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The molecular basis of hemophilia B : identification of the defect in factor IXVancouver Geddes, Valerie Anne 1987

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THE MOLECULAR BASIS OF HEMOPHILIA B: IDENTIFICATION OF THE DEFECT IN FACTOR IXVANCOUVER By VALERIE ANNE GEDDES B.Sc. (Hons) Simon Fraser University, 1984 A THESIS SUBMITTED LN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Biochemistry (Genetics Program) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA February 1987 © Valerie Anne Geddes, 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Biochemistry The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 „ . 8 7 : 0 3 : 2 0 Date DE-6(3/81) A B S T R A C T Hemophilia B . , is a moderately severe hereditary disorder in which the factor r Vancouver • J LX antigen is present in relatively normal amounts but the biological activity of factor I X is markedly reduced. Previous studies have demonstrated that although the patient has 62% of the normal factor LX antigen level in his plasma, he shows only 2.6% of normal factor IX procoagulant activity. In addition, radioimmunoassays have shown that epitopes on both the heavy and light chains of activated factor LX are present. These two results were taken as an indication that the molecular defect causing the hemophilia may be a point mutation involving an amino acid change in the protein. In order to identify the mutation involved, D N A was isolated from lymphocytes in a blood sample from the patient. This D N A was used to construct a genomic library in the A. vector E M B L 3 . One mil l ion of the resultant clones were screened with a labeled factor I X c D N A probe to identify those clones containing portions of the factor LX gene. D N A inserts from three X clones, which together span the entire gene, were subcloned to facilitate sequence analysis of the exons and intron / exon junctions of the factor LX gene. The nucleotide sequences of the coding regions were found to match the published sequence of the normal gene, except for one nucleotide. A single mutation was found at nucleotide 31,311 of the factor LX gene (Yoshitake et al., 1985), corresponding to amino acid 397 of the mature protein. This alteration, which changes an isoleucine codon, A T A , to a threonine codon, A C A , is novel among the mutations which have been reported to cause hemophilia B. A three dimensional model of the protease domain of factor LX , which was prepared on the basis of its homology to the pancreatic serine proteases, was examined in the vicinity of residue 397. The position of threonine 397 in this model suggests that this mutation could alter the hydrogen bonding between factor I X and its substrate, factor X . Taken together, these data suggest that this mutation is the cause of the hemophilia in this patient. TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iii LIST OF TABLES v LIST OF FIGURES vi LIST OF ABBREVIATIONS vii ACKNOWLEDGEMENTS ix INTRODUCTION p.l 1) The Role of Factor IX in Blood Coagulation p.l 2) The Structure of the Factor LX Gene p.2 3) The Structure of the Factor DC Protein p.4 4) Hemophilia B as a Heterogeneous Disorder p.8 5) Classifications of Hemophilia B p.9 6) Mutations Known to Cause Hemophilia B p.10 7) Profile of the Subject's Family: A. Pedigree p. 13 B. Factor LX Activity and Antigen Assays p.13 C. Classification of the Subject's Hemophilia p. 16 8) Objectives of This Study p.17 MATERIALS & METHODS p. 19 1) Materials p. 19 2) Strains, Vectors, and Media p.20 A. Bacterial Strains p.20 B. Vectors p.20 C. Media..... p.20 3) Basic Molecular Biological Techniques p.21 A. Gel Electrophoresis p.21 B. DNA Isolations p.22 C. Production of DNA Fragments for Ligations p.25 D. Ligation of DNA into Ml3 Vectors p.26 E. Transformation of DNA into Bacteria p.27 F. Klenow Labeling of DNA Fragments p.28 4) Construction of the Genomic Library p.28 A. Isolation of DNA from Lymphocyte Nuclei p.28 B. Partial Digestions of Genomic DNA with Sau3A p.30 C. Ligations into X EMBL3 Arms p.31 D. Packaging of Reconstituted EMBL3 p.32 5) Screening of the Genomic Library p.32 A. Plating the Phage Library p.32 B. Hybridizations and Washing p.33 MATERIALS AND METHODS (cont'd) 6) Mapping of the Factor IX EMBL3 Clones p.34 7) Subcloning into M13 vectors for Sequencing p.35 A. Construction of M13 Clones p.35 B. Screening of M13 Clones p.37 C. M13 DNA Isolation p.37 8) DNA Sequencing p.38 9) Genomic Southern Blot p.40 RESULTS p.42 1) Southern Blot Analysis of the SB LX-4 Factor IX Gene p.42 2) Genomic Factor LX Clones Isolated from the SB IX-4 Library p.42 3) M13 Subclones Containing SB LX-4 Factor IX Exons p.44 4) Nucleotide Sequence of SB IX-4 Activation Peptide and Intron / Exon Junctions p.44 5) Nucleotide Sequence Alteration in SB IX-4 Factor IX Gene p.49 DISCUSSION p.52 1) Summary of Data Concerning Patient SB LX-4 p.52 2) Naming of the Mutation Found in SB LX-4 p.52 3) Implications for Carrier Detection and Prenatal Diagnosis p.52 4) Possible Effect of the He„0„ to Thr„0„ Substitution on Factor LX Activity: jy I Dy I a A. Structure of Normal Factor LX p.54 a B. Structural Homology Between the Trypsin-Like Serine Proteases.. ..p.54 C. Model of the Complex Between Factor IX and Its Substrate Peptidep.58 D. Hydrogen Bonding in the Normal Factor LX - Substrate Complex . .p.62 E. Hydrogen Bonding in the Factor LX a V a n c o u v e r - Substrate Complex .p.62 5) Directions for Further Research p.63 6) Summary p.65 REFERENCES p.66 LIST OF TABLES TABLE I. Point mutations known to cause hemophilia B p.11 TABLE II. Factor IX clotting activity and antigen assays of the subject's family p.15 TABLE HI. Composition of dideoxy / deoxy sequencing mixes p.39 TABLE IV. Nucleotide sequences of the SB IX-4 activation peptide and intron / exon junctions p.48 TABLE V. Amino acid sequence alignment of normal human factor IX and the pancreatic serine proteases p.55 v LIST OF FIGURES FIGURE 1. Structure of the human factor IX gene, mRNA and protein p.3 FIGURE 2. Pedigree of the subject's family with respect to hemophilia B p. 14 FIGURE 3. Southern blot analysis of the SB IX-4 factor IX gene p.43 FIGURE 4. Genomic factor LX clones isolated from the SB IX-4 phage library ...p.45 FIGURE 5. M13 subclones containing SB IX-4 factor IX exons p.46 FIGURE 6. Nucleotide sequence alteration in the SB IX-4 factor IX gene p.50 FIGURE 7. Hydrogen bonding in the factor IX - substrate complex models p.60 vi LIST OF ABBREVIATIONS A Adenosine ATP Adenosine triphosphate bp Base pair(s) BSA Bovine serum albumin C Cytidine dH 2 0 Distilled water DMSO Dimethylsulfoxide dNTP Deoxyribonucleoside triphosphate ddNTP Dideoxyribonucleoside triphosphate DNA Deoxyribonucleic acid DNase Deoxyribonuclease DTT Dithiothreitol EDTA Ethylenediaminetetraacetic acid EtBr Ethidium bromide G Guanosine Gla y - carboxyglutamic acid HEPES N-2-Hydroxyethylpiperazine-N'-2-ethane sulfonic acid IPTG Isopropyl-p-D-Thiogalactopyranoside kb Kilobase pairs rpm Revolutions per minute mA Milliamperes mRNA Messenger RNA N Nucleoside (G, A, T, or C) OD Optical density PEG Polyethylene glycol vii pfu Plaque forming unit RNA Ribonucleic acid RNase Ribonuclease SDS Sodium dodecyl sulfate TEMED N, N, N', N'-Tetramethylethylenediamine Tris Tri (hydroxymethyl) aminomethane tRNA Transfer RNA U Units of enzyme activity U V Ultra violet V Volts T Thymidine W Watts Xgal 5-Bromo-4-chloro-3-indolyl-P-D-galactopyranoside viii ACKNOWLEDGEMENTS I offer special thanks to the following friends and colleagues for their gifts of advice, reagents, encouragement and humor: Gordon Louie Bruce Tiberiis Debbie Cool Colin Hay Dave Irwin Kathy Robertson Gary Brayer I am indebted to Ross MacGillivray for his colossal generosity. My most earnest thanks to my extraordinary mate, Chris Dewhurst. INTRODUCTION The arrest of bleeding following trauma is controlled by three interrelated factors: the constriction of blood vessels at the site of injury, the formation of a platelet plug, and the coagulation of blood. The process of coagulation occurs as a series of complex steps which terminate in the formation of a fibrin clot. Coagulation involves at least 2+ 14 plasma proteins, Ca , phospholipid membrane surfaces, at least one tissue protein, and platelets (Jackson and Nemerson, 1980). Two systems exist for the activation of blood coagulation: the intrinsic pathway, which is a relatively slow process, and the extrinsic pathway, which is a much faster process (Hirsch and Brain, 1979). The protein coagulation factors, which circulate in the form of inactive precursors, are converted to their active forms in a cascade of sequential reactions. The coagulation disorder known as hemophilia B is inherited as an X-linked recessive trait occurring approximately once in every 25,000 male births (Thompson, 1986). The disease is also known as Christmas disease and as plasma thromboplastin component deficiency. The clinical symptoms of hemorrhage in hemophilia B patients are due to the absence or decreased activity of blood coagulation factor IX. 1. The Role of Factor IX in Blood Coagulation Factor LX circulates in plasma as a 54 kDa glycoprotein (Thompson, 1986), which constitutes 1/10,000 of the total circulating protein (Hedner and Davie, 1982). During the middle phase of blood coagulation, factor IX is converted from its zymogen form to an active serine protease called factor LX . This activation can be 3. achieved in two ways. As part of the intrinsic pathway of blood coagulation, factor IX is activated in a reaction with factor XI a in the presence of calcium. This is the 2+ earliest Ca dependent reaction in the intrinsic clotting system. An alternate route to 1 activation has been identified and involves the extrinsic coagulation system (Osterud and Rapaport, 1977). In this reaction, factor VII , in the presence of the lipoprotein known as tissue factor, activates factor IX. The relative importance of these two activation mechanisms is not understood (Thompson, 1986). Both of these reactions have the same biochemical result: the removal of an activation peptide leaves two polypeptide chains held together by disulfide bond(s). Activated factor LX acts as a 2+ serine protease to cleave factor X in the presence of Ca , phospholipid and factor VJTIa. The chain of activation reactions then continues until finally a fibrin clot is formed. 2. The Structure of the Factor IX Gene The genomic and protein structures of factor LX are presented schematically in Figure 1. The gene for factor IX spans approximately 34 kilobases of DNA. Cytogenetic studies have localized the gene to the subtelomeric region of the long arm of the X chromosome. The factor LX gene is proximal to the gene for blood coagulation factor VIII and these two genes flank the site associated with fragile X mental retardation, atXq27.3 (Purrello et al, 1985; Camerino et al., 1984; Chancer al., 1983; and Boyd et al, 1984). The cloning and sequencing of the entire human factor DC gene (Yoshitake et al, 1985; earlier work by Anson et al, 1984; Kurachi and Davie, 1982; and Choo et al, 1982) have shown that the mRNA is encoded in eight separate exons. The gene codes for a 2802 nucleotide mRNA (Anson et al, 1984), of which about one half is a long 3' untranslated tail. Analysis of the genetic control elements by Yoshitake et al (1985) resulted in the following observations. Putative promoter sites 5' to the factor IX coding region include residues -27 to -24. The sequence TGTA, which may 2 Figure 1 Structure of the human factor IX gene, m R N A and protein DNA The genomic structure of human factor IX (Yoshitake et al., 1985; Thompson, 1986) is represented on the top line from 5' (left) to 3' (right). Numbers below the line refer to kilobases. Exons 1 through 8 are indicated above the gene. Introns are indicated by capital letters. The m R N A is presented in the central diagram. The lighter section of the m R N A signifies the translated region and the darker section the long 3' untranslated tail. The translation product of this m R N A is shown in the lower diagram. Amino acids of the mature protein are numbered below the line. The contributions from each of the eight exons are indicated above the protein. Abbreviations used: "prepro" refers to the 46 amino acids between the translation initiation methionine and the processing cleavage site; "g la" refers to the domain containing gamma-carboxyglutamic acid residues; " E G F " stands for the epidermal growth factor homologous regions; " A P " for the activation peptide which contains asparagine-linked carbohydrate moieties and the two factor LX activation cleavage sites (indicated by arrows); and "tryptic" for the catalytic portion, including the active site serine, " S " . 3 represent a TATA box (Goldberg-Hogness box; Breathnach and Chambon, 1981), is found at this site. This occurs 27 bp upstream of the initiation site as determined by nuclease SI mapping analysis (Anson et al., 1984). Two other sites further upstream of this region also show TATA homology similar to that observed in other genes, such as factor V m . It has not been established whether these sites are associated with RNA polymerase II binding and the start of transcription. None of these sequences displays a nearby CCAAT regulator sequence (Breathnach and Chambon, 1981) nor upstream G/C rich regions (McKnight and Kingsbury, 1982). The intron / exon junctions in the factor IX gene have been shown to conform to the GT / AG rule of donor and acceptor splice sites (Breathnach and Chambon, 1981) and to be very similar to the consensus sequence summarized by Mount (1982). A recognition sequence for poly(A) addition is found at position 32,741. It is identical to the AATAAA consensus sequence (Proudfoot and Brownlee, 1976) found to be necessary but not sufficient for poly(A) primary site selection. In the factor IX gene, the AATAAA sequence is found 39 bp upstream of the sequence CATTG. This sequence is homologous to the consensus sequence CA(T/C)TG, which may provide further specificity for cleavage site selection (Berget, 1984; Birnstiel et al., 1985). 3. The Structure of the Factor I X Protein The factor IX mRNA codes for a protein containing a prepro leader sequence which is not found in the mature plasma protein. Both the pre and pro sequences are removed during the biosynthesis of factor LX in hepatocytes. The hydrophobic pre sequence (residues -46 to -19) has limited homology to the signal sequences observed in other vitamin K - dependent clotting factors (Fung et al., 1985). It is thought to be removed by a signal peptidase during translocation of the nascent protein across the rough endoplasmic reticulum (von Henji, 1983 and 1985). The pro region (residues -18 to -1) is thought to be removed by an unidentified processing peptidase (Steiner et al., 1980), after further modification of the protein. The amino terminal domain of the mature plasma protein comprises the gla region, which takes its name from the y - carboxyglutamic acid residues it contains. A vitamin K - dependent carboxylase system in the liver converts 12 glutamic acid residues to y - carboxyglutamic acid. Factor VII, factor X, prothrombin, protein C, protein S and protein Z also contain gla domains (Suttie, 1985). The gla residues are 2+ associated with the sixteen Ca binding sites observed in human factor IX (Bajaj, 1982), although one of these sixteen sites has been shown to be gla-independent 2+ (Morita et al., 1984). The binding of Ca to the strongly acidic y - carboxyglutamic acid residues is a cooperative phenomenon which results in a conformational change 2+ in the molecule (Bajaj, 1982). The association of Ca with factor LX greatly increases the rate of activation of factor IX by factor XI . Factor IX must interact J a a 2+ with a phospholipid membrane, as well as with factor VIII and Ca , in order to activate factor X. The y - carboxyglutamic acid residues are thought to allow factor 2+ IX and other blood coagulation factors to form Ca bridges to phospholipid membranes (Suttie, 1985). The region of the protein directly following the gla residues contains two copies of a region showing homology to the murine epidermal growth factor (EGF) protein (Doolittle et al, 1984; Blomquist et al., 1984). These two factor IX regions are similar to 10 internally homologous subregions of the EGF protein. The same type of domain is found in several of the blood coagulation factors, including factors VII, X, XI and XII. It has been postulated that this region might have a role in the 5 interaction of factor LX with cell surfaces, since epidermal growth factor has this property in vivo (Anson et al., 1984), but no evidence of this has been reported. The unusual amino acid (3 - hydroxyaspartate is also found in the EGF region. Its position at residue 64 is homologous to its position in protein C, factor X, and factor VH (McMullen et al., 1983). The significance of this modification of an aspartate residue is unknown. It is possible that the P - hydroxyaspartate may be associated 2+ with the high affinity gla-independent Ca binding site (Morita et al., 1984). The sites for the activation cleavages of factor IX are found in the region of the protein downstream of the EGF domain. The bonds cleaved are those between arginine145 and alanine146 and between arginine18Q and valine^. After these cleavages occur, the amino terminal chain and the carboxy terminal chain are held together by a disulfide bond or bonds. The activation peptide removed during this process is 35 amino acids in length and contains two of the asparagine-linked carbohydrate moieties found in factor LX. During the activation of factor LX by factor XI , the arginine - alanine bond is cleaved first. The product of this cleavage, factor IXa, has no proteolytic activity. Some in vitro activators of factor LX, such as Russell's viper venom, cleave the arginine-valine bond at a greater rate than the arginine-alanine bond. The product of the cleavage of the arginine-valine bond only, factor IX , has some activity (Lindquist et al., 1978). The fully active species H O C generated when both of the bonds have been cleaved is referred to as factor LXap. By analogy with the pancreatic serine proteases, the free amino group generated by the activation reaction (valine 181) is likely to be involved in an ion pair interaction with the free carboxyl group of the aspartate residue at position 364 (Thompson, 1986).This association probably induces a conformational change in the catalytic 6 subunit of factor LXap. The change in conformation may consolidate the molecule into a configuration which increases the reactivity of the active site serine (Fujikawa et al, 197'4; Furie et al, 1982). The serine protease domain is found in the heavy chain of factor LX . The catalytic subunit includes the active site serine (position 195 in chymotrypsin), aspartic acid (102) and histidine (57) residues which are thought to contribute to the charge relay system (Kraut, 1977). There is a close evolutionary relationship between the factor IX catalytic subunit and members of the trypsin-like serine protease family. This 3, group includes the pancreatic serine proteases chymotrypsin, trypsin, and elastase as well as kallikrein, plasmin, thrombin, and blood clotting factors XII , X , VII and 3 3. 3 XIa (Jackson and Nemerson, 1980). In addition to having the ability to cleave a specific peptide bond, there are a number of other requirements for the full biological activity of factor IX. The pre and pro leader sequences must be removed and the protein must be secreted into the bloodstream. Modifications of the gla residues and the P - hydroxyaspartate residue must occur during maturation of the protein in hepatocytes. The gla region must 2+ effectively bind Ca and must be able to associate with phospholipid membranes. Factor IX must be recognized by factor XI . Both activation cleavages must occur to obtain a fully active protease. Finally, factor IX must be able to interact in some 3 manner with its protein cofactor, factor VIII . 7 4. Hemophilia B as a Heterogeneous Disorder Since factor LX has a complex a role in coagulation, it is not surprising that the profiles of patients with hemophilia B vary widely with respect to clinical severity, clotting factor activities and antigen levels (Thompson, 1986). The wide heterogeneity of clinical symptoms was first recognized by Fantl (1956) and Roberts et al. (1968). These authors suggested that the disease existed in at least two forms. Roberts found that for 10% of his patients, hemophiliac plasma could neutralize factor LX inhibitors. This result suggests that their plasma contained significant amounts of factor IX antigen. The remaining 90% of his patients did not display such neutralization of inhibitors. Further investigations (Hougie and Twomey, 1967; Gray et al., 1968) identified a group of patients exhibiting a prolonged prothrombin time when ox brain, rather than human brain, was used as a source of thromboplastin reagent. Such hemophilias were referred to as hemophilia Bm The prothrombin time assay (abbreviated PT) is a standard hematological measure of the activity of the extrinsic pathway. The majority of hemophilia B patients give a normal prothrombin time with both human and ox brain thromboplastin. Brown et al. (1970) noted that hemophiliacs giving a prolonged ox brain PT were severely affected and clinically indistinguishable from one another. Those patients not exhibiting the prolonged ox brain PT time were distinguishable in terms of clinical profile. A more extensive survey of hemophilia B patients by Kasper et al. (1977) showed that hemophilia B is even more heterogeneous a disease than was previously recognized. Of 92 patients surveyed, 36 showed a discrepancy between antigen and activity levels, while 56 showed a proportional decrease in these two parameters. The 8 authors divided their patients into six groups with respect to their clinical severity, factor I X activity, antigen levels, and ox brain P T results. Within several of these groups, however, there was further evidence of heterogeneity. A l l hemophilia B m patients in this study had an excess of antigen over activity. However, there was evidence of heterogeneity within the B m group with respect to the degree of prolongation of the ox brain PT time. Another study has shown that one variant of factor I X f i m acts as a competitive inhibitor of factor X when the latter is activated by the reaction product of bovine tissue factor and human factor VII (Osterud et al, 1981). A recent paper reported three variants of factor L X f i m which all exhibit different rates of activation cleavage (Yoshioka et al., 1986). Thus, the B m phenotype does not appear to define a specific subgroup within the disease. 5. Classifications of Hemophilia B Studies such as the above have resulted in the development of several classification systems for hemophilia B variants. Hemophilias are identified as mild (over 4% of normal factor I X clotting activity), moderate (2 - 4%) or severe (less than 2%). Hemophilias also vary with respect to the presence or absence of circulating inhibitors of factor DC. These inhibitors are thought to be alloantibodies raised during treatment with exogenous factor LX. They are presumed to arise when the patient's own factor I X is either present at a very low level, contains deletions, or is heavily modified (Ratnoff, 1978). Another classification of hemophilia B variants is based on the presence or absence of factor I X antigen (Roberts et al., 1968). Those patients displaying normal levels of antigen but reduced levels of factor I X activity are designated c r m + (cross reacting material positive); those displaying reductions in both red activity and antigen, crm ; and those showing undetectable antigen and severely reduced activity, crm". C r m + mutations and those c r m r e d hemophilias with a large 9 discrepancy between antigen and activity levels are of particular interest, since these mutations must result in critical changes to the factor IX protein which affect its biological function. 6. Mutations Known to Cause Hemophilia B The heterogeneity in the clinical profiles of hemophilia B is a strong indication that the disease is caused by a variety of genetic mutations. Knowledge of the sequence of the factor LX gene and of the sequence and characteristics of the protein has led to investigations into the relationship between structure and function in factor IX. Table 1 presents the point mutations causing hemophilia B which have been identified at the amino acid or nucleotide level. Two mutations have been identified which involve alterations in the prepro leader sequence. Factor LX 0 x f o r d , associated with a severe form of hemophilia B, exhibits an amino acid change at residue -4 from an arginine residue to a glutamine (Bentley et al., 1986). This alteration appears to preclude the removal of the 18 amino acid propeptide and results in decreased factor IX activity in the patient's plasma. Factor LXoxford was found to bind calcium normally and to be y carboxylated to a normal degree. The authors suggest that either the basic charge on the propeptide or steric interference due to the extra 18 amino acids could alter phospholipid binding or other properties. Factor ^ -Q^^^gQ contains an alteration from an arginine at position -1 to a serine residue (Diuguid et al., 1986). Like Factor IXQxford, this mutation results in a factor IX which is extended by the 18 amino acids of the propeptide. The failure to remove the leader peptide appears to have a profound effect on the activity of factor 10 Table I. Point mutations known to cause hemophilia B 12 +1 prepro gla egf AP tryptlc S Mutation name residue mutation region Factor IX levels antigen activity 1) Factor IX 0 x f o r d aa - 4 arg to gin prepro 2) F a c t o r I X C a m b r i d g e a a " 1 « * t o s e r 3) Factor LX M a h a m a aa 47 asp to gly EGF 89% <0.5% 80% <1% moderate 4) Factor LX Chapel Hin aa 145 arg to his AP normal 8% 5) donor splice site n 21,165 GT to TT exon 6 0.3% <0.5% The location of each mutation is indicated in the second column, followed by the amino acid and/or nucleotide change observed (aa = amino acid, n = nucleotide). The region of the factor IX protein in which the mutation occurs is also indicated. Clinical symptoms, as far as they were reported in the original papers, are also recorded. Antigen levels were measured by immunoassay and activity levels by standard one or two stage clotting time assays. References: 1) Bentley et al., 1986; 2) Diuguid et al., 1986; 3) Davis et al., 1984; 4) Noyes et al., 1983; and 5) Rees et al., 1985. 11 ^Cambridge* The presence of the abnormal amino terminal extension appears to affect the carboxylation of the gla residues, suggesting that the sequence prior to the gla domain is important in recognition by the vitamin K - dependent carboxylase system (Bentley etal., 1986). The molecular defect in factor LX., , has been reported as an aspartate to histidine Alabama change in the EGF region of the protein. When the sequence of the protein was analyzed (Davis et al., 1984), no other change from the normal sequence was observed. The amino acid change results in the loss of a negative charge. As the role of the EGF domain itself is not understood, the role of this residue in the protein is not clear. In the hemophilia associated with factor LX C h ap e l H i u , amino acid 145 has been changed from an arginine residue to a histidine residue. Arginine 1 4 5 is the site of one of the activation cleavages of factor LX. Both factor XI and VII require the arginine side chain in this position to recognize the cleavage site. Thus, the change to a histidine precludes activation. Since the partially activated factor LX has reduced biological activity, this mutation lowers factor IX activity to 8% of normal. A splicing mutation is responsible for the decreased activity of factor LX in another case of hemophilia B (Rees et al, 1985). DNA analysis has shown that in this variant, the donor splice site GT of exon 6 has been altered to a TT dinucleotide. Such a mutation would be expected to result in an altered mRNA. Several different types of RNA can result from splicing mutations: either the entire intron is retained, or cryptic splice sites are used. Often splicing mutations result in truncated messages or in mRNAs which are unstable. Such mutations have previously been observed in the P° thalassemias (Weatherall et al., 1984). The precise effect of the splicing mutation in this hemophilia has not been determined. However, it is known that the mutation results in an almost undetectable factor IX antigen level in the plasma. Many more variants of hemophilia B have been identified which involve partial or gross deletions of the factor IX gene. Some of these studies have identified the boundaries of these deletions, while others have identified them only in a qualitative way (Bray and Thompson, 1985,1986; Hassan etal, 1985; Vidaud et al, 1986; Giannelli et al., 1983). In addition to these mutations, there is a further group of patients for which a molecular diagnosis has not been obtained (Thompson, 1983; Mertens et al., 1983; Bertina and van der Linden, 1982a; Usharani et al., 1985; Briet et al., 1982; Osterud et al., 1979). For these patients, analyses of blood clotting characteristics and of the presence or absence of inhibitors of factor IX are often the only data available. These patients present a wide variety of clinical profiles. 7. Profile of the Subject's Family: A. Pedigree A British Columbian family with a history of hemophilia B has undergone carrier detection using restriction fragment length polymorphisms (Hay et al., 1986). The pedigree of this family with respect to hemophilia is found is Figure 2. Hemophilia B has been observed over several generations, indicating that the mutation in patient SB IX-4 is more likely to have been inherited than to be a new mutation. B. Factor IX Activity and Antigen Assays During the carrier detection study, factor LX clotting activity and factor IX antigen levels were measured for several members of the family. Table II lists the results of these assays. In each case, the values are expressed as a percentage of the 'normal' 13 Figure 2 Pedigree of the subject's family with respect to hemophilia B m 6 c T i dTo SB IX-4 Affected males are indicated by filled squares and suspected carrier females by dotted circles. Normal individuals are indicated by white circles (females) or white squares (males). Question marks indicate individuals for whom carrier status has not been established. The hemophiliac whose gene is under study has been indicated by an arrow and identified as individual SB IX-4. Table II Factor IX clotting activity and antigen assays of the subject's family Subject IX: C IX:Ag a IX:Ag b Father 110 98 113 Mother (carrier) 18 73 70 Son (hemophiliac), SB IX-4 2.6 61 63 Daughter 100 92 90 Control 108 104 121 The factor IX clotting activities (IX:C) of members of the hemophilia B family and an unrelated, unaffected male were measured with a one stage assay using factor IX deficient plasma. The factor IX antigen levels (IX:Ag) were assayed using two methods: a, using a polyclonal non-calcium-dependent preparation for both the coating and the labeled species; and b, using a monoclonal antibody to an epitope on the heavy chain of factor IXa (Thompson, 1983) as the solid phase and a calcium-dependent polyclonal fraction which recognizes a light chain epitope (Bray et al., 1986) as the labeled species. Results are expressed as percent of normal values. From Hay et al., 1986. value. 'Normal' refers to the mean value from many unaffected individuals. The immunoassays show that the hemophiliac son's circulating factor LX contains epitopes from both the light and heavy chains of factor IX (column LX:Ag b). In this fl assay, a monoclonal antibody to an epitope on the heavy chain of factor IX fl (Thompson, 1983) was used as the solid phase attached to microtitre wells. Added factor IXa would therefore not be retained in the wells unless it contained the heavy chain epitope. A calcium - dependent polyclonal antibody fraction, which recognizes a light chain epitope (Bray et al, 1986), was used as the labeled species. When this labeled preparation was applied, radioactivity would not be associated with the wells unless the retained factor IX presented the light chain epitope. While the patient's fl factor LX clotting activity was measured to be 2.6% of the normal level, his antigen level was shown to be considerably higher, at approximately 62% of normal. C. Classification of the Subject's Hemophilia The above assay results place the patient SB IX-4 in the moderate, crmred class of hemophilia B variants. No ox brain prothrombin test has been performed on the patient's plasma to date. Plasma from SB IX-4 does not contain circulating inhibitors to factor IX (G. Growe, personal communication). The assay results suggest that the molecular defect responsible for hemophilia B in this family is likely to be a small deletion or point mutation within the protein coding region of the factor IX gene. Changes such as major gene deletions, splicing mutations, and mutations in transcriptional control elements would be expected to reduce antigen and activity levels proportionately. The reason for the reduction in the factor IX antigen level in SB IX-4 to 62% of normal was not under investigation in this study. Unequal reduction in activity and antigen level is a common feature in patients with hemophilia B (Kasper et al., 1977; 16 25 patients of 92 investigated). In addition, there is a very wide range of factor IX activity values even among normal individuals: a range of 37 to 170% was noted in one survey (Ratnoff, 1978). Within the family under study, however, clotting activities correlate with carrier or hemophiliac status. Hemophilias with unequal reduction in activity and antigen are not well understood. The suggestion has been that an amino acid change in the protein could either alter the clearance of circulating factor IX from the blood or reduce the ability of the antibodies used in the immunoassay to recognize factor LX . Two distinct radioimmunoassays, which used antibody preparations recognizing different epitopes on the factor LX molecule, were performed. Since these assays gave similar results, it seems unlikely that epitope changes could explain the reduced antigen value. The reduction in factor LX antigen level is probably due to an altered halflife for the mutant protein. 8. Objectives of This Study The purpose of the present study was to determine the molecular defect resulting in decreased specific coagulation activity of the factor IX found in SB IX-4. An analysis of such a defect can be performed in several ways. One method to pinpoint a mutation is to prepare a cDNA library and sequence the factor IX cDNA. This approach, however, is impractical in a human study. Since factor LX is synthesized in hepatocytes, a liver sample would be required to provide an RNA source containing factor IX mRNA. Another approach is to isolate and sequence the mutant protein. The third approach, and the one chosen for this study, is to prepare a genomic library from the patient's DNA. DNA isolated from peripheral lymphocytes in a blood sample from SB LX-4 was used to prepare a genomic library. The library was screened by using a normal human factor LX cDNA as a hybridization probe. 17 The DNA inserts from positive clones were then subcloned and the exons and intron / exon boundaries of the gene were sequenced. In this manner, a coding region mutation in the serine protease subunit of factor IX& was identified. 18 MATERIALS and METHODS 1. Materials Agarose, acrylamide, bisacrylamide, urea, ammonium sulfate and TEMED were obtained from Bio-Rad Laboratories. Yeast extract, casamino acids, bacto-tryptone, and bacto-agar were Difco grade and obtained from the Grand Island Biological Company. Nitrocellulose sheets and circles (82 and 132 mm, 0.45 u.m pore size) 32 were obtained from Millipore or Schleicher & Schuell. P - labeled nucleotides were obtained from Amersham. Phenol was obtained from the British Drug Houses Ltd and was redistilled before use. The fraction distilled at 179° C was collected and frozen in aliquots at - 20 °C. Deoxy- and dideoxy-ribonucleotides were purchased from PL-Pharmacia. IPTG, Xgal, EtBr, DMSO, yeast tRNA, chloramphenicol and ribonuclease A were obtained from Sigma. Cesium chloride was purchased from Cabot Berylco Ltd. Ultrogel AcA54 was purchased from LKB. All other chemicals were of reagent grade or higher and were purchased from either Sigma Chemical Co., Fisher Scientific, or British Drug Houses Limited. Restriction endonucleases, T4 DNA ligase, T4 DNA polymerase and nuclease-free BSA were obtained from New England Biolabs, Bethesda Research Laboratories, or PL-Pharmacia. DNA Polymerase I Klenow fragment was obtained from PL-Pharmacia. Blood samples were taken by Dr. G. H. Growe, Department of Medicine, Vancouver General Hospital. 19 2. Strains, Vectors and Media A. Bacterial Strains E. coli LE 392 and its P2 lysogen P2 392 (F", hsd R514, (rK", mK+), sup E44, sup F58, (lacYl or A (lac IZY)), galK2, galT22, metBl, trpR55, X-; Maniatis et al, 1982) were hosts for the screening and isolation of clones in EMBL3. E. coli JM103 (Alacpro, strA, thi", supE, sbcB15, endA, hsdR", F, traD36, proAB, laclq, lacZAM15; Messing, 1983) was the host for transformation and isolation of clones in M13. E. coli MC1061 was used as the host for the factor IX cDNA clone (pCHLX). B. Vectors The genomic library was constructed in the phage X vector EMBL3 (Frischauf et al, 1983) . For DNA sequence analysis, the M13 vectors mplO, 18 and 19 were used (Messing, 1983). The factor LX cDNA (pCHIX) was previously cloned into the plasmid vector pKT218. C. Media The medium for growth and screening of X clones and hosts was NZYM (Maniatis et al, 1982; 10 g NZ Amine type A, 2 g MgCL 5 g NaCl and 5 g yeast extract per liter, pH adjusted to 7.5 with NaOH). For screening X libraries, the phage were plated on NZYM agar (1.5%, w/v) plates with an overlay of NZYM agarose (0.7%, w/v). For titering of phage stocks, the overlay consisted of NZYM agar in place of NZYM agarose. Bacteria containing M13 clones were grown in YT medium (5 g yeast extract, 8 g bacto-tryptone, and 5 g NaCl per liter, pH 7.5). Phage M13 transformants were plated on YT agar (1.5%, w/v) plates overlayed with YT agarose (0.7%, w/v). E. 20 coli JM103, the host for M13 vectors, was maintained on minimal medium plates (Messing, 1983). Minimal medium was prepared as follows: 3 g of agar in 160 ml dH20 was autoclaved, cooled to 55 °C, and was mixed with 40 ml 5X salts (2.1 g K2HP04, 0.9 g KH2P04, 0.2 g (NH4)2S02> 0.1 g Na citrate7H20 per 40 ml). Autoclaved solutions of 20% glucose (2 ml), 20% MgS04 (0.2 ml), and a filter sterilized solution of 10 mg/ml thiamine (0.1 ml) were added. Plates were poured immediately. 3. Basic Molecu lar Biology Techniques A . Ge l Electrophoresis DNA fragments were separated according to size by electrophoresis in agarose or polyacrylamide gels. The buffer for agarose gel electrophoresis was IX TAE (50X TAE contains 2 M Tris base, pH 7.5,1 M glacial acetic acid, and 0.1 M EDTA; Maniatis et al, 1982). Agarose gel electrophoresis was performed in a horizontal apparatus. DNA fragments in these gels were visualized by fluoresence in the presence of EtBr (0.5 |ig/ml) under UV irradiation at 260 nm. The buffer for polyacrylamide gel electrophoresis was IX TBE (10X TBE contains 0.89 M Tris base, 0.89 M boric acid, 25 mM EDTA, pH 8.3; Maniatis et al, 1982). Denaturing polyacrylamide gels contained 8.3 M urea; non-denaturing polyacrylamide gels did not contain urea. For non-denaturing gels, acrylamide (added to the appropriate concentration from a stock solution of acrylamide:bisacrylamide in dH20 (38% : 2%, w/v) and buffer were mixed with the appropriate volume of dH20, and degassed. Polymerization was initiated by the addition of ammonium persulfate and TEMED to final concentrations of 0.066% (w/v) and 0.04% (w/v), respectively. The gels were stained with 0.5 u,g/ml EtBr in dH20 for 10 min, and the DNA was visualized under 21 UV irradiation. Polyacrylamide gels in TBE buffer containing urea were prepared, degassed and polymerized in the same manner except that the final concentration of TEMED was 0.024% (w/v). Polyacrylamide gel electrophoresis was performed in a vertical apparatus without pre-electrophoresis. DNA in denaturing gels was visualized by autoradiography after drying the gels under vacuum in a Bio-Rad gel drier at 80 °C for 20 - 30 min, followed by exposure to Kodak XK-1 film, with or without intensifying screens. B. DNA Isolations Precipitations of DNA from aqueous solution was achieved by the addition of 1/10 volume of 3 M NaOAc and either 2 volumes of - 20 °C 95% ethanol or 1 volume of room temperature isopropanol. The resulting solution was mixed thoroughly and incubated at either 0 °C, - 20 °C, or - 70 °C unless otherwise specified. Incubation time at these temperatures varied from 30 min to overnight. After incubation, the solutions were centrifuged in either a microfuge at top speed or a Sorvall centrifuge at 10 krpm for 30 min. Nucleic acid pellets were washed once with - 20 °C 75% ethanol and allowed to air dry. Double stranded replicative form Ml3 DNA was isolated as described by Messing (1983). A single plaque was used to inoculate 2 ml of YT containing 10 u.1 of an overnight JM103 culture. Small amounts of M13 replicative form DNA were prepared by a modification of the method of Birnboim and Doly (1979) (Maniatis et al., 1982). An aliquot (1.5 ml) of the 6 h culture of the clone of interest was placed in a microfuge tube and the bacteria were collected by centrifugation for 1 min in a microfuge. The cell pellet was resuspended in 100 ul of an ice cold solution containing 50 mM glucose, 10 mM EDTA, 25 mM Tris-HCl, pH 8.0, and 4 mg/ml 22 lysozyme. After the suspension was incubated for 5 min at room temperature, 200 u.1 of a solution containing 0.2 N NaOH-1% SDS was added. The mixture was incubated for 5 min on ice, and 150 |il of potassium acetate solution (60 ml 5 M KOAc, 11.5 ml glacial acetic acid, 28.5 ml dH20, pH 4.8) was added. After vortexing, the suspension was incubated at 4 °C for 5 min. Cellular debris was removed by centrifugation in a microfuge for 5 min at 4 °C. The supernatant was removed and extracted with an equal volume of phenolxhloroform (1:1, v/v). Nucleic acids were precipitated with ethanol at room temperature. The nucleic acids were resuspended in 50 ul of TE buffer (10 mM Tris-HCl, pH 8.0, ImM EDTA) containing 20 Ug/ml pancreatic RNase A . A larger scale preparation of some Ml3 replicative form DNA was also performed. In this case, the 6 h culture grown from the single plaque was used to inoculate 100 ml of fresh YT. One milliliter of a culture of uninfected JM103 cells grown to log phase was added. The 100 ml culture was grown for a further 6 h. The previously described extraction procedure, scaled up by a factor of 6, was used to isolate the cloned DNA. A large scale alkali lysis procedure was used to purify plasmid DNA. An aliquot from an overnight culture (5 ml) of pCHLX grown in E. coli MC1061 was used to inoculate 11 of YT. The culture was incubated at 37 °C until the OD at 400 nm was > 0.4. At this time, 5 ml of chloramphenicol solution (34 mg/ml in ethanol) was added and the culture was incubated overnight at 37 °C. The extraction procedure is a scaled-up version of the previously described method with the following modifications. After the addition of potassium acetate, the debris was removed by centrifugation at 35 krpm in a Ti60 rotor for 30 min at 4 °C. Plasmid DNA was 23 separated from chromosomal DNA and RNA by isopycnic centrifugation using a cesium chloride gradient. The gradient solutions were made by the addition of 3.9 g CsCl and 0.3 ml EtBr (10 mg/ml) to 3.8 ml of the resuspended DNA. The gradient was then formed by centrifugation at 50 krpm using a Ti70.1 Beckman rotor for 20 h. The bright red band was removed from the tube with a syringe. This solution was then extracted 5 times with equal volumes of 2-butanol and dialyzed for 3 h against several changes of 11 TE at 4 °C. The nucleic acids were then precipitated from ethanol at - 20 °C overnight and resuspended into 300 u.1 of TE. For large scale preparations of EMBL3 clones (Maniatis et al. 1982), 2 X 1010 host bacterial cells grown in YT with 0.2% maltose were collected by centrifugation and resuspended in 4 ml of SM buffer (5.8 g NaCl, 2 g MgSO47H20, 50 ml 1 M Tris-HC1, pH 7.5, 5 ml 2% gelatin (w/v) per liter). The cloned EMBL3 phage (2 X 107 pfu) were added to the cells, and the phage were allowed to adsorb to the cells during an incubation at 37 °C for 20 min. This mixture was used to inoculate 400 ml of prewarmed NZ medium and the culture was incubated at 37 °C until lysis, approximately 4 h. Chloroform (4 ml) was added to lyse the remainder of the cells by incubation at 37 °C for 10 min. The solution was brought to 1 M NaCl by the addition of 80 ml of 5 M NaCl. Bacterial debris was removed by centrifugation at 5 krpm in a GSA rotor for 10 min. The supernatant was then digested with DNase I (800 ul of 1 mg/ml solution) and RNase A (80 ul of 10 mg/ml solution) for 30 min at 4 °C. Phage particles were precipitated by the addition of 80 g of polyethylene glycol 6000 (Carbowax 8000) followed by incubation at 4 °C overnight. Phage particles were collected by centrifugation at 5 krpm in a GSA rotor for 10 min at 4 °C. After removal of the PEG / NaCl solution, the phage particles were gently resuspended in 25 ml SM. An extraction with an equal volume of CHCL was followed by 2 4 centrifugation at 8 krpm for 10 min. Phage were purified from the aqueous supernatant in CsCl gradients. Gradients were made by the addition of 18 g CsCl per 25 ml of phage solution. Centrifugation at 60 krpm was continued for 16 - 20 h at 20 °C in a Ti70.1 rotor. Phage were removed from the gradient after visualization as a blue band visible in white light. The solution of recovered phage was made 0.02% with respect to gelatin. CsCl was removed by dialysis of the solution against 10 mM Tris base, pH 8.0,25 mM NaCl, and 10 mM MgCl2 for 2 h at 4 °C. SDS was added to 0.05% (w/v), EDTA to 20 mM, and proteinase K to 50 Lig/ml. The solution was incubated at 68 °C for 1 h. The DNA was purified by extraction with phenolxhloroform (1:1, v/v), followed by an extraction with chloroform alone. The solution was dialyzed overnight against TE at 4 °C. Phage DNA was precipitated from ethanol at 0 °C for 30 min. The DNA was resuspended in 500 LLI TE. Small scale preparations of EMBL3 clones were scaled down from the large preparation described above (Maniatis et al., 1982). Eluted phage from a single plaque were adsorbed to 100 ul of host cells at 37 °C for 10 min and used to inoculate 20 ml of NZ medium. DNA isolation followed the above protocol until the CsCl gradient step. At this point, the phage suspension was digested with proteinase K by the addition of SDS to 1%, EDTA to 5 mM, and proteinase K to 0.15 mg/ml. DNA was precipitated from isopropanol at 0 °C for 1 h and resuspended in 100 u.1 of TE buffer. C. Production of DNA Fragments for Ligation In order to isolate specific fragments or sizes of fragments for ligations or other manipulations, restriction enzyme digests were electrophoresed on 0.7% low melting 25 point agarose gels. For the long genomic fragments (10-20 kb), the volume of the gel slice was estimated and 5 volumes of 20 mM Tris base, pH 8.0,1 mM EDTA was added to the same tube. This mixture was heated in a 68 °C bath until the gel had completely melted. When the solution had returned to room temperature, it was extracted with equal volumes of phenol, then a 1:1 mixture of phenol:sevag (24:1 chloroform : iosamylalcohol), and finally with sevag alone. The DNA was then precipitated from ethanol at room temperature for 20 min and resuspended in an appropriate volume of dFLTj. For shorter fragments, a modification of this procedure was used. In this case, 500 u.1 or less of the agarose slice was placed in an eppendorf tube and melted by incubation at 68 °C. When the temperature of the gel had returned to approximately 40 °C, an equal volume of phenol was added to the tube, and the mixture was vigorously vortexed. The tube was then placed in a dry ice - ethanol bath at - 70 °C for 15 min. After the tube was removed and the solution completely thawed, the tube was centrifuged for 15 min in a microfuge. The aqueous layer was removed into a fresh tube. Approximately 150 ul of TE was added to the phenol layer in the original tube. This tube was then vortexed and centrifuged in the microfuge as previously described. The two aqueous layers were then pooled, and extracted once with an equal volume of phenol and once with sevag. After the extractions, the aqueous layer was transferred to a fresh eppendorf tube. The DNA was precipitated from ethanol at room temperature for 20 min. D. Ligation of DNA into M13 Vectors DNA fragments were ligated into vector DNA in 10 -15 ul of a buffer containing 66 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 5 mM DTT, and 0.4 - 1 mM ATP. For 26 ligations, 10 - 20 ng vector DNA was incubated with a 1 - 5 molar excess of insert DNA. T4 DNA ligase was added at 1 U for blunt ended ligations and 0.1 U for sticky ended ligations (Maniatis et al, 1982). Ligations were allowed to proceed overnight at 15 °C. E. Transformation of DNA into Bacteria Host bacteria for M13 transformations were made competent by treatment with CaCl 2 (Dagert and Ehrlich, 1979; Messing, 1983). Fifty millilitres of YT were inoculated with a host colony from a minimal glucose plate and incubated at 37 °C with vigorous shaking until the C © 6 0 0 of the culture was 0.4 to 0.6. Cells were collected by centrifugation at 2.5 krpm in an HB-4 rotor at 4 °C for 5 min, and were gently resuspended in 1/2 of the starting volume of ice cold 50 mM CaCl. The cells were incubated on ice for 25 min and were again collected by centrifugation in an HB-4 rotor at 2.5 krpm for 5 min at 4 °C. The bacteria were gently resuspended in 1/10 of the starting volume in ice cold 50 mM CaCl 2. The highest transformation efficiency was obtained when the cells were stored at 4 °C for 24 h before use (Dagert and Ehrlich, 1979). Aliquots (0.3 ml) of competent cells were transformed with 2 ul of ligated DNA (see previous section). Cells were incubated with DNA in 13 X 100 mm glass tubes at 4 °C for 40 min and then heat shocked at 42 °C for 2 min. M13 transformants were plated immediately with the addition of 10 ul 100 mM IPTG, 50 ul Xgal (10 mg/ml in dimethylformamide), 0.2 ml host cells in log phase, and 4 ml soft YT agarose, and spread on YT plates. The plates were then incubated at 37 °C overnight. Recombinants were identified as colorless plaques in the presence of Xgal and IPTG (Messing, 1983). 27 F. Klenow Labeling of DNA Fragments DNA was labeled using the method of Feinberg and Vogelstein (1983). Typical reaction mixtures contained 100 - 200 ng DNA in a volume of 50 ul. DNA in 32 ul of dH20 was denatured by boiling for 10 min, and was cooled to 37°C for 15 - 30 min. Labeling was set up in a final volume of 50 ul of 50 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 10 mM (3 mercaptoethanol, 20 uM dCTP, 20 uM dGTP, 20 uM dTTP, 50 uCi a - 3 2P dATP (3000 Ci/mmole), 200 mM HEPES, pH 6.6, 60 A26Q/ml p(dN6) (random primers), 0.4 mg/ml BSA and 0.1 U/ul E. coli DNA Polymerase I (Klenow fragment). Extension was allowed to occur overnight at 37 °C. The reaction was terminated by the addition of SDS to 0.7% and EDTA to 7.5 mM. The total volume was brought to 200 ul with dH20. Labeled DNA was separated from unincorporated labeled nucleotides by chromatography on an Ultrogel AcA54 column by elution from the column with a solution of 10 mM Tris-HCl, pH 7.5, 200 mM NaCl, and 0.25 mM EDTA. The specific activity of the probes labeled in this o manner was between 0.5 - 4 X 10 cpm/u.g. Labeled DNA was denatured immediately before use by boiling for 10 min or by addition of 1/10 volume 1 N NaOH and incubation at 68°C for 10 min. If the DNA was denatured with alkali, 1/10 volume of 1.5 M NaH2P04 was added prior to the hybridization reaction. 4. Construction of the Genomic Library A. Isolation of DNA from Lymphocyte Nuclei The protocol for isolating DNA from peripheral lymphocytes was derived from that of Bell et al. (1981). Frozen whole blood collected in tubes containing EDTA was frozen at -20 °C in 20 ml aliquots. The blood was thawed by standing at room temperature for 30 min. It was then diluted with 200 ml of sucrose buffer (0.32 M 28 sucrose, 10 mM Tris-HC,l pH 7.5, 5 mM MgCl 2) and transferred to 250 ml centrifuge bottles. The solution was mixed gently for 5 min at room temperature, then centrifuged at 4 krpm , 4 °C, in a Sorvall GSA rotor for 10 min. The supernatant was carefully removed by aspiration. Sucrose buffer (25 ml) was then added to the pellet and the white blood cells and red blood cell debris were resuspended using a sterile pasteur pipet. The centrifugation step was repeated, and again the supernatant fraction was removed by aspiration. The pellet was resuspended in 9 ml of nuclei buffer (75 mM NaCl, 24 mM EDTA, pH 8.0). The solution was transferred to a sterile 50 ml tube, and 225 ul of 20% SDS solution was added. The mixture was gently inverted and left to stand for 15 min at room temperature. A small amount of proteinase K powder was added to the solution (10 mg). The mixture was then incubated overnight at 37 °C. Each sample was extracted 3 times with equal volumes of phenol. The tube was rocked gently for 30 min at room temperature. The mixtures were then centrifuged at 4 krpm in a Sorvall HB-4 rotor for 10 min at room temperature. The aqueous supernates were removed to a fresh tube. Two further phenol extractions were performed in the same manner. Finally, a sevag extraction was performed. One tenth volume (800 ul) of 4 M NH 4OAc was added and the solution was thoroughly mixed for 45 min. An equal volume of isopropanol was added and the tube gently inverted. The filamentous DNA precipitate was collected on the end of a micropipet. The pellet was washed twice with 95% ethanol and allowed to air dry. The DNA was resuspended in 200 ul TE (10 mM Tris base, pH 8.0, 1 mM EDTA), and was allowed to rehydrate for one week before use. 29 B. Partial Digestions of Genomic DNA with Sau3A Fresh Sau3A restriction enzyme was obtained from Biolabs. In order to assay the activity of Sau3A required to give an appropriate degree of partial digestion (Maniatis et al., 1982), 10 ul of concentrated genomic DNA (approximately 15 |ig) was dissolved in 150 ul of the buffer recommended by the manufacturer. The DNA was mixed by inverting and the tube was left to sit at room temperature for approximately 1 h. The mixture was then divided into nine aliquots labeled #1 to #9. Tube 1 received 30 ul, while tubes #2 through #9 received 15 ul each. Four units (4 U, 1 ul) of Sau3A were then added to tube #1 and the contents of the tube mixed thoroughly. An aliquot (15 ul) of this reaction mixture was then transferred to tube #2 and mixed well. An aliquot of tube #2 was then transferred to tube #3, etc. until all but tube #9 had received a dilution of the enzyme. Tube #9 received no enzyme. Tubes #1 to #9 were incubated at 37 °C for 1 h. At this time, 1 ul of 0.5 M EDTA and 2 ul of 10X ficoll load mix (25% ficoll (type 400), 0.25% bromophenol blue, 0.25% xylene cyanol) was added to each tube. The reactions were applied to the wells of a 0.5% agarose gel, and electrophoresed at 100 V for approximately 90 min. The resulting pattern of digestion revealed the amount of enzyme giving the best partial digestion in the range of 10 to 20 kb. The preparative digest was performed in 3 aliquots, in order to achieve equal representation of sequences cut by the enzyme more or less readily. Forty microlitres of concentrated genomic DNA were diluted to 600 ul in IX Sau3A buffer. This was separated into 3 aliquots of 200 ul. The aliquots were treated with either 1, 2, or 4 U of Sau 3A (4 U/ul). The reactions were then incubated at 37 °C for 1 h, 30 when 50 ul of 0.5 M EDTA and 70 ul of 10X ficoll load mix was added. The samples were applied to a low melting point agarose gel (0.6%). When the fragments had separated sufficiently by electrophoresis, the region of the gel containing fragments 10 to 20 kb in length was cut out. The DNA was extracted from the gel slice by phenol extraction as described in the section on basic techniques. Precipitated DNA was resuspended in 150 ul of TE. C. Ligations into X E M B L 3 Arms Before ligations into EMBL3 arms, the genomic DNA fragments resulting from the partial Sau3A digest were tested for their ability to ligate to each other. Samples of the fragments were ligated in a 5 ul volume. Ligation buffer for BRL T4 DNA ligase (10X) contains 500 mM Tris base, pH 8.0, 70 mM MgCl2> and 10 mM DTT. The test ligations contained 0.5 |ig partially digested DNA, 0.5 ul 10 mM ATP (freshly diluted from 100 mM), 0.5 ul 10X ligation buffer, and dH20 to 5 ul. One Weiss unit (0.5 ul) of T4 DNA ligase was used. The reaction mixture was thoroughly mixed and transferred to a siliconized micropipet. Both ends of the pipet were then sealed over a flame. The pipet was incubated overnight in a tube of water at 4 °C. The degree of ligation was assessed by agarose gel electrophoresis against an unligated control sample. After determining that self-ligation of the partial digestion fragments was adequate, a preparative reaction was carried out. The EMBL3 arms obtained from the supplier had been digested with 2 enzymes. Digestion with BamFfl provided sticky ends homologous to those of the Sau3A fragments. Digestion with EcoRI served to remove the X stuffer fragment and reduce the probability of stuffer religation. The EMBL3 ligation reactions contained lu l (l|J.g) pre-digested EMBL3 arms, 0.4 \ig 31 partial digest fragments in 2.5 ul or less, 0.5 ul 10 mM ATP, 0.5 ul 10X ligation buffer, and dH20 to 4.5 ul. T4 DNA ligase was added (0.5 ul, 1 Weiss unit). The mixture was thoroughly mixed and transferred to a siliconized micropipet and sealed as for the test ligation. The pipet was incubated overnight at 4 °C. D. Packaging of Reconstituted EMBL3 Gigapack packaging extracts were obtained from Vector Cloning Systems Ltd. Extracts were kept frozen at - 70 °C until immediately prior to use. The freeze / thaw extract tube (red) was thawed between the fingers and returned immediately to ice. The sonic extract tube (yellow) was thawed as above and returned to ice. The ligated DNA (4 ul) was added to the freeze / thaw tube (red). A 15 ul aliquot of the sonic extract was added to the freeze / thaw extract and mixed gently. The reaction was incubated at room temperature for 2 h. At this time 500 ul sterile SM (0.1 M NaCl, 8 mM MgCl2, 50 mM Tris-HCl, pH 7.5, and 0.01% gelatin) and 20 ul CHC13 were added to the tube and the contents mixed well. The reaction was then stored at 4 °C until it was plated onto E. coli LE 392 and its P2 lysogen derivative P2 392 in order to determine the titre. The packaging reaction in SM was diluted by factors of 4 X 102, 4 X 103, and 4 X 104. The phage were adsorbed to host cells for 20 min at 37 °C and plated onto NZ plates. 5. Screening of the Genomic L i b r a r y A . Plating the Phage L i b r a r y The genomic library prepared above was screened by the procedure of Benton and Davis (1977). The library was initially screened at a high density of 104 plaques per 4 100 mm petri dish or 5 X 10 plaques per 150 mm petri dish. Aliquots of appropriate 32 dilutions of the packaging solution were incubated with host cells at 37 °C for 10 min and then plated on NZ plates with the addition of soft NZ agarose. Plates were incubated at 37 °C until the phage plaques were visible but not touching one another. The plates were then incubated at 4 °C for 1 h. Replicas of the plaques were transferred to nitrocellulose circles. In order to allow amplification of the phage plaques, the circles were inverted on fresh NZ plates and incubated at 37 °C overnight. Master plates were stored at 4 °C. For further screens, the amplification step was omitted. B. Hybridizations and Washing The DNA on the nitrocellulose filters was denatured by treatment with 0.5 M NaOH, 1.5 M NaCl for 5 min. The pH of the nitrocellulose filters was neutralized by treatment with 1 M Tris-HCl, pH 7.5 for 5 min, followed by 0.5 M Tris-HCl, pH 7.5, 1.5 M NaCl for 5 min. After air drying, the filters were baked at 68 °C for 2 h. Recombinant phage of interest were detected by hybridization to labeled probes and autoradiography. Prior to hybridization, filters were washed with 6X SSC (20X SSC contains 3 M NaCl, 0.4 M Na citrate, pH 7.0), then prehybridized with 6X SSC, 2X Denhardt's (Denhardt's solution contains 0.02% BSA, 0.02% ficoll, and 0.02% polypyrrolidine) at 68 °C for 1 to 4 h. Filters were then hybridized overnight at 68 °C in 6X SSC, 2X Denhardt's, 1 mM EDTA, 0.5% SDS, and denatured, labeled probe. The probe used for screens (pCHIX) was at least 1 X 106 cpm/ml in Q the hybridization mix, with a specific activity of >0.5 X 10 cpm/Ltg. After hybridization, the filters were washed twice at room temperature in 2X SSC followed by three washes at 68 °C in IX SSC, 0.5% SDS for 30 - 40 min, and finally rinsed in IX SSC at room temperature. After air drying, the filters were exposed to Kodak XK-1 film overnight at -70 °C with intensifying screens. When the film was developed, the outlines and orientation markers on the filters were traced onto the film and the plates were oriented using these guides. The appearance of a comet shaped area of exposure revealed the presence of a hybridizing phage clone. For the initial screen, the area of the agar identified by this procedure was lifted out using the large end of a pasteur pipet. For the second and third screens, care was taken to try to isolate as few surrounding phage as possible, so the narrow end of a pasteur pipet was used to isolate the plaques. In both cases the plaques were eluted into 500 ul of SM in a 1.5 ml microfuge tube. The tube was vigorously vortexed to free the phage from the agar and the mixture was incubated for a minimum of 6 h at 4 °C . After this stock was titred for phage, appropriate dilutions were made and new plates of the isolate were grown. The screening process was then repeated for these new plates. 6. M a p p i n g of the Factor I X E M B L 3 Clones When EMBL3 clones containing regions of the factor LX gene had been isolated, fresh plates were prepared from single, isolated plaques, and stocks of cell lysate were frozen at -70 ° C for future use. DNA was isolated from each clone as described in the section on basic techniques. In order to orient each clone with respect to the map of the factor LX gene, various digestions with restriction enzymes were performed on each of the eight EMBL3 clones isolated during the screen. The restriction enzymes used for these digestions included Hindlll, EcoRI, BamHI, Sail, Bglll, and KpnI. Southern blots were prepared for several of these gels in order to identify bands containing exon sequence (as described in Part 9). The factor IX cDNA was used as a probe for these blots 34 (0.8 X 10° cpm/ml). The blots were wetted in 6X SSC. Prehybridization solution contained 6X SSC, 0.5% SDS, 100 ug/ml salmon sperm DNA (denatured), and 2X Denhardt's solution. The blots were prehybridized for 2 h at 68 °C. Hybridization solution contained 6X SSC, 0.5% SDS, 2X Denhardt's, 10 mM EDTA, 100 Ug/ml salmon sperm DNA (denatured), and denatured, labeled probe. The blots were hybridized overnight at 68 °C. The first two washes of 10 min each were performed at room temperature in 2X SSC, 0.5% SDS and in 2X SSC, 0.1% SDS, respectively. Three 30 min washes in 0.1X SSC, 0.5% SDS followed. The blots were air dried and exposed to X-ray film overnight with intensifying screens. 7. Subcloning into M13 Vectors for Sequencing A. Construction of M13 Clones DNA was sequenced by the chain termination method (Sanger et al., 1977) using the M13 sequencing vectors (Messing et al., 1981; Messing, 1983). DNA to be cloned into M13 vectors was produced by restriction enzyme digestion and recovery of the fragment from low melting point agarose gels (Messing, 1983) or by sonication and end repair (Deininger, 1983). Fragments produced with restriction enzymes were digested under the conditions suggested by the manufacturers. If fragments or mixtures of fragments were to be put directly into a ligation reaction, the digestion mix was heated to 68 °C for 10 min to inactivate the enzymes. The mixture was then extracted with phenol and precipitated from ethanol before ligation. Purified restriction enzyme fragments were isolated from low melting point agarose or polyacrylamide gels. Random DNA fragments of the factor IX EMBL3 clones were produced by sonication (Deininger, 1983), using a Heat Systems Sonifier at output level 2. DNA 35 (10 - 20 |ig in 0.5 ml of 0.5 M NaCl, 0.1 M Tris-HCl, pH 7.4, 10 mM EDTA) was sonicated in 5 pulses of 5 seconds each. The DNA solution was cooled on ice during the sonication, and was thoroughly mixed between pulses. After the 5 pulses, the DNA was precipitated from solution with ethanol at - 20 °C for 30 min. The precipitated DNA was resuspended into 24 ul of dKL^O. After the addition of 3 ul each of 10X ficoll load mix and 10X TBE, the solution was loaded onto a 5% polyacrylamide non-denaturing gel. The samples were electrophoresed at 50 V for approximately 50 min. After the gel was stained for 10 min in 0.5X TBE with 2 Ltg/ml of EtBr, the region of the gel containing fragments between 300 and 500 bp was excised from the gel. The DNA contained in the gel fragment was collected by electroelution into IX TBE at 100 V and precipitated from ethanol at - 70 °C for 1 h. The DNA was resuspended and the solution was extracted 6 times with phenol (IX) and ether (5X). The DNA was again precipitated from ethanol at - 20 C for 30 min, and was resuspended in 46 ul of IX SI buffer (0.25 M NaCl, 30 mM KOAc, 20 mM ZnS04, and 10% glycerol). The ends of the DNA fragments were repaired by incubation with nuclease SI followed by end repair with the Klenow fragment of DNA Polymerase I. The nuclease SI reaction consisted of 46 nl DNA solution from above, 4 u.110X dilute S1 nuclease (5.3 U/ul) for 30 min at room temperature. The reaction was stopped with the addition of Tris base, pH 8.0 to 80 mM and EDTA to 20 mM. The solution was extracted once with a mixture of phenol and chloroform, and precipitated from ethanol at 0 °C for 30 min. The DNA was resuspended in 46 ul of Klenow buffer (20 mM Tris base, pH 8.0, 7 mM MgCl2). Enzyme (DNA Polymerase 1, Klenow fragment, 7.5 U/ul, O.lul used) was added to the reaction and preincubated for 37 °C for 10 min. After 1 ul of each of the dNTPs (10 mM solutions of each) was added, 36 the reaction was allowed to proceed for another 5 min at 37 0 C. Finally, the reaction mixture was incubated at 68°C for 10 min. The concentration of DNA in the solution was then adjusted to 10 ng/ul with TE. B. Screening of M13 Clones On some occasions, a purified fragment was used to ligate into Ml3 vectors. On other occasions, a mixture of fragments was used. In the latter case, those clones containing exon sequence were identified before sequence analysis. The plaques were screened by plaque hybridization exactly as described for the library screening. In order to screen for clones containing similar inserts to a clone already isolated, but in the opposite orientation, figure eights between the clones were prepared. An aliquot (1 ul) of DNA from of each of two clones to be tested (DNA isolation described below) was added to 14 ul of a solution containing 100 mM Tris-HCl, pH 7.5, 500 mM NaCl, 16% glycerol, 0.2% SDS, and 0.04% bromophenol blue in an 0.5 ml tube. The contents of the tubes were thoroughly vortexed and centrifuged for 1 min in a microfuge. The tubes were incubated for at least 1 h at 68 °C. After the solutions had cooled on ice for 10 min, they were loaded onto 0.7% agarose gels and electrophoresed for 1 h at 100 V. The presence of a clone with a similar insert to the selecting clone, but in the opposite orientation, resulted in the formation of a figure eight structure. This type of complex is retarded in its movement through the gel when compared to the individual clones. C. M13 DNA Isolation DNA from clones identified in the previous section was prepared as described by Messing (1983). M13 clones were grown as 2 ml cultures in YT medium in 15 ml Falcon 2059 tubes using one plaque and 20 ul of fresh JM103 culture as inoculum. The cultures were incubated at 37 °C for 6 - 16 h. Clones containing larger inserts, > 1 kb, were grown for 6 h. Host cells were removed by centrifugation in a 1.5 ml microfuge tube. Phage particles in 1.3 ml of supernatant were precipitated by the addition of 0.3 ml of 20% PEG, 2.5 M NaCl, and incubation at room temperature for 15 min. M13 phage were collected by centrifugation in an microfuge for 5 min . After removal of all the supernatant, the phage particles were resuspended in 200 ul of low tris buffer (50 mM NaCl, 10 mM Tris-HCl, pH 7.5,1 mM EDTA). DNA was purified by successive extractions with phenol, phenohchloroform (1:1, v/v), and chloroform. DNA was precipitated twice from ethanol for 30 min at 0 °C. The final DNA pellet was resuspended in 50 u.1 of low tris buffer. 8. DNA Sequencing DNA in M13 clones was sequenced by the chain termination method (Sanger et al., 1977) as modified for phage M13 templates (Messing et al., 1981). Sequencing reactions were carried out using dideoxy- and deoxy-ribonucleotide solutions of the relative concentrations shown in Table III. Sequencing was performed by hybridizing 4 ul of template (isolation method in the previous section) with 1 ul of primer (0.03 OD 2 6 0 n m/ml, 17-mer: 5' -GTAAAACGACGGCCAG- 3"), 1 ul dH 20, and 2 ul 10X Hin buffer (600 mM NaCl, 100 mM Tris-HCl, pH 7.5, 70 mM MgCl 2) at 68 °C for 10 min. After the hybridization mix was allowed to cool to room temperature (20 - 30 min), 1 ul of 15 uM dATP and 1.5 ul of a - 3 2P dATP (10 uCi/ul, 3000 Ci/mmole) were added to the tube. An aliquot (2.5 ul) of this template / primer mix was mixed with 1.5 ul of the appropriate deoxy / dideoxy solution (see Table III) in a fresh tube. DNA Polymerase I Klenow fragment (lu.1 of 0.2 U/ul) was added. After 15-20 min of incubation at room temperature, 1 ul of 0.5 mM dATP was added. After a further 15 to 20 min at room temperature, 5 ul of stop / dye mix (98% formamide, 10 mM EDTA pH 8.0,0.02% xylene cyanole, 0.02% bromophenol blue) was combined with the contents of the tube. The extended products were denatured by heating to 92 °C for 3 min. Aliquots (1 - 2 |il) of these products were analyzed on 6% and 8% denaturing polyacrylamide gels (50 cm long) at 52 W in IX TBE. After electrophoresis, the gels were dried at 80 °C under vacuum on a Bio Rad gel drier for 20 - 30 min, and autoradiographed on Kodak XK-1 film overnight at room temperature Table III. Composition of dideoxy / deoxy sequencing solutions Solutions d/ddG d/ddA d/ddT d/ddC Nucleotide dG 7.9 109.4 158.7 157.9 dT 157.6 109.4 7.9 157.9 dC 157.6 109.4 158.7 10.5 ddG 157.4 - - -ddA - 116.7 - -ddT - - 550.3 -ddC _ _ _ 191.6 The concentrations of the dideoxyribonucleotide (dd) and deoxy-ribonucleotide (d) triphosphates use in the sequencing solutions for M 1 3 DNA sequencing are shown. Concentrations are given in L I M . 39 9. Genomic Southern Blot The genomic Southern blot was transferred to nitrocellulose essentially as described by Southern (1975), and blots were hybridized and washed as described by Kan and Dozy (1978). Genomic DNA (10 jig) was digested with restriction endonucleases (20 - 30 U) in a volume of 40 ul under the conditions suggested by the enzyme manufacturers. DNA fragments were separated by electrophoresis for approximately 16 h at 20 mA in a submerged agarose gel. After electrophoresis, the gel was stained with EtBr so that the DNA in the lanes could be visualized. The gel was exposed to UV light of 260 nm for 90 s to introduce single strand nicks in the DNA, which promote more efficient transfer of the larger fragments. DNA in the gel was then denatured for 45 min in 0.5 N NaOH, 0.6 M NaCl and was then neutralized for 2 periods of 30 min in 1 M Tris-HCl, pH 7.5, 0.6 M NaCl. DNA was transferred to nitrocellulose membranes with 10X SSC for 52 h. After the transfer, the nitrocellulose filter was washed in 3X SSC to remove any agarose, air dried, and then baked at 68 °C for 6 h. 32 DNA fragments were detected by hybridization to P - labeled probes. The nitrocellulose filter was first wetted with 3X SSC and then prehybridized for 15 h in a solution containing 50% formamide, 3X SSC, 1 mM EDTA, 0.1% SDS, 10 mM Tris-HCl, pH 7.5, 10X Denhardt's solution, 0.05% sodium pyrophosphate, 100 Lig/ml denatured herring sperm DNA, 2.5 |ig/ml poly(A), and 50 Ltg/ml tRNA. Hybridizations were carried out in a buffer similar to the above, but lacking the tRNA and containing 6 X 106 cpm/ml of denatured, labeled pCHLX insert. The hybridization reaction was allowed to proceed for 48 h at 37 °C. After hybridization, 40 the blot was washed for 1 h at room temperature in 2X SSC, IX Denhardt's, and then washed twice for 90 min at 50 °C in 0. IX SSC, 0.1% SDS. The blot was then rinsed twice at room temperature in 0.1XSSC, 0.1% SDS, followed by four rinses at room temperature in 0.1X SSC. After air drying, blots were exposed to Kodak XK-1 film with intensifying screen for 10 days at - 70 °C. 41 RESULTS 1. Southern Blot Analysis of the SB IX-4 Factor IX Gene As a preliminary step in the analysis of the patient's factor LX gene, it was of interest to investigate whether or not gross deletions of the gene had occurred. To address this question, a genomic Southern blot was prepared. A photograph of the autoradiograph from the blot is presented in Figure 3. In this figure, DNA from the patient's father, who is unaffected by hemophilia, was included as a control. The presence of partial digest bands (EcoRI lane, 8.4 kb; Bglll lane, 9.5 kb; Hindlll lane, 8.5 kb and 14.5 kb) resulted in some differences in intensity between the normal and hemophiliac bands within a given digest. Results from three different restriction enzymes showed that each of the father's factor LX - homologous bands had a similar counterpart in the son's lane. The bands present on the blot were consistent with the fragment sizes predicted by the published factor LX gene map (Yoshitake et al., 1985). These results suggested that no gross deletions or insertions had occurred in the SB LX-4 factor LX gene. 2. Genomic Factor IX Clones Isolated from the SB IX-4 Library A genomic library of DNA from the patient was then prepared. DNA fragments (10 -20 kb) from a partial digest of SB LX-4 DNA with the restriction enzyme Sau3A were isolated. The fragments were then introduced into BamHI - digested X EMBL3 arms. One million independently derived X clones were screened, using a radioactively 42 Figure 3 Southern blot analysis of the SB LX-4 factor LX gene Bglll EcoRI ffindHI 1 2 3 4 5 6 The restriction enzymes Bglll, EcoRI and Hindlll were used to digest genomic DNA as indicated. Lanes 1,3 and 5: DNA from the unaffected father of the hemophiliac, representing the normal gene. Lanes 2,4 and 6: SB IX-4 DNA, from the hemophiliac. The normal human factor IX cDNA was used as a hybridization probe. The sizes of selected bands are shown to the right of the blot labeled factor DC cDNA as a hybridization probe. Three successive screens of this library yielded eight factor DC containing clones. The map of the restriction enzyme sites of these clones is presented in Figure 4. The EMBL3 clones designated X LX-12, X DC-10, and XIX-6 were selected for further analysis, since together they span the entire gene (Figure 4). 3. M13 Subclones Containing SB IX-4 Factor IX Exons To facilitate sequence analysis, the DNA inserts from X clones DC-12,10, and 6 were fragmented using either restriction enzymes or sonication treatment. These fragments were inserted into Ml3 vectors. Screens of the subclones with the factor DC cDNA as a hybridization probe allowed identification of those phage inserts containing exons. Positive clones were sequenced using the dideoxy method of Sanger et al. (1977). A map of these M13 subclones is presented in Figure 5. Approximately 50% of the coding region sequence was obtained from both coding and non-coding strands. Greater than 80% of the 3' untranslated region was also sequenced. Results from the sequencing of the Ml3 subclones are presented in Table IV. 4. Nucleotide Sequence of SB IX-4 Activation Peptide and Intron / Exon Junctions An amino acid polymorphism has been reported in the activation peptide of the factor IX protein (McGraw et al, 1985). Patient SB DC-4 has the ACT codon at amino acid position 3 of the activation peptide (amino acid 148 of the mature protein), as reported by McGraw et al (1985), while others have indicated a GCT codon at this position (Yoshitake et al, 1985) (Table IV). Since the activation peptide is completely removed from factor IX upon activation, this polymorphism is thought to have little effect on the activity of factor LX . Both codons have been observed in normal 44 Figure 4 Genomic factor FX clones isolated from the SB IX-4 library IX-10 IX-6 IX-12 E E E E E E B B B E DB E E E 5' 1 2 3 4 5 6 7 8 ' I 11 ij 1 l' 10 20 3" The lower diagram is a representation of the human factor IX gene (Yoshitake et al., 1985). Exons 1 through 8 are indicated as in Figure 1. EMBL3 clones containing exon sequence were mapped with several restriction enzymes to position the clones relative to the published map of the gene. Each dark line represents a single clone. The sites of cleavage for the restriction enzymes BamFfl (B) and EcoRI (E) have been indicated on the line above the gene. The boundaries of the clones extending 3 to the gene were not precisely mapped. Those clones chosen for subcloning are labeled as LX-12, 10 and 6. Figure 5 M13 subclones containing SB IX-4 factor IX exons Ml3 subclones containing exons from the SB IX-4 library are indicated by arrows beneath the exons. Due to the length of the gene (34 kb), only the protein coding regions have been shown. The arrow points in the direction in which the clone was sequenced, from the Ml 3 primer through the clone. Leftward arrows indicate that the coding strand has been sequenced. Rightward arrows indicate that the noncoding strand has been sequenced. Each clone is labeled with its name. Only the translated region has been presented, although clones containing 3' untranslated region were also obtained. Restriction enzyme sites used in cloning are indicated below the exons. Scale: I 1 100 bp. 46 EX0N1 -c StuI 12-1 1-1 EXON2 EXON3 —T EcoRI AM 3G-7 3F-U EXON4 n-PvuII 4-1 5-2 EXON5 Xbal 6-2 EX ON 6 BgUI Rsal Mbol Rsal Mbol Bell Table IV Nucleotide sequences of the SB IX-4 activation peptide and intron / exon junctions ACTIVATION PEPTIDE N H 2 - - AAG CTC ACC CGT / GCT GAG GCT GTT TTT CCT - - COOH lys leu thr arg a] a glu ala val phe pro 145 146 - - AAG CTC ACC CGT / GCT GAG ACT GTT TTT CCT - -th r INTRON / EXON JUNCTIONS intron exon/5' intron 3'/exon A CAG/GTTTGT TTTCAG/T B ACA/GTGAGT TTATAG/A C TTG/GTAAGC TCAAAG/A D TAG/GTAAGT TTTTAG/A E CAG/GTCATA TTCTAG/T F CAG/GTACTT TCACAG/G G CAG/GTAAAT TAATAG/G consensus CAG/GT AAGT T TN TAG/G A G CC C after Yoshitake et al., 1985 Yoshitake et al., 1985 SB1X-4 McGraw etal, 1985 In the first section, the polymorphic activation peptide residue is shown. The N-terminal activation cleavage site, between amino acids 145 and 146, is shown by a slash. The polymorphic amino acid is shown in bold type: the upper line represents the most commonly observed sequence, and the lower line the sequence found by McGraw et al., 1985 and in SB IX-4. The lower table lists the sequences found at the factor DC intron / exon junctions and lists the consensus sequences identified by Mount, 1982, and Sharp, 1981. In each case the slash represents the site of splicing. All intron /exon junctions sequences in SB IX-4 were identical to those identified by Yoshitake et al., 1985. individuals. Knowledge of this polymorphism has been exploited in carrier detection studies using an oligonuleotide probe (Winship and Brownlee, 1986) and an antibody (Wallmark et al., 1985) specific to this region. In addition to the coding regions, flanking portions of introns and intron / exon junctions were compared to the normal sequence. Table IV notes the sequences observed in these areas. All intron / exon boundaries displayed the invariant GT / AG dinucleotides associated with introns (Breathnach and Chambon, 1981). The sequence of the introns extending from such junctions was determined for a minimum of 25 base pairs. In each case the sequence observed was identical to that reported by Yoshitake etal. (1985). The putative sites for transcript cleavage and poly(A) addition were sequenced and were found to be identical to the sequence presented by Yoshitake et al. (1985). The putative promoter region was found to be identical to the normal sequence for 225 base pairs upstream of exon 1. 5. Nucleotide Sequence Alteration in the SB IX-4 Factor IX Gene One nucleotide change relative to the normal protein coding sequence was observed a nucleotide 31,311 of the factor LX gene (numbering system of Yoshitake et al, 1985). This corresponds to amino acid 397 of the mature protein, which is found in the serine protease subunit of factor IXa. The normal isoleucine codon, ATA, has been changed to a threonine codon, ACA. This sequence has been observed in two independently obtained non-coding strand clones (8C-5 and 8C-17), and from one coding strand clone derived by reversing the insert of 8C-5. Figure 6 shows photographs of the sequencing gel autoradiographs. The DNA sequence of this region and the nucleotide change relative to the normal sequence are shown. 49 Figure 6 Nucleotide sequence alteration in SB LX-4 factor IX gene The sequence of the alteration at nucleotide 31,311, corresponding to amino acid 397, is given for both the coding and noncoding strands of DNA. The sequence of interest is given to the left of the autoradiogram and the horizontal arrow points directly toward the mutation. The amino acid sequences given in the boxes show the direction of sequencing. The direction of the arrow indicates the way the sequence is to be read from the bottom to the top of the autoradiogram. 5 0 CODING STRAND SB LX-4 A A C A 5 ' GGAATA TAT 3 ' Gly De Tyr 396 397 398 t ACA thr normal A T A A NONCODING STRAND normal SB IX-4 T T T T A G T T 3 ' CCT TAT ATA 5 ' t TGT DISCUSSION 1. Summary of Data Concerning Patient SB IX-4 A significant array of data have now been compiled about the molecular defect causing decreased factor LX clotting activity in the patient SB IX-4. Antigen and activity assay results indicate a significant reduction in factor LX activity with respect to factor IX antigen levels. A Southern blot has suggested that no major deletions of the factor LX gene are present. All eight exons were isolated from the genomic library prepared from the patient's DNA. Within the coding regions, a single nucleotide change relative to the normal sequence (Yoshitake et al., 1985) was observed. This change results in the substitution of a threonine codon, ACA, for the normal isoleucine codon, ATA. It should result in an amino acid alteration at residue 397 of the protein. These results are consistent with each other and suggest that the observed nucleotide change is the cause of the decrease in specific activity in the factor IX of SB IX-4. 2. Naming of the Mutation Found in SB IX-4 No other mutation in the serine protease region of factor LX has been identified at the molecular level. In keeping with the tradition of naming a newly identified mutation of factor LX by a subscript naming the place of birth of the patient, we have given the name factor LX.. to the abnormal protein found in patient SB IX-4. Vancouver r r 3. Implications for Carrier Detection and Prenatal Diagnosis Several restriction fragment length polymorphisms (RFLP's) have been identified in the factor IX gene (Camerino et al., 1985; Camerino et ai, 1984; Winship et al., 1984; Hay et al., 1986; Giannelli et al., 1984). These polymorphisms have been used extensively in the diagnosis of hemophilia B carrier status and in prenatal diagnosis. Due to linkage disequilibrium between several of these RFLP sites, however, diagnosis is not possible in all cases. Several studies have shown that knowledge of a point mutation can be used in the diagnosis of human diseases. Such diagnoses were first obtained for sickle cell anemia (Conner et al, 1983). Recently, the method has been applied to hemophilia B (Rees et al., 1985; Winship and Brownlee, 1986). In these studies, pairs of 19 base long oligonucleotide probes were prepared. One probe contained a region of the normal sequence, and the other contained the same region but the abnormal or polymorphic sequence. By careful manipulation of hybridization and washing conditions, it was possible to distinguish between the normal and mutant alleles on Southern blots. In the case of factor ^ Vancouver' oligonucleotide probes could be synthesized which match the normal and mutant sequences between amino acids 394 to 400 (nucleotides 31,302 to 31,320). These probes could be used in carrier detection and prenatal diagnosis. Since the proportion of hemophilia B variants involving a mutation at isoleucine 397 may be small, this analysis might be of limited general usefulness. However, if a series of such specific probes were to become available, it might be possible to obtain molecular diagnoses for many variants of hemophilia B. 53 4. Possible Effects of the I'e 3 9 7 to Thr 3 9 7 Substitution on Factor IXa Activity: A. Structure of Normal Factor IXa The three dimensional structures of factor LX and its substrate, factor X, have not a been determined by X-ray crystallography since, like many of the blood clotting factors, these proteins have not been crystallized. Therefore, the role of isoleucine 397 in the activity of normal factor LX is not known. Factor IX activates the next J a a clotting factor in the cascade, factor X, by cleaving the peptide bond between arginine194 and alanine .^ The precise points of interaction of the proteins in the factor LXa - factor X complex, however, are not known. B. Structural Homology Between the Trypsin-Like Serine Proteases The term 'serine protease' is applied to a group of structurally related endoproteases which have a serine residue at the active site. These enzymes have been isolated from a variety of organisms. On the basis of amino acid sequence homologies, serine proteases are divided into two major families, the subtilisins and the trypsin-like enzymes. Factor IX is a member of the trypsin-like family. Trypsin is one of several well known serine proteases which are involved in the process of digestion. Two other digestive proteases, a chymotrypsin and elastase, have also been studied in detail. The three dimensional structures of all three of these enzymes have been determined by X-ray crystallography. An alignment of the amino acid sequences of these three serine proteases with the sequence of factor LX (Table V), shows the the striking similarity between these enzymes. Since it is traditionally applied to all the enzymes in order to simplify comparisons (Hartley and Shotton, 1971), the chymotrypsin numbering system has 54 Table V Amino acid sequence alignment of normal human factor LX a and the pancreatic serine proteases The amino acid sequences of the catalytic regions of normal human factor IXa, bovine trypsin (TRYP), porcine elastase (ELAS) and bovine chymotrypsin (CHYM) are shown. The sequences were taken from Yoshitake et al.. (1985), Marquart et al. (1983), Sawyer et al. (1978) and Cohen et al. (1981), respectively. The numbering system shown here is based on the system developed for chymotrypsinogen A (Hartley and Kauffman, 1966), with insertions in the sequences of related enzymes denoted by letters (i.e. 61a, 61b). Deletions are indicated by dashes. Identical residues in corresponding positions of all four proteins are boxed. The standard single letter code for amino acids is used. 55 16 17 18 19 20 21 22 23 24 25 TRYP I V G G Y T C G A N ELAS V V G G T E A Q R N CHYM I V N G E E A V P G FIXa V V G G E D A K P G a b c d 26 27 28 29 30 31 32 33 34 35 36 36 36 36 T V P Y Q V S L N S W P S Q I S L Q Y R S G S S W P W Q V S L Q D K - - -Q F P W Q V V L N G K - - -37 38 39 40 41 42 43 44 45 46 47 TRYP S G Y H F C G G S L I ELAS S W A H T C G G T L I CHYM T G F H F c G G S L I FIXa V D - A F c G G S I V a b a b 60 61 61 62 63 64 65 65 66 67 68 TRYP K S - G I Q V R L - -ELAS D R - E K T F R V V V CHYM V T - T S D V V V A FTXa E T G V K I T - V V A 81 82 83 84 85 86 87 88 89 90 91 TRYP Q F I S A S K S I V H ELAS Q Y V G V Q K I V V H CHYM Q K L K I A K V F K N FIXa $1 K R N V I R I I P H 48 49 50 51 52 53 54 55 56 57 58 59 N S Q W V V S A A H C Y R Q N W V M T A A H C V N E N W V V T A A H c G N E K W I V T A A H c F 69 70 71 72 73 74 75 76 77 78 79 80 G E D N I N V V E G N E G E H N L N Q N N G T E G E F D Q G s S S E K I G E H N I E E T E H T E "IT b c a b c 92 93 94 95 95 95 96 97 98 99 99 99 P S Y N - - S N T L - -P Y W N - - T D D V A A S K Y N A A S L T I - -H N Y N - - I N K Y - -100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 TRYP N N D I M L I K L K S A A S L N S R V A S I ELAS G Y D I A L L R L A Q S V T L N S Y V Q L G CHYM N N D I T L L K L S T A A S F S Q T V S A V FIXa N H D I A L L E L D E P L V L N S Y V T P I a b c 122 123 124 125 126 126 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 TRYP S L P T - - - S C A S - A G T Q c L I S G W G ELAS V L P R A - - G T I L A N N S P C Y I T G W G CHYM C L P S A - S D D F A A G T T c V T T G W G FIXa C I M D K E Y T N I F L K F G S G Y V S G W G 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 TRYP N T K S S G T S Y P D V L K c L K A P I L S N ELAS L T R - T N G Q L A Q T L Q Q A Y L P T V D Y CHYM L T R Y T N A N T P D R L Q Q A S L P L L S N FIXa R V F H K G R S A L V L Q Y L R V P L V D R a b c a b 166 167 168 169 170 170 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 184 185 TRYP S S c K S - A Y - P G Q I T S N M F c A G Y L ELAS A I C S S S S Y W G S T V K N S M V c A G - G CHYM T N c K K - Y w G T K I K D A M I c A G - A FIXa A T c L R - S T K F T I Y N N M F c A G - F TRYP EI-AS CHYM FIXa TRYP ELAS CHYM FIXa a b c 186 186 186 E - -N - -S - -H E G a b 188 188 189 190 G K D S V R S G V - S S R - D S 206 207 208 209 210 G K L Q Q Y A V H A W T L V T S F L T 211 G G G G 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 C Q G D S G G P V V C S - - . c Q G D S G G P L H c L V N G c M G D S G G P L V c K K N G c Q G D S G G P H V T E V E G a b b 214 215 216 217 217 218 219 220 221 221 222 223 224 225 226 S W G S - - G C A Q K N K P G S F V S R L G c N V T R K P T S W G S S T c S . T S T P G S W G E - - E c A M K G K Y G 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 TRYP V Y T K V c N Y V S W I K Q T I A S N ELAS V F T R V s A Y I S W I N N V I A S N CHYM V Y A R V T A L V N w V Q Q T L A A N FTXa I Y T K V S R Y V N w I K E K T K L T 56 been used. Insertions and deletions relative to the chymotypsin sequence have been accommodated by giving the same number but different letters to each residue in the inserted regions. Those positions at which there is identity between the residues of all four of the proteins have been enclosed in boxes. When the amino acid sequences of the pancreatic serine proteases are compared to one another, there is approximately 41% identity of amino acids between them (James et al., 1978). When the known three dimensional structures of the pancreatic enzymes are compared by superposition of the backbone chains, however, an even greater similarity is evident (~ 85% of the a - carbon atoms are topologically equivalent; James et al.., 1978). The levels of primary sequence identity among these enzymes (~ 41%), and between them and human factor FX (~ 36%), are similar. 3. Therefore it is reasonable to expect a corresponding level of tertiary structure similarity between factor IX a and the pancreatic serine proteases. In addition to the structural similarity of the proteases, the three dimensional structures of portions of several of the known inhibitors of serine proteases are similar. Bovine pancreatic trypsin inhibitor (BPTT) and soy bean trypsin inhibitor (SBTI) are two well known examples. Those regions of the inhibitors which interact directly with the active site show as much structural similarity between them as do the structures of the proteases themselves (Chen and Bode, 1983). While the binding in the complexes formed between these inhibitors and the proteases is thought to be stronger than that between the proteases and their substrates, they have been shown to react with the enzymes by the same pathway. The overall structure of the two kinds of complexes is thought to be similar (Chen and Bode, 1983; and references therein). 57 C. Model of the Complex Between Factor IXfl and Its Substrate Peptide The similarity with the pancreatic serine proteases and others, such as kallikrein, can be exploited to prepare models of the approximate structure of the blood clotting serine proteases. Building on the known coordinates of the polypeptide chain atoms of the pancreatic enzymes, several models of the three dimensional structure of human factor FX have been prepared (Furie et al., 1985; Greer, 1984; and G. Louie and G. fl Brayer, personal communication). Models have also been developed for prothrombin, factor X and factor XII (Furie et al., 1982; Cool et al., 1985). These models have allowed identification of regions of conserved structure and regions of variability between the enzymes (Furie et al., 1982). While the cores of the molecules appear to be highly conserved, the surface features are variable between the enzymes. The blood clotting serine proteases are highly specific for their protein substrates. Since the active sites of these enzymes are nearly identical, it is the protein-protein interactions outside the direct substrate binding regions which are thought to allow specific recognition of the protein substrate (Craik et al., 1985). In addition to the models of factor LXa, a model of the structure of an eight residue peptide containing the activation cleavage sequence of factor X has been prepared. This model is based on the conformation of that portion of the bovine pancreatic trypsin inhibitor which is bound in the active site of trypsin (Louie and Brayer, personal communication). The bond cleaved by factor LXo is part of the sequence a. N-191asn-leu-thr-arg==ile-val-gly-gly19g-C in the 55 kDa factor X protein (Fung et al., 1984). The model of this substrate peptide can be inserted into the model of factor IXa> The resulting complex will be referred to as the factor IXa - substrate complex. Using this model, interactions between these proteins can be studied. 58 The model of the factor IX - substrate complex prepared by Louie and Brayer 3 (personal communication) has been analyzed with respect to the position of the factor ^Vancouver m u t a t i o n a t residue 397. In the chymotrypsin numbering system, amino acid 397 is referred to as amino acid 227. As shown in the three dimensional model of factor IX (Figure 7A), isoleucine 227 (LX 397) occupies a position adjacent to the active site of the enzyme. Five structural features of serine proteases are thought to be crucial to their unique reactivity (Kraut, 1977). The active site serine is in a position to form a covalent bond with the carbonyl carbon atom of the susceptible bond. There is a site (termed the 'oxyanion hole') for binding of the carbonyl oxygen atom when the carbonyl is in the tetrahedral transition state. An S1 binding pocket accomodates the side chain of the amino acid on the acyl side of the bond to be cleaved and thus provides substrate specificity. On the amino side of the susceptible bond, the polypeptide chain of the substrate is weakly bound to the enzyme by hydrogen bonds. The weakness of this binding facilitates the removal of the first product of the reaction from the active site. Finally, there is extensive binding between that portion of the substrate polypeptide chain acyl to the susceptible bond and the surface of the serine protease. Residue 397 of factor LX is located near the surface involved in this extensive a hydrogen bonding with the acyl portion of the bound substrate. In the region of factor LXa around residue 397, therefore, there are believed to be significant interactions between factor LX and its substrate, factor X (Chen and Bode, 1983). 3 By analogy to the bonding of the other serine proteases with their various inhibitors, these interactions would be expected to involve hydrogen bonding and / or hydrophobic interactions (Kraut 1977). 59 Figure 7 Hydrogen bonding in the factor FX - substrate complex models Photographs of the computer screen show the three dimensional structure of the factor IX a model's active site (Louie and Brayer, personal communication). The catalytic triad residues are shown in red and are labelled with their names and amino acid number. They are serine 195, histidine 57 and aspartate 102. The side chains of isoleucine 227 (LX 397) of the normal molecule and threonine 227 (IX 397) of factor L X y a n c o u v e r are drawn in blue. The relevant hydrogen bonds are indicated as dashed yellow lines. The numbers on these lines refers to the interatomic distance, in Angstroms. The atoms involved in the hydrogen bonds have been labeled in white. The factor X subtrate peptide has been indicated in green. A) The position of ile 227 (LX 397) relative to the active site residues in the normal factor IX model, a B) Two of the potential hydrogen bonds in the model of the complex formed between normal factor LX and the factor X substrate peptide are shown. C) The potential hydrogen bond unique to factor ^Vancouver * s indicated. The formation of this bond may interupt the normal hydrogen bonding shown in part B. D. Hydrogen Bonding in the Normal Factor IX -Substrate Complex Examination of the model of the normal factor IX - factor X peptide complex reveals 3 several potential sites for hydrogen bonding. The presence of donor and acceptor ligands, an appropriate geometry for interaction, and the close proximity (~ 2.5 to 3.5A) of these ligands to one another are the factors which determine the possibility of hydrogen bonding. One of the possible interchain hydrogen bonds in the normal factor LX - factor X peptide complex appears between the amide nitrogen of glycine 3. 216 (LX 386) of factor LX and the carbonyl oxygen of leucine 192 (also called a leucine 13) of the factor X peptide (Figure 7B). E. Hydrogen Bonding in the Factor IX - Substrate Complex J & * a Vancouver r From the model of the factor FX,, - substrate complex, it appears that an Vancouver r r r additional intramolecular hydrogen bond could form between two factor IX a atoms. An extra bond is possible since the non-hydrogen bonding side chain of isoleucine 397 is replaced by a hydroxyl group in the threonine side chain. The additional hydrogen bond could form between the hydroxyl group on the side chain of threonine 227 (IX 397) and the carbonyl oxygen of the peptide bond of tryptophan 215 (IX 385) (Figure 7C). If this abnormal intrachain bond was to form, the normal hydrogen bonding between leucine 192 of factor X and glycine 216 (IX 386) of factor IX (described above) could be disrupted. It is possible that such a change in 3 the hydrogen bonding between the enzyme and its substrate could lead to a less productive orientation of the scissile bond of factor X in the active site of factor IXa. This could result in a significantly reduced rate of factor LX catalytic activity. Thus, 3 the three dimensional model of factor IX , although approximate, has provided a possible rationale for the observed decrease in specific coagulant activity associated with factor LX,, Vancouver 5. Directions for Further Research Further studies on the structure of factor FX,, are possible. The patient under Vancouver r r study has sufficient antigen in his plasma to allow the purification of factor IX,, . Protein analysis could be used to compare characteristics of factor Vancouver J ^ FX,, with normal factor LX: calcium binding could be quantitated, activation Vancouver ° n cleavages could be performed and compared to those of normal factor LX, and the molecular weight of the mutant protein could be estimated (Bertina and van der Linden, 1982b). A more complete understanding of the efffects of the isoleucine to threonine mutation at amino acid 397 may be more difficult to obtain. Since the mutation in factor LX„ is thought to affect binding of factor X to factor LX , a direct test of the Vancouver ° ° a mutation would involve some form of kinetic measurements. Coagulation assays measure the rate of formation of a fibrin clot. Since a great many enzymes and cofactors are involved in the formation of a clot, coagulation assays are heterogeneous, difficult to standardize, and unsuitable for kinetic studies of the individual enzymes (Stormorken, 1979). Kinetic experiments on isolated factor LX,, may be hampered by the fact that Vancouver J r J highly reactive in vitro substrates for factor LX are not yet available. A number of 3 synthetic substrates for the blood clotting serine proteases have been prepared. Substrates tested for activity with bovine factor LX include ethyl ester, thioester, 3 4 - nitroanilide, and fluorogenic peptides and amino acids (Lindquist et al., 1978; Byrne and Castellino, 1978; Byrne et al., 1980; McRae et al., 1981; Link and Castellino, 1983; Castillo et al., 1983; and Cho etal., 1984). Factor IX has been 3 63 identified as the least reactive and most specific of the blood clotting serine proteases with regard to these synthetic substrates. In addition to the problem of low reactivity of the substrates, their limited size may hinder demonstration of altered substrate binding by factor IXy a n c o u v e r- It is possible that less direct, two stage assays, in which the activation of factor X by factor LX is followed with chromogenic substrates of factor X , could be used to study the effect of the factor IX.. a J Vancouver mutation (Griffith et al., 1982; Suomela et al., 1977). Several synthetic and natural inhibitors of factor LX have been reported (Rosenberg et al., 1975; Kurachi et al., cl 1976; Lollar and Fass, 1984; and Turner et al., 1986) which can be used in the identification or quantitation of the factor IX a active site. A detailed analysis of the interaction of factor IX,. with its substrate may thus Vancouver J not be feasible until more reactive synthetic substrates are prepared or until factor IX cl is crystallized for X-ray diffraction studies. Indirect lines of investigation, however, are open. This mutation will be of interest to enzymologists due to the fact that few mutations in serine proteases have been reported. Site directed mutagenesis of the homologous amino acid in trypsin, chymotrypsin or elastase may allow testing of the possibility of altered substrate binding. Since it is easier to solve the structures of molecules differing by only one residue from a known structure, the exact structures of the mutant molecules could be determined. Kinetic studies of normal and altered molecules could be performed if suitable substrates are available. 6 4 6. Summary A nucleotide alteration has been identified in the factor LX gene isolated from patient SB LX-4. The alteration changes an amino acid in the serine protease subunit of factor LX . Since this is the only observed departure from the normal sequence of the protein coding region of the gene, it is likely to be the molecular defect responsible for the decrease in specific coagulant activity of factor IX in this hemophiliac. A three dimensional model of human factor LX , which is based on its homology to the pancreatic serine proteases, shows that the altered amino acid is very close to the active site. The change from an isoleucine side chain, which cannot participate in hydrogen bonding, to a threonine side chain, which carries a hydrogen bonding ligand, could disrupt the normal hydrogen bonding between factor LX and its substrate, factor X. This disruption could result in the reduced specific activity observed in the SB LX-4 factor LX a molecule. In accordance with standard practice, the mutation has been given the name factor LX Vancouver* 65 REFERENCES Anson, D.S., Choo, K.H., Rees, D.J.G., Giannelli, F., Gould, K., Huddleston, J.A. and Brownlee, G.G. (1984). 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