T H E J O U R N A L OF

PEDIATRIC S MARCH

1992

Volume 120

Number 3

MEDICAL PROGRESS Cystic fibrosis: Beyond the gene to therapy E d u a r d o F, Tizzano, MD, a n d M a n u e l B u c h w a l d , PhD From the Department of Genetics, Research Institute, Hospital for Sick Children, and the Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada

Cystic fibrosis is a complex, inherited disorder and is the most common severe autosomal recessive disease in white populations (1 in 2500 live births). Dysfunction of exocrine glands appears to be the major pathogenic mechanism and is responsible for a wide and variable group of clinical manifestations and complications. Remarkable advances have been made in the treatment of affected individuals during the past two decades; as a result, the median age at death has increased from less than 10 years to more than 30 years.l Progress is also being made in understanding the metabolic abnormality of the disease. Electrophysiologic studies show decreased permeability of ionized chloride in the secretory epithelia of CF organs. 2 Most important, the gene responsible for this disease has been localized to the long arm of chromosome 7 (7q31.3) and subsequently isolated by molecular cloning strategies. 3' 4 The major mutation causing CF has been defined at the DNA sequence level, 5 and this has already had a significant impact on genetic counseling and prenatal diagnosis. The identification of the gene thus serves as a new entry point for CF research. The elucidation of the primary cel-

Supported in part by grants from the Canadian Cystic Fibrosis Foundation, the Cystic Fibrosis Foundation (United States), and the National Institutes of Health (United States). Dr. Tizzano is a Postdoctoral Fellowof the Ca.nadianCystic Fibrosis Foundation. The work from our laboratory is part of the Research Development Programme at the Hospital for Sick Children. Reprint requests: Manuel Buchwald, PhD, Department of Genetics, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada. 9/18/34710

lular defect, which will certainly follow, should have major therapeutic implications. This review summarizes the steps taken, after the cloning of the gene, to understand the function and regulation of the gene and its product, and discusses how these advances are leading to new prospects for diagnosis and therapy. ISOLATION AND CHARACTERIZATION T H E CF G E N E

OF

Cloning the CF gene. Together, the autosomal recessive mode of inheritance of CF and the results of extensive population analyses led to the assumption that CF is due to a mutation in a single gene (the CF gene). Affected individbp cAMP cDNA CF CFTR DNase kb mRNA RFLP

Base pair Cyclicadenosine monophosphate Complementary DNA Cystic fibrosis Cysticfibrosis transmembrane regulator Deoxyribonuclease Kilobase Messengerribonucleic acid Restrictionfragment length polymorphism

uals have two abnormal copies of this defective gene. Carriers have one normal and one abnormal version of the gene, are symptom free, and are not known to be at increased risk for any disease. However, the high frequency of the CF gene in the population may occur because carriers have a selective advantage (greater fitness) relative to homozygous normal individuals. In particular, it has been suggested that CF carriers have an increased resistance to C1- secreting diarrhea, 6 an attractive hypothesis given the recent evidence that the CF gene product may be involved in C1- transport 337

338

Tizzano and Buchwald

The Journal of Pediatrics March 1992

Entire Genome

Chromosome7q region Chromosomal localization by linkage analysis and molecular cytogenetics

l

Mapping and refining localizationwith RFLPs

I Long range restriction map

j

Closemarkers as starting points for chromosome walking and jumping I

D7S411 Cloning of candidate genes

D7S399

r

Sequence conservation across species Sequencing and comparison of normal vs. disease gene

Pattern of tissue expression Properties of the predicted protein

GENE

]

~ CF GENE

Fig. 1. Steps in positional cloning of the CG gene (left side) and of region on chromosome 7 containing CF gene and flanking DNA markers (right side). Starting from entire genome (3 x 109 bp), investigatorsfirst localizedthe gene to 7q31 region of chromosome 7 near marker D7S15 (3 X 107 bp). Discoveryof two closelylinked markers on either side of the gene (D7S8 and MET) shortened the distance under study to approximately 1.5 X 106 bp. Isolation of markers closer to the gene allowed cloning of Candidategenes, one of which spanned 2.5 • 105 bp and was identifiedas the CF gene. 7", Telomere; C, centromere. (see the section on CFTR and the molecular basis of chloride channel dysregulation, below). The development of molecular genetic techniques has made it possible to use the "reverse genetics," or "positional cloning," approach to finding the CF gene (Fig. 1). The gene was first localized to chromosome 7 by linkage analysis using DNA markers defined by restriction fragment length polymorphisms. The first linkage between the CF gene and a random DNA segment (D7S15) was found in 1985. The position on chromosome 7 was further refined by the discovery of two closely linked markers, the met oncogene and the random DNA segment D7S8, that flanked the gene. The distance between these two markers was estimated at 1500 kb, representing approximately 1% to 2% recombination.7 Additional markers between these probes, and therefore closer to the CF gene, were subsequently found and used as the starting point in the molecular search for the gene. The physical relationships among these markers were examined

by long-range restriction maps of the region. Molecular cloning techniques (such as chromosome walking and jumping) were then used to isolate the DNA in the interval under study. As the search proceeded, candidate genes were detected by finding DNA segments that had sequences conserved between species. The conservation of a sequence through evolution suggests that it has an essential function. Three DNA segments were identified with particularly strong conservation between human and bovine DNA; two of them were eliminated as candidates by genetic and DNA analyses. About 280 kb of genomic sequence were cloned before the 5' end of the CF gene was found. 3As the CF gene was presumed to be expressed in sweat gland cells, a complementary DNA library was constructed from such ribonucleic acid. The third conserved fragment identified a clone from the sweat gland cDNA library and was used as the starting point to clone the entire gene. 4 Features of the CF gene and its predicted protein. The gene extends over approximately 250 kb of genomie DNA,

Volume 120 Number 3

Cystic fibrosis therapy

339

A. CF gene coding region and CFTR protein EXONS

1 2 3

4

5 6a 6b

7

8

9

10 11 12

13

14a14b15 16 17a17b 18

19

2021 2223 24

aa2-I I I ~ ~ I I~N IU//,I I ~ ~ - I~11~ I~.~,1 ~1~1 ~ 1 I II I I I I 1 I I DOMAINS membranespanning nucleetide regulatory(R) membranespanning nucleotide binding fold binding fold

I-COOH

B. Protein model CHO I.~,,_CHO ! ~ outside ~ ::i ~ i~'~i ::ili ii iiliii ::i ;:i :;i iiili ~' ~ i i ~i ::~i i ::i i iii ::iiii i ::!ii ii Membrane ' :' :': '" ....... ' ................... inside OOH F i g . 2. CF gene and its protein product, CFTR. A, Correlation between exons in the gene and predicted domain structure of CFTR. B, Model of CFTR and its localization in membrane. Each of the two membrane-spanning domains has three exterior loops. First loop in second membrane-spanningdomain is predicted to have two N-linked glycosylationsites. Two nucleotide-bindingdomains and a regulatory domain are also present in CFTR. Greek letter Zxrepresents location of ~F508 mutation. (Modified from Tsui LC, Buchwatd M [Adv Hum Genet 1991;20:153-266].)

with 27 coding regions (exons) separated by noncoding regions (introns). The messenger ribonucleic acid is approximately 6.2 kb in length and contains an open reading frame capable of encoding a polypeptide of 1480 amino acids (Fig. 2). The tissue distribution of the CF mRNA was consistent with that predicted by the pathogenesis of the disease. The putative protein was called the cystic fibrosis transmembrane regulator. 4 The structure of CFTR predicted from the nucleotide sequence was that of a membrane protein with structural similarity to a variety of bacterial, yeast, and mammalian transport proteins. Notable examples are P-glycoproteins (also known as multiple drug resistance proteins). Increased expression of P-glycoprotein is a major cause of drug resistance in cancer chemotherapy. 8 Like P-glycoprotein, CFTR contains two different domains that are duplicated. In addition, CFTR contains a novel region (R domain) with an abundance of potential sites for phosphorylation by protein kinase A or C (Fig. 2). Thus the properties expected for CFTR are consistent with those of a membrane protein with a role in the regulation of ion transport. This was the second evidence that CFTR was the defective gene causing CF. 4 The third and most compelling Evidence that the CF gene had been isolated was obtained from correlating sequence data with genetic analysis, A specific deletion of three base pairs was found in nearly 70% of CF chromosomes but not in normal chromosomes. This mutation was localized in exon 10, and as a result the corresponding protein was predicted to lack the amino acid phenylalanine at position 508 (AF508). 5 Since that time the worldwide frequency of

AF508 has been extensively characterized. The Cystic Fibrosis Genetic Analysis Consortium reported that 68% of a total of 17,000 CF chromosomes contain the AF508 mutation. A generally higher frequency of AF508 has been observed in northern European countries than in southern European countries, ranging from 30% to 35% in Turkey and Israel, 50% in Greece, Italy, and Spain, and up to 85% in Denmark. 9 The remaining CF alleles appear diverse. More than 100 different mutations have been reported to the consortium. Approximately half of these are missense mutations, leading to substitutions of single amino acids in the protein. Some of these changes occur at residues highly conserved among the nucleotide binding folds found in various adenosine triphosphate-binding proteins. This result is consistent with the importance of these residues for CFTR function. Another significant proportion are nonsense mutations, expected to cause premature termination of the CFTR polypeptide and resulting in a truncated, nonfunctional protein. The remainder of the mutations include deletions and insertions of 1 or 2 bp and those affecting splice junctions. Even though the gene is large (250 kb), no gross deletions have been detected, as has been the case in other larger genes (for example, the Duchenne muscular dystrophy gene). Most of the non-AF508 mutations are relatively rare, ranging from