Pan-ethnic carrier screening and prenatal diagnosis for spinal muscular atrophy: clinical laboratory analysis of >72,400 specimens.

Spinal muscular atrophy (SMA) is a leading inherited cause of infant death with a reported incidence of ~1 in 10,000 live births and is second to cystic fibrosis as a common, life-shortening autosomal recessive disorder. The American College of Medical Genetics has recommended population carrier screening for SMA, regardless of race or ethnicity, to facilitate informed reproductive options, although other organizations have cited the need for additional large-scale studies before widespread implementation. We report our data from carrier testing (n = 72,453) and prenatal diagnosis (n = 121) for this condition. Our analysis of large-scale population carrier screening data (n = 68,471) demonstrates the technical feasibility of high throughput testing and provides mutation carrier and allele frequencies at a level of accuracy afforded by large data sets. In our United States pan-ethnic population, the calculated a priori carrier frequency of SMA is 1/54 with a detection rate of 91.2%, and the pan-ethnic disease incidence is calculated to be 1/11,000. Carrier frequency and detection rates provided for six major ethnic groups in the United States range from 1/47 and 94.8% in the Caucasian population to 1/72 and 70.5% in the African American population, respectively. This collective experience can be utilized to facilitate accurate pre- and post-test counseling in the settings of carrier screening and prenatal diagnosis for SMA.

Cystic fibrosis carrier testing in an ethnically diverse US population.

BACKGROUND: The incidence of cystic fibrosis (CF) and the frequency of specific disease-causing mutations vary among populations. Affected individuals experience a range of serious clinical consequences, notably lung and pancreatic disease, which are only partially dependent on genotype. METHODS: An allele-specific primer-extension reaction, liquid-phase hybridization to a bead array, and subsequent fluorescence detection were used in testing for carriers of 98 CFTR [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] mutations among 364 890 referred individuals with no family history of CF. RESULTS: One in 38 individuals carried one of the 98 CFTR mutations included in this panel. Of the 87 different mutations detected, 18 were limited to a single ethnic group. African American, Hispanic, and Asian individuals accounted for 33% of the individuals tested. The mutation frequency distribution of Caucasians was significantly different from that of each of these ethnic groups (P < 1 × 10⁻¹°). CONCLUSIONS: Carrier testing using a broad mutation panel detects differences in the distribution of mutations among ethnic groups in the US.

Development of genomic DNA reference materials for genetic testing of disorders common in people of ashkenazi jewish descent.

Many recessive genetic disorders are found at a higher incidence in people of Ashkenazi Jewish (AJ) descent than in the general population. The American College of Medical Genetics and the American College of Obstetricians and Gynecologists have recommended that individuals of AJ descent undergo carrier screening for Tay Sachs disease, Canavan disease, familial dysautonomia, mucolipidosis IV, Niemann-Pick disease type A, Fanconi anemia type C, Bloom syndrome, and Gaucher disease. Although these recommendations have led to increased test volumes and number of laboratories offering AJ screening, well-characterized genomic reference materials are not publicly available. The Centers for Disease Control and Prevention-based Genetic Testing Reference Materials Coordination Program, in collaboration with members of the genetic testing community and Coriell Cell Repositories, have developed a panel of characterized genomic reference materials for AJ genetic testing. DNA from 31 cell lines, representing many of the common alleles for Tay Sachs disease, Canavan disease, familial dysautonomia, mucolipidosis IV, Niemann-Pick disease type A, Fanconi anemia type C, Bloom syndrome, Gaucher disease, and glycogen storage disease, was prepared by the Repository and tested in six clinical laboratories using three different PCR-based assay platforms. A total of 33 disease alleles was assayed and 25 different alleles were identified. These characterized materials are publicly available from Coriell and may be used for quality control, proficiency testing, test development, and research.

Development of genomic reference materials for cystic fibrosis genetic testing.

The number of different laboratories that perform genetic testing for cystic fibrosis is increasing. However, there are a limited number of quality control and other reference materials available, none of which cover all of the alleles included in commercially available reagents or platforms. The alleles in many publicly available cell lines that could serve as reference materials have neither been confirmed nor characterized. The Centers for Disease Control and Prevention-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the genetic testing community as well as Coriell Cell Repositories, have characterized an extended panel of publicly available genomic DNA samples that could serve as reference materials for cystic fibrosis testing. Six cell lines [containing the following mutations: E60X (c.178G>T), 444delA (c.312delA), G178R (c.532G>C), 1812-1G>A (c.1680-1G>A), P574H (c.1721C>A), Y1092X (c.3277C>A), and M1101K (c.3302T>A)] were selected from those existing at Coriell, and seven [containing the following mutations: R75X (c.223C>T), R347H (c.1040G>A), 3876delA (c.3744delA), S549R (c.1646A>C), S549N (c.1647G>A), 3905insT (c.3773_3774insT), and I507V (c.1519A>G)] were created. The alleles in these materials were confirmed by testing in six different volunteer laboratories. These genomic DNA reference materials will be useful for quality assurance, proficiency testing, test development, and research and should help to assure the accuracy of cystic fibrosis genetic testing in the future. The reference materials described in this study are all currently available from Coriell Cell Repositories.

Prevalence and instability of fragile X alleles: implications for offering fragile X prenatal diagnosis.

OBJECTIVE: To document fragile X allele frequencies in a national referral population and evaluate CGG repeat expansion in mother-offspring transmissions. METHODS: Fragile X DNA analysis by Southern blot and polymerase chain reaction was completed for 14,675 women, aged 18 years or older, and 238 mother-offspring pairs between January 1999 and June 2004. Carrier frequencies were compared between groups referred for different clinical indications. Direct comparison of the FMR1 gene CGG repeat size in mother-offspring pairs determined intermediate and premutation allele stability. RESULTS: Intermediate fragile X alleles (45-54 CGG repeats) occurred in 257 (1 in 57). The combined total number of patients with a premutation (55-200 CGG repeats) or full mutation (more than 200 CGG repeats) numbered 208 (1 in 71). One in 3.5 women with a family history of fragile X and 1 in 10 with premature ovarian failure had a FMR1 mutation. This compared with 1 in 86 for those with a family history of mental retardation and 1 in 257 for women with no known risk factors for fragile X. Among 238 mother-offspring pairings, the smallest allele to expand to a full mutation in one generation contained 60 CGG repeats. Although 6.6% (4 of 60) of intermediate repeat alleles did expand, none jumped to a clinically significant full mutation-sized allele. CONCLUSION: Based on these data and other published literature, offering invasive prenatal diagnosis for fragile X syndrome is not indicated for women with intermediate alleles. Invasive prenatal diagnosis is warranted for those women with a fragile X allele containing 55 or more CGG repeats.

Consensus characterization of 16 FMR1 reference materials: a consortium study.

Fragile X syndrome, which is caused by expansion of a (CGG)(n) repeat in the FMR1 gene, occurs in approximately 1:3500 males and causes mental retardation/behavioral problems. Smaller (CGG)(n) repeat expansions in FMR1, premutations, are associated with premature ovarian failure and fragile X-associated tremor/ataxia syndrome. An FMR1-sizing assay is technically challenging because of high GC content of the (CGG)(n) repeat, the size limitations of conventional PCR, and a lack of reference materials available for test development/validation and routine quality control. The Centers for Disease Control and Prevention and the Association for Molecular Pathology, together with the genetic testing community, have addressed the need for characterized fragile X mutation reference materials by developing characterized DNA samples from 16 cell lines with repeat lengths representing important phenotypic classes and diagnostic cutoffs. The alleles in these materials were characterized by consensus analysis in nine clinical laboratories. The information generated from this study is available on the Centers for Disease Control and Prevention and Coriell Cell Repositories websites. DNA purified from these cell lines is available to the genetics community through the Coriell Cell Repositories. The public availability of these reference materials should help support accurate clinical fragile X syndrome testing.

Development of genomic reference materials for Huntington disease genetic testing.

PURPOSE: Diagnostic and predictive testing for Huntington disease requires an accurate measurement of CAG repeats in the HD (IT15) gene. However, precise repeat sizing can be technically challenging, and is complicated by the lack of quality control and reference materials (RM). The aim of this study was to characterize genomic DNA from 14 Huntington cell lines available from the National Institute of General Medical Sciences Human Genetic Cell Repository at the Coriell Cell Repositories for use as reference materials for CAG repeat sizing. METHODS: Fourteen Huntington cell lines were selected for study. The alleles in these materials represent a large range of sizes that include important diagnostic cutoffs and allele combinations. The allele measurement study was conducted by ten volunteer laboratories using a variety of polymerase chain reaction-based in-house developed methods and by DNA sequence analysis. RESULTS: The Huntington alleles in the 14 genomic DNA samples range in size from 15 to 100 CAG repeats. There was good agreement among the ten laboratories, and thus, the 95% confidence interval was small for each measurement. The allele size determined by DNA sequence analysis agreed with the laboratory developed tests. CONCLUSION: These DNA materials, which are available from Coriell Cell Repositories, will facilitate accurate and reliable Huntington genetic testing.

Genetically characterized positive control cell lines derived from residual clinical blood samples.

BACKGROUND: Positive control materials for clinical diagnostic molecular genetic testing are in critically short supply. High-quality DNA that closely resembles DNA isolated from patient specimens can be obtained from Epstein-Barr virus (EBV)-transformed peripheral blood lymphocyte cell lines. Here we report the development of a process to (a) recover residual blood samples with clinically important mutations detected during routine medical care, (b) select samples likely to provide viable lymphocytes for EBV transformation, (c) establish stable cell lines and confirm the reported mutation(s), and (d) validate the cell lines for use as positive controls in clinical molecular genetic testing applications. METHODS: A network of 32 genetic testing laboratories was established to obtain anonymous, residual clinical samples for transformation and to validate resulting cell lines for use as positive controls. Three panel meetings with experts in molecular genetic testing were held to evaluate results and formulate a process that could function in the context of current common practices in molecular diagnostic testing. RESULTS: Thirteen laboratories submitted a total of 113 residual clinical blood samples with mutations for 14 genetic disorders. Forty-one EBV-transformed cell lines were established. Thirty-five individual point and deletion mutations were shown to be stable after 20 population doublings in culture. Thirty-three cell lines were characterized for specific mutations and validated for use as positive controls in clinical diagnostic applications. CONCLUSIONS: A process for producing and validating positive control cell lines from residual clinical blood samples has been developed. Sustainable implementation of the process could help alleviate the current shortage of positive control materials.

Analysis of 3208 cystic fibrosis prenatal diagnoses: impact of carrier screening guidelines on distribution of indications for CFTR mutation and IVS-8 poly(T) analyses.

PURPOSE: To evaluate and quantify indications for CFTR mutation analysis of prenatal specimens, and to determine if a significant portion of tests are performed only for the identification of 5T alleles, we surveyed our laboratory data over a 3-year time period that spanned the issuance of the cystic fibrosis (CF) carrier screening guidelines. METHODS: Referral indications for 3208 prenatal specimens were compared for an 18-month period before (April 2000 to September 2001) and after (October 2001 to April 2003) publication of the ACMG/ACOG statement regarding prenatal and preconception testing for CF. RESULTS: The frequency of cases received for testing when one or both parents were CF mutation carriers did not change significantly after publication of the guidelines. The most frequent indication during the entire 3-year period was fetal ultrasound abnormality, yet in the post-ACMG/ACOG period the percentage decreased significantly due to an increase in the number of prenatal screening cases. Testing indications related to parental 5T status also increased significantly in the post-ACMG/ACOG period and accounted for 2.9% of testing over the 3-year period. A small subset (1.6%) of prenatal specimens were tested for poly(T) even though the parents did not carry 5T allele(s). However, more than 40% of these cases could be attributed to parental R117H mutations. CONCLUSION: These data indicate that although indications for prenatal testing shifted after the issuance of carrier screening guidelines, prenatal testing related to parental 5T alleles comprised < 3% of the total referral indications.

CFTR mutation distribution among U.S. Hispanic and African American individuals: evaluation in cystic fibrosis patient and carrier screening populations.

PURPOSE: We reviewed CFTR mutation distribution among Hispanic and African American individuals referred for CF carrier screening and compared mutation frequencies to those derived from CF patient samples. METHODS: Results from CFTR mutation analyses received from January 2001 through September 2003, were analyzed for four populations: Hispanic individuals with a CF diagnosis (n = 159) or carrier screening indication (n = 15,333) and African American individuals with a CF diagnosis (n = 108) or carrier screening indication (n = 8,973). All samples were tested for the same 87 mutation panel. RESULTS: In the Hispanic population, 42 mutations were identified: 30 in the patient population (77.5% detection rate) and 33 among carrier screening referrals. Five mutations not included in the ACMG/ACOG carrier screening panel (3876delA, W1089X, R1066C, S549N, 1949del84) accounted for 7.55% detection in patients and 5.58% among carriers. Among African American referrals, 33 different mutations were identified: 21 in the patient population (74.4% detection) and 23 in the carrier screening population. Together, A559T and 711+5G>A were observed at a detection rate of 3.71% in CF patients and 6.38% in carriers. The mutation distribution seen in both the carrier screening populations reflected an increased frequency of mutations with variable expression such as D1152H, R117H, and L206W. CONCLUSIONS: A detailed analysis of CFTR mutation distribution in the Hispanic and African American patient and carrier screening populations demonstrates that a diverse group of mutations is most appropriate for diagnostic and carrier screening in these populations. To best serve the increasingly diverse U.S. population, ethnic-specific mutations should be included in mutation panels.

Simultaneous detection of multiple point mutations using allele-specific oligonucleotides.

This unit describes high-throughput mutation analysis using hybridization with pooled allele-specific oligonucleotide (ASO) probes. The approach can be used to screen one gene for many allelic mutations or to screen several loci for several allelic mutations each. Because tetramethyl ammonium chloride (TMAC) is added to the hybridization solution, the melting temperature of each oligonucleotide is independent of G-C content and oligonucleotides of the same length can be hybridized simultaneously. The pooled probes will give a positive hybridization signal from any PCR-amplified DNA sample containing a sequence complementary to any of the ASOs in the pool of oligonucleotide sequences. If many PCR-amplified samples are spotted onto a single membrane, multiple individuals can then be screened simultaneously for many mutant sequences. This multiple ASO hybridization technique is appropriate only for circumstances when hybridization with any one of the pooled probes is expected to be uncommon.

Association between hemochromatosis (HFE) gene mutation carrier status and the risk of colon cancer.

BACKGROUND: Iron is a pro-oxidant that may promote carcinogenesis. Mutations in the hemochromatosis (HFE) gene are associated with increased total body iron stores in some individuals. We assessed the risk of colon cancer among individuals with and without HFE gene mutations. METHODS: We performed a population-based, case-control study in North Carolina. Case patients with colon cancer and control subjects provided information on multiple environmental exposures, including total iron intake and nonsteroidal anti-inflammatory drug (NSAID) use. They also provided a venous blood sample, from which DNA was extracted, amplified, and subjected to diagnostic restriction enzyme mapping to detect two major HFE gene mutations, C282Y and H63D. Data were analyzed with Fisher's exact test and logistic regression. All statistical tests were two-sided. RESULTS: Thirteen hundred and eight subjects participated (475 case patients, 833 control subjects). The allele frequencies of the H63D and C282Y mutations were greater among case patients (0.11 and 0.046, respectively) than among control subjects (0.09 and 0.044, respectively; P =.14 and P =.85, respectively). When we controlled for age, race, sex, red meat consumption, NSAID use, and total iron intake, subjects with any HFE gene mutation were more likely to have colon cancer than subjects with no HFE gene mutations (adjusted odds ratio [OR] = 1.40, 95% confidence interval [CI] = 1.07 to 1.87). The magnitude of the effect was similar for both the H63D (adjusted OR = 1.44, 95% CI = 1.04 to 1.98) and C282Y mutations (adjusted OR = 1.39, 95% CI = 0.88 to 2.19). The risk of colon cancer associated with an HFE gene mutation was similar for those who did and did not have a family history of colon cancer. Among those with HFE mutations, cancer risk increased with increasing age and total iron intake. CONCLUSIONS: HFE gene mutations are associated with an increased risk of colon cancer. Cancer risk is greatest in mutation carriers who are older or consume high quantities of iron.

The I148T CFTR allele occurs on multiple haplotypes: a complex allele is associated with cystic fibrosis.

PURPOSE: To determine whether intragenic changes modulate the cystic fibrosis (CF) phenotype in individuals who are positive for the I148T allele. METHODS: The genes from individuals who carried at least one copy of the I148T allele were analyzed for additional changes that may be acting as genetic modifiers. RESULTS: Seven of eight individuals with a known or suspected diagnosis of CF who carried I148T in combination with a severe CF mutation also carried 3199del6. Eight apparently healthy adult individuals who were compound heterozygous for I148T and a severe CF mutation or homozygous for I148T did not carry the deletion ( = 0.0014). The I148T allele occurs on at least three haplotypes: an IVS-8 9T background, a 7T background, or a 9T + 3199del6 background. The 3199del6 allele was not identified in 386 non-CF chromosomes. CONCLUSIONS: It is concluded that I148T occurs on at least three haplotypes and the complex allele I148T + 9T + 3199del6 is associated with a classic CF phenotype.