Molecular Basis for Type 1 Antithrombin Deficiency - Blood

Molecular Basis for Type 1 Antithrombin Deficiency: Identification of Two Novel Point Mutations and Evidence for a De Novo Splice Site Mutation

By Kristin Jochmans, Willy Lissens, Ting Yin, Jan Jacques Michiels, Leonie van der Luit, Kathelijne Peerlinck, Marc De Waele, and lnge Liebaers

Inherited type 1 antithrombin (AT) deficiency is characterized by a reduction in both immunologically and functionally detectable protein. The disorder is associated with a high risk of thromboembolic disease. We have investigated the molecular basis of type 1 AT deficiency in three unrelated families. We have used the polymerase chain reaction single- strand conformation polymorphism (PCR-SSCP) analysis, followed by direct sequencing of the seven exons and the intron-exon junctions of the AT gene. Two novel point mutations were identified. A T to C single-base substitution was found in codon 421 in exon 6 (nucleotide position 133801, leading to an AT 421 isoleucine to threonine substitution. In

NTITHROMBIN (AT) is the major physiologic inhibitor of thrombin and other proteases of the coagulation

system. The rate of inhibition is markedly increased in the presence of heparin. The single-chain plasma glycoprotein consists of 432 amino acids and belongs to the serine prote- ase inhibitor (serpin) superfamily of proteins.’

Hereditary AT deficiency was first described by Egeberg’ as an autosomal dominant inherited disease. Affected individuals have an increased risk of venous thrombosis and embolic complications.3.4 Immunologic and functional assays allow the identification of different types of inherited AT deficiencies. In the first update of the AT mutation data base, a preferred revised classification is proposed.’ Classical type 1 or quantitative deficiency is characterized by low functional and immunologic levels of normal AT protein. Type 2, or qualitative deficiency, represents a group of defects resulting in the presence of variant AT proteins. De- pending on the localization of the mutation, there is an effect on the reactive site or heparin binding site, or a pleiotropic effect. The gene for human AT maps on chromosome lq23- 25 and contains 7 exons distributed over a 14-kb DNA sequence.‘,’ The complete nucleotide (nt) sequence shows a gene spanning 13477 bp from the transcription start site to the poly(A) addition signal.x The AT mutation data base reports 39 distinct molecular defects and nine whole or partial gene deletions in type l AT deficiency.’ We have investigated the genetic basis for type 1 AT deficiency in three unrelated families, using polymerase chain reaction single- strand conformation polymorphism (PCR-SSCP) analysis

A

From the Departments of Hematology and Medicul Genetics, A u - demic Hospital of the Free University of Brussels, Brussels, Belgium: Department of Hematology, University Hospital Rotterdam-Dijkzigt, Rotterdam, The Netherlands; and the Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium.

Submitted May 19, 1994; accepted August 8, 1994. Address reprint requests to Kristin Jochmans, MD, Laboratory oj

Hematology, Academic Hospital of the Free Universiry of Brussels, Laarbeeklaan 101, B-1090 Brussels, Belgium.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with IS U.S.C. section 1734 solely to indicate this fuct. 0 1994 by The American Socieg oj Hematology. 0006-4971/94/841 I -0007$3.00/0

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another kindred, one of three CS at nucleotide (ntl positions 5448 to 5450 in exon 3A (codon 151 or 152) was deleted, resulting in a frameshift mutation and predicting premature termination of protein translation at codon 251. In a third family, a previously reported G to A substitution, at nt position 9788 in intron 4, 14 bp in front of exon 5, was found. We have demonstrated the creation of a de novo exon 5 splice site by ectopic transcript analysis of lymphocyte mRNA. In all cases, the affected individuals were heterozygous for the mutation and no variant AT protein was detected. 0 1994 by The American Society of Hematology.

and direct DNA sequencing. We have detected a novel point mutation in exon 6 (AT 421 Ile to Thr) and a single-base deletion in exon 3A (AT 151 [-C] or AT 152 [-C]). The third case involves an already described single-base substitution in intron 4. Using ectopic transcript analysis, we could demonstrate the abnormal mRNA, resulting from the de novo-created exon 5 splice site.

MATERIALS AND METHODS

Patients

Family dM (The Netherlands). The proposita was born in 1936. At the age of 27 years, she developed deep venous thrombosis (DVT) and pulmonary embolism (PE) during the postpartum period of her first pregnancy. Similar episodes occurred after a second and third delivery. A fourth and fifth pregnancy were complicated by thrombosis before the partus. Her daughter suffered from DVT with PE after appendectomy at the age of 23 years. Six other relatives were found to be AT-deficient: four of them had venous thrombotic episodes involving the legs.

Furnily H (Belgium). The proposita, 10 weeks‘ pregnant, presented with sudden abdominal pain at the age of 26 years. At laparot- omy, mesenteric venous thrombosis was diagnosed. Resection of a part of the small bowel was necessary, and the pregnancy is currently continued under heparin therapy. AT activity and antigen levels determined on plasma samples, taken during heparin therapy, were 26% and 25%, respectively. At the age of 19 years, she had already developed a DVT with PE, without a search for AT deficiency at that time. Her sister had a DVT after an episode of immobilization, but had no problems during her two pregnancies. AT activity levels were 56%. The mother developed DVT during nearly every pregnancy (four of six). Her AT levels were 56%. Further family studies are in progress.

Fumily dV (The Netherlands). The propositus is a 5 I -year-old man with a history of DVT at the age of 19 years. A second DVT developed 25 years later, complicated with PE. His daughter presented with a first thrombotic event at the age of 18 years, after taking oral contraceptives. In the large kindred, we diagnosed six more AT deficiencies. All affected members suffered from recurrent thromboembolic events, such as DVT after pregnancy or immobilization, or cerebral thrombosis after pregnancy. An episode of PE in a 20-year-old woman had resulted in death.

Hernostutic Tests and AT Assays

Investigation of different hemostatic parameters was performed on plasma samples from patients of the three families. Activated partial thromboplastin time and prothrombin time, and fibrinoge11,

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MOLECULAR BASIS FOR TYPE 1 AT DEFICIENCY 37 43

protein C, protein S, and plasminogen levels were assayed as described previously? Functional AT activity was measured using an amidolytic heparin cofactor assay with Coatest reagents (chromogenic substrate S-2238) for anti-IIa activity and Coamate reagents (chromogenic substrate S-2765) for anti-Xa activity, both from Chro- mogenix, Molndal, Sweden. Immunoreactive AT levels were evalu- ated using the Laurel1 technique” with Assera-Plate AT III (Stago, Gennevilliers, France). Crossed immunoelectrophoresis (CIE) of plasma, in the absence and presence of heparin (20 U/mL), was performed according to the method reported by Sas et al.’’

DNA Studies of the AT Gene General. PCR-SSCP analysis and DNA sequencing of the seven

AT exons, as well as Southern blot analysis of the AT gene, have previously been described in full detail? Restriction enzyme digestion of PCR products was performed without prior purification. For each individual enzyme, the optimal salt concentrations, as indicated by the manufacturer, were adjusted by addition of a 1OX concentrate, taking into account the concentration of the different components already present in the PCR mixture. All products used in this study were from Amersham, Buckinghamshire, UK, except for the Taq polymerase (Perkin-Elmer Cetus Instrument, Norwalk, CT), the restriction enzyme E s d I (New England Biolabs, Beverly, MA) and the First-Strand cDNA Synthesis Kit (Pharmacia, Brussels, Belgium).

Study of the exon 4/exon 5 boundary by ectopic PCR. Total RNA was isolated from Percoll-purified white blood cells from hepa- rinized blood of patient dV and a normal control.’* One microgram of RNA was used in reverse-transcription reactions (First Strand cDNA Synthesis Kit) in a total volume of 33 pL. Five microliters of both patient and control reverse-transcribed RNA was used then in the first of two consecutive PCR reactions with nested primers. The first oligonucleotide primer pair consisted of AT3.82 (5”TGG- TCCTCATCTTCCCCAAG-3’; positions 7553-7572 in exon 4)’ and PS12B9 in exon 6. Five microliters of the first PCR was used in the second PCR with nested primers AT3.83 (5’-AGGTGGAGAAGG- AACTCACC-3‘; positions 7592-7611 in exon 4) and AT3.81 (5’- AACACAAGGGTTGGCTACTCTG-3’; positions 13407-13390 in exon 6). The composition of the PCR mixtures was the same as previously described for sequencing of the AT exons, except for the DNA template and the thermal cycling conditions: denaturation at 94°C for 1 minute, annealing at 55°C for l minute, and extension at 72°C for 2 minutes for 30 cycles, and a final extension at 72°C for 5 minutes. The fragment lengths of the PCR products using primer combinations AT 3.82PS12B and AT3.83/AT3.81 are 485 bp and 413 bp, respectively. For DNA sequencing of the exon 4/ exon 5 boundary of patient dV, 10 pL of the PCR-amplified AT3.83/ AT3.81 fragment was incubated with 10 U of restriction enzyme EsaJI for 2 hours at 37T , loaded on a 5% nondenaturing polyacryl- amide gel, and electrophoresed for 4 hours at 200 V. The gel was stained with ethidium bromide, and the undigested fragment was cut out and eluted overnight in 100 pL of water. Ten microliters was used to reamplify the fragment with primers AT3.83/AT3.81. This PCR mixture was then purified on a Centricon 100 microconcentrator (Amicon, Beverly, MA) and directly sequenced as described previously with an internal primer AT3.S (5”AGCAATCACAAC- AGCGGTAC-3‘; positions 13291-13272 in exon 6). For DNA sequencing of the same region of the control, the PCR product obtained after the second PCR was immediately purified on a Centricon 100 microconcentrator and sequenced as described for the patient.

RESULTS

Hemostatic Tests and AT Assays

Immunoreactive and functional AT activity levels, determined in affected individuals, were equally decreased (Table

Table 1. AT Functional and Immunologic Quantitation

Heparin Cofactor Activity (%)

Subiect Anti-lla Anti-Xa Antigen (%)

Family dM Proposita 68 68 68 Daughter 72 63 64

Proposita ND 26* 25* Sister 65 56 52

Family H

Family dV Propositus 65 58 64

Daughter 68 64 59 Normal 70-120 70-120 80-1 20

Abbreviation: ND, not determined. x Samples taken during heparin therapy.

1). The results in the three unrelated families were consistent with type 1 AT deficiency. AT-CIE, in the absence and the presence of heparin, was performed on plasma samples of AT-deficient subjects in the three kindreds. In family dM (Fig lA), family H (Fig lB), and family dV (Fig lC), the CIE with heparin in the first dimension showed no abnormal migrating component. The characteristic fast-moving anodal peak was present in reduced amounts as compared with the normal control. All other hemostatic parameters were normal (data not shown).

DNA Studies

Southern blot analysis of PstI and BarnHI digests of the AT gene of all individuals described in Table 1 did not show any abnormalities (data not shown). In PCR-SSCP analysis of the seven AT exons, abnormal migrating bands, as compared with the patterns observed in two non-AT-deficient controls, were found for exon 6 in family dM, for exon 3A in family H, and for exon 5 in family dV. For each family, the corresponding exon was sequenced.

In Family dM, a T to C substitution was found at position 13380 in one of the alleles (Fig 2A), predicting an isoleucine (ATC) to threonine (ACC) substitution at position 421 (I421T) of the AT gene product. The presence of this mutation was confirmed by digestion of the PCR product of exon 6 (210 bp) of both affected family members with restriction enzyme MboII. In normal individuals, this enzyme generates four fragments of lengths 114, 59, 24, and 13 bp, whereas the mutation described in this family destroys one site, generating fragments of 173 (114 bp + 59 bp), 24, and 13 bp. The two patients were indeed heterozygous for the 173-bp and the 114-bp and 59-bp fragments, whereas in 115 normal unrelated controls only bands of 114 and 59 bp were found (data not shown). To further exclude that the T to C substitution represents a natural polymorphism, the six other exons of the proposita were sequenced, but no other abnormalities compared with the published sequence were found.

In family H, sequencing of exon 3A showed a deletion of a C in one allele; the exact location of this deletion cannot be determined because it occurs in a stretch of three CS at positions 5448 to 5450 (Fig 2B), amino acid positions 15 1 or 152. The deletion results in a frameshift, predicting a




3744 JOCHMANS ET AL

highly truncated protein from amino acid position 153, and a premature stop codon at position 25 1 . This mutation does not create or destroy a restriction enzyme site.

In family dV, a G to A substitution was found in one allele, 14 bp in front of exon S (position 9788; Fig 2C). The mutation destroys the unique Mspl restriction site in the 1 8 1 bp exon S PCR fragment. The propositus and his symptom- atic daughter were heterozygous for MspI fragments of 18 1 bp. and 135 bp and 46 bp. whereas normal controls only showed 135-bp and 46-bp fragments (data not shown). This mutation has been described previously.".'J The creation of a new splice site in front of the usual exon S splice site has been suggested. To verify this hypothesis, we have developed an ectopic PCR, using RNA from white blood cells, which allows the amplification of part of exon 4, exon S and part of exon 6. The new splice site would predict the insertion of 12 bp of intron 4 into the AT cDNA. In the normal cDNA, a unique BsnJl site ( ENNm: two CS and two Gs, interrupted by two random bases) is present in the AT3.83/ AT3.8 1 PCR fragment, spanning the exon 4/exon S boundary (=CAS exon 41s exon S ) . In the alternatively spliced boundary, this site would be destroyed by the insertion of 12 bp of intron 4 DNA (=CAS exon 4hcttccttccag intron 41s exon S ) . PCR amplification of the fragment, followed by digestion with BscrJI, indeed confirmed that the patient was heterozygous for fragments of 413 bp, and 175 bp and 238 bp, instead of being homozygous for the 175-bp and 238-bp fragments, as normally predicted (Fig 3).

Densitometric scanning of the bands from the patient and the normal control showed that almost equal amounts of uncut (48%) and cut (52%) fragments were present in the patient. The uncut fragment was isolated from a polyacryl- amide gel, reamplified, and sequenced. The sequencing showed that the 12 bp from intron 4, just in front of exon S , were present, whereas in the normal control the expected sequence was found (Fig 4).

DISCUSSION

Hereditary AT deficiency is a well recognized cause of thrombophilia.'' It is associated with a high risk of recurrent venous thrombosis and PE. Type I or classical type is characterized by low functional and immunologic protein activity without variant AT protein on CIE. This type accounts for the majority of clinical problems in affected individuals. We studied three unrelated kindreds with type 1 AT deficiency and found a high incidence of thrombotic disease.

In 1991, an AT mutation data base was created. which compiled the molecular defects responsible for AT deficiency.'" During the following years, several new genetic abnormalities have been described. The content of the first update of the data base' shows an expansion of the listings of type I deficiencies, and more recent reports can be added." These data indicate that type 1 deficiency is caused by het- erogeneous molecular defects, with few partial or whole gene deletions, and a majority of point substitutions, minor inser- tions, or minor deletions. Most of these mutations are unique events that are characteristic for one kindred. We studied

deficient member of thethree different families Propos- three different families. and two of the three mutations iden- itus from family dM. (B) Sister of proposita from family H. (C) Propos- tified have not been described before now. itus from family dV. Using PCR-SSCP analysis, followed by direct DNA se-

Fig 1. CIE in heparin-agarose: normal control (top) and an AT-




MOLECULAR BASIS FOR TYPE 1 AT DEFICIENCY 31 45

T G C A

4

A B C

N I

T

Fig 2. Direct genomic sequencing of PCR-amplified exon 6(A), exon 3A (B), and exon 5(C) from the propositae of the families dM, H, and dV, respectively. (A) The presence of both a T (normal) and a C (mutant) in the second position of codon 421 is indicated by an arrow. (B) Arrow indicates the start of the frameshift in this family: deletion of a C in one allele. Below the arrow the sequences of both alleles are identical, whereas above the arrow the normal and the deleted sequences are intermixed. (C) Arrow at the left points at the presence of both a G (normal) and an A (mutant) at a position 14 bp in front of exon 5.

quencing of the seven exons and intronkxon junction regions, we were able to detect the different abnormalities. SSCP analysis has proven to be a rapid and sensitive screening method for the detection of base changes in given sequences of genomic DNA.” This technique is based on the detection of conformational changes of single-stranded DNA in nondenaturing gels, caused by modifications of size or sequence. In our patients, SSCP analysis disclosed aberrant patterns in exon 3A, intron 4, and exon 6.

In family dM, the SSCP gels showed abnormal bands in exon 6 not found in healthy controls. On DNA sequencing, we detected a single-base substitution in codon 421. At nt position 13380. a heterozygous T to C transition was demon-

strated, which converted an ATC codon for isoleucine to an ACC codon for threonine. This T to C substitution removes a cutting site for the enzyme MhoII. Restriction analysis with this enzyme allowed confirmation of the sequencing data. The substitution has not been described previously, neither as a mutation nor as a polymorphism, and it was not found in the AT gene of 115 normal unrelated controls. DNA sequencing of the other six exons showed no further abnormalities. Referring to the proposed three-dimensional structure of the serpins,’’ isoleucine 421 is located in the AT protein in sheet strand SB. At the carboxy-terminal end of the molecule, sheet strands 4B and SB represent highly hydrophobic regions, which are close together in the tertiary structure of




3746 JOCHMANS ET AL

fetus remained in good condition and the pregnancy is currently continued under subcutaneous heparin therapy. The patient had a history of previous DVT and PE, without diag- nosis of AT deficiency. Very low levels of functional and immunologic AT activity were found, probably influenced by the continuous heparin therapy. The mother and a sister, both with a history of DVT, had equally deficient levels of functional and immunologic AT protein. CIE assays, in the absence and the presence of heparin, showed a single, small, fast-moving peak, with normal AT protein mobility. No variant protein was demonstrated. Studying the molecular basis for this type I AT deficiency, PCR-SSCP gels demonstrated a disturbed pattern for exon 3A. DNA sequencing showed a single-base deletion in this exon. In the normal nucleotide sequence of the AT gene,' three consecutive CS are found at positions S448 to S450 (codon 15 I T G and codon 1 S2

a frameshift. In protein translation, codon 15 1 remains unaf- fected (TCC), whereas codon IS2 changes from CTT to TTA, both coding for a leucine. Starting from codon 153, the translation of an altered amino acid sequence of approxi- mately 1 0 0 residues is predicted. In the new reading frame, a premature stop codon (TGA) is encountered at amino acid position 251 in exon 4. There was no detectable truncated protein in the plasma. As suggested previously, the absence of the gene product in the plasma may result from different mechanisms: transcription of unstable mRNA, translational or posttranscriptional defects, or intracellular degradation of the translated p~lypeptide.'~.''

Family dV presented with an interesting mutation, re- cently reported by Vidaud et al'? and Chowdhury et located in the intron 4/exon S junction region. In this large kindred, we diagnosed type 1 AT deficiency in several members, all of whom had a history of thromboembolic events at a young age. PCR-SSCP analysis demonstrated an abnormal pattern for exon S. DNA sequencing of the seven exons and

- CTT). In our case, a deletion of one C was found, generating

Fig 3. 6saJl digestion of PCR product, spanning exons 4 through 6. The PCR product, generated after reverse transcription of leuko- cyte RNA from a control and the propositus of family dV, followed by nested PCR, was cut with restriction enzyme 8saJl and run on a 2% agarose gel (lanes 1 and 3). Uncut fragments are shown in lanes 2 and 4. Lanes 1 and 2, normal control; lanes 3 and 4, propositus dV family.

the AT protein. Consensus sequences become apparent by aligning the amino acids of the serpin superfamily members." Highly conserved nonpolar residues are present in both strand 4B (positions 408 to 410) and strand 5B (positions 422, 424. and 426). Frameshift mutations, deletions, and two single-amino acid substitutions (Gly 424 Arg" and Ala 427 Asp'") have been idcntified in this part of the gene, and all are associated with type I AT deficiencies.' Appar- ently, the substitution of isoleucine at position 421 by threonine belongs to the same group of mutations. Antigenic and functional AT assays were decreased to the same extent and no variant AT protein could be detected by CIE. The three single-amino acid substitutions, so far detected in this region, drastically influence the nature of the SB sheet strand: hydrophobic nonpolar side groups are replaced by polar (1421T). acid (G424R). or basic (A427D) side groups. Proba- bly, these changes have an important influence on the tertiary structure of the molecule by disturbing the alignment of sheet strand 4B and SB, with consequent impairment of the stability of the mutant AT protein. Lane et al" and Watton et al" studied different mutations in or around this part of the gene. These investigators demonstrated that point mutations, leading to single-amino acid substitutions in the 402 to 407 region of AT strand IC, have pleiotropic effects on the resulting variant proteins. They decrease the amount of circulating variant protein in most cases, and they affect both the reactive site and the heparin-binding properties. The P429L substitution in the AT Budapest variant exhibits the same pleiotropic effect." The proline at position 429 lies just out of sheet strand SB and is involved, together with N428, in the initiation of a one-turn C-terminal helix (h13). The reported defect maps close to 142 IT, G424R, and A427D in the primary structure of the AT molecule. The difference in expression between the mutations underlying the pleitropic defects, and the substitutions in sheet strand SB, can probably be explained by their position in the native molecule. Moreover, in the AT Budapest variant, the nonpolar proline is substituted by another nonpolar leucine.

A second novel mutation was found in family H. The proposita presented with a major thrombotic event during her first pregnancy. She developed a mesenterial thrombosis, which necessitated partial resection of the small bowel. The

P "

C ' T G c A " T G C A '

d

Fig 4. Sequencing of the exon 4/exon 5 boundary in the propositus of the family dV (P) and a normal control (Cl. The cDNAs were sequenced from the antisense strand (direction from exon 6 to exon 41. In the control, the lower arrow indicates the last base of exon 4, and exon 5 is to the bottom of the figure. In the patient, the upper and lower arrows indicate the 12-bp insertion of intron 4 DNA between exon 4 (top) and exon 5 (bottom).




MOLECULAR BASIS FOR TYPE 1 AT DEFICIENCY 3747

the flanking regions showed a single-base substitution in intron 4, in one allele. At nt position 9788, 14 nt 5‘ to exon 5, a G to A substitution was detected. This mutation occurs in a CpG dinucleotide. The localization of an A at this position leads to a new nt sequence, mimicking in a perfect way the 3’ splice site consensus sequence.26 This finding suggests that the mutation might create an abnormal splice site for exon 5. If so, 12 bp would be added to the coding part of the gene, resulting in an altered mRNA. This mRNA would code for four extra amino acids in the polypeptide chain. The equally low functional and immunologic AT levels, indi- cating a type 1 deficiency, together with the absence of a variant AT protein in CIE, support the idea that the mutant allele is not expressed at the protein level. We have investigated the phenotypic expression of the mutation at the mRNA level. The AT gene is normally transcribed in liver tissue, which is poorly accessible for obtaining study mate- rial. Many tissue-specific genes have been found to be transcribed ectopically in various tissues, including lymphocytes. These ectopic transcripts facilitate the study of the effect of certain mutations on the transcript struct~re.~’ Berg et a12* also used ectopic transcript analysis to investigate a putative splicing defect involving the exon 3A donor splice site and demonstrated the splicing out of exon 3A. We isolated total RNA from the propositus’ peripheral blood lymphocytes and reverse-transcribed it. PCR was repeatedly performed with nested primers, spanning the studied region. Restriction enzyme analysis of the obtained cDNA showed the heterozygous absence of the normal BsaJI restriction site. Subse- quently, the mutant fragment was isolated and sequenced, confirming the insertion of 12 bp in exon 5. Our studies indicate that, although apparently no mutant protein is formed, there is expression of the mutant allele at the mRNA level with nearly exclusive utilization of the new splice site. At the protein level, glycine at position 353 is normally coded in part by exon 4 (G) and exon 5 (GT). If the insertion of 12 bp would happen, this glycine would be lacking and be replaced by valine-phenylalanine-leucine-proline-glycine. In the tertiary structure, glycine 353 is close to glycine 93, located at the end of helix B, Addition of side chains at these positions would disrupt this structure. The substitution of glycine 353 by valine, together with the insertion of four extra amino acids, probably leads to major alterations of the normal tertiary structure, which results in an unstable protein.

In the present report, we studied three unrelated kindreds with hereditary type 1 AT deficiency, associated with famil- ial occurrence of serious thrombotic complications. We identified two novel point mutations. A single-base substitution (nt position 13380, T to C) in exon 6 resulted in a single- amino acid change at a highly conserved residue, probably inducing an important change in the tertiary structure of the molecule. A single-base deletion in exon 3A (nt position 5448,5449, or 5450) introduced a frameshift with premature stop at codon 251. The third point mutation, in intron 4 (nt position 9788, G to A) was suggested to create a new splice site for exon 5. Using ectopic transcript analysis, we determined the phenotypic consequences of this genetic defect at the mRNA level by demonstrating the presence of an abnormal mRNA, including 12 extra bp between exon 4 and 5 , predicting an unstable protein with four extra amino acids.

ACKNOWLEDGMENT

We thank the staff of the Laboratories of Coagulation and Medical Genetics for technical support and Brigitte Guns for secretarial assis- tance. We are grateful to A. de Oude and K. Van Assche for services rendered.

REFERENCES 1. F‘ratt CW, Church FC: Antithrombin: structure and function.

Semin Hematol 28:3, 1991 2. Egeberg 0: Inherited antithrombin 111 deficiency causing

thrombophilia. Thromb Diath Haemorrh 13516, 1965 3. Thaler E, Lechner K: Antithrombin 111 deficiency and thrombo-

embolism. Clin Haematol 10:369, 1981 4. Michiels JJ, Van Vliet HH: Hereditary antithrombin 111 defi-

ciency and venous thrombosis. Neth J Med 27:226, 1984 5. Lane DA, Olds W, Boisclair M, Chowdhury V, Thein SL,

Cooper DN, Blajchman M, Perry D, Emmerich J, Aiach M: Anti- thrombin 111 mutation database: First update. Thromb Haemost 70:361, 1993

6. Bock SC, Harris JF, Balazs I, Trent JM: Assignment of the human antithrombin 111 structural gene to chromosome 1q23-25. Cytogenet Cell Genet 39:67, 1985

7. Bock SC, Marrinan JA, Radziejewska E: Antithrombin 111 Utah: Proline-407 to leucine mutation in a highly conserved region near the inhibitor reactive site. Biochemistry 27:6171, 1988

8. Olds RJ, Lane DA, Chowdhury V, De Stefan0 V, Leone G, Thein SL: Complete nucleotide sequence of the antithrombin gene: Evidence for homologous recombination causing thrombophilia. Biochemistry 32:4216, 1993

9. Jochmans K, Lissens W, Vervoort R, Peeters S, De Waele M, Liebaers I: Antithrombin-gly424arg: A novel point mutation responsible for type 1 antithrombin deficiency and neonatal thrombosis. Blood 83:146, 1994

10. Laurel1 CB: Electroimmunoassay. Scand J Clin Lab Invest 125:21, 1972

11. Sas G, Pepper D, Cash J: Investigations on antithrombin 111 in normal plasma and serum. Br J Haematol 30:265, 1975

12. Chomczynski P, Sacchi N: Single-step method of RNA isola- tion by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156, 1987

13. Vidaud D, Gandrille S, Emmerich J, Sirieix ME, Alhenc- Gelas M, Fiessinger JN, Si6 P, Gouault-Heilman M, Aiach M: Identi- fication of 6 novel mutations responsible for type 1 AT deficiencies. Thromb Haemostas 65:762, 1991 (abstr)

14. Chowdhury V, Olds RJ, Lane DA, Conard J, Pabinger I, Ryan K, Bauer KA, Bhavani M, Abildgaard U, Finazzi G, Castaman G, Mannucci PM, Thein SL: Identification of nine novel mutations in type 1 AT deficiency by heteroduplex screening. Br J Haematol 84:656, 1993

15. Demers C, Ginsberg JS, Hirsh J, Henderson P, Blajchman MA: Thrombosis in antithrombin 111 deficient persons-Report of a large kindred and literature review. Ann Intern Med 116:754, 1992

16. Lane DA, Ireland H, Olds RJ, Thein SL, Peny DJ, Aiach M: Antithrombin 111: A database of mutations. Thromb Haemost 66657, 1991

17. Orita M, Suzuki Y, Sekiya T, Hayashi K: Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5:874, 1989

18. Huber R, Carrell RW: Implications of the three-dimensional structure of a,-antitrypsin for structure and function of serpins. Bio- chemistry 28:8951, 1989

19. Lane D, Caso R: Antithrombin: Structure, genomic organiza- tion, function and inherited deficiency. Bailliere Clin Haematol 2:961, 1989

20. Millar DS, Lopez A, White D, Abraham G, Laursen B, Hold-




3748 JOCHMANS ET AL

ing S, Reverter JC, Reynaud J, Martinowitz U, Hayes JP, Kakkar VV, Cooper DN: Screening for mutations in the antithrombin I11 gene causing recurrent venous thrombosis by single-strand conformation polymorphism analysis. Hum Mutat 2:324, 1993

2 I . Lane DA, Olds RJ, Conard J, Boisclair M, Bock SC, Hultin M, Abildgaard U, Ireland H, Thompson E, Sas G, Horellou M, Tamponi G, Thein S: Pleiotropic effects of antithrombin strand IC substitution mutations. J Clin Invest 90:2422, 1992

22. Watton J, Longstaff C, Lane DA, Barrowcliffe TW. Heparin binding affinity of normal and genetically modified antithrombin 111 measured using a monoclonal antibody to the heparin binding site of antithrombin 111. Biochemistry 32:7286, 1993

23. Olds RJ, Lane DA, Caso R, Panico M, Morris HR, Sas G, Dawes J, Thein SL: Antithrombin 111 Budapest: A single amino acid substitution (429 Pro to Leu) in a region highly conserved in the serpin superfamily. Blood 79:1206, 1992

24. Vidaud D, Emmerich J, Sirieix ME, Sit P, Alhenc-Gelas M,

Aiach M: Molecular basis of antithrombin 111 type 1 deficiency: Three novel mutations located in exon 1V. Blood 78:2305, 1991

25. Gandrille S, Vidaud D, Emmerich J, Clauser E, Sit P, Fiessinger JN, Alhenc-Gelas M, Priollet P, Aiach M: Molecular basis for hereditary antithrombin I11 quantitative deficlencies: A stop codon i n exon IIIa and a frameshift in exon VI. Br J Haematol 78:414, 1991

26. Shapiro MB, Senapathy P: RNA splice junctions of different classes of eukaryotes: Sequence statistics and functional implications in gene expression. Nucleic Acids Res 15:7155, 1987

27. Roberts RG, Bobrow M, Bentley DR: Point mutations i n the dystrophin gene. Proc Natl Acad Sci USA 89:233 I , 1992

28. Berg L-P, Grundy CB, Thomas F, Millar DS, Green PJ, Slom- ski R, Reiss J, Kakkar VV, Cooper DN: De novo splice site mutation in the antithrombin 111 gene causing recurrent venous thrombosis: Demonstration of exon skipping by ectopic transcript analysis. Geno- mics t3:1359, 1992




1994 84: 3742-3748

LiebaersK Jochmans, W Lissens, T Yin, JJ Michiels, L van der Luit, K Peerlinck, M De Waele and I mutationtwo novel point mutations and evidence for a de novo splice site Molecular basis for type 1 antithrombin deficiency: identification of

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