C. Sue Richards

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.

Population-specific screening by mutation analysis for diseases frequent in Ashkenazi Jews

We describe a partially automated DNA mutation assay for detecting the most frequent mutations in the α‐subunit of β‐hexosaminidase A, the acid β‐glucosidase and the cystic fibrosis transmembrane conductance regulator genes for the Ashkenazi Jewish population. The assay detects carriers for Tay‐Sachs disease, Gaucher disease, and cystic fibrosis with sensitivities of at least 92%, 96%, and 97%, respectively. Among 1,364 young adults of Ashkenazic ancestry in the Dor Yeshurim community who were tested, 52 were Tay‐Sachs carriers, 110 were Gaucher carriers, and 62 were cystic fibrosis carriers. Ten individuals were carriers for two diseases, and three unsuspected cases were diagnosed with Gaucher disease based on mutation test results. In addition to Tay‐Sachs mutation data, results for hexosaminidase A activity were also available. All of 1,254 samples normal by enzyme quantitation were also negative for the three α‐subunit mutations tested, and all of 43 samples with ‘inconclusive’ enzyme results were negative by DNA. Only 52 of 67 samples positive by enzyme assay were also positive for one of the three mutations tested for Tay‐Sachs disease. The data suggest a high degree of false positivity inherent in enzyme identification of carriers. There are no correlative methods to assess the sensitivity of Gaucher and CF carrier testing. The results show that population screening can be carried out efficiently by DNA analysis, with the accrual of carrier information for three separate diseases conducted as a single test. Furthermore, the DNA method for Tay‐Sachs screening appears to exceed the specificity of hexosaminidase A enzyme testing.

Analytic validity of cystic fibrosis testing: A preliminary estimate

Purpose: Derive estimates of analytic sensitivity and specificity of DNA testing for cystic fibrosis in the United States.Methods: Analyze published results of the American College of Medical Genetics (ACMG)/College of American Pathologists (CAP) Molecular Genetics Survey between 1996 and 2001, taking into account difficult, simulated clinical samples included for educational purposes.Results: Analytic sensitivity is 97.9% [95% confidence interval (CI) 96.8–98.7%], and analytic specificity is 99.4% (95% CI 98.7–99.9%) after removing challenges involving delI507 and performing other adjustments. Analytic sensitivity is consistent over the 6 years. Specificity was lower in 1997.Conclusion: These preliminary estimates indicate that analytic validity of cystic fibrosis testing is very good and can likely be improved. To date, fewer than half of the mutations in the panel recommended for preconceptional or prenatal screening have been challenged. The present study highlights the value of performing confirmatory testing when a mutation is identified to reduce false-positive results.

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

Estimated analytic validity of HFE C282Y mutation testing in population screening: the potential value of confirmatory testing.

Purpose The purpose of this study was to estimate analytic sensitivity and specificity of HFE testing for C282Y homozygosity in the hypothetical setting of population screening for hemochromatosis.Methods We analyzed published results of the Molecular Genetics Survey performed by the American College of Medical Genetics/College of American Pathologists between 1998 and 2002, taking into account its educational nature.Results Analytic sensitivity for C282Y homozygosity is 98.4% (95% CI 95.9%–99.5%). The analytic specificity is 99.8% (99.4%–99.9%). At a frequency of 40 per 10,000 for the homozygous genotype, the analytic positive predictive value is 66%.Conclusion HFE testing for C282Y homozygosity is highly reliable. Homozygosity is uncommon in population screening, however, and confirmatory testing should be considered.

Alternative Approaches to Proficiency Testing in Molecular Genetics

Clinical genetic testing laboratories have come under scrutiny in the US and Europe with increasing public awareness of genomic research. Such increased publicly driven demand for quality can improve laboratories and encourage high standards of excellence. The Department of Health and Human Services Secretary’s Advisory Committee on Genetic Testing (1) was instrumental in placing genetic testing in the public eye and exerted pressure on genetic providers to organize our profession and address public concerns, particularly concerns about laboratory quality. Are safeguards in place to prevent poor-quality genetic testing? Multiple federal and state agencies as well as professional organizations have developed guidelines, recommendations, and checklists with which laboratories must comply. In the US these include the Clinical Laboratory Improvement Amendments (CLIA) of 1988 (2), Genetic Testing Under the Clinical Laboratory Improvement Amendments (3), New York State Department of Health Laboratory Standards (4), the College of American Pathologists (CAP) checklist for molecular pathology laboratories (5), the American College of Medical Genetics (ACMG) Standards and Guidelines for Clinical Genetics Laboratories (6), and a NCCLS guideline (7). A recent review of quality assurance in genetic testing has been addressed in the ACCE [analytical validity, clinical validity, clinical utility, and ELSI (ethical, legal, and social issues of genetic testing)] report on carrier screening for cystic fibrosis, a CDC-sponsored project (8). Internal and external quality assurance and quality-control programs have been established to ensure that laboratories can reliably produce (and reproduce) high-quality results in a timely manner and with clinical utility for patients and healthcare providers. Proficiency testing (PT) identifies weaknesses and provides guidance for improvement. The ACMG/CAP PT program has been providing a molecular genetics laboratory survey with challenges on multiple genetic disorders since 1995, and CAP has provided on-site laboratory inspection and a certification …

Mammography behavior after receiving a negative BRCA1 mutation test result in the Ashkenazim: a community-based study.

Purpose: To define the impact of a negative BRCA1 test result on subsequent breast cancer screening behavior in women.Methods: Longitudinal study of a community-based sample of Ashkenazi Jews offered testing for the 185delAG BRCA1 mutation in 1996. Of 309 participants, 118 women were mutation negative, of average risk (based on family history of cancer), unaffected with breast cancer, and provided complete data at baseline, and Year 1 and Year 2 follow-up questionnaires.Results: Women age 50 and older had 91.7% compliance with mammography for the year prior to entry (baseline), 88.3% during Year 1, 91.7% during Year 2 (no significant change; P = 0.775). Women under age 50 demonstrated an increase in mammography (49.2% at baseline, 62.7% Year 1, and 67.1% Year 2; P = 0.035). Both groups demonstrated significant decreases in breast cancer worry and perceived risk. Logistic regression analysis on having a mammogram at Year 2 showed that age, physician recommendation, worry, and perceived risk were all significant.Conclusion: Receipt of negative BRCA1 test results in a cohort of Ashkenazi Jewish women did not have a negative impact on mammography behavior 2 years after genetic testing.

Prenatal diagnosis of a fetus with a homologous Robertsonian translocation of chromosomes 15

We present a prenatal diagnosis of a de novo homologous Robertsonian translocation involving both chromosomes 15. Amniocentesis was performed on a 36-year-old woman at 16.5 weeks of gestation. Chromosome analysis documented a 45,XX,der(15;15) (q10;q10) chromosome pattern. No evidence of a deletion was observed by FISH using a SNRPN DNA probe associated with the Prader-Willi/Angelman syndrome critical region. Molecular studies in the family using six polymorphic markers for chromosome 15 and Southern blot analysis of DNA methylation for the CpG island near the SNRPN gene showed normal biparental inheritance of chromosome 15, excluding uniparental disomy. The patient was counseled that her child would not be able to bear off-spring without clinical assistance. Otherwise the health and intellect of her child were not expected to be affected by the translocation. We consider this to be the first prenatal case identified with a balanced der(15;15)(q10;q10) Robertsonian translocation and a phenotypically normal female outcome. Prenatally identified cases of der(15;15)(q10;q10) warrant further investigation by molecular methodology.

Molecular Diagnostic Testing

Molecular diagnostic testing is a rapidly evolving field that currently includes analysis for single-gene disorders, multifactorial disorders, some forms of cancer, infectious disease, microbial epidemiology, and personal identification. The era of molecular medicine began in the late 1970s with the cloning of the β-globin gene and identification of the point mutation responsible for sickle-cell anemia. At the same time, population-based carrier testing, using other technologies, for common genetic diseases such as Tay-Sachs and sickle-cell disease and prenatal diagnosis for chromosomal abnormalities were becoming widely available. The diagnostic capability of molecular technology has expanded tremendously, and there are now tests available for more than 300 genetic diseases being performed in greater than 200 diagnostic laboratories, academic and commercial, throughout the country (information provided by Helix; Seattle, Washington). The completion of the Human Genome Project will facilitate the identification of all disease genes, making a greater number of molecular diagnostic tests possible. While improved technological tools are essential for molecular diagnostics to move forward, the basic strategies for designing and implementing testing, which we describe in this chapter, will be long-lasting. To illustrate these principles, we will focus on the application of molecular diagnostic testing for single-gene disorders using DNA technology.