Antithrombin-Gly 424 Arg: A Novel Point Mutation ...Antithrombin-Gly 424 Arg: A Novel Point Mutation Responsible for Type 1 Antithrombin Deficiency and Neonatal Thrombosis By Kristin

Antithrombin-Gly 424 Arg: A Nove l Po in t Mutat ion Respons ib le for Type 1 Antithrombin Deficiency and Neonatal Thrombosis

By Kristin Jochmans, Willy Lissens, Raf Vervoort, Stefaan Peelers, Marc De Waele, and lnge Liebaers

Inherited type 1 antithrombin (AT) 111 deficiency is characterized by a decrease of immunoreactive and functional protein levels toabout 50%. The disorder is associated with a significantly increased risk of thromboembolism. We have investigated the molecular basis of type 1 AT deficiency in a Belgian family. The diagnosis of the disease was primarily made in a newborn girl with unusually severe thrombotic complications. Using the polymerase chain reaction and single-strand conformation polymorphism analysis, followed by direct sequencing of AT gene fragments, we identified a novel point mutation in exon 6. We de-

NTITHROMBIN (AT) 111 is a single-chain glycoprotein consisting of 432 amino acids. It belongs to the family

of serine protease inhibitors (serpins) and has a principal role in inactivating thrombin and other activated proteases of the coagulation system. The rate of this inhibition is strongly accelerated by heparin. Hereditary deficiency of AT, first described by Egeberg’ is inherited in an autosomal dominant way and associated with an increased risk of recurrent thromboembolism. The prevalence of AT deficiency is estimated to be about 2% to 6% in patients with history of thrombosis’ and 15,000 to 1:2,000, or even higher. in the general population.’

Multiple attempts to classify inherited AT deficiency were based on the presence of quantitative and/or qualitative abnormalities detected in plasma, using immunologic and functional assay^.^-^ Recently, a subdivision of the variants of the disease with regard to the molecular defects was pro- posed,’ resulting in four types. Type 1 is characterized by a 50% decrease in antigenic and functional levels. Type 2 has a gene defect affecting the thrombin-binding domain, whereas in type 3 the heparin-binding domain is altered. Plasma AT antigen levels are normal in types 2 and 3. Type 4 consists of a miscellaneous group of unclassifiable mutations. The gene for AT lies on the long arm of chromosome 1 and contains seven exons distributed over a 19-kb DNA sequence.’ Six DNA-sequence polymorphisms have been described’ and two of these have often been used for genetic analysis. Nearly 30 different single-base mutations associated with immunoreactive variant AT molecules have been reported.’ Approximately 10 novel mutations associated with “classical” type 1 AT deficiency have more recently

A

From the Depurtment.s ofHemalologv, Medical Gcnetic.Y, and PP diatrics, Academic Ho.~,niiaioflhe Fret Universiny qfBntssc.ls. B n w sel.~, Belgium.

Suhmitted.dApril8, 1993; atreptedAzrgtrst 26, 1993. Address reprint requests to Kristin Jochtnans, MD, Laboratory (?l

Hematology, Academic Hospital of the Free Universit,v of Brussels, Luarheeklaan 101, B-1090, Brussels, Belgium.

The publication costs ofthis article were defiayed in part by page churge payment. This article must therethe be hereby marked “advertisement” in accordancc1 “ith I8 L!S.C. section I734 solely t o indicate thisfact. 0 I994 by The American Society o f Hematology. 0006-4971/94/8301-0010$3.00/0

146

tected a G to C substitution in the first position of codon 424 leading to a glycine to arginine substitution. The mod- ification at this highly conserved position in the serine protease inhibitor gene family probably leads to an unstable mutant-gene product. The mutation creates a unique restriction site for the enzyme Hha I in exon 6. This change permitted a rapid and accurate screening of the kindred with identification of the molecular defect in five otherfam- ily members. 0 1994 by The American Society of Hematology.

been We report a case of an unusual manifestation of hereditary type 1 AT deficiency in a newborn girl. We studied the molecular basis of the defect in her family and detected six affected members. Applying polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis and DNA sequencing, we found a novel point mutation located in exon 6 (exon numbering as designated by Bock et aI9). A G to C transition in the first position of codon 424 (GGC to CGC) leads to a Gly 424 Arg substitution. The changed nucleotide sequence creates a unique restriction site for the enzyme Hha I, which allows us to easily screen other family members.

MATERIALS AND METHODS

Patients. The proposita was born in August 1990, after a normal pregnancy of 40 weeks. Delivery was uneventful except for a shoulderdystocia that led to a fractured right clavicle. The baby was in good health at birth. During the immediate postnatal period, the baby’s clinical status deteriorated progressively and within a few days, multiple thrombotic eventsoccurred. Adocumented myocardial infarction led to cardiac failure, and thrombi were formed at venous or arterial puncture sites. Finally, a cerebral thrombosis developed in the dural venous sinuses. There were no infectious proh- lems. Low levels of AT were repeatedly found, but were difficult to assess because normal values of many hemostatic parameters are low in newborn infants. However. during the further evolution, AT levels remained low. whereas other parameters increased to normal adult values. A family study led to the finding of low antigen and functional levels of AT in six members, three of them with no history ofthromboembolism. The family tree is shown in Fig 1. Patient I3 developed deep vein thrombosis after delivery. The history of patient IIs showed an episode of severe thromboembolic complications after a surgical intervention at the age of 20. Cardiologic and psychomotoric follow-up of the baby showed good recuperation of all functions and the child is now in good physical condition.

Ifemostutic t w s . Activated partial thromboplastin time (APTT). prothrombin time(PT), and fibrinogen levels wereassayed by standard techniques. Protein C and plasminogen levels were measured by chromogenic substrate assay (Behringwerke, Marburg, Germany). Protein S levels were assayed by electroimmunodiffusion technique using Assera-Plate Protein S (Stago, Gennevilliers, France).

,4 T a.ssa~,s. Plasma samples were obtained from the proposita and different family members. Functional AT 111 activity was measured in the presence of heparin (heparin cofactor activity) using the Coatest reagents with the chromogenic substrate S-2238 (Kabi Diagnostica. Molndal. Sweden). Anti-Xa activity was tested with

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ANTITHROMBIN-Gly 424 Arg 147

I

m 1 2 3

Fig 1. Family tree. (0, 0) Subjects with normal AT act ivi i and antigen levels; (l&@) subjects with low AT activity and antigen levels; (m, 01 subjects with low AT activity and antigen levels, and with thrombotic episodes; (El, 01 subjects not tested. The arrow points at the proposita (ill,).

heparin using the Coamate reagents with the chromogenic substrate S-2765 (Chromogenix, Molndal, Sweden). Immunoreactive AT levels were evaluated using the electroimmunodiffusion method of Laurel1 with Assera-Plate AT 111 (Stago). Crossed immunoelectro- phoresis (CIE) was performed according to Sas et al,” in the presence of heparin (20 U/mL). The second dimension run was performed in agarose containing 1% rabbit anti-AT 111 antiserum (Behring, Marburg, Germany).

PCR-SSCP analysis of the AT gene. DNA was isolated from peripheral blood collected on EDTA, as previously described.” All seven exons of the AT gene were amplified by E R L 2 using oligonu- cleotide primer pairs AT 1.5 (5”GAGATTTAGAGGAAAGAACC- 3’) and AT 1.3 (S-TTGGAGGTCATTCCTGTGAGTC-3’) for exon I , PI and P2 for exon AT 3.5 (S-ACCACCCATGTTAAC- TAGGC-3’) and AT3.3 (S-CTCCAGCAGTCTTCAGCAGC-3’) for exon 3A, E3-5 and E3-3 for exon 3B,14 E4-5 and E4-3 for exon 4,14 E5-5 and E5-3 for exon 5,14 and PS1 IC and PSl2B for exon 6.14 All reactions were performed in a final volume of 50 pL containing 100 ng of genomic DNA, IO pmol of each primer, 50 mmol/ LKCI, 10 mmol/L Tris-HCI pH 8.3, 2 mmol/L MgCI2, 12.5 pmol/L of each Z’deoxyadenosine 5’-triphosphate (dATP), 2” deoxycytidine S-triphosphate (dCTP), 2’-deoxyguanosine 5‘-triphosphate (dGTP), 2”deoxythymidine S-triphosphate (dTTP), 2.5 U of Taq polymerase (Perkin-Elmer Cetus Instrument, Nonvalk, CT), I O pCi ”S-dATP (1 ,OOO Ci/mmol; Amersham, Buckingham- shire, UK), IO pCi ”S-dCTP (1,000 Ci/mmol; Amersham), and 0.01% (wt/vol) gelatin. Thermal cycling conditions were denatur- ation at 94°C for 30 seconds, annealing at 57°C for 30 seconds and extension at 72’C for 1 minute for 30 cycles, and a final extension at 72°C for 5 minutes.

For SSCP analysis,15.’6 5 pL of each PCR product was mixed with 20 pL of a solution containing 0.1% sodium dodecyl sulfate (SDS), I O mmol/L EDTA pH 8, and 20 pL of 95% formamide, 0.05% bro- mophenol blue, and 0.05% xylene cyano1 FF and 20 mmol/L EDTA pH 8. Five microliters was removed for the identification of double-stranded fragments, and the remaining 40 pL was heated to 85°C for 5 minutes and then immediately placed on ice. Nondena- tured and denatured samples, 3.5 pL, were then loaded on SSCP gels. All the different conditions ofelectrophoresis, described by Mi- chaud et al,” were tested except that either 6% or 8% polyacrylamide gels (acrylamide: bis ratio 29: I and 3731) were used. After electrophoresis the gels were fixed in 10% (vol/vol) of acetic acid/ 10% (vol/vol) of methanol and dried on filter paper. Autoradiogra- phy was performed for 24 hours.

DNA sequencing of the seven AT exons. The seven AT exons were PCR amplified in a total volume of 100 pL as described for SSCP analysis except that 1 pg of genomic DNA, 200 pmol/L of each dNTP, and 1 0 0 pmol of each primer were used. Radioactive nucleotides were omitted from the reaction mixtures. After ampli-

fication, the PCR products were purified on a Centricon 100 micro- concentrator (Amicon, Beverly, MA) and sequenced in both direc- tions by dideoxynucleotide chain termination with the PCR primers, using the Sequenase Version 2.0 kit (US Biochemical Corp, Beverly, MA) as described.’*

Restriction analysis of the ATgene. Southern blot analysis of 5 p g samples of genomic DNA of patients and controls was performed as previously described.lg Pst I and BarnHI were used as restriction enzymes and a full-length human AT cDNA was used as a probe (pAT3; American Type Culture Collection, Rockville, MD). Exon 6 of the AT gene was PCR amplified as described in the previous section. Ten microliters of unpurified product was added to I O pL of a solution containing 50 mmol/L KCI, IO mmol/L Tris-HCI pH 7.5, 18 mmol/L MgCI2, 2 mmol/L dithiothreitol, and 0.2 mg/mL bovine serum albumin. Five units of Hha I (New England Biolabs, Beverly, MA) was then added to each tube and the tubes were incu- bated at 37’C for 2 hours. The whole samples were then applied to a 2% agarose mini-gel and run for 1 hour at 100 V. The gel was stained with ethidium bromide and visualized under UV light at 312 nm.

RESULTS

Hemostatic tests and AT assays. Laboratory investigations of the baby’s hemostatic parameters are shown in Ta- ble l . As expected in newborns, a few values were low, shortly after birth. Determinations later in life showed normal adult levels for all parameters except for AT. Protein S , protein C, and plasminogen levels were normal in both parents and in other family members (data not shown). The results of the familial AT assays are presented in Table 2. The functional and immunologic assays of AT in the affected family members are consistent with type 1 AT deficiency.

AT-CIE in heparin-agarose, performed on plasma samples of the AT-deficient subjects, showed no abnormal mi- grating component. A single fast-moving anodal peak was displayed, showing the same mobility as that of the control plasma (Fig 2) , and a normal binding to heparin. The areas under the anodal peaks were substantially smaller in the affected individuals, corresponding to the lower levels of circulating AT protein.

DNA studies. Southern blot analysis of Pst I and BamHI digests of the AT gene of the subjects Iz , 114, 116, II,, 111, , 1112,1113 and normal controls with a full-length cDNA probe did not show any abnormalities (data not shown). On Pst I digestions, constant fragments of 1.8 kb (encompassing exon 2) , 2.5 kb (exon 6) and polymorphic fragments of 10.5 kb and 5.5 and 5.0 kb (exons 3A to 5) were seen in the pa-

Table l. Hemostatic Parameters of the Proposita

Neonatal Values

Values Normal Adult at 1 mo Values

PT (S) 13.9 12.1 10.8-13.3 APTT (S) 44.2 37.3 26-38.5 Fibrinogen (g/L) 2.94 2.25 1.50-4.00 AT (96) 37 42 70-1 20 Protein C (%) 45 67 65-1 20 Protein S (%) 80 113 70-1 20 Plasminogen (%) 73 95 80-1 20




148 JOCHMANS ET AL

Table 2. AT Functional and Immunologic Quantitation

Heparin Cofactor Activity (%)

Subject Anti-lla Anti-Xa Antigen 1%)

12 62 64 46 13 56 56 45 111 105 105 85 112 101 101 92 113 94 ND 88 114 92 110 95 115 51 51 45 1lS 45 50 40 117 94 ND 97 118 60 50 45 Ill, 37'159t 48t 30'151 t 1112 115 ND 117

1113 92 110 110 Normal 70-1 20 70-1 20 80-1 20

Numbering refers to the family tree represented in Fig 1 Abbreviation: ND, not determined.

Neonatal value. t Value at 1 year of age.

and 35 years old24 or I5 and 50 years old.2 We describe the case of a full-term neonate, apparently in good health at birth, except for a shoulder dystocia and fractured right clavicle. Predisposing factors for neonatal thrombosis, such as severe birth asphyxia, shock syndrome, or vessel catheter- ization were not present initially. There were no congenital malformations. The baby's clinical condition rapidly deteriorated. Multiple thrombotic events did occur during the first days of life, involving venous and arterial sites. A well- documented myocardial infarction was diagnosed 12 hours after birth. Subsequently, thrombi developed in the right atrium, at vein puncture places, in the radial artery after blood-gas monitoring. and finally in the dural venous sinuses. Low functional and antigenic levels of AT were found and the diagnosis of hereditary AT deficiency was confirmed after repeated examinations ofthe child's plasma (until 1 year of age) and after detecting the deficiency in the father and grandmother. Other hemostatic parameters were

tient. BurnHI digestion showed constant fragments of 10.5 kb (exons 2 to 5) , 5.0 kb (exon 6) and polymorphic 1.44F allele) and 1.5-kb (S allele) fragments (exon l).2o From the family study we deduced that the disease segregated with the presence of the Psf I site and the S allele. Moreover, because no difference in the intensity ofbands was observed between the patient and normal controls, it was concluded that the mutation leading to an AT deficiency in this family should be caused by a minor change in the gene.

SSCP analysis of the seven exons showed normal patterns in exons I to 5 and a modified pattern in exon 6 (Fig 3A). The exon 6 fragment was PCR amplified, purified, and sequenced. The normal sequence was found except for the first position of codon 424 of the AT cDNA, where both a G and a C were apparent (Fig 3B). The latter G to C conversion predicts a glycine to arginine substitution in the mutated protein. This G to C transition creates a unique Hhu I restriction site in the exon 6 PCR-amplified fragment (210 bp), resulting in fragments of 154 bp and 56 bp on restriction analysis. The results of this analysis for all family members is shown in Fig 4. The substitution was found in all members with reduced AT activity, whereas it was absent in all family members with normal AT levels.

DNA sequencing of the six other AT exons did not show any abnormalities in comparison with the published AT se-

or -8 in the 3' end of intron 4 just in front of exon 5 (ttcs- ttccadexon 5). This insertion is not disease causing, because it was found in homozygous form in the patient and also in two normal controls.

quence,8.9.2 1.22 except for the insertion of a C at position -7

DISCUSSION

Congenital AT deficiency is associated with an increased risk of recurrent thromboembolism. The incidence of thrombotic events is minimal in pediatric age,23 the first e p ~i~ 2. CIE in hepa~n-agame of plasma AT antigen from a con- isode most frequently occumng in individuals between 15 trol adult (top) and from the proposita (bottom).




ANTITHROMBIN-Gly 424 Arg

P C

4

c* c - 4

Fig 3. (A) Single-strand conformation polymorphism analysis of exon 6 of the AT gene. The patterns of the proposita (P) and a normal control (C) are shown. Abnormal fragments in the proposita are indicated by arrows. The band at the bottom of the figure represents double-stranded (nondenatured) DNA. The samples were run in 8% 0.4-millimeter-thick polyacrylamide gels (acrylamide: bis ratio 37.5:l) at 2.5 W constant power for 16 hours at room tempera- ture in an LKB 2001 Vertical Electrophoresis Unit (Pharmacia LKB Biotechnology, Bromma, Sweden). (B) Direct genomic sequencing of the antisense strand of the amplified exon 6 of the AT gene from the proposita. The presence of both a G (normal) and a C (mutant) in the first position of codon 424 is indicated by arrows.

normal in the proposita and different family members. A few cases of inherited AT deficiency, diagnosed in newborns, have been rep~rted.’~”~ only some of them with severe thrombotic complications. On the basis of the results of a literature review, the pooled prevalence of arterial thrombotic disease in AT-deficient subjects seems much less than the pooled prevalence of venous thrombosis: 2 8 versus

149

5 I %.l4 These findings suggest that arterial disease is rather uncommon.

Assessment of AT levels in different members of the baby’s family confirmed the inheritance of type I AT deficiency. In six subjects. A T levels were approximately 50% of normal in both antigenic and functional assays. No variant A T protein with altered heparin-binding properties could be detected in the plasma of the affected individuals by CIE in the presence of heparin. Southern blot analysis after digestion with restriction enzymes Pst I and BurnHI showed no gross rearrangements within an allele and indicated the presence of both alleles. Moreover, we could conclude that the disease cosegregated with the presence of the polymorphic PSI I site in exon 4 and the larger fragment ofthe length polymorphism in exon 1 (S allele).

We decided to search for the molecular basis of the deficiency by PCR-SSCP analysis and DNA sequencing. SSCP analysis of the seven exons showed a modified pattern in exon 6, which was then PCR amplified and sequenced. We detected a yet-undescribed point mutation in the first position of codon 424 in exon 6. Codon GGC of glycine-424 is replaced by a codon CGC, coding for an arginine residue at this position. Both normal and mutant sequences were found, indicating a heterozygous defect. Further sequencing of the other six exons of the AT gene did not show any further abnormalities.

Considerable heterogeneity is observed in the genetic abnormalities of type 1 AT deficiencies caused by miscellaneous gene alterations. Gross gene deletions seem infre- quent.’3.30 so that in the majority of cases, Southern blot analysis may not be the indicated technique to detect such minor mutations. Only very few cases of complete- or partial-AT gene deletions have been described. There have been several reports of frameshift mutations (resulting from small nucleotide insertions or deletions), or single-base sub- stitutions leading to stop codons.’.* In the kindred we studied, a single-base substitution was found, leading to a single amino-acid replacement.

Interestingly, the nucleotide substitution in our newly characterized AT-GIy 424 Arg mutation creates a unique restriction site for the enzyme Hha I in exon 6. This change

Fig 4. Hhe I restriction analysis of amplified DNA from AT exon 6. The G to C mutation in the first position of codon 424 creates a unique Hhe I cutting site, such that the presence of the mutation results in two fragments of 154 bp and 56 bp, instead of 21 0 bp for the normal fragment. The affected members in this family are all heterozygous for the mutation, because they all have bands of 21 0 bp, 154 bp, and 56 bp. The numbers above refer to the family members in Fig. 1. Cl and C2 are normal unrelated controls.




150 JOCHMANS ET AL

permits rapid and accurate identification of the mutation in Hha I digests of PCR-amplified exon 6 genomic DNA and is useful in screening members of the affected family for the molecular defect.

Different serpins have close similarities considering their tertiary structures. aI Antitrypsin, usually considered as the model, appears as a highly ordered globular molecule consisting of three sheets surrounded by nine he lice^.^,^' Align- ment of the amino-acid sequences of members of the serpin superfamily shows some highly conserved residues4 In our case, glycine 424 represents a nearly invariant residue (in 15 of 17 serpin sequences), structurally located in sheet strand 5B at the C-terminal end of the molecule. The substitution of glycine, the smallest amino acid, by arginine, an amino acid with a long side chain, will undoubtedly influence the sterical model of this part of the protein. Moreover, hydro- phobicity and charge (neutral v basic) are very different in these two amino acids, probably leading to an unstable variant of the normal protein. Lane et a13’ described six different substitution mutations in the 402 through 407 region of the AT protein. In contrast to their findings, we could not detect variant AT proteins with heparin-binding abnormalities. As suggested this study confirms that point mutations located in exon 6 can lead to important conformational abnormalities in terms of protein folding. However, the precise mechanism, linking the small molecular modi- fication that we detected to the disturbed AT gene expres- sion with important decrease in circulating protein and severe clinical implications, is not known.

ACKNOWLEDGMENT

We thank the staff of the Laboratories of Coagulation and Medi- cal Genetics for technical support and Brigitte Guns for secretarial assistance.

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1994 83: 146-151

K Jochmans, W Lissens, R Vervoort, S Peeters, M De Waele and I Liebaers type 1 antithrombin deficiency and neonatal thrombosisAntithrombin-Gly 424 Arg: a novel point mutation responsible for

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Antithrombin-Gly 424 Arg: A Novel Point Mutation ...Antithrombin-Gly 424 Arg: A Novel Point Mutation Responsible for Type 1 Antithrombin Deficiency and Neonatal Thrombosis By Kristin