HUMAN MUTATION 1:491-500 (1992)
RESEARCH ARTICLE
Two Novel Mutations Responsible for Hereditary Type I Protein C Deficiency: Characterization by Denaturing Gradient Gel E l e c t r ~ p h ~ r e ~ i ~ S. Gandrille, * M. Vidaud, M. Aiach, M. Alhenc-Gelas, A.M. Fischer, M. Gouault-Heilman, P. Toulon, J.N. Fiessinger, and M. Goossens INSERM U.91 and Laboratoire d'He'matologie, HGpital Henri-Mondm, 94010 Cre'teil (S.G., M.V., M.G.-H., M.G.), Group de Recherche sur la Thrombose, lNSERM CJF 91-01, Faculte' des Sciences Pharmaceutiques et Biologiques and Centre Claude Bernard de Recherche en Pathologie Vasculaire, HGpital Broussais, 75006 Paris (S. G.,M.A., M. A. -G., J. N. F.), Laboratoire d'He'matologie, HGpital Necker, 75015 Paris (A.M. F.), and Laboratoire d'He'mtologie, HGpital Cochin, 75014 Paris (I? T. 1, France Communicated by Jean-Claude Kaphn
Hereditary protein C (PC) deficiency is usually associated with a high risk of thrombosis. We report the results of a study undertaken to screen for molecular defects in families with hereditary quantitative PC deficiency. Using a strategy combining polymerase chain reaction amplification of selected gene fragments, denaturing gradient gel electrophoresis of the amplification products, and direct sequencing of fragments with altered melting behavior, we studied the PC gene exons and exodintron junctions of subjects with hereditary type I PC deficiency. Computer simulation of DNA melting was used to design several sets of primers, each containing a GC-clamp, permitting the complete analysis of each amplified exon sequence. Using this procedure, we identified two previously undescribed mutations located in exon VII: a C-to-T substitution generating a nonsense codon in place of Arg 157 in the mature PC and a G-to-A substitution converting Arg 178 to GIn. The two mutations were detected in, respectively, 3 and 2 apparently independent families. This strategy is therefore a valuable tool for screening patients, and the results emphasize its advantages over plasma assays in individuals with a family history of thrombosis. o 1992 Wiley-Liss, Inc. KEY WORDS:
Protein C deficiency, Mutations, Thrombosis
INTRODUCTION Protein C (PC) is a vitamin K-dependent plasma glycoprotein which is converted into a serine protease after activation by thrombin complexed to thrombomodulin, a vascular receptor. Protein S, another vitamin K-dependent protein, regulates the reaction by assembling protein S-protein C complexes on phospholipid surfaces. These complexes inactivate the two coagulation factors F VIII and F V, thus reducing thrombin generation (for review see Clouse and Comp, 1986; Esmon, 1989). The clinical expression of hereditary PC deficiency is variable. A mild fall in the PC concentration is associated with thrombotic complications in about 50% of apparently heterozygous individuals (Horellou et al., 1984; Bovill et al., 1989). T h e frequency of this dominant clinical 0 1992 WILEY-LISS,INC.
trait is estimated at 1 in 15,000 in the general population (Miletich, 1990). Patients with undetectable levels of circulating PC, thought to be either homozygotes or compound heterozygotes, develop massive thrombosis during the neonatal period (Marciniak et al., 1985). The parents and some family members of these homozygous subjects are usually asymptomatic but present heterozygous plasma phenotype (Marlar et al., 1989). The frequency of such asymptomatic heterozygous subjects is estimated at 1 in Received July 2, 1992; accepted September 30, 1992. *To whom reprint requestskorrespondence should be addressed. This work was presented as a preliminay report at the 32nd Annual Meeting of the American Society of Hematology, Boston, MA, December 1990, and has appeared in abstracted form in Blood 76:507a, 1990 (Abstr, Suppl 1).
492
GANDRILLE ET AL.
200 or 300 in the general population (Miletich et al., 1987). These data suggest that only 1 in 50-80 patients with heterozygous plasma phenotype develops clinical symptoms and, in the absence of objective criteria to differentiate dominant and recessive clinical traits, it is impossible to evaluate the thrombotic risk associated with a mild deficiency in a given patient. Moreover, the overlapping concentration ranges in normal subjects and deficient patients (Broekmans and Conard, 1988) make it difficult to identify plasma heterozygosity in patients with PC borderline values. Identification of the molecular abnormality at the gene level is the only way of clarifying the heterogeneous presentations in hereditary PC deficiency. Direct analysis of genomic DNA could help to establish the diagnosis and to identify patients at risk of thrombosis. The PC gene, which maps to chromosome 2 (Rocchi et al., 1986; Kato et al., 1988; Patracchini et al., 1989), was sequenced by Foster e t al. (1985) and comprises 9 exons (Plutzky et al., 1986). Until 1991, few examples of genomic abnormalities had been described in type I deficiency (characterized by a simultaneous decrease in PC antigen levels and activity). Apart from 3 cases of large insertions or deletions (Crabtree et al., 1985), most patients with heterozygous genotype had point mutations. A Trp 402 to Cys substitution and a nonsense codon at position 306 have been described by Romeo et al. (1987). Of 30 novel mutations recently reported (Reitsma et al., 1991; Gandrille et al., 1991; Grundy et al., 1991a; Yamamoto e t al., 1991; Tsuda e t al., 1991; Sala et al., 1991), only 6 were due to microdeletions or insertions (Yamamoto et al., 1991; Tsuda et al., 1991; Sala et al., 1991). Four families with homozygous genotype have also been described (Grundy et al., 1991b; Conard et al., 1992). In an attempt to identify the mutations responsible for PC deficiency in a large series of patients, we have developed a strategy for rapid screening of amplified gene fragments. It involves amplification of genomic DNA targets by the polymerase chain reaction (PCR), followed by denaturing gradient gel electrophoresis (DGGE). In carefully chosen conditions, fragments bearing mutations display a shift in mobility on DGGE. We applied this strategy to the analysis of exon VII in 25 families with type I PC deficiency and we detected 2 novel mutations in 5 apparently independent families. In 3 cases, a C-to-T substitution generated a nonsense codon at position 157 of the protein, while in the
Main Features in Terms of Thrombotic Risk of the 12 Subjects Presenting a Mutation in PC Exon VII
TABLE 1.
Subject
Year of birth
Clinical presentation (age in years at the first thrombotic event)
Family A
11.4 11.6 11.9
1953 1958 1971
Arterial thrombosis (32) Asymptomatic Asymptomatic
1941 1967 1968
Asymptomatic Asymptomatic Pulmonary embolism (20)
1945 1966 1968
Asymptomatic Asymptomatic Pulmonary embolism (21)
1951 1984 1941
Placental infarction (36) Asymptomatic Deep and superficial venous thrombosis (18)
Family B
1.2 11.1 11.2 Family C 1.2
11.2 11.3 Family D 1.2
11.1 Patient E
remaining 2 cases, a G-to-A substitution converted Arg 178 to GIn. MATERIALS AND METHODS Patients
Exon VII from subjects belonging to 25 families presenting with hereditary type I PC deficiency were studied. Five apparently independent families presented with a mutation in this exon. The main clinical features of these five family members are described in Table 1, and 4 of the 5 pedigrees are presented in Figure 1. Unfortunately, only the propositus of family E could be explored (an acquired PC deficiency was excluded by normal hemostasis test results). One of the 3 affected members in each family A or B presented unusual thrombosis. In the propositus of family A, several episodes of lower-limb arterial thrombosis with no apparent cause were observed. Subject 11.2 of family B suffered a pulmonary embolism after starting oral contraception. In family C, patient 11.3 suffered from pulmonary embolism at the age of 21 years, whereas the other 2 patients were asymptomatic. The propositus of family D (1.2) was investigated following a fetal death due to placental infarction. Patient E had presented recurrent deep-vein thrombosis since the age of 18 years. Seven of the 12 heterozygous patients were asymptomatic; the first thrombotic complication in the other 5 patients (Table 1) occurred between
TWO NOVEL MUTATIONS IN TYPE I PC DEFICIENCY
493
FAMILY A
I 1
6
f
7
8
9
53%
FAMILY C
FAMILY B 94 010
100%
65%
55 010
I 19
2
FAMILY D
41 % FIGURE 1. Pedigrees of families with a mutation in exon VII.
Propositi are indicated by arrows. PC levels (percentages) were measured using the 3 different assay kits described in Materials and Methods except for 1 patient (*antigen level).
the ages of 18 and 26 years, in accordance with previous findings (Marlar, 1985; Broekmans,
1988). The diagnosis of PC deficiency was based o n low PC activities, measured using an amidolytic assay (Stachrom Prot C, Stago, Asniere, France, or Berichrom Behring, Rueil, France), a coagulation assay (Staclot, Stago), and low antigen levels (Asserachrom Prot C, Stago). The normal range was 70-130% in the 3 commercial techniques. Venous blood samples were collected in 0.11 M trisodium citrate (1:lO) for PC assays and the plasma was kept frozen until use. Blood was collected in ethylene diamine tetracetic acid for the DNA studies and kept at VC. Leukocytes were isolated within 48 hr and stored frozen until DNA extraction.
As the results of the 3 kits were similar, only the results obtained with the amidolytic assay are shown. 00 = normal male/female; 0 @ = subjects bearing a mutation; El 0 = not studied; = deceased.
Molecular Biology Techniques Materia1s Themus aquaticus (Taq) polymerase (5 U/p,l) was from Perkin-Elmer Cetus Instruments (Norwalk, CT). The deoxynucleotides deoxyadenosine triphosphate (dATP), deoxycytosine triphosphate (dCTP), deoxythymidine triphosphate (dTTP), and deoxyguanosine triphosphate (dGTP) were from Pharmacia Fine Chemicals (Uppsala, Sweden). The OPC columns were from Applied Biosystems (Roissy, France). The sequenase kit was from the U.S. Biochemical Corporation (Cleveland, OH) and the Centricon 100 apparatus from Amicon (Denver, CO). C X - ~ ~ S ( ~ A and T PY) - ~ ~ P (dATP) were from Amersham (Buckinghamshire,
UK).
494
GANDRILLE ET AL.
TABLE 2. Oligonucleotide Primers for
Exon
I I1 I11
IV ivsb D
V VI
VII VIIl IX
PCR Amplification of PC Exons and Allele-SpecificOligonucleotide (ASO)Probes
Primer name PCPROMA GCPCPROMB GCPC 2A PC 2B GCPC 3 PC 3A PC 3B PC 45A PC 45B PC 6A GCPC 6B PC 7A GCPC 7B GCPC 8A PC 8B PC 9A1 GCPC 9A2 GCPC 9B1 PC 9B2 GCPC 9C1 PC 9C2 PC 9c3
AS0 name 157 N CGA 157 M TGA 178 N CGG 178 M CAG
Oligonucleotide sequences 5’-AGAGGTGGGCTTCGGGCAGA-3’
5’4G/C),,-GGACCTGAGACTGTGGCTGC-3‘ 5’4G/C),,-GCAGAGCAGCAGCGGGGGTA-3’ 5’-GAGGCCACAAGGGTCCCGGG-3‘ 5’-(G/C),o-TGCTTCCTCAGACCCCCTCA-3’
5’-CCTCGAAGTCACAGATCTCC-3‘ 5’-TCGAGGCCCCTGCTGGTTAC-3’ 5’-ACACCGGCTGCAGGAGCCTG-3‘
5’-TGCTGGTGCCGCGCCCCCAA-3’ 5‘-GGCGCGGCACCAGCACCAGC-3‘
5’-(G/C),,-AACCCTCCTGAGCCCCCCGC-3’ 5’-GACCAAGACAGGAGGGCAGT-3‘ 5’4G/C),,-CCCAGCACGTGAGCAGCCGG-3’
5’4G/C),o-ATATGAAACCCAGGTGCCCT-3’
5’-TCCCCCTACCCAGGCCTCTG-3’ 5’-TGGGTGCACAGTCTCCGGGT-3‘ 5’4G/C),,-GGGAGGCAGATGGGCACTAT-3’ 5‘4G/C),,-GAGCACCACCGACAATGACA-3’ 5’-GCATCCTGCCGGTCCCCGAG-3’
5’4G/C),,-TGAGCAACATGGTGTCTGAG-3’ 5’-CGCCGTAGTTGTGAAGGAGC-3’ 5’-GCTTGTTACATGTCCCTTTA-3’
Nucleotide numbering” - 1637 to - 1618 - 1268 to - 1287 -72 to -53 111 to 92 1256 to 1275 1465 to 1446 1565 to 1546 2921 to 2940 3270 to 3289 3275 to 3294 3546 to 3527 6055 to 6074 6303to 6284 7063 to 7082 7308 to 7289 8247 to 8266 8551 to 8532 8468to 8487 8779 to 8760 8719 to 8738 8889 to 8870 9043 to 9024
Sequence 5’-TACCCTGAAACGAGACACAGA-3’
5‘-TCTGTGTCTCATTTCAGGTGA-3’
5’-GCTGTCTCCCCGCCTGGTCAT-3’ 5’-ATGACCAGGCAGGGAGACAGC-3’
”Nucleotidenumbering as in Foster et al. (1985). bivs = intervening sequence.
Methods
DNA was extracted from the patients’ leukocytes and purified as previously described (MolhoSabatier et al., 1989). Oligonucleotides were synthesized on a gene assembler (381A DNA synthesizer, Applied Biosysterns). The 20-mers (i.e., PC7) were purified by ethanol precipitation, while the GC-clamps (i. e., the 55-mers GCPC7 containing a 35 nucleotide GC-clamp) were purified on an OPC column prior to ethanol precipitation. General strategy
Enzymatic amplification of each PC gene exon and its splice junctions was performed essentially as described by Saiki e t al. (1988), using a set of oligonucleotides comprising a 20-mers and a primer to which a G C-rich sequence (GCclamp) comprising various numbers of nucleotides was added (Myers et al. , 1985a,b; Sheffield e t al. , 1989). This GC-rich region allows amplified fragments to be screened for sequence variations by
+
DGGE. All primer sequences are presented in Table 2 and their locations are presented in Figure 2. The locations of the PCR primers were chosen using the computer programs MELT 87 and SQHTX, written and kindly provided by Drs. Lerman and Silverstein (1987). These softwares simulate the melting behavior of DNA fragments according to their nucleotide sequence and base composition. The information thus acquired was used to select positions for PCR primers allowing the generation of fragments suitable for DGGE analysis and to determine the range of denaturant concentrations and electrophoresis times giving maximum gel resolution. In every instance, one of the amplification primers carried at its 5‘ end an additional G C-rich nucleotide fragment in order to create a high temperature melting domain (Sheffield et al., 1989) and to position the sequence of interest within the first melting domain. Because of their length, exons 111 and IX were studied using 2 and 3 sets of primers, respectively. DGGE could not be used for exons IV and V be-
+
TWO NOVEL MUTATIONS IN TYPE I PC DEFICIENCY IVS A
IVS B
IVS
C
-
IVS E
IVS F
495
IVS G
PC9C2
4
Pc9c3 GCPCPA P C P B
pc3B
L PCBA
s C 6 B
GCPCBA
EBB
GCPC~BI
FC~BZ
FIGURE2. Location of the amplification primers on the PC gene. The GC-clamps are sym-
bolized by the open bars.
90
cause of their very high melting temperatures. These exons were studied for each patient by direct sequencing after asymmetric PCR.
GCPC7
DGGE and sequencing of the DNA domain bearing the mutation
PC gene exon VII amplification was performed using PC7 and GCPC7 as extension primers. As shown in Figure 3, PC7 was located in IVS F and GC-PC 7 in IVS G. Amplification was performed as follows: each PCR mixture contained 30 pmol of each primer, 200 p M of each dNTP, 1 pg of genomic DNA, 1 x PCR buffer [I0 mM Tris-HC1, pH 8.3, 50 mM KC1, 1 mM MgCl,, 0.01% (w/v) gelatin], and 2.5 U of Tag polymerase in a final volume of 100 ELI. The reactions were run in a Perkin-Elmer Cetus DNA thermal cycler. The thermal profile included 5 min denaturation at 94"C, followed by 30 cycles consisting of denaturation for 1 min at 94"C, annealing for 1 min at 60"C, and extension for 1 min at 72°C. Samples were held at 72°C for 10 min, and the PCR cycles were followed by 10 min denaturation at 94°C and 45 min at 56°C to favor heteroduplex formation. The 284-bp PCR product obtained was analyzed on a 6% polyacrylamide gel. 0 DGGE was performed as described by Attree et al. (1989). Amplified DNA fragments were subjected to electrophoresis for 5 hr at 160 V in a 6.5% polyacrylamide gel containing a 30-80% denaturant gradient (100% denaturant = 7 M urea and 40% formamide in TAE buffer) and stained with e thidium bromide. 0 T h e asymmetric PCR method developed by Gyllensten and Erlich (1988) was performed using 5 pmol of PC7 and 50 pmol of GCPC7, as described above, except that the thermal profile involved 60 cycles. This reaction leads to the preferential amplification and enrichment of the noncoding strand of exon VII. The single-stranded templates were sequenced using the sequenase kit with PC7 as the sequencing primer. Oligoprobe hybridization: To confirm the
a2 (7
2
5 78
74
1 0
50 100 150 200 250 300 BASE POSITION
FIGURE 3. Melting map of PC exon VII. Positions of primers PC7 and GCPC7 are indicated. The GC-clampis symbolized by the open bar.
mutations detected by direct sequencing, four 21mer oligonucleotides were synthesized (Table 2); 157 N CGA and 178 N CGG correspond to the wild-type sequence, and 157 M TGA and 178 M CAG correspond to the identified mutations. Amplified exon VII DNA fragments from each member of families A-E and from normal subjects were blotted onto nitrocellulose membranes, hybridized with the 5 '-end labeled allele-specific oligonucleotide (ASO) probes, and washed in stringent conditions. RESULTS Using DGGE, we analyzed all the coding regions and splice junctions of the PC gene. For exons 1-111 and VI-IX, computer prediction of DNA melting behavior enabled us to design a set of 2 primers. The first contained a GC-rich region (GC-clamp) at its 5' end, permitting the complete analysis of each amplified PC gene exon and its flanking splice-site sequences by DGGE. The
496
GANDRILLE ET AL.
FIGURE4. DGGE patterns of amplified PC exon VII with abnormal melting behavior from subjects of
families with the Arg 178 to Gln mutation (a) or the Arg 157 to nonsense codon (b). Subjects are designated by the numbering system used in Figure 1. NS = normal subject.
other exons (IV and V) were analyzed by direct sequencing after asymmetric PCR amplification. In the 5 families with mild PC deficiency depicted in Figure 1, the only abnormality was located in exon VII. Figure 3 depicts the position of the primers (PC7 and GCPC7) used to amplify a 284-bp fragment. The melting curve of the resulting amplified fragment confirmed that the sequence of interest (exon VII and its associated splice region) was located within the first melting domain of the DNA fragment. Two different DGGE patterns were observed, suggesting the existence of 2 different exon VII mutations: 1 in families A and B and 1 in families
GE. The normal and mutant homoduplex fragments were not separated in families A and B, but the presence of heteroduplexes (upper 2 bands) was typical of a nucleotide substitution in 1 of the alleles (Fig. 4a). Heteroduplexes, generated during the last step of PCR, are due to mismatches of normal alleles with mutant alleles and melt at a lower denaturant concentration. Patients 11.6 and 11.9 (family A) and 1.2 (family B) were carriers, since their abnormal DGGE pattern was identical to that of the propositus.
In families C and D (Fig. 4b) and in patient E, the DGGE patterns contained 4 bands, with the upper 2 bands constituting a very close doublet. These patterns are also typical of heterozygosity: band 4 migrated like DNA fragments from normal subjects and represents normal homoduplexes. Band 3 corresponded to homoduplexes of the mutant allele. Bands 1 and 2 resulted from the generation of heteroduplexes. To check that the observed abnormalities were not neutral polymorphisms, we tested 60 individuals with normal PC levels; the DGGE pattern was normal in all cases. The genomic abnormalities were further characterized by direct sequencing of the singlestranded fragment obtained by asymmetric amplification. In families A and B (Fig. 5a), a G-to-A transition converted Arg 178 to Gln. In the other three families, a C-to-T substitution generated a nonsense codon in place of the Arg at position 157 (Fig. 5b). Hybridization of amplified exon VII fragments with AS 0 corresponding to either the normal or abnormal sequences (Fig. 6) confirmed that all the patients with PC deficiency were heterozygous for the defect, as both normal and mutant AS0 hybridized with amplified DNA.
TWO NOVEL MUTATIONS IN TYPE 1 PC DEFICIENCY
497
FIGURE5. (a) Sequence of exon VII of patients with the Arg 178 to Gln mutation. As the patients were heterozygous for the mutation, both normal (CGG) and mutated (CAG) codons are present. (b) Sequence of exon VII of patients with the Arg 157 to nonsense mutation. As the patients were heterozygous for the mutation, both normal (CGA) and mutated (TGA) codons are present.
DISCUSSION Some of the difficulties encountered in the diagnosis of hereditary PC deficiencies can be overcome by using DNA analysis, and DGGE proved to be a valuable tool in this setting. This electrophoretic method enables DNA fragments to be separated according to changes in the melting properties produced by simple nucleotide substitutions (Fischer and Lerman, 1983; Myers et al., 198%). The technique permitted rapid screening of our patients, restricting the need for DNA sequencing to the samples showing abnormal electrophoretic mobility. We detected 2 novel mutations in exon VII which would alter expression of the protein in some of our patients. A missense mutation (in 2 families) or a nonsense codon (in the other 3 fam-
ilies) were located in this critical domain which contains 2 cleavage sites essential for correct maturation of the protein and its activation by thrombin. In the former case, the mutation led to the replacement of the Arg at position 178 by a Gln. PC is a vitamin K-dependent zymogen presenting structural similarities with clotting factors 11, VII, IX, and X. Arg 178 is located within a domain containing several highly conserved amino acids and is adjacent to Gly 179, a residue found at this position in most vitamin K-dependent proteins (when sequences are aligned). In factor IX, the substitution of Gln 191, which is located immediately after the corresponding Gly, is associated with severe antigen-negative hemophilia B (Giannelli e t al., 1990). The Arg 178 to Gln muta-
498
-
GANDRILLE ET AL.
normal despite a borderline PC level. These results confirm that the available plasma assays (3 of NS 12 I 1 112 NS NS 115 116 117 118 119 which were used in this study) can often fail to 0 ~ 0 ~ 0 0 a ~ 178 0 N C0G G 0 identify PC deficiency in heterozygous subjects. Although our population of patients is too small 0 0 0 0 178 M CAG to draw any conclusion with regard to phenotype/ genotype correlation, some of the data deserve attention. In both families bearing the Arg 178 to Gln mutation, some of the subjects had borderline plasma PC values. In subject 11.5 of family A, the FAMILY D FAMILY C Subject I 2 Subject E DNA study enabled us to exclude the presence of / the mutation. In family B, the propositus (11.2) NS I 1 12 112 113 had a PC level of 72% in the amidolytic assay and 157NCGA 0 0 0 0 0 0 65% in the immunoenzymatic assay. Unfortunately, no samples were available for DNA studies for this patient. The mother carried the mutation, 8 0 0 0 157MTGA despite borderline PC levels with respective values FIGURE 6. Dot blots of samples from family members with of 65 and 78% with the 2 assays. It is possible that normal (157 N CGA or 178 N CGG) and mutant (157 M TGA the Arg 178 to Gln mutation would lead to an or 178 M CAG) probes. For details, see Materials and Methincreased turnover of PC or render it unstable, ods. resulting in a moderate decrease in its plasma level and a thrombotic tendency. tion is therefore likely to have a deleterious effect The mutation observed in the other 3 families on PC folding. The fact that the DGGE pattern gave rise t o a stop codon which was associated with characteristic of this mutation was not observed in clearly low levels of both PC activity and antigen amplified DNA fragments from 60 control subjects in all the patients explored, regardless of the assays makes it unlikely that this nucleotide substitution used. This mutation introduces a nonsense codon is a polymorphism. The value of DNA analysis is at position 157 which gives rise to a truncated illustrated by the fact that 1 of these patients famprotein lacking two-thirds of the polypeptide ily B, 11.2 was considered normal in terms of the chain. Two hypotheses may explain the absence of plasma assay results whereas he carried the mutathe abnormal protein in the circulation: either the tion. In another subject with no thrombotic commRNA is unstable and thus cannot be translated plications family A, 11.5, the DGGE pattern was into protein or the truncated protein is not seFAMILY B
FAMILY A
I
I
14
I
I1
Ill
IV
v
VII
VI
lea im
147
vK
IX
Vlll
F P C G R P w KQM E K KQS H L KQDT E
1c
1
STOP
DQEDQv D
~ I D GLK M T RQG D s P w Q
11 L W
1
Q
w
FIGURE 7. Schematic representationof the mutations described on CpG of the PC gene exon VI1. The size of the entire gene is 11.6 kb. Sizes of the exons are indicated in base pairs below each exon. Amino acids correspondingto hot-spots for mutation are circled.
TWO NOVEL MUTATIONS IN TYPE I PC DEFICIENCY
creted. However, it is interesting to note that in the 3 family C members bearing the mutation, PC levels varied over a wide range. The 2 mutations described here involve CpG dinucleotides which are likely to be hot-spots for mutation in vertebrates (Bird, 1980) by methylation-mediated deamination of 5-methylcytosine, the most frequent cause of recurrent mutation in man (Cooper and Krawczak, 1990). Four previously described mutations, Arg 169 to Trp causing type I1 qualitative hereditary PC deficiency (Matsuda et al., 1987; Grundy et al., 1989), and 3 mutations responsible for quantitative deficiencies, Arg 152 + Cys, Pro 168 + Leu, and Arg 178 + Trp (Gandrille et al., 1991; Reitsma et al., 1991; Conard et al., 1992), also involved CpG dimers. Therefore, 5 of the 6 CpG dimers present in exon VII have so far been shown to be the sites of mutations, as summarized in Figure 7. These hot-spots for mutation may explain the recurrence of the mutations of our families; however, as our patients were all European Caucasian, 1 family being Portuguese, and the others being French families, we cannot exclude an identity by descent, as described in 3 families by Reitsma et al. (1991).
ACKNOWLEDGMENTS
S.G. was the recipient of a grant from La Fondation pour la Recherche Medicale. This work was supported by 1’Institut National pour la SantC et la Recherche Medicale (INSERM) , by 1’Association Claude Bernard, and by a grant from 1’Association Franqaise contre les Myopathies. We thank Drs. Lerman and Silverstein, who kindly provided the computer programs MELT 87 and SQHTX. REFERENCES Attree 0, Vidaud D, Vidaud M, Amselem S,Lavergne JM, Goossens M (1989) Mutations in the catalytic domain of human coagulation factor IX: Rapid characterization by direct genomic sequencing of DNA fragments displaying an altered melting behavior. Genomics 4:266-272. Bird AP (1980) DNA methylation and the frequency of CpG in animal DNA. Nucleic Acids Res 8:1499-1504. 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. Broekmans AW, Conard] (1988) Hereditary protein C deficiency. In Bertina RM (ed): Protein C and Related Proteins. New York: Churchill Livingstone, pp 160-181. Clouse LH, Comp PC (1986) The regulation of hemostasis: The protein C system. N Engl J Med 314:1298-1304. Conard J , Horellou MH, Van Dreden P, Samama M, Reitsma PH, Poort S,Bertina RM (1992) Homozygous protein C deficiency with late onset and recurrent coumarin-induced skin necrosis. Lancet 339: 743-744. Cooper DN, Krawczak M (1990) The mutational spectrum of sin-
499
gle base-pair substitutions causing human genetic disease: Patterns and predictions. Hum Genet 85:55-74. Crabtree GR, Plutzky J, Marler R, Bertina RM, Broekmans AW, Griffin J, Zarcharski L, Gruppo R, Sala N, Long G (1985) The range of genotypes underlying human protein C deficiency. Thromb Haemostas 5456 (Abstr S331). Esmon CIT (1989) The roles of protein C and thrombomodulin in the regulation of blood coagulation. J Biol Chem 264:47434746. Fischer SG, Lerman LS (1983) DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels: Correspondence with melting theory. Proc Natl Acad Sci USA 80:1579-1583. Foster DC, Yoshitake S, Davie EW (1985) The nucleotide sequence 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, Gouault-Heilman M, Toulon P, Goossens M (1991) Six previously undescribed mutations in 9 families with protein C quantitative deficiency. Thromb Haemostas 65:646 (Abstr 3). Giannelli F, Green PM, High KA, Lozier JN, Lillicrap DP, Ludwig M, Olek K, Reitsma PH, Goossens M, Yoshioka A, Sommer S, Brownle G G (1990) Haemophilia B: Data base of point mutations and short additions and deletions. Nucleic Acids Res 18:4053-4059. Grundy CB, Chitolie A, Talbot S,Bevan I), Kakkar V, Cooper DN (1989) Protein C London 1: A recurrent mutation at Arg 169 (CGG+TGG) in the protein C gene causing thrombosis. Nucl Acids Res 17:10513. Grundy CB, Melissari E, Kakkar VV, Cooper DN (1991a) A molecular genetic study of protein C deficiency. Thromb Haemostas 65:646 (Abstr 2). Grundy CB, Lindo V, Kakkar VV, Melissari E, Scully MF, Cooper DN (1991b) Late-onset homozygous protein C deficiency. Lancet 338:575-576. Gyllensten UB, Erlich H A (1988) Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequending of the HLA-DQA locus. Proc Nit1 Acad Sci USA 85:7652-7656. Horellou MH, Conard J, Bertina RM, Samama M (1984) Congenital protein C deficiency and thrombotic disease in nine French families. Br Med J 289:1285-1287. Kato A, Miura 0 , Sumi Y, Aoki N (1988) Assignment of the human protein C gene (PROC) to chromosome region 2q14---q21 by in situ hybridization. Cytogen Cell Genet 47: 46-47. Lerman LS, Silverstein K (1987) Computational simulation of DNA melting and its application to denaturing gradient gel electrophoresis. Methods Enzymol 155:482-501. Marciniak E, Wilson HD, Marlar RA (1985) Neonatal purpura fulminans: A genetic disorder related to the absence of protein C in blood. Blood 65:15-20. Marlar RA (1985) Protein C in thromboembolic disease. Semin Thromb Haemostas 11:389-395. Marlar RA, Montgomery RR, Broekmans AW, and the Working Party (1989) Diagnosis and treatment of homozygous protein C deficiency. J Pediatr 114:528-534. Matsuda M, Sugo T, Sakata Y, Murayama H, Mimuro J, Tanabe S, Yoshitake S (1987) A thrombotic state due to an abnormal protein C. N Engl J Med 319:1265-1268. Miletich JP, Sherman L, Broze G (1987) Absence of thrombosis in subjects with heterozygous protein C deficiency. New Engl J Med 317:991-996. Miletich JP (1990) Laboratory diagnostic of protein C deficiency. Semin Thromb Haemostas 16:169-1 76. Molho-Sabatier P, Aiach M, Gaillard I, Fiessinger IN,Fischer
500
GANDRILLE ET AL.
AM, Chadeuf G, Clauser E (1989) Molecular characterization of seven AT 111 variants using PCR. Identification of a new mutation: Ala 384-Pro. J Clin Invest 84:1236-1242. Myers RM, Fischer SG, Lerman LS, Maniatis T (1985a) Nearly all single base substitutions in DNA fragments joined to a GCclamp can be detected by denaturing gradient gel electrophoresis. Nucleic Acids Res 13:3131-3145. Myers RM, Fischer SG, Maniatis T, Lerman LS (1985b) Modification of the melting properties of duplex DNA by attachment C-rich DNA sequence as determined by denaturing of a G gradient gel electrophoresis. Nucleic Acids Res 13:3111-3129. ) Myers RM, Lumelsky N, Lerman LS, Maniatis T ( 1 9 8 5 ~ Detection of single base substitutions in total genomic DNA. Nature 3 13495-497. Patracchini P, Aiello V, Palazzi P, Calzolari E, Bernardi F (1989) Subclonalization of the human protein C gene on chromosome 2q13-ql4. Hum Genet 81~191-192. Plutzky 1, Hoskins JH, Long GL, Crabtree OR (1986) Evolution and organization of the human protein C gene. Proc Natl Acad Sci USA 83546-550. 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 cY1-glycoprotein to chromosome 9. Hum Genet 74:30-33.
+
Romeo G, Hassan HJ, Staempfli S, Roncuzzi L, Cianetti L, Leonardi A, Vicente V, Mannucci PM, Bertina RM, Peschle C, Cortese R (1987) Hereditary thrombophilia: Identification of nonsense and missense mutations in the protein C gene. Proc Natl Acad Sci USA 84:2829-2832. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487-491. Sala N , Poort SR, Bertina RM, Soria JM, Fonrcuberta 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 1809). Shefield VC, Cox DR, Lerman LS, Myers RM (1989) Attachment of a 40-base-pair G C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes. Proc Natl Acad Sci USA 86~232-236.
+
Tsuda S, Reitsma P, Miletich J (1991) Molecular defect causing heterozygous protein C deficiency in three asymptomatic kindreds. Thromb Haemostas 65:647 (Abstr 7). Yamamoto K, Tanimoto M, Matsushita T, Sigiura I, Takamatsu J, Saito H (1991) Hereditary protein C deficiency due to a frameshift mutation in exon IX of the protein C gene. Thromb Haemostas 65:646 (Abstr 4).
RESEARCH ARTICLE
Two Novel Mutations Responsible for Hereditary Type I Protein C Deficiency: Characterization by Denaturing Gradient Gel E l e c t r ~ p h ~ r e ~ i ~ S. Gandrille, * M. Vidaud, M. Aiach, M. Alhenc-Gelas, A.M. Fischer, M. Gouault-Heilman, P. Toulon, J.N. Fiessinger, and M. Goossens INSERM U.91 and Laboratoire d'He'matologie, HGpital Henri-Mondm, 94010 Cre'teil (S.G., M.V., M.G.-H., M.G.), Group de Recherche sur la Thrombose, lNSERM CJF 91-01, Faculte' des Sciences Pharmaceutiques et Biologiques and Centre Claude Bernard de Recherche en Pathologie Vasculaire, HGpital Broussais, 75006 Paris (S. G.,M.A., M. A. -G., J. N. F.), Laboratoire d'He'matologie, HGpital Necker, 75015 Paris (A.M. F.), and Laboratoire d'He'mtologie, HGpital Cochin, 75014 Paris (I? T. 1, France Communicated by Jean-Claude Kaphn
Hereditary protein C (PC) deficiency is usually associated with a high risk of thrombosis. We report the results of a study undertaken to screen for molecular defects in families with hereditary quantitative PC deficiency. Using a strategy combining polymerase chain reaction amplification of selected gene fragments, denaturing gradient gel electrophoresis of the amplification products, and direct sequencing of fragments with altered melting behavior, we studied the PC gene exons and exodintron junctions of subjects with hereditary type I PC deficiency. Computer simulation of DNA melting was used to design several sets of primers, each containing a GC-clamp, permitting the complete analysis of each amplified exon sequence. Using this procedure, we identified two previously undescribed mutations located in exon VII: a C-to-T substitution generating a nonsense codon in place of Arg 157 in the mature PC and a G-to-A substitution converting Arg 178 to GIn. The two mutations were detected in, respectively, 3 and 2 apparently independent families. This strategy is therefore a valuable tool for screening patients, and the results emphasize its advantages over plasma assays in individuals with a family history of thrombosis. o 1992 Wiley-Liss, Inc. KEY WORDS:
Protein C deficiency, Mutations, Thrombosis
INTRODUCTION Protein C (PC) is a vitamin K-dependent plasma glycoprotein which is converted into a serine protease after activation by thrombin complexed to thrombomodulin, a vascular receptor. Protein S, another vitamin K-dependent protein, regulates the reaction by assembling protein S-protein C complexes on phospholipid surfaces. These complexes inactivate the two coagulation factors F VIII and F V, thus reducing thrombin generation (for review see Clouse and Comp, 1986; Esmon, 1989). The clinical expression of hereditary PC deficiency is variable. A mild fall in the PC concentration is associated with thrombotic complications in about 50% of apparently heterozygous individuals (Horellou et al., 1984; Bovill et al., 1989). T h e frequency of this dominant clinical 0 1992 WILEY-LISS,INC.
trait is estimated at 1 in 15,000 in the general population (Miletich, 1990). Patients with undetectable levels of circulating PC, thought to be either homozygotes or compound heterozygotes, develop massive thrombosis during the neonatal period (Marciniak et al., 1985). The parents and some family members of these homozygous subjects are usually asymptomatic but present heterozygous plasma phenotype (Marlar et al., 1989). The frequency of such asymptomatic heterozygous subjects is estimated at 1 in Received July 2, 1992; accepted September 30, 1992. *To whom reprint requestskorrespondence should be addressed. This work was presented as a preliminay report at the 32nd Annual Meeting of the American Society of Hematology, Boston, MA, December 1990, and has appeared in abstracted form in Blood 76:507a, 1990 (Abstr, Suppl 1).
492
GANDRILLE ET AL.
200 or 300 in the general population (Miletich et al., 1987). These data suggest that only 1 in 50-80 patients with heterozygous plasma phenotype develops clinical symptoms and, in the absence of objective criteria to differentiate dominant and recessive clinical traits, it is impossible to evaluate the thrombotic risk associated with a mild deficiency in a given patient. Moreover, the overlapping concentration ranges in normal subjects and deficient patients (Broekmans and Conard, 1988) make it difficult to identify plasma heterozygosity in patients with PC borderline values. Identification of the molecular abnormality at the gene level is the only way of clarifying the heterogeneous presentations in hereditary PC deficiency. Direct analysis of genomic DNA could help to establish the diagnosis and to identify patients at risk of thrombosis. The PC gene, which maps to chromosome 2 (Rocchi et al., 1986; Kato et al., 1988; Patracchini et al., 1989), was sequenced by Foster e t al. (1985) and comprises 9 exons (Plutzky et al., 1986). Until 1991, few examples of genomic abnormalities had been described in type I deficiency (characterized by a simultaneous decrease in PC antigen levels and activity). Apart from 3 cases of large insertions or deletions (Crabtree et al., 1985), most patients with heterozygous genotype had point mutations. A Trp 402 to Cys substitution and a nonsense codon at position 306 have been described by Romeo et al. (1987). Of 30 novel mutations recently reported (Reitsma et al., 1991; Gandrille et al., 1991; Grundy et al., 1991a; Yamamoto e t al., 1991; Tsuda e t al., 1991; Sala et al., 1991), only 6 were due to microdeletions or insertions (Yamamoto et al., 1991; Tsuda et al., 1991; Sala et al., 1991). Four families with homozygous genotype have also been described (Grundy et al., 1991b; Conard et al., 1992). In an attempt to identify the mutations responsible for PC deficiency in a large series of patients, we have developed a strategy for rapid screening of amplified gene fragments. It involves amplification of genomic DNA targets by the polymerase chain reaction (PCR), followed by denaturing gradient gel electrophoresis (DGGE). In carefully chosen conditions, fragments bearing mutations display a shift in mobility on DGGE. We applied this strategy to the analysis of exon VII in 25 families with type I PC deficiency and we detected 2 novel mutations in 5 apparently independent families. In 3 cases, a C-to-T substitution generated a nonsense codon at position 157 of the protein, while in the
Main Features in Terms of Thrombotic Risk of the 12 Subjects Presenting a Mutation in PC Exon VII
TABLE 1.
Subject
Year of birth
Clinical presentation (age in years at the first thrombotic event)
Family A
11.4 11.6 11.9
1953 1958 1971
Arterial thrombosis (32) Asymptomatic Asymptomatic
1941 1967 1968
Asymptomatic Asymptomatic Pulmonary embolism (20)
1945 1966 1968
Asymptomatic Asymptomatic Pulmonary embolism (21)
1951 1984 1941
Placental infarction (36) Asymptomatic Deep and superficial venous thrombosis (18)
Family B
1.2 11.1 11.2 Family C 1.2
11.2 11.3 Family D 1.2
11.1 Patient E
remaining 2 cases, a G-to-A substitution converted Arg 178 to GIn. MATERIALS AND METHODS Patients
Exon VII from subjects belonging to 25 families presenting with hereditary type I PC deficiency were studied. Five apparently independent families presented with a mutation in this exon. The main clinical features of these five family members are described in Table 1, and 4 of the 5 pedigrees are presented in Figure 1. Unfortunately, only the propositus of family E could be explored (an acquired PC deficiency was excluded by normal hemostasis test results). One of the 3 affected members in each family A or B presented unusual thrombosis. In the propositus of family A, several episodes of lower-limb arterial thrombosis with no apparent cause were observed. Subject 11.2 of family B suffered a pulmonary embolism after starting oral contraception. In family C, patient 11.3 suffered from pulmonary embolism at the age of 21 years, whereas the other 2 patients were asymptomatic. The propositus of family D (1.2) was investigated following a fetal death due to placental infarction. Patient E had presented recurrent deep-vein thrombosis since the age of 18 years. Seven of the 12 heterozygous patients were asymptomatic; the first thrombotic complication in the other 5 patients (Table 1) occurred between
TWO NOVEL MUTATIONS IN TYPE I PC DEFICIENCY
493
FAMILY A
I 1
6
f
7
8
9
53%
FAMILY C
FAMILY B 94 010
100%
65%
55 010
I 19
2
FAMILY D
41 % FIGURE 1. Pedigrees of families with a mutation in exon VII.
Propositi are indicated by arrows. PC levels (percentages) were measured using the 3 different assay kits described in Materials and Methods except for 1 patient (*antigen level).
the ages of 18 and 26 years, in accordance with previous findings (Marlar, 1985; Broekmans,
1988). The diagnosis of PC deficiency was based o n low PC activities, measured using an amidolytic assay (Stachrom Prot C, Stago, Asniere, France, or Berichrom Behring, Rueil, France), a coagulation assay (Staclot, Stago), and low antigen levels (Asserachrom Prot C, Stago). The normal range was 70-130% in the 3 commercial techniques. Venous blood samples were collected in 0.11 M trisodium citrate (1:lO) for PC assays and the plasma was kept frozen until use. Blood was collected in ethylene diamine tetracetic acid for the DNA studies and kept at VC. Leukocytes were isolated within 48 hr and stored frozen until DNA extraction.
As the results of the 3 kits were similar, only the results obtained with the amidolytic assay are shown. 00 = normal male/female; 0 @ = subjects bearing a mutation; El 0 = not studied; = deceased.
Molecular Biology Techniques Materia1s Themus aquaticus (Taq) polymerase (5 U/p,l) was from Perkin-Elmer Cetus Instruments (Norwalk, CT). The deoxynucleotides deoxyadenosine triphosphate (dATP), deoxycytosine triphosphate (dCTP), deoxythymidine triphosphate (dTTP), and deoxyguanosine triphosphate (dGTP) were from Pharmacia Fine Chemicals (Uppsala, Sweden). The OPC columns were from Applied Biosystems (Roissy, France). The sequenase kit was from the U.S. Biochemical Corporation (Cleveland, OH) and the Centricon 100 apparatus from Amicon (Denver, CO). C X - ~ ~ S ( ~ A and T PY) - ~ ~ P (dATP) were from Amersham (Buckinghamshire,
UK).
494
GANDRILLE ET AL.
TABLE 2. Oligonucleotide Primers for
Exon
I I1 I11
IV ivsb D
V VI
VII VIIl IX
PCR Amplification of PC Exons and Allele-SpecificOligonucleotide (ASO)Probes
Primer name PCPROMA GCPCPROMB GCPC 2A PC 2B GCPC 3 PC 3A PC 3B PC 45A PC 45B PC 6A GCPC 6B PC 7A GCPC 7B GCPC 8A PC 8B PC 9A1 GCPC 9A2 GCPC 9B1 PC 9B2 GCPC 9C1 PC 9C2 PC 9c3
AS0 name 157 N CGA 157 M TGA 178 N CGG 178 M CAG
Oligonucleotide sequences 5’-AGAGGTGGGCTTCGGGCAGA-3’
5’4G/C),,-GGACCTGAGACTGTGGCTGC-3‘ 5’4G/C),,-GCAGAGCAGCAGCGGGGGTA-3’ 5’-GAGGCCACAAGGGTCCCGGG-3‘ 5’-(G/C),o-TGCTTCCTCAGACCCCCTCA-3’
5’-CCTCGAAGTCACAGATCTCC-3‘ 5’-TCGAGGCCCCTGCTGGTTAC-3’ 5’-ACACCGGCTGCAGGAGCCTG-3‘
5’-TGCTGGTGCCGCGCCCCCAA-3’ 5‘-GGCGCGGCACCAGCACCAGC-3‘
5’-(G/C),,-AACCCTCCTGAGCCCCCCGC-3’ 5’-GACCAAGACAGGAGGGCAGT-3‘ 5’4G/C),,-CCCAGCACGTGAGCAGCCGG-3’
5’4G/C),o-ATATGAAACCCAGGTGCCCT-3’
5’-TCCCCCTACCCAGGCCTCTG-3’ 5’-TGGGTGCACAGTCTCCGGGT-3‘ 5’4G/C),,-GGGAGGCAGATGGGCACTAT-3’ 5‘4G/C),,-GAGCACCACCGACAATGACA-3’ 5’-GCATCCTGCCGGTCCCCGAG-3’
5’4G/C),,-TGAGCAACATGGTGTCTGAG-3’ 5’-CGCCGTAGTTGTGAAGGAGC-3’ 5’-GCTTGTTACATGTCCCTTTA-3’
Nucleotide numbering” - 1637 to - 1618 - 1268 to - 1287 -72 to -53 111 to 92 1256 to 1275 1465 to 1446 1565 to 1546 2921 to 2940 3270 to 3289 3275 to 3294 3546 to 3527 6055 to 6074 6303to 6284 7063 to 7082 7308 to 7289 8247 to 8266 8551 to 8532 8468to 8487 8779 to 8760 8719 to 8738 8889 to 8870 9043 to 9024
Sequence 5’-TACCCTGAAACGAGACACAGA-3’
5‘-TCTGTGTCTCATTTCAGGTGA-3’
5’-GCTGTCTCCCCGCCTGGTCAT-3’ 5’-ATGACCAGGCAGGGAGACAGC-3’
”Nucleotidenumbering as in Foster et al. (1985). bivs = intervening sequence.
Methods
DNA was extracted from the patients’ leukocytes and purified as previously described (MolhoSabatier et al., 1989). Oligonucleotides were synthesized on a gene assembler (381A DNA synthesizer, Applied Biosysterns). The 20-mers (i.e., PC7) were purified by ethanol precipitation, while the GC-clamps (i. e., the 55-mers GCPC7 containing a 35 nucleotide GC-clamp) were purified on an OPC column prior to ethanol precipitation. General strategy
Enzymatic amplification of each PC gene exon and its splice junctions was performed essentially as described by Saiki e t al. (1988), using a set of oligonucleotides comprising a 20-mers and a primer to which a G C-rich sequence (GCclamp) comprising various numbers of nucleotides was added (Myers et al. , 1985a,b; Sheffield e t al. , 1989). This GC-rich region allows amplified fragments to be screened for sequence variations by
+
DGGE. All primer sequences are presented in Table 2 and their locations are presented in Figure 2. The locations of the PCR primers were chosen using the computer programs MELT 87 and SQHTX, written and kindly provided by Drs. Lerman and Silverstein (1987). These softwares simulate the melting behavior of DNA fragments according to their nucleotide sequence and base composition. The information thus acquired was used to select positions for PCR primers allowing the generation of fragments suitable for DGGE analysis and to determine the range of denaturant concentrations and electrophoresis times giving maximum gel resolution. In every instance, one of the amplification primers carried at its 5‘ end an additional G C-rich nucleotide fragment in order to create a high temperature melting domain (Sheffield et al., 1989) and to position the sequence of interest within the first melting domain. Because of their length, exons 111 and IX were studied using 2 and 3 sets of primers, respectively. DGGE could not be used for exons IV and V be-
+
TWO NOVEL MUTATIONS IN TYPE I PC DEFICIENCY IVS A
IVS B
IVS
C
-
IVS E
IVS F
495
IVS G
PC9C2
4
Pc9c3 GCPCPA P C P B
pc3B
L PCBA
s C 6 B
GCPCBA
EBB
GCPC~BI
FC~BZ
FIGURE2. Location of the amplification primers on the PC gene. The GC-clamps are sym-
bolized by the open bars.
90
cause of their very high melting temperatures. These exons were studied for each patient by direct sequencing after asymmetric PCR.
GCPC7
DGGE and sequencing of the DNA domain bearing the mutation
PC gene exon VII amplification was performed using PC7 and GCPC7 as extension primers. As shown in Figure 3, PC7 was located in IVS F and GC-PC 7 in IVS G. Amplification was performed as follows: each PCR mixture contained 30 pmol of each primer, 200 p M of each dNTP, 1 pg of genomic DNA, 1 x PCR buffer [I0 mM Tris-HC1, pH 8.3, 50 mM KC1, 1 mM MgCl,, 0.01% (w/v) gelatin], and 2.5 U of Tag polymerase in a final volume of 100 ELI. The reactions were run in a Perkin-Elmer Cetus DNA thermal cycler. The thermal profile included 5 min denaturation at 94"C, followed by 30 cycles consisting of denaturation for 1 min at 94"C, annealing for 1 min at 60"C, and extension for 1 min at 72°C. Samples were held at 72°C for 10 min, and the PCR cycles were followed by 10 min denaturation at 94°C and 45 min at 56°C to favor heteroduplex formation. The 284-bp PCR product obtained was analyzed on a 6% polyacrylamide gel. 0 DGGE was performed as described by Attree et al. (1989). Amplified DNA fragments were subjected to electrophoresis for 5 hr at 160 V in a 6.5% polyacrylamide gel containing a 30-80% denaturant gradient (100% denaturant = 7 M urea and 40% formamide in TAE buffer) and stained with e thidium bromide. 0 T h e asymmetric PCR method developed by Gyllensten and Erlich (1988) was performed using 5 pmol of PC7 and 50 pmol of GCPC7, as described above, except that the thermal profile involved 60 cycles. This reaction leads to the preferential amplification and enrichment of the noncoding strand of exon VII. The single-stranded templates were sequenced using the sequenase kit with PC7 as the sequencing primer. Oligoprobe hybridization: To confirm the
a2 (7
2
5 78
74
1 0
50 100 150 200 250 300 BASE POSITION
FIGURE 3. Melting map of PC exon VII. Positions of primers PC7 and GCPC7 are indicated. The GC-clampis symbolized by the open bar.
mutations detected by direct sequencing, four 21mer oligonucleotides were synthesized (Table 2); 157 N CGA and 178 N CGG correspond to the wild-type sequence, and 157 M TGA and 178 M CAG correspond to the identified mutations. Amplified exon VII DNA fragments from each member of families A-E and from normal subjects were blotted onto nitrocellulose membranes, hybridized with the 5 '-end labeled allele-specific oligonucleotide (ASO) probes, and washed in stringent conditions. RESULTS Using DGGE, we analyzed all the coding regions and splice junctions of the PC gene. For exons 1-111 and VI-IX, computer prediction of DNA melting behavior enabled us to design a set of 2 primers. The first contained a GC-rich region (GC-clamp) at its 5' end, permitting the complete analysis of each amplified PC gene exon and its flanking splice-site sequences by DGGE. The
496
GANDRILLE ET AL.
FIGURE4. DGGE patterns of amplified PC exon VII with abnormal melting behavior from subjects of
families with the Arg 178 to Gln mutation (a) or the Arg 157 to nonsense codon (b). Subjects are designated by the numbering system used in Figure 1. NS = normal subject.
other exons (IV and V) were analyzed by direct sequencing after asymmetric PCR amplification. In the 5 families with mild PC deficiency depicted in Figure 1, the only abnormality was located in exon VII. Figure 3 depicts the position of the primers (PC7 and GCPC7) used to amplify a 284-bp fragment. The melting curve of the resulting amplified fragment confirmed that the sequence of interest (exon VII and its associated splice region) was located within the first melting domain of the DNA fragment. Two different DGGE patterns were observed, suggesting the existence of 2 different exon VII mutations: 1 in families A and B and 1 in families
GE. The normal and mutant homoduplex fragments were not separated in families A and B, but the presence of heteroduplexes (upper 2 bands) was typical of a nucleotide substitution in 1 of the alleles (Fig. 4a). Heteroduplexes, generated during the last step of PCR, are due to mismatches of normal alleles with mutant alleles and melt at a lower denaturant concentration. Patients 11.6 and 11.9 (family A) and 1.2 (family B) were carriers, since their abnormal DGGE pattern was identical to that of the propositus.
In families C and D (Fig. 4b) and in patient E, the DGGE patterns contained 4 bands, with the upper 2 bands constituting a very close doublet. These patterns are also typical of heterozygosity: band 4 migrated like DNA fragments from normal subjects and represents normal homoduplexes. Band 3 corresponded to homoduplexes of the mutant allele. Bands 1 and 2 resulted from the generation of heteroduplexes. To check that the observed abnormalities were not neutral polymorphisms, we tested 60 individuals with normal PC levels; the DGGE pattern was normal in all cases. The genomic abnormalities were further characterized by direct sequencing of the singlestranded fragment obtained by asymmetric amplification. In families A and B (Fig. 5a), a G-to-A transition converted Arg 178 to Gln. In the other three families, a C-to-T substitution generated a nonsense codon in place of the Arg at position 157 (Fig. 5b). Hybridization of amplified exon VII fragments with AS 0 corresponding to either the normal or abnormal sequences (Fig. 6) confirmed that all the patients with PC deficiency were heterozygous for the defect, as both normal and mutant AS0 hybridized with amplified DNA.
TWO NOVEL MUTATIONS IN TYPE 1 PC DEFICIENCY
497
FIGURE5. (a) Sequence of exon VII of patients with the Arg 178 to Gln mutation. As the patients were heterozygous for the mutation, both normal (CGG) and mutated (CAG) codons are present. (b) Sequence of exon VII of patients with the Arg 157 to nonsense mutation. As the patients were heterozygous for the mutation, both normal (CGA) and mutated (TGA) codons are present.
DISCUSSION Some of the difficulties encountered in the diagnosis of hereditary PC deficiencies can be overcome by using DNA analysis, and DGGE proved to be a valuable tool in this setting. This electrophoretic method enables DNA fragments to be separated according to changes in the melting properties produced by simple nucleotide substitutions (Fischer and Lerman, 1983; Myers et al., 198%). The technique permitted rapid screening of our patients, restricting the need for DNA sequencing to the samples showing abnormal electrophoretic mobility. We detected 2 novel mutations in exon VII which would alter expression of the protein in some of our patients. A missense mutation (in 2 families) or a nonsense codon (in the other 3 fam-
ilies) were located in this critical domain which contains 2 cleavage sites essential for correct maturation of the protein and its activation by thrombin. In the former case, the mutation led to the replacement of the Arg at position 178 by a Gln. PC is a vitamin K-dependent zymogen presenting structural similarities with clotting factors 11, VII, IX, and X. Arg 178 is located within a domain containing several highly conserved amino acids and is adjacent to Gly 179, a residue found at this position in most vitamin K-dependent proteins (when sequences are aligned). In factor IX, the substitution of Gln 191, which is located immediately after the corresponding Gly, is associated with severe antigen-negative hemophilia B (Giannelli e t al., 1990). The Arg 178 to Gln muta-
498
-
GANDRILLE ET AL.
normal despite a borderline PC level. These results confirm that the available plasma assays (3 of NS 12 I 1 112 NS NS 115 116 117 118 119 which were used in this study) can often fail to 0 ~ 0 ~ 0 0 a ~ 178 0 N C0G G 0 identify PC deficiency in heterozygous subjects. Although our population of patients is too small 0 0 0 0 178 M CAG to draw any conclusion with regard to phenotype/ genotype correlation, some of the data deserve attention. In both families bearing the Arg 178 to Gln mutation, some of the subjects had borderline plasma PC values. In subject 11.5 of family A, the FAMILY D FAMILY C Subject I 2 Subject E DNA study enabled us to exclude the presence of / the mutation. In family B, the propositus (11.2) NS I 1 12 112 113 had a PC level of 72% in the amidolytic assay and 157NCGA 0 0 0 0 0 0 65% in the immunoenzymatic assay. Unfortunately, no samples were available for DNA studies for this patient. The mother carried the mutation, 8 0 0 0 157MTGA despite borderline PC levels with respective values FIGURE 6. Dot blots of samples from family members with of 65 and 78% with the 2 assays. It is possible that normal (157 N CGA or 178 N CGG) and mutant (157 M TGA the Arg 178 to Gln mutation would lead to an or 178 M CAG) probes. For details, see Materials and Methincreased turnover of PC or render it unstable, ods. resulting in a moderate decrease in its plasma level and a thrombotic tendency. tion is therefore likely to have a deleterious effect The mutation observed in the other 3 families on PC folding. The fact that the DGGE pattern gave rise t o a stop codon which was associated with characteristic of this mutation was not observed in clearly low levels of both PC activity and antigen amplified DNA fragments from 60 control subjects in all the patients explored, regardless of the assays makes it unlikely that this nucleotide substitution used. This mutation introduces a nonsense codon is a polymorphism. The value of DNA analysis is at position 157 which gives rise to a truncated illustrated by the fact that 1 of these patients famprotein lacking two-thirds of the polypeptide ily B, 11.2 was considered normal in terms of the chain. Two hypotheses may explain the absence of plasma assay results whereas he carried the mutathe abnormal protein in the circulation: either the tion. In another subject with no thrombotic commRNA is unstable and thus cannot be translated plications family A, 11.5, the DGGE pattern was into protein or the truncated protein is not seFAMILY B
FAMILY A
I
I
14
I
I1
Ill
IV
v
VII
VI
lea im
147
vK
IX
Vlll
F P C G R P w KQM E K KQS H L KQDT E
1c
1
STOP
DQEDQv D
~ I D GLK M T RQG D s P w Q
11 L W
1
Q
w
FIGURE 7. Schematic representationof the mutations described on CpG of the PC gene exon VI1. The size of the entire gene is 11.6 kb. Sizes of the exons are indicated in base pairs below each exon. Amino acids correspondingto hot-spots for mutation are circled.
TWO NOVEL MUTATIONS IN TYPE I PC DEFICIENCY
creted. However, it is interesting to note that in the 3 family C members bearing the mutation, PC levels varied over a wide range. The 2 mutations described here involve CpG dinucleotides which are likely to be hot-spots for mutation in vertebrates (Bird, 1980) by methylation-mediated deamination of 5-methylcytosine, the most frequent cause of recurrent mutation in man (Cooper and Krawczak, 1990). Four previously described mutations, Arg 169 to Trp causing type I1 qualitative hereditary PC deficiency (Matsuda et al., 1987; Grundy et al., 1989), and 3 mutations responsible for quantitative deficiencies, Arg 152 + Cys, Pro 168 + Leu, and Arg 178 + Trp (Gandrille et al., 1991; Reitsma et al., 1991; Conard et al., 1992), also involved CpG dimers. Therefore, 5 of the 6 CpG dimers present in exon VII have so far been shown to be the sites of mutations, as summarized in Figure 7. These hot-spots for mutation may explain the recurrence of the mutations of our families; however, as our patients were all European Caucasian, 1 family being Portuguese, and the others being French families, we cannot exclude an identity by descent, as described in 3 families by Reitsma et al. (1991).
ACKNOWLEDGMENTS
S.G. was the recipient of a grant from La Fondation pour la Recherche Medicale. This work was supported by 1’Institut National pour la SantC et la Recherche Medicale (INSERM) , by 1’Association Claude Bernard, and by a grant from 1’Association Franqaise contre les Myopathies. We thank Drs. Lerman and Silverstein, who kindly provided the computer programs MELT 87 and SQHTX. REFERENCES Attree 0, Vidaud D, Vidaud M, Amselem S,Lavergne JM, Goossens M (1989) Mutations in the catalytic domain of human coagulation factor IX: Rapid characterization by direct genomic sequencing of DNA fragments displaying an altered melting behavior. Genomics 4:266-272. Bird AP (1980) DNA methylation and the frequency of CpG in animal DNA. Nucleic Acids Res 8:1499-1504. 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. Broekmans AW, Conard] (1988) Hereditary protein C deficiency. In Bertina RM (ed): Protein C and Related Proteins. New York: Churchill Livingstone, pp 160-181. Clouse LH, Comp PC (1986) The regulation of hemostasis: The protein C system. N Engl J Med 314:1298-1304. Conard J , Horellou MH, Van Dreden P, Samama M, Reitsma PH, Poort S,Bertina RM (1992) Homozygous protein C deficiency with late onset and recurrent coumarin-induced skin necrosis. Lancet 339: 743-744. Cooper DN, Krawczak M (1990) The mutational spectrum of sin-
499
gle base-pair substitutions causing human genetic disease: Patterns and predictions. Hum Genet 85:55-74. Crabtree GR, Plutzky J, Marler R, Bertina RM, Broekmans AW, Griffin J, Zarcharski L, Gruppo R, Sala N, Long G (1985) The range of genotypes underlying human protein C deficiency. Thromb Haemostas 5456 (Abstr S331). Esmon CIT (1989) The roles of protein C and thrombomodulin in the regulation of blood coagulation. J Biol Chem 264:47434746. Fischer SG, Lerman LS (1983) DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels: Correspondence with melting theory. Proc Natl Acad Sci USA 80:1579-1583. Foster DC, Yoshitake S, Davie EW (1985) The nucleotide sequence 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, Gouault-Heilman M, Toulon P, Goossens M (1991) Six previously undescribed mutations in 9 families with protein C quantitative deficiency. Thromb Haemostas 65:646 (Abstr 3). Giannelli F, Green PM, High KA, Lozier JN, Lillicrap DP, Ludwig M, Olek K, Reitsma PH, Goossens M, Yoshioka A, Sommer S, Brownle G G (1990) Haemophilia B: Data base of point mutations and short additions and deletions. Nucleic Acids Res 18:4053-4059. Grundy CB, Chitolie A, Talbot S,Bevan I), Kakkar V, Cooper DN (1989) Protein C London 1: A recurrent mutation at Arg 169 (CGG+TGG) in the protein C gene causing thrombosis. Nucl Acids Res 17:10513. Grundy CB, Melissari E, Kakkar VV, Cooper DN (1991a) A molecular genetic study of protein C deficiency. Thromb Haemostas 65:646 (Abstr 2). Grundy CB, Lindo V, Kakkar VV, Melissari E, Scully MF, Cooper DN (1991b) Late-onset homozygous protein C deficiency. Lancet 338:575-576. Gyllensten UB, Erlich H A (1988) Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequending of the HLA-DQA locus. Proc Nit1 Acad Sci USA 85:7652-7656. Horellou MH, Conard J, Bertina RM, Samama M (1984) Congenital protein C deficiency and thrombotic disease in nine French families. Br Med J 289:1285-1287. Kato A, Miura 0 , Sumi Y, Aoki N (1988) Assignment of the human protein C gene (PROC) to chromosome region 2q14---q21 by in situ hybridization. Cytogen Cell Genet 47: 46-47. Lerman LS, Silverstein K (1987) Computational simulation of DNA melting and its application to denaturing gradient gel electrophoresis. Methods Enzymol 155:482-501. Marciniak E, Wilson HD, Marlar RA (1985) Neonatal purpura fulminans: A genetic disorder related to the absence of protein C in blood. Blood 65:15-20. Marlar RA (1985) Protein C in thromboembolic disease. Semin Thromb Haemostas 11:389-395. Marlar RA, Montgomery RR, Broekmans AW, and the Working Party (1989) Diagnosis and treatment of homozygous protein C deficiency. J Pediatr 114:528-534. Matsuda M, Sugo T, Sakata Y, Murayama H, Mimuro J, Tanabe S, Yoshitake S (1987) A thrombotic state due to an abnormal protein C. N Engl J Med 319:1265-1268. Miletich JP, Sherman L, Broze G (1987) Absence of thrombosis in subjects with heterozygous protein C deficiency. New Engl J Med 317:991-996. Miletich JP (1990) Laboratory diagnostic of protein C deficiency. Semin Thromb Haemostas 16:169-1 76. Molho-Sabatier P, Aiach M, Gaillard I, Fiessinger IN,Fischer
500
GANDRILLE ET AL.
AM, Chadeuf G, Clauser E (1989) Molecular characterization of seven AT 111 variants using PCR. Identification of a new mutation: Ala 384-Pro. J Clin Invest 84:1236-1242. Myers RM, Fischer SG, Lerman LS, Maniatis T (1985a) Nearly all single base substitutions in DNA fragments joined to a GCclamp can be detected by denaturing gradient gel electrophoresis. Nucleic Acids Res 13:3131-3145. Myers RM, Fischer SG, Maniatis T, Lerman LS (1985b) Modification of the melting properties of duplex DNA by attachment C-rich DNA sequence as determined by denaturing of a G gradient gel electrophoresis. Nucleic Acids Res 13:3111-3129. ) Myers RM, Lumelsky N, Lerman LS, Maniatis T ( 1 9 8 5 ~ Detection of single base substitutions in total genomic DNA. Nature 3 13495-497. Patracchini P, Aiello V, Palazzi P, Calzolari E, Bernardi F (1989) Subclonalization of the human protein C gene on chromosome 2q13-ql4. Hum Genet 81~191-192. Plutzky 1, Hoskins JH, Long GL, Crabtree OR (1986) Evolution and organization of the human protein C gene. Proc Natl Acad Sci USA 83546-550. 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 cY1-glycoprotein to chromosome 9. Hum Genet 74:30-33.
+
Romeo G, Hassan HJ, Staempfli S, Roncuzzi L, Cianetti L, Leonardi A, Vicente V, Mannucci PM, Bertina RM, Peschle C, Cortese R (1987) Hereditary thrombophilia: Identification of nonsense and missense mutations in the protein C gene. Proc Natl Acad Sci USA 84:2829-2832. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487-491. Sala N , Poort SR, Bertina RM, Soria JM, Fonrcuberta 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 1809). Shefield VC, Cox DR, Lerman LS, Myers RM (1989) Attachment of a 40-base-pair G C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes. Proc Natl Acad Sci USA 86~232-236.
+
Tsuda S, Reitsma P, Miletich J (1991) Molecular defect causing heterozygous protein C deficiency in three asymptomatic kindreds. Thromb Haemostas 65:647 (Abstr 7). Yamamoto K, Tanimoto M, Matsushita T, Sigiura I, Takamatsu J, Saito H (1991) Hereditary protein C deficiency due to a frameshift mutation in exon IX of the protein C gene. Thromb Haemostas 65:646 (Abstr 4).
