotide hybridization. 6 polymorphisms (intron 8 5/7/9T, I506V, I507V, F508C) were also tested.

GENERAL DISCLAIMER: DNA studies do not constitute a definitive carrier test for CF in all individuals. Thus, interpretation is given as a probability. Risk calculation requires accurate family history and ethnicity information. While DNA analysis is highly accurate, technical sources of error include rare genetic variants that interfere with the analysis.

This test or one or more of its components was developed and its performance characteristics determined by Specialty Laboratories.It has not been cleared of approved by the U.S. Food and Drug Administration (FDA). The FDA has determined that such clearance or approval is not necessary. This test is used for clinical purposes. It should not be regarded as investigational or for research. This laboratory is certified under the Clinical Laboratory Improvement Amendments of 1988 ("CLIA") as qualified to perform high-complexity clinical laboratory testing.

Laboratory results and interpretation reviewed by jean Amos, PhD FACAG

Figure 10-1. Template report for a patient without a family history of CF and a negative test. a, the patient's ethnicity as listed in Table 10-3 (a different template is used when the patient's ethnicity is Asian,not provided,or complex); b,the prior risk as listed in Table 10-3, no affected relative; c, the revised risk as listed in Table 10-3, no affected relative; d, the mutation detection in the specific ethnic group as listed in Table 10-3; e, the revised risk, expressed as a percentage.

tion."). When the familial mutation(s) is known and analyzed, the residual risk for the consultand is very low, on the order of the false-negative rate of the assay.

Reporting for most of the possible positive results in a carrier test is straightforward and, as recommended by ACMG/ACOG, should always contain a recommendation for genetic counseling and a suggestion that at-risk relatives also may benefit from genetic counseling and mutation analysis for carrier risk revision. As recommended by ACMG/ACOG, interpretation of positive results for the R117H mutation should always be made in the context of the patient genotype for the intron 8 5T/7T/9T sequence, since expression of the R117H mutation in a compound heterozygous child is variable, depending on the specific genotypic combination. The R117H mutation is most severe when on the same allele as the 5T variant. The reflex of an unexpected AF508 homozygous result to testing for other exon 10 sequence variations (I506V, I507V, and F508C) is necessary to prevent false-positive results,since some detection platforms (e.g., probe hybridization) do not discriminate the AF508 mutation from these polymorphisms.

The laboratory also must be informed when partners are tested, particularly when one or both are found to be carriers. Partners often have different last names, they may be tested at different times or in different laboratories, or the laboratory volume can be high enough that partners are not recognized during CF testing. The nongeneticist physician usually can interpret a 1-in-4-risk for couples who are both carriers, but may not have the experience to provide often are referred for carrier risk testing, and the same screening mutation panel is used. A footnote can be provided in the negative-test report for a patient without a family history for CF to alert the physician that residual carrier risk after a negative test is modified by this history. The footnote may also include a recommendation that the physician refer patients with a positive family history for genetic counseling and Bayesian calculation of revised carrier risk. Several template reports may be needed for patients with a positive family history and a negative test; the variable portion of such a template is shown in Figure 10-2. All such reports include a patient-specific revised risk calculation and a recommendation for genetic counseling; the prior and residual risks for patients with a positive family history are also shown in Table 10-3.

Often the affected family member has not been tested and the familial mutation(s) is not known, or the mutation has been identified but the information is not available to the at-risk relative. In such cases, the report should state that the familial mutation(s) is not known, provide a patient-specific Bayesian risk, and make a specific recommendation for testing the closest known carrier relative (e.g., "We recommend that this patient's father's sister be referred for mutation analysis to identify the familial muta

CYSTIC FIBROSIS GENOTYPR®

TEST COD 5356: CARRIER STUDY

SOURCE:

Blood a

INDICATION:

Positive family history; DETAILS3

ETHNICITY:

ETHNIcCITYb

PRIOR CARRIER RISK:

PRISKc; familial mutation unknown

RESULT:

Negative for the mutations analyzed

REVISED CARRIER RISK:

RRISKd

Additional studies and genetic counseling are recommended; see interpretation.

INTERPRETATION: This patient has a family history of cystic fibrosis (CF). We are not aware whether any members of the family have had mutation analysis. Thus, the familial mutation(s) are currently unknown. Based on family history, the prior risk that he/she is a CF carrier is PRISKc

INTERPRETATION: This patient has a family history of cystic fibrosis (CF). We are not aware whether any members of the family have had mutation analysis. Thus, the familial mutation(s) are currently unknown. Based on family history, the prior risk that he/she is a CF carrier is PRISKc

Molecular analysis revealed that this patient is negative (normal) with respect to common mutations which comprise PERCENT1e% of CF mutations in his/her ethnic group. Based on positive family history,ethnicity, and this negative mutation analysis, the revised risk that he/she is a CF carrier is RRISK1 (0.PERCENT2f%).

It would be possible to further revise this patient's camier risk if the familial mutation(s) were identified. To this end, we recommend that this patient,s RELATIVE9, who is an obligate CF carrier, be referred for mutation analysis.

The risk that this patient will have a child with CF is decreased by this result. This risk, however, is also dependent on the carrier status of his/her partner. We recommend that this patient and partner be referred for genetic counseling.

Figure 10-2. Template report for a patient with a family history of CF and a negative test. a,a phrase that describes the closest carrier relative, for example,"patient's mother's sibling is reportedly an obligate carrier"; b,the patient's ethnicity as listed in Table 10-3 (a different template is used when the patient's ethnicity is Asian, not provided, or complex c, the prior risk based on pedigree analysis, as listed in Table 10-3; d,the revised risk,as listed in Table 10-3; e,the mutation detection in the specific ethnic group,as listed in Table 10-3; f, the revised risk, expressed as a percentage; g, the specific relative recommended to be tested, for example, "mother's sibling."

a fetal risk assessment when only one member of the couple is a carrier. Here, the referral for genetic counseling is critical for the single carrier couple. Heathcare providers are very likely to inform the laboratory that one member of the couple has already tested positive when referring the second member for testing, but less likely to alert the laboratory when the couple is tested simultaneously. Again, this information can be solicited on the requisition and follow-up questionnaire, but this is the most difficult pre-analytic information to obtain. Healthcare providers need additional education to enhance the probability that they will provide accurate and complete information needed for interpretation of CF test results. Clients must be educated to provide this information so that residual risks to a current or future pregnancy can be stated in the report. Residual fetal risks for couples in a variety of testing circumstances are available in the technical standards and guidelines for CF mutation testing (http://www.acmg.net).

Couples with negative or positive test results may have an affected child in the setting of one parent with a known mutation and the other parent with a rare mutation that is not included in any CF mutation test panel. These births are not avoidable using current technology. Anecdotal reports from the genetic counselors that have seen these patients after the unexpected diagnosis after birth inform us that the couple was not aware of their residual risk after carrier testing. Genetic counseling for couples with one identified CF mutation prior to conception or birth of a child would provide this information to couples.

CF carrier screening is the first large genetic screening program based on DNA analysis in the United States. Little data is available regarding the nongeneticist physician's ability to interpret a complex laboratory report, communicate the results and interpretations to his or her patients, and make appropriate management decisions. The success of this program will depend on many factors, the most important of which is physician education to react appropriately to the complex scenarios that occur in patients and couples.

0 0

Post a comment