Rapid Screening for Five Major Cystic
Fibrosis Mutations by Melting Peak Analysis
Using Fluorogenic Hybridization Probes
SIEGFRIED BURGGRAF*, NAEEM MALIK,
EDITH SCHUHMACHER, BERNHARD OLGEMOLLER
Introduction
Cystic fibrosis (CF), also known as mucoviscidosis, is a disorder of the exocrine
glands [1]. Symptoms of CF are secretion of abnormally viscous mucus and elevated sweat electrolytes. The accumulation of thick mucus in the epithelium of the
respiratory system and digestive tract causes progressive respiratory and gastrointestinal problems, including liver disease and diabetes mellitus, resulting in
high morbidity and reduced life expectancy of CF patients. Variations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic adenosine
monophosphate regulated chloride channel, are responsible for CF [2]. CF is one
of the most common autosomal single gene disorders in Caucasian populations.
The CFTR gene has a size of 250,000 bp separated into 27 exons, most of which are
not larger than 200 bp [3]. So far, almost 1,000 CF-causing mutations, more or less
scattered over the whole CFTR gene, have been registered by the Cystic Fibrosis
Genetic Analysis Consortium (http://www.genet.sickkids.on.ca/cftrJ). Only persons with homozygote or compound heterozygote CFTR gene mutations suffer
from CF. The prevalence of CF in North America and Europe is between 112,400
and 111,600 and 4%-5% of the population are healthy carriers of one CF mutation.
The size of the gene and the large number of mutations make genetic CF
screening complicated. However, the majority of the genetic variations are
extremely rare. The most frequent genetic variation (e.g., in 77% of the CF cases
in Bavaria, Germany [4, 5]) is a 3-bp deletion encoding phenylalanine at residue
508 (.1F508). The frequency of specific mutations varies between different ethnic
groups. Usually, however, only four to five further mutant alleles occur at a relative frequency of around 1 %, whereas for all other mutations the frequency is significantly lower. A screening for the five or six most frequent mutations thus
results in a sensitivity of about 95%. This means that in CF patients, at least one
of the mutated alleles is detected by the test. Adding more mutations, e.g., the 25
next frequent mutations worldwide, to the screening program only marginally
improves this sensitivity.
Since there is strong evidence that CF patients benefit from early detection and
treatment [6), a neonatal screening for CF is already performed in several countries.
* Siegfried Burggraf (~) (e-mail: burggraf@labor-bo.de)
Labor Becker, Olgemiiller und Kollegen, Fiihrichstrasse 70,81671 Munich, Germany
n
Fibrosis Mutations by Melting Peak Analysis
Using Fluorogenic Hybridization Probes
SIEGFRIED BURGGRAF*, NAEEM MALIK,
EDITH SCHUHMACHER, BERNHARD OLGEMOLLER
Introduction
Cystic fibrosis (CF), also known as mucoviscidosis, is a disorder of the exocrine
glands [1]. Symptoms of CF are secretion of abnormally viscous mucus and elevated sweat electrolytes. The accumulation of thick mucus in the epithelium of the
respiratory system and digestive tract causes progressive respiratory and gastrointestinal problems, including liver disease and diabetes mellitus, resulting in
high morbidity and reduced life expectancy of CF patients. Variations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic adenosine
monophosphate regulated chloride channel, are responsible for CF [2]. CF is one
of the most common autosomal single gene disorders in Caucasian populations.
The CFTR gene has a size of 250,000 bp separated into 27 exons, most of which are
not larger than 200 bp [3]. So far, almost 1,000 CF-causing mutations, more or less
scattered over the whole CFTR gene, have been registered by the Cystic Fibrosis
Genetic Analysis Consortium (http://www.genet.sickkids.on.ca/cftrJ). Only persons with homozygote or compound heterozygote CFTR gene mutations suffer
from CF. The prevalence of CF in North America and Europe is between 112,400
and 111,600 and 4%-5% of the population are healthy carriers of one CF mutation.
The size of the gene and the large number of mutations make genetic CF
screening complicated. However, the majority of the genetic variations are
extremely rare. The most frequent genetic variation (e.g., in 77% of the CF cases
in Bavaria, Germany [4, 5]) is a 3-bp deletion encoding phenylalanine at residue
508 (.1F508). The frequency of specific mutations varies between different ethnic
groups. Usually, however, only four to five further mutant alleles occur at a relative frequency of around 1 %, whereas for all other mutations the frequency is significantly lower. A screening for the five or six most frequent mutations thus
results in a sensitivity of about 95%. This means that in CF patients, at least one
of the mutated alleles is detected by the test. Adding more mutations, e.g., the 25
next frequent mutations worldwide, to the screening program only marginally
improves this sensitivity.
Since there is strong evidence that CF patients benefit from early detection and
treatment [6), a neonatal screening for CF is already performed in several countries.
* Siegfried Burggraf (~) (e-mail: burggraf@labor-bo.de)
Labor Becker, Olgemiiller und Kollegen, Fiihrichstrasse 70,81671 Munich, Germany
n