Cystic Fibrosis Carrier Testing in an Ethnically Diverse US Population E.M. Rohlfs, Z. Zhou, R.A. Heim, N. Nagan, L.S. Rosenblum, K. Flynn, T. Scholl, V.R. Akmaev, D.A. Sirko-Osadsa, B.A. Allitto, and E.A. Sugarman June 2011 © Copyright 2011 by the American Association for Clinical Chemistry

© Copyright 2009 by the American Association for Clinical Chemistry Introduction  Cystic fibrosis (CF) is a pan-ethnic autosomal recessive disease. –Symptoms include obstructive lung disease, exocrine pancreatic insufficiency, diabetes, liver disease and male infertility.  Age of onset and clinical expression of CF is variable and only partially dependent on CFTR genotype. –Clinical expression is modified by other genes and environmental factors.  >1600 CFTR mutations have been identified.

© Copyright 2009 by the American Association for Clinical Chemistry Introduction  Some mutations occur in multiple populations but at varying frequencies, for example: –p.F508del frequency is 80% in Danes, 30% in Ashkenazi Jews, 18% in Iranians.  Some mutations appear to be limited to specific ethnic groups, for example: –c.3744delA is found in Hispanics and p.Y1092X is found in Iraqi Jews.  A standard panel of 23 mutations common among Caucasians and Ashkenazi Jews was recommended by the ACMG.

© Copyright 2009 by the American Association for Clinical Chemistry Questions  What factors, in addition to genotype, affect the age of onset and clinical expression of CF?  What are some of the challenges to the design of a CF mutation panel for the US population?  The standard panel provides a high detection rate for which populations?

© Copyright 2009 by the American Association for Clinical Chemistry Materials & Methods  Study population –364,890 individuals referred for carrier testing. –Over 1400 different ethnicities, combinations of ethnicities or countries of origin.  Mutation panel of 98 mutations selected based on: –The reported frequency in the literature, –The association with CF disease, –The predicted effect on the CFTR protein.

© Copyright 2009 by the American Association for Clinical Chemistry Materials & Methods  Mutation detection –DNA was extracted from patient specimens, amplified by PCR, and subjected to multiplex allele-specific primer extension. –Products were hybridized to a Luminex xMAP® bead array and fluorescence was detected with the Luminex 100™ instrument. –Some mutations were identified from the ratio of the mutant signal to the wild-type signal. –Other mutations were identified from absolute median fluorescence intensity, with zygosity determined by bi-directional sequencing.

© Copyright 2009 by the American Association for Clinical Chemistry Materials & Methods  Statistical Analysis –Differences between corrected carrier frequencies and carrier frequencies calculated from CF incidence were tested with a 2- population Z-test. –The P value for the Pearson x 2 statistic was used to test the difference between mutation frequencies for the different ethnic groups.

© Copyright 2009 by the American Association for Clinical Chemistry Questions  What types of patient samples can be tested using this method?  What type of array is used for the hybridization step?  Which factors should be considered when selecting mutations for a panel?

© Copyright 2009 by the American Association for Clinical Chemistry Results  Carrier frequency –Among 364,890 individuals with no family history, 1 in 38 carried a CF mutation.  Mutations identified –13% of all mutations identified were not mutations that are included in the standard mutation panel. –p.F508 was the most frequent (57.7% of all mutations). –22 mutations were more common than mutations in the standard mutation panel. Ex: c _ del21kb (CFTRdel2,3) accounted for 0.45% of all mutations

© Copyright 2009 by the American Association for Clinical Chemistry Table 2

© Copyright 2009 by the American Association for Clinical Chemistry Table 3

© Copyright 2009 by the American Association for Clinical Chemistry Questions  Which ethnic groups have the highest carrier frequency?  How can the finding of certain mutations in only one ethnic group be explained?  What value can be compared to the observed carrier frequency to rule out the presence of benign alleles in the panel?

© Copyright 2009 by the American Association for Clinical Chemistry Discussion  The primary ethnic groups in the US share many mutations in common but there is a significant difference in the mutation distribution. –Some mutations appear to be limited to certain ethnic groups. Ex: p.S1255X accounted for >1% of mutations in African Americans.  Ethnic limited mutations may make a substantial contribution to the mutation spectrum in a single group but not to the overall population.

© Copyright 2009 by the American Association for Clinical Chemistry Discussion  All of the major US ethnic groups are accepting CF testing.  The limitations of predicting clinical presentation from CFTR genotype should be discussed during genetic counseling and informed consent process.  These data may be considered when developing mutation panels for diverse populations.

© Copyright 2009 by the American Association for Clinical Chemistry Discussion  Editorial by Iris Schrijver –The increased frequency of certain alleles in different ethnic populations is an important consideration in the design of a screening program or diagnostic test. –CF screening has been widely implemented across the US. –To assess the utility of expanded panels, the application, composition and populations to be studies should be taken into account.

© Copyright 2009 by the American Association for Clinical Chemistry Thank you for participating in this month’s Clinical Chemistry Journal Club. Additional Journal Clubs are available at Follow us