Hum Genet (1991) 87:751-752
9 Springer-Verlag1991
D N A variants S s t l l polymorphic sites in the promoter region
of the human cystatin C gene Milagros Balbin and Magnus Abrahamson Department of Clinical Chemistry,Universityof Lund, UniversityHospital, S-22185 Lund, Sweden Received March, 5, 1991 / Revised March 19, 1991
Summary. By direct sequencing of polymerase chain reaction (PCR) amplified DNA from different individuals, three point mutations have been found in a 220-bp fragment from the promoter region of the human cystatin C gene. The three mutations are all localized within a short segment of 85bp on the same allele. One of the base substitutions results in the generation of a novel SstII restriction site and another in the loss of the commonly occurring SstII restriction site. A PCR-based assay for analysis of the two SstII sites was designed and used to demonstrate Mendelian inheritance of the polymorphism. This SstII restriction fragment lenght polymorphism offers a new probe-independent marker for chromosome 20 linkage studies.
Introduction Human cystatin C is a cysteine-proteinase inhibitor with widespread distribution in body fluids (Abrahamson et al. 1986). It plays an important role in the development of brain haemorrhage in patients suffering from hereditary cystatin C amyloid angiopathy (HCCAA), in which the protein is deposited as amyloid in the walls of the rupturing cerebral arteries (Cohen et al. 1983; Grubb et al. 1984). In a previous restriction fragment length polymorphism (RFLP) study with a cystatin C cDNA (Abrahamson et al 1987) as probe, a HCCAA-specific point mutation causing an amino acid substitution in cystatin C (Ghiso et al. 1986) has been demonstrated (Palsdottir et al. 1988). The human cystatin C gene, called CST3, has been sequenced entirely and assigned to chromosome 20 (Abrahamson et al. 1989, 1990).
Materials and methods By use of the polymerasechain reaction (PCR), we have amplified a 220-bp segmentin the putative promoter region of the cystatinC Offprint requests to: M. Balbin
gene, immediately upstream of the coding sequence of exon 1 (Fig. la). The primers used had sequences 5'-[GGGAATTC]TGGGAGGGACGAGGCGTI'CC-3'(024) and 5'-[GGTCTAGA]CCGGCCATGGTCGGCTAGGA-3'(028) (parts given in brackets are recognition sites for EcoRI and XbaI). Amplificationwas carried out for 30 cycles(96~ 1 min; 60~ 1min; 72~ 1min) in a Perkin Elmer-CetusThermal Cyclerusing 2.5 units of Taq polymerase in the buffer recommendedby the supplier (Perkin ElmerCetus).
Results and discussion During direct DNA sequencing of PCR products amplified from genomic DNA of different individuals, we observed base variability in three positions within the studied fragments; at positions -72 (A in the published sequence [Abrahamson et al. 1990], also C), -153 (T, also G) and -157 (G, also C) (Fig. la). Out of 12 directly sequenced PCR products from different individuals, 6 displayed just the normal sequence, i.e. were homozygous for the previously published sequence (here called the AA genotype), 5 exhibited heterozygosity in all three positions and one was homozygous for the three base changes (BB genotype). These results clearly indicated that the three mutations were present on the same allele (allele B). In addition, one of the observed base changes (-72) generated a new SstI1 restriction site and another change (-157) destroyed a commonly occuring SstlI site (see scheme in Fig. 1a), so that the distribution of these mutations could easily be studied in a larger number of samples by SstII digestion of PCR products. The sizes of the SstII fragments expected from the A allele, and from the B allele are given in Fig. la. Figure lb shows an agarose gel electrophoresis of SstII-digested PCR fragments amplified from six different individual DNAs; the three expected genotypes were indeed detected. The AA and BB genotypes gave a 172/48-bp and 134/86bp pattern, respectively, whereas the AB genotype gave a 172/134/ 86/48-bp pattern plus an 220-bp band corresponding to undigested heteroduplex DNA formed upon annealing of DNA strands of both A and B alleles.
752 1
Allele A
primer 024 ~
2
primer 028 CCGCGGTG Sst II 48
Allele B
-
-
CAGCGG 172
157
-153
Y
T
CCCCGGGG
q g
3
72
Y
CCGCGG Sst I1 IB
136
~
M e n d e l i a n i n h e r i t a n c e of this p o l y m o r p h i s m was demo n s t r a t e d in four families (26 meioses). T h e d i s t r i b u t i o n of the g e n o t y p e s studied in 67 u n r e l a t e d C a u c a s i a n individuals was 42 A A , 20 A B a n d 5 BB, with the f r e q u e n cies of alleles A a n d B b e i n g 0.71 a n d 0.29, respectively. F r o m the data o b t a i n e d with the SstII R F L P in the studied p o p u l a t i o n , it can be c o n c l u d e d that this polym o r p h i s m in the cystatin C p r o m o t e r region involves three l i n k e d m u t a t i o n s , since alleles carrying only o n e of the three base changes have n o t b e e n found. T h e described R F L P offers a n e w p r o b e - i n d e p e n d e n t m a r k e r for c h r o m o s o m e 20 linkage studies.
B
84
Acknowledgements. This work was supported by grants from the Segerfalk Foundation, the Medical Faculty of the University of Lund, the University of Oviedo (Spain), T. Nilson's Foundation and the Swedish Medical Research Council (project nos. 05196 & 09291). The authors thank Professor Anders Grubb for stimulating comments.
References
Fig.1. a Location of the PCR-amplified segment in the cystatin C gene, and the three mutations found within the segment. The human cystatin C gene is schematically illustrated at the top, with the exons numbered and shown as filled boxes. The amplified 220-bp fragments from the normal (A) and mutated (B) alleles are shown below. Nucleotide sequences around the mutations are given, with nucleotide numbering for the three base substitutions relating to the start site for cystatin C translation (+1 to +3 equals initiator methionine codon). The polymorphic SstII sites are underlined, and expected lengths of DNA fragments after SstII cleavage are indicated, b Agarose gel electropherogram of amplified and SstIIdigested DNA. PCR products were purified and concentrated by ethanol precipitation in the presence of ammonium acetate, digested with SstII and loaded on a 2% agarose gel. Individuals homozygous (AA and BB) and heterozygous (AB) for the two alleles were analysed. The sizes of relevant fragments from HaeIII-digested ~X174 DNA are indicated
Abrahamson M, Barrett AJ, Salvesen G, Grubb A (1986) Isolation of six cysteine proteinase inhibitors from human urine: their physicochemical and enzyme kinetic properties and concentrations in biological fluids. J Biol Chem 261:11282-11289 Abrahamson M, Grubb A, Olafsson I, Lundwall A (1987) Molecular cloning and sequence analysis of cDNA coding for the precursor of the human cysteine proteinase inhibitor cystatin C. FEBS Lett 216:229-233 Abrahamson M, Islam MQ, Szpirer J, Szpirer C, Levan G (1989) The human cystatin C gene (CST3), mutated in hereditary cystatin C amyloid angiopathy, is located on chromosome 20. Hum Genet 82 : 223-226 Abrahamson M, Olafsson I, Palsdottir A, Ulvsbfick M, Lundwall A, Jensson O, Grubb A (1990) Structure and expression of the human cystatin C gene. Biochem J 268 : 287-294 Cohen DH, Feiner H, Jensson O, Frangione B (1983) Amyloid fibril in hereditary cerebral hemorrhage with amyloidosis (HCHWA) is related to the gastro-entero-pancreatic neuroendocrine protein, 7-trace. J Exp Med 158:623-628 Ghiso J, Jensson O, Frangione B (1986) Amyloid fibrils in hereditary cerebral hemorrhage with amyloidosis of Icelandic type is a variant of 7-trace basic protein (cystatin C). Proc Natl Acad Sci USA 83 : 2974-2978 Grubb A, Jensson O, Gudmunsson G, Arnason A, L6fberg H, Maim J (1984) Abnormal metabolism of 7-trace microprotein. The basic defect in hereditary cerebral hemorrhage with amyloidosis. N Engl J Med 311 : 1547-1549 Palsdottir A, Abrahamson M, Thorsteinsson L, Arnason A, Olafsson I, Grubb A, Jensson O (1988) Mutation in cystatin C gene causes hereditary brain haemorrhage. Lancet I1:603-604
9 Springer-Verlag1991
D N A variants S s t l l polymorphic sites in the promoter region
of the human cystatin C gene Milagros Balbin and Magnus Abrahamson Department of Clinical Chemistry,Universityof Lund, UniversityHospital, S-22185 Lund, Sweden Received March, 5, 1991 / Revised March 19, 1991
Summary. By direct sequencing of polymerase chain reaction (PCR) amplified DNA from different individuals, three point mutations have been found in a 220-bp fragment from the promoter region of the human cystatin C gene. The three mutations are all localized within a short segment of 85bp on the same allele. One of the base substitutions results in the generation of a novel SstII restriction site and another in the loss of the commonly occurring SstII restriction site. A PCR-based assay for analysis of the two SstII sites was designed and used to demonstrate Mendelian inheritance of the polymorphism. This SstII restriction fragment lenght polymorphism offers a new probe-independent marker for chromosome 20 linkage studies.
Introduction Human cystatin C is a cysteine-proteinase inhibitor with widespread distribution in body fluids (Abrahamson et al. 1986). It plays an important role in the development of brain haemorrhage in patients suffering from hereditary cystatin C amyloid angiopathy (HCCAA), in which the protein is deposited as amyloid in the walls of the rupturing cerebral arteries (Cohen et al. 1983; Grubb et al. 1984). In a previous restriction fragment length polymorphism (RFLP) study with a cystatin C cDNA (Abrahamson et al 1987) as probe, a HCCAA-specific point mutation causing an amino acid substitution in cystatin C (Ghiso et al. 1986) has been demonstrated (Palsdottir et al. 1988). The human cystatin C gene, called CST3, has been sequenced entirely and assigned to chromosome 20 (Abrahamson et al. 1989, 1990).
Materials and methods By use of the polymerasechain reaction (PCR), we have amplified a 220-bp segmentin the putative promoter region of the cystatinC Offprint requests to: M. Balbin
gene, immediately upstream of the coding sequence of exon 1 (Fig. la). The primers used had sequences 5'-[GGGAATTC]TGGGAGGGACGAGGCGTI'CC-3'(024) and 5'-[GGTCTAGA]CCGGCCATGGTCGGCTAGGA-3'(028) (parts given in brackets are recognition sites for EcoRI and XbaI). Amplificationwas carried out for 30 cycles(96~ 1 min; 60~ 1min; 72~ 1min) in a Perkin Elmer-CetusThermal Cyclerusing 2.5 units of Taq polymerase in the buffer recommendedby the supplier (Perkin ElmerCetus).
Results and discussion During direct DNA sequencing of PCR products amplified from genomic DNA of different individuals, we observed base variability in three positions within the studied fragments; at positions -72 (A in the published sequence [Abrahamson et al. 1990], also C), -153 (T, also G) and -157 (G, also C) (Fig. la). Out of 12 directly sequenced PCR products from different individuals, 6 displayed just the normal sequence, i.e. were homozygous for the previously published sequence (here called the AA genotype), 5 exhibited heterozygosity in all three positions and one was homozygous for the three base changes (BB genotype). These results clearly indicated that the three mutations were present on the same allele (allele B). In addition, one of the observed base changes (-72) generated a new SstI1 restriction site and another change (-157) destroyed a commonly occuring SstlI site (see scheme in Fig. 1a), so that the distribution of these mutations could easily be studied in a larger number of samples by SstII digestion of PCR products. The sizes of the SstII fragments expected from the A allele, and from the B allele are given in Fig. la. Figure lb shows an agarose gel electrophoresis of SstII-digested PCR fragments amplified from six different individual DNAs; the three expected genotypes were indeed detected. The AA and BB genotypes gave a 172/48-bp and 134/86bp pattern, respectively, whereas the AB genotype gave a 172/134/ 86/48-bp pattern plus an 220-bp band corresponding to undigested heteroduplex DNA formed upon annealing of DNA strands of both A and B alleles.
752 1
Allele A
primer 024 ~
2
primer 028 CCGCGGTG Sst II 48
Allele B
-
-
CAGCGG 172
157
-153
Y
T
CCCCGGGG
q g
3
72
Y
CCGCGG Sst I1 IB
136
~
M e n d e l i a n i n h e r i t a n c e of this p o l y m o r p h i s m was demo n s t r a t e d in four families (26 meioses). T h e d i s t r i b u t i o n of the g e n o t y p e s studied in 67 u n r e l a t e d C a u c a s i a n individuals was 42 A A , 20 A B a n d 5 BB, with the f r e q u e n cies of alleles A a n d B b e i n g 0.71 a n d 0.29, respectively. F r o m the data o b t a i n e d with the SstII R F L P in the studied p o p u l a t i o n , it can be c o n c l u d e d that this polym o r p h i s m in the cystatin C p r o m o t e r region involves three l i n k e d m u t a t i o n s , since alleles carrying only o n e of the three base changes have n o t b e e n found. T h e described R F L P offers a n e w p r o b e - i n d e p e n d e n t m a r k e r for c h r o m o s o m e 20 linkage studies.
B
84
Acknowledgements. This work was supported by grants from the Segerfalk Foundation, the Medical Faculty of the University of Lund, the University of Oviedo (Spain), T. Nilson's Foundation and the Swedish Medical Research Council (project nos. 05196 & 09291). The authors thank Professor Anders Grubb for stimulating comments.
References
Fig.1. a Location of the PCR-amplified segment in the cystatin C gene, and the three mutations found within the segment. The human cystatin C gene is schematically illustrated at the top, with the exons numbered and shown as filled boxes. The amplified 220-bp fragments from the normal (A) and mutated (B) alleles are shown below. Nucleotide sequences around the mutations are given, with nucleotide numbering for the three base substitutions relating to the start site for cystatin C translation (+1 to +3 equals initiator methionine codon). The polymorphic SstII sites are underlined, and expected lengths of DNA fragments after SstII cleavage are indicated, b Agarose gel electropherogram of amplified and SstIIdigested DNA. PCR products were purified and concentrated by ethanol precipitation in the presence of ammonium acetate, digested with SstII and loaded on a 2% agarose gel. Individuals homozygous (AA and BB) and heterozygous (AB) for the two alleles were analysed. The sizes of relevant fragments from HaeIII-digested ~X174 DNA are indicated
Abrahamson M, Barrett AJ, Salvesen G, Grubb A (1986) Isolation of six cysteine proteinase inhibitors from human urine: their physicochemical and enzyme kinetic properties and concentrations in biological fluids. J Biol Chem 261:11282-11289 Abrahamson M, Grubb A, Olafsson I, Lundwall A (1987) Molecular cloning and sequence analysis of cDNA coding for the precursor of the human cysteine proteinase inhibitor cystatin C. FEBS Lett 216:229-233 Abrahamson M, Islam MQ, Szpirer J, Szpirer C, Levan G (1989) The human cystatin C gene (CST3), mutated in hereditary cystatin C amyloid angiopathy, is located on chromosome 20. Hum Genet 82 : 223-226 Abrahamson M, Olafsson I, Palsdottir A, Ulvsbfick M, Lundwall A, Jensson O, Grubb A (1990) Structure and expression of the human cystatin C gene. Biochem J 268 : 287-294 Cohen DH, Feiner H, Jensson O, Frangione B (1983) Amyloid fibril in hereditary cerebral hemorrhage with amyloidosis (HCHWA) is related to the gastro-entero-pancreatic neuroendocrine protein, 7-trace. J Exp Med 158:623-628 Ghiso J, Jensson O, Frangione B (1986) Amyloid fibrils in hereditary cerebral hemorrhage with amyloidosis of Icelandic type is a variant of 7-trace basic protein (cystatin C). Proc Natl Acad Sci USA 83 : 2974-2978 Grubb A, Jensson O, Gudmunsson G, Arnason A, L6fberg H, Maim J (1984) Abnormal metabolism of 7-trace microprotein. The basic defect in hereditary cerebral hemorrhage with amyloidosis. N Engl J Med 311 : 1547-1549 Palsdottir A, Abrahamson M, Thorsteinsson L, Arnason A, Olafsson I, Grubb A, Jensson O (1988) Mutation in cystatin C gene causes hereditary brain haemorrhage. Lancet I1:603-604
