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From www.bloodjournal.org by guest on June 18, 2017. For personal use only. Type I Factor XI11 Deficiency Is Caused By a Genetic Defect of Its b Subunit: Insertion of Triplet AAC in Exon I11 Leads to Premature Termination in the Second Sushi Domain By Tornonori Izurni, Teruto Hashiguchi, Giancarlo Castaman, Alberto Tosetto, Francesco Rodeghiero, Antonio Girolarni, and Akitada lchinose Factor Xlll deficiency has been classified into two categories: type I deficiency, characterized by the lack of both thea and b subunits; and type II deficiency, characterized by the lack of the a subunit alone. To clarify the genetic bases of these diseases, previously reported cases of the type I deficiency were examined at the DNA level. DNA sequence analysis showed that a nucleotide triplet (AAC) was inserted within the codon for Tyr-80 in exon 111 of the gene for a female proband‘s b subunit, resulting in the creation of a stop codon. Restriction digestion of amplified DNAs confirmed that the proband and hersister were homozygotes, and their family members were heterozygotes of this mutant allele. A truncated protein composed of 79 amino acids could be synthesized by these homozygotes; however, such aprotein would not besecreted or it would degrade quickly, because there were normal amounts of the mutant mRNA, but no b subunit was detected in these patients. The a subunit deficiency of these patients must be secondary to theb subunit deficiency, as their gene for the a subunit was intact, and the a subunit in their platelets was present within normal levels. 0 1996 by The American Societyof Hematology. F a change of Cys430 to Phe in the seventh Sushi domain causes impaired transport of the mutant b subunit, resulting in its deficiency.” In this study, to clarify the genetic basis of type I factor XI11 deficiency, we examined the two original patients6.*’ at the DNA level and found that they are homozygotes of a defective b subunit allele. Accordingly, a new genetic classification for factor XI11 deficiency is proposed. ACTOR XI11 deficiency is an inherited hemorrhagic disease characterized by a life-long bleeding tendency and abnormal wound healing in affected patients and spontaneous abortion in affected fernales.l~*Coagulation factor XI11 is a plasma transglutaminase consisting of two catalytic a and two noncatalytic b s~bunits.’.~.~ The a subunit contains an active site region of transglutaminases, and the b subunit is composed of 10 tandem repeats called “Sushi domain^"^ because of their shape, or “short consensus repeats (SCRs)”, which are also called “GP-l structures” because they were identified first in P*-glycoprotein I. Factor XI11 activated by thrombin catalyzes intermolecular cross-linking reactions between various proteins in plasma and an extracellular matrix. Thus, deficiency of factor XI11 results in defective crosslinking reactions. More than 200 cases of factor XI11 deficiency have been identified. Its frequency is calculated to be one in 10,000.* In most cases, the diagnosis for factor XI11 deficiency is made by the measurement of factor XI11 activity, which represents the amount of the functional a subunit. It had been proposed by Girolami et aP7that factor XI11 deficiency is to be classified by the presence or absence of antigens into two groups: type I deficiency, characterized by the lack of both a and b subunits, and type I1 by the lack of a subunit alone. This classification canbemadeonlyby a precise immunologic examination employing monospecific antibodies against the a and b subunits, separately. Unfortunately, an exact classification for factor XI11 deficiency has been difficult and confusing because at least one commercial antiserum against the b subunit crossreacted with another plasma protein as reported in 19786and several vials of commercial antibodies have done so during the last 5 years in our laboratories. Thus, some of the patients of real type I deficiency could have been misdiagnosed as having type I1 deficiency by erroneous immunologic measurements. Determination of the genomic sequence for both subunits’”’ has made it possible to characterize factor XI11 deficiency decisively at the DNA level.‘*The several examples of type II factor XIII deficiencyinwhichthemutationis knownl*-“j all result from genetic defects of the a subunit. An inherited quantitative abnormality of the b subunit also leads to factor XI11 deficiency,17in which two genetic mutations in the gene for the b subunit have now been identified.” In this patient, Blood, Vol 87, No 7 (April l ) , 1996: pp 2769-2774 MATERIALSANDMETHODS Study subjects. Venous blood was drawn from normal individuals, two previously reported patients with type I factor XIII deficiency‘,*” and their family members (Fig 1) after informed consent had been obtained. Factor XI11 activity was determined by the standard dansylcadaverine-incorporation assay?’ and the a and b subunits in plasma were measured by enzyme-linked immunosorbent assay (ELISA)?~ In vitro amplification ofthe b subunit gene. Genomic DNA samples were prepared from leukocytes by standard techniques. A total of 1 pg of a genomic DNA was amplified in a 100-pL reaction mixture employing 2.5 to 5.0 U of Thermus aquaticus DNA polymerase (Promega, Madison, W1 or AGS GmbH, Heidelberg, Germany). After 30 cycles of amplification, 9 pL of each reaction mixture was applied to a 0.8% agarose gel containing ethidium bromide From the Department of Molecular Patho-Biochemistry, Yamagata University School of Medicine, Yamagata, Japan; the Department of Haematology, San Bortolo Hospital, Vicenza, Italy; and the Institute of Medical Semeiotics, University of Padua Medical School, Padua, Italy. Submitted May 22, 1995; accepted November 13, 1995. Supported in part by research grants from the Mochida Memorial Foundation (Japan), the Ichiro Kanehara Foundation (Japan), and the Japan Research Foundation for Clinical Pharmacology. Presented in part at the 36th Annual Meeting of the American Society of Hematology, Nashville, TN, December 2-6, 1994. Address reprint requests to Akitada Ichinose, MD, PhD, Department of Molecular Patho-Biochemistry, Yamagata University School of Medicine, Iida-Nishi 2-2-2, Yamagata, 990-23 Japan. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1996 by The American Society of Hematology. 0006-4971/96/8707-0$3.00/0 2769 From www.bloodjournal.org by guest on June 18, 2017. For personal use only. IZUMI ET AL 2770 (0.5 pg/mL) in 1 X TBE buffer (89 mmol/L Tris base189 m m o K boric acid/:! m m o K EDTA, pH 7.8). Oligonucleotides were prepared with an Applied Biosystems’ synthesizer. For amplification, a total of six pairs of gene-specific primers were designed from the nucleotide sequence of the normal 6 subunit gene” as listed in Table 1 of reference 18 with a slight modification; pair 2R for Exons I1 and 111, S’-side, the sense primer of former pair 3’’ and 5”TCAGGATCCTGCATTGTAGACATAATGAAAAATAA-3’ (antisense); pair 3R for Exons 111, IV and V, 5”GATGGATCCAAAATGAAATCGCCAATAATAACATT-3’ (sense) and the antisense primer of former pair 3.” Nucleotide sequence analysis. The amplified DNA samples were digested with restriction enzymes to generate the proper ends for ligation into sequencing vectors. The digested samples were applied to a 0.8% agarose gel, electroeluted, and then subcloned into M13mp18 or M13mp19 (GIBCO-BRL, Gathersburg, MD or Toyobo, Tokyo, Japan) with restriction sites to obtain discrete sequences. The DNA sequence of an insert was then obtained using the dideoxynucleotide method” with deoxyadenosine S’-[a-[’sS]thio]triphosphate (Amersham, Arlington Heights, IL). Tominimize the possibilityof obtaining the DNA sequences with misincorporation by the Taq DNA polymerase, 10 or more samples of each amplified region of the b subunit gene were examined. Detection of the insertion mutation by restriction digestion. For restriction enzyme analysis for the insertion of a triplet AAC in exon 111, 15 pL of the DNA sample amplified by employing the 5”primer (sense) of pair 3R and the 3”primer (antisense) of pair 2R was incubated with 2 U of Mae111 endonuclease (Boehringer Mannheim GmbH, Germany) for 2 hours at 45°C. The amplified DNA also was incubated with 1.S U of Tru91 endonuclease (Boehringer, Mannheim, Germany) for 2 hours at 65°C. After digestion, half of each sample was applied to a 2% agarose gel. Immuno6lotting of the b subunit. For Western blotting of the 6 subunit, plasma was mixed with sample buffer (125 mmoUL TrisHCI, pH 6.8, 4% sodium dodecyl sulfate [SDS], SO% glycerol, and 1 mg/mL bromophenol blue) and deionized water at a ratio of 1:5:4, and then heat-denatured. Samples were electrophoresed on a 10% polyacrylamide gel. The separated proteins were transferred electrophoretically onto a nitrocellulose membrane, and the membrane was blocked at 4°C overnight with the blocking buffer containing 0.5% nonfat dry milk and a washing buffer (1 0 mmol/L Tris-HCI, pH 7.4, and 150 mmolk NaCI). The blocked membrane was incubated at room temperature for 1 hour with rabbit antiserum against the 6 subunit (Calbiochem, La Jolla, CA), rinsed three times, and incubated at room temperature with a goat antirabbit IgG (H + L) conjugated with horseradish peroxidase (Bio-Rad, Richmond, CA). The band for the b subunit was detected by adding diaminobenzidine and H202. Reverse transcription-polymerase chain reaction (RT-PCR) assay of’ aberrant mRNA for the b subunit. Total mRNA was extracted by the standard guanidinium thiocyanate method from leukocytes followed by ultracentrifugation on a cesium chloride gradient. The isolated RNA was stored at -80°C until use. RT of the total RNA ( I .7 pg) was performed using an oligo dT (dT,,) primer and Superscript 11 RNase H- (GIBCO-BRL, Gathersburg, MD). A total of l / SO of the synthesized first-strand cDNA was used for PCRin a reaction mixture of 50 pL by employing 0.5 yL of a-[’*P]-dCTP ( 1 0 mCi/mL, ICN Biomedicals Inc, Costa Mesa, CA) and two pairs of primers separately; for the b subunit, 5”ATCTGTGCAGTGCAACAGAGG-3‘ (sense) and S’-CATTGAATTCTATGTTCTTAAGGG’ITC’ITGATAA-3’ (antisense, the underlined nucleotides were introduced to create an EcoRI site); for human glyceraldehyde-3phosphate dehydrogenase (GAPDH) as an internal control, 5’-CATCACCATCTTCCAGGAGC-3’ (sense) and S’-TAAGCAGTTGGTGGTGCAGG-3’ (antisense). After 20 cycles (for GAPDH) or 25 cycles (for the 6 subunit), 8 p L of the reaction mixture (GAPDH) or 15 pL (the 6 subunit) were applied to a 2.5% agarose gel. After electrophoresis, the gel was dried and subjected to quantitation of the amplified products by BAS1000 Mac Bio-Imaging Analyzer (FUJIX, Tokyo, Japan). The fluorescence intensity of the PCR product for the 6 subunit was normalized to that for GAPDH. The dried gel was also exposed to Kodak XAR autoradiography film (Eastman Kodak CO, Rochester, NY) at -80°C. Exarninution of the a subunit. All regions, including exons and exonhtron boundaries of the a subunit gene, were amplified by employing 16 pairs of primers as described.” Thesingle-strand conformation polymorphism (SSCP) analyses of the amplified DNAs were performed as des~ribed.*~.*~ At10°C , a SSCP gelwasrun in the buffer cooled by Model 1000 Mini Chiller (Bio-Rad). Nucleotide sequences of the amplified DNA samples were also determined as described above. To quantitate the a subunit in platelets by ELBA,’? platelets were isolated from citrated blood by centrifugation on Ficoll-Hypaque gradients andwashedin Tris-EDTA-saline buffer.’” The platelets were lysed by incubation with 1/40 volume of 20% wt/wt Triton X100. RESULTS Insertion of U triplet AAC i n exon III of the gene , f . r the b subunit. Six pairs of specific amplification primers were employed for in vitro amplification of all 12 exons andtheir boundaries of the gene for theb subunit (Fig 2) in the DNA of theproband’ssister (11-1 in Fig 1). Pairs 1-6 produced 2.1, 1 S , 1.8, 2.0, 2.3, and 1.7 kb DNA fragments, respectively (data not shown). The sizes of these fragments were exactly the same as predicted from the normal genomic sequence.” These resultsconfirmedthat there was no gross change(s) in the patient’s gene for the b subunit. Nucleotide sequences of the six amplified fragments were then examined to find any possible small difference(s) from the normalsequence. When exon I11 of the probandwas sequenced, all of the 15 ssDNA samples showed ATGGTTA U C A T C (Fig 3), while the normal sequence is ATGGTTACATC. The insertion of this AAC triplet falls within a codon forTyr-80: between its first and secondnucleotides, -adenosine-5285 and cytidine-5286,” resulting in the creation of a stop codon at amino acid position 80. Another mutation, adenosine-27970to guanosine, was also found in the 3’-noncoding region. This is one of the same polymorphisms that was found in the Japanese patient with complete h subunit deficiency and also in normal individuals.’* No other change in the nucleotide sequence of the proband’s gene for the b subunit was detected by sequence analysis after the remaining exons andtheir flanking regions were compared with thenormal gene.“ Thus, it is concluded that the only genetic defect regarding this patient’s b subunit is the insertion of the triplet in exon I11 of the gene. Detection of the insertion by restriction enzyme digestion. Because insertion of the nucleotide triplet (AAC) in the proband’s DNA destroys an intrinsic Mae111 site (GTNAC) in exon I11 and creates a new Tru9I site (TTAA), a fragment spanning the entire region of exon 111 was amplifiedand digestedwith Mae111 or Tru9I endonuclease. The MueIII digest of the 430-bp fragment from oneof the family members (111-5) and from a normal individual yielded two fragments of 285 bp and 145 bp (Fig 4, top), whereas the 433- From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 2771 GENETIC BASIS OF TYPE I FXlll DEFICIENCY G I A N123456 T C / N123456 /I I T I1 A 111 50, 54/52 90,54159 58, 60155 50. 50/47 74. 81/55 121, 103/106 Fig 1. Family pedigree of typeI factor Xlll deficiency. Closed circles represent homozygotes, and half-closed circles (females1and squares (males) stand for heterozygotes. An arrow indicates the proband (112); her father (1-1) died before the present study. The first, second, and third numbers indicate the b subunit antigen, the activityof the B subunit, and its antigen levels in plasma, respectively, in each family member. bp fragments of the DNAs from the proband and her sister (11-2 and 11-1) remained unchanged. The remaining five of their family members (1-2.11-3.111-1,111-2. and 111-3) showed both cleaved and uncleaved bands. These results indicated that the two patients of type I deficiency were homozygotes and five of their family members were heterozygotes of this mutant allele, which is not found in normal individuals. The Tr~r91digest of the 430-bp fragments from a normal individual and a member (111-5) (Fig 4, bottom)yielded five fragments of 293 bp. 47 bp, 44 bp, 41 bp, and 5 bp (the last fourfragmentsare unresolved because ofsmall size). In contrast, the digest of the 433-bp fragment fromthe proband and her sister yielded six fragments of 242 bp, 54 bp, 47 bp, 44 bp, 41 bp, and 5 bp(the last five fragmentsare unresolved). Theremaining five oftheirfamily members showed both 293-bp and 242-bp bands. Thus, the genotype of each individual was confirmed by digesting the fragment with both endonucleases. This is consistent with the phenotype determined by ELISA (Fig l ) . Search for the truncated b subunit in plasma. The insertion ofthe AAC triplet falls within a codonforTyr-80, resulting in the creation of a stop codon at amino acid posi- . Sushi Domain 519~1 Gm€ frapmanla 8 5 6 7 I 1- VI I1 I V I I I V PR- 4- m- * Vlll VI1 IX 5- and samples 1 through 6 are from the proband (11-21. Sequences of all samples were thesame as those of normal except for an insertion of AAC, shown in a square. tion 80 (Fig S. top). This would lead to the formation of a peptide consisting of only79aminoacids andincluding only a single Sushi domain (Fig 5, bottom). To investigate whether thetruncatedhsubunit is present in circulation, plasma obtained from two patients of type I deficiency (11-1 and 11-2) and their family members was analyzed by Western blotting using anti-h subunit antiserum. A band for the normal h subunit of about 70 kD was observed in normal individuals and seven family members. but not in the patients (data not shown); or were bands for the truncated h subunit patients' including possible oligomers detected in the plasma. It is very likely that the resulting truncated protein does not reach circulation, as no h subunit antigen was detected by more sensitive ELISA in either the proband or her sister." Semiquantitation of mRNA for the bsubunit.Because noprotein for themutant b subunit was detected in the Mae 111 digest C-lermlnal 9 ::: c-)"-HH Exons PCR 1 2 3 4 Fig 3. Nucleotide sequence of part of exon 111. The left-end lanes (NI in nucleotide G, A, T, C groups are from the normal individual, IO II LI'NC m m l , , I x X I XI1 e0 For diammair U Fig 2. Strategy for in vitro amplification of the gene for the b subunit and the insertiondetected. All 12 exons and theirboundaries in the gene for the b subunit were amplified employingsix pairs of oligonucleotide primers shown byarrows. For analysis of the insertion of AAC between adenosine-5285 and cytidine-5286 shown by a closed circle, a region was amplified employing the 5"primer of pair 3R and the 3"primer of pair 2R. One of genetic polymorphisms is shown by an open circle. 100 digest Fig 4. (Top) Restriction digestionwith Madllendonuclease of the amplified products of exon 111. (Bottom) Restriction digestion with TrusI endonuclease. Molecular weight standards are from Bethesda Research Laboratories. Samples 1-2, 11-1, 11-2, 11-3, 111-1. 111-2, 111-3, and 111-5 refer t o the proband's mother, the probands elder sister, the proband, her younger sister, et al. as indicated in Fig 1. From www.bloodjournal.org by guest on June 18, 2017. For personal use only. IZUMI ET AL 2772 Exon 111 Normal 1.....77 78 79 8081 82 83......641 H G Y l S D .,.... T STP G A A . . . . A G T ART GGT TRC ATCTCT GAT.. ACA TAG E ..... S .... T AAC Mutant 1.....77 78 79 H G STP S GAR RGT ART GGT TA E..... .... ATC TCT G A T . . . . . . . ... was detected by agarose gel electrophoresis (data not shown); neither was any mobility shift foundby SSCP analyses of the amplified DNAs when compared with normal (data not shown). In addition, no nucleotide substitutions were detected by sequencing analysis of the amplified DNAsafter they were compared with the normal genomic sequence of the n subunit.“’ Furthermore, normal amounts of the n subunit were detected by ELlSA in platelets obtained from the members of this family (Table I), whereas two patients with type I1 deficiency from two other independent families had no n subunit in their platelets. Thus, it was concluded that the gene for the CI subunit is intact in all members of the family examined in the present study. DISCUSSION The only significant mutation detected in this patient’s h subunit was the insertion of thetriplet in exon 111 of the 1st Sushi Domain L 1 II Xlllb 300 tlmc T STP Fig 5. (Top) Amino acid sequence of the normal and mutant b subunits. Amino acid sequence of a part of Exon 111 is shownin detail. Amino acid residues are indicated by a one-letter code. STPstands for an in-frame stop codon, created by the mutation. (Bottom) Schematic representation of the mutant b subunit. A truncated protein composed of only 79 amino acids would make up a single (first) Sushi domain, while oneCys residue (with -?l would remainunpaired. The signal peptide region has been enclosed. Arrows indicate positions of introns A and B. patients’ plasma, its mRNA was examined. A RT-PCR assay was performed to estimate the amount of aberrantly expressed mRNA for the h subunit in leukocytes, as samples of hepatocytes from these patients were not available. Both a 493-bp fragment for the h subunit and a 253-bp fragment for GAPDH were obtained by RT-PCR for a normal Japanese, a homozygote (11-l), and a normal Italian individual (Fig 6, top). Under experimental conditions, a linear relationship was observed between the amounts of total RNA employed and the fluorescence intensity of these bands (Fig 6, bottom). The ratio of the h subunit mRNA/GAPDH mRNA was 0.7, 0.6, and 0.7 for the normal Japanese, the homozygote (11-l), and the normalItalian. respectively. Thus, the amount of the patient’s mRNA was comparable to that of normal individuals, strongly suggesting that the mutant mRNA is synthesized as stable as normal. Examination of the a subunit. All 15 exons of the gene for the n subunit of the proband and her sister were successfully amplified by PCR, and no size difference from normal 3ooJ GAPDH 200 U, U 0 10 20 30 40 50 Total RNA (ng) Fig 6. (Top) RT-PCR assay for the mRNAs of the b subunit (Xlllb) and GAPDH. A radioactive nucleotide was incorporated into PCR products t o estimate the amounts of mRNAs for both genes. Four left of the panel contain amplified products of lanes (Control) on the diluted (1 t o 1/81 samples from a normal individual. Normal4 and -I stand for samples from normal Japanese and Italian individuals, respectively, and 11-1 for a homozygote of the typeI deficiency. (Bottom) Linear relationship between the amounts of total RNA used and the fluorescence intensity of the radioactive bands. Amounts of the amplified product roughlyrepresent the amounts of mRNAs for the b subunit (0) and for GAPDH (01,respectively. From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 2773 GENETIC BASIS OF TYPE I FXlll DEFICIENCY Table 1. Amounts of a Subunit in Platelets a Subunit Subject Type I deficiency 1-2 11-3 55 1g/i09 platelets 65 58 66 111-1 49 11-1 11-2 111-3 Type II deficiency 60 Case 1 Case 2 Normalrange Average (n = 10) Absent* Absent* 34-78 50 * Lower than detection limit. gene. Both the proband and her sister were homozygotes, and five of their family members were heterozygotes of this mutant allele. Neither normal nor truncated b subunit was detected in the homozygotes’ plasma. These results suggest either that the aberrant b subunit is not synthesized or that it quickly degrades. The mutant mRNAmaybe unstable; however, it is notpossible to study the effect of this mutation on the in vivo mRNA for the b subunit because it is synthesizedin the liver and mRNAs of hepatocytes cannot be obtained from these patients. As an alternative, the amount of aberrantly expressed mRNA in leukocytes was measured by a RT-PCR assay. The amount of the patient’s mRNA was comparable to that of normal individuals, suggesting that transcription of the gene for the b subunit and stability of its mRNA are not affected by the mutation. The truncated peptide contains Cys-71, butnot its counterpart Cys-126, which would be expected based on its homology with p2glycoprotein I’7 and C4 binding protein.’* Therefore, it is very likely that this Cys residue in the second Sushi domain remains unpaired (Fig 5, bottom), whichmay render the mutant peptide unstable and/or decrease its biosynthesis or secretion from hepatocytes. A number of cases where deficiencies of various proteins are due to the appearance of unpaired Cys residues support this conclusion.19~29~3n In contrast to the genetic defect of the patients’ b subunit, the genes for the a subunit were intact and expressed in normal amounts. Accordingly, it is concluded that the a subunit deficiency of these patients is secondary to the b subunit deficiency; that is, the b subunit deficiency is the genetic basis for type I factor XI11 deficiency. Before the first case of complete b subunit deficiency was found in Japan,” at least four patients with type I factor XI11 deficiency, demonstrating similar laboratory findings, had been reported.6,3’,3z The present study clarified that two of them were cases of the b subunit deficiency, but the remaining two cases have not been examined for their genetic defects. They may have separate defects in both the a and b genes by chance, resulting in real combined deficiency of both subunits. Alternatively, the lack of the a subunit in plasma in these cases maybe secondary to the b subunit deficiency, in the same manner as the Japanese In the past, only the enzymatic activity of factor XI11 could be used for diagnosis of its deficiency, then immunological assays of each antigen became available, and finally genetic analyses for both subunits have been developed for precise diagnosis. When patients with the a subunit deficiency who are diagnosed by the factor XI11 enzymatic activity are examined, the amount of the b subunit antigen must be determined because the a subunit deficiency can be caused by genetic defects either of the a or b subunit. Measurements of the a subunit in platelets (or placenta, if available) are also useful’7 because, if there is no a subunit in platelets, it may be concluded that the a subunit deficiency is independent of the b subunit deficiency. Moreover, if normal amounts of the a subunit are present in platelets, it may be concluded that the gene for the a subunit is normal and that the gene for the b subunit must have some defect(s). The present study showed that the two original patients of type I deficiency are the second and third cases of complete b subunit deficiency to be reported thus far. In addition, the first case with complete b subunit deficiency demonstrated the a subunit deficiency as well,17 indicating that this patient falls into the category of type I deficiency. Thus, we concluded that congenital deficiency of the b subunit is the genetic basis of type I factor XI11 deficiency. On the other hand, many patients of type I1 factor XI11 deficiency inwhich the mutation is all result from genetic defects of the a subunit. It is, therefore, quite reasonable to conclude that congenital deficiency of the a subunit is the genetic basis of type I1 factor XI11 deficiency. There is, as yet, no example containing real combined genetic defects for both the a and b subunits. Such a patient would show the same phenotype as for type I deficiency. Although the combined genetic deficiency of both subunits could occur, it would be extremely rare because these genes are localized to separate chromosomes.” Therefore, we propose a new genetic classification for factor XI11 deficiency in threeparts: the a subunit deficiency (former type I1 deficiency), the b subunit deficiency (former type I deficiency), and a possible combined deficiency of both the a and b subunits. ACKNOWLEDGMENT The authors thank DrsM. Togashi and S. Tsutsumi for performing the PCR-SSCP analysis for the a subunit gene and L. Boba for her help in preparation of the manuscript. REFERENCES 1 . Lorand L, Losowsky MS, Miloszewski KJM:Human factor XIII. Fibrin-stabilizing factor. Prog Thromb Hemostas 5:245, 1980 2. Duckert F: Documentation of the plasma factor XI11 deficiency in man. Ann N Y Acad Sci 202:190, 1972 3. Folk JE, Chung SI: Blood coagulation factor XIII: Relationship of some biological properties to subunit structure,in Reich E, Rifken DB, Shaw E (eds): Proteases and Biological Control. New York, NY, Cold SpringHarborLaboratory, 1975, p 157 4. Ichinose A: Physiology and biochemistry of factor XIII, in Bloom AL, Forbes CD, Thomas DP, Tuddenham EGD (eds): Haemostasis and Thrombosis. Edinburgh, UK, Churchill Livingstone, 1995, p S31 S. Ichinose A, Bottenus RE, Davie EW: Structure of transglutaminases. J Biol Chem 265:13411, 1990 6. Girolami A, Burul A, Fabris F, Cappellato G, Betterle C: Stud- From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 2774 IZUMI ET AL ies on factor XI11 antigen in congenital factor XI11 deficiency. A 20. Rodeghiero F, Tosetto A, Di Bona E, Castaman G: Clinical tentative classification of the disease in two groups. Folia Haematol pharmacokinetics of a placenta-derived factor XI11 concentrate i n 105:131, 1978 Type I and Type 11 factor XI11 deficiency. Am J Hematol 36:30, 7. Girolami A, Cappellato MC, Lazzaro AR, Boscaro M: Type I 1991 and TypeI1 disease in congenital factor XI11 deficiency. Blut53:41 I , 21. Lorand L, Urayama T, De Kiewet JWC, Nossel HL: Diagnos1986 tic and genetic studies on fibrin-stabilizing factor with a new aswy 8. Ichinose A, Hendrickson LE, Fujikawa K, Davie EW: Amino based on amine incorporation. J Clin Invest 48: 1054. I969 acid sequence of the a subunit of human factor XIII. Biochemistry F: Abnormalplasma 22.TosettoA,CastamanG.Rodeghiero factor XI11 subunit A with fully functional platelet FXllI ill a family 25:6900,1986 9.IchinoseA,McMullenBA,FujikawaK,DavieEW:Amino with congenitaldeficiency of FXllIsubunit B. ThrombHaemost acidsequenceofthe b subunit of humanfactorXIII,aprotein 69: 1295, I993 (abstr) composed of ten repetitive segments. Biochemistry 25:4633, 1986 23.Sanger F, Nicklen S, Coulson AR: DNAsequencing with IO. Ichinose A, Davie EW: Characterization of the gene for the chain-termination inhibitors. Proc Natl Acad Sci USA 745463, 1977 a subunit of human factor XI11 (plasma transglutaminase), a blood 24. Orita M, Suzuki Y, Sekiya T, Hayashi K: Rapid and sensitive coagulation factor. Proc Natl Acad Sci USA 855829, 1988 detection of pointmutationsandDNApolymorphismsusing the I I . Bottenus RE, Ichinose A, Davie EW: Nucleotide sequence of polymerase chain reactions. Genomics 5:874, 1989 thegeneforthe b subunitofhumanfactor XIII. Biochemistry RJ, WeghorstCM:‘Cold 25. Hongyo T, BuzardCS.Calvert 29:11195,1990 SSCP’: A simple, rapid and non-radioactive method lor optimized 12. Ichinose A, Kaetsu H: Molecular approach to the structuresingle-strand conformation polymorphism analyses. Nucleic Acids function relationship of human coagulation factor XIII. Method EnRes 21:3637, 1993 zymol 222:36, 1993 26. Rodeghiero F, Castaman G, Di Bona E, Ruggeri M, Lombardi 13. Kamura T, Okamura T, Murakawa M, Tsuda H, Teshima T, R, MannucciPM:Hyper-responsiveness to DDAVPforpatients Shibuya T, Harada M, Niho Y: Deficiency of coagulation factor XI11 with type I von Willebrand’s disease and normal intra-platelet von A subunit caused by the dinucleotide deletion at the S’ end of exon Willebrand factor. Eur J Haematol 40:163, 1988 111. J Clin Invest 90:315, 1992 27. Kdto H, Enjoji K: Amino acid sequence and location of the 14. Board P, Coggan M, Miloszewski K: Identification of a point disulfide bonds in bovine &glycoprotein I: The presence of tive mutation in factor XI11 A subunit deficiency. Blood 80:937, 1992 sushi domains. Biochemistry 30:11687, l99 I 15. Standen GR, Bowen DJ: Factor XI11 ABristol 1: detection of 28. Janatova J. Reid KGM, Willis AC: Disulfide bonds are locala nonsense mutation (Arg”’-stop codon) in factor XI11 A subunit ized within the short consensus repeat units of complement reguladeficiency. Br J Haematol 85:769, 1993 tory proteins: C4b-binding protein. Biochemistry 28:4754, 1989 29. Green PM, Bentley DR, Mibashan RS, Nilsson IM, Giannelli 16. Mikkola H, SyrjalaM, Rasi V,Vahtere E, Hamalainen E, Peltonen L, Palotie A: Deficiency in the A-subunit of coagulation F: Molecular pathology of haemophilia B. EMBO J 8: 1067, 1989 factor XIII: Two novel point mutations demonstrate different effects 30. Romeo G, Hassan HJ, Staempfli S, Roncuzzi L. Cianetti L. Leonardi A, VicenteV, Mannucci PM, BertinaR, Peschle C. Cortese on transcript levels. Blood 84517, 1994 R: Hereditarythrombophilia: Identification of nonsenseandmis17. Saito M, Asakura H, Yoshida T, Ito K, Okafuji K, Yoshida sensemutations in theproteinCgene. Proc Natl Acad Sci USA T,MatsudaT:Afamilialfactor XIII subunitBdeficiency.BrJ 842829, 1987 Haematol 74:290, 1990 3 I . Kiesselbach TH, Wagner RH: Demonstration of factor XL11 18. Hashiguchi T, Saito M, Morishita E, Matsuda T, Ichinose A: in human megakaryocytes by a fluorescent antibody technique. Ann Two genetic defects in a patient with complete deficiency of the h subunit for factor XIII. Blood 82:145, 1993 NY Acad Sci 2O2:318, 1972 32. Cappellato MG, Lazzaro AR, Marafioti F, Polato G, Girolami 19. HashiguchiT,IchinoseA:Molecularandcellularbasis of A: Anewfamily with congenitalfactor XIII deficiency showing deficiency of the b subunit for factor XI11 secondary to a Cys430a deficitof both subunit U and b. Type I factor XI11 deficiency. Phe mutation in the seventh Sushi domain. J Clin Invest 95:1002, Haematologia 20: 179, 1987 199s From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 1996 87: 2769-2774 Type I factor XIII deficiency is caused by a genetic defect of its b subunit: insertion of triplet AAC in exon III leads to premature termination in the second Sushi domain T Izumi, T Hashiguchi, G Castaman, A Tosetto, F Rodeghiero, A Girolami and A Ichinose Updated information and services can be found at: http://www.bloodjournal.org/content/87/7/2769.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. 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