© 1996 American Heart Association, Inc.
Articles |
Identification of Two Novel Point Mutations in the Human Protein S Gene Associated With Familial Protein S Deficiency and Thrombosis
the Department of Biochemistry, College of Medicine, University of Vermont, Burlington.
Correspondence to George L. Long, PhD, Department of Biochemistry, Given Bldg Room B418A, University of Vermont College of Medicine, Burlington, VT 05405. E-mail glong@zoo.uvm.edu.
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Abstract |
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Individuals with thrombosis who were believed to possess associated familial protein S deficiency were analyzed for mutations in the protein S gene by a two-step process. First, the individuals were analyzed for protein S Pro626 A/G dimorphism in both their genomic DNA and reverse-transcribed (RT) polymerase chain reaction (PCR)–amplified cDNA from peripheral blood cell mRNA. If a heterozygote expressed both alleles at the mRNA level at this site in genomic DNA, a search for point mutations was made by direct cDNA sequencing. RT-PCR amplification of exons 1-6 with mRNA from two twin sisters, each of whom has severe type I protein S deficiency, revealed both larger and smaller fragments in addition to the expected 504–base pair fragment in normal individuals. A donor splice-site mutation at position +4 of the 5' end of intron A was subsequently identified in both sisters and their mother. This mutation would lead to incorrect precursor mRNA splicing and the observed cDNA products. Translation of the altered mRNAs would result in a truncated protein without biological activity. In a second family, cDNA sequencing revealed a T
G mutation at codon 603 (IleSer) in exon 15 of the protein S gene in an individual with protein S deficiency (mixed type) and a history of thrombosis. The same mutation was also detected in the proband's mother and grandmother, both of whom also exhibit protein S deficiency and thrombotic disease. This mutation occurs within a disulfide loop of protein S that is believed to be responsible for binding to C4b binding protein and may result in greater affinity between protein S and C4b, consequently leading to thrombotic disease.
Key Words: protein S • thrombosis • mRNA processing • C4b binding protein
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Introduction |
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Protein S, a single-chain glycoprotein, is a vitamin K–dependent plasma protein that plays an important role in the protein C anticoagulant pathway. Protein S is a cofactor to activated protein C and thereby enhances inactivation of FVa and VIIIa and stimulates fibrinolysis. Plasma protein S circulates in two forms: a free form and complexed with the complement component C4bBP. Only the free form of protein S has cofactor activity.
The expressed (
) PROS gene contains 15 exons and is >80 kb long,1 2 3 and the protein S pseudogene (ß) is homologous to exons 2-15 of the PROS- gene.1 2 3 Both genes are closely linked on chromosome 3p11.1-3q11.2.4 In 5% to 8% of patients with a history of thrombotic disease, protein S deficiency (ie, reduced levels of free and/or total protein S) is the only clinical finding and strongly suggests that this deficiency is an important risk factor for thrombosis.5 6
Two large deletions in PROS- have been shown to be associated with protein S deficiency and thrombophilia.7 8 The first, reported by Ploos van Amstel et al,7 is located in the central portion of the PROS- gene and at least partially includes the coding segments for amino acids 
316-405. The second deletion (5.3 kb), reported by Schmidel et al,8 has been mapped at the DNA sequence level (within introns L and M) and results in deletion of coding exon XIII and a truncated protein product. Family members with either deletion exhibit associated protein S deficiency and thrombophilia.7 8
Unlike protein C and antithrombin III deficiencies, for which 160 different mutations from 315 unrelated probands9 and 59 different mutations from 177 individuals,10 respectively, have been reported, few genetic studies on protein S deficiencies have appeared in the literature.6 7 11 This is partly due to the complexity of the PROS- gene and the existence of the very similar pseudogene (>95% nucleic acid identity).1 A cDNA (mRNA)-dependent PCR method was developed to circumvent problems associated with both the complex gene structure and the presence of the pseudogene.12 However, several cases have been reported of markedly reduced mutant mRNA levels of protein S ("allelic exclusion"), which severely limits or prevents detection of mutations. An alternative but more burdensome strategy of amplifying each of the 15 exons and exon/intron boundaries of only the PROS- gene by choosing primers at positions where nucleotide differences exist between PROS- and PROS-ß has been recently employed.13 14 This approach, followed by denaturing gradient gel electrophoresis screening14 or direct sequencing,15 16 17 has resulted in the identification of 37 point mutations and 4 neutral polymorphisms to date: 3 splice-site abnormalities, 7 frameshift deletions, 3 frameshift insertions, 3 nonsense mutations, and 21 missense mutations. However, this approach is technically difficult and somewhat limited because of the high degree of sequence identity between the two genes (97% for coding regions and 95% for introns).18 Although most of the PROS- gene exons can be amplified by this approach, some (eg, 3 and 14) are resistant to specific amplification because of the lack of sequence differences.
In this article, we describe a procedure in which both cDNA (mRNA) and genomic DNA were employed in our studies. Key to the strategy is the frequent presence of the Pro626 (A/G) dimorphism18 at both the genomic DNA and cDNA (mRNA) levels. This dimorphism occurs at position 3 of the Pro codon and is therefore "silent" in terms of altering protein sequence. The allele frequency is .48G and .52A18 ; both are widely distributed and result in about half of the population being heterozygous. These aspects make this dimorphism an excellent genetic marker independent of protein S deficiency and the associated thrombosis. This convenient screening method identifies candidates for further detailed mutation analysis and allows assessment of the possible contribution of allelic exclusion to protein S deficiency. Using this strategy, we studied four unrelated families with hereditary protein S deficiency and found that two of them carry novel mutations.
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Methods |
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Patients
Blood samples were obtained from families with hereditary protein S deficiency. Total protein S antigen was measured with a commercial enzyme-linked immunosorbent assay19 (Diagnostica Stago), and protein S functional activity was determined by a prothrombin time–based clotting assay20 from Diagnostica Stago. The ratio of free to total protein S was determined by the Laurell rocket electrophoretic method as described by Comp et al.21 Clinical and laboratory data for the two families described in this article are summarized in Table 1
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RNA and DNA Extraction
Two 10-mL volumes of anticoagulant (EDTA)-treated blood were obtained from individuals for separate RNA and DNA extraction. For RNA extraction, lymphocytes were separated with lymphocyte separation medium (LSM, Organon Teknika Corp) according to the supplier's directions, and total RNA was isolated from lymphocytes by the method of Chomczynski and Sacchi.22 For DNA extraction, a simple salting-out procedure from whole blood was performed according to Miller et al.23
RT-PCR
RT-PCR amplification was performed with a thermal cycler (model 480, Perkin-Elmer) and GeneAmp RNA PCR kit components (Perkin-Elmer). Total RNA (1 to 3 µg) was incubated for 60 minutes at 42°C in 20 µL RT-Master mix (including 50 U MMLV reverse transcriptase) and 1 µL of 20 µmol/L RT primer (No. 699, 114, or 29; Table 2) for reverse transcription. This was followed by adding 78 µL PCR-Master mix containing 2.5 U Taq DNA polymerase (AmpliTaq) and 1 µL of 20 µmol/L forward primer (No. 313 or 698; Table 2). The first round of PCR was performed as follows: denaturation for 1 minute at 95°C, "touchdown"25 annealing for 1 minute at 67°C to 55°C (the temperature was decreased 2°C every 2 cycles to 57°C, followed by 25 cycles at 55°C), and elongation for 2 minutes at 72°C for a total of 37 cycles; final elongation was 10 minutes at 72°C. The second round of PCR was performed by adding 1 to 6 µL of the products obtained during round one to a 100-µL reaction mixture containing 200 µmol/L of each dNTP, 1.25 mmol/L MgCl2, and standard reaction buffer. After a 7-minute denaturation at 95°C, 2.5 U Taq DNA polymerase (Boehringer Mannheim Corp) was added, and PCR was performed as described above.
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Genomic DNA Amplification for the PROS- Gene
The 5' noncoding region, individual exons, and intron/exon junctions were amplified by PCR, with primers chosen specifically for the PROS- gene (Table 3). PCR conditions were the same as those described above for the second round. Amplification specificity was verified by digesting the reaction products with restriction endonucleases that specifically cleave either PROS- or PROS-ß14 (results not shown).
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Direct Sequencing of PCR Products
RT-PCR and genomic DNA PCR products were observed on 1% agarose gels and purified directly with the QIAquick kit (QIAGEN Inc); alternatively, individual bands were cut from the agarose gel and isolated by electroelution. DNA sequencing was performed with a sequencing kit (Taq Dye Deoxy Terminator Cycle, Applied Biosystems) and the products resolved and analyzed on an automated DNA sequencer (ABI model 373, Applied Biosystems). DNA sequences for the 5' noncoding region, all exons, and intron/exon junctions were identical to those previously reported for wt PROS- unless specifically noted.
Pro626 Dimorphism Analysis
The CCA/CCG neutral dimorphism at Pro626 in exon 15 of the PROS- gene was determined by BstXI digestion of amplified genomic DNA and cDNA. Exon 15 was amplified from genomic DNA with primers 626 and 757 (see Table 3
) as described above, and the PCR products (10 µL) were digested with BstXI (Life Technologies–GIBCO BRL) and observed on a 1.5% agarose gel. For the G allele (no digestion), a 700-bp fragment was observed, whereas the A allele resulted in the appearance of 480- and 220-bp fragments. Exon 14-15 was amplified from cDNA with primers 756 and 29 (Table 2), and PCR products (10 µL) were either digested with BstXI or directly sequenced after purification. Digestion of the PCR product yielded 167-, 152-, and 45-bp bands for the A allele and 197- and 167-bp bands for the G allele.
Restriction Endonuclease Detection of Mutation
Restriction endonuclease (Vsp I, Sca I, and BstEII [Life Technologies–GIBCO BRL]) digestion of amplified DNA was used to confirm sequence-identified mutations and to analyze other family members. Ten microliters (10% of total volume) of PCR product from cDNA or genomic DNA amplification was mixed with reaction buffer and enzyme in a total final volume of 100 µL and digested according to the suppliers' directions. Digestion products were precipitated with isopropanol, and the fragments were resolved on 1.5% to 2.5% agarose gels, followed by ethidium bromide staining and visualization.
Analysis for FV Arg506Gln Mutation
Genomic DNA from affected individuals was used to amplify by PCR a region of the human FV gene containing a mutation (Arg506Gln) associated with activated protein C resistance and thrombosis.26 Primers and amplification conditions initially described by Bertina et al26 were used. The expected 267-bp product was digested with Mnl I to distinguish wt from mutant (loss of digestion site) gene products and analyzed after agarose gel electrophoresis.
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Results |
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Analysis of Pro626 A/G Dimorphosim in Genomic DNA and RT-PCR cDNA
Initially, genomic DNA from 6 unrelated individuals who were believed to have familial thrombosis and protein S deficiency were analyzed. Two of them did not have the Pro626 dimorphism and consequently were not studied further. Two other individuals with the dimorphism at both the genomic DNA and mRNA levels were analyzed for mutations in the 5' noncoding region and all coding exons. No mutations were identified in either individual, and they were not studied further for the protein S gene. The results of Pro626 dimorphism analysis for the families of the two remaining individuals in whom mutations were identified are presented in Table 4
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FV Arg506Gln Mutation
PCR amplification of genomic DNA and Mnl I digestion product revealed that 5 of the 6 unrelated individuals had two normal copies of the FV gene. The sixth individual, who had no identified mutations in the PROS- gene, was heterozygous for the FV Arg506Gln mutation (data not shown). This individual was initially suspected of having type II protein S deficiency on the basis of disproportionately low protein S functional activity. The absence of any mutations in the PROS gene and recent reports of interference by the Arg506Gln variant of FV in functional protein S assays27 28 suggest that mutant FV and not protein S is responsible for the reduced protein S functional activity in this individual.
RNA and DNA Analyses for Family I
RT-PCR amplification with primers 698 (within exon 1) and 699 (within exon 6) resulted in multiple products for both twin sisters. In addition to the expected 504-bp fragment in other family members and unrelated normal individuals, both larger and smaller fragments were observed (Fig 1). DNA sequencing of the 234-bp cDNA fragment S2 revealed that it lacked exons 2, 3, and 4 (Fig 2A). Sequencing of the 615-bp fragment B1 showed that it contained 111 bp of the 5' end of intron A between exons 1 and 2 (Fig 2B) and ended just before a second putative cryptic 5' splice site with the recognition sequence GTAAGT (Fig 2C). Sequencing of the 337-bp fragment S1 revealed that it contained the expected portion of exon 1 followed by 316 bp of the 5' intron A sequence (Fig 2C). We believe that this product was generated by "false priming" of primer 699 to a segment of intron A (nucleotides 296-306) in the abnormal cDNA, which is identical to the primer sequence. This observation and the large, unidentified products shown in Fig 1 suggest that much larger intron insertions generated by other cryptic splice sites in intron A are also present. The possibility that the larger bands are the result of contaminating protein S genomic DNA amplification is ruled out by the fact that the distance from exon 1 to exon 6 in the PROS- gene is >49 kb (References 1 through 3 and G. Long, unpublished results, 1995) and that the PROS-ß pseudogene does not possess a region homologous to exon 1.1 2 3 Also, RT-PCR with RNA from many individuals believed to have a normal PROS- gene does not result in the appearance of these anomalous bands (data not presented). Results from sequencing of the aberrant cDNA product are schematically summarized in Fig 3. Analysis of the 5' end of intron A in fragments B1 (Fig 2B) and S1 (Fig 2C) reveals an AG mutation at position +4 from the precursor mRNA normal 5' splice site.
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) at position +4 from the normal splice site and the resulting premature translation stop site. Also shown are two additional nucleotides (underlined) not reported in our earlier publication.
) designate the proposed cryptic 5' splice site, resulting in fragment B1 (Fig 1B
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Taken together, sequence analysis of abnormal cDNA products suggests that they arose from aberrantly spliced precursor mRNA, resulting from the AG mutation in the intron A 5' splice-site recognition sequence. Subsequent genomic DNA sequencing for twin 1 confirmed an AG transition at position +4 of the 5' end of intron A (data not shown). Her twin sister and mother were also determined to have this mutation by PCR-enabled mutagenic RFLP (Fig 4).9 An inverse complement mutagenesis primer mut2 was designed (5'-GAAGAGACGCTATTGgTTAC-3'), so that an AG change at nucleotide +4 from the 5' splice site, in concert with the induced mutation (Ag), would create a BstEII site (GGTNACC) for the mutant allele. The results of RFLP after BstEII digestion of PCR products from family members is shown in Fig 4. To discount the possibility that this donor splice-site mutation might represent a common polymorphism, we examined DNA from 30 unrelated control individuals by PCR-enabled mutagenic RFLP analysis and found no occurrences.
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As indicated in Fig 1, the expected normal 504-bp PCR fragment was always observed as the most abundant product. However, the relative abundance of other PCR products varied for separate identical PCR amplifications and between two independent mRNA preparations. We cannot offer a clear explanation for this observation but note that caution should be taken when only a single PCR is performed or alternatively, that replicate amplifications should be performed. Replicate amplifications from family II mRNA and normal individuals showed no variation, suggesting that the variation may be partially related to the AG mutation in family I.
RNA and DNA Analyses for Family II
RT-PCR amplification was used to amplify a region of exons 14 and 15 with primers 756 and 29 (Table 2). cDNA sequencing of the PCR product revealed a TG transversion in codon 603 (IleSer) within exon 15 (Fig 5) and consequential loss of a Vsp I restriction endonuclease digestion site (ATTAAT). Vsp I analysis of the cDNA fragment clearly distinguished normal from heterozygous subjects despite incomplete digestion by this enzyme (Fig 6A). To confirm this mutation, the inverse complement mutagenesis primer mut1 (5'-ACCCAGATCCAACTGTACACCAgTA-3') was used. The induced mutation (Tg) in concert with the natural mutation created a Sca I digestion site (AGTACT), as demonstrated in Fig 6B.
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) in both alleles at position 2077 (numbering from Reference 24). Shown below is the sequence from the grandmother in family II, indicating both T and G at the corresponding position.
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The aforementioned mutation was confirmed in other affected family members by PCR amplification of genomic DNA with primers 626 and 757 (see Table 3) and subsequent Vsp I digestion (data not shown). To eliminate the unlikely possibility that the primers had inadvertently amplified the PROS-ß pseudogene region, differential digestion with Ban I was performed on products from the mother, wt genomic DNA (no digestion site), and the corresponding amplified region of cloned PROS-ß DNA (pPTS92-22 from 
22 in Reference 11), which contains a Ban I site. Ban I completely digested the PCR product from amplified, cloned PROS-ß DNA to the predicted 453- and 247-bp fragments, whereas digestion of amplified product from total genomic DNA (from the mother and an unrelated wt individual) resulted in only very faint bands at the locations of the digestion fragments (data not shown).
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Discussion |
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To screen for mutations in the PROS-
gene responsible for protein S deficiencies, an RT-PCR method combined with genomic DNA analysis was adopted. In family I, a donor splice-site AG mutation at position +4 of the 5' end of intron A was found. The normal donor splice-site sequence GTGAGT of protein S intron A resembles the eukaryotic consensus donor site sequence (GT GAAGT).30 Statistically, A is present 71% of the time at position +4, whereas G appears only 11% of the time, suggesting that A at position +4 is generally more effective than G in directing correct splicing. RT-PCR and DNA sequencing results indicate that the mRNA of affected members of family I consists of multiple species of transcripts presumably generated from aberrant splicing. Our results, which reveal exon skipping (fragment S2) and proximal cryptic 5' intronic splice sites (fragments S1 and B1), are consistent with the "exon definition" (recognition) model for splicing of vertebrate genes with large introns, as reviewed by Berget.31 In the case of PROS-, intron A is >32 kb (References 1 through 3 and G. Long, unpublished data, 1996). One of these, the exon 2-4 deletion, would result in the loss of Gla- and thrombin-sensitive domains required for biological activity but might not alter the level of circulating total or C4bBP-bound mutant protein S. On the other hand, proposed intron A insertions would result in a frameshift and creation of a new stop codon after the 2 amino acids indicated in Fig 3. Consequently, no portion of protein S would be produced other than the signal peptide.
In family II, the point mutation at amino acid 603 (IleSer) in exon 15 changes a large, hydrophobic residue to a small, polar one and may affect the overall structure. Unfortunately, no three-dimensional structure for this part of the molecule has yet been deduced. Comparison of homologous amino acid sequences among species (humans, monkeys, cows, pigs, mice, rats, and rabbits), however, does indicate that Ile603 has been conserved during evolution.32 33 This dramatic amino acid substitution may alter the overall structure and stability of the protein and may account for the moderately low levels of total circulating protein S (see Table 1). Alternatively or additionally, this residue is within one of the two carboxy-terminal disulfide loops (residues 598-625) reported to be important for binding to C4bBP and immediately adjacent to a 10–amino acid segment (residues 605-614) reported to directly interact with C4bBP on the basis of synthetic peptide studies34 35 and site-directed mutagenesis.36 Consequently, the Ile603Ser mutation may result in higher affinity between protein S and C4bBP and therefore lower levels of free, functionally active protein S, consistent with the disproportionately low levels of protein S functional activity. On the basis of the identified mutation and its possible effect on structure and function, it would be useful to measure the ratio of free to bound protein S in family members. Unfortunately, the family has been uncooperative for follow-up blood studies. Consequently, resolution of this issue must await the generation of recombinant, mutant protein S in culture and in vitro testing of C4bBP binding, both of which are currently under way in our laboratory.
As indicated in Table 1, affected members of both families appear to have disproportionately low protein S functional activity. Members of both families were also shown to be free of the FV Arg506Gln mutation associated with activated protein C resistance, thereby excluding this mutation as the cause of the observed lower functional protein S activity.
Several groups have recently reported the lack of protein S mutant mRNA expression, termed allelic exclusion. This phenomenon limits the ability to detect mutations based on RT-PCR amplification from circulating blood cell mRNA. However, a two-step analysis involving initial genomic DNA and RT-PCR cDNA screening for the Pro626 A/G dimorphism, followed by detailed sequence analysis in candidate individuals, can be applied in a straightforward fashion as reported in this article. One drawback of this new strategy, however, is that it excludes recognition of individuals who are homozygous for this dimorphism. The molecular basis for mutant mRNA allelic exclusion is unclear. Recently, a number of nonsense mutations (stop codon) have been reported to cause considerable reduction in mRNA levels.37 38 In the case of protein S, identified nonsense mutations (at the genomic DNA level) have been reported to result in allelic exclusion by several investigators.12 13 16 17 39 In the present report, allelic exclusion was observed in family I (with a truncation stop codon in splicing variants containing the 5' end of intron A) but not in family II. The allelic exclusion observed in family I appears to vary in degree, depending on the particular mRNA preparation. We also wish to point out that the father in family I appears to display allelic exclusion despite the absence of any identified mutation in the protein S gene, suggesting that caution should be taken in any attempt to determine the cause of observed allelic exclusion.
Note added in proof: Two additional fragment polymorphisms in the PROS- gene have recently been described,40 which will provide for wider application of the strategy presented in this article.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported in part by US Public Health Service, National Institutes of Health (Bethesda, Md) grants RO1-HL38899 and CO6-HL39745 (to G.L.L.).We thank Drs Oscar D. Ratnoff (Case Western Reserve University, Cleveland, Ohio) and Eric Pillemer (Southwestern Vermont Medical Center, Bennington) for providing us with blood samples and the initial clinical histories for families I and II, respectively. We are also grateful to the families for providing blood samples and family history. The technical assistance of Patricia Poundstone in DNA sequencing, Liz Golden (Fletcher Allen Hospital clinical laboratory) for family I protein S antigen determinations, and Julia Valliere in FV Arg506
Gln analyses is also gratefully acknowledged. |
Footnotes |
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Presented in part in abstract form at the 37th annual meeting of the American Society of Hematology, December 1-5, 1995, Seattle, Wash, and published in Blood. 1995;86:201a.
Received June 24, 1996;
revision received September 17, 1996;
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