Presence of C1-Inhibitor Polymers in a Subset of Patients Suffering from Hereditary Angioedema

Hereditary angioedema (HAE) is a potentially life-threatening disease caused by mutations in the gene encoding the serine protease inhibitor (serpin) C1 inhibitor (C1-inh). The mutations cause decreased functional plasma levels of C1-inh, which triggers unpredictable recurrent edema attacks. Subjects suffering from HAE have been classified in type I patients with decreased functional and antigenic levels of C1-inh, and type II patients with decreased functional but normal antigenic C1-inh levels. However, a few reports have demonstrated that some mutations cause C1-inh polymerization in vitro, and it is speculated that C1-inh polymers may exist in patient plasma, challenging the current classification of HAE patients. To investigate the presence of C1-inh polymers in patient plasma samples, we developed an immunological method, where monoclonal antibodies produced against polymerized C1-inh were applied in native PAGE western blotting. Using this approach we analyzed genuine plasma samples from 31 Danish HAE families, and found that plasma samples from three genotypically distinct HAE type I families (classified upon C1-inh plasma concentrations) contained C1-inh polymers. Identical C1-inh polymerization phenotypes were observed in four affected family members from one of these families. Genotyping of the families revealed that the polymerogenic mutations of two families were located in proximity to the reactive center loop insertion site in C1-inh (p.Ile271Thr and p.Ser258_Pro260del),and one mutation affected helix C (p.Thr167Asn). In conclusion, we demonstrate that C1-inh polymers are present in the plasma of a subgroup of HAE type I patients.     

Molecular analysis of three novel G6PD variants: G6PD Pedoplis-Ckaro, G6PD Piotrkow and G6PD Krakow

We present three novel mutations in the G6PD gene and discuss the changes they cause in the 3-dimensional structure of the enzyme: 573C-->G substitution that predicts Phe to Leu at position 191 in the C-terminus of helix alphae, 851T-->C mutation which results in the substitution 284Val--> -->Ala in the beta+alpha domain close to the C-terminal part of helix alphaj, and 1175T-->C substitution that predicts Ile to Thr change at position 392.     

An in vivo approach to identifying sequence context of 8-oxoguanine mutagenesis

Base substitution mutations are not distributed randomly in that most are located at a few specific hotspots sites. We have been studying 7,8-dihydro-8-oxoguanine mutagenesis in Escherichia coli in the supF gene carried in a plasmid. Among hotspots, guanine within the 5'-AGA-3' located in the anticodon site was susceptible to the induction of G:C-->T:A transversion. In this study, we constructed variants of the supF gene in which the hotspot 5'-AGA-3' was modified to 5'-AGT-3', 5'-AGG-3' and 5'-AGC-3' to determine the influence of 3' neighboring base on G:C-->T:A mutational activity. Using these variant supF genes propagated in a 7,8-dihydro-8-oxoguanine repair-deficient host, we found that guanine within 5'-AGA-3' and 5'-AGG-3' produce G:C-->T:A, but guanine within 5'-AGT-3' and 5'-AGC-3' reduce the formation of G:C-->T:A. These changes were thus due to the effect of sequence context on the efficiency of mutation formation at the sites of 7,8-dihydro-8-oxoguanine. We also observed a longer range base-pair effect on hotspot formation.     

A mutation unique in serine protease inhibitors (serpins) identified in a family with type II hereditary angioneurotic edema.

BACKGROUND: Hereditary angioneurotic edema (HANE) is an autosomal dominant disease due to genetic alterations at the C1 inhibitor gene. Mutations within the C1 inhibitor gene are responsible for the molecular defect in type II HANE. Most of the dysfunctional proteins result from mutations involving the Arg-444 (the P-1 site of the reactive center) or amino acids NH2-terminal to the reactive center. MATERIALS AND METHODS: We have studied a Spanish family with type II HANE by using polymerase chain reaction (PCR) to amplify the exon eight of the C1 inhibitor gene. The purified 338-bp PCR product was subcloned and transformed into competent cells. After overnight cultures, we extracted the cloning vector from the positive colonies and sequenced both strands of the PCR product from each patient and healthy members of the family. RESULTS: We show that affected individuals in this family have a missense mutation, changing an adenine to cytosine in the codon 445. This substitution changes threonine at the P-1' site of the reactive center to a proline. This mutation generates a new restriction site, recognized by Bsi YI. CONCLUSIONS: To our knowledge, this is the first molecular defect characterized in a Spanish family with type II HANE, and to date, this is the first reported mutation at the P-1' site of the reactive center in individuals with type II HANE. This new mutation located at the reactive center emphasizes once more time the enormous heterogeneity of this gene.     

Identification of a new P1 residue mutation (444Arg----Ser) in a dysfunctional C1 inhibitor protein contained in a type II hereditary angioedema plasma

A new reactive-centre P1 residue mutation (444Arg----Ser), has been identified in a dysfunctional C1 inhibitor protein, C1 inhibitor(Ba), contained in a type II hereditary angioedema plasma. This substitution is compatible with a point mutation of the 444Arg codon (CGC----AGC), and represents the first non-histidine, non-cysteine P1 residue mutant described for C1 inhibitor.     

Hereditary Angioedema Due to C1 Inhibitor Deficiency in Serbia: Two Novel Mutations and Evidence of Genotype-Phenotype Association

Hereditary angioedema due to C1 inhibitor deficiency (C1-INH-HAE) is a rare autosomal dominant disease characterized by recurrent life-threatening oedemas and/or abdominal pain and caused by mutations affecting the C1 inhibitor gene, SERPING1. We sought to investigate the spectrum of SERPING1 mutations in Serbia and the possible genotype-phenotype association. C1-INH-HAE was diagnosed on the basis of clinical and laboratory criteria in 40 patients from 27 families; four were asymptomatic. Mutational analysis of the SERPING1 gene was performed by sequencing and multiplex ligation-dependent probe amplification. Disease-causing mutations in SERPING1 were identified in all patients. In C1-INH-HAE type I, we identified 19 different mutations, including 6 missense mutations, 6 nonsense mutations, 2 small deletions, 1 small insertion, 2 splicing defects and 2 large deletions. Two of the mutations (c.300C>T and c.1184_1185insTA) are reported here for the first time. All C1-INH-HAE type II patients from three families harboured the same substitution (c.1396C>T). Based on the type of mutation identified in the SERPING1 gene, patients were divided into two groups: group 1 (nonsense, frameshift, large deletions/insertions, splicing defect, and mutations at Arg444) or group 2 (missense, excluding mutations at Arg444). Significant differences were found in the clinical severity score (P = 0.005), prevalence of laryngeal (P = 0.040) and facial (P = 0.013) oedema, and long-term prophylaxis (P = 0.023) between the groups with different types of mutations. Because our population consisted of related subjects, differences in the severity score between mutation groups were further confirmed using the generalized estimating equation (P = 0.038). Our study identified 20 different disease-causing mutations, including two novel mutations, in all C1-INH-HAE patients, highlighting the heterogeneity of mutations in the SERPING1 gene. Furthermore, it appears that mutations with a clear effect on C1-INH function might be responsible for a more severe disease phenotype.     

Recombinational biases in the rearranged C1-inhibitor genes of hereditary angioedema patients

DNA structural changes responsible for hereditary angioedema were sought in the C1-inhibitor gene, which contains unusually dense clusters of Alu repeats in various orientations. Among patients belonging to 45 unrelated families, eight partial C1-inhibitor gene deletions and a partial duplication were found. Four deletions had one of the boundaries within the gene and the other in extragenic regions--in three cases 5' of the gene and in one case 3' of the gene. The boundaries of the partial duplication and of the remaining four deletions mapped instead within a few kilobases of exon 4. The same element--Alu 1--the first of three tandem Alu repeats preceding exon 4, contained one of the breakpoints of each of these five rearrangements. Moreover, these recombination breakpoints spread over the entire length of Alu 1, in contrast with the tight clustering observed near the 5' end of Alu sequences rearranged in other human genes. Thus, two uncommon recombinational biases are observed in the Alu rearrangements of hereditary angioedema patients; one promotes the occurrence of intragenic breakpoints in a single Alu repeat, and the other allows the breaks to be distributed over the entire Alu structure rather than within the hot spot of the left Alu monomer. A region of potential Z-DNA structure, located 1.7 kb upstream of Alu 1, may contribute to both peculiarities.     

SURF1 gene mutations in Polish patients with COX-deficient Leigh syndrome

One of the most frequent forms of Leigh syndrome (LS), a severe neurodegenerative, genetically heterogenous disease, is associated with cytochrome c oxidase (COX) deficiency. No mutations in any of the 13 polypeptide subunits of human COX have been detected in LS patients. Recently, SURF1, a positional candidate gene for LS has been identified on chromosome 9q34. We present the identification of SURF1 mutations in a randomly chosen group of Polish patients with a classical form of LS. Sequence analysis revealed the presence of a novel 704T-->C transition (Met235Thr), and two recurrent dinucleotide deletions (758delCA, 845delCT), as well as one novel polymorphic 573C-->G transversion (Thr191Thr). 845delCT was identified in 66% of all our patients in homozygous or heterozygous form. Our study confirms the recent observations that SURF1 is consistently involved in disorders of the mitochondrial respiratory chain in patients with typical Leigh syndrome.     

Genetic suppression of intronic +1G mutations by compensatory U1 snRNA changes in Caenorhabditis elegans

Mutations to the canonical +1G of introns, which are commonly found in many human inherited disease alleles, invariably result in aberrant splicing. Here we report genetic findings in C. elegans that aberrant splicing due to +1G mutations can be suppressed by U1 snRNA mutations. An intronic +1G-to-U mutation, e936, in the C. elegans unc-73 gene causes aberrant splicing and loss of gene function. We previously showed that mutation of the sup-39 gene promotes splicing at the mutant splice donor in e936 mutants. We demonstrate here that sup-39 is a U1 snRNA gene; suppressor mutations in sup-39 are compensatory substitutions in the 5' end, which enhance recognition of the mutant splice donor. sup-6(st19) is an allele-specific suppressor of unc-13(e309), which contains an intronic +1G-to-A transition. The e309 mutation activates a cryptic splice site, and sup-6(st19) restores splicing to the mutant splice donor. sup-6 also encodes a U1 snRNA and the mutant contains a compensatory substitution at its 5' end. This is the first demonstration that U1 snRNAs can act to suppress the effects of mutations to the invariant +1G of introns. These findings are suggestive of a potential treatment of certain alleles of inherited human genetic diseases.     

山羊生长激素基因5调控区的多态性分析

以鲁北白山羊、引进波尔山羊、纯繁波尔山羊以及鲁北白山羊与波尔山羊的杂交一代、回交一代共计274个个体为研究材料,用两对引物分别扩增山羊生长激素(GH)基因5^'区的26－239bp以及225－429bp片段,扩增产物经SSCP分析发现均存在多态性。在26－239bp片段上,波尔山羊及杂交后代以 AA型个体占多数,而鲁北白山羊则BB型个体较多;在225－429bp片段上,所有种群均以 CC型个体较多。对两个片段的纯合型（AA,BB;CC,DD）分别克隆测序发现：（1）26－239bp片段上AA型在第60位发生了C→T的突变,第211位发生碱基C的丢失,（2）225－429bp片段上,DD型存在3处突变,分别为264位由T→C,292位由T→A,372位由C→T。上述结果为首次实验证实山羊生长激素5^'调控区存在序列多态性。     

Specificity of spontaneous mutations induced in mutA mutator cells

Escherichia coli cells expressing the mutA allele of a glyV (glycine tRNA) gene express a strong mutator phenotype. The mutA allele differs from the wild type glyV gene by a base substitution in the anticodon such that the resulting tRNA misreads certain aspartate codons as glycine, resulting in random, low-level Asp-->Gly substitutions in proteins. Subsequent work showed that many types of mistranslation can lead to a very similar phenotype, named TSM for translational stress-induced mutagenesis. Here, we have determined the specificity of forward mutations occurring in the lacI gene in mutA cells as well as in wild type cells. Our results show that in comparison to wild type cells, base substitutions are elevated 23-fold in mutA cells, as against a eight-fold increase in insertions and a five-fold increase in deletions. Among base substitutions, transitions are elevated 13-fold, with both G:C-->A:T and A:T-->G:C mutations showing roughly similar increases. Transversions are elevated 35-fold, with G:C-->T:A, G:C-->C:G and A:T-->C:G elevated 28-, 13- and 27-fold, respectively. A:T-->T:A mutations increase a striking 348-fold over parental cells, with most occurring at two hotspot sequences that share the G:C-rich sequence 5'-CCGCGTGG. The increase in transversion mutations is similar to that observed in cells defective for dnaQ, the gene encoding the proofreading function of DNA polymerase III. In particular, the relative proportions and sites of occurrence of A:T-->T:A transversions are similar in mutA and mutD5 (an allele of dnaQ) cells. Interestingly, transversions are also the predominant base substitutions induced in dnaE173 cells in which a missense mutation in the alpha subunit of polymerase III abolishes proofreading without affecting the 3'-->5' exonuclease activity of the epsilon subunit.     

Analyses of genetic abnormalities in type I CD36 deficiency in Japan: identification and cell biological characterization of two novel mutations that cause CD36 deficiency in man

To elucidate genetic abnormalities in type I CD36 deficiency, we analyzed 28 Japanese subjects whose platelets and monocytes/macrophages lacked CD36 on their surface. We identified two novel mutations in the CD36 gene. One was a complex deletion/insertion mutation, in which 3 bp, GAG, were deleted at nucleotide (nt) 839-841, and 5 bp, AAAAC, were inserted at the same position (839-841del-->insAAAAC). Mutation 839-841del-->insAAAAC led to a frameshift and appearance of a premature stop codon; it was also accompanied with a marked reduction in the amount of CD36 mRNA. The other was a 12-bp deletion at nt 1438-1449 (1438-1449del) accompanied with or without skipping of exon 9 (nt 959-1028). Mutation 1438-1449del led to an inframe 4-amino-acid deletion, whereas exon 9 skipping led to a frameshift and the appearance of a premature stop codon. Expression assay revealed that both 1438-1449del and exon 9 skipping directly caused impairment of the surface expression of CD36. A survey of the five known mutations including 839-841del-->insAAAAC and 1438-1449del in type I CD36-deficient subjects demonstrated that the five mutations covered more than 90% of genetic defects among them and that the substitution of T for C at nt 478 (478C-->T) was the most common mutation with more than 50% frequency. However, none of the four subjects that possessed isoantibodies against CD36 had 478C-->T, suggesting that 478C-->T prevents the production of isoantibodies against CD36.     

Association of the polymorphisms in the 5'-untranslated region of PTEN gene with type 2 diabetes in a Japanese population

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is known to act as a lipid phosphatase hydrolyzing phosphatidylinositol (PI)(3,4,5)P(3) to PI(4,5)P(2). Since the PI3-kinase product, PI(3,4,5)P(3), is an important second messenger leading to the metabolic action of insulin, PTEN functions as a potent negative regulator of insulin signaling and its gene is one of the possible candidates involved in susceptibility to the development of type 2 (non-insulin-dependent) diabetes. In the present study, we investigated the polymorphisms of the PTEN gene in Japanese patients with type 2 diabetes and non-diabetic control subjects. We identified three mutations of the gene in the type 2 diabetes patients. Among these mutations, the frequency of the substitution of C with G at position -9 (-9C-->G) (SNP1), located in the untranslated region of exon 1, was significantly higher in type 2 diabetic patients than in control subjects. In addition, transfection of the PTEN gene with SNP1 resulted in a significantly higher expression level of PTEN protein compared with that of the wild-type PTEN gene in Cos1 and Rat1 cells. Furthermore, insulin-induced phosphorylation of Akt in HIRc cells was decreased more greatly by transfection of SNP1 PTEN gene than that of wild-type PTEN gene. These findings suggest that the change of C to G at position -9 of the PTEN gene is associated with the insulin resistance of type 2 diabetes due possibly to a potentiated hydrolysis of the PI3-kinase product.     

Characterization of missense mutations and large deletions in the ALPL gene by sequencing and quantitative multiplex PCR of short fragments

Hypophosphatasia is a rare inherited bone disorder characterized by defective bone and dental mineralization and deficiency of serum and liver/bone/kidney alkaline phosphatase activity. The disease is due to mutations in the alkaline phosphatase liver-type (ALPL) gene. Gross deletions or insertions have not previously been reported in this gene. We report here the characterization of nine novel ALPL gene mutations in a series of 8 patients affected by various forms of hypophosphatasia. The newly discovered mutations included five missense mutations (c.368C --> A, c.814C--> T, c.1196C--> T, c.1199C--> T, c.1283G--> C), two small deletions (c.797_802del, c.1044_1055del), and two large deletions. The large deletions were detected by quantitative multiplex polymerase chain reaction (PCR) of short fluorescent fragments (QMPSF). We conclude that QMPSF slightly reduces the proportion of undetected mutations in hypophosphatasia and improves genetic counselling in the affected families.     

Mutation screening of the FSH receptor gene in infertile men.

Follicle stimulating hormone (FSH) is important for controlling spermatogenesis through binding with its receptor. However, little information is available on mutations of the FSH and its receptor gene in infertile men. To study the genetic defects, which caused problems in spermatogenesis, we screened the point mutations of the FSH receptor gene in infertile men with high serum FSH concentrations. Seventy male infertile patients with high FHS levels (> 12 mIU/ml) were screened for mutations in each of the 10 exons of the FSH receptor gene, using genomic DNA PCR and a single-strand conformation polymorphism (SSCP) analysis. From this study, three shifted bands were detected by SSCP. The first shifted band was found in the PCR product of exon 4, including the exon-intron boundary sequence in only one patient. The sequence analysis revealed a nucleotide A to T substitution in intron 3 (IVS3-4A-->T). The second shifted band was detected in exon 10 with high frequency (33%). A nucleotide A to G substitution was found at the position of the 994th nucleotide, predicting a Thr to Ala substitution at the position of the 307th amino acid (Thr307Ala). The third shifted band in the 3' region of exon 10 was detected frequently in infertile patient and normal groups. It was tightly linked to the Thr307Ala variant. Thus, all of the abnormalities represent neutral polymorphisms, and not pathological mutations of the FSH receptor gene. In conclusion, we did not confirm that the genomic mutation of the FSH receptor is a major genetic cause in Korean infertile patients with high FSH levels.     

Identification of mutations in cystatin B, the gene responsible for the Unverricht-Lundborg type of progressive myoclonus epilepsy (EPM1).

Progressive myoclonus epilepsy (EPM1) is an autosomal recessive disorder, characterized by severe, stimulus-sensitive myoclonus and tonic-clonic seizures. The EPM1 locus was mapped to within 0.3 cM from PFKL in chromosome 21q22.3. The gene for the proteinase inhibitor cystatin B was recently localized in the EPM1 critical region, and mutations were identified in two EPM1 families. We have identified six nucleotide changes in the cystatin B gene of non-Finnish EPM1 families from northern Africa and Europe. The 426G-->C change in exon 1 results in a Gly4Arg substitution and is the first missense mutation described that is associated with EPM1. Molecular modeling predicts that this substitution severely affects the contact of cystatin B with papain. Mutations in the invariant AG dinucleotides of the acceptor sites of introns 1 and 2 probably result in abnormal splicing. A deletion of two nucleotides in exon 3 produces a frameshift and truncates the protein. Therefore, these four mutations are all predicted to impair the production of functional protein. These mutations were found in 7 of the 29 unrelated EPM1 patients analyzed, in homozygosity in 1, and in heterozygosity in the others. The remaining two sequence changes, 431G-->T and 2575A-->G, probably represent polymorphic variants. In addition, a tandem repeat in the 5' UTR (CCCCGCCCCGCG) is present two or three times in normal alleles. It is peculiar that in the majority of patients no mutations exist within the exons and splice sites of the cystatin B gene.     

Mutational analysis of the defective protease in classic late-infantile neuronal ceroid lipofuscinosis, a neurodegenerative lysosomal storage disorder.

The late-infantile form of neuronal ceroid lipofuscinosis (LINCL) is a progressive and ultimately fatal neurodegenerative disease of childhood. The defective gene in this hereditary disorder, CLN2, encodes a recently identified lysosomal pepstatin-insensitive acid protease. To better understand the molecular pathology of LINCL, we conducted a genetic survey of CLN2 in 74 LINCL families. In 14 patients, CLN2 protease activities were normal and no mutations were identified, suggesting other forms of NCL. Both pathogenic alleles were identified in 57 of the other 60 LINCL families studied. In total, 24 mutations were associated with LINCL, comprising six splice-junction mutations, 11 missense mutations, 3 nonsense mutations, 3 small deletions, and 1 single-nucleotide insertion. Two mutations were particularly common: an intronic G-->C transversion in the invariant AG of a 3' splice junction, found in 38 of 115 alleles, and a C-->T transition in 32 of 115 alleles, which prematurely terminates translation at amino acid 208 of 563. An Arg-->His substitution was identified, which was associated with a late age at onset and protracted clinical phenotype, in a number of other patients originally diagnosed with juvenile NCL.