The Challenges of Applying Massively Parallel Sequencing to Newborn Screening for Cystic Fibrosis.

This Commentary highlights the article by Lefterova et al that describes newborn screening of cystic fibrosis using massively parallel sequencing.

Chronotropic incompetence precedes silent pulmonary embolism.

A patient with recurrent pulmonary emboli collected heart rate data during exercise, which provided important premorbid clues to changes in cardiopulmonary exercise tolerance coincident with accrual of thrombus in the central circulation. On both occasions, chronotropic incompetence (CI) preceded the pulmonary emboli events. When patients with programmed exercise goals notice CI, they should seek professional guidance.

Evaluation of high resolution gel ß(2)-transferrin for detection of cerebrospinal fluid leak.

BACKGROUND: Cerebrospinal fluid (CSF) leaks are potentially life-threatening conditions that can be diagnosed by detection of ß(2)-transferrin using protein electrophoresis. Another less commonly available test is ß-trace protein quantitation using immunoassay. The objectives of this study were to evaluate a new immunofixation-based ß(2)-transferrin test for detection of CSF leaks and to compare it to an existing agarose gel electrophoresis test and ß-trace protein immunoassay. METHODS: For method comparison, 63 consecutive samples from physician-ordered ß(2)-transferrin tests were analyzed using two different electrophoresis methods, agarose gel fractionation followed by acid-violet staining, and high resolution agarose gel electrophoresis followed by ß(2)-transferrin immunofixation. A subset of samples (16/63) were analyzed for ß-trace protein. Results were compared against patient chart data for the presence of a CSF leak. Additional studies were performed to assess the stability, detection limit, and analytical specificity of the ß(2)-transferrin immunofixation test. RESULTS: The ß(2)-transferrin immunofixation test had a sensitivity of 100 % (40/40) and specificity of 71 % (12/17) for detection of CSF leaks. By comparison, the agarose gel test had a sensitivity of 87 % (35/40) and specificity of 94 % (16/17). ß-trace protein had a sensitivity of 100 % (10/10) and specificity of 86 % (5/6). Serum and saliva could be differentiated from CSF by the ß(2)-transferrin immunofixation test based on their migration patterns. However, whole blood samples appeared positive for ß(2)-transferrin at a threshold of ~ 4 g/L hemoglobin. At a cut-off of 3 mg/L, ß-trace protein was increased in 10/10 cases with documented CSF leak and in 1/6 patients without CSF leak. CONCLUSIONS: Both the new immunofixation test for ß(2)-transferrin and the ß-trace protein were effective at detecting CSF leaks. Users of the ß(2)-transferrin immunofixation test should be cautioned against interpreting samples with blood contamination.

Elevated prevalence of 35-44 FMR1 trinucleotide repeats in women with diminished ovarian reserve.

INTRODUCTION: Fragile X premutations are associated with primary ovarian insufficiency when the patient presents with amenorrhea, but the fragile X mental retardation 1 (FMR1) CGG repeat count among cycling women with low ovarian reserve (diminished ovarian reserve [DOR]) is not yet established. PATIENTS AND METHODS: Sixty-two infertile DOR patients were recruited from 4 US private and academic fertility centers. RESULTS: The prevalence of 35-44 FMR1 CGG repeats was 14.5%. Compared with the general female population estimate from the literature, infertile women with DOR were more likely to have 35-44 FMR1 CGG repeats (14.5% and 3.9%, respectively, P = .0003). Similar findings were noted by 5-repeat bandwidth: 35-39 CGG repeats (9.7% DOR vs 3.2% comparison, P = .012) or 40-44 CGG repeats (4.8% DOR vs 0.7% comparison, P = .024). CONCLUSIONS: These data suggest that CGG repeats of 35-44 may be markedly overrepresented in women with DOR, whereas the current FMR1 reference range indicates that there is no clinical phenotype with <45 CGG repeats.

Genetic modifiers of liver disease in cystic fibrosis.

CONTEXT: A subset (approximately 3%-5%) of patients with cystic fibrosis (CF) develops severe liver disease with portal hypertension. OBJECTIVE: To assess whether any of 9 polymorphisms in 5 candidate genes (alpha(1)-antitrypsin or alpha(1)-antiprotease [SERPINA1], angiotensin-converting enzyme [ACE], glutathione S-transferase [GSTP1], mannose-binding lectin 2 [MBL2], and transforming growth factor beta1 [TGFB1]) are associated with severe liver disease in patients with CF. DESIGN, SETTING, AND PARTICIPANTS: Two-stage case-control study enrolling patients with CF and severe liver disease with portal hypertension (CFLD) from 63 CF centers in the United States as well as 32 in Canada and 18 outside of North America, with the University of North Carolina at Chapel Hill as the coordinating site. In the initial study, 124 patients with CFLD (enrolled January 1999-December 2004) and 843 control patients without CFLD were studied by genotyping 9 polymorphisms in 5 genes previously studied as modifiers of liver disease in CF. In the second stage, the SERPINA1 Z allele and TGFB1 codon 10 genotype were tested in an additional 136 patients with CFLD (enrolled January 2005-February 2007) and 1088 with no CFLD. MAIN OUTCOME MEASURES: Differences in distribution of genotypes in patients with CFLD vs patients without CFLD. RESULTS: The initial study showed CFLD to be associated with the SERPINA1 Z allele (odds ratio [OR], 4.72; 95% confidence interval [CI], 2.31-9.61; P = 3.3 x 10(-6)) and with TGFB1 codon 10 CC genotype (OR, 1.53; 95% CI, 1.16-2.03; P = 2.8 x 10(-3)). In the replication study, CFLD was associated with the SERPINA1 Z allele (OR, 3.42; 95% CI, 1.54-7.59; P = 1.4 x 10(-3)) but not with TGFB1 codon 10. A combined analysis of the initial and replication studies by logistic regression showed CFLD to be associated with SERPINA1 Z allele (OR, 5.04; 95% CI, 2.88-8.83; P = 1.5 x 10(-8)). CONCLUSIONS: The SERPINA1 Z allele is a risk factor for liver disease in CF. Patients who carry the Z allele are at greater risk (OR, approximately 5) of developing severe liver disease with portal hypertension.

Detection of immunoglobulin heavy chain gene rearrangements in classic hodgkin lymphoma using commercially available BIOMED-2 primers.

CONTEXT: Classic Hodgkin lymphoma (cHL) is regarded as a clonal B-cell neoplasm. The BIOMED-2 group recently validated a set of immunoglobulin heavy chain (IGH) multiplex primers with high sensitivity in B-cell non-Hodgkin lymphomas. We postulated that after using these primers, a higher proportion of the cHLs would have detectable rearrangements without microdissection. DESIGN: Forty-two patients with cHL were selected. The densities of Reed-Sternberg cells/10 high-power field and CD30+ cells/10 high-power field were classified as low, intermediate, or high. The quantities of background CD20+ B cells were classified as low or high. DNA from formalin-fixed, paraffin-embedded sections was used to perform polymerase chain reactions with the InVivoScribe IGH Gene Clonality Assay for ABI detection. Dominant peaks were considered to be monoclonal if they were >3x the height of the polyclonal background, and borderline monoclonal if between 2 and 3x. RESULT: Overall, 10/42 (24%) of the cHL samples were monoclonal, and 7/42 (17%) were borderline monoclonal. Higher densities of CD30+ cells and lower background B cells were statistically correlated with clonality. CONCLUSIONS: The BIOMED-2 primers demonstrate IGH gene clonality in 24% to 40% of cHLs without microdissection. In a subset of the cHL, the IGH gene rearrangement analysis might be useful for diagnosis, but can lead to confusion between cHLs and non-Hodgkin lymphomas if used as a discriminative criterion.

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.

Molecular diagnostics: a historical perspective.

The rapid growth in molecular diagnostic testing, which has averaged between 10% and 20% per year for the past 5 years, is largely attributable to both breakthroughs in our basic understanding (i.e., the Human Genome Project) and in applied technology. In the past decade, molecular applications have moved from labor-intensive and manual to rapid and automated due to improvements in sample extraction, target amplification, and sensitive and specific detection schema. This review describes some of the more significant technological milestones of the past 10 years and, when tied to basic and applied research, how these have led to important clinical applications. The next decade promises even more exciting technologies and applications for the field of molecular laboratory medicine.

Acceptance of fragile X premutation genetic screening in women with ovarian dysfunction.

OBJECTIVE: The purpose of this study was to assess patient perception of fragile X premutation genetic testing (FRAX). STUDY DESIGN: This was a cross-sectional survey of women with elevated follicle stimulating hormone levels with (premature ovarian failure or early menopause [POF/EM], n = 20) or without (diminished ovarian reserve [DOR], n = 20) amenorrhea. Seventy-five percent participated. RESULTS: Seventy-five percent of the DOR group and 43% of the POF/EM group desired FRAX testing. Eighty-three percent wanted to assist the scientific knowledge of FRAX, even if they did not want to know their own results. POF/EM women were more concerned than DOR women about paying out-of-pocket (P = .001) and maintaining confidentiality insurance-wise (P = .07). Primary motivations for women who wanted testing were the desire to know if they have FRAX, and wanting to determine if FRAX is the cause of their ovarian dysfunction. The primary decision factor for those declining testing was unwillingness to pay out-of-pocket (75%). CONCLUSION: Women with ovarian dysfunction are interested in FRAX testing. Cost, confidentiality, and the implications for relatives are their key concerns.

Developing a sustainable process to provide quality control materials for genetic testing.

PURPOSE: To provide a summary of the outcomes of two working conferences organized by the Centers for Disease Control and Prevention (CDC), to develop recommendations for practical, sustainable mechanisms to make quality control (QC) materials available to the genetic testing community. METHODS: Participants were selected to include experts in genetic testing and molecular diagnostics from professional organizations, government agencies, industry, laboratories, academic institutions, cell repositories, and proficiency testing (PT)/external Quality Assessment (EQA) programs. Current efforts to develop QC materials for genetic tests were reviewed; key issues and areas of need were identified; and workgroups were formed to address each area of need and to formulate recommendations and next steps. RESULTS: Recommendations were developed toward establishing a sustainable process to improve the availability of appropriate QC materials for genetic testing, with an emphasis on molecular genetic testing as an initial step. CONCLUSIONS: Improving the availability of appropriate QC materials is of critical importance for assuring the quality of genetic testing, enhancing performance evaluation and PT/EQA programs, and facilitating new test development. To meet the needs of the rapidly expanding capacity of genetic testing in clinical and public health settings, a comprehensive, coordinated program should be developed. A Genetic Testing Quality Control Materials Program has therefore been established by CDC in March 2005 to serve these needs.

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.

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.

A novel mutation in exon 5 of the ALAS2 gene results in X-linked sideroblastic anemia.

BACKGROUND: Mutations in the erythroid-specific 5-aminolevulinate-synthase gene (ALAS2) have been identified in many cases of X-linked sideroblastic anemia (XLSA). METHODS: A polymerase chain reaction-mediated restriction fragment length polymorphism (RFLP) assay was used. RESULTS: A G527T point mutation was identified. This resulted in a substitution of tyrosine for asparagine at residue 159 (D159Y). This mutation was also identified in the mother of the two probands. Mutations in all three individuals were confirmed by DNA sequencing analysis. CONCLUSIONS: We identified a missense mutation in exon 5 of the ALAS2 gene in two brothers of a consanguineous marriage, who were clinically pyridoxine-responsive.