Dig Dis 1991;9:179-188
© 1991 S. Kaiger AG. Basel 0257-2753/91/0093-01 79S2.75/0
Genetics of Cystic Fibrosis Paolo Gasparini
Cystic fibrosis (CF) is one of the most common genetic disorders in the Caucasian population. It affects about 1 in 2,000 new borns. The disease is transmitted as an au tosomal recessive trait; therefore, a person has to inherit two affected genes to get the disease. The carrier of one defective gene copy does not have any symptoms. The car rier frequency is about 5%. The high fre quency of the CF gene is one of the most puzzling problems in genetic studies of this lethal disease. It cannot be explained by mu tation rate alone, so heterozygote advantage and genetic drift may play a role. CF is a chronic disease of children and young adults which affects several organs including the lung, the exocrine pancreas, and the sweat glands. Respiratory complica tions account for much of the morbidity and for more than 95% of the mortality. Survival has increased in the last years into the 2nd and even the 3rd decades of life. In 1953 a consistent abnormality in elec trolyte content of sweat was found in af fected individuals [1], This alteration corre lated with the high electrical potential differ ence detected across epithelial surfaces, in cluding the respiratory tract and the walls of sweat gland secretory coils and reabsorptive ducts [2], Therefore, the basic defect was related to decreased chloride ion conduc
tance across the apical membrane of the epi thelial cell [3], A possible failure of the out wardly rectifying anion channel to respond to phosphorylation by protein kinases was recently demonstrated by patch clamp analy ses [4], Some polypeptide components of an epithelial chloride channel that mediates conductance have been recently isolated [5], but their relation to the CF defect was not determined. In parallel with these biochemical and electrophysiological studies, other re searchers have utilized an alternative ap proach in order to understand the nature of the molecular defect, involving the cloning of the responsible gene on the basis of its chromosomal location. In fact, DNA analy sis, has given in several instances molecular reality to a gene, making it possible first to localize and then to define the functional unit. This way, several DNA fragments pro gressively closer to the CF gene were identi fied, and polymorphic markers that segre gate with CF in affected families (i.e. in link age disequilibrium) were found. Linkage analysis based on a number of polymorphic DNA markers has assigned the CF locus to the long arm of chromosome 7, band q31 [68]. These defined DNA sequences close to the CF gene have made it possible to use dif ferent gene localization strategies, such as
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/15/2019 3:06:37 AM
Institute of Biological Sciences, School of Medicine. University of Verona. Italy
Gasparini
180
other known proteins may help indicating its function. Practically, it is a genetic analysis that proceeds, as shown in figure 1. from the bottom up. rather than from the top down. In the case of CF. this approach was success ful. as in September 1989 the identification of the cystic fibrosis transmembrane regula tor (CFTR) gene was described [9-11].
Fig. 1. Different approaches to the genotype/phenotype correlation analysis. ( I) Mcndelian approach: from the phenotype to the gene, without knowing how the gene action is mediated. (2) Step by step ap proach: from the phenotype to the gene, each passage being the consequence of a previous one. (3) Reverse genetics approach: from the gene to the phenotype. The sequence of a gene suggests a protein which can be involved in some homeostatic systems accounting for the phenotype. This is a bottom-up approach, that follows gene expression and protein function path ways.
chromosome walking and jumping, to pin point the gene. Further genetic analysis with linked DNA markers in many CF families was then helpful in indicating the position of the gene among several DNA markers. This approach to the study of genetic dis ease is called ‘reverse genetics’, as it proceeds in the reverse order of classical genetics. The protein product is therefore inferred from the DNA sequence of the gene, and conclu sions are then drawn as to the role of the former in homeostasis. A comparison of the sequence of its amino acids with those of
The recent identification of the CF gene marked the end of a long search which inten sified 5 years ago, at the end of 1985, when the gene was localized on chromosome 7 [6-8]. A CF ‘candidate’ gene was isolated in 1987 by selective cloning of Hpall tiny-fragment islands, which usually mark expressed genes [12]. Several DNA polymorphisms from this region, in linkage disequilibrium with the CF locus, were isolated. Extensive family studies utilizing these linked markers have resulted in the identification of a few families in which there was evidence of re combination between the candidate gene and CF [ 13], The existence of these recombi nants demonstrated that the CF gene was not the candidate gene, even if it was close to it. These data were confirmed by the cDNA cloning, which showed that the encoded pro tein had a growth factor-like structure, show ing homology to the murine and human pro to-oncogene int-1. As a consequence this coding region was called 1RP (int-1 -related protein) gene. At last the identification of the CF gene by chromosome walking and jumping proce dures was described in 1989 [9-11]. The gene extends across nearly 250 kb of ge nomic DNA. It consists, like other genes of higher organisms, of protein-coding exons.
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/15/2019 3:06:37 AM
The CF Gene
181
Genetics of Cystic Fibrosis
Fig. 2. CF gene structure and predicted protein with functional domains [ 10], Numbers 1-24 indicate the 27 exons of the gene. TMD = Transmembrane domain 1 and II: NBF = nucleotide binding fold I and 2; R = regulatory region. The elements are not in scale.
Linked Restriction Fragment Length Polymorphisms, F508 and Other Mutations Many nonpathological DNA sequence variations are present in the human genome. In most cases these variations are due to sin gle base substitutions, which often create or destroy a cleavage site of a specific restric tion enzyme, producing DNA fragments which differ in length. The fragments are identified by specific DNA probes with the method of Southern blotting [14], This kind of DNA polymorphism is called restriction fragment length polymorphism (RFLP) [15], The use of DNA probes in families where the disease segregates allows the determination of linkage between a restriction fragment and the disease locus. This approach was first successfully utilized to analyze sexlinked diseases such as Duchenne muscular dystrophy [16] or to study still unassigned autosomal dominant diseases such as Hun tington's chorea [17] and adult polycystic kidney disease [18]. Many RFLPs recog nized by DNA probes tightly linked to CF were discovered. The early CF locus markers
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/15/2019 3:06:37 AM
27 in this case, separated by noncoding introns, as reported in figure 2. Sequence anal ysis revealed that the protein encoded by the gene should contain about 1,480 amino acids and that it had all the earmarks of a membrane protein, possibly related to ion secretion. The protein was called CFTR [ 10]. On the basis of its structure, the transmem brane regulator protein may be the channel itself or a direct or indirect regulator of ion conductance in epithelial cells. A mutation of the gene coding portion was already de scribed [11]. Three specific nucleotides in exon 10 were found to be deleted in several CF chromosomes but never in normal chro mosomes. As a result, the corresponding pro tein lacks the amino acid phenylalanine in position 508. This deletion (delta F508) af fects the region containing an apparent bind ing site for ATP or other cyclic nucleotides, compounds that provide energy for several cellular functions. The loss of phenylalanine may therefore interfere with chloride ion transport by preventing cyclic nucleotide binding to the CFTR protein, and depriving it of the energy it needs for performing its function.
182
Gasparini
Table I. RFLPs associated with the CF locus Probe
Enzyme
Fragments
Source
PCR
10-1X6 10-1X6 TS/20 H I.3 CE 1.0 G2 H80
Acc 1 Hae 111 Msp 1 Nco I Ndc 1 Xba I Pst I
6.5/3.5/3.0 12.0/0.6 8.0/4.3 2.4/1.4/1.0 5.5/4.7/0.8 1.6/0.8 8.8/2.6
TS TS TS TS TS RW RW
_
Met D Met D Met H J.3.11 J.29 7.C.22 B79a
Ban I Taq 1 Taq I Msp I Pvu II EcoR I Hind III
7.6/6.8 6.2/4.8 7.5/4.0 4.2/1.8 9.0/6.0 7.2/5.1 8.1/4.3
RWh RWh RWh RW RW RW RW
E6 E7 PH131 W3D14 Xv-2c CS.7 E9
Taq I Taq 1 Hinf I Hind III Taq 1 Cfo I Msp 1
4.4/3.6 39.0/3.0/0.9 0.4/0.3 20.0/10.0 2.1/1.4 0.7/0.5 13.0/8.5
TS TS TS TS XE XE TS
—
MP6d-9 J.44
Msp 1 Xba I
12.0/8.5/3.5 15.3/15.0/0.3
XE TS
+ +
-
_ -
+ + -
-
+ + -
met [7] and J.3.11 [8] show about I % recom bination with the CF locus. A second gener ation of probes. X V-2c [ 19], KM 19 [ 12] and C5.7 [12], define the locus D7S23 which lies much closer to the CF locus and corresponds to the IRP gene. Finally, a third generation of probes. MP6d9 [20] and J44 [11], located right next to the gene, was described. All the above-mentioned markers lie outside of the gene at decreasing distances from it. To date some new gene probes which recognize in tragenic sequences are also available [11]. A
list of the most important CF gene markers presently available is reported in table 1. Utilizing these RFLPs. haplotypes were defined before the gene was identified, and their frequency determined in North Euro pean countries [19, 20], in Italy [21], in Ger many [22], in France [23], in Finland [24], in Poland and Holland [25], and in North American populations [26-28]. Significant differences were found in the frequency of these haplotypes in CF patients from South (particularly Italian and Spanish) and North
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/15/2019 3:06:37 AM
The first group represents intragenic probes, the other three groups linked, closely linked (
© 1991 S. Kaiger AG. Basel 0257-2753/91/0093-01 79S2.75/0
Genetics of Cystic Fibrosis Paolo Gasparini
Cystic fibrosis (CF) is one of the most common genetic disorders in the Caucasian population. It affects about 1 in 2,000 new borns. The disease is transmitted as an au tosomal recessive trait; therefore, a person has to inherit two affected genes to get the disease. The carrier of one defective gene copy does not have any symptoms. The car rier frequency is about 5%. The high fre quency of the CF gene is one of the most puzzling problems in genetic studies of this lethal disease. It cannot be explained by mu tation rate alone, so heterozygote advantage and genetic drift may play a role. CF is a chronic disease of children and young adults which affects several organs including the lung, the exocrine pancreas, and the sweat glands. Respiratory complica tions account for much of the morbidity and for more than 95% of the mortality. Survival has increased in the last years into the 2nd and even the 3rd decades of life. In 1953 a consistent abnormality in elec trolyte content of sweat was found in af fected individuals [1], This alteration corre lated with the high electrical potential differ ence detected across epithelial surfaces, in cluding the respiratory tract and the walls of sweat gland secretory coils and reabsorptive ducts [2], Therefore, the basic defect was related to decreased chloride ion conduc
tance across the apical membrane of the epi thelial cell [3], A possible failure of the out wardly rectifying anion channel to respond to phosphorylation by protein kinases was recently demonstrated by patch clamp analy ses [4], Some polypeptide components of an epithelial chloride channel that mediates conductance have been recently isolated [5], but their relation to the CF defect was not determined. In parallel with these biochemical and electrophysiological studies, other re searchers have utilized an alternative ap proach in order to understand the nature of the molecular defect, involving the cloning of the responsible gene on the basis of its chromosomal location. In fact, DNA analy sis, has given in several instances molecular reality to a gene, making it possible first to localize and then to define the functional unit. This way, several DNA fragments pro gressively closer to the CF gene were identi fied, and polymorphic markers that segre gate with CF in affected families (i.e. in link age disequilibrium) were found. Linkage analysis based on a number of polymorphic DNA markers has assigned the CF locus to the long arm of chromosome 7, band q31 [68]. These defined DNA sequences close to the CF gene have made it possible to use dif ferent gene localization strategies, such as
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/15/2019 3:06:37 AM
Institute of Biological Sciences, School of Medicine. University of Verona. Italy
Gasparini
180
other known proteins may help indicating its function. Practically, it is a genetic analysis that proceeds, as shown in figure 1. from the bottom up. rather than from the top down. In the case of CF. this approach was success ful. as in September 1989 the identification of the cystic fibrosis transmembrane regula tor (CFTR) gene was described [9-11].
Fig. 1. Different approaches to the genotype/phenotype correlation analysis. ( I) Mcndelian approach: from the phenotype to the gene, without knowing how the gene action is mediated. (2) Step by step ap proach: from the phenotype to the gene, each passage being the consequence of a previous one. (3) Reverse genetics approach: from the gene to the phenotype. The sequence of a gene suggests a protein which can be involved in some homeostatic systems accounting for the phenotype. This is a bottom-up approach, that follows gene expression and protein function path ways.
chromosome walking and jumping, to pin point the gene. Further genetic analysis with linked DNA markers in many CF families was then helpful in indicating the position of the gene among several DNA markers. This approach to the study of genetic dis ease is called ‘reverse genetics’, as it proceeds in the reverse order of classical genetics. The protein product is therefore inferred from the DNA sequence of the gene, and conclu sions are then drawn as to the role of the former in homeostasis. A comparison of the sequence of its amino acids with those of
The recent identification of the CF gene marked the end of a long search which inten sified 5 years ago, at the end of 1985, when the gene was localized on chromosome 7 [6-8]. A CF ‘candidate’ gene was isolated in 1987 by selective cloning of Hpall tiny-fragment islands, which usually mark expressed genes [12]. Several DNA polymorphisms from this region, in linkage disequilibrium with the CF locus, were isolated. Extensive family studies utilizing these linked markers have resulted in the identification of a few families in which there was evidence of re combination between the candidate gene and CF [ 13], The existence of these recombi nants demonstrated that the CF gene was not the candidate gene, even if it was close to it. These data were confirmed by the cDNA cloning, which showed that the encoded pro tein had a growth factor-like structure, show ing homology to the murine and human pro to-oncogene int-1. As a consequence this coding region was called 1RP (int-1 -related protein) gene. At last the identification of the CF gene by chromosome walking and jumping proce dures was described in 1989 [9-11]. The gene extends across nearly 250 kb of ge nomic DNA. It consists, like other genes of higher organisms, of protein-coding exons.
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/15/2019 3:06:37 AM
The CF Gene
181
Genetics of Cystic Fibrosis
Fig. 2. CF gene structure and predicted protein with functional domains [ 10], Numbers 1-24 indicate the 27 exons of the gene. TMD = Transmembrane domain 1 and II: NBF = nucleotide binding fold I and 2; R = regulatory region. The elements are not in scale.
Linked Restriction Fragment Length Polymorphisms, F508 and Other Mutations Many nonpathological DNA sequence variations are present in the human genome. In most cases these variations are due to sin gle base substitutions, which often create or destroy a cleavage site of a specific restric tion enzyme, producing DNA fragments which differ in length. The fragments are identified by specific DNA probes with the method of Southern blotting [14], This kind of DNA polymorphism is called restriction fragment length polymorphism (RFLP) [15], The use of DNA probes in families where the disease segregates allows the determination of linkage between a restriction fragment and the disease locus. This approach was first successfully utilized to analyze sexlinked diseases such as Duchenne muscular dystrophy [16] or to study still unassigned autosomal dominant diseases such as Hun tington's chorea [17] and adult polycystic kidney disease [18]. Many RFLPs recog nized by DNA probes tightly linked to CF were discovered. The early CF locus markers
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/15/2019 3:06:37 AM
27 in this case, separated by noncoding introns, as reported in figure 2. Sequence anal ysis revealed that the protein encoded by the gene should contain about 1,480 amino acids and that it had all the earmarks of a membrane protein, possibly related to ion secretion. The protein was called CFTR [ 10]. On the basis of its structure, the transmem brane regulator protein may be the channel itself or a direct or indirect regulator of ion conductance in epithelial cells. A mutation of the gene coding portion was already de scribed [11]. Three specific nucleotides in exon 10 were found to be deleted in several CF chromosomes but never in normal chro mosomes. As a result, the corresponding pro tein lacks the amino acid phenylalanine in position 508. This deletion (delta F508) af fects the region containing an apparent bind ing site for ATP or other cyclic nucleotides, compounds that provide energy for several cellular functions. The loss of phenylalanine may therefore interfere with chloride ion transport by preventing cyclic nucleotide binding to the CFTR protein, and depriving it of the energy it needs for performing its function.
182
Gasparini
Table I. RFLPs associated with the CF locus Probe
Enzyme
Fragments
Source
PCR
10-1X6 10-1X6 TS/20 H I.3 CE 1.0 G2 H80
Acc 1 Hae 111 Msp 1 Nco I Ndc 1 Xba I Pst I
6.5/3.5/3.0 12.0/0.6 8.0/4.3 2.4/1.4/1.0 5.5/4.7/0.8 1.6/0.8 8.8/2.6
TS TS TS TS TS RW RW
_
Met D Met D Met H J.3.11 J.29 7.C.22 B79a
Ban I Taq 1 Taq I Msp I Pvu II EcoR I Hind III
7.6/6.8 6.2/4.8 7.5/4.0 4.2/1.8 9.0/6.0 7.2/5.1 8.1/4.3
RWh RWh RWh RW RW RW RW
E6 E7 PH131 W3D14 Xv-2c CS.7 E9
Taq I Taq 1 Hinf I Hind III Taq 1 Cfo I Msp 1
4.4/3.6 39.0/3.0/0.9 0.4/0.3 20.0/10.0 2.1/1.4 0.7/0.5 13.0/8.5
TS TS TS TS XE XE TS
—
MP6d-9 J.44
Msp 1 Xba I
12.0/8.5/3.5 15.3/15.0/0.3
XE TS
+ +
-
_ -
+ + -
-
+ + -
met [7] and J.3.11 [8] show about I % recom bination with the CF locus. A second gener ation of probes. X V-2c [ 19], KM 19 [ 12] and C5.7 [12], define the locus D7S23 which lies much closer to the CF locus and corresponds to the IRP gene. Finally, a third generation of probes. MP6d9 [20] and J44 [11], located right next to the gene, was described. All the above-mentioned markers lie outside of the gene at decreasing distances from it. To date some new gene probes which recognize in tragenic sequences are also available [11]. A
list of the most important CF gene markers presently available is reported in table 1. Utilizing these RFLPs. haplotypes were defined before the gene was identified, and their frequency determined in North Euro pean countries [19, 20], in Italy [21], in Ger many [22], in France [23], in Finland [24], in Poland and Holland [25], and in North American populations [26-28]. Significant differences were found in the frequency of these haplotypes in CF patients from South (particularly Italian and Spanish) and North
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/15/2019 3:06:37 AM
The first group represents intragenic probes, the other three groups linked, closely linked (
