Original Paper
Hum Hcred 1992;42:231-234
Z. Siddique3 A.R. McPhadena D. McCluskeyb K. Whaleyc
A Single Base Deletion from the Cl-Inhibitor Gene Causes Type I Hereditary Angio-Oedema
a University Department of Pathology, Western Infirmary, Glasgow; b Department of Medicine, Royal Victoria Hospital, Belfast; c Department of Immunology, Leicester Royal Infirmary, Leicester, UK
Key Words
Abstract
Hereditary angio-oedema Cl-inhibitor gene mutation
RFLP analysis, the polymerase chain reaction and nucleotide sequencing have been used to characterise a Cl-inhibitor gene mutation responsible for type I hereditary angio-oedema (HAE). A single base deletion (C-16698) from the eighth exon of the Cl-inhibitor gene alters the reading frame of the exon and generates a premature translation termination codon. This represents the first report of this form of Cl-inhibitor gene mutation in type I HAE.
Cl-inhibitor is an important inhibitor of the complement system of proteins and of other serine protease dependent plasma mediator systems [1], Hereditary Cl-inhibitor defi ciency, or hereditary angio-oedema (HAE), is an autosomal dominant disease which is char acterised by recurrent episodes of subcutane ous and submucous oedema [2], Biochem ically, HAE can be subdivided into type I HAE in which there are low serum levels of functionally normal Cl-inhibitor, and type II
HAE with normal or elevated serum levels mainly of a dysfunctional Cl-inhibitor protein [3], Recently, two categories of Cl-inhibitor gene mutations responsible for type I HAE have been described (4,5]. We now describe a third category.
Materials and Methods Peripheral blood leukocyte genomic DNA was iso lated from normal and affected family members of a kindred with type I HAE [5]. Restriction fragment length polymorphism (RFLP) analysis of the Cl-inhib-
Prof. K. Whaley Department of Immunology Leicester Royal Infirmary Leicester LEI 5WW (UK)
© 1992 S. Karger AG, Basel 0001-5652/92/ 0424-0231 $2.75/0
Downloaded by: Thomas Jefferson University Scott Library 147.140.27.100 - 8/24/2018 9:53:14 PM
Introduction
PCR product to ensure the presence of the mutation in the patient samples, both strands of the PCR product were then subjected to direct di-dcoxy nucleotide se quence analysis using internal oligonucleotide primers for the sequencing reaction [6].
Results and Discussion
itor gene in this kindred had previously pinpointed a mutation within the eight exon of the Cl-inhihitor gene, a mutation which co-segregated absolutely with the disease (5). In order to characterise the mutation, polymerase chain reaction (PCR) amplification of the eighth exon of all family members has now been car ried out using Taq polymerase and two oligonucleotide primers: 5' TGATCAGGATGAACCCAGAGA 3' (Clinhibitor gene nucleotides 16548-16568) and 5' TCTAGAGAAAGTCATGGTCTG 3' (nucleotides 1715217172) [6, 7J. Following Bgl II digestion of the 626 bp
232
Acknowlegements This study was supported by grant 960 from the Scottish Hospital Endowment Research Trust.
Note added in proof The results of direct sequencing have since been confirmed by single stranded sequencing in the M13 mpl8 vector.
Siddique/McPhaden/McCluskey/ Whaley
Cl-Inhibitor Deletion in Angio-Oedema
Downloaded by: Thomas Jefferson University Scott Library 147.140.27.100 - 8/24/2018 9:53:14 PM
Fig. 1. Southern blot analysis of Bgl II digested ge nomic DNA probed with the eighth exon of the Clinhibitor gene. The 6.5-kh and 1.9-kb fragments are generated in normal family members. The additional 8.4-kb fragment present in the middle lane indicates heterozygous loss of the Bgl II site between the 6.5-kh and 1.9-kb fragments (6.5 +1.9 = 8.4) in a patient DNA sample.
The Bgl II RFLP shown in figure 1 indi cates loss of the Bgl II recognition site within the eighth exon of one allele of the Cl-inhibitor gene, without apparent deletion of a sig nificant length of DNA. Subsequent exon am plification and direct nucleotide sequencing of the 626-bp PCR product demonstrates the deletion of a single cytosine base (C-16698) from the Bgl II hexanucleotide recognition motif (AGATCT ->AGATT) within the exon (fig. 2). This deletion mutation alters the read ing frame of the eighth exon downstream of the deletion and so generates a premature translation termination codon (fig. 2). To date, the deletion of whole exons, and a point mutation which destroys the niRNA do nor splice site recognition motif for the sixth intron, represent the Cl-inhibitor gene muta tions that have been shown to cause type I HAE [4,5]. The exon eight mutation describ ed in this study therefore represents a third category of gene mutation that produces type I HAE. Although not described previously in type I HAE, this form of mutation is recog nised as a cause of other genetic diseases such as [5-thalassaemia and haemophilia B[8, 9],
Amino acid Patient Amino acid
A G A T C T T CAGGT T T C T G C G A T G C A G C A C C A G A C A G T G C T G G A A C T G ACAG D
L
Q
V
S
A
M
Q
H
Q
T
V
L
E
L
T
AGATTTCAGGTTTCTGCGATGCAGCACCAGACAGTGCTGGAACTGACAG D
F
R
F
L
R
C
S
T
R
Q
C
W
N
l Q Termination codon
Fig. 2. Nucleotide and amino acid sequences of the normal and mutant Cl-inhibitor alleles. The position of the C-16698 deletion and resultant frameshift is shown. Due to heterozygosity, doublets arc seen at each nucleotide position downstream of the frameshift in this direct sequencing reaction of the whole PCR product. The prema ture translation termination codon TGA is generated 40 nucleotides downstream of the frameshift. In addition a completely altered amino acid sequence is predicted 3' to the mutation as shown.
233
Downloaded by: Thomas Jefferson University Scott Library 147.140.27.100 - 8/24/2018 9:53:14 PM
Normal
1 Davis AE III: Cl -inhibitor and hereditary angioneurotic edema. Ann Rev Immunol 1988:6595-628. 2 Cicardi M. Bcrgamaschini L. Marasini B. Boccassini G. Tucci A. Agos tini A: Hereditary angioedema: An appraisal of 104 cases. Am J Med Sci 1982;284:2-9. 3 Kerr MA. Yeung-Laiwah AAC; CTinhibitor deficiency and angiooedema; in Whaley K (ed): Comple ment in Health and Disease. Lan caster. MTP Press. 1987. pp 53-78. 4 Stoppa-Lyonnct D, Carter PE. Meo T, Tosi, M: Clusters of intragenic Alu repeats predispose the human Cl-inhibitor locus to deleterious re arrangements. Proc Natl Acad Sci USA 1990;87:1551-1555.
5 McPhaden AR. Birnie GD, Whaley K; Restriction fragment length poly morphism analysis of the Cl-inhib itor gene in hereditary Cl-inhibitor deficiency. Clin Genet 1991:39:161171. 6 Saiki RK, Gyllenstein VB, Erlich HA: The polymerase chain reaction; in Davies KE (ed): Genome Analy sis. A Practical Approach. Oxford. IRL Press, 1988. pp 141-152. 7 Carter PE. Duponchcl C, Tosi M. Fothergill JE: Complete nucleotide sequence o f the gene for human Cl inhibitor with an unusually high density of Alu elements. Eur J Biochcm 1991:197:301-308.
8 Kazazian HH Jr. Boehm CD: Mo lecular basis and prenatal diagnosis of |S-thalassnemia. Blood 1988:72: 1107-1116. 9 Green PM. Bentley DR. Mibashan RS. Nilsson IM. Giannclli F: Molec ular pathology of haemophilia B. EM BO J 1989;8:1067-1072.
234
Siddique/McPhaden/McCluskcy' Whaley
Cl-inhibitor Deletion in Angio-Oedema
Downloaded by: Thomas Jefferson University Scott Library 147.140.27.100 - 8/24/2018 9:53:14 PM
References
Hum Hcred 1992;42:231-234
Z. Siddique3 A.R. McPhadena D. McCluskeyb K. Whaleyc
A Single Base Deletion from the Cl-Inhibitor Gene Causes Type I Hereditary Angio-Oedema
a University Department of Pathology, Western Infirmary, Glasgow; b Department of Medicine, Royal Victoria Hospital, Belfast; c Department of Immunology, Leicester Royal Infirmary, Leicester, UK
Key Words
Abstract
Hereditary angio-oedema Cl-inhibitor gene mutation
RFLP analysis, the polymerase chain reaction and nucleotide sequencing have been used to characterise a Cl-inhibitor gene mutation responsible for type I hereditary angio-oedema (HAE). A single base deletion (C-16698) from the eighth exon of the Cl-inhibitor gene alters the reading frame of the exon and generates a premature translation termination codon. This represents the first report of this form of Cl-inhibitor gene mutation in type I HAE.
Cl-inhibitor is an important inhibitor of the complement system of proteins and of other serine protease dependent plasma mediator systems [1], Hereditary Cl-inhibitor defi ciency, or hereditary angio-oedema (HAE), is an autosomal dominant disease which is char acterised by recurrent episodes of subcutane ous and submucous oedema [2], Biochem ically, HAE can be subdivided into type I HAE in which there are low serum levels of functionally normal Cl-inhibitor, and type II
HAE with normal or elevated serum levels mainly of a dysfunctional Cl-inhibitor protein [3], Recently, two categories of Cl-inhibitor gene mutations responsible for type I HAE have been described (4,5]. We now describe a third category.
Materials and Methods Peripheral blood leukocyte genomic DNA was iso lated from normal and affected family members of a kindred with type I HAE [5]. Restriction fragment length polymorphism (RFLP) analysis of the Cl-inhib-
Prof. K. Whaley Department of Immunology Leicester Royal Infirmary Leicester LEI 5WW (UK)
© 1992 S. Karger AG, Basel 0001-5652/92/ 0424-0231 $2.75/0
Downloaded by: Thomas Jefferson University Scott Library 147.140.27.100 - 8/24/2018 9:53:14 PM
Introduction
PCR product to ensure the presence of the mutation in the patient samples, both strands of the PCR product were then subjected to direct di-dcoxy nucleotide se quence analysis using internal oligonucleotide primers for the sequencing reaction [6].
Results and Discussion
itor gene in this kindred had previously pinpointed a mutation within the eight exon of the Cl-inhihitor gene, a mutation which co-segregated absolutely with the disease (5). In order to characterise the mutation, polymerase chain reaction (PCR) amplification of the eighth exon of all family members has now been car ried out using Taq polymerase and two oligonucleotide primers: 5' TGATCAGGATGAACCCAGAGA 3' (Clinhibitor gene nucleotides 16548-16568) and 5' TCTAGAGAAAGTCATGGTCTG 3' (nucleotides 1715217172) [6, 7J. Following Bgl II digestion of the 626 bp
232
Acknowlegements This study was supported by grant 960 from the Scottish Hospital Endowment Research Trust.
Note added in proof The results of direct sequencing have since been confirmed by single stranded sequencing in the M13 mpl8 vector.
Siddique/McPhaden/McCluskey/ Whaley
Cl-Inhibitor Deletion in Angio-Oedema
Downloaded by: Thomas Jefferson University Scott Library 147.140.27.100 - 8/24/2018 9:53:14 PM
Fig. 1. Southern blot analysis of Bgl II digested ge nomic DNA probed with the eighth exon of the Clinhibitor gene. The 6.5-kh and 1.9-kb fragments are generated in normal family members. The additional 8.4-kb fragment present in the middle lane indicates heterozygous loss of the Bgl II site between the 6.5-kh and 1.9-kb fragments (6.5 +1.9 = 8.4) in a patient DNA sample.
The Bgl II RFLP shown in figure 1 indi cates loss of the Bgl II recognition site within the eighth exon of one allele of the Cl-inhibitor gene, without apparent deletion of a sig nificant length of DNA. Subsequent exon am plification and direct nucleotide sequencing of the 626-bp PCR product demonstrates the deletion of a single cytosine base (C-16698) from the Bgl II hexanucleotide recognition motif (AGATCT ->AGATT) within the exon (fig. 2). This deletion mutation alters the read ing frame of the eighth exon downstream of the deletion and so generates a premature translation termination codon (fig. 2). To date, the deletion of whole exons, and a point mutation which destroys the niRNA do nor splice site recognition motif for the sixth intron, represent the Cl-inhibitor gene muta tions that have been shown to cause type I HAE [4,5]. The exon eight mutation describ ed in this study therefore represents a third category of gene mutation that produces type I HAE. Although not described previously in type I HAE, this form of mutation is recog nised as a cause of other genetic diseases such as [5-thalassaemia and haemophilia B[8, 9],
Amino acid Patient Amino acid
A G A T C T T CAGGT T T C T G C G A T G C A G C A C C A G A C A G T G C T G G A A C T G ACAG D
L
Q
V
S
A
M
Q
H
Q
T
V
L
E
L
T
AGATTTCAGGTTTCTGCGATGCAGCACCAGACAGTGCTGGAACTGACAG D
F
R
F
L
R
C
S
T
R
Q
C
W
N
l Q Termination codon
Fig. 2. Nucleotide and amino acid sequences of the normal and mutant Cl-inhibitor alleles. The position of the C-16698 deletion and resultant frameshift is shown. Due to heterozygosity, doublets arc seen at each nucleotide position downstream of the frameshift in this direct sequencing reaction of the whole PCR product. The prema ture translation termination codon TGA is generated 40 nucleotides downstream of the frameshift. In addition a completely altered amino acid sequence is predicted 3' to the mutation as shown.
233
Downloaded by: Thomas Jefferson University Scott Library 147.140.27.100 - 8/24/2018 9:53:14 PM
Normal
1 Davis AE III: Cl -inhibitor and hereditary angioneurotic edema. Ann Rev Immunol 1988:6595-628. 2 Cicardi M. Bcrgamaschini L. Marasini B. Boccassini G. Tucci A. Agos tini A: Hereditary angioedema: An appraisal of 104 cases. Am J Med Sci 1982;284:2-9. 3 Kerr MA. Yeung-Laiwah AAC; CTinhibitor deficiency and angiooedema; in Whaley K (ed): Comple ment in Health and Disease. Lan caster. MTP Press. 1987. pp 53-78. 4 Stoppa-Lyonnct D, Carter PE. Meo T, Tosi, M: Clusters of intragenic Alu repeats predispose the human Cl-inhibitor locus to deleterious re arrangements. Proc Natl Acad Sci USA 1990;87:1551-1555.
5 McPhaden AR. Birnie GD, Whaley K; Restriction fragment length poly morphism analysis of the Cl-inhib itor gene in hereditary Cl-inhibitor deficiency. Clin Genet 1991:39:161171. 6 Saiki RK, Gyllenstein VB, Erlich HA: The polymerase chain reaction; in Davies KE (ed): Genome Analy sis. A Practical Approach. Oxford. IRL Press, 1988. pp 141-152. 7 Carter PE. Duponchcl C, Tosi M. Fothergill JE: Complete nucleotide sequence o f the gene for human Cl inhibitor with an unusually high density of Alu elements. Eur J Biochcm 1991:197:301-308.
8 Kazazian HH Jr. Boehm CD: Mo lecular basis and prenatal diagnosis of |S-thalassnemia. Blood 1988:72: 1107-1116. 9 Green PM. Bentley DR. Mibashan RS. Nilsson IM. Giannclli F: Molec ular pathology of haemophilia B. EM BO J 1989;8:1067-1072.
234
Siddique/McPhaden/McCluskcy' Whaley
Cl-inhibitor Deletion in Angio-Oedema
Downloaded by: Thomas Jefferson University Scott Library 147.140.27.100 - 8/24/2018 9:53:14 PM
References
