AMERICAN COLLEGE OF MEDICAL GENETICS
Technical Standards and Guidelines for CFTR Mutation Testing
2006 Edition.
 
  Technical Standards and Guidelines for CFTR Mutation Testing

Standards and Guidelines for Clinical Genetics Laboratories 2002
Supercedes Technical Standards and Guidelines for CFTR Mutation Testing

Approved by the Board of Directors of the American College of Medical Genetics
October 26, 2002. Genetics in Medicine 2002;3 (5).

Reviewed and Revised: 2005 by the Molecular Subcommittee of the Laboratory Quality Assurance Committee

Jean Amos, PhD
Specialty Laboratories2

Gerald L. Feldman, MD, PhD
Wayne State University/Detroit Medical Center1-3

Wayne W. Grody, MD, PhD
UCLA1-3

Kristin Monaghan, PhD
Henry Ford Hospital1-3

Glenn E. Palomaki
Foundation for Blood Research2,3

Thomas W. Prior, PhD
Ohio State University1,3

C. Sue Richards, PhD
Oregon Health & Science University1-3

Michael S. Watson, PhD
ACMG3

1Molecular Subcommittee of the Laboratory Quality Assurance Committee
2Cystic Fibrosis Molecular Working Group
3American College of Medical Genetics

Disclaimer These standards and guidelines are designed primarily as an educational resource for clinical laboratory geneticists to help them provide quality clinical laboratory genetic services. Adherence to these standards and guidelines does not necessarily ensure a successful medical outcome. These standards and guidelines should not be considered inclusive of all proper procedures and tests or exclusive of other procedures and tests that are reasonably directed to obtaining the same results. In determining the propriety of any specific procedure or test, the clinical molecular geneticist should apply his or her own professional judgment to the specific clinical circumstances presented by the individual patient or specimen. It may be prudent, however, to document in the laboratory record the rationale for any significant deviation from these standards and guidelines.
Preface One mission of the ACMG Laboratory Quality Assurance (Lab QA) Committee is to develop standards and guidelines for clinical genetic laboratories, including cytogenetics, biochemical, and molecular genetics specialties. This document was developed under the auspices of the Molecular Subcommittee of the Lab QA Committee by the Cystic Fibrosis (CF) Working Group. It was placed on the "fast track" to address the pre-analytical, analytical, and post-analytical quality assurance practices of laboratories currently providing testing for CF. Due to the impact of the ACMG statement endorsing carrier testing of reproductive couples,1 CF testing had increased in volume and the number of laboratories offering CF testing has also increased. Therefore, this document was drafted with the premise of providing useful information gained by experienced laboratory directors who have provided such testing for many years. In many instances, "tips" are given. However, these guidelines are not to be interpreted as restrictive or the only approach but to provide a helpful guide. Certainly, appropriately trained and credentialed laboratory directors have flexibility to utilize various testing platforms and design testing strategies with considerable latitude. We felt that it was essential to include technique-specific guidelines for several current technologies commonly used in laboratories providing CF testing, since most of the technologies discussed are available commercially and are widely utilized. We take the view that these technologies will change and thus, this document will change with future review. In response to the revised mutation panel recommendations and based on data collected during the previous two years of cystic fibrosis transmembrane conductance regulator (CFTR) screening experience, the following updates have been added as of October 27, 2005.
CF 1

INTRODUCTION

Disease-specific statements are intended to augment the current general ACMG Standards and Guidelines for Clinical Genetics Laboratories (http://www.acmg.net). This document is intended to enhance the ACMG statement on "Laboratory Standards and Guidelines for Population-Based Cystic Fibrosis Carrier Screening"1 by inclusion of more technical laboratory issues related to CF testing and the inclusion of diagnostic and prenatal diagnostic testing as well as prenatal carrier screening. It is intended for genetic testing professionals who are already familiar with the disease and the methods of analysis. Issues related to the trypsinogen component of newborn screening are not addressed, but these guidelines are applicable to subsequent molecular analysis of newborns.

Individual laboratories are responsible for meeting the CLIA/CAP quality assurance standards with respect to appropriate sample documentation, assay validation, general proficiency and quality control measures.

CF 2

BACKGROUND ON CYSTIC FIBROSIS

CF 2.1

Gene Symbol/Chromosome Locus

CFTR on chromosome 7q31.2 was positionally cloned in 1989.2-4 CFTR contains 27 coding exons; genomic sequence is ~230 kb; mRNA is ~6.5 kb.

CF 2.2

OMIM Number: 602421

CF 2.3

Brief Clinical Description

Cystic fibrosis is one of the most common autosomal recessive diseases in the Caucasian population with a prevalence estimate of 1 in 2500 to 3300 live births. CF is characterized by viscous mucus in the lungs with involvement of digestive and reproductive systems as well as sweat glands (excess salt loss). Pulmonary disease is the critical factor in prognosis/survival but both pancreatic sufficient and insufficient forms exist. Recurrent and persistent pulmonary infections are common and lead to respiratory failure. Pancreatic insufficiency occurs in 85% of affected individuals. Neonatal meconium ileus occurs in 10% to 20% of newborns with CF. Other manifestations include chronic sinusitis, nasal polyps, liver disease, pancreatitis and congenital absence of the vas deferens. The overall average survival of CF patients, including those with milder presentation, is approximately 30 years. Treatment for CF patients is palliative and includes control of infections, clearance of mucus in the lung and improvement of nutrition through pancreatic enzymatic replacement. Somatic gene therapy is a research focus. For more information see the online GeneClinics profile at www.geneclinics.org and the National Cystic Fibrosis Foundation at www.cff.org. Newborn screening programs for CF measure immunoreactive trypsinogen, often with follow-up DNA testing. Differential diagnosis is by sweat chloride testing (>60 mM/L).

CF 2.4

Mode of Inheritance: Autosomal Recessive

CF 2.5

Gene Description/Normal Gene Product

CFTR is 1480 amino acids with a mass of ~170,000 daltons. CFTR is in the ATP-binding cassette family of transporter proteins. The CFTR protein contains five domains including two membrane-spanning domains, a regulatory domain, and two nucleotide-binding domains that interact with ATP.

CF 2.6

Mutational Mechanism/Abnormal Gene Product

An abnormal CFTR protein results in defective electrolyte transport and defective chloride ion transport in the apical membrane epithelial cells of the sweat gland, airway, pancreas, and intestine. There are four classes of CFTR mutations: Class I mutations lead to defective protein products, Class II mutations result in defective protein processing, Class III mutations have a defect in the channel regulation, Class IV mutations are defective in conductance through the channel and represent milder mutations, and Class V mutations of abnormal splicing. Mutations in CFTR can affect the function of the cAMP-regulated chloride channel membrane-spanning domains of the CFTR that form the channel pore or the channel opening, which is controlled by phosphorylation of the regulatory domain residues.

CF 2.7

Mutation Spectrum

A complete list of all mutations can be found in the CF Mutation Database at www.genet.sickkids.on.ca/cftr/. Over 1000 mutations have been identified in the CFTR gene. However, the vast majority of mutations are at frequencies of <0.1% or represent private mutations. The major mutation, ΔF508, accounts for 31% to 72% of CF chromosomes, depending upon ethnicity/race.

CF 2.8

Racial/Ethnic Association of Common Mutations

The ACMG recommended carrier screening panel, while panethnic, is primarily based on mutation frequency in the non-Hispanic Caucasian and Ashkenazi Jewish populations due to the high frequency of the disease in these groups, which represent about 57% of the U.S. population. Laboratories providing testing for African American, Hispanic or other ethnic groups should be aware of mutation frequencies as they apply to their testing population. Depending upon the ethnic group, these mutation frequencies may be difficult to obtain (see Table 1).

Self-reported race is an important consideration when interpreting CFTR mutation test results used in prenatal screening for cystic fibrosis. This is also true for ethnicity. Most studies in the United States use Census Bureau racial/ethnic categories, in which race is divided into Caucasian, African American and Asian American. These are further stratified into Hispanic/non-Hispanic ethnicity among Caucasians and African Americans. Another Caucasian ethnic subgroup (Ashkenazi Jewish) is usually not collected by the government, but can be useful when testing for CF. While all of these categories may include relatively distinct subgroups and are less than ideal, they nonetheless provide practical information for individuals and couples.

 

Table 1.
Risk that both partners carry a CFTR mutation and the risk for them to have a CF child based on their test results and race/ethnicity


Race/Ethnic Group (Carrier Rate, Mutations Identified by the Panel %)

Test Results for the Couple1


Risk of Being a Carrier Couple
(1 in N)2

Risk of Having a CF Child
(1 in N)3

Not Tested

Negative

Positive


Ashkenazi Jewish

XX

   

570

2,270

(1/23.8, 94.04%)

 

XX

 

147,000

490,000

     

XX

1

4

 

X

X

 

9,140

36,600

 

X

 

X

23.8

95.2

   

X

X

384

1,540


Non-Hispanic Caucasian

XX

   

625

2,500

(1/25.0, 88.29%)

 

XX

 

42,400

170,000

     

XX

1

4

 

X

X

 

5,150

20,600

 

X

 

X

25

100

   

X

X

206

824


African American

XX

   

3,770

15,100

(1/61.4, 64.46%)

 

XX

 

29,200

117,000

     

XX

1

4

 

X

X

 

10,500

42,000

 

X

 

X

61.4

246

   

X

X

171

684


Hispanic Caucasian

XX

   

3,380

13,500

(1/58.2, 71.72%)

 

XX

 

41,200

165,000

     

XX

1

4

 

X

X

 

11,800

47,200

 

X

 

X

58.1

232

   

X

X

203

812


Asian American

XX

   

8,780

35,100

(1/93.7, 48.93%)

 

XX

 

33,500

134,000

     

XX

1

4

 

X

X

 

17,100

68,600

 

X

 

X

93.7

375

   

X

X

183

732


1 X indicates the result for one of the partners
2 The product of each partner’s carrier risk
3 The couples’ carrier risk times ¼
 
Numbers rounded to 3 significant digits after computations are completed.

CF 2.8.1

The most common mutations in the Ashkenazi Jewish population have been described.5-9 In the recent ACMG policy statement,8 the 5 most commonly identified CF mutations in that population were: W1282X (45.92%), ΔF508 (31.41%), G542X (7.55%), 3842+10kbC>T (4.77%), and N1303K (2.78%). They account for 94% to 97% of the mutant alleles in Ashkenazi Jewish CF patients.5,8 The new ACMG panel of 23 mutations accounts for 94.04% of detectable mutations.

A report by Orgad et. al.10 indicated that additional mutations were found in Jewish Israeli populations, including D1152H, 405+1G>A, W1089X, and S549R. In a large screening program in this population, the D1152H mutation had a carrier frequency of 1/190 and represented 12% of CF carrier alleles.11 Other identified mutations included 1717-1G>A, R117H, R334W, A455E, G551D and R553X. Functional studies suggest that D1152H may be a pathogenic mutation.12 At least 1 study recommended including the D1152H mutation in a CF screening panel if an individual is 100% Ashkenazi Jewish.11 However the actual percentage of D1152H disease-causing mutations in this population and its frequency in other populations is still unknown. Furthermore, the severity of this allele remains to be determined, as some reported D1152H patients in combination with a classic CF mutation such as ΔF508 have had mild disease, while 1 fetus with G542X/D1152H had hyperechogenic bowel loops and meconium ileus. 13-16

CF 2.8.2

The most common mutations responsible for cystic fibrosis in non-Hispanic Caucasians have been described. The ∆F508 allele ranks as the most common CFTR mutation although the frequency varies among subgroups of non-Hispanic Caucasians. In a heterogeneous North American non-Hispanic Caucasian population, the frequency of ∆F508 was estimated to be 68.94% (using the CF Consortium data) and 75.90 (using the CF Foundation data). Various sources of under- or over-ascertainment are likely to be responsible for these differences and the average of the two may be the most reliable estimate. The next 5 most common mutations identified are G542X, G551D, 621+1G>T, W1282X and N1303K.17 The updated ACMG panel of 23 mutations accounts for at least 88.29% of detectable mutations. Only a marginal gain in mutation detection is generated when additional mutations are added to the ACMG panel in this population.

CF 2.8.3

The most frequent mutations identified in the African-American CF population using the ACMG recommended CF carrier screening panel 8,17 were: ΔF508 (44.07%), 3120+1G>A (9.57%), R553X (2.32%) ΔI507 (1.89%), G542X (1.45%), G551D (1.21%) and 621+1G>T (1.11%). The new ACMG- recommended CF carrier screening panel would detect 64.46% of CF mutations in African-American CF patients.8 In a recent study, additional mutations, not included in the ACMG revised panel, were detected in African-American CF patients, including 2307insA and A559T.18 In addition, in CF carrier screening among African-Americans, R117H and G622D were identified most commonly after ΔF508 in one study.19 What seems apparent is that a number of less frequent CF mutations occur in this population, and that ethnic-specific detection panels may be used by some laboratories to increase their mutation detection rates.

CF 2.8.4

In general, the term "Hispanic" refers to persons from Latin America, a wide geographic area of significant racial and ethnic diversity. Latin American countries include the Caribbean (e.g., Jamaica, Puerto Rico, Cuba), Central America (e.g., Mexico, Costa Rica) and South America. Populations from these areas include descendants of Europeans, native people and Africans/Blacks, with variable levels of admixture. For many of these geographical areas, data on mutation frequencies are unavailable; others are based on small studies or limited testing panels. The most frequent CF mutations identified in the Hispanic-Caucasian CF patient population in the recent ACMG revised carrier screening panel were Δ F508 (54.38%), G542X (5.10%), R553X (2.81%), R334W (1.78%), N1303K (1.66%), and 3849+10kbC>T (1.57%), with 71.72 % of CF mutations detectable in the revised panel.8,17 A number of mutations not included in the ACMG recommended panel have been identified in other studies,21-23,18 including D1270N, 3876delA, W1089X, R1066C, S549N and 1949del84.

CF 2.8.5

Forty eight point ninety three percent (48.93%) of CF mutations in the Asian American population were identified using the ACMG panel in the recently published revised ACMG recommendations.8 A total of 86 CF chromosomes was used for the Asian American computation.24-28 The proportion of detectable mutations and the prevalence of CF in this group is poorly defined. CF is very rare in native Asians (about 1 in 900,000 births) and the higher rate of 1 in 35,000 found in Asian Americans (Table 1) is likely due to admixture. Although these data are less reliable, it is clear that Asian Americans have the lowest proportion of detectable mutations, and the lowest birth prevalence of CF, making them the least likely to benefit from prenatal screening via carrier testing.

CF 2.9

Indications for Testing

  • Diagnostic Testing, possible diagnosis of CF
  • Diagnostic Testing, definite diagnosis of CF
  • Diagnostic Testing, infants with meconium ileus
  • Diagnostic Testing, congenital bilateral absence of the vas deferens (CBAVD) in males
  • Carrier Testing, partners of individuals with positive family history
  • Carrier Testing, partners of CBAVD males
  • Carrier Testing, general population of reproductive couples
  • Carrier Testing, premarital population, to assist in selection of a mate
  • Carrier Testing, positive family history
  • Carrier Testing, gamete donors
  • Preimplantation Testing
  • Prenatal Diagnostic Testing, positive family history or for couples having a CF mutation in both partners
  • Prenatal Diagnostic Testing, echogenic bowel in fetus during second trimester
  • Newborn Screening

CF 2.10

Genotype-Phenotype Considerations

Genotype-phenotype correlations are imprecise and should not be used clinically in predicting lung involvement or survival. Mutations in CFTR have been classified based on association with pancreatic sufficient or insufficient phenotype, with non-classic or atypical CF presentation, including borderline to normal sweat chloride levels, pancreatic sufficiency, male infertility, or mild pulmonary disease. Examples of such mutations include: R117H, 3849+10kbC>T, A455E, 2789+5G>A, G85E, and R334W. However, no significant correlation with genotype or concordance within sibships has been demonstrated for pulmonary disease. While there is variability in pulmonary phenotype, the majority of individuals with CF have serious, progressive lung disease. Approximately 85% of CF patients are pancreatic insufficient. The remainder of CF patients are pancreatic sufficient and usually have at least one mutation associated with a milder phenotype. It is important to note that there are a number of exceptions to these generalizations.

CF 2.11

Special Testing Considerations

CF 2.11.1

Clinical Validation: Clinical Sensitivity and Specificity, and Other Test Performance Characteristics

The clinical sensitivity of CFTR testing varies depending upon several factors, including the mutation panel being used, the race/ethnicity of the population being tested, and the clinical setting. Therefore, it is important that the laboratories request and obtain information about race/ethnicity, family history, and reason for testing. The following sections provide estimates of clinical sensitivity and specificity for non-Hispanic Caucasians (hereafter referred to as Caucasians, including individuals of American Caucasian, Caucasian with mixed European ancestry, and Caucasian with Northern European ancestry), using the ACMG recommended mutation panel for carrier screening in a prenatal and preconceptual setting.

Clinical Sensitivity: Clinical sensitivity is defined as the proportion of individuals who have CF and also have a positive CFTR test with two identifiable mutations. Most laboratories will rely on the literature. For example, the panel of 23 mutations proposed by the ACMG will identify about 88% of carriers in Caucasians.25 Thus, about 77% (88% X 88%) of Caucasians with CF (or the same proportion of carrier couples) will have a positive test result (two mutations identified). Laboratories should also be able to provide estimated clinical sensitivities for other defined racial/ethnic groups that may be tested. Estimates of clinical sensitivity could also take into account published estimates of analytic sensitivity.

Clinical Specificity: Clinical specificity can be defined as the proportion of negative test results among individuals who do not have CF. Analytic error or variable expressivity of certain mutations can reduce the clinical specificity of the test. Although the clinical expression of most of the 23 recommended mutations is known to be highly consistent with a classic CF phenotype, there may be some exceptions. For example, the R117H mutation may produce a more variable clinical phenotype, depending upon genetic modifiers, some of which may not be well defined. Analytic false positive errors may occur at the rate of about 6 per 1000 carrier tests. If confirmatory testing is routinely performed, however, this rate is likely to be 1 per 1000 or lower.28-29

Prevalence: The birth prevalence of CF varies by ethnicity/race. Based on a literature review of prenatal screening trials, newborn screening trials and systematic registries, the birth prevalence of CF in Caucasians is about 1:2500 (carrier rate 1/25). Ashkenazi Jewish individuals have a carrier frequency that is slightly higher than that in Caucasians. Less data are available for other racial ethnic groups and thus the estimates are less confident (Table 1).

Clinical Positive Predictive Value: In this setting, the clinical positive predictive value can be defined as the proportion of couples with positive test results who are at a 25% risk of having an affected child. This value can be computed by knowing the analytic and clinical sensitivity and specificity as well as prevalence of the disorder. The major CF mutations are expected to produce a CF clinical phenotype so the clinical positive predictive value will be high (most carrier couples will have the 25% reproductive risk). Exceptions will occur, however, because of analytic false positives and because of variable expressivity of some CFTR mutations. Because of this latter group, laboratories should consider confirmatory testing of carrier couples prior to offering amniocentesis.

Clinical Negative Predictive Value: In this setting, the clinical negative predictive value can be defined as the proportion of couples with negative test results who are not at a 25% risk of having an affected child. The clinical negative predictive value is high because the disorder is rare. Results that compromise negative predictive value occur due to analytical errors and because the panel cannot detect 23% of carrier couples. A reasonable estimate for clinical negative predictive value is 99.96 % (1 in 2500 negative couples are actually at a 25% risk compared to 1 in 625 prior to testing). (See Table 1.)

CF 2.11.2

Test Validation Requirements: The laboratory should satisfy the test validation criteria described by ACMG and any and all state and federal applicable guidelines. Guidance is available from ACMG and other agencies, including the New York State Department of Health (www.wadsworth.org/labcert), the Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards (NCCLS)), MM1-A Vol 20 #7, MM9-A and College of American Pathologists Checklist (CAP, www.cap.org).30-32

CF 2.12

Prenatal Diagnostic Testing and Prenatal Screening: CFTR mutation testing is used for both confirmative diagnosis and for carrier detection as part of prenatal screening. A positive prenatal diagnostic test result is considered to be definitive rather than predictive since the penetrances for these 23 mutations are known to be high. Carriers identified as part of the screening process are expected to be asymptomatic. However, the process will occasionally identify individuals who carry two mutations but are asymptomatic, present with non-classical symptoms or have a late onset presentation. There are no reports of de novo CFTR mutations. A larger number of mutations (>23) is generally appropriate for diagnostic testing in order to achieve the highest possible clinical sensitivity, but care should be taken to ensure that the penetrance of tested mutations is known.

CF 2.12.1

Prenatal Diagnostic Testing: CF mutation analysis can be used for prenatal diagnosis in both direct and cultured amniotic fluid cells (AFC) and chorionic villus samples. It is recommended that both parents be tested prior to testing of fetal specimens, preferably within the same laboratory. As appropriate, parents and fetus should be tested (or re-tested) within the same laboratory. The laboratory must specify the amount of material required for testing and provide referring professionals with appropriate instructions. Laboratories must have a prenatal follow-up program in place to verify diagnostic accuracy.

CF 2.12.2

Indications for Prenatal Diagnosis: Indications for prenatal diagnosis are known parental mutations, family history of CF and echogenic bowel at ultrasound during the second trimester.

CF 2.12.3

Maternal Cell Contamination (MCC): All prenatal samples should be examined in parallel with a maternal sample to rule out error due to maternal cell contamination. A combination of several polymorphic short tandem repeat (STR) sites is recommended. Sensitivity studies should be included in the assay validation in order to determine the acceptable detection level of MCC.

CF 3

GUIDELINES

CF 3.1

Pre-Test Considerations

CF 3.1.1

Informed consent is recommended for prenatal screening for CF via carrier testing. It is the duty of the healthcare professional, not the laboratory, to obtain informed consent. It is the laboratory’s responsibility to explain CF testing to the healthcare provider such that meaningful informed consent may be obtained.

CF 3.1.2

Laboratories should have a mechanism to collect pre-test clinical information that includes patient date of birth, indication for testing (see section CF 2.9), racial/ethnic background, and specific family history of CF. If the patient has a positive family history, the laboratory should determine if the familial mutation(s) is (are) known. Pre-test information can be solicited using a specialized test requisition or questionnaire. The physician should be contacted if the pre-analytical information does not accompany the specimen. If the laboratory is unable to obtain this information, the written report should contain language or tabular information to assist clients in interpreting the results. For example, a report for carrier risk revision may contain tables that allow the ordering physician to interpret carrier studies with negative finding, tabulated by ethnicity and family history. If the laboratory determines that the requested test is inappropriate, the ordering physician should be contacted immediately.

CF 3.2

Methodological Considerations

All general guidelines for Polymerase Chain Reactions (PCR) in the ACMG Standards and Guidelines apply. The following additional details are specific for cystic fibrosis. For this test, there are many valid methods with different strengths and weaknesses.

CF 3.2.1

Positive Controls: Mutation-positive controls for all of the ACMG 23 mutations can be obtained from the NIGMS Human Genetic Cell Repository (http://locus.umdnj.edu/nigms/) as either cell lines or DNA. Synthetic super controls that include all of the ACMG 23 alleles are available from several vendors. However, many mutations commonly included in testing panels are not commercially available, which presents a problem for the laboratory validating their test. For mutations that are unavailable commercially, one option for the laboratory is to produce synthetic controls using PCR or by oligonucleotide synthesis protocols. All synthetic controls produced in the laboratory, however, must be validated by sequence analysis in both forward and reverse directions to confirm the specific mutation. The amount used should be empirically determined and ideally be less than expected from a genomic sample. It is recommended that once the laboratory identifies a patient positive for such a mutation and provided that the patient has previously consented for re-use of his/her DNA, that genomic DNA be used as a positive control in future CF assays. If positive controls are generated using PCR, it is important that the laboratory take appropriate precautions to avoid contamination of patient assays with control PCR product. Although it is desirable that all positive controls be included in each assay, given the large number of CF mutations in the standard test, it is not always practical to run all positive controls on every assay, particularly depending upon the laboratory and the specific technology used. At a minimum, during routine testing, it is recommended that each run include at least one positive assay control and that all positive controls be tested on a rotating basis. Thus, in each specific technology section, we address the issue of positive controls.

CF 3.2.2

Sample Preparation: Multiplex PCR detection is amenable to the use of DNA prepared from blood using a variety of extraction protocols, ranging from crude lysates to highly purified DNA depending on the sizes of the amplicons. This procedure also accommodates DNA prepared from buccal samples (i.e., brushes, swabs and mouth washings). It is recommended that DNA from prenatal samples, amniocytes and chorionic villi be highly purified in order to be sufficient in quality and quantity for any additional testing that may be required. Typically, 10 to 50 ng of patient DNA is adequate for a robust amplification reaction. For information on how to control for maternal cell contamination, refer to the ACMG Standards and Guidelines for Clinical Genetics Laboratories Section G19.

CF 3.2.3

Validation of Methods: For CF mutation analysis, laboratories can currently choose between development of in-house developed testing methodologies or use of commercial analyte specific reagents (ASRs). Laboratories offering genetic tests for clinical use, independent as to whether they are in-house developed or purchased kits, are regulated under the provisions of CLIA '88. CLIA '88 requires collection of in-house data to validate test performance prior to reporting results, but provides little detailed guidance that is relevant to DNA-based testing. Whether the laboratory chooses to develop CF testing as an in-house developed test or use commercially available ASR reagents, it is the laboratory’s responsibility to validate assay performance and provide other information such as intended use of the test, methodology and reporting formats. The State of New York Department of Health currently provides a helpful checklist for the preparation of genetic testing validation packages and other guidelines are under review.30 For additional information on test validation procedures, refer to the ACMG Standards and Guidelines for Clinical Genetic Laboratories, Section C8.

CF 3.2.3.1

Forward Allele-Specific Oligonucleotide (ASO)

CF 3.2.3.1.1

Overview: The ASO method is based upon hybridization of a labeled oligonucleotide probe containing either wild-type sequence or known mutant sequence to the target, patient DNA. This method has been described and applied to high spectrum cystic fibrosis mutation analysis in a clinical laboratory setting.33-35 Generally, PCR products from multiplex PCR reactions of patient DNAs are manually or robotically spotted onto replicate filters (dot blots) and then hybridized to labeled ASOs under specific conditions. Design of the multiplex PCR conditions, ASOs, hybridization and wash conditions, and detection is complex. There are no commercial "kits" or ASRs currently available. An advantage of this method is that mutations can be readily added to an already existing panel. There are a number of issues that must be considered in the development of this test platform.

CF 3.2.3.1.2

Design and Labeling of ASO Probes: ASOs for the normal and mutant sequence pair should be derived from the same DNA strand. Since G:T and G:A mismatches are less destabilizing during hybridization reactions, it is important to avoid a G:T or G:A mismatch between the mutant oligonucleotide and the normal template. ASO probes are labeled for radioactive or chemiluminescent detection. If radioactively labeled, the laboratory determines the need for purification and quantification prior to use.

CF 3.2.3.1.3

Multiplex PCR Amplification: Various parameters can be employed which allow the use of one PCR program for a combination of primer sets. One method is touchdown annealing cycling. Others may depend on primer design.

CF 3.2.3.1.4

Dot-Blot Membranes: To prepare replicate filters, the use of a robotic system or a multichannel pipetting device is recommended to ensure that the same patient PCR product is placed at the same position on each filter. This is critical to the interpretation of the results of this assay.

CF 3.2.3.1.5

Hybridization: For radioactively labeled probes, it is recommended that an optimized and constant number of counts per minute, per milliliter (cpm/ml) be consistently used from run to run in order to obtain consistent quality of results. In addition, it is recommended that a non-labeled competitive probe be included at an increased molar concentration (about 10- to 20-fold higher) in order to eliminate non-specific signal (i.e., increased signal to noise ratio). The optimum conditions for hybridization must be determined by the laboratory. Optimal pooling strategies for combining probes should be determined by the laboratory if pooling is performed. Calculation of melting temperature (Tm) for each oligonucleotide is insufficient to predict the correct conditions for hybridization, which must be empirically determined. Protocols describing a pooled hybridization condition have been described.33-35

CF 3.2.3.1.6

Interpretation of Results: Comparison of the autoradiograph of the wild-type filter and the mutant filter based upon position is necessary for interpretation of test results. In general, a positive result at a given position only on the wild-type filter is interpreted as normal, a positive only on the mutant filter is interpreted as homozygous for the mutation, and a positive on both filters is interpreted as heterozygous for the mutation. For CF analysis, a number of filters are necessary to obtain the minimum panel of the 23 recommended mutations. Thus, it is important that results from all filters be read prior to interpretation, particularly when two different mutations are detected in the same patient, such as in diagnostic testing. A grid placed over the filters is recommended for location of exact position, particularly when the analysis is performed in a 96-well format. It is also recommended that at least two (or more) individuals read the results and concur prior to reporting.

CF 3.2.3.1.7

Reflex Testing: Rare DNA variants can cause failure of amplification or failure of the ASO to hybridize. Of particular concern is the presence of apparent homozygosity for the ΔF508 mutation by ASO analysis. It is critical that laboratories include known variants in the mutation panel to prevent mistyping of compound genotypes such as F508C/ΔF508. Laboratories may wish to confirm all ΔF508 homozygous results, particularly unexpected homozygous results, by another type of analysis. Laboratories should be aware that failure of one allele to amplify can also lead to apparent homozygosity. In certain cases of unusual findings, such as homozygosity for rare mutations, laboratories may consider testing parents in order to confirm the genotype.

CF 3.2.3.2

Reverse Dot Blot Hybridization (RDB)

CF 3.2.3.2.1

Overview: An alternative approach to ASO is reverse dot-blot (RDB) hybridization. In this method, the roles of the oligonucleotide probe and the target amplified DNA are reversed. Probe pairs, complementary to mutant and normal DNA sequences, are bound to nylon membranes in the form of dots or slots. DNA that has been amplified in multiplex reaction(s) and labeled using end-labeled primers or internal incorporation of biotinylated dUTP, is hybridized to the membrane. This procedure is very amenable to high throughput analysis of high mutation spectrum genes and has been applied to the detection of β-thalassemia and CFTR mutations.36-38 Although probe design and production of the spotted membranes may be complex, mutation detection using this method is non-radioactive, convenient, rapid, robust and requires no specialized interpretation skills. Commercial sets of ASRs are available and sufficient published information exists so that laboratories can develop in-house developed test assays. Two colorimetric and one chemiluminescent biotin-based detection systems have been reported. This technology, while robust, is relatively inflexible and not easily expanded to include additional mutations.

CF 3.2.3.2.2

Oligonucleotide Probe Design: Probes are conjugated at the 5′ end by an amino linker group, added by an aminophosphoramidite during synthesis, for subsequent covalent linkage to the carboxyl group of the activated nylon membrane. Length of the allele-specific primer and base composition must be optimized so that the final optimal hybridization and washing conditions for all detected alleles are identical. Probes lengths 15 to 17 nucleotides with 30% to 50% guanine-cytosine (GC) content are adequate to discriminate point mutations. Otherwise, the same guidelines apply as for probe preparation for forward ASO hybridization. However, despite these general rules, probe design for adequate detection may also involve trial and error.

CF 3.2.3.2.3

Strip Layout, Manufacture and Quality Control: Covalent linkage of the amino-modified oligonucleotide to the membrane-bound activated carboxyl group increases the sensitivity of the assay relative to previous enzymatic probe tailing methods. Each oligonucleotide solution should contain a dye such as phenol red to allow for visual inspection of the spotted membranes. The arrangement of oligonucleotides on the strip is a matter of personal preference; wild type and mutant probes can be spotted in separate rows or groups, or interspersed among each other. Manual production of RDB strips is described in Cai et. al.37 This process is amenable to robotic production of large strip lots that can then be stored at room temperature until use. Each lot of strips should be compared to a previous lot to verify consistency with respect to each allele detected in the assay as well as a negative (no DNA) control. For in-house developed strip production, it is often necessary to adjust the amount of new lots of probe that is applied to the strips in order to optimize hybridization signal.

CF 3.2.3.2.4

Multiplex PCR Amplification: All general guidelines for multiplex PCR amplification apply to RDB detection. It has been reported that semi-nested PCR may increase hybridization signal for some mutations.38 It is useful to design the primers so that each product differs by at least 10 bp in length so that robustness of amplification can be visualized on a check gel prior to hybridization. The choice of probe labeling depends on the detection system; primers are biotinylated at the 5′ terminus for subsequent strepavidin-horseradish peroxidase detection.

CF 3.2.3.2.5

Controls: While the laboratory may determine that it is not feasible to include each positive assay control in each run due to batch size limitations, QC on a new lot of RDB should include testing for each mutation. At a minimum, during routine testing, it is recommended that each run include at least one positive assay control and that all positive controls be tested on a rotating basis. The number of positive controls can also be minimized by using genomic or synthetic compound heterozygotes.

CF 3.2.3.2.6

Hybridization, Detection and Interpretation: Hybridization and detection are straightforward and require minimal labor. Care should be taken to protect light sensitive reagents. The genotype of the patient is easily read from the array of hybridization signal on each strip. Individual test results should be read by two reviewers who concur prior to reporting. Since the hybridization signal fades over time, the strips should be photocopied, photographed, digitized, or scanned in order to keep a permanent result record for each patient.

CF 3.2.3.2.7

Reflex Testing: One of the strengths of the RDB method is the ability to test simultaneously for a high mutation spectrum. However, additional labor is incurred when mutations are tested only as a reflex. As for ASO typing, it is necessary to include frequent polymorphisms in the coding region of the CFTR gene (e.g., F508C, to prevent mistypings of polymorphism/mutant compound heterozygous genotypes such as F508C/ΔF508). As described below, however, it is desirable to determine the 5/7/9T genotype only for diagnostic cases or carriers positive for the R117H mutation.

CF 3.2.3.3

Amplification Refractory Mutation System (ARMS)

CF 3.2.3.3.1

Overview: ARMS, or allele specific amplification, is the PCR equivalent of allele specific hybridization with ASO probes. Worldwide, ARMS is one of the most frequently used methods for multiplex detection of common CFTR mutations, partly due to the commercial availability of kits and ASR reagents. Advantages of the ARMS method are that it is rapid (results can be obtained in one working day), reportedly reliable and nonisotopic. In addition, analytic validity and other performance characteristics of ARMS for the specific application of CF carrier testing can be estimated using data from eight published reports.39-46 Most of these studies utilized primers obtained from the same commercial source.

PCR reactions depend on two oligonucleotide primers that bind to the complementary strands at either end of the DNA segment to be amplified. ARMS is based on the observation that oligonucleotide primers that are complementary to a given DNA sequence except for a mismatch (typically at the 3' OH residue) will not, under appropriate conditions, function as primers in a PCR reaction. For genotyping, paired PCR reactions are performed for each mutation tested. One primer (common primer) is used in both reactions, while the other is either specific for the mutant or wild-type sequence. In principle, ARMS tests can be developed for any single base pair change or small deletions/insertions. Achieving acceptable specificity is dependent on primer selection and concentration. Use of longer primers (e.g., 30 vs. 20 bp) and inclusion of control reactions have been reported to improve specificity. Primers and conditions for multiplex reactions must be selected so that the relative yields of PCR products are balanced and the PCR products can be adequately separated on agarose gels. Detection of 23 mutations is likely to require two or more multiplex reactions.

In-house developed primer sets must be validated to ensure desired performance characteristics, and new reagent lots should be compared to a previous lot to ensure consistency in performance and robustness. One commercial set of ASR ARMS reagents for detecting 29 CF mutations is available in the U.S. Although the manufacturer performs a level of performance evaluation on these reagents, the laboratory must also complete an internal validation to assess proficiency prior to use on patient samples.

CF 3.2.3.3.2

Controls: Internal control reactions are not required if mutant and wild-type ARMS reactions are combined in the same test. However, for screening purposes, multiplexing mutant ARMS reactions without paired wild-type reactions can result in significant cost savings. Internal controls (additional control primers that amplify unrelated sequences) can be included in each multiplex reaction to ensure that DNA samples will generate at least one PCR product in each tube and reduce the likelihood of false negative results. Negative and positive control samples must be run with each assay but the laboratory may determine that it is not feasible to include all 23 mutation controls in each run due to batch size limitations. Pooled positive DNA control samples can be utilized to allow efficient inclusion of the most common mutation controls in each run. Remaining positive controls can be tested on a rotating basis.

CF 3.2.3.3.3

Visualization and Interpretation of Results: Non fluorescent PCR products are separated by electrophoresis through an agarose gel containing ethidium bromide and visualized by UV transillumination. Individual test results are interpreted by analysis of the banding pattern by two reviewers in comparison with a molecular weight standard. PCR products generated by fluorescent ARMS technology are resolved using capillary electrophoresis and sized using fragment analysis software. Assays without paired wild-type reactions will not discriminate between the heterozygous and homozygous state. Therefore, reflex testing by another method must be performed in order to accurately interpret the results..

CF 3.2.3.4

Oligonucleotide Ligation Assay (OLA)

CF 3.2.3.4.1

Overview: The oligonucleotide ligation assay (OLA) is a novel approach to mutation detection of point mutations, small deletions and small insertions, and consists of two phases. The first phase involves, allele-specific PCR, which is designed to support hybridization of exact-match PCR primers to target sequences within the CFTR gene. During the second phase, a ligation reaction is performed in which two oligonucleotide proves are hybridzed to adjacent sequences of the resulting ampliccons. The 5' probe is an allele-specific oligonucleotide (ASO) designed with either the normal or the mutant nucleotide(s) at its ultimate 3' end as well as a mobility modifying tail at its 5', which allows electrophoretic separation by size and differentiation between normal and mutant alleles. The 3' probe is a ligation-specific oligonucleotide (LSO) that consists of a dye-labeled common sequence immediately adjacent to the mutation site. The ligation occurs only if both the ASO and LSO probes are perfectly hybridized. The common probe is phosphorylated at the 5' end to allow for the ligation reaction and contains a fluorescent dye marker at the 3' end to allow detection upon separation. Detection requires the use of an automated sequencer capable of multi-fluorescence detection, and may be performed in a gel or capillary format. The normal and mutant peaks are identified based upon their product size and fluorescent tag. A properly designed OLA gives only the appropriate normal or mutant product(s). CF PCR and CF OLA Analyte-Specific Reagents (ASRs) are commercially available that have been used to construct protocols that detect and identify 30 normal and 32 mutant alleles (including I148T) within the CFTR gene, variants to the poly-T tract, and potential interfering single nucleotide polymorphisms (SNPs) within the exon 10 hotspot. Protocols constructed with the CF ASRs and general purpose reagents (GPRs) have been described.47 The entire procedure (PCR through OLA) is performed in a single tube. In addition, CF-specific templates have been developed that support results interpretation. The templates were developed by sites that have generated and validated ASR-based CF protocols, and are intended for use with commercially available software to analyze data and create summary reports. Since the reagents are ASRs, it is important that the laboratory perform a minimal internal validation to assess proficiency prior to use on patient samples. (See Section CF 3.2.3.)

CF 3.2.3.4.2

Controls: If practical for the laboratory, it is desirable to include all positive controls in each assay. However, it may not be feasible to include numerous positive controls in each assay run. Minimally, negative controls, a heterozygous and homozygous positive control for ΔF508, and several "no DNA" controls should be included in each run. Additional positive controls should be rotated among assay runs. The assay should be robust and give consistent results with relative peak heights and mutation assignment. While the software automatically interprets the data, it is important that the results are confirmed visually by the laboratory director or designee.

CF 3.2.3.4.3

Visualization and Interpretation of Results: Fluorescently labeled PCR products are separated by electrophoresis using an automated sequencer, either gel or capillary-based. The data is analyzed using commercially available software that has been configured with protocol specific parameters, which support the generation of results. Samples that are homozygous normal will generate a single peak since both alleles will migrate to the position of the normal sequence. Likewise, homozygous mutant samples will generate a single peak at the position of the mutant sequence. The mobility modifiers separate normal from mutant peaks by size, with mutant alleles appearing approximately two bases longer than the corresponding normal allele. Thus samples that are heterozygous for a particular locus will produce two peaks with the normal peak always appearing to the left of its longer, mutant counterpart. Additionally, the peak heights for heterozygous loci will be half the intensity of the homozygous (normal or mutant) peaks. Since many mutations can be analyzed simultaneously in one reaction tube, it is critical that the position of migration for each allele is appropriately validated to ensure accurate interpretation of patient results. It is also important that the laboratory set thresholds for peak height to avoid pitfalls of misinterpretation due to background noise. It is recommended that the laboratory verify that the multiplex reaction, which includes all alleles to be analyzed, both normal and mutant, is robust and reproducible. Automated peak assignment is an attractive feature of the software, which is desirable for quality assurance issues. Visual inspection, however, is recommended.

CF 3.2.3.5

Liquid Bead

CF 3.2.3.5.1

Overview: Liquid bead arrays provide simple and high-throughput analysis of DNA polymorphisms with discrete detection of wild-type and mutant alleles in a complex genetic assay. Bead-array platforms use either universal tags or allele specific capture probes that are covalently immobilized on spectrally distinct microspheres. Because microsphere sets can be distinguished by their spectral addresses, they can be combined, allowing as many as 100 analytes to be measured simultaneously in a single-reaction vessel. A third fluorochrome coupled to a reporter molecule quantifies the molecular interaction that has occurred at the microsphere surface. The microspheres, or beads, are dyed internally with one or more fluorophores, the ratio of which can be combined to make multiple bead sets. Capture probes are covalently attached to beads via a terminal amine modification. Bead arrays offer significant advantages over other array technologies in that hybridization occurs rapidly in a single tube, the testing volume scales to a microtiter plate, and unlike glass or membrane microarrays, bead solutions can be quality tested as individual components.48-51

CF 3.2.3.5.2

Multiplex PCR Amplification: All general guidelines for multiplex PCR amplification apply to liquid bead array-based detection. All commercial products use a single multiplex PCR with proprietary primers designed to accommodate the hybridization and detection system being used. Since liquid bead arrays work well with various front-end chemistries, including oligonucleotide ligation, allele-specific single base extension, ASO hybridization and allele-specific primer extension (ASPE), the detection chemistry of the particular detection format can be incorporated into the PCR and/or subsequent amplicon modification steps.

CF 3.2.3.5.3

Hybridization and detection: One commercial platform uses biotin-modified PCR products that are hybridized to allele-specific capture probes on different beads.48-50 Another uses allele-specific primer extension of the PCR product such that "universal tags" are incorporated into the product for allele discrimination.51 The biotinylated PCR product or extended PCR product is then hybridized to either capture probes or "universal anti-tags," respectively, that are covalently bound to the beads. Both platforms use a reporter fluorophore, streptavidin-phycoerythrin, in or before the hybridization reaction. After hybridization, the modified amplicon is bound to a reporter substrate and transferred directly to a detection instrument without post-hybridization purification. The sample genotype is assigned by comparing the relative hybridization signal between the wild-type and mutant alleles. The generation of electronic data facilitates the development of automated analysis software and database archiving. The reaction is analyzed for bead identity and associated hybridization signal intensity. Lasers interrogate hybridized microspheres individually as they pass, single file, in a rapidly flowing stream. Thousands of microspheres are interrogated per second, resulting in an analysis system capable of analyzing and reporting up to 100 different hybridization reactions in a single well of a 96-well plate in just a few seconds.

CF 3.2.3.5.4

Visualization and interpretation of results: Output files generated during detection are automatically processed and made available in a report format through customized software. The software should allow for controlled access to data, patient reports, comments and sample history. Electronic data output is archived into a database format for data integrity, quality control tracking, and result trending and incorporates batch processing of results, highlighting samples with mutations and genotype calling.

CF 3.2.3.5.5

QC and Controls: It may not be feasible to include a genomic DNA (gDNA) for each positive assay control in each run due to reagent cost and batch size limitations. However, QC on a new lot of beads should include gDNA-based testing for each mutation. At a minimum, during routine testing, it is recommended that each run include at least one positive assay control and that all positive controls be tested on a rotating basis. The use of either genomic or synthetic compound heterozygotes can also maximize the number of positive controls while limiting the number of reaction wells used. The last sample in each batch should be a no-template control, to assess for reagent contamination by previous or current amplicons. The ratio of wild type to mutant signal, adjusted for background for each control, should fall into previously set ranges that maximize the signal to noise ratio and the no-template controls should fall below an arbitrary pre-set detection limit.

CF 3.2.3.5.6

Reflex Testing: All commercial liquid bead array assays include PCR and ultimate detection of the reflex polymorphisms. However, one of the strengths of customized software is data masking, such that the data for the polymorphisms are revealed only as appropriate or on demand. Thus, for example, no additional labor is required for reporting of the intron 8-polyT track in the presence of an R117H allele. The laboratory can also choose to report the exon 10 polymorphisms for each patient or to reveal only the polymorphism genotype in the presence of a ΔF508 allele.

CF 3.2.3.6

Fluorescence Resonance Energy Transfer (FRET)

CF 3.2.3.6.1

Overview: The fluorescence resonance energy transfer (FRET) assay involves two concurrent reactions in a single well on a 96-well plate. The primary reaction utilizes two different oligonucleotide probes, one specific for the normal sequence and the other specific for the mutant sequence. Both probes hybridize to the target genomic DNA, forming an overlapping structure. This structure is recognized by a proprietary enzyme, resulting in the release of a DNA fragment, which forms the substrate for the secondary reaction. The secondary reaction involves the binding of the released DNA fragment to a FRET cassette containing a fluorescent reporter and quencher molecule. The overlapping structure created by the binding of the released DNA fragment to the cassette is recognized by the same enzyme as the primary reaction. The second structure is cleaved, separating the fluorophore and quencher, generating a detectable fluorescence signal. Mismatch between the mutant probe and wild-type target DNA or wild-type probe and mutant target DNA in the primary reaction prevents the formation of the overlapping structure and the generation of the subsequent fluorescent signal. By utilizing two different allele-specific (normal and mutant) probes in the primary reaction, with each binding to a different FRET cassette with a unique spectral fluorophore, 2 sequence variants (normal and mutant) at a single site can be detected in the same well.

A CF ASR platform run on a microfluidics card utilizing the FRET assay is commercially available. This format enables the user to run multiplex FRET assays. The heat-stable card contains 8 raised samples lanes (1 lane per sample) with each lane subdivided into 48 separate reaction chambers. This allows for a single pipetting step of reagents into up to 48 different reactions. The CF assay involves PCR amplification of the target DNA using a limited number of cycles. The amplified DNA is transferred to the card, which contains dried down oligonucleotide probes and FRET cassettes in each chamber. After the addition enzyme, the cards are sealed using a scoring device, and incubated. After the incubation is complete, the fluorescence generated from each sample is read by a fluorescent plate reader that can accommodate a 96-well format and is equipped with the appropriate filters.

The ASR tests for 42 mutations, including the original ACMG-recommended panel, plus reflex polymorphisms. One advantage of this assay is that the user has the ability to display the genotype for mutations or reflex polymorphisms as determined to be appropriate.

CF 3.2.3.6.2

Controls: Due to the nature of the assay, it is not practical to run genomic DNA positive controls for each mutation analyzed using this assay. However, it may be possible to run several positive controls for each run. At a minimum, a normal (wild-type), heterozygous mutant, and negative (no DNA) control should be included in each run. Positive controls could be rotated among each assay run. Failure of any control to give the expected result invalidates that particular run and the assay must be repeated.

CF 3.2.3.6.3

Interpretation of Results: The genotype of the sample is determined using software-generated calculations. The ratio of each fluorescent signal compared to the negative (no DNA) control determines the net signal for each probe. Based on the ratio of the net signals for each sample (wild type: mutant), the genotype is determined to be homozygous wild-type, heterozygous, or homozygous mutant for each analyte. Samples that do not fall into the predetermined ranges for each genotype are flagged as equivocal and must be repeated. Samples that generate low counts are flagged as ‘low signal’ and must be repeated. Results for each sample are reported on an easy to read summary page. Results for each mutation analyzed are available in greater detail in a separate report.

CF 3.2.3.6.4

Reflex Testing: The user has the ability to pre-select which analytes are reported for each sample. In the event that genotyping at the polyT locus is desired or if a homozygous delta ΔF508 genotype is generated, reflex testing for 5T, 7T, 9T or F508C, respectively, can be performed without additional reagents or labor.

CF 3.2.3.7

Additional Methods: Additional methods for performing high mutation spectrum and high throughput single nucleotide polymorphism (SNP) analyses exist, although few are currently in use in clinical molecular genetic laboratories in the U.S. These methods (which are not all-inclusive) include flow-cytometry-based detection of bead-coupled ASOs, various arrayed primer extension methods, mass spectrometry detection methods, oligonucleotide array approaches and mini-sequencing of target regions. However, at the present time, most (but not all) of these technologies are severely limited in ability to perform multiplex analysis. Thus, while they may be applicable for use in testing for a small number of variants, such as hemochromatosis or factor V Leiden, they currently have not been applied to the detection of a large number of mutations as required in the CF analysis. We anticipate that future improvements in these technologies or others will make them adaptable to CF analysis. Thus, they will be included in these guidelines at a later date.

CF 3.2.4

Guidelines for Development of Primers and Probes:

General considerations include:

  • sequence composition
  • Tm
  • GC content
  • size of desired product
  • intron/exon boundary inclusions to detect splice-site mutations
  • avoidance of polymorphisms at the primer site

Any in-house developed primer sets should be thoroughly tested to ensure desired performance characteristics.

CF 3.2.4.1

Published Lists of Primers: Published lists of primers are available. 5,52 Several sets of primers, PCR conditions and methods of separation and detection have been published.6,33 Other primers and methods can be used, if adequate validation is performed.

CF 3.2.4.2

In-House Developed Primers: In-house developed primers can be developed using any commercially available primer design software package that helps to select optimum sets of primers based on Tm and salt concentration.

CF 3.2.4.3

Multiplex Considerations:

General issues to consider in designing a multiplex PCR analysis include:

  • optimum design of several sets of primers for amplification under a single set of conditions including the same Tm
  • length of primer
  • compatibility of primers (avoidance of primer interactions)
  • specificity of primers
  • avoidance of pseudogenes and known polymorphisms
  • similar GC content
  • optimizing salt concentration
  • determining concentration of each primer to use in reaction (trial and error)
  • unifying the annealing temperature by using commercially available buffers (such as Q solution) or DMSO53
  • type of Taq (i.e., Taq Gold, Hot-start, etc.)

Generally, for multiplex PCR reactions, lower primer concentrations are recommended and higher dNTP concentrations are required. For the CF gene, the 23 recommended mutations are found in 15 exons (or intronic regions). Thus, a 15-plex reaction would be required for amplification of all in a single tube. For the complete CF gene analysis including 27 exons, the laboratory may perform multiple multiplex PCR reactions. It is the laboratory’s responsibility to validate all assays in which PCR primers are designed in-house. For troubleshooting assay failures it is recommended that multiplex assays be designed with each PCR product of a different length and sufficient to visualize on an agarose gel to determine the presence and amount of product. Commercial PCR optimizing kits are available to aid laboratories in development efforts.

CF 3.2.4.4

Setting Optimum Reaction Conditions: Factors to consider include optimization of salt concentration and primer concentration, choice of buffer, and choice of Taq Polyamerase. Single PCR reactions will have different reaction conditions from multiplex PCR reactions. It is important to set these conditions to obtain a robust PCR product reproducibly, yet to avoid spurious results.

CF 3.2.4.5

Setting Optimum Cycling Conditions: Various approaches exist in setting these conditions. Step-down conditions have been described and are particularly useful for multiplex reactions when primers anneal at various temperatures.33 Generally, cycling conditions should include no more than 35 cycles in order to avoid introduction of errors. The cycling conditions should be set for high stringency to obtain pure products. Annealing temperature should be closely determined by Tm of primers. It is advisable for the laboratory to develop primers that use the same set of reactions and cycling conditions. Following PCR the laboratory may or may not choose to examine the PCR product on an agarose gel.

CF 3.2.4.6

General Disclaimer about Primer-Binding/Probe-Annealing Regions: It should be realized that there are many sources of diagnostic errors. Genotyping errors can result from trace contamination of PCR reactions and from rare genetic variants that interfere with analysis. Additionally, polymorphisms in targeted regions (primer-binding or probe-annealing) can lead to testing errors and result in failure of one allele to amplify (allele drop-out).

CF 3.3

Mutation Panel

CF 3.3.1

Minimum Mutation Panel for Population-Based Carrier Screening Purposes:1 Different testing panels might be employed for identification of CFTR mutations in patients diagnosed with CF, in relatives of CF patients, or in newborn screening. It is important to recognize that this panel is subject to change as new information becomes available. Consequently, with the emergence of a vast amount of new data from multiple laboratories using this initial mutation panel, data evaluation has resulted in this revised panel.17

ΔF508

R553X

R1162X

2184delA

3120+1G>A

ΔI507

G542X

G551D

W1282X

N1303K

621+1G>T

R117H

1717-1G>A

A455E

R560T

G85E

R334W

R347P

711+1G>T

1898+1G>A

3849+10kbC>T

2789+5G>A

3659delC

   

CF 3.3.2

Inclusion of the Common R117H Mutation in the Test Panel Screens for Congenital Bilateral Absence of the Vas Deferens (CBAVD) as well as for CF: The phenotypic consequences of the R117H mutation are modulated in cis by the 5/7/9T polypyrimidine tract in intron 8 such that R117H/7T is associated with CBAVD and R117H/5T is associated with CF.54 Moreover, the 5T allele is associated as a trans mutation in CBAVD.55 It is recommended that the 5/7/9T variant be excluded from the routine carrier screen but tested as a reflex for carriers shown to be heterozygous for the R117H mutation. The 5/7/9T variant should be included for diagnostic panels to distinguish the genotypes of R117H associated with CF from those associated with CBAVD and as a potential pathogenic mutation for CBAVD.

CF 3.3.3

Issues of Unexpected Homozygosity Due to Polymorphisms: Tests may not distinguish between a CF mutation and benign variants. For example, I506V, I507V and F508C are performed as reflex tests for ΔF508 positives unless it is proven that these variants do not cause assay interference.

CF 3.3.3.1

Incorrect Assignment of Homozygosity: Deletions, polymorphisms and benign variants can lead to incorrect assignment of homozygosity when a benign variant is present in at the same site on the second allele. Parental testing to confirm homozygosity is recommended for rare mutations. Clinical indication can suggest potential false positive homozygosity when the indication is carrier testing.

CF 3.3.4

Controls: Controls representing the mutations to be tested should be run on each assay, if feasible, based upon the testing method. Laboratories should validate their control DNA by sequencing, by exchange with another laboratory or by using consensus-validated material (for more information see http://www.phppo.cdc.gov/dls/genetics/qcmaterials/materialsavailability.aspx).

CF 3.3.5

Laboratories that service a particular ethnic population based on geography may consider including additional mutations in the testing panel that are specific to that particular population. Every effort should be made to determine the frequency of specific CF mutations within the target population and to provide testing at reasonably high sensitivity levels.

CF 3.3.6

An extended panethnic mutation panel may be appropriate for certain diagnostic testing purposes but it is not currently recommended by ACMG for routine carrier screening of reproductive couples.1 If a laboratory offers an extended panel, it is important that the composition be determined based on frequency of the mutation within the target population. The 23-mutation panel was based upon a 0.1% frequency worldwide. An extended panel would go beyond that requirement for some mutations and expand its scope to the population of service, such as the U.S. population. Additional mutations of >0.1% frequency in the U.S. population that laboratories may wish to consider adding to the minimum panel have been recently described.24

CF 3.3.7

Testing for Unknown Mutations Using Scanning Technology/Sequence Analysis: All scanning methodologies described in the ACMG Standards and Guidelines for Clinical Genetics Laboratories apply. Detection of a sequence alteration by a scanning technology must be confirmed by sequence analysis and interpreted according to the ACMG "Standards and Guidelines for Interpretation of Sequence Variation." In addition, the alteration must be named according to the accepted guidelines for mutation nomenclature. The nomenclature developed by the Ad Hoc Committee on Mutation Nomenclature56 and Antonarakis et. al.57 is recommended. The nomenclature established for CFTR mutations follows these guidelines and is found in the CF mutation database at http://www.genet.sickkids.on.ca/.

CF 3.3.8

Linkage Analysis in CF Families in which One or No CFTR Mutations Have Been Identified: Multiple informative markers are available within the CFTR gene and flanking the gene. It is recommended that more than one marker be included in the analysis and that the laboratory follows standard linkage analysis procedures in pre-analytical, analytical, and post-analytical testing. The use of intragenic markers is preferred over the previously used extragenic markers. Prior to performing linkage analysis, it is recommended that the laboratory obtain confirmation of the clinical diagnosis of CF in the family.

CF 3.4

Quality Assurance

Laboratories should follow the ACMG/CAP checklists, be in compliance with the NIH-DOE Task Force on Genetic Testing (http://www.genome.gov/10001808) and follow the ACMG Standards and Guidelines for Clinical Genetics Laboratories. Laboratories should also participate in the CAP/ACMG Proficiency Testing Program or other inter-laboratory proficiency testing program. All aspects of testing, including pre- and post-analytical, must be in full compliance with regards to appropriateness of test ordering, interpretation, reporting and counseling. Laboratories must validate their CF assays, whether in-house developed or commercial kit, as well as state the analytical and clinical sensitivity and specificity according to the ACMG guidelines.

CF 3.5

Interpretations (Post-Analytical)

CF 3.5.1

The following elements should be included in the report, in addition to the items described in the current general Standards and Guidelines:

CF 3.5.1.1

Ethnicity, family history, indication for testing, test method with the FDA statement regarding the use of ASRs, test result, mutations tested and residual risk based on ethnicity should be included.

CF 3.5.1.2

Labs should include clear interpretation of the patient result as homozygous for a mutation (predicted affected with CF), a compound heterozygote (predicted affected with CF), heterozygous carrier (interpretation depends on whether this is carrier testing or diagnostic testing) or negative (interpretation depends on whether this is carrier testing, presence or absence of family history or diagnostic). In cases where mutations have been identified, the mutation(s) name should be included. For examples of appropriate report components, laboratories should refer to the CF report templates for carrier screening as described by Grody et. al.1

CF 3.5.1.3

All positive results for diagnostic tests or for positive/positive couple screening should state that genetic counseling is indicated and testing is appropriate for at-risk family members. When sequential carrier testing is done, a positive result on one partner should include the recommendation of testing the other partner and at-risk family members. All individuals who have a family history of CF should receive genetic counseling. All CFTR carriers, including healthy males who have mutations associated with infertility, should also be referred for genetic counseling.

CF 3.5.2

Comments on Phenotype Issues with CBAVD, R117H and 5T, 7T Background: ACMG recommends that all R117H positive results require reflex testing for the 5T/7T/9T variant in the polythymidine tract at intron 8 in the CFTR gene. Refer to model reports for carrier screening presented in the ACMG statement.1 For R117H /5T positive heterozygotes, testing of parents is recommended in order to determine the inheritance of the R117H and the 5T variant (i.e., cis vs. trans position). If the R117H and 5T variant are determined to be in cis, then the report should reflect that this mutation has been associated with a variable phenotype when R117H/5T (cis) or another CFTR mutation is present in CF patients. If the R117H mutation and 5T are determined to be in trans, the report should indicate that the individual carries a relatively benign CF mutation that is not generally associated with the phenotype of typical CF patients but has been associated with CBAVD, leading to infertility in males and no known clinical features in females. In addition, the report should reflect that the 5T variant on one chromosome, in combination with a CFTR mutation on the opposite chromosome, may lead to male infertility due to CBAVD, with or without mild or atypical symptoms of CF, and that there is no known clinical significance of 5T in females. The penetrance of 5T is reduced and thus it is difficult to predict the clinical significance of the 5T variant. For individuals who are R117H positive and 5T negative, the report should indicate that the R117H mutation is not expected to lead to a typical CF clinical phenotype. However, R117H has been associated with CBAVD. In all above cases, genetic counseling is recommended. For diagnostic testing, and particularly for testing for CBAVD in males with infertility, it is recommended that the intron 8 variant be included in the testing panel.58

CF 3.5.3

Comments on individual residual risk and reproductive risk for couples (when appropriate) should be included in the patient report or provided to the referring healthcare professional. Comments should be written to be consistent with current HIPPA guidelines. Table 1 is given as an example and is not intended to be all-inclusive of every ethnic group. Several assumptions were used in developing the risk values in this table, including carrier frequencies of various ethnic/racial groups and sensitivities of the minimum mutation panel of 23 mutations in these various populations. This table is intended for use in CF screening of reproductive couples who have no family history of CF. For individuals with a family history of CF, the calculations would be different and would be based upon pedigree information. It is the laboratory’s responsibility to provide this type of information, specific for the population it serves. Negative results should be interpreted within the context of patient personal/family history and ethnicity and both prior and revised carrier risks should be stated.

CF 3.5.4

Residual Risk for Fetus with Echogenic Bowel: Echogenic bowel in the fetus based upon ultrasound, present in 0.1% of all pregnancies, can be due to CF or may be associated with normal variation, chromosome abnormality, or congenital viral infection.59 There have been relatively few studies to determine the frequency with which echogenic bowel in the fetus correlates with CF. Thus, it is difficult to determine a prior risk when echogenic bowel is identified in a second trimester fetus upon ultrasound. Collective data suggests a risk of approximately 1%, which has been used in calculating posterior CF risk in a fetus with echogenic bowel and heterozygous for a CF mutation.59-62 There are two publications describing echogenic bowel calculations, one using Bayesian analysis63 and one using a complex probability calculation62 which laboratories may use. In calculating risk, carrier frequency and the test sensitivity in the specific ethnic/racial population must be considered. Whether or not to provide residual risk information for these cases is left to the laboratory’s discretion, as the literature is limited and additional data collection is desirable in order to provide accuracy in risk assessment. Some laboratories, however, will take the view that even limited information can be useful for these families. For such laboratories, we provide the following information.

CF 3.5.4.1

Example of a Laboratory Report for a Fetus with Echogenic Bowel: It is important to recognize that there is considerable heterogeneity in ultrasound findings reported by different examiners. This report addresses the situation of typical echogenic bowel in the second trimester. It should be recognized that calcifications in the liver and findings suggestive of peritonitis in the third trimester are significantly different. There is a published report60 indicating that there may be an empirical risk that 13% of such fetuses prove to have CF. This number may, in fact, be as low as 3% (Baylor DNA Diagnostic Laboratory, unpublished data). Calculations can be made using a range of empirical risk for these fetuses between 3% and 13%. Obviously, if the fetus has two CF mutations, this is diagnostic of CF. A fetus with echogenic bowel and one identified CF mutation represents the most difficult counseling circumstance. The risk of such a fetus to be affected with CF can be calculated to be within a range of 13% to 43%, depending upon the assumption regarding the prior empirical risk. If no mutation is detected in the fetus, the risk for the fetus to be affected with CF would be equal to or less than 1 in 645. These risks are calculated based upon the assumption of a Caucasian fetus of Northern European ancestry, a carrier frequency of 1 in 25, and a test sensitivity of 90%. The calculations would be different for a fetus of Ashkenazi Jewish, African American, Hispanic, or other ethnic background, based on differences in the test sensitivities and the carrier frequencies for each of these populations. It is also important to consider other pathology in such cases, such as chromosome abnormality, intestinal malformation, and congenital infection (particularly if calcification is present).

CF 3.5.5

5T/7T/9T Reporting Issues: Commercial products, including RDB hybridization and or other platforms, generally contain all of the alleles on a single strip.  Thus, in some cases, additional data that was not requested and may not be desired is obtained. This has been a matter of practical experience of several CFTR testing laboratories that have routinely tested for 5T because they were using a commercial ASR that did not support reflex testing. While some newer testing technologies have developed software to filter out unwanted results, and thus avoid the issue of how to report it, other technologies, such as the reverse dot blot, routinely include 5T in the panel, thus generating a 5T result for all samples tested.  This often presents a dilemma in reporting of results.  Accordingly, when unwanted 5T results are generated, some laboratories choose to blind their interpretation to the 5T results, and thus, eliminate the reporting of the unwanted result.  In other cases, laboratories cautiously report the 5T result with (or without) a well-thought-out interpretation, often leaving the genetic counselor or clinician in a difficult counseling session.

 

State laws vary with respect to the duty of the laboratory to fully disclose all test results, even when a specific test was not ordered. Moreover there may be CLIA implications of reporting, or not reporting, such results.  Given both the clinical and legal uncertainties in this area, the ACMG recommends that each institution consult with their legal counsel for guidance on the best practice laboratory policy to handle this difficult issue.  At a minimum, however, it would seem wise to clarify, on both the test request form and the report of results, the disorder (e.g., CF versus infertility) rather than the allele (e.g., 5T) for which testing is performed.

CF 4

POLICY STATEMENTS

CF 4.1

The NIH Consensus Conference64 issued a statement that CF mutation testing should be made available to all pregnant couples.

CF 4.2

The American College of Medical Genetics issued a policy statement entitled "Laboratory Standards and Guidelines for Population-based Cystic Fibrosis Carrier Screening."1

CF 4.3

The American College of Obstetricians and Gynecologists (ACOG), in collaboration with ACMG and the National Human Genome Research Institute, has developed and distributed clinical and laboratory guidelines (October 2001). One document entitled "Preconception and Prenatal Carrier Screening for Cystic Fibrosis: Clinical and Laboratory Guidelines" provides information for providers. Two patient educational brochures entitled "Cystic Fibrosis Carrier Testing: The Decision Is Yours" and "Cystic Fibrosis Testing: What Happens If Both My Partner and I Are Carriers?" were developed to help patients with their decisions.

CF 4.3.1

In December 2005, the ACOG Committee on Genetics issued an Update on Carrier Screening for Cystic Fibrosis. A critical new recommendation is as follows: "Information about cystic fibrosis screening should be made available to all couples. It is reasonable to offer CF Carrier screening to all couples, regardless of race or ethnicity as an alternative to selective screening." Additional recommendations are also included in that document. Please refer to the document for further details.65

CF 5

ACKNOWLEDGMENTS

The authors offer sincere thanks to Dr. Larry Silverman for helpful discussions and review of the final manuscript. We also wish to thank Dr. Madhuri Hegde for providing helpful information and discussions regarding the use of OLA for CF analysis, and the Baylor DNA Diagnostic Laboratory for use of their report as an example. The revised document was reviewed and endorsed by the Molecular Working Group of the ACMG Quality Assurance Committee including several of the authors on this guideline and others, including Elaine Spector, Linda Bradley and Dan Bellissimo.

CF 6

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Updated: 4/26/2006 7:00 PM