HUMAN MUTATION 1:428-431 (1992)
MUTATION IN BRIEF
Protein C Deficiency: Identification of a Novel Two-Base Pair Insertion and Two Point Mutations in Exon 7 of the Protein C Gene in Spanish Families Jost Manuel Soria, Jordi Fontcuberta, Montserrat Borrell, Xavier EstivilI, and Niiria &la* Molecular Genetics Departament, IRO Cancer Research Institute, Hospital Duran i Reynals, Barcelona, Spain (lM. . S., X.E., N.S.) and Haematology Departament, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain (J.F., M.B.) Communicated by Haig H. Kurarian, Jr
We have applied single-strand conformation polymorphism (SSCP) to the analysis of exon 7 of the anticoagulant protein C (PC) gene, in 13 PC-deficient Spanish families. Abnormal patterns were visualized in three samples from type I or quantitative PC deficient proposita. A previously undescribed mutation due to a TT insertion after nucleotide 6139, between codons Gly-142 and Arg-143 was found in one family. T h e mutation (6139,ins TT) should result in a frameshift with a stop at codon 156, which agrees with the presence of a type I or quantitative PC deficiency in the affected members of the family. T h e second mutation identified was a C to T transition at nucleotide 6274, 9 base pairs into intron G. This mutation (6274,C+T), found for the first time in a Spanish family, is identical to the previously characterized PC Sant Louis. T h e third mutation was a G to A transition that replaces arginine 1 7 8 with glutamine (178,R-Q). This is the third case of 178,R-Q mutation in 17 apparently unrelated Spanish families with type I PC deficiency. Furthermore, SSCP analysis allowed the detection of another previously described mutation in a PC-deficient Spanish family (178,R-W). 0 1992 Wiley-Liss, Inc. KEY WORDS:
Protein C deficiency, Frameshift mutation, Missense mutation, SSCP analysis
INTRODUCTION
Protein C (PC) is the vitamin K-dependent plasma zymogen of a serine protease that regulates blood coagulation by inactivating the procoagulant cofactors Va and VIIIa (Clouse and Comp, 1986; Greengard and Griffin, 1988). The physiological importance of the PC anticogulant pathway is illustrated by the relationship existing between hereditary PC deficiency and thromboembolic disease (Bovill et al., 1989). The human protein C gene consists of 9 exons, spanning about 11 kb of DNA on chromosome 2 (Foster et al., 1985; Plutzky et al., 1986; Rocchi et al., 1986). In a previous study (Sala et al., 1991) we found that three out of five unrelated type I or quantitative PC-deficient Spanish families had a mutation in exon 7 of the PC gene, affecting the codon for arginine 178. In two cases this arginine was replaced by glutamine (178,R-+Q), while in the third case it was replaced by tryptophan (178 ,R-+W) . 0 1992
WILEY-LISS,INC.
In order to study the presence of the 178,R+Q and 178,R-+W mutations in a further 13 PC-deficient Spanish familes, we developed conditions for single strand conformation polymorphism (SSCP) analysis of exon 7 of the PC gene. We report here the identification of a novel frameshift mutation in one patient, a further case of 178,R+Q mutation, and one case of the previously identified PC Sant Louis I (6274,C-tT) (Tsuda et al., 1991). MATERIALS AND METHODS Protein C-Deficient Families
Exon 7 of the PC gene has been analysed in the proposita of 13 Spanish families with hereditary P C deficiency (families PC7 to PC16 and PC18 to PC20). In all cases except one (PCZO), patients Received July 1, 1992; accepted September 2, 1992 'To whom reprint requestsicorrespondence should be addressed.
EXON 7 PROTEIN C GENE MUTATlONS IN SPANlSH FAMlLlES
429
94, 61, and 74°C for 30, 30, and 45 sec, respectively, were performed. Amplified samples were diluted 1/50 in distilled water and 1/2 in stop solution containing 10 mM NaOH and 6 pl of each dilution were loaded in a 6% acrylamide gel containing 5% glycerol in TBE. Electrophoresis was performed at 10 W for 24 hr a t room temperature. DNA SEQUENCING
DNA sequencing of PCR amplified fragments, purified according to Reitsma et al. (1991), was performed in an Applied Biosystems 370A DNA sequencer (Chill6n et al., 1992), using PC711 or PC7 12 primers.
FIGURE 1. SSCP analysis of
exon 7 of PC gene. The number of each lane corresponds to the PC deficient patient analysed. Abnormal bands are indicated by an arrow. (A) Lanes 1.1 and 1.2 correspond to the previously identified 178,R-W mutation in family PC1; lanes 4 and 6 correspond to the 178,R-Q mutation; lanes 2, 3, and 5 correspond to normal controls for exon 7. Lanes 7 to 20 correspond to the proposita of the 13 PC deficient families analysed in this study. (9) Further analysis of PC6 (178,R-tQ mutation) and PC15.
had been classified as heterozygous for a type I or quantitative PC deficiency, with reduced levels of PC antigen (ELISA Protein C, Boehringer Mannheim-Hemodiagnostica Stago, Germany) as well as amidolytic (Coamate protein C, KabiVitrum, Stockholm, Sweden) and anticoagulant (Protein C Reagent, coagulometric, Behringwerke AG, Marburg, Germany) activities. PC-deficient patients from family PC20 are heterozygous for a type I1 or qualitative PC deficiency, with normal levels of PC antigen but reduced PC amidolytic and anticoagulant activities. SSCP Analysis (Orita et al., 1989)
Five picomoles of each oligonucleotide primers
PC7 11
(5’-CTTGAACCCTGCACTGTGGC-
3’) and PC7 12 (5’-CGCTTCCCTCTCGGTTTCTG-3’) (Reitsma et al., 1991), 100 ng of DNA, and 70 pM of each dNTP in a final reaction volume of 10 p l (Chill6n et al., 1992) were used to PCR amplify a 355 bp ~ x - ~ ~ P - l a b e DNA l e d fragment containing the entire exon 7 of the PC gene, as well as its splice junctions. Thirty-five cycles at
RESULTS Figure 1 shows the results of SSCP analysis of exon 7 of the PC gene of the proposita of the studied families. Abnormal bands, different from those of control samples for normal exon 7 (lanes 2, 3, and 5), were seen for the previously characterized 178,R-+W mutation (lanes 1.1 and 1.2, Fig. IA), as well as in lanes 9 and 19, corresponding to patients from families PC9 and PC19, with unknown mutations. In lanes 4 (178,R-Q mutation) and 15 a very faint band was detected. Further analysis of PC6 (also known to have the 178,R+Q mutation) and PC15 confirmed the presence of a similar abnormal band in both families (Fig. 1B). Sequencing of the PCR amplified exon 7 of PC9, PC15, and PC19 showed that PC15 was heterozygous for a new case of 178,R-+Q mutation due to a G to A transition at nucleotide 6246 of the PC gene, according to the numbering of Foster et al. (1985) (Fig. 2a). PC19 was found to be het. erozygous for a C to T transition at nucleotide 6274, 9 base pairs into intron G (Fig. 2b). This mutation (6274,C-+T), identical to the one previously described as PC Sant Louis I (Tsuda et al., 1991), creates an alternative splice junction which results in a frameshift with a stop, 30 codons into exon 8. The presence of this mutation in patient PC19 was furthermore confirmed by restriction analysis with HphI of PCR amplified exon 7 (results not shown). DNA sequencing of the excised abnormal band from the propositus of PC9 in the SSCP gel showed the presence of a TT insertion between nucleotides 6139,G and 6140,A, corresponding to codons Gly- 142 and Arg- 143, respectively (Fig. 2c, SSCP band). The same insertion in the heterozygous state was seen after sequencing of the amplified exon 7 from genomic DNA of the pa-
430
SORIA ET AL.
L P€ 15a1
tient (Fig. 2c, PC9). This mutation (6139,ins TT) should result in a frameshift with a stop at codon 156 and eliminates a restriction site for MnlI. Further SSCP analysis of exon 7 from PC9 family members, as well as restriction analysis with MnlI, indicated the presence of the TT insertion in the sister and nephew of the propositus, both classified as PC deficient (results not shown). The wife and daughter of the propositus, with normal PC levels, were also normal for exon 7.
+
NORMAL
~A E D C E E ~ E A
A 8 6 EWE 8 8 A
DISCUSSION AGG CGG GGA
AGG C i G GGA
Arg Gly 177 178 179
Arg Gln Gly
plrg
NORMAL
GGGAGGCGAG
NORMAL
--0 6 8 C C T C C C
177 170 179
F€ 19.1
b
C GGGAGGTGAG
Fc
SSCP BAND
9.1
C 41
11 -I____
U 6 0 C C TI
A C C
c
o 0 Q C CTCCC,
We have used SSCP to analyse exon 7 of the PC gene of 13 PC-deficient Spanish families and found three different mutations related to type I or quanp titative PC deficiency. One of them is a new case of 1 7 8 , R j Q mutation, which is the third among 17 apparently unrelated type I PC-deficient Spanish families. The second, 6274,C+T, 9 bp into intron G, was found to be the same as PC Sant Louis (Tsuda e t al., 1991). The other mutation identified was a previously undescribed frameshift mutation due to a double T insertion between codons Gly-142 and Arg-143 (6139,ins TT mutation). Cosegregation of this mutation with PC deficiency in family members has been shown by SSCP and restriction analysis. The three mutations identified here were found in the heterozygous state in proposita who had suffered at least one episode of venous thrombosis. In family PC9, with the 6139,ins TT mutation, there is a clear history of recurrent venous thromboembolism in the propositus and his mother, while his affected sister and nephew remain asymptomatic. Affected patients with any of these three mutations had already been diagnosed as heterozygous for a type I PC deficiency, due to the presence in their plasma of similarly reduced PC antigen and activity levels of around 50%. The relationship between type I PC deficiency and the 178,R-+Q and 6274,C+T mutations has already been found by other authors (Grundy et al., 1991; Gandrille et
sequencing of exon 7 from the propositus of family PC15. The primer used was the direct one (PC711). The mutation is indicated by an arrow. (b) DNA sequencing of exon 7 from the propositus of family PC19. The primer used was the reverse one (PC712). The corresponding direct sequence is written underneath. The mutation is indicated by an arrow. (c) DNA sequencing of exon 7 from the excised abnormal allele on the SSCP gel (SSCP band), and from genomic DNA (PC9.1), of the propositus of family PC9. The primer used was the reverse one (PC712).The corresponding direct sequence is written underneath. The mutation is indicated by arrows. FIGURE 2. (a) DNA
GGG AGG CCC
CGGTPAGGCCC
G l y Arg P r o 1 4 2 1 4 3 144
G l y Leu Gly 1 4 2 1 4 3 144
EXON 7 PROTEIN C GENE MUTATIONS IN SPANISH FAMILIES
al., 1991; Tsuda et al., 1991). As far as we know, the 6139,ins TT mutation found in family PC9 is the first case described of an insertion in the PC gene causing PC deficiency. In this case the presence of a type I PC deficiency is explained by the fact that this insertion should result in a frameshift and a termination codon for the protein C synthesis.
ACKNOWLEDGMENTS We express our gratitude to the participants of the Spanish protocol for the biological study of venous thrombosis for providing us with blood samples from the PC-deficient patients analysed; to Drs. P.H. Reitsma, and R.M. Bertina for their comments and encouragement; to H. Kruyer for help with the manuscript; and to the Spanish “Die recci6n General de Investigacih Cientifica y Ticnica” for Grant DGICYT PM90-54.
REFERENCES Bovill EG, Bauer KA, Dickerman JD, Callas P, West B (1989) The clinical spectrum of heterozygous protein C deficiency in a large New England kindred. Blood 73:712-717. Chilldn M, Palacio A, Nunes V, Casals T, Giminez J, Estivill X (1992) Identification of a frameshift mutation (1609delCA) in exon 10 of the CFTR gene in seven Spanish cystic fibrosis patients. Hum Mut 1:75-76. Clouse LH, Comp PC (1986) The regulation of haemostasis: The protein C system. N Engl J Med 314:1298-1304. Foster DC, Yoshitake S, Davie EW (1985) The nucleotide see
431
quence of the gene for human protein C. Proc Natl Acad Sci USA 82~4673-4677. Gandrille S, Vidaud M, Aiach M, Alhenc-Gelas M, Fischer AM, Gouauk-Heilman M, Toulon P, Goossens M (1991) Six previously undescribed mutations in 9 families with protein C quantitative deficiency. Thromb Haemostas 65:646 (abstr). Greengard JS, Griffin ]H (1988) Protein C pathways. I n Hoffbrand AV (ed): Recent Advances in Haematology, Vol. 5. London: Churchill Livingstone, pp. 275-289. Grundy CB, Melissari E, Kakkar VV, Cooper DN (1991) A molecular genetic study of protein C deficiency. Thromb Haemostas 65:646 (abstr). Orita M, Hiroyuki I, Hiroshi K (1989) Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polimorphisms. Proc Natl Acad Sci USA 86: 2766-2770. Plutzky J, Hoskins JA, Long GL, Crabtree GR (1986) Evolution and organisation of the human protein C gene. Proc Natl Acad Sci USA 83:546-550. Reitsma PH, Poort SR, Allaart CF, Briet E, Bertina RM (1991) The spectrum of genetic defects in a panel of 40 Dutch families with symptomatic protein C deficiency type I: Heterogeneity and founder effects. Blood 78:890-894. Rocchi M, Roncuzzi L, Santamaria R, Archidiacono N, Dente L, Romeo G (1986) Mapping through somatic cell hybrids and cDNA probes of protein C to chromosome 2, factor X to chromosome 13 and al-acid glycoprotein to chromosome 9. Hum Genet 74:30-33. Sala N , Poort SR, Bertina RM, Soria JM, Fontcuberta J, Reitsma PH (1991) Identification of two deletions and four point mutations in the protein C gene of 6 unrelated Spanish patients with hereditary protein C deficiency. Thromb Haemostas 65: 1197 (abstr). Tsuda S , Reitsma P, Miletich J (1991) Molecular defects causing heterozygous protein C deficiency in 3 asymptornacic kindreds. Thromb Haemostas 65:647 (Abstr).
MUTATION IN BRIEF
Protein C Deficiency: Identification of a Novel Two-Base Pair Insertion and Two Point Mutations in Exon 7 of the Protein C Gene in Spanish Families Jost Manuel Soria, Jordi Fontcuberta, Montserrat Borrell, Xavier EstivilI, and Niiria &la* Molecular Genetics Departament, IRO Cancer Research Institute, Hospital Duran i Reynals, Barcelona, Spain (lM. . S., X.E., N.S.) and Haematology Departament, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain (J.F., M.B.) Communicated by Haig H. Kurarian, Jr
We have applied single-strand conformation polymorphism (SSCP) to the analysis of exon 7 of the anticoagulant protein C (PC) gene, in 13 PC-deficient Spanish families. Abnormal patterns were visualized in three samples from type I or quantitative PC deficient proposita. A previously undescribed mutation due to a TT insertion after nucleotide 6139, between codons Gly-142 and Arg-143 was found in one family. T h e mutation (6139,ins TT) should result in a frameshift with a stop at codon 156, which agrees with the presence of a type I or quantitative PC deficiency in the affected members of the family. T h e second mutation identified was a C to T transition at nucleotide 6274, 9 base pairs into intron G. This mutation (6274,C+T), found for the first time in a Spanish family, is identical to the previously characterized PC Sant Louis. T h e third mutation was a G to A transition that replaces arginine 1 7 8 with glutamine (178,R-Q). This is the third case of 178,R-Q mutation in 17 apparently unrelated Spanish families with type I PC deficiency. Furthermore, SSCP analysis allowed the detection of another previously described mutation in a PC-deficient Spanish family (178,R-W). 0 1992 Wiley-Liss, Inc. KEY WORDS:
Protein C deficiency, Frameshift mutation, Missense mutation, SSCP analysis
INTRODUCTION
Protein C (PC) is the vitamin K-dependent plasma zymogen of a serine protease that regulates blood coagulation by inactivating the procoagulant cofactors Va and VIIIa (Clouse and Comp, 1986; Greengard and Griffin, 1988). The physiological importance of the PC anticogulant pathway is illustrated by the relationship existing between hereditary PC deficiency and thromboembolic disease (Bovill et al., 1989). The human protein C gene consists of 9 exons, spanning about 11 kb of DNA on chromosome 2 (Foster et al., 1985; Plutzky et al., 1986; Rocchi et al., 1986). In a previous study (Sala et al., 1991) we found that three out of five unrelated type I or quantitative PC-deficient Spanish families had a mutation in exon 7 of the PC gene, affecting the codon for arginine 178. In two cases this arginine was replaced by glutamine (178,R-+Q), while in the third case it was replaced by tryptophan (178 ,R-+W) . 0 1992
WILEY-LISS,INC.
In order to study the presence of the 178,R+Q and 178,R-+W mutations in a further 13 PC-deficient Spanish familes, we developed conditions for single strand conformation polymorphism (SSCP) analysis of exon 7 of the PC gene. We report here the identification of a novel frameshift mutation in one patient, a further case of 178,R+Q mutation, and one case of the previously identified PC Sant Louis I (6274,C-tT) (Tsuda et al., 1991). MATERIALS AND METHODS Protein C-Deficient Families
Exon 7 of the PC gene has been analysed in the proposita of 13 Spanish families with hereditary P C deficiency (families PC7 to PC16 and PC18 to PC20). In all cases except one (PCZO), patients Received July 1, 1992; accepted September 2, 1992 'To whom reprint requestsicorrespondence should be addressed.
EXON 7 PROTEIN C GENE MUTATlONS IN SPANlSH FAMlLlES
429
94, 61, and 74°C for 30, 30, and 45 sec, respectively, were performed. Amplified samples were diluted 1/50 in distilled water and 1/2 in stop solution containing 10 mM NaOH and 6 pl of each dilution were loaded in a 6% acrylamide gel containing 5% glycerol in TBE. Electrophoresis was performed at 10 W for 24 hr a t room temperature. DNA SEQUENCING
DNA sequencing of PCR amplified fragments, purified according to Reitsma et al. (1991), was performed in an Applied Biosystems 370A DNA sequencer (Chill6n et al., 1992), using PC711 or PC7 12 primers.
FIGURE 1. SSCP analysis of
exon 7 of PC gene. The number of each lane corresponds to the PC deficient patient analysed. Abnormal bands are indicated by an arrow. (A) Lanes 1.1 and 1.2 correspond to the previously identified 178,R-W mutation in family PC1; lanes 4 and 6 correspond to the 178,R-Q mutation; lanes 2, 3, and 5 correspond to normal controls for exon 7. Lanes 7 to 20 correspond to the proposita of the 13 PC deficient families analysed in this study. (9) Further analysis of PC6 (178,R-tQ mutation) and PC15.
had been classified as heterozygous for a type I or quantitative PC deficiency, with reduced levels of PC antigen (ELISA Protein C, Boehringer Mannheim-Hemodiagnostica Stago, Germany) as well as amidolytic (Coamate protein C, KabiVitrum, Stockholm, Sweden) and anticoagulant (Protein C Reagent, coagulometric, Behringwerke AG, Marburg, Germany) activities. PC-deficient patients from family PC20 are heterozygous for a type I1 or qualitative PC deficiency, with normal levels of PC antigen but reduced PC amidolytic and anticoagulant activities. SSCP Analysis (Orita et al., 1989)
Five picomoles of each oligonucleotide primers
PC7 11
(5’-CTTGAACCCTGCACTGTGGC-
3’) and PC7 12 (5’-CGCTTCCCTCTCGGTTTCTG-3’) (Reitsma et al., 1991), 100 ng of DNA, and 70 pM of each dNTP in a final reaction volume of 10 p l (Chill6n et al., 1992) were used to PCR amplify a 355 bp ~ x - ~ ~ P - l a b e DNA l e d fragment containing the entire exon 7 of the PC gene, as well as its splice junctions. Thirty-five cycles at
RESULTS Figure 1 shows the results of SSCP analysis of exon 7 of the PC gene of the proposita of the studied families. Abnormal bands, different from those of control samples for normal exon 7 (lanes 2, 3, and 5), were seen for the previously characterized 178,R-+W mutation (lanes 1.1 and 1.2, Fig. IA), as well as in lanes 9 and 19, corresponding to patients from families PC9 and PC19, with unknown mutations. In lanes 4 (178,R-Q mutation) and 15 a very faint band was detected. Further analysis of PC6 (also known to have the 178,R+Q mutation) and PC15 confirmed the presence of a similar abnormal band in both families (Fig. 1B). Sequencing of the PCR amplified exon 7 of PC9, PC15, and PC19 showed that PC15 was heterozygous for a new case of 178,R-+Q mutation due to a G to A transition at nucleotide 6246 of the PC gene, according to the numbering of Foster et al. (1985) (Fig. 2a). PC19 was found to be het. erozygous for a C to T transition at nucleotide 6274, 9 base pairs into intron G (Fig. 2b). This mutation (6274,C-+T), identical to the one previously described as PC Sant Louis I (Tsuda et al., 1991), creates an alternative splice junction which results in a frameshift with a stop, 30 codons into exon 8. The presence of this mutation in patient PC19 was furthermore confirmed by restriction analysis with HphI of PCR amplified exon 7 (results not shown). DNA sequencing of the excised abnormal band from the propositus of PC9 in the SSCP gel showed the presence of a TT insertion between nucleotides 6139,G and 6140,A, corresponding to codons Gly- 142 and Arg- 143, respectively (Fig. 2c, SSCP band). The same insertion in the heterozygous state was seen after sequencing of the amplified exon 7 from genomic DNA of the pa-
430
SORIA ET AL.
L P€ 15a1
tient (Fig. 2c, PC9). This mutation (6139,ins TT) should result in a frameshift with a stop at codon 156 and eliminates a restriction site for MnlI. Further SSCP analysis of exon 7 from PC9 family members, as well as restriction analysis with MnlI, indicated the presence of the TT insertion in the sister and nephew of the propositus, both classified as PC deficient (results not shown). The wife and daughter of the propositus, with normal PC levels, were also normal for exon 7.
+
NORMAL
~A E D C E E ~ E A
A 8 6 EWE 8 8 A
DISCUSSION AGG CGG GGA
AGG C i G GGA
Arg Gly 177 178 179
Arg Gln Gly
plrg
NORMAL
GGGAGGCGAG
NORMAL
--0 6 8 C C T C C C
177 170 179
F€ 19.1
b
C GGGAGGTGAG
Fc
SSCP BAND
9.1
C 41
11 -I____
U 6 0 C C TI
A C C
c
o 0 Q C CTCCC,
We have used SSCP to analyse exon 7 of the PC gene of 13 PC-deficient Spanish families and found three different mutations related to type I or quanp titative PC deficiency. One of them is a new case of 1 7 8 , R j Q mutation, which is the third among 17 apparently unrelated type I PC-deficient Spanish families. The second, 6274,C+T, 9 bp into intron G, was found to be the same as PC Sant Louis (Tsuda e t al., 1991). The other mutation identified was a previously undescribed frameshift mutation due to a double T insertion between codons Gly-142 and Arg-143 (6139,ins TT mutation). Cosegregation of this mutation with PC deficiency in family members has been shown by SSCP and restriction analysis. The three mutations identified here were found in the heterozygous state in proposita who had suffered at least one episode of venous thrombosis. In family PC9, with the 6139,ins TT mutation, there is a clear history of recurrent venous thromboembolism in the propositus and his mother, while his affected sister and nephew remain asymptomatic. Affected patients with any of these three mutations had already been diagnosed as heterozygous for a type I PC deficiency, due to the presence in their plasma of similarly reduced PC antigen and activity levels of around 50%. The relationship between type I PC deficiency and the 178,R-+Q and 6274,C+T mutations has already been found by other authors (Grundy et al., 1991; Gandrille et
sequencing of exon 7 from the propositus of family PC15. The primer used was the direct one (PC711). The mutation is indicated by an arrow. (b) DNA sequencing of exon 7 from the propositus of family PC19. The primer used was the reverse one (PC712). The corresponding direct sequence is written underneath. The mutation is indicated by an arrow. (c) DNA sequencing of exon 7 from the excised abnormal allele on the SSCP gel (SSCP band), and from genomic DNA (PC9.1), of the propositus of family PC9. The primer used was the reverse one (PC712).The corresponding direct sequence is written underneath. The mutation is indicated by arrows. FIGURE 2. (a) DNA
GGG AGG CCC
CGGTPAGGCCC
G l y Arg P r o 1 4 2 1 4 3 144
G l y Leu Gly 1 4 2 1 4 3 144
EXON 7 PROTEIN C GENE MUTATIONS IN SPANISH FAMILIES
al., 1991; Tsuda et al., 1991). As far as we know, the 6139,ins TT mutation found in family PC9 is the first case described of an insertion in the PC gene causing PC deficiency. In this case the presence of a type I PC deficiency is explained by the fact that this insertion should result in a frameshift and a termination codon for the protein C synthesis.
ACKNOWLEDGMENTS We express our gratitude to the participants of the Spanish protocol for the biological study of venous thrombosis for providing us with blood samples from the PC-deficient patients analysed; to Drs. P.H. Reitsma, and R.M. Bertina for their comments and encouragement; to H. Kruyer for help with the manuscript; and to the Spanish “Die recci6n General de Investigacih Cientifica y Ticnica” for Grant DGICYT PM90-54.
REFERENCES Bovill EG, Bauer KA, Dickerman JD, Callas P, West B (1989) The clinical spectrum of heterozygous protein C deficiency in a large New England kindred. Blood 73:712-717. Chilldn M, Palacio A, Nunes V, Casals T, Giminez J, Estivill X (1992) Identification of a frameshift mutation (1609delCA) in exon 10 of the CFTR gene in seven Spanish cystic fibrosis patients. Hum Mut 1:75-76. Clouse LH, Comp PC (1986) The regulation of haemostasis: The protein C system. N Engl J Med 314:1298-1304. Foster DC, Yoshitake S, Davie EW (1985) The nucleotide see
431
quence of the gene for human protein C. Proc Natl Acad Sci USA 82~4673-4677. Gandrille S, Vidaud M, Aiach M, Alhenc-Gelas M, Fischer AM, Gouauk-Heilman M, Toulon P, Goossens M (1991) Six previously undescribed mutations in 9 families with protein C quantitative deficiency. Thromb Haemostas 65:646 (abstr). Greengard JS, Griffin ]H (1988) Protein C pathways. I n Hoffbrand AV (ed): Recent Advances in Haematology, Vol. 5. London: Churchill Livingstone, pp. 275-289. Grundy CB, Melissari E, Kakkar VV, Cooper DN (1991) A molecular genetic study of protein C deficiency. Thromb Haemostas 65:646 (abstr). Orita M, Hiroyuki I, Hiroshi K (1989) Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polimorphisms. Proc Natl Acad Sci USA 86: 2766-2770. Plutzky J, Hoskins JA, Long GL, Crabtree GR (1986) Evolution and organisation of the human protein C gene. Proc Natl Acad Sci USA 83:546-550. Reitsma PH, Poort SR, Allaart CF, Briet E, Bertina RM (1991) The spectrum of genetic defects in a panel of 40 Dutch families with symptomatic protein C deficiency type I: Heterogeneity and founder effects. Blood 78:890-894. Rocchi M, Roncuzzi L, Santamaria R, Archidiacono N, Dente L, Romeo G (1986) Mapping through somatic cell hybrids and cDNA probes of protein C to chromosome 2, factor X to chromosome 13 and al-acid glycoprotein to chromosome 9. Hum Genet 74:30-33. Sala N , Poort SR, Bertina RM, Soria JM, Fontcuberta J, Reitsma PH (1991) Identification of two deletions and four point mutations in the protein C gene of 6 unrelated Spanish patients with hereditary protein C deficiency. Thromb Haemostas 65: 1197 (abstr). Tsuda S , Reitsma P, Miletich J (1991) Molecular defects causing heterozygous protein C deficiency in 3 asymptornacic kindreds. Thromb Haemostas 65:647 (Abstr).
