From www.bloodjournal.org by guest on November 18, 2014. For personal use only. 1995 86: 2632-2641 Detection and characterization of seven novel protein S (PROS) gene lesions: evaluation of reverse transcript-polymerase chain reaction as a mutation screening strategy CJ Formstone, AI Wacey, LP Berg, S Rahman, D Bevan, M Rowley, J Voke, F Bernardi, C Legnani and P Simioni Updated information and services can be found at: http://www.bloodjournal.org/content/86/7/2632.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved. From www.bloodjournal.org by guest on November 18, 2014. For personal use only. Detection and Characterization of Seven Novel Protein S ( P R O S ) Gene Lesions: Evaluation of Reverse Transcript-Polymerase Chain Reaction as a Mutation Screening Strategy By Caroline J. Formstone, Adam I. Wacey, Lutz-Peter Berg, Salman Rahman, David Bevan, Megan Rowley, Jennifer Voke, Francesco Bernardi, Cristina Legnani, Paolo Simioni, Antonio Girolami, Edward G.D. Tuddenham, Vijay V. Kakkar, and David N. Cooper The molecular genetic analysis of protein S deficiency has been hampered by the complexity of the protein S (PROS) gene andby the existence of a homologous pseudogene. In an attempt t o overcome these problems, a reverse transcript-polymerasechain reaction (RT-PCR) mutation screening procedure was developed. However, the application of this mRNA-based strategy t o the detection of gene lesions causing heterozygous type I protein S deficiency appears limited owing to the high proportionof patients exhibiting absence of mRNA derived from the mutation-bearing allele (“allelic exclusion“). Nevertheless,this strategy remains extremely effective for rapid mutation detection in type 11/111 protein S deficiency. Using the RT-PCR technique, a G-to-A transition was detected at position + l of the exon IV donor splice site, which was associated with type I deficiency and resulted in bothexon skipping and cryptic splice site utiliration. No abnormal protein S was detected in plasma from this patient. A missense mutation (Asn 217 t o Ser), which may interfere with calcium binding, was also detected in exon Vlll in a patient with type 111 protein S deficiency. A further three PROS gene lesionswere detected in three patients with type l deficiency by direct sequencing of exon containing genomic PCR fragments: a single base-pair (bp] deletion in exon XIV, a 2-bp deletion in exon VIII, and a Gto-A transition at position-1 of the exon X donor splice site all resulted in the absence of mRNA expressed from the disease allele. Thus, the RT-PCR methodology proved effective for further analysis of the resulting protein S-deficienl phenotypes. A missense mutation (Met570t o Thr) in exon XIV of afurther type Ill-deficient proband was subsequently detected in this patient‘s cDNA. No PROS gene abnormalities were found in the remaining four subjects, three 01 whom exhibited allelic exclusion. However,the father of one such patient exhibiting allelic exclusion was subsequently shown to carry a nonsense mutation (Gly448to Term) within exon XII. 0 1995 by The American Society of Hematology. T tinguished phenotypically”: Type I deficiency is characterized by reduced total and free antigen levels together with reduced anticoagulant activity. In type I1 deficiency, protein S activity is reduced, although total and free antigen levels are normal, whereas in type I11 deficiency, the total protein S antigen level isnormalbut free protein S antigen and activity are reduced. The diagnosis and classification of protein S deficiency are complicated both by the considerable overlap in protein S antigen levels (free and total) between protein S-deficient heterozygotes and normal controls” and by the unreliability of protein S assays (antigen and activity). The effect of oral anticoagulant treatment and the existence of acquired protein S deficiency also contribute to diagnostic uncertainty.” The study and characterization of mutations underlying protein S deficiency promise to allow the construction of a rational classificatory system as well as improvement of diagnostic procedures. Previously reported “small” protein S (PROS) gene lesions have been identified by polymerase chain reaction (PCR)-direct sequencing of exon-containing DNA fragments.””* Such a strategy is rather laborious on account of the structural complexity of the PROS gene (15 exons) and complicated technically owing to the existence of a homologous pseudogene that exhibits 97% similarity to the PROS In this report, we describe seven novel PROS gene lesions, detected using a combination of two mutation screening strategies: (1) PCWdirect sequencing and (2) an alternative mRNA-based strategy. HE IMPORTANT REGULATORY role of protein S in hemostasis is reflected by the association of inherited protein S deficiency with recurrent venous thrombosis.’ This vitamin K-dependent protein serves as a cofactor to activated protein C (APC), enhancing the rate of inactivation of factors Va and VIIIa in the coagulation ca~cade.’.~ Protein S synthesis has been shown in a number of tissues, including megakaryocytes and liver.637 Protein S occurs in two forms: approximately 60% of protein S in plasma is noncovalently complexed with C4bbinding protein (C4bBP), a regulatory protein from the classical complement pathway.* The remainder (40%) is unbound (‘ ‘free”). The interaction of functional free protein S with C4bBP abolishes the former’s anticoagulant activity.’ Three types of inherited protein S deficiency have been disFrom Charter Molecular Genetics Laboratory, Thrombosis Research Institute; the Haematology Department,St Georges Hospital: Haemostasis Research Group, Clinical Sciences Centre, Hammersmith Hospital, London; the Haematology Department, Luton and Dunstable Hospital, Luton, UK; Centro di Studi Biochimici della Patologie del Genoma Umano, Universita di Ferrara, Ferrara; Servizio di Angiologia e Malattie della Coagulazione, Ospedale San Orsola, Bologna; and Istituto di Semeiotica Medica, Universitci di Padova, Padova, Italy. Submitted January 17, 1995; accepted June 5, 1995. Supported by the Thrombosis Research Trust and Charter plc. Address reprint requests to David N. Cooper, PhD, Institute of Medical Genetics, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN. UK. The publication costsof this article were defrayedin part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1995 by The American Society of Hematology. 0006-4971/95/8607-0029$3.00/0 2632 MATERIALS AND METHODS Patients Initial laboratory diagnosis of protein S deficiency was made bq assay of antigen levels (total and free) using a standard enzyme. Blood, Vol 86, No 7 (October l ) , 1995 pp 2632-2641 From www.bloodjournal.org by guest on November 18, 2014. For personal use only. MOLECULARGENETICS A 2633 OF PROTEIN S DEFICIENCY C B 0& @ k i i x O ~ ~ & * ~ t : T-14% @ *b * T-70% F-16% D M E 1- - AG F-30% T-74% F-%% A49% F-18% , *Il"o J- / G 6 F-13% F * * IA QE?% AG - ! ~ 6 ~ F-B% AG A allele lost 66 G allele lost "p' *!IOJpTfg A allele lost T-64% F-35% H G allele &C M [ 1c : GF-1 Fallele M ~ I I%"JLJ" oflp I% %g%"2% I w p u up^ I0 Gg$bTb h' F - w + F-l6 A-58% F-50% NT ._. AG T-B% " i t l F 4 9 % F49% A49% wi"4"s0 I * F 0 0 ;:?:%bp F?? or E:00 T-13& F-S% T-75% - F-10% T-21% < l296 F- GT-37% allele lost F-24% F-10% AG F-11% 0666 O W bb6 G allele lost F-=% F-17% M F-%% F-11% AG Fig 1. Family pedigrees of patients 1 through 10 (A through J, respectively). Pedigree (B) has been published previously.m Semi-shaded symbols denote clinically affected individuals. T, total protein S antigen. F, free protein S antigen. A, protein S activity. *Proven carrier of mutation. (0)Proband studied by RT-PCR, PCRldirect sequencing of PROS gene exons, and Pro626 RFLP analysis ofplatelet cDNA. ( ) Patient on anticoagulant therapy. AG denotes heterozygos" at Pro626 locus. AAlGG denote homozygosity at Pro626 locus. NT, not tested by PCRl direct sequencing analysis. Data on allelic exclusion are also provided. C4b-BP levels for family C ware within thenormal range. + linked immunosorbent assay (ELISA, Dako, High Wycombe, UK) and a rabbit polyclonal antibody. Protein S activity was measured using the Stago kit (Diagnostica Stago, Asnitres, France). The hereditary nature of the deficiency state was established by identification of additional family members with abnormally low levels of protein S antigen (total and/or free) and activity. Twenty probands with an inherited protein S deficiency and a personal and/or family history of venous thrombosis were initially chosen for study. Ten of these were studied in detail (Fig 1). Plasma Blood was collected in a tube with 1/10 v01 3.8% sodium citrate and centrifuged at 2,OOOg for 20 minutes. The plasma was stored at -80°C until use. Pooled normal plasma was prepared from eight normal individuals. Strategies for Mutation Screening and Analysis Southern blot analysis. Southern blotting was performed as described." DNA was restricted with Sst I (Promega, Southampton, UK) as recommended by the manufacturer. Bands homologous to the PROS gene and its pseudogene were visualized using a full- lengthPROS cDNA probe (gift from P.H. Reitsma, Leiden, The Netherlands). Pro626 RFLP analysis. Exon XVi3 and cDNA fragment PS9 (Table 1) were PCR amplified as described and digested with BstXI (Boehringer Mannheim, Mannheim, Germany) according tothe manufacturers' instructions. Restriction products were visualized as for PCR fragments. The exon XV PCR fragment migrated at 415 bp.BsfXI restriction of an exon XV PCR fragment carrying the Pro626 CCA allele yielded restriction fragments of 180 bp and 225 bp. An exon XV PCR fragment carrying the Pro626 CCG allele was unaltered by BsfXI restriction and remained at 415 bp. Similarly for PS9, the PCR fragment migrated at 180 bp. BstXI restriction of a CCA-bearing PCR fragment yielded fragments of 130 bp and 50 bp. Reverse Transcriptase-PCR (RT-PCR) Platelets from protein S4eficient patients were separated from whole blood" and RNA extracted from these ~ e l l s . Specific ~ ~ . ~ ~reverse transcription of platelet mRNA was performed using the Superscript RNase H RT kit (GIBCO-BRL, Middlesex, UK). Two distinct cDNA templates were transcribed (PROS-specific primers 15B and RevII; Fig 2, Table 1) from mRNA to avoid the possibility of ineffi- From www.bloodjournal.org by guest on November 18, 2014. For personal use only. 2634 FORMSTONE ET AL Table 1. Oligonucleotide Primers for RT of PROS cDNA, Amplification of First- (PSA-C) and Second-Round (PSI-9)PCR Fragments, and Amplification Of the Donor Splice Siteof Exon 4, Exon 14, and the5' Region of the Human PROS Gene otide Sequence Oligonucleotide Primer Fragment or cDNA(i) cDNA(ii) A B C PS1 PS2 PS3 PS4 PS5 PS6 PS7 PS8 PS9 4 PSPl5 14 "C 5' Rev II 5' AACTGCTCCGCCAAGTAA 3' 15Bt 5' GATAGCAAGAGAAGTAAGAATTTC 3' PSlNP CGCCTCCGCGCCTTCGAA -18 3' PS3A 5' AGCTGTGCCACAAATGCTTGG 3' PS3 5' CCATTCCAGACCAGTGTAGT 3' PS6 5' ATCCATCTAGACGAGGGT 3' PS6A 5' TGGCGGAGCAGTTTGCAGG 3' ATAAGCAGAGAAAAGATGCC Rev 5' end 3' PSlNP 5' CGCCTCCGCGCCTTCGAA3' PSlA 5' AATTTGCACGACGCTTCCTA 3' PSZN 5' TTCCCGTCTCAGAGGCAAAC 3' PS2A 5' TTCTCCTTGCCAACCTGGTT 3' PS3A2 5' CTAAGAAGCTGTGTCAATG 3' PS3N TCAAAGAGCATKATCCACAT 5' 3' PS4A 5' AGTTACCACTGTTCCTGTAA 3' PS4N GTTCAAGGGAAGGCACACTG 5' 876 3' PS5N 5' GCTCAGCTTTGTGTCAATT 3' PS5A 5' GTGATCGATAGATTCTGCGT 3' PS6A 5' TGGCGGAGCAGTTTGCAGG 3' PS6 5' ATCCATCTAGACGAGGGT 3' 5' GAAGCTGTGATGGATATA 3' PS7A 5' CAGATGAGATTGTTGATC 3' PS8A 5' CTGGTAACAACACAGTGCCCT 3' PS8 5' TTCACTTCCATGCAGCCATT 3' PS9A 5' TCCATTCAGTGCCACACCAGT 3' ATAAGCAGAGAAAAGATGCC Rev 5' end 2051 3' Exon Splice TAGTTTATATTACCATGG site 5' 3' AH4 5' to ATG PSP23 codon PSP35 PSP43 Exon DC1 DC4 916 to 899 205 to 182t to 1 - 645 to 625 347 to 366 1345 t o 905 t o 2051 to --- 18 to 130 to 111 56 t o 75 456 t o 437 328 to 622 to 540 to to 54 757 to 1038 to 905 to 1345 t o 1204 to 1725 t o 1583 t o 1928 to 1875 to -95P 371V5' TGGAAGTTGTCTTGACCAGT to -391 3' 5' AGGAGACCGCCCGCTCCCAG -160 3' 5' CTATCACCATAGTTCTTCC -514 3' 5' AGACATCCTTCTTCACCA 3' 5' AACTCAAAAGTCACTCTTAA 3' 5' AAATATTATCGGTTTGAT 3' 1328 923 2032 1 346 602 560 857 715 1019 923 1328 1221 1708 1603 1909 1895 to 2032 to 56 to 7 5 t a - 60 50 50 60 62 62 56 52 52 58 58 48 60 -179t' to t o -496$" -240 t o -257$" -53 to -73*b 43 to 60*b 52 49 * Nucleotide numbering of cDNA primers is derived from the published PROScDNA sequence4' except where indicatedtS. Position1 denotes A nucleotide of ATG (Met). t Data from Reitsma et al.13 Numbering of position for genomic DNA primers is relative to (a) the 5' or 3' boundary of the exon 4 invariant GT of the donor splice site (b) the 5' or 3' boundary of the donor or acceptor splice sites of exon 14, respectively, and (c) theATG translation initiation codon. * cient reverse transcription whichwouldleadtothe production of less than full-length cDNA species. RevII cDNA comprised only exons I to IX of the gene (Fig 2). Two successive rounds of PCR amplification (Fig 2) were used to generate nine overlapping cDNA fragments, which together spanned the entire PROS gene coding region. PCR primers were specifically chosen to allow identification of aberrant mRNA splicing involving any one of the PROS gene exons. The three large overlapping first-round PCR cDNA fragments PSA, B, and C (Fig 2) were used as templates to generate the smaller second-round fragments PSI-3, PS4,5, and PS6-9, respectively. PSA was amplified using RevII cDNA as template. PSB and C were bothamplified from cDNA generated using primer 15B. The primer sequences used and their locations are presented in Table I . PCR was performed in a total volume of 100 pL containing template DNA (typically 3 pL of reverse transcription reaction [cDNA products derived from 2 pg RNA] or I to 5 pL first-round PCR product), 500 ng appropriate primer, 100 p m o L each dNTP (Pharmacia, Uppsala, Sweden), 2 U Tuq DNA polymerase (Promega), and Tuq polymerase buffer (1.5 mmoVL MgCI2; Promega). PCR reactions were performed using either a Perkin Elmer-Cetus (Beaconsfield, UK) or Hybaid (Teddington, UK) Omnigene thermal cycler. Forty cycles of PCR (denaturation at 94°C for 1 minute, annealingat the appropriate temperature for I minute,and extension at 72°C for I minute) were then followed by a final elongation step at 72°C for 4 minutes. Templates for amplification of PSA and PSI were initially denatured at 99°C for 15minutes, followed by 2 minutes at 94°C. At this point, dNTPs and Tuq DNA polymerase were added. Annealing temperatures for each PCR reaction are presented in Table 1. Second-round PCR fragments were visualized byUV transillumination of ethidium bromide-stained agarose gels (2%). Purification ofPCR products was performed using Geneclean I1 (BiolOl, Vista, CA). Purified PCR fragments were sequenced using either the Sequenase kit (Amersham, Amersham, UK) or a cycle sequencing system (GIBCO-BRL). For cycle sequencing, PCR products were purified by excision from a 1 % low-melting point agarose gel and then added directly to the sequencing mix. PCWdirect sequencing of exon-containing DNA fragments. Genomic DNA was extracted from whole blood*' and DNA amplification was performed essentially as described." PCR amplification From www.bloodjournal.org by guest on November 18, 2014. For personal use only. MOLECULARGENETICS UT PRE nR TSR EGF PWGLP, I a n ~ I II 111 IV 2635 OF PROTEIN S DEFICIENCY WJT SHBG II Ill IV a n v VIIVlll VI c b a n x IX XI e d ~ Q f a ~ m m Rev 1 I PS A 160 PS c FIRST ROUND PCR PS B l48 380 295 ~ xv XI1 XIV Xlll of factor X (A. Tulinsky, personal communication, November 1994) as described previo~sly?~Residues (311120.5 (E205) and Am217 (N217) were rotamerized to reproduce the geometry of the analogous calcium-binding side-chain carbonyl groups of Gln49 (449) and Hya 63 (P-hydroxylated Asp) of the first EGF domain of factor X. A calcium ion was then introduced and orientated into the putative binding pocket of the model. ~ n r r 346 -__ - _ ROUND 336 253 U1 522 PS2 PS3 PS. PS5 PS6 SECOND PS, ~ 175 " RESULTS PCR PS1 a PS8 PS9 Southern blot analysis of genomic DNA derived from the 20 protein S-deficient patients under initial study failed to Fig 2. Schematic representation of the RT-PCR strategy for the show any gross abnormalities of the PROS gene. detection of mutations in the PROS gene; shown are the location of primers used for cDNA synthesis and the products of first-(PS A In an attempt to avoid the laborious and technically probthrough Cl andsecond-round IPS 1 through 91 PCR amplification. lematic method of screening each individual exon of the The structure of the human protein S (PROSI gene coding region, PROS gene, a two-step mRNA-based protocol was devised comprising exons I through XV, is shown: 5'UT. 5' untranslated reand used to screen the 20 protein S-deficient patients. Table gion; PRE, prepeptide; PRO, propeptide; G M , Gla domain; HR. hydrophobic region; TSR, thrombin-sensitive region; EGF, epidermal 2 (patients 1 , 2, 3, and 8) summarizes the results of this growth factor-like modulesI through W ; SHBG, sexhormone binding analysis. globulin-like domains athrough g; 3'UT, 3' untranslated region. (I) Positions of alternative primers for cDNA synthesis. Fragment sizes are given in bp. First-Stage RT-PCR of overlapping DNA fragments encompassing DNA sequence 515 bp upstream of the translation initiation codon (ATG) was performed using primers PSP35, PSP43 and PSP15, PSP23 (Table 1). DNA template was initially denatured as for fragments PSA andPS1 above. Exons IV and XIV were amplified using primers PS4-5I3 and AH4 and DC1 and DC4 (Table l), respectively. Annealing temperatures were as shown in Table 1. PClUdirect sequencing was performed as described for RT-PCR. Mutations detected were confirmed by direct sequencing of the other strand. Screening for factor V Leiden. APC resistance assays and screening for the common factor V Leiden lesion (CGA to CAA converting Arg506 to Gln) were performed essentially as de~cribed.~'~" Immunoblot Analysis of Protein S Protein S was partially purified from plasma essentially as in the method of Friedberg and Pizzo2'except that 20 mmoVL benzamidine (Sigma, Poole, Dorset, UK) was included in BaCI, precipitation and washing procedures. The washed pellet was dissolved in 0.1 molL Tris-HCI, 0.1 mom EDTA, 20 mmoVL benzamidine (pH 7.3, and dialyzed overnight against 50 mmol/LTris-HC1, 0.1 m o a NaCl ( ~ H 7 . 3 Purified . ~ ~ protein S (Calbiochem, Nottingham, UK) and both pooled control and patient BaClz precipitated protein S fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)30using a 10% separating gel and transferred to nitrocellulose. Protein S was identified using a monoclonal anti-protein S antibody (HS-4) (Sigma) and the presence of abnormal protein S was evaluated using a polyclonal protein S antiserum (Sigma). Molecular Modeling of the Fourth EGF-like Domain of Protein S Molecular modeling of the fourth EGF-like domain of protein S was performed on a Silicon Graphics Indigo Elan R4000 (Mountain View, CA) using the BIOSYM software suite (BIOSYM Technologies Inc, San Diego, CA) and Ribbons 2.5 (M. Car~on).~' Template structures were derived from the calcium-binding residues of the factor X first EGF-like domain (PDB accession number 1CCF32) and the serine proteinase binding region of the second EGF domain Nine overlapping PROS cDNA fragments PS1-9 (Fig 2) were PCR amplified individually. For 18 of the 20 individuals, PS1-9 were of the expected size (380 bp). However, for patient 1 two abnormally sized PS2 fragments of 428 bp and 294 bp were observed in addition to the 380-bp wild-type fragment (Fig 3, tracks 2, 3, and 4). Some variation in the extent of amplification from different PSA templates of the smaller 294-bp PS2 fragment, and occasionally of the wildtype PS2 fragment, wasnoted. However, an intense PS2 band at 428 bp was consistently observed irrespective of the fragment A template used. PS2 amplified directly from patient l Rev11 cDNA confirmed that this larger abnormal fragment at 428 bp was the predominant aberrant mRNA species (Fig 3, track 5). The smaller 294-bp PS2 band was detected at a significantly lower level than either the wild-type band or the larger abnormal band. Second-round PCR amplification of fragment PS3 showed the existence ofan aberrant Table 2. Summary of Mutations Detected in 11 Protein S-Deficient Individuals Mutation Screening Strategy RT-PCR Patient 1 2 3 492 + 1 G + A AAT AGT Asn217 + Ser + ? 1908 del AC (Thr547/Pro548) ACG (Met570 Thr) 1301 - l G + A 927 del G (Gly220) 4* 5 ATG 6' 7* ' 8 9* 9a 10' Exon Sequencing Insertion in intron 3? 4 + ? ? GGA 4 TGA (Gly448 + Term) ? Mutations are designated by reference to the PROSgene sequence presented by Schmidel et alZoaccording to the nomenclature recornmended by Beaudet and Tsui." Patient 9a is the father of patient 9. * Exhibit allelic exclusion. From www.bloodjournal.org by guest on November 18, 2014. For personal use only. FORMSTONE ET AL 2636 1 2 3 4 5 PS3 band at 343 bp in addition to the wild-type (Fig 3, tracks 6 and 7). PCWdirect sequencing of the two aberrant PS2 cDNA fragments from patient 1 showed the loss of the 87-bp exon IV from the smaller 294-bp fragment and inclusion of an extra 48 bp of DNA sequence from the 5’ end of the intervening intron D, interposed between exons IV and V (data not shown). Inspection of the sequence of the donor splice site of exon IV within the 428-bp PS2 fragment showed a heterozygous G to A transition within the invariant GT dinucleotide. The abnormal PS3 fragment was also found to contain a 48-bp intronic sequence identical to that observed in PS2 and inserted at the same position between exons IV and V (data not shown). The sequences of the normal-sized PS2 and PS3 fragments were identical to that of the wild-type. Confirmation of this putative PROS gene lesion was obtained by PCWdirect sequencing of genomic DNA from patient 1 across the donor splice site region (data not shown). Inspection of the published PROS gene sequence” indicated the presence of a cryptic splice site (taggtaaa) in intron D, 48 bp 3’ to the normal donor splice site. The first three bases of this cryptic splice site (tag) were present at the junction between the inserted intronic sequence and exon V in both the aberrant 428-bp and 343-bp PS2and PS3 fragments, respectively. This PROS gene lesion cosegregated with the type I protein S-deficient phenotype in the family (Fig IA). PS2 amplification from a second patient (patient 8, Fig IH) also showed an abnormal band of slightly larger size than wild-type (data not shown). PCWdirect sequencing of this aberrant PS2 fragment showed the presence of a stretch of DNA sequence between exons 111 and IV, the origin of which remains unclear. The molecular basis of this rearrangement and its possible causative role in the deficiency state are under further study. Second-Stage RT-PCR The second step in the RT-PCR mutation screening strategy used direct sequencing of nine PCR-amplified overlapping cDNA fragments. However, expression of both PROS mRNA alleles is a requirement for successful mutation detection using this approach. To assess allele-specific mRNA 6 7 Fig 3. Agarose gel (2%) of PS2 (tracks 2 through 5) and PS3 (tracks 6 and 7) cDNA fragments. cDNA synthesis was performed using Redl primer. Firstround PCR primers were PSlNP and PS3A. Primers for PS2 and PS3 fragment PCR amplification were PS2, PS2N and PS3N. PS3A2, respectively. Track 2 contains a PS2 fragment of the expected size (380 bp). Tracks 3 and 4 are second-round PS2fragments amplified from different patient 1 fragment A templates and contain aberrantly sized DNA fragments. Track 5 also shows PS2 PCR fragments amplified directlyfrom patient 1 Rewil cDNA. Tracks6 and 7 show fragment PS3 amplified from control and patient 1 fragment A, respectively. For complete explanation, see text. Track 1 is a d, X174/Hadll size marker. Numbers for size markers are 1353, 1078, 872, 603, 310, 281, and 271 bp when reading down from the top of the gel. expression in each of the 20 protein S-deficient individuals, a silent restriction fragment length polymorphism (RFLP) at Pro626 in exon XVof the PROS gene“4 wasused. This variant represents the only reported RFLP within the PROS coding region. However, only eight probands were heterozygous for the Pro626 RFLP at the genomic DNA level (patients 2 through 4, 6 through 10; data not shown). Of these eight probands, only two (patients 2 and 3) were heterozygous at the cDNA level, ie, they exhibited mRNA expression from both PROS alleles (mRNA analysis for patient 2 has been reported p r e v i o ~ s l y ~The ~ ) . other six individuals exhibited loss ofmRNAfrom one allele (“allelic exclusion”). Analysis of platelet cDNA from five normalcontrols heterozygous for the CCA/CCG polymorphism indicated that the two alleles were normallyand codominantly transcribed (data not shown). Patients 2 and 3 were subsequently analyzed by second-stage mutation screening. PCWdirect sequencing of fragment PS4 amplified from patient 2 cDNA showed an AAT to AGT transition within exon VI11 of the PROS gene converting Asn217 to Ser (data not shown). Confirmation of this missensemutation was obtained by PCWdirect sequencing of exon VI11 from patient 2 genomic DNA. This lesion served to create an Mae111 restriction site (recognition sequence, GTNAC). Cosegregation of this lesion with the type 111 protein S deficiency in this familyz4was confirmed by screening 23 family members by Mae111 digestion; eight individuals were found to be heterozygous for the missense mutation. PCWdirect sequencing of the remaining coding sequence of the patient’s PROS genes failed to detect any other mutational lesion. No mutation was detected in the other patient (patient 3). PCWDirect Sequencing of Exon-Containing DNA Fragments mRNA analysis of 20 protein S-deficient patients showed the limited applicability of the RT-PCR technique to the screening of inherited PROS gene lesions. Because PCW direct sequencing of the PROS genes from the remaining 17 patients would clearly have been laborious, only certain individuals were included in this nextphase of mutation screening. Selection was made principally on the basis of From www.bloodjournal.org by guest on November 18, 2014. For personal use only. 2637 MOLECULARGENETICS OF PROTEIN S DEFICIENCY allelic exclusion, since the presence of a PROS gene lesion could be inferred in these patients. The absence of mRNA from one PROS allele was noted in six of the eight probands (patients 4 , 6 through 10; Table 2) with CCNCCG heterozygosity. All six individuals manifested a type I deficiency. One further proband (patient 5 ) was also included in this analysis as part of a family study involving a case of compound heterozygosity. PCR amplification of exons I through XV from patients 4 through 10 generated fragments of the expected size.I3 Table 2 summarizes the results of the subsequent PCWdirect sequencing analysis (patients 4 through 10). Patient 7 exhibited a single base-pair deletion involving a G nucleotide in codon Gly220 (exon VIII). This deletion creates an EcoRII site [t CC(m)GG]. Cosegregation of this lesion with the clinical phenotype in the affected family was confirmed by EcoRII restriction enzyme digestion of exon VI11 PCR fragments from two family members (Fig 1G). A further deletion of an AC dinucleotide, encompassing Thr547 andor Pro548 (exon XIV) of patient 4, was shown in one other affected family member (Fig 1D) by PCWdirect sequencing analysis of exon XIV amplified from the genomic DNA of this individual. Sequence analysis of PS4 and PS8, PCR amplified during the RT-PCR study from the cDNA of patients 7 and 4, respectively, showed the absence, in both individuals, of the deletion-bearing PROS allele (data not shown). A G-to-A transition at position -1 (nucleotide 130lZ0)of the donor splice site of exon X was detected in patient 6 and PCWdirect sequencing was again used to confirm the presence of this lesion in two other affected family members (Fig 1F). PCR amplification of fragment PS6 from patient 6 cDNA, again performed previously as part of the RT-PCR study, had not shown the existence of an aberrant cDNA/ mRNA species. Finally, patient 5 was shown to carry an ATG to ACG transition in exon XIV converting Met570 to Thr. PCWdirect sequencing of cDNA fragment PS8, amplified from patient 5 cDNA during the first stage of the RT-PCR protocol, showed the existence of this missense mutation at the mRNA level. PCWdirect sequencing was used to confirm the presence of this lesion within exon 14 from one other protein Sdeficient family member (Fig 1E). Therefore, patient 5 has inherited a PROS gene lesion from his father not his mother. PCWdirect sequencing of the remaining 14 PROS exons failed to show a further PROS gene mutation. The phase between the disease-associated allele, as determined by Pro626 RFLP analysis of genomic DNA from different family members, and the mRNA species exhibiting allelic exclusion (patients 4,and 6 through 10) was established by reference to the PS9 Pro626 cDNA RELP data (Fig lD, F, G, H, I, and J). In patients 8, 9, and 10, no abnormality within the PROS gene sequence was identified. PCWdirect sequencing of a patient 8 exon 11-containing PCR fragment showed a 3-bp (tct) deletion at nucleotide position 74 bp 5’ to the acceptor splice site of exon I1 [Intron A; 223-74 del TCT]. However, this deletion didnot cosegregate with the protein S deficiency phenotype in the affected family (Fig 1H) and presumably represents a neutral polymorphism. DNA sequence up to 515 bp 5‘ to the PROS transcription initiation sitezo was PCR amplified from the genomic DNA of patients 8, 9, and 10 and sequenced directly. No deviations from the wildtype DNA sequence were detected. For both patients 9 and 10, the CCG PROS gene allele, which was absent at the mRNA level, did not cosegregate with the protein S-deficient phenotype in other family members (Fig l , I and J). PCW direct sequencing of 15 PROS gene exons amplified from the genomic DNA of the father of patient 9 showed the presence of a nonsense mutation (GGA to TGA) within exon XI1 (Table 2, 9a). This Gly448 to Term conversion was consistent with the type I deficiency state in this individual (Fig 11). cDNA analysis was not undertaken for this individual. No further family members were available for genetic analysis. Conversely, the sister and niece of patient 10 (Fig 1J) carried no abnormalities within the PROS gene sequence. Factor V Leiden Screening Because APC r e ~ i s t a n c ecould ~ ~ increase the probability of protein S-deficient individuals coming to clinical attention, the presence of APC resistance in the 10 patients studied in detail was excluded either by screening for the causative factor V Leiden mutation directly (patients 1 and 3 through 10) or by means of the APC resistance assay (patient 2). Analysis of Plasma Protein S From Patient I SDS-PAGE of pure human protein S and BaCl, precipitated fractions of pooled control and patient plasma under reducing conditions was followed by Western blot analysis using a protein S monoclonal antibody (HS-4). A migrating at 86 kD and 76 kD was revealed in all cases (data not shown). A similar pattern was observed with a protein S polyclonal antiserum. No abnormally sized band(s) were detected in patient plasma (data not shown). DISCUSSION Using a combination of two mutation screening protocols, seven novel PROS gene lesions were identified during the detailed study of 10 unrelated protein S-deficient patients. The RT-PCR strategy was originally adopted to avoid the potential problems stemming from the presence of the protein S pseudogene” (this homologous sequence is not transcribed37),and to circumvent the laborious task of sequencing all 15 PROS exons individually. The protocol was devised to allow the identification of intronic mutations that might alter mRNA splicing as well as mutations within the coding sequence of the PROS gene. Indeed, the first PROS gene lesion to be identified, a point mutation that abolished the donor splice site consensus sequence of exon IV, was detected initially by PCR amplification ofan aberrant PS2 cDNA fragment. Thus, the effectiveness of the RT-PCR strategy for the detection of splice site mutations that do not influence the abundance of PROS mRNA was realized early in the analysis. An initial group of 20 protein S-deficient individuals (mainly type I) was chosen for mutation screening analysis. Progressively, however, during the different phases of the From www.bloodjournal.org by guest on November 18, 2014. For personal use only. 2638 screening process, certain individuals were singled out for further detailed study. Obviously, expression of the mutation-bearing allele is necessary for successful mutation detection when using the RT-PCR technique. At present, the Pro626 RFLP provides the only available means to determine allelic heterozygosity at both genomic DNA and cDNA levels, thereby demonstrating the existence of mRNA species fromboth PROS alleles. From our studyof 20 protein Sdeficient individuals, only 8 were found to be heterozygous (CCNCCG) at the Pro626 position. Further reports of informative intragenic PROS RFLPs in linkage disequilibrium with Pro626 should ease the identification of potentialcandidates for mutation analysis using RT-PCR. However, more seriouswas the subsequentdemonstration of the loss of mRNA from one allele for 6 of the 8 CCNCCG heterozygotes (“allelic exclusion”). All six individuals manifested a type 1 deficiency. In a similar study reported by Reitsma et al,” three mutations associated with type I protein S deficiencyweredetected by PCWdirectsequencing of PROS exons, but only one of these lesions was identified using an mRNA-basedmutation screeningprotocol. Theothertwo type I mutations resulted inthe absenceof mRNA transcripts derived from the diseaseallele. Thus, of 7 type 1 PROS gene lesionsreported to date (for whichanassociatedstudyof mRNA abundance had been performed), 5 affect the level of themRNAspecies bearing the PROS mutation.Such a high frequency of allelic exclusion clearly questions the feasibility of using the RT-PCR technique successfully as a screening strategy in type I protein S deficiency. However, using the RT-PCR protocol can provide valuable information on both the biochemical phenotype and the molecularbasis of thetype I protein S deficiencystates studied. For example, thepoint mutation at the invariant + 1 position of theexon IV donorsplice siteresultedin the generationof twoabnormal yetin-frame mRNA species. The predominant mRNA resulted from utilization of a cryptic splice site 48 bp 3’ to the donor splicesite itself, whereas the rarer mRNA species was generated by the skipping of exon IV. Abnormal protein was not detected in plasma from thispatient. The type I deficiency phenotype of patient 1 must therefore result from the inability of protein, translated from either one orboth aberrant mRNA species, to fold into a stable conformation. A mutation at position - 1 within the donor splicesite of exon X (patient 6) resulted in the production of anunstable mRNA. Aberranttranscripts fromthe mutation-bearing allele could not be detected when cDNA from thispatient was analyzed by PCR amplification offragment PS6 (Fig 2). Reitsma et all3 have also reported an exon X donor splice site mutation at position +5 that is associated with decreased mRNA stability. Two short PROS gene deletions resulting in frameshifts were also foundto influence mRNA stability. Thesingle base-pair deletion within exon VI11 (patient 7) gave rise to an in-frame TGA termination codon at amino acid position 250, whereas the 2-bp deletionin exon XIV (patient 4) generated an in-frame TAA termination codon just three amino acids C-terminal to the lesion. Neither lesion was detected when PCWdirect sequencing of second-round cDNA fragments, PCR amplified from patient cDNA during the RT- FORMSTONE ET AL PCR procedure, was performed. This is consistent with the previously observed loss, in patients 4 and 7, of one PROS mRNA allele. In eukaryotes, mRNAs that harbor a nonsense codon often exhibit an abnormally low abundance.3x.3” We report here the loss of stability of PROS mRNA associated with the generation of a termination codon close to the 3’ terminus of the PROS gene coding region. The RT-PCR strategy may still be considered to be extremely effective when screening for lesions causing type I1 and I11 protein S deficiency. Second-stage RT-PCR analysis enabled us to identify rapidly a missense mutation in fragment PS4 (exon VIII) which converted Asn2 l7 to Ser (patient 2). Classification of the protein S deficiency type resulting from this PROS gene lesion had originally proved difficult owing to variation in protein S antigen levels24 exhibited by eight different members of the family. Theirmean protein S activity, free antigen, and total antigen values were 45%, 58%, and 76%,respectively (free andtotal values were incorrectly interchanged in the legend to Fig 1 in the article cited”).By comparison, 15 clinicallyandphenotypically normal family members exhibited mean values of84%. 9 l%, and 89%, respectively, whereas 9 unrelatedsubjects with type I protein S deficiency exhibited protein S activity and free and total antigen values of 25%. 21%, and 4596, respectively. From these data, we may conclude that patient 2 and other affected family members manifest a type 111 protein S deficiency. It is known that protein S contains four very high-affinity calcium-binding sites:” one or more of which have been localized tothe third and fourth Asn217 (N217)-bearing EGF-like domains.Byanalogy with the calcium-binding pockets of the first EGF domain of factors IX and X, the mutated N217 residue is believed to play a significant role in the binding of (Fig The 4). substitution of Asn217 by Ser resultsin theloss of a y-carbonand the replacement of the apical amide and reactive carbonyl group by a less reactive hydroxyl group. The resultant reduction in both steric and polaric contact between the mutated residue and the calcium ion would be expected to lead to loss of calcium bindingaffinity. This proposal remainstobe tested by the biochemicalcharacterization of an in vitroexpressed protein. Previously, the allelic status of the mRNA from patient 5 was unknown; this patient is a Pro626 CCA homozygote. However, patient 5 would have been a candidate for rapid second-stage RT-PCR analysis because the Met570 to Thr missense mutation was detected in his mRNA. Our inability to incorporate this patient into the RT-PCR study highlights the requirement for further informative PROS RFLPs. Because patient 5 is undergoing anticoagulant therapy, we must assume that his protein S-deficient phenotype will resemble that of his father, ie, type 111, who also carries the Met570 PROS gene lesion. Met570 is conserved in both rabbit4’ and bovine protein S4’ and in the human sex hormone binding globulin (SHBG).& Such sequenceconservation merits speculation as to the structural importance of this residue. The amino acid environment of Met570 appears hydrophobic, which suggests thatthis region may beinternal to theprotein. The type I11 deficiency indicates an effect on C4bBPinterac- From www.bloodjournal.org by guest on November 18, 2014. For personal use only. MOLECULARGENETICS OF PROTEIN S DEFICIENCY 2639 A 1 . / F. 1 L Y218 Fig 4. Molecular model of the fourth EGF-like domain of protein S and its putative Ca" binding pocket. (A) The secondarystructure of the fourth EGF domain of protein S. p-Pleated sheets areshown as green arrows, random coil as a blue ribbon, disulphide bridges in gold, and cysteine SG atoms in yellow. The side chainof residue Asn217is a ball and stick representation. (B) Close-up view of the wild-type protein S Ca2+binding pocket. The backboneis represented by a cyan ribbon; residues areshown as stick and are coloredby atom. (C) Close-upview of the mutant protein S C#+ binding pocket. The backboneis represented by a blue ribbon; residues are shown as stick and are colored by atom. (D) Close-up view of the wild-type factor X Ca2+binding pocket. The backboneis represented by a purple ribbon; residues areshown as stick and are colored by atom. The calcium-binding geometry ofthe fourthEGF-like domain has been inferred from NMR analysis of the first EGF domain of factor X.= The backbone carbonyl of Gly47 and Gly64 and the side-chain carbonyl oxygens ofHya63 and Gln49together form a pocket capable of binding calcium (inset D). The replacement of Glu49 and Hya63 of factor X by the analogous Glu205 (E2051 and Asn217 (N2171residues ofprotein S preservesthe location of the apical reactive groups the of side chainwhile rotatingtheir geometry through 180' (inset B). This observation is consistent with experiments performed on the calcium-binding domain of factor IX EGF domain l?' The orientation of the reactive side chains ofN217 and E205 may bring the carbonyl group of N217 into closer contact with thecalcium ion than is observed in theNMR structure of factor X. This may accountfor the increased binding capacity of the protein Winding pocket overthat of factor X. Substitution of Asn217 by Ser results in the loss of a pcarbon and the replacement of the apical amide and reactive carbonyl group by a less reactivehydroxyl group. The resultant reduction in both steric and polariccontact between the mutated residue and calcium ion wouldbe expected to lead to a loss of calcium binding affinity in this pocket consistent with the phenotype observed. tion. Indeed, Lys571 is predicted to interfere with protein S binding to C4bBP." However, further assessment of the phenotypic effectof this mutation must again await the biochemical characterization of an in vitro-expressed protein together with the determination of a crystallographic structure for the SHBG domain of protein S . Our failure to detect PROS lesions in four patients (probands 3, 8, 9, and 10) is intriguing, but not without precedent.I3 Each proband exhibited a family history of protein S deficiency and venous thrombosis (Fig 1, C,H, I, and J). We may speculate from the type E l deficiency exhibited by proband 3 and his family (Fig 1C; normal total antigen and reduced free antigen and activity), that the genetic lesion responsible may be nonallelic, eg, it could reside in one of the genes encoding the two chains of C4bBp47 (C4bBP levels, for all family members analyzed, were found to be within the normal range). However, patients 8, 9, and 10 exhibited the lossof mRNA from one specificPROS allele, which implies the existence of a PROS gene lesion. Firststage RT-PCR showed that patient 8 exhibited a low-level aberrant mRNA transcript. The additional DNA sequence interposed between exons 3 and 4 was of unknown origin. This short stretchof DNA may represent unmapped protein S intronicsequence,ahypothesisthatiscurrentlyunder investigation. The loss of the Pro626 CCG allele in patient 8 (Fig1H)wasinaccordancewiththedemonstrationof From www.bloodjournal.org by guest on November 18, 2014. For personal use only. 2640 FORMSTONE ET AL cosegregation of the CCG allele and the disease phenotype in affected family members. For patients 9 and 10, however, loss of the CCG allele was observed, but the disease phenotype in other family members was found to cosegregate with the CCA allele (Fig 1, I and J). For patient 9, PCWdirect sequencing of his father’s PROS genes showed a nonsense mutation (Gly448 to Term) within exon XI1 that was consistent with this individual’s type I deficiency state (Fig 11). However, the explanation for the lack of concordance in family H is unclear. Platelet mRNA may not be representative of liver PROS mRNA. However, Reitsma et all3 have shown that the abundance of PROS mRNA is comparable in liver, endothelial cells, and blood platelets. Furthermore, analysis of the PS Heerlen polymorphismz9in one informative individual showed codominant transcription of the platelet PROS gene. We have confirmed this observation in five normal individuals by studying the Pro626 polymorphism. This study also provides data that argue against the use of RT-PCR as a method for quantifying mRNA levels, particularly when employing a two-stage analy~is.~’ Patients 1 and 8 exhibited variation in the quantity of aberrant secondstage PCR fragments amplified from patient cDNA and, i n the case of patient 1, variation in PCR amplification of the wild-type fragment. However, such a bias in the amplification of different cDNA templates may prove advantageous in the amplification of low-abundance mRNA species, such as those evident in some cases of allelic exclusion. In conclusion, RT-PCR provides a rapid effective methodology for mutation detection in type I1 and I11 protein S deficiency states, but is inappropriate for screening for inherited type I protein S deficiency. Exon-by-exon sequencing remains the most efficient and reliable screening protocol for type I protein S deficiency. Indeed, our initial argument against the use of exon-by-exon sequencing for reasons of technical complexity has been weakened recentlyby a report describing a homozygous exon I11 PROS gene mutation.” Finally, analysis of mRNA abundance intypeI-deficient patients using the RT-PCR strategy has been shown to be a useful tool for further analysis of the resulting phenotype manifested by the PROS gene lesions identified. ACKNOWLEDGMENT We thank P.H. Reitsma for providing thePROS gene cDNA clone used in these studies and for PROS gene primer sequences, A.M.R. Taylor for collecting normal blood samples, V. Lindo for performing protein S antigen assays on patient 7,P.J. Hallam for factor VLeiden analysis of patient 5, G. Patel and C. Goodwin for advice on plasma protein S analysis, A. Tulinsky for pre-release factor X coordinates (EGFI), and D.S. Millar for critical reading of the manuscript. REFERENCES l . Henkens CMA, van der Meer J, Hillege JL, van der Schaaf W, Bom VJJ, Halie MR: The clinical expression of hereditary protein risk C and protein S deficiency: A relation to clinical thrombotic S? Blood Coag Fibrinol factors and to levels of protein C and protein 4555, 1993 2. Dahlback B: Protein S and C4b-binding protein: Components involved in the regulation of the protein C anticoagulant system. Thromb Haemost 66:49, 1991 3. Heeb MJ, Mester RM, Tans G, Rosing J. Griffin JH: Binding of proteinS to factor Va associated with inhibition of prothrombinase that is independent of activated protein C. J Biol Chem 268:2872, 1993 4. Heeb MJ, RosingJ, Bakker HM, Fernandez JA, Tans G, Griffin JH: Protein S binds to and inhibits Factor Xa. Proc Natl Acad Sci USA 91: 2728, 1994 S. ShenL, Dtihlback B:FactorVandprotein S as synergistic cofactors to activated protein C in degradation of factor VIIIa. J Biol Chem 269: 18735, 1994 6. Schwarz HP: Extra-hepaticsynthesis of protein S. Thromb Haemost 64:333, 1990 7. Maillard C, Berruyer M, Serre CM, DeChavanne M, Delmas PD: Protein S, avitaminK-dependantprotein, is a bonematrix protein component synthesized and secretedby osteoblasts. Endocrinology 130:1.599, 1992 8.DahlbackB,StenfloJ:Highmolecularweightcomplex in human plasma between vitamin K-dependent protein S and complementcomponentC4b-bindingprotein.ProcNatlAcadSci USA 78:2512, 1981 9. Dahlback B: Inhibitionof protein C cofactor functionof human and bovine protein S by C4b-binding protein. J Biol Chem 26 1:12022, 1986 IO. Scientific and Standardization Committee of the International Society of Thrombosis and Haemostasis Subcommittee on protein C and protein S, Munchen, Germany, July 1992. Stuttgart, Germany. Schattauer 11. Engesser L, Broekmans AW, Briet E, Brommer EP, Bertina RM: Hereditary protein S deficiency: Clinical manifestations. Ann Intern Med 106:677, 1987 12. Malm J, Laurel1 M, Dahlback B: Changes in the plasma levels of vitamin K-dependent proteins C andS and of C4b-binding protein duringpregnancyandoralcontraception.BrJHaematol68:437, 1988 13. Reitsma PH, Ploos van Amstel HK, Bertina RM: Three novel mutations in five unrelated subjects with hereditary protein S deficiency type I . J Clin Invest 93:486, 1994 14. Hayashi T, Nishioka J, Shigekiyo T. Saito S, Suzuki K: Protein S Tokushima: Abnormal molecule with a substitution of Glu of for Lys-IS5 in the second epidermal growth factor-like domain protein S. Blood 83:683, 1994 IS. Yamazalu T, Sugiura 1, Matsushita T, Kojima T, Kagami K, Takamatsu J, Saito H: A phenotypically neutral dimorphism of protein S: The substitution of Lys IS5 by Glu in the second EGF domain Res 70:39S, predicted by an A to G base change in the gene. Thromb 1993 16. Borgel D, Gandrille S, Gouault-Heilmann M, Aiach M: First frameshift mutation in the active protein S gene associated with a quantitative hereditary deficiency. Blood Coag Fibrinol 5:593, 1994 17. Gomez E, Ledford MR, Pegelow CH, Reitsma PH, Bertina RM: Homozygous protein S deficiency due to a one base-pair deletion that leads to stopcodon in exon Ill of the protein S gene. Thromb Haemost 7 I :723, I994 I 8. Gandrille S, Borgel D, Eschwege-GuffletV, Aillaud M, Dreyfus M, Matheron C, Gaussem P, Abgrall IF, Jude B, Sie P, Toulon P, Aiach M: Identification of IS differentcandidatecausal point mutationsandthreepolymorphisms in 19patientswithprotein S deficiency using a scanning method for the analysis of the protein S active gene. Blood SS:130, 1995 19. Ploos van Amstel HK , Reitsma PH, van der Logt PE, Bertina RM: Intron-exon organisation of the active human protein S gene PS (Y anditspseudogene PS p: Duplicationandsilencingduring primate evolution. Biochemistry 29:7853, 1990 20. Schmidel DK, Tatro AV, Phelps LC, Tomczak JA, Long GL: Organisation of the human protein S genes. Biochemistry 29:7845, 1990 From www.bloodjournal.org by guest on November 18, 2014. For personal use only. MOLECULARGENETICS OF PROTEIN S DEFICIENCY 2 I . Southern EM: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol98:503, 1975 22. Ginsberg D, Konkle BA, Gill JC, Montgomery RR, Bockenstedt PL, Johnson TA, Yang AY: Molecular basis of human von Willebrand disease: Analysis of platelet von Willebrand factor mRNA. Proc Natl Acad Sci USA 86:3723, 1989 23. Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156, 1987 24. Marchetti G, Legnani C, Patracchini P, Gemmati D, Ferrati M, Palareti G, Coccheri S, Bernardi F: Study of a protein S gene polymorphism at DNA and mRNA level in a family with symptomatic protein S deficiency. Br J Haematol 85:173, 1993 25. Kunkel LM, Smith KP, Boyer SH, Borganokar DS, Wachtel SF, Miller OJ, Boseg WR, Jones HW, Rory RM: Analysis of human Y-chromosome-specific reiterated DNAin chromosome variants. Proc Natl Acad Sci USA 74:1245, 1975 26. Bertina R M , Koeleman BPC, Koster T, Rosendal FR, Dirren RI, de Ronde H, van der Velden PA, Reitsma PH: Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 36954, 1994 27. Koster T, Rosendaal FR: Activated protein C resistance in venous thrombosis. Lancet 342: 1503, 1994 28. Friedberg RC, Pizzo SV: Large scale purification of factor X by hydrophobic chromatography. Prep Biochem 18:303, 1988 29. dertina RM, Ploos van Amstel HK, van Wijngaarden A, Coenen J, Leemhuis MP, Deutz-Terlouw PP, van der Linden IK, Reitsma PH: Heerlen polymorphism of protein S, an immunologic polymorphism due to dimorphism of residue 460. Blood 76:538, 1990 30. Laemli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680, 1970 3 1. Carson M: Ribbon models of macromolecules. J Mol Graphics 5:103, 1987 32. Selander-Sunnerhagen M, Ullner M, Person E, Teleman 0, Stenflo J, Drakenberg T: How an epidermal growth factor (EGF)like domain binds calcium. J Biol Chem 267:19642, 1992 33. Wacey AI, Krawczak M, Kakkar VV, Cooper DN: Determinants of the factor IX mutational spectrum in haemophilia B: An analysis of missense mutations using a multi-domain molecular model of the activated protein. Hum Genet 94:594, 1994 34. Diepstraten CM, Ploos van Amstel HK, Reitsma PH, Bertina RM: A CCNCCG neutral dimorphism in the codon for Pro 626 of thehuman protein S gene PS alpha (PROS1). Nucleic Acids Res 195091, 1991 35. Svensson PJ, Dahlback B: Resistance to activated protein C is a basis for venous thrombosis. N Engl J Med 330:517, 1994 2641 36. Dahlback B: Purification of human vitamin K-dependant protein S and its limited proteolysis by thrombin. Biochem J 209:837, 1983 37. Ploos van Amstel HK, Diepstraten CM, Reitsma PH, Bertina RM: Analysis of platelet protein S mRNA suggests silent alleles as a frequent cause of hereditary protein S deficiency type I. Thromb Haemost 659308, 1991 38. Belgrader P, Maquat LE: Nonsense but not missense mutations can decrease the abundance of nuclear mRNA for the mouse major urinary protein, while both types of mutation can facilitate exon skipping. Mol Cell Biol 14:6326, 1994 39. Cheng J, Belgrader P, Zhou X, Maquat LE: Introns are cis effectors of the nonsense-codon-mediated reduction in nuclear mRNA abundance. Mol Cell Biol 14:6317, 1994 40. Dahlback B, Hildebrand B, Linse S: Novel t y F of very high affinity calcium-binding sites in 8-hydroxyasparagine-containing epidermal growth factor-like domains in vitamin K-dependent protein S. J Biol Chem 265:18481, 1990 41. Handford PA, Mayhew M, Baron M, Winship PR, Campbell ID, Brownlee GG: Key residues involved in calcium-binding motifs in EGF-like domains. Nature 351:164, 1991 42. He X, Dahlback B: Molecular cloning, expression and functional characterization of rabbit anticoagulant vitamin K-dependent protein S. Eur J Biochem 217:857, 1993 43. Dahlback B, Lundwall A, Stenflo J: Primary structure of bovine vitamin K-dependent protein S. Proc NatlAcad Sci USA 83:4199, 1986 44. Gershagen S, Fernlund P, Lundwall A: A cDNA coding for humansex hormone binding globulin. Homology to vitamin Kdependant protein S. FEBS Lett 220:129, 1987 45. Greengard JS, Fernandez JA, Radtke K-P, Griffin JH: Identification of candidate residues for interaction of protein S with C4b binding protein and activated protein C. Biochem J 305:397, 1995 46. Hillarp A, Pardo-Manuel F, Ramos Ruiz R, Rodriguez de Cordoba S, Dahlback B: The human C4b-binding protein 8-chain gene. J Biol Chem 268:15017, 1993 47. Femandez JA, Griffin JH: A protein S binding site on C4bbinding protein involves 8-chain residues. J Biol Chem 269:2535, 1994 48. Slomski R, Schlosser M, Berg L-P, Wagner M, Kakkar VV, Cooper DN, Reiss J: Omission of exon 12 in CFTR gene transcripts. Hum Genet 89:615, 1992 49. Hoskins JA, Norman DK, Beckmann RJ, Long GL: Cloning and characterisation ofhuman liver cDNA encoding a protein S precursor. Proc Natl Acad Sci USA 84:349, 1987 50. Beaudet AL, Tsui L-C: A suggested nomenclature for designating mutations. Hum Mutation 2:245, 1993
© Copyright 2024