Cystic Fibrosis Carrier Testing in anEthnically 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, andE.A. Sugarman

www.clinchem.org/cgi/content/article/57/6/841

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 x2 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.54-5940_273+10250del21kb (CFTRdel2,3) accounted for

0.45% of all mutations

© Copyright 2009 by the American Association for Clinical Chemistry

Table 2

Table 2. Ethnicity-specific data for individuals w ith no history of CF referred for carrier screening.

Proportion Observed Corrected Calculated of tested carrier carrier from

Ethnic group Individuals, n individuals, % frequency frequency incidencea P

African American 40 071 11.0 1 in 84 1 in 61b; 1 in 63c 1 in 61 0.88; 0.57

Ashkenazi Jewish 15 244 4.2 1 in 29 1 in 27c; 1 in 28d 1 in 24 0.12; 0.052

Asian 19 344 5.3 1 in 242 1 in 176c 1 in 94 0.11

Caucasian 158 658 43.5 1 in 28 1 in 25e 1 in 25 0.63

Hispanic 60 930 16.7 1 in 59 1 in 45b; 1 in 49c 1 in 58 0.11; 0.3

Jewish 1887 0.5 1 in 32

M iddle Eastern 1726 0.5 1 in 91

Native American 491 0.1 1 in 70

South Asian 2365 0.7 1 in 118

Other ethnicity 1663 0.5 1 in 111

> 1 Ethnicity 8514 2.3 1 in 34

Part African American 1238 0.3 1 in 56

Part Caucasian 7510 2.1 1 in 32

Part Hispanic 2380 0.7 1 in 51

Not provided 53 997 14.8 1 in 37

All individuals 364 890 100 1 in 38 l. (11 ). (7 ) (Cystic Fibrosis Foundation data). et al. (15 ). t al. (10 ) (Cystic Fibrosis Foundation data).

a Palomaki et al. (7 ). b Carrier frequency corrected with detection rate reported by Sugarman et a c Carrier frequency corrected with detection rate reported by Palomaki et al. d Carrier frequency corrected with the detection rate reported by Abeliovich e Carrier frequency corrected with the detection rate reported by Palomaki e

© Copyright 2009 by the American Association for Clinical Chemistry

Table 3

Table 3. CFTR mutations detected in individuals referred for carrier screening with no history of CF.

Total individual Non-ACMG/ACOG ACMG/ACOG Total mutant Non-ACMG/ACOG

Ethnic group mutations, n mutations, n mutations, n alleles, n alleles, n (%)

African American 46 28 18 476 108 (22.7)

Ashkenazi Jewish 18 5 13 539 70 (13.0)

Asian 24 15 9 81 30 (37.0)

Caucasian 67 44 23 5763 536 (9.3)

Hispanic 53 34 19 1046 281 (26.9)

Jewish 11 3 8 59 14 (23.7)

M iddle Eastern 9 3 6 19 4 (21.0)

Native American 1 0 1 7 0

Southern Asian 8 4 4 20 5 (25.0)

Other ethnicity 8 3 5 15 3 (20.0)

> 1 Ethnicity 30 12 18 248 33 (13.3)

Part African American 10 4 6 22 4 (18.2)

Part Caucasian 29 11 18 236 30 (12.7)

Part Hispanic 14 4 10 47 8 (17.0)

Not provided 61 39 22 1454 180 (12.4)

All individuals 87 64 23 9727 1264 (13.0)

© 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

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