Survey of CF mutations in the clinical laboratory Part 1

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Survey of CF mutations in the clinical laboratory

Klaus Roland Huber1 , Borka Mirkovic1 , Rhea Nersesian2 , Myers2 ,

Randall Saiki2  and Kurt Bauer1

1Ludwig Boltzmann Institute for moleculargenetic laboratory diagnostics,

Donauspital, Vienna, Austria

2Roche Molecular Systems, Inc., Alameda, CA, USA

BMC Clinical Pathology 2002 2:4

This article is available from:

http://www.biomedcentral.com/1472-6890/2/4

Received   2 Sep 2002

Accepted   19 Nov 2002

Published   19 Nov 2002

© 2002 Huber et al; licensee BioMed Central Ltd. This is an Open Access

article: verbatim copying and redistribution of this article are

permitted in all media for any purpose, provided this notice is preserved

along with the article's original URL.

Background Since it is impossible to sequence the complete CFTR gene

routinely, clinical laboratories must rely on test systems that screen

for a panel of the most frequent mutations causing disease in a high

percentage of patients. Thus, in a cohort of 257 persons that were

referred to our laboratory for analysis of CF gene mutations, reverse

line probe assays for the most common CF mutations were performed. These

techniques were evaluated as routine first-line analyses of the CFTR gene

status.MethodsDNA from whole blood specimens was extracted and subjected

to PCR amplification of 9 exons and 6 introns of the CFTR gene. The

resulting amplicons were hybridised to probes for CF mutations and

polymorphisms, immobilised on membranes supplied by Roche Molecular

Systems, Inc. and Innogenetics, Inc.. Denaturing gradient gel

electrophoresis and sequencing of suspicious fragments indicating

mutations were done with CF exon and intron specific primers.ResultsOf

the 257 persons tested over the last three years (referrals based on 1)

clinical symptoms typical for/indicative of CF, 2) indication for in

vitro fertilisation, and 3) gene status determination because of

anticipated parenthood and partners or relatives affected by CF), the

reverse line blots detected heterozygote or homozygote mutations in the

CFTR gene in 68 persons (26%). Eighty-three percent of those affected

were heterozygous (47 persons) or homozygous (10 persons) for the ?F508

allele. The only other CF-alleles that we found with these tests were the

G542X allele (3 persons), the G551D allele (3 persons), the 3849+10kb C-T

allele (2 persons) the R117H allele (2 persons) and the 621+1G-T allele

(1 person).Of the fifteen IVS8-5T-polymorphisms detected in intron 8,

seven (47%) were found in males referred to us from IVF clinics. These

seven 5T-alleles were all coupled with a heterozygous ?F508 allele, they

make up 35% of the males with fertility problems (20 men) referred to

us.ConclusionsIn summary, the frequency of CF chromosomes in the cohort

examined with these tests was 26%, with the ?F508 allele affecting 83% of

the CF chromosomes. It is a substantial improvement for routine CF

diagnostics to have available a test system for 30 mutations plus the

polypyrimidine length variants in intron 8. Our results show that this

test system allows a routine first-line analyses of the CFTR gene status.

Outline   Background

Tables

Table 1 Exons and introns that are amplified with the line probe assay,

and the mutations they encompass

Table 2 Genotypes of patients with mutations, final results

Diagnosis of hereditary human disease to date requires arduous techniques

and intense manual handling. When a disorder is caused by mutations in a

large gene and at multiple loci, the demands pose considerable challenges

to the testing laboratory. In some cases, the underlying mutations can

not be found at all, because it is impossible to sequence complete genes

routinely. Consequently, clinical laboratories must rely on test systems

that screen for a panel of the most frequent mutations causing disease in

a high percentage of patients, in order to minimise the need for further

elaborate protocols.Accordingly, for the diagnosis of mutations that lead

to cystic fibrosis, a panel of methods is needed, i.e. PCR-based

techniques, reverse blots, DGGE, sequencing a.s.o [1]. The demand for

improved tools for CF diagnostics is substantial, because cystic fibrosis

is among the most common autosomal recessive diseases in the Caucasian

population. It affects 1:2000–1:3000 births each year and results in

pulmonary failure and death in most cases at around the third decade of

life [2]. The molecular defect in CF was elucidated following the cloning

of the cystic fibrosis transmembrane conductance regulator (CFTR) gene

[3]. So far, over 800 different mutations are recorded that lie in

introns and exons. Recently, association of CFTR mutations with other

diseases – such as congenital bilateral absence of vasa deferentia

(CBAVD) [4,5] disseminated bronchiectasis [6], or allergic

bronchopulmonary aspergillosis [7] – have emerged. This is not surprising

considering the cellular heterogeneity of CFTR expression and the

multiple mutations in this gene [8-10]. In the case of CBAVD a strong

association between the repeat number of a polypyrimidine tract in intron

8 of the CF gene with the absence of the vas deferens was found

[11,12].Our laboratory performs CF diagnostics for the north-eastern part

of Austria. In this area – including and surrounding Vienna – reside

approximately three million individuals. We have been using reverse line

blot assays for CF mutations that encompass up to 29 mutations, 3

polymorphisms, and 3 alleles of the intron 8 polypyrimidine tract for a

rapid first screening of patient samples (table 1) complemented by DGGE

and sequencing as indicated (table 2). The test panel fared well in the

ECCACF (European Community Concerted Action for Cystic Fibrosis) Quality

Control Trial of 1997, 1998, 1999, 2000, and 2001.The aim of this study

was the exploration of the screening procedure for CF mutations employed

in our laboratory as a first-line diagnostic instrument for Cystic

Fibrosis. Here, we report our experiences with various test systems and

the resultant CF mutations in persons from north-eastern Austria,

referred to our laboratory for CF diagnosis since 1999.

Outline   Methods

PatientsIn all, 135 men and 122 women were analysed. The male group

consisted of 64 children (< 1–18 yrs) and 71 adults (mean age of 32 yrs,

range 19–69). The group of females included 56 children (< 1–18 yrs) and

66 adults (mean age of 31 yrs, range 19–65). All probands (or their

parents, respectively) had given informed consent for genetic analysis as

required by the Austrian law. Patients and probands referrals to our

laboratory for CF mutation analyses were based on1) clinical symptoms

typical for/indicative of CF,2) indication for in vitro fertilisation

(IVF – group 2a: men, 2b: their spouses), and3) gene status determination

because of anticipated parenthood and partners or relatives affected by

CF.Of all 257 persons analysed for CF mutations, 105 were examined for 16

mutations, 152 for 29 mutations, 31 individuals were tested further by

DGGE of all exons and sequencing of suspicious fragments.DNA studiesDNA

was extracted routinely from blood using the Kristalâ„¢ DNA extraction kit

(Cambridge Molecular Technologies, Cambridge, England). The INNO-LiPa

CFTR17+Tn (INNOGENETICS, Ghent, Belgium) assay was performed according to

the recommendation of the supplier.For the evaluation of the Amplicor®

Cystic Fibrosis kit (Roche Molecular Systems, Alameda, CA, USA), the

extraction protocol included in the kit was tested, as well. For this

test, 100 µL blood was incubated 5 to 10 min with 1 ml " Specimen Wash

Solution " as supplied in the test kit in Eppendorf vials to lyse red

blood cells. The solution was centrifuged for 1 min at maximum speed in a

microfuge and the supernatant was discarded. The leucocyte pellet was

treated once more with " Specimen Wash Solution " as above. The final

pellet was taken up in " Extraction Reagent " and the subsequent steps were

performed analogous to Kristal kit-extracted DNA: Cell pellets or DNA

(minimum of 400 ng in a volume of 50 µL or less) were incubated in 200 µL

Extraction Reagent for 30 min at 100°C. Twenty-five µL of this solution

were PCR-amplified with 25 µL of 16.5 mmol/L Magnesium Chloride solution

and 50 µL PCR " Mastermix " according to the manufacturers specification.

The resulting amplicons were denatured immediately with " Denaturing

Solution " to inhibit the action of uracyl-N-glycosylase (Amperase)

present in the Mastermix. The panels with immobilised probes for 16 CFTR

mutations and some polymorphisms were prehybridised for 10 min with

" Hybridisation Buffer " (3× SSPE + 0.5% SDS). Then, 100 µL denatured

amplicons were hybridised to the probes for 20 min at 50°C in

hybridisation buffer. After a stringent wash for 12 min at 50°C with

" Wash Buffer " (2× SSPE + 0.3% SDS) the panels were incubated with

Streptavidin-Horseradish-Peroxidase-Conjugate. After further wash steps,

addition of substrate (hydrogen peroxide + tetramethylbenzidine) produces

a blue precipitate at the regions of hybridisation. Individual bands can

be identified by superimposing a transparent foil with the identification

codes imprinted.DGGE analyses and sequencingFor the individuals specified

in table 2), all exons were analysed by DGGE according to Audrezet et al.

[20]. Suspicious fragments indicating mutations were sequenced on a Licor

4000L sequencing machine

Becki

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