Three novel PROC gene lesions causing protein C deficiency

Hallam PJ, Mannucci P, Tripodi A, Bevan D, Laursen B, Tengborn L, Wacey A, Cooper DN. Three novel PROC gene lesions causing protein C deficiency. Clin Genet 1998: 54: 231–233. 0 Munksgaard, 1998
Missense mutations. three of them novel (Am210→Val, Asn248→ Ile, Ah355→Val), were found in the protein c ( PROC ) genes of 7 patients with inherited protein C deficiency associated with venous thrombosis. Comparison with the phenotypic effects of mutations in the analogous residues of factor IX causing hdernophilia B and the use of molecular modelling has provided explanations as to how these lesions might alter either the structure, function or secretion of the protein C molecules encoded.     

Analysis of phenotypes and genetic mutations in two pedigrees affected with hereditary protein C deficiency北大核心CSCD

Objective To explore the pathogenesis of protein C deficiency in two pedigrees through mutation detectionand model analysis. Methods Chromogenic substrate method and enzyme linked immunosorbent assay (ELISA) wereused to determine the plasma protein C activity (PC : A) and protein C antigen (PC : Ag) in the two probandsand their family members. All of the 9 exons and intron-exon boundaries of the PROC gene were amplified by PCRand analyzed with Sanger sequencing after purification. Corresponding mutate sites of the family members werealso amplified and sequenced. The PolyPhen-2 software was used to analyze the perniciousness of the mutationsand Clustal X was to analyze the conservatism. The protein model and amino acids interaction of the mutationswere analyzed by Swiss-PdbViewer software. Results The PC : A and PC : Ag of proband 1 was 30% and 35%, whilePC : A of his father, mother and aunt were all slightly under the reference range. Two heterozygous missensemutations were found in exons 7 and 5 of the PROC gene, namely c. 565 C>T (p. Argl47Trp) and c. 383 G >A (p. Gly86Asp). His father and aunt were carriers for c. 565 C>T, while his mother had carried c. 383 G>A. The PC : A of proband 2 and his son were 50% and 64% , respectively. And they were both positive for p. Argl47Trp. Analysis of PolyPhen-2 indicated that p. Argl47Trp was benign, while p. Gly86Asp was damaging. Clustal X analysis indicated that the p. Argl47Trp was non-conservative, while the p. Gly86Asp was highly conservative. Modeling for the mutant proteins revealed that the simple aromatic ring of Trpl47 in p.Argl47Trp destroyed the two hydrogen bonds between Argl47-Lysl46 and Argl47-Lysl51, and steric hindranted withArgl78. The side chain of Asp86 extended and generated steric clash with Gln90 with the occurrence of p.Gly86Asp. The change of hydrogen bonds and steric effects has altered the spatial configuration of amino acids, which led to unstable mutate proteins and interfered with the secretion. Conclusion Both probands hadhereditary protein C deficiencies, for which their parents were all carriers. The heterozygous mutations p.Argl47Trp and p. Gly86Asp were the main cause for PC : A activity decrease. Among these, p. Gly86Asp wasdiscovered for the first time.     

Inherited prothrombotic defects in Budd-Chiari syndrome and portal vein thrombosis: a study from North India

We studied 57 patients with Budd-Chiari syndrome (BCS) and 48 with portal vein thrombosis (PVT) for underlying inherited prothrombotic defects such as protein C, protein S, and antithrombin III deficiencies. Genetic mutations for factor V Leiden, prothrombin gene 20210A, and methyltetrahydrofolate reductase (MTHFR) C677T were studied in 29 patients in each group. Inherited prothrombotic defects were detected in 16 (28%) of 57 patients with BCS and 7 (15%) of 48 patients with PVT. Factor V Leiden mutation was the most common prothrombotic defect in BCS (5/29 [17%]) followed by protein C deficiency (7/57 [12%]) and protein S deficiency (4/57 [7%]), whereas in PVT, protein C deficiency was the most common inherited prothrombotic defect (4/48 [8%]) followed by protein S deficiency (2/48 [4%]). The factor V Leiden mutation was detected in only 1 (3%) of 29 cases of PVT. The heterozygous MTHFR C677T mutation was detected in 7 (24%) of 29 patients with BCS and 6 (21%) of 29 patients with PVT. Antithrombin III deficiency, homozygous MTHFR C677T mutation, and prothrombin G20210A mutation were not detected in any patients.     

A phenylalanine hydroxylase amino acid polymorphism with implications for molecular diagnostics.

Mutations in the gene encoding phenylalanine hydroxylase (PAH, EC 1.14.16.1) are associated with various degrees of hyperphenylalaninemia, including classical phenylketonuria (PKU). We examined the PAH gene in a Brazilian PKU family of African origin and identified three missense variants, R252W (c.754C --> T), K274E (c.820A --> G), and I318T (c.953T --> C), the two latter of which were transmitted in cis. Expression analyses in two different in vitro systems showed that I318T is associated with profoundly decreased enzyme activity, whereas the enzyme activity of K274E is indistinguishable from that of the wild-type protein. Detailed kinetic analyses of PAH expressed in E. coli showed that the K274E mutant protein has kinetic properties similar to that of the wild-type protein. Population studies have suggested that the K274E variant occurs on approximately 4% of African-American PAH alleles, whereas the neonatal screening incidence of PKU among African Americans is only 1:100,000. This is to our knowledge the first demonstration of a PAH missense variant with no apparent association to PAH deficiency. Awareness of this common variant may be helpful to laboratories that perform molecular diagnosis of PAH deficiency in populations of African origin.     

云南籍G6PD缺乏症病例的基因型分析

目的：确定23例云南籍G6PD缺乏症病例的基因突变类型,了解G6PD基因突变的分子遗传特性。方法：运用扩增阻滞突变系统、自然及错配引物PCR／限制性酶切、PCR－单链构象多态分析及DNA自动测序技术,检测分析了23例云南G6PD缺乏症病例第2－12共11个G6PD基因外显子和部分内含子的基因突变。结果：23例中共发现5种突变,形成了5种突变基因型：G487A10例（7例汉族、3例傣族）、IVS－11T39C复合C1311T7、IVS－5636或637T→del4例、G871A1例、G487A／T93C／C1311T1例。云南人群中存在93C／1311T、487A／93C／1311T两种单体型。IVS－11T93C突变产生的多态性NlaⅢ酶切位点与1311T呈现紧密关联。首次报道了IVS－5636或637T→del在中国人群中、G871A在中国大陆地区以及G487A在云南汉族的存在。结论：云南省G6PD缺乏症具有明显的异质性;G487A突变是该地区的常见G6PD基因突变,并可能有不同的起源;IVS－11T93C突变可能起源于非洲以外地区,并与C1311T共同作用而导致G6PD活性降低。G6PD基因突变型研究对疾病防治、民族起源及迁移等研究具有重要价值。     

Identification and computationally-based structural interpretation of naturally occurring variants of human protein C

Protein C (PC) is a key regulator of blood clotting and inflammation. Its inherited deficiency is associated with venous thromboembolism, and recombinant activated PC is currently used to increase survival in severe sepsis. The molecular basis of inherited PC deficiency is heterogeneous. Due to its multiple physiologic interactions and functions, and its modular structure, natural variants aid in the understanding of the relationship between critical residues and discrete functions. This knowledge has important therapeutic implications in the planning of a recombinant activated PC with a specific therapeutic target and devoid of major collateral effects. A way of predicting important functional consequences of residue variation is the use of molecular modeling and structural interpretation of amino acidic substitutions. A study of 21 out of 32 identified PC gene (PROC) variants is presented. For three of them, localized in the active site, electrostatic potential variation was calculated. For more than half of the studied variants, an explanation for the functional impairment could be derived from computational analysis, allowing a focused choice of which variants it is worthwhile pursuing.     

Molecular bases of antithrombin deficiency: twenty-two novel mutations in the antithrombin gene

Antithrombin (AT) is a major physiological inhibitor of hemostasis. We report 22 novel antithrombin gene (SERPINC1) mutations associated with antithrombin deficiency in 17 French and five German families. They were all present at the heterozygous state. Nine missense mutations accounted for type I deficiency, defined by equally low antithrombin activity and antigen level. Most of them (7/9) affected highly conserved serpin residues and were associated with venous thrombosis occurring at a young age (before age 32). One splice site, one nonsense mutation, three small deletions and one insertion were also identified as a cause for type I antithrombin deficiency. Seven other missense mutations were identified in type II or unclassified AT deficiency; g.5270C>T (p.T147I, T115I) and g.5281A>T (p.I151F, I119F) change residues in the heparin binding region, g.13267C>G (p.P439A, P407A) and g.13271T>C (p.F440S, F408S) affect amino acids in the pleiotropic region, g.2372G>A (p.G25D, G-8D) changes a signal peptide amino acid, g.2456G>C (p.C53S, C21S) affects one of the three disulfide bonds of the protein, and g.7585A>T (p.M347K, M315K) changes a nonconserved residue on strand 2C.     

DR3 and nonDR3 associated complement component C4A deficiency in systemic lupus erythematosus

The molecular basis of complement component C4A deficiency in white U.S. and Mexican patients with systemic lupus erythematosus (SLE) was studied. Genomic DNA from SLE patients and non-SLE controls was analyzed for restriction fragments using HindIII and a 5' C4 cDNA probe. C4A gene deletion was recognized by the loss of a 15-kb restriction fragment and the appearance of a 8.5-kb fragment. Thirty-two selected U.S. SLE patients, 7 nonSLE controls, and 11 Mexican SLE patients and 9 relatives were studied. The deletion was recognized in all of the 14 HLA-B8;DR3 SLE patients with a C4A protein deficiency. Two SLE patients with DR3 but without B8 also had this gene deletion. None of the 3 U.S. SLE nonDR3, C4A protein deficient patients nor 20 C4A protein deficient Mexican individuals (11 SLE patients and 9 relatives; none had B8 and/or DR3) showed this deletion. Thus the C4A gene deletion failed to account for the C4A protein deficiency in all the nonDR3 Mexicans and in some U.S. SLE patients. To determine whether gene conversion at the C4A locus would encode a C4B-like protein and be responsible for the C4A protein deficiency (in nonDR3 patients), the C4d region of the gene was amplified by polymerase chain reaction and subjected to Nla IV digestion, and restriction fragment analysis was performed using a C4d region-specific probe. The resulting restriction fragment length polymorphism pattern revealed no changes in the isotype-specific region of the gene as characterized by C4A-specific 276- and 191-bp fragments in Dr3 or nonDR3 individuals. Thus, homoexpression of C4B at both loci was not responsible for C4A deficiency in nonDR3 SLE patients without C4A gene deletion.     

The frequency of short-chain acyl-CoA dehydrogenase gene variants in the US population and correlation with the C(4)-acylcarnitine concentration in newborn blood spots

Short-chain acyl-CoA dehydrogenase (SCAD) deficiency is a clinically heterogeneous disorder. The clinical phenotype varies from fatal metabolic decompensation in early life to subtle adult onset, some patients remain asymptomatic. Two mutations (511C>T; 625G>A) have been described in exons 5 and 6 of the SCAD gene. Although they alter the structural and catalytic properties of the SCAD protein, these variants are not true disease-causing mutations but confer disease susceptibility. Previous studies found these gene variants to be common in Europeans. We aimed to establish the frequency of these variants in the US population and to determine whether the presence of these variants correlates with elevated butyrylcarnitine (C(4)-acylcarnitine) concentrations in newborn blood spots. Based on the analysis of 694 samples, we found that the allele frequency of the 625G>A variant was significantly higher (22%) than that of the 511C>T variant (3%). These gene variants were detected in either homozygous or compound heterozygous form in 7% of the study population. Additionally, the frequency of the 625G>A allele in the Hispanic population (30%) was significantly higher than that of the African-American (9%) and Asian (13%) subpopulations. A previously unreported variant, IVS 5 (-10) C>T, was identified in three African-American newborns (0.3%). The C(4)-acylcarnitine concentration in blood spots was significantly higher in subjects homozygous for the 625A variant when compared to those homozygous for the wild type (p<0.0001). However, none of the observed genotypes was associated with a concentration of C(4)-acylcarnitine that would be consistent with a biochemical diagnosis of SCAD deficiency.     

Hereditary protein C deficiency with recurrent thrombosis: identification of a missense mutation (C6218T).

Protein C is the central component of a major anti-thrombotic regulatory system and individuals with hereditary protein C deficiency (PCD) tend to have an increased risk of thromboembolism. During the last several years, mutations causing PCD have been identified in Western countries and in Japanese. In the present study, we report a case of hereditary PCD with a missense mutation (C6218T) in a 44 year old female with recurrent pulmonary thromboembolism. The protein C activity (35%) and antigen (58%) levels in this patient were decreased. Furthermore, we have identified the same molecular defect and PCD in other asymptomatic family members including proband''s mother and two daughters and one son. To our knowledge, this is the first case of hereditary PCD with identified genetic defect in the Korean population, which may be one of hot spots for mutation in the protein C gene.     

Analysis of Ig gene hypermutation in Ung−/−Polh−/− mice suggests that UNG and A:T mutagenesis pathway target different U:G lesions

The activation-induced cytidine deaminase (AID) initiates Ig gene hypermutation by converting cytosine to uracil (U) and generating a U:G lesion. Genetic and biochemical studies suggest that the AID-triggered U:G lesions are processed by three mutagenic pathways to induce mutations at both C:G and A:T pairs. First, direct replication of the U:G lesion leads to C to T and G to A transitions. Second, U can be excised by the uracil DNA glycosylase (UNG) and the replication/processing of the resulting abasic site leads to transversions and transitions at C:G pairs. Third, the U:G lesion is recognized by an atypical mismatch repair (MMR) pathway which generates mutations at A:T pairs in a DNA polymerase η (POLH)-dependent manner. To further explore whether these three mutagenic pathways function competitively or independently, we have analyzed Ig gene hypermutation in mice deficient in both UNG and POLH. Compared with WT mice, UNG deficiency caused elevated frequency of C:G mutations, suggesting that UNG-mediated U excision led to error-free as well as error-prone repair. In contrast, UNG deficiency did not affect the frequency and patterns of A:T mutations, suggesting that the MMR did not target U:G lesions normally recognized and processed by UNG. In addition, POLH deficiency did not affect the frequency and patterns of C:G mutations and UNG POLH double deficiency showed an additive effect of single deficiency. Based on these observations and previous results, along with the recent finding that UNG excises AID-triggered U predominantly during G1 phase of the cell cycle, it appears that UNG and MMR targets U:G lesions generated during G1 and S phases of the cell cycle, respectively.     

Analysis of Ig gene hypermutation in Ung(-/-)Polh(-/-) mice suggests that UNG and A:T mutagenesis pathway target different U:G lesions

The activation-induced cytidine deaminase (AID) initiates Ig gene hypermutation by converting cytosine to uracil (U) and generating a U:G lesion. Genetic and biochemical studies suggest that the AID-triggered U:G lesions are processed by three mutagenic pathways to induce mutations at both C:G and A:T pairs. First, direct replication of the U:G lesion leads to C to T and G to A transitions. Second, U can be excised by the uracil DNA glycosylase (UNG) and the replication/processing of the resulting abasic site leads to transversions and transitions at C:G pairs. Third, the U:G lesion is recognized by an atypical mismatch repair (MMR) pathway which generates mutations at A:T pairs in a DNA polymerase η (POLH)-dependent manner. To further explore whether these three mutagenic pathways function competitively or independently, we have analyzed Ig gene hypermutation in mice deficient in both UNG and POLH. Compared with WT mice, UNG deficiency caused elevated frequency of C:G mutations, suggesting that UNG-mediated U excision led to error-free as well as error-prone repair. In contrast, UNG deficiency did not affect the frequency and patterns of A:T mutations, suggesting that the MMR did not target U:G lesions normally recognized and processed by UNG. In addition, POLH deficiency did not affect the frequency and patterns of C:G mutations and UNG POLH double deficiency showed an additive effect of single deficiency. Based on these observations and previous results, along with the recent finding that UNG excises AID-triggered U predominantly during G1 phase of the cell cycle, it appears that UNG and MMR targets U:G lesions generated during G1 and S phases of the cell cycle, respectively.     

Molecular effects of the tissue-nonspecific alkaline phosphatase gene polymorphism (787T > C) associated with bone mineral density

Based on studies of hypophosphatasia, which is a systemic skeletal disorder resulting from tissuenonspecific alkaline phosphatase (TNSALP) deficiency, TNSALP was suggested to be indispensable for bone mineralization. Recently, we demonstrated that there was a significant difference in bone mineral density (BMD) among haplotypes, which was lowest among TNSALP (787T [Tyr-246Tyr]) homozygotes, highest among TNSALP (787T > C [Tyr246His]) homozygotes, and intermediate among heterozygotes. To analyze protein translated from the TNSALP gene 787T > C, we performed the biosynthesis of TNSALPs using TNSALP cDNA expression vectors. TNSALP (787T) and TNSALP (787T > C) were synthesized similarly as a high-mannose-type 66-kDa form, becoming an 80-kDa form. Expression of the human 787T > C TNSALP gene using the cultured mouse marrow stromal cell line ST2 demonstrated that the protein translated from 787T > C exhibited an ALP-specific activity similarly to that of 787T. Interestingly, the Km value for TNSALP in ST2 cells transfected with the 787T > C TNSALP gene was decreased significantly compared to that of cells carrying the 787T gene (P < 0.01). These results suggest that the significant difference in Km values between the proteins translated from 787T > C and 787T may contribute to regulatory effects on bone metabolism.     

Two novel mutations in the C7 gene in a Korean patient with complement C7 deficiency

Complement C7 deficiency is an autosomal recessive disorder well known to be associated with increased susceptibility to meningococcal infection and has mostly been reported in Caucasians. In the Korean population, no case of C7 deficiency has been reported to date. Recently we experienced an 11-yr-old girl with meningococcal meningitis who was diagnosed as having C7 deficiency based upon the undetectable serum C7 protein on radial immunodiffusion and the undetectable serum total and C7 hemolytic activities. To identify the genetic basis of the C7 deficiency of the patient, we performed a mutation analysis for the C7 gene and found two novel mutations; a point mutation at the 3'' splice acceptor site of intron 4 (c.281-1G>T) and a large deletion mutation encompassing almost the whole C7 gene from exon 1 to exon 17 (c.1-?_2350+?del). A haplotype analysis showed that the large deletion mutation was inherited from the patient''s father. To the best of our knowledge, this is the first confirmed case of C7 deficiency in Korea.     

Lack of evidence of a specific role for C4A gene deficiency in determining disease susceptibility among C4-deficient patients with systemic lupus erythematosus (SLE)

The aim of the present study was to investigate the prevalence of C4 and C2 deficiencies and to characterize genomic alterations in C4 genes in a large cohort of 125 unselected patients with SLE. We determined the protein concentration and functional activity of C2 and C4, as well as the C4 phenotype. C4 genotyping included Taq 1 restricted fragment lengh polymorphism (RFLP) analysis and polymerase chain reaction using sequence-specific primers (SSP-PCR). Type I C2 deficiency was diagnosed by PCR. Overall, 79.2% of the patients exhibited abnormalities of the C4 genes including deletion, non-expression, gene conversion and duplication. Among C4-deficient patients (n = 66, 52.8% prevalence), 41.0% of the patients exhibited a C4A deficiency and 59.0% a C4B deficiency. Half of the C4 deficiencies were due to a gene deletion. There was a strong association between C4A and C4B gene deletion and the presence of the DRB1*03 allele. Among the silent C4A genes, only two cases were related to a 2-bp insertion in exon 29 of the C4A gene. A gene conversion was demonstrated in eight patients (6.4%). One patient had a homozygous C4A deficiency. Three (2.4%) patients presented with a heterozygous type I C2 deficiency and none with homozygous deficiency. Our results argue against a specific role for C4A gene deficiency in determining disease susceptibility among patients with SLE that are C4-deficient.     

Molecular defects of the C7 gene in two patients with complement C7 deficiency

Different genetic mutations have been described in complement components resulting in total or subtotal deficiency states. In this work we report the genetic basis of C7 deficiency in a previously reported Spanish patient exhibiting a combined total deficiency of C7 and C4B associated with systemic lupus erythematosus. Exon-specific polymerase chain reaction and sequencing revealed a not previously described single base mutation in exon 10 (T1458A) leading to a stop codon that causes the premature truncation of the C7 protein (C464X). Additionally, a C to A transversion at position 1561 (exon 11) was found in the patient resulting in an amino acid change (R499S). This latter mutation has been previously reported in individuals with subtotal C7 deficiency or with combined subtotal C6/C7 deficiency from widely spaced geographical areas. Another novel mutation was found in a second patient with meningococcal meningitis of Bolivian and Czech origin; a 11-base pair deletion of nucleotides 631-641 in exon 6 leading to the generation of a downstream stop codon causing the premature truncation of the C7 protein product (T189 x 193). This patient was found to be a heterozygous compound for another mutation in C7; a two-base pair deletion of nucleotides 1922 and 1923, 1923 and 1924 or 1924 and 1925 in exon 14 (1922delAG/1923delGA/1924delAG), leading again to the generation of a downstream stop codon that provokes the truncation of the C7 protein (S620x630). This latter mutation has been recently reported by our group in another Spanish family. Our results provide more evidences for the heterogeneous molecular basis of C7 deficiency.     

C6 haplotypes: associations of a Dde I site polymorphism to complement deficiency genes and the Msp I restriction fragment length polymorphism (RFLP)

Complement C6 has a common charge polymorphism designated A and B with gene frequencies of 0.65 and 0.35. The probable molecular basis for this is a Glu (C6A) for Ala (C6B) substitution at amino acid position 98, and is detected by digestion with the restriction enzyme Dde I of a polymerase chain reaction (PCR)-amplified fragment of genomic DNA. C6A was found to be Dde I-positive and C6B corresponds to Dde I-negative. We have applied our Dde I A/B polymorphism genotyping method to the investigation of C6-deficient individuals with complete (C6Q0) and sub-total deficiency (C6SD) protein phenotypes, including members of four families. We have also investigated the RFLP detected by digestion of genomic DNA with the enzyme Msp I, which is due to a polymorphic site located in the 5' section of the gene, the variable sequence of which has yet to be determined. Sixteen out of seventeen unrelated C6Q0 subjects were found to be genotypically Dde I B/Msp I-negative; the remaining subject was heterozygous at both the loci under investigation. The C6SD phenotype was found to be associated with the Dde I A/Msp I-positive genotype in two families with combined C6/C7 subtotal deficiency and two with C6SD. It can be concluded that the two forms of C6 deficiency, C6Q0 and C6SD, arose independently on two different C6 allelic backgrounds. These associations have allowed the genotyping of the rare families that contain both types of deficiency. We have also defined a number of normal C6 Dde I/Msp I haplotypes in Caucasians and Cape Coloured populations.     

Protein C deficiency: identification of a novel two-base pair insertion and two point mutations in exon 7 of the protein C gene in Spanish families.

We have applied single-strand conformation polymorphism (SSCP) to the analysis of exon 7 of the anticoagulant protein C (PC) gene, in 13 PC-deficient Spanish families. Abnormal patterns were visualized in three samples from type I or quantitative PC deficient proposita. A previously undescribed mutation due to a TT insertion after nucleotide 6139, between codons Gly-142 and Arg-143 was found in one family. The mutation (6139,ins TT) should result in a frameshift with a stop at codon 156, which agrees with the presence of a type I or quantitative PC deficiency in the affected members of the family. The second mutation identified was a C to T transition at nucleotide 6274, 9 base pairs into intron G. This mutation (6274,C-->T), found for the first time in a Spanish family, is identical to the previously characterized PC Sant Louis. The third mutation was a G to A transition that replaces arginine 178 with glutamine (178,R-->Q). This is the third case of 178,R-->Q mutation in 17 apparently unrelated Spanish families with type I PC deficiency. Furthermore, SSCP analysis allowed the detection of another previously described mutation in a PC-deficient Spanish family (178,R-->W).