Bradley W. Popovich

Cystic fibrosis population carrier screening: 2004 revision of American College of Medical Genetics mutation panel

Cystic fibrosis population carrier screening: 2004 revision of American College of Medical Genetics mutation panel

Standards and Guidelines for CFTR Mutation Testing

One mission of the ACMG Laboratory Quality Assurance (QA) Committee is to develop standards and guidelines for clinical genetics laboratories, including cytogenetics, biochemical, and molecular genetics specialties. This document was developed under the auspices of the Molecular Subcommittee of the Laboratory QA Committee by the Cystic Fibrosis (CF) Working Group. It was placed on the “fast track” to address the preanalytical, analytical, and postanalytical quality assurance practices of laboratories currently providing testing for CF. Due to the anticipated impact of the ACMG recommendation statement endorsing carrier testing of reproductive couples, it was viewed that CF testing would increase in volume and that the number of laboratories offering CF testing would also likely increase. 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 of several current technologies commonly used in laboratories providing CF testing, since three of the four 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.

Real-Time Polymerase Chain Reaction With Fluorescent Hybridization Probes for the Detection of Prevalent Mutations Causing Common Thrombophilic and Iron Overload Phenotypes

We evaluated more than 450 patients with thrombophilia or iron overload for the presence of a factor V Leiden (R506Q), prothrombin G20210A, or HFE C282Y mutation using a standard method (polymerase chain reaction [PCR]-restriction fragment length polymorphism) and a comparative real-time PCR fluorescent resonance energy transfer (FRET) hybridization probe melting curve method. There was 100% concordance between the genotypes ascertained by the 2 methods (at each loci). In addition, phenotypic biochemical laboratory parameters measured on a subset of referred patients correlated with their respective genotypes. In the iron overload cohort, HFE C282Y homozygotes (n = 74) had significantly higher (P < .0001) transferrin saturation levels (74% +/- 25%) than did nonhomozygotes (n = 340; 51.4% +/- 28%), suggesting a genotype-dependent increase in body iron loads. In the thrombophilic cohort, the degree of activated protein C resistance (APCR), measured by a clotting time-based test, was associated significantly with the presence of 0 (n = 255; APCR = 2.59 +/- 0.26), 1 (n = 84; APCR = 1.61 +/- 0.13), or 2 (n = 5; APCR = 1.16 +/- 0.04) copies of the mutant factor V Leiden allele. As the fluorescent genotyping method required no postamplification manipulation, genotypes could be determined more quickly and with minimized risk of handling errors or amplicon contamination. In addition to these practical advantages, the FRET method is diagnostically accurate and clinically predictive of phenotypic, disease-associated manifestations.

Technical standards and guidelines: venous thromboembolism (Factor V Leiden and prothrombin 20210G >A testing): a disease-specific supplement to the standards and guidelines for clinical genetics laboratories.

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 this statement 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.

Hypomethylation of an Expanded FMR1 Allele Is Not Associated with a Global DNA Methylation Defect

The vast majority of fragile-X full mutations are heavily methylated throughout the expanded CGG repeat and the surrounding CpG island. Hypermethylation initiates and/or stabilizes transcriptional inactivation of the FMR1 gene, which causes the fragile X-syndrome phenotype characterized, primarily, by mental retardation. The relation between repeat expansion and hypermethylation is not well understood nor is it absolute, as demonstrated by the identification of nonretarded males who carry hypomethylated full mutations. To better characterize the methylation pattern in a patient who carries a hypomethylated full mutation of approximately 60-700 repeats, we have evaluated methylation with the McrBC endonuclease, which allows analysis of numerous sites in the FMR1 CpG island, including those located within the CGG repeat. We report that the expanded-repeat region is completely free of methylation in this full-mutation male. Significantly, this lack of methylation appears to be specific to the expanded FMR1 CGG-repeat region, because various linked and unlinked repetitive-element loci are methylated normally. This finding demonstrates that the lack of methylation in the expanded CGG-repeat region is not associated with a global defect in methylation of highly repeated DNA sequences. We also report that de novo methylation of the expanded CGG-repeat region does not occur when it is moved via microcell-mediated chromosome transfer into a de novo methylation-competent mouse embryonal carcinoma cell line.

Cystic fibrosis carrier screening: Issues in implementation

The transition of testing in the cystic fibrosis transmembrane conductance regulator (CFTR) gene to a prenatal and preconceptional carrier screening program1,2 has been both interesting and informative. The long-held sense in the medical genetics community that there is significant genetic variability in the population has been reinforced as more people are tested and we continue to learn more. It is also apparent that there are varying perspectives about how testing should be done and how results should be communicated, as well as about ongoing program updates and education of providers and patients. In 1997, a National Institutes of Health Consensus Development Conference3 recommended the implementation of prenatal and preconceptional carrier screening for cystic fibrosis (CF). However, it was immediately clear that laboratory testing for CFTR gene mutations and variations was not standardized and that few educational materials to support such a new program were available to providers on the front lines (obstetricians and family physicians) and their patients. A workshop in 1998 was the genesis of a collaborative effort involving the American College of Medical Genetics (ACMG), the American College of Obstetricians and Gynecologists (ACOG), and the National Human Genome Research Institute’s Ethical, Legal and Social Implications program to develop the materials and standards needed to ensure the appropriate implementation of this program. Over the next 2 years, ACMG used existing CF patient and family data to identify a set of mutations that should be a core component of a screening program. ACOG, in collaboration with the National Human Genome Research Institute, worked to develop educational materials for providers and patients. The program was announced in September 2001.2 At the time the mutation panel was set, it was clear that the inherent variation between ethnic groups in both disease incidence and the clinical sensitivity of the panel was dramatic. For the ACMG mutation panel, this is apparent in updated data from a Centers for Disease Control and Prevention–sponsored study (www.cdc.gov/genomics/info/reports/research/FBR/ ACCE.htm) showing the differences between non-Hispanic Caucasians (CF incidence 1/2,500) and Chinese Americans (CF incidence 1/31,000). Inasmuch as more data come from the ethnic groups most affected, it is apparent that the panel is going to best represent the spectrum of mutations in those groups, and this contributes to the differences in clinical sensitivity in various ethnic groups (e.g., Ashkenazim carrier detection rate 97% versus 56% in Hispanic Americans). Furthermore, data were based on a population defined by a disease for which money was most expended for basic research and for clinical testing, with the long-term clinical investigative work being done in the course of service provision. Some information on CF carrier screening in a prenatal diagnostic setting was available.3– 8 However, little genetics research money is expended on the “normal” or general population; its genetic characteristics remain poorly understood. CF represents a classic example of the way genetic information evolves. Genetic testing typically begins with the inherent biases of gene identification studies in a group with the most classic and severe presentations. It then moves to studies within these families that are often biased by similar genetic backgrounds on which mutations sit that can account for the extreme presentations in the initial patients and their families. However, intrafamilial variation begins to break down many of the most extreme biases. Studies progress to less severe presentations of the condition under consideration, specific phenotypes, or other factors that identify higher-risk groups (e.g., ethnicity). The last phase involves very large or general population studies that ultimately provide the unbiased perspective. When a test is considered part of a public health activity, it is usually pilottested with state-supported funding to accumulate a body of data about the particular genetic disease marker in a general population. However, these uses are generally aimed only toward the diagnosis of affected individuals. Although some general population data were available for the CFTR gene, very large, general population studies for most genes have not been among the beneficiaries of such support and are left to evolve in the private sector marketplace environment. Another side result of these services being predominantly in the health care marketplace rather than in public health programs is that the full breadth of a screening program must evolve as well. Many of the required ancillary services were highlighted in the announcement of the screening program. Many require additional support to fully evolve. The initial CFTR mutation and variant panel was determined on the basis of the prevalence of mutations in more than 20,000 classical CF patients,1 a seemingly large, although geographically diverse, population. Any mutation representing 0.1% or more of CFTR alleles in a pan-ethnic population was included. This resulted in 25 mutations and 4 variants known to modify the expression of one of the mutations. Insufficient data on specific ethnic groups were available and the inherent complexity of targeting screening to specific ethnic groups resulted in the initial panel’s being limited and pan-ethnic. In the ensuing year since implementation, both anticipated and new problems have been recognized and a somewhat typical course of adoption of the recommendations has been experienced. CFTR mutation testing has increased by as much as November/December 2002 Vol. 4 No. 6 e d i t o r i a l

Naming and Counting Disorders (Conditions) Included in Newborn Screening Panels

The rapid introduction of new technologies for newborn screening is affecting decisions about the disorders (conditions) that are required or offered as an option through public and private newborn screening. An American College of Medical Genetics report to the Health Resources and Services Administration summarized an extensive effort by a group of experts, with diverse expertise within the newborn screening system, to determine a process for selecting a uniform panel of newborn screening disorders. The expert panel did not propose a mechanism for counting or naming conditions. Differences in the nomenclature used to identify disorders have resulted in difficulties in developing a consensus listing and counting scheme for the disorders in the recommended uniform panel. We suggest a system of nomenclature that correlates the screening panel of disorders recommended in the American College of Medical Genetics report with the screening analyte and accepted standardized nomenclature. This nomenclature system is proposed to remove ambiguity and to increase national uniformity in naming and counting screening disorders.

Fragile X full mutations are more similar in siblings than in unrelated patients: further evidence for a familial factor in CGG repeat dynamics.

Purpose: We sought to compare patterns of full mutation repeat-length variability in the peripheral blood DNA of patients with fragile X syndrome to determine whether siblings possess mutation patterns more similar than those of unrelated patients.Methods: Mutation patterns were visualized by Southern blot analysis and captured digitally with a phosphor imager. Novel comparison strategies based on overlapping profile plots and calculation of weighted mean CGG repeat values were used to assess mutation pattern similarity.Results: Within the population that we analyzed of 56 patients with full mutation, mutation patterns were found to be more similar in siblings than in unrelated patients.Conclusion: These results indicate that repeat-length variability may be generated in a nonrandom manner and that familial factors influence this process.