1 Program Resources/DynCorp, Frederick Cancer Research
and Development Center, Frederick, MD 21702-1201, USA.
2 Laboratory of Viral Carcinogenesis, National Cancer Institute,
Frederick Cancer Research and Development Center, Frederick, MD
3 Hanneman University, Philadelphia, USA.
4 Hospital for Obstetrics and Gynecology, Leningrad, USSR.
5 Academy of Medical Sciences, Moscow, USSR.
6 University of Michigan, Ann Arbor, USA.
7 University of Naples, Italy.
8 University of Utah, Salt Lake City, USA.
9 Boston University, Boston, Mass., USA
Cystic fibrosis (CF) is an autosomal recessive disorder, and di
Sant' Agnese et al.  found that the sweat of CF patients contains
an excess of sodium and chloride ions. Defects in the regulation
of chloride ion transport have been documented in CF epithelial
cells [5, 10, 14, 16]. The chloride channel normally responds to
ßadrenergic agents, but CF cells are defective in this response
[6,9, 14]. It has been proposed that the CF defect involves a pathway
whereby cAMP regulates ion transport. The symptoms of CF patients
are heterogeneous between and within families . Although most
individuals are diagnosed by the time they reach the age of ten,
a few remain undiagnosed until adulthood [2, 15]. Approximately
15% of CF patients do not require supplemental pancreatic enzymes
and are designated as pancreatic sufficient (PS) . PS is typically
concordant within families, suggesting that PS patients may have
less severe mutations in the CF gene. However, the heterogeneity
within families suggests that additional genetic and environmental
factors contribute to the severity of the disease. The molecular
cloning of the CF gene has provided additional research strategies
to further understand the disease, and the regulation of ion transport
in secretory cells. The gene encodes a 170 kDa polypeptide that
is a member of a superfamily of membrane-bound active transport
molecules [4, 11, 12]. A threenucleotide deletion in a putative
A TP binding domain has been found in 70 % of CF chromosomes; this
alteration removes a phenylalanine codon at position 508 (delta
F 508). To further understand the relationship between mutations
in the gene and the phenotype of patients, we have examined a group
of patients who do not contain the common mutation on both chromosomes.
Methods and Results
To identify mutations in the CF gene, specific regions were amplified
by the polymerase chain reaction (PCR) and assayed for single-stranded
conformation polymorphisms (SSCPs). This newly described method
allows the rapid screening of samples for the presence of genetic
variation . SSCPs are detected by denaturing the DNA and resolving
it on nondenaturing acrylamide gels. Each strand of the DNA fragment
can potentially form a unique conformation (and have a distinct
mobility), and any mutation within that segment can potentially
affect the mobility.
Table I. Detection of mutations using
the SSCP technique
We screened 150 CF patients who have at least one chromosome that
does not contain the common (F 508) mutation (Table 1 ). Primers
were chosen to individually amplify coding regions of the gene.
Each patient that displayed an aberrantly migrating fragment on
an SSCP gel was chosen for the subsequent direct sequence analysis.
Alterations were classified as CF mutations based on the following
criteria: (1) The alteration shifts the reading frame and causes
premature termination of the protein; (2) An amino acid is replaced
with a dissimilar residue, and this alteration does not occur on
a large number of normal chromosomes with the same haplotype. Eleven
separate CF mutations have been identified, eight of which are frameshift
or nonsense mutations and three that replace amino acids (Table
2). Each of the frameshift mutations has been found in only a single
family, whereas two of the three-point mutations are found in multiple
families. The phenotypes of the patients are summarized in Table
2. All individuals that we have examined that are homozygous for
the deltaF508 mutation are pancreatic insufficient (PI) and have
moderate to severe disease (data not shown). Most of the patients
with the frameshift mutations are homozygous for the absence of
the common mutation, and therefore must contain an additional, unidentified
mutation. These patients are clinically
Table 2. CFTR mutations
Fig. I. Sequence of a portion of the CFTR transmembrane
region from exon 7. The nucleotide sequence is shown from humans,
as well as other vertebrate species, with numbering of the human
sequence as in Riordan et al. . The underlined codon is arginine
347, which was found mutated in several CF patients (mutation G
1172C, Table 2). LRO: lion tamarin; WHALE: humpback whale; FROG:
Xenopus; MOUSE: Balb/c
heterogeneous; they typically have moderate to severe disease. The
majority of the patients with CF point mutations have delta F 508
on their other chromosome, are diagnosed as PS, and have mild disease.
Therefore, the type of mutation at the CF locus appears to play
an important role in the clinical presentation of the patient. To
further explore the role of the missense mutations in the function
of the CF Transmembrane conductance regulator (CFTR) we have amplified
the region surrounding these mutations from DNA from a variety of
species. The sequence of the species obtained is displayed in Fig.
1. All of the residues that we have found mutated are conserved
in all of the species examined. Overall, these regions of the gene
show a high degree of conservation, suggesting that alterations
in the transmembrane domain are poorly tolerated.
Because the most common mutation accounts for only 70% of CF chromosomes
, a large proportion of CF patients (40 % -50% ) are compound
heterozygotes, i.e., they have two different mutations in the gene.
Thus there is a large number of possible genotypes found in CF patients.
This appears to account, in part, for the variation observed in
the phenotype of patients. However, within families affected individuals
can show differences in sweat chloride levels and severity, demonstrating
that additional genetic and/or environmental factors contribute
to these phenotypes. The clearest correlation between the patient's
genotype and phenotype is seen in the pancreas. All patients we
have observed that are homozygous for the delta F 508 deletion are
PI. However, even in these patients, genetically identical at the
CF locus, there is considerable variation in clinical outcome. This
variation is expressed in the age of diagnosis, pulmonary function,
and sweat chloride value. In the lungs of CF patients, damage is
principally caused by bacterial infection. These infections are
believed to be secondary to the abnormal mucus present in patients.
Furthermore, immune function genes such as the human lymphocyte
antigens (HLA) and/or the T cell receptor locus could playa role
in the susceptibility and/or response to bacterial infection.
We thank McNeil Pharmaceuticals for support of research in the
USSR. Marga Belle White is supported by a postdoctoral fellowship
from the US Cystic Fibrosis Foundation. This project has been funded
at least in part with Federal funds from the Department of Health
and Human Services under con tract n um ber N O 1-CO- 74102 with
Program Resources, Inc. The content of this publication does not
necessarily reflect the views or policies of the Department of Health
and Human Services, nor does mention of trade names, commercial
products, or organizations imply endorsement by the US Government.
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