Title: Prekallikrein (PK) Tokushima: PK deficiency caused by a Gly401→Glu mutation
Abstract: Dear Sir, Prekallikrein (PK) deficiency was first reported in 1965 by Hathaway et al.[1Hathaway W.E. Belhasen L.P. Hathaway H.S. Evidence for a new plasma thromboplastin factor I. Case report, coagulation studies and physicochemical properties.Blood. 1965; 26: 521-32Crossref PubMed Google Scholar], and more than 20 families with this deficiency have been reported. The majority of the patients had no bleeding diatheses and been incidentally discovered during routine preoperative clotting studies [2Saito H. Contact factors in health and disease.Semin Thromb Hemost. 1987; 13: 36-49Crossref PubMed Scopus (62) Google Scholar]. In 1981, Saito et al. demonstrated the molecular heterogeneity of human PK deficiency and showed that persons with the CRM + variant have a non-functional form of PK in their plasma [3Saito H. Goodnough L.T. Soria J. Soria C. Aznar J. España F. Heterogeneity of human prekallikrein deficiency (Fletcher trait). Evidence that five of 18 cases are positive for cross-reacting material.N Engl J Med. 1981; 305: 910-4Crossref PubMed Scopus (36) Google Scholar]. Subsequently, the functional characterization of abnormal PK has been reported in two cases [4Bouma B.N. Kerbiriou D.M. Baker J. Griffin J.H. Characterization of a variant prekallikrein, prekallikrein Long Beach, from a family with mixed cross-reacting material-positive and cross-reacting material-negative prekallikrein deficiency.J Clin Invest. 1986; 78: 170-6Crossref PubMed Scopus (16) Google Scholar, 5Wuillemin W.A. Furlan M. Von Felten A.L. Lämmle B. Functional characterization of a variant prekallikrein (PK Zürich).Thromb Haemost. 1993; 70: 427-32Crossref PubMed Scopus (14) Google Scholar]. However, no DNA alterations underlying PK deficiency have yet been reported. In this study, we identified the missense mutation responsible for PK deficiency inherited in a Japanese family. Clinical and laboratory data relevant to the PK deficiency in this report have previously been presented [6Shigekiyo T. Fletcher factor deficiency: 2 homozygotes and 12 heterozygotes in a family.Shikoku Acta Med. 1981; 37: 541-55Google Scholar]. In brief, the proband, a 47-year-old male, was referred to us because of prolonged coagulation time found during hemostasis screening before surgery for sinusitis. The proband and his younger sister were homozygotes and 12 heterozygotes, including the parents, were discovered over three generations. Bleeding episodes were not obtained from family members except the proband, who gave a history of nasal bleeding at the age of 44 years. PK clotting activity was 0.2% and 1% in two homozygotes and ranged 23–50% (mean, 38%) in 12 heterozygotes and 88–150% (mean, 115%) in normal family members. PK antigen level determined by Laurell's method with rabbit antihuman plasma kallikrein sera (Behringwerke AG, Marburg, Germany) was 25 and 21% in homozygotes and ranged 50–76% (mean, 59%) in heterozygotes and 82–140% (mean, 115%) in normal family members. These results suggested that CRM + PK deficiency is hereditary in this family. We designated this case PK Tokushima after the city where this family's PK deficiency was detected. In the present study, blood was drawn from all individuals after obtaining informed consent according to the Helsinki Declaration and following the institutional guidelines of the University of Tokushima. Genomic DNA was isolated from whole blood by the standard phenol/chloroform extraction method. To elucidate the genetic basis of PK deficiency, we examined the nucleotide sequences of all 15 exons of the proband's PK gene. Polymerase chain reaction (PCR) and DNA sequence analysis were performed as previously described [7Shigekiyo T. Yoshida H. Kanagawa Y. Satoh K. Wakabayashi S. Matsumoto T. Koide T. Histidine-rich glycoprotein (HRG) Tokushima 2: novel HRG deficiency, molecular and cellular characterization.Thromb Haemost. 2000; 84: 675-9Crossref PubMed Scopus (19) Google Scholar]. The sequences of all primers used for PCR were the same as those used by Yu et al.[8Yu H. Anderson P.J. Freedman B.I. Rich S.S. Bowden D.W. Genomic structure of the human plasma prekallikrein gene, identification of allelic variants, and analysis in end-stage renal disease.Genomics. 2000; 69: 225-34Crossref PubMed Scopus (46) Google Scholar]. One single-base substitution was found in exon 11, and no substitution was found in the remaining exons. The amino acid residues of eight polymorphic sites shown by Yu et al.[8Yu H. Anderson P.J. Freedman B.I. Rich S.S. Bowden D.W. Genomic structure of the human plasma prekallikrein gene, identification of allelic variants, and analysis in end-stage renal disease.Genomics. 2000; 69: 225-34Crossref PubMed Scopus (46) Google Scholar] were identified as Asn124, His183, His189, Lys229, Gln300, Thr339, Ser362, and Gln541. A single G to A substitution at nucleotide position 1352, resulting in a change of Gly401 to Glu in the catalytic domain, was found in all of the 12 different clones analyzed (Fig. 1). These results indicate that the proband carried a homozygous deficiency. To confirm the genetic basis for the phenotype observed in this family, we performed allele-specific oligonucleotide (ASO) hybridization analysis of the amplified PCR fragments of exon 11 from the proband and other available family members (Fig. 2b). ASO hybridization analysis was performed as previously described [7Shigekiyo T. Yoshida H. Kanagawa Y. Satoh K. Wakabayashi S. Matsumoto T. Koide T. Histidine-rich glycoprotein (HRG) Tokushima 2: novel HRG deficiency, molecular and cellular characterization.Thromb Haemost. 2000; 84: 675-9Crossref PubMed Scopus (19) Google Scholar]. ASO probes employed in this study contained the normal sequence 5′-ACCTGTGTGGAGGGTCACT-3′ and the mutant sequence 5′-ACCTGTGTGAAGGGTCACT-3′ (base changes underlined). The proband (IV-1 in Fig. 2a) and his younger sister (IV-5), who had homozygous PK deficiency, were homozygous for the mutation (1352G→A). The proband's next younger brother (IV-6) and two sons (V-1 and V-2), who had heterozygous PK deficiency, were heterozygous for this mutation. The proband's youngest brother (IV-8), having a normal PK level, was homozygous normal with respect to this mutation. These results indicate that a G to A substitution in exon 11 found in the proband is responsible for the PK deficiency inherited in this family. We then studied the ASO hybridization with 50 unrelated healthy Japanese individuals and did not find any mutations in exon 11 in the population (data not shown). This study eliminates the possibility that this mutation is a common polymorphism.Figure 2(a) Pedigree of the family with PK deficiency. Arrow, the proband; ▪●, homozygous PK deficiency; ◒, heterozygous PK deficiency; ⊟⊖, subjects with normal PK levels; ⍁, deceased family members; □○, unexplored subjects. (b) ASO hybridization analysis. The proband (IV-1) and his younger sister (IV-5) were homozygous for the mutation (1352G→A). The proband's next younger brother (IV-6) and two sons (V-1 and V-2) were heterozygous for this mutation. The proband's youngest brother (IV-8) was homozygous normal with respect to this mutation.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Gly401 and its surrounding sequence in PK are rather conserved among species, including human [9Chung D.W. Fujikawa K. McMullen B.A. Davie E.W. Human plasma prekallikrein, a zymogen to a serine protease that contains four tandem repeats.Biochemistry. 1986; 25: 2410-7Crossref PubMed Scopus (189) Google Scholar], rat [10Beaubien G. Rosinski-Chupin I. Mattei M.G. Mbikay M. Chrétien M. Seidah N.G. Gene structure and chromosomal localization of plasma kallikrein.Biochemistry. 1991; 30: 1628-35Crossref PubMed Scopus (69) Google Scholar], mouse [11Seidah N.G. Sawyer N. Hamelin J. Mion P. Beaubien G. Brachpapa L. Rochemont J. Mbikay M. Chrétien M. Mouse plasma kallikrein: cDNA structure, enzyme characterization, and comparison of protein and mRNA levels among species.DNA Cell Biol. 1990; 9: 737-48Crossref PubMed Scopus (17) Google Scholar], guinea-pig [12Shibuya Y. Semba U. Nishino N. Khan M.M.H. Tanase S. Okabe H. Yamamoto T. Primary structure of guinea pig plasma prekallikrein.Immunopharmacology. 1999; 45: 127-34Crossref PubMed Scopus (5) Google Scholar], and pig [13Kimura A. Kihara T. Okimura H. Hamabata T. Ohnishi J. Moriyama A. Takahashi K. Takahashi T. Identification of porcine follipsin as plasma kallikrein, and its possible involvement in the production of bradykinin within the follicles of porcine ovaries.Mol Reprod Dev. 2000; 57: 79-87Crossref PubMed Scopus (8) Google Scholar] (Fig. 3). In general, glycine is known to be one of the conservative amino acids and is important to support the correct conformation of proteins [14Schulz G.E. Schirmer R.H. Amino acids.in: Canter CR Principles of Protein Structure. Spring-Verlag, 1978: 1-16Google Scholar]. Since there is a disulfide bridge between Cys400 and Cys416 [15McMullen B.A. Fujikawa K. Davie E.W. Location of the disulfide bonds in human plasma prekallikrein: the presence of four novel apple domains in the amino-terminal portion of the molecule.Biochemistry. 1991; 30: 2050-6Crossref PubMed Scopus (76) Google Scholar], Gly401 is located near His415, part of the reactive site triad. Therefore, the mutation (Gly401→Glu) may lead to loss of the enzyme activity. However, since the mutation is located next to Cys400, we cannot exclude the possibility that the disulfide bridge cannot be formed. Since His415 is located next to Cys416, lack of the disulfide bridge can lead to loss of the enzyme activity. Bouma et al. characterized a variant PK, PK Long Beach [4Bouma B.N. Kerbiriou D.M. Baker J. Griffin J.H. Characterization of a variant prekallikrein, prekallikrein Long Beach, from a family with mixed cross-reacting material-positive and cross-reacting material-negative prekallikrein deficiency.J Clin Invest. 1986; 78: 170-6Crossref PubMed Scopus (16) Google Scholar]. The variant was cleaved by β-factor XIIa 200 times slower than the normal molecule, and no amidolytic activity was detected for the cleaved variant. Isoelectric focusing studies suggested a difference of one charged amino acid residue between the variant and normal molecules. From these data and other observations, it was suggested that an amino acid was substituted in the variant near the N-terminal end of the kallikrein light chain. The mutation site of PK Long Beach may be close to that of PK Tokushima. In conclusion, we identified a genetic abnormality in a Japanese family with PK deficiency. This is the first report in which a mutation causing PK deficiency was identified. We thank Dr Kensaku Takase for help in submitting this manuscript online.