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      Nationwide genetic analysis for molecularly unresolved cystic fibrosis patients in a multiethnic society: implications for preconception carrier screening

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          Abstract

          Background

          Preconception carrier screening for cystic fibrosis ( CF) is usually performed using ethnically targeted panels of selected mutations. This has been recently challenged by the use of expanded, ethnically indifferent, pan‐population panels. Israel is characterized by genetically heterogeneous populations carrying a wide range of CFTR mutations. To assess the potential of expanding the current Israeli preconception screening program, we sought the subset of molecularly unresolved CF patients listed in the Israeli CF data registry comprising ~650 patients.

          Methods

          An Israeli nationwide genotyping of 152 CF cases, representing 176 patients lacking molecular diagnosis, was conducted. Molecular analysis included Sanger sequencing for all exons and splice sites, multiplex ligation probe amplification (MLPA), and next‐generation sequencing of the poly‐T/ TG tracts.

          Results

          We identified 54 different mutations, of which only 16 overlapped the 22 mutations included in the Israeli preconception screening program. A total of 29/54 (53.7%) mutations were already listed as CF causing by the CFTR2 database, and only 4/54 (7.4%) were novel. Molecular diagnosis was reached in 78/152 (51.3%) cases. Prenatal diagnosis of 24/78 (30.8%) cases could have been achieved by including all CFTR2‐causing mutations in the Israeli panel.

          Conclusions

          Our data reveal an overwhelming hidden abundance of CFTR gene mutations suggesting that expanded preconception carrier screening might achieve higher preconception detection rates.

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          Most cited references48

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          Carrier testing for severe childhood recessive diseases by next-generation sequencing.

          Of 7028 disorders with suspected Mendelian inheritance, 1139 are recessive and have an established molecular basis. Although individually uncommon, Mendelian diseases collectively account for ~20% of infant mortality and ~10% of pediatric hospitalizations. Preconception screening, together with genetic counseling of carriers, has resulted in remarkable declines in the incidence of several severe recessive diseases including Tay-Sachs disease and cystic fibrosis. However, extension of preconception screening to most severe disease genes has hitherto been impractical. Here, we report a preconception carrier screen for 448 severe recessive childhood diseases. Rather than costly, complete sequencing of the human genome, 7717 regions from 437 target genes were enriched by hybrid capture or microdroplet polymerase chain reaction, sequenced by next-generation sequencing (NGS) to a depth of up to 2.7 gigabases, and assessed with stringent bioinformatic filters. At a resultant 160x average target coverage, 93% of nucleotides had at least 20x coverage, and mutation detection/genotyping had ~95% sensitivity and ~100% specificity for substitution, insertion/deletion, splicing, and gross deletion mutations and single-nucleotide polymorphisms. In 104 unrelated DNA samples, the average genomic carrier burden for severe pediatric recessive mutations was 2.8 and ranged from 0 to 7. The distribution of mutations among sequenced samples appeared random. Twenty-seven percent of mutations cited in the literature were found to be common polymorphisms or misannotated, underscoring the need for better mutation databases as part of a comprehensive carrier testing strategy. Given the magnitude of carrier burden and the lower cost of testing compared to treating these conditions, carrier screening by NGS made available to the general population may be an economical way to reduce the incidence of and ameliorate suffering associated with severe recessive childhood disorders.
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            Cystic fibrosis population carrier screening: 2004 revision of American College of Medical Genetics mutation panel

            In April 2001, the American College of Medical Genetics (ACMG) Cystic Fibrosis (CF) Carrier Screening Working Group recommended a panel of mutations and variants that should be tested to determine carrier status within the CFTR gene as a part of population screening programs.1,2 This was initially done in response to the recommendations of an NIH CF Consensus Conference that CF carrier screening be considered by all couples for use before conception or prenatally.3 At that time, the Working Group recognized limitations in our understanding of the population frequencies of several CF alleles and proposed to review mutation distribution data after the first two years of the program. In 2002, as part of an ongoing effort to ensure that the cystic fibrosis carrier screening programs are current with respect to the scientific literature and other available data and practices, we initiated a second review of data on the distribution of mutations in different ethnic groups and we began to assess whether providers were experiencing challenges in delivering this service.4 The current CF Foundation patient mutation database includes nearly double the number of CF patient chromosomes available for analysis in 2000. This report summarizes the major recommendations of our Working Group with the supporting justification for these decisions. A number of articles in this issue of Genetics in Medicine provide some of the data on which our decisions were made, whereas others provide new information related to this topic. QUESTIONS ADDRESSED The questions addressed in this reevaluation of data were as follows: Has the observed frequency of any CF mutation changed significantly since 1999? Should mutations not meeting the prior standard of > 0.1% frequency in CF patients be removed? Should mutations that now have a frequency of 0.1% or greater but that were 400,000 individuals screened was provided by Kaiser Permanente of Northern California, Quest Diagnostics, Laboratory Corporation of America (LabCorp), and Genzyme Genetics (data not shown). General population data are mostly derived from testing that uses the ACMG 25 mutation panel. Hence, general population data for mutations not included in that panel was limited to information provided by Genzyme Genetics and Kaiser Permanente. Organization of data Data were stratified by self-reported race/ethnicity when available. CF patients self-identified as either non-Hispanic Caucasian, African American, Hispanic, Asian, or other. There were 37,263 non-Hispanic Caucasian CF chromosomes, 1,350 from African Americans, 2,718 from Hispanics, 125 from Asians, and 108 from others (American Indians and Aleutians). An additional 1,173 CF chromosomes were from individuals of unknown ethnicity. A subset of patients from the general population study self-identified as either non-Hispanic Caucasian, African American, Hispanic, Asian, or American Indian/Aleutian. Mutation selection standards As in the initial recommendations of a CF mutation screening panel, a standard was set that a mutation should be present in at least 0.1% of CF patient chromosomes. Further, the mutations chosen should be associated with classical CF rather than with milder phenotypes because the decision-making process largely impacts reproductive decisions. Revising the CF carrier screening panel Table 1 lists the mutations found in CF patient chromosomes in descending order of their occurrence in a pan-ethnic population. Mutation frequencies are listed by ethnic group to provide laboratories testing local populations with data to determine an appropriate mutation panel for their test population. Has the observed frequency of any CF mutation changed significantly since 1999? 1078delT was found to occur in 0.03% of CF cases in the current analysis. Technical issues of removing a mutation from a panel, from the perspective of assay platform development, are thought to be minimal. Although there may be other issues associated with changing a “standard,” it was felt that changes should be implemented on the basis of the substantially enlarged data set available to the Working Group. It was decided that any mutation that has prevalence <0.1% should be removed from the screening panel, but that henceforth, decisions would be based on the benefits and costs of incremental gain in the performance characteristics of the screening test as defined here. Thus, the ACMG recommends that 1078delT be removed from the panel. Six mutations not included in the original panel occurred at frequencies ranging from 0.1% to 0.17% in CF patients in the 2003 data (Table 2). Together the six could account for approximately 0.77% of CF alleles. Several issues arise when considering adding mutations to a carrier screening mutation panel. We chose to give minimal consideration to the adaptability of current testing platforms. Rather, we focused on improved clinical utility and have previously addressed issues of analytical quality.6 We also weighed the incremental gain that would be achieved by adding these six mutations against the potential increase in cost and errors associated with the changes.7 The Working Group recommends no additions to the general population screening panel be made at this time. Additions may be considered in the future as more data become available for different ethnic groups or as new technologies emerge. The Working Group recognizes that local demographics may suggest the need to add mutations specific to some groups or to compress the panel to an ethnic-specific subset as was stated in the original recommendations. Table 2 shows the rates of occurrence of an additional 14 of the 53 mutations for which data were available. These 14 mutations occurred in 0.01% to 0.09% of CF patient chromosomes and may be considered when supplementing our recommended panel to improve its sensitivity in other ethnic groups. However, their relationship to disease severity was not assessed in our analysis. Is the prevalence of CF mutations in the general population the same as that predicted from their frequency in CF patients? The mutation/variant I148T occurs at rates 50 to 100 times higher than in the general population being tested for carrier status than in patients.8,9 It was shown that CFTR genes bearing I148T in CF patients have a second mutation termed 3199del6. The vast majority of individuals in the general population with I148T do not have the 3199del6 mutation. Several lines of evidence indicate that 3199del6 is the disease causing mutation. One, CF patients have been described who lack I148T but have 3199del6 in association with another CF causing mutation.10 Two, unaffected individuals have been described who have a CF mutation associated with severe CF and I148T but lacking 3199del6.7–11 Because the frequency of I148T alone is 0.05% and I148T with 3199del6 in this analysis is considerably lower than 0.1% and because I148T does not cause classical CF by itself, we recommend removing I148T from the CF carrier screening panel. The rarity of 3199del6 does not support its addition to the panel as a disease associated mutation and we recommend against it being added as a reflex test for carrier testing. Is there evidence of consistent and recurring challenges with interpretation of some of the mutations in the CF panel? R117Hwasconsideredproblematicininterpretationduetothe complexity of its association with the 5T/7T/9T variant and others have suggested that it may not be appropriate for carrier screening.12 Based on assessment of this situation, the Working Group decided that interpretive problems would have been avoided if 5T had been tested only as a reflex to a finding of R117H, as originally recommended. Furthermore, individuals with R117H and 5T are at risk of having offspring with CF if their partner is also a CF carrier and should be counseled accordingly. Phasing of these sequence changes may be uncertain and these patients may benefit from genetic counseling to discuss the risk and prenatal testing options. Because the frequency of R117H-5T is appreciable, the Working Group recommends retaining R117H, whereas emphasizing the need to perform a screening test for 5T only as a reflex when R117H is present. DISCUSSION In addition to the issues already discussed, several related issues have been raised by others. Warner et al.12 suggest that it is inappropriate to screen for mutations such as R117H for which a definitive prediction of clinical outcome can not be provided. With appropriate informed consent, the limitations inherent in predicting specific phenotypes resulting from any mutations in a CF carrier screening panel should be discussed. Although our recommendations are pertinent to classical CF risk rather than the milder phenotypes discussed, there are some mutations that may be associated with mild or severe forms of CF depending on the mutations with which they are paired, thereby complicating the process of selecting mutation panels. A goal of informed consent for CF carrier testing is to make couples aware that there is a range of clinical outcomes that cannot be predicted, and that that, in and of itself, constitutes informed consent. An additional issue that may be amenable to the informed consent process is the reporting of results. It has been argued that a laboratory is obligated to report any and all information that is gleaned from a test system, however, there is no regulatory requirement and practice varies. Because the CF mutation testing platforms included the reflex tests this led to the reporting of the 5T allele in the absence of R117H by some laboratories. It is not an uncommon practice for clinical chemistry testing platforms to bundle many analytes into a single test but it poses a dilemma for laboratories. Laboratories using existing reagents and tests may be uncomfortable not reporting the 5T/7T/9T alleles and sequences that are no longer considered appropriate for CF carrier screening. The informed consent process should emphasize that CF carrier screening is not designed to detect all of the mutations that cause classical CF or the milder phenotypes. As a result of this process, specification of which results would be reported may help laboratories decide to only report the test results that comprise the recommended panel. However, this may be difficult to implement. CF carrier screening is among the first general population genetic screening tests. Our experience with CF carrier screening offers a potential prototype for the development of other genetic screening programs. Recent experience with I148T serves to demonstrate the importance of evaluating distribution among both affected and carrier screening populations to discern discrepancies before inclusion in a screening panel.
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              ACOG Committee Opinion No. 486: Update on carrier screening for cystic fibrosis.

              (2011)
              In 2001, the American College of Obstetricians and Gynecologists and the American College of Medical Genetics introduced guidelines for prenatal and preconception carrier screening for cystic fibrosis. The American College of Obstetricians and Gynecologists' Committee on Genetics has updated current guidelines for cystic fibrosis screening practices among obstetrician-gynecologists.
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                Author and article information

                Contributors
                cmddoron@clalit.org.il
                Journal
                Mol Genet Genomic Med
                Mol Genet Genomic Med
                10.1002/(ISSN)2324-9269
                MGG3
                Molecular Genetics & Genomic Medicine
                John Wiley and Sons Inc. (Hoboken )
                2324-9269
                19 February 2017
                May 2017
                : 5
                : 3 ( doiID: 10.1002/mgg3.2017.5.issue-3 )
                : 223-236
                Affiliations
                [ 1 ] Clalit National Personalized Medicine Program Department of Community Medicine and EpidemiologyCarmel Medical Center HaifaIsrael
                [ 2 ] Bruce Rappaport Faculty of MedicineTechnion‐Israel Institute of Technology HaifaIsrael
                [ 3 ] Gene by GeneGenomic Research Center Houston Texas
                [ 4 ]The Cystic Fibrosis Foundation of Israel Ramat GanIsrael
                [ 5 ] Pulmonology Institute and CF CenterCarmel Medical Center HaifaIsrael
                [ 6 ] Pediatric Pulmonary Institute and CF CenterRappaport Children's Hospital Rambam Health Care Campus HaifaIsrael
                [ 7 ] Kathy and Lee Graub Cystic Fibrosis CenterSchneider Children's Medical Center of Israel Petach TikvaIsrael
                [ 8 ] Sackler Faculty of MedicineTel Aviv University Ramat AvivIsrael
                [ 9 ] Cystic Fibrosis CenterHadassah‐Hebrew University Medical Center JerusalemIsrael
                [ 10 ] Cystic Fibrosis CenterSheba Medical Center Ramat GanIsrael
                [ 11 ] Cystic Fibrosis CenterSoroka Medical Center BeershevaIsrael
                [ 12 ] Cystic Fibrosis CenterShaare Zedek Medical Center Hebrew University Medical Center JerusalemIsrael
                [ 13 ] Medical GeneticsBarzilai Medical Center AshkelonIsrael
                Author notes
                [*] [* ] Correspondence

                Doron M. Behar, Clalit National Cancer Control and Personalized Medicine Program, Carmel Medical Center, Haifa 3436212, Israel. Tel: 972‐4‐8250474; Fax: 972‐4‐8344358; E‐mail: cmddoron@ 123456clalit.org.il

                Author information
                http://orcid.org/0000-0002-2623-4599
                Article
                MGG3278
                10.1002/mgg3.278
                5441412
                28546993
                4e46e643-7eef-409a-bcae-cb4d4b85d039
                © 2017 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.

                This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 10 November 2016
                : 07 January 2017
                : 13 January 2017
                Page count
                Figures: 1, Tables: 4, Pages: 14, Words: 10450
                Funding
                Funded by: Medison Pharma Corporation
                Categories
                Original Article
                Original Articles
                Custom metadata
                2.0
                mgg3278
                May 2017
                Converter:WILEY_ML3GV2_TO_NLMPMC version:5.0.9 mode:remove_FC converted:23.05.2017

                carrier screening,cystic fibrosis,detection rate,preconception

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