Factor XIII ABristol 1: detection of a nonsense mutation (Arg171→+stop codon) in factor XIII A subunit deficiency

Summary Molecular analysis has been performed on a patient with coagulation factor XIII A subunit deficiency. A previously published genomic sequence indicates that exon 3 of the factor XIII A subunit gene contains two TaqI restriction sites within which arginine (CGA)→stop (TGA) nonsense mutations are possible. Oligonucleotide primers were therefore used to amplify exon 4 by the polymerase chain reaction. TaqI digestion of the 326 base pair (bp) product derived from normal genomic DNA yielded expected fragments of 244, 73 and 9 bp in size. In the case of the patient, however, an additional fragment of 253 bp was present. Direct sequence analysis showed that the 5' TaqI site had been lost from one allele by a C→T transition at nucleotide 598. Family studies demonstrated the mutation in the patient's father but no other first-degree relatives. This is the third independent mutation described in the factor XIII A subunit gene and the first to be identified in a patient compound heterozygous for the disorder.     

Structural analysis of a missense mutation (Val414Phe) in the catalytic core domain of the factor XIIIA subunit

Molecular analysis has been performed on a Malaysian patient with a severe bleeding disorder due to factor XIII_A subunit deficiency. Total mRNA was isolated from the patient's leucocytes and four overlapping segments corresponding to the entire coding region of the A subunit cDNA were amplified by RT-PCR. The cDNA segments amplified efficiently and were of expected size. Direct sequencing of the complete reading frame revealed a single homozygous base change (nt 1327G→T) in exon 10 corresponding to a missense mutation, Val414Phe, in the catalytic core domain of the A subunit monomer. The mutation eliminates a BsaJ1 restriction site and family screening showed that both parents were heterozygous for the defect. The base substitution was absent in 55 normal individuals. Val414 is a highly conserved residue in the calcium-dependent transglutaminase enzyme family. Computer modelling based on 3D crystallographic data predicts that the bulky aromatic side chain of the substituted phenylalanine residue distorts protein folding and destabilizes the molecule. In addition, conformation changes in the adjacent catalytic and calcium binding regions of the A subunit are likely to impair the enzymatic activity of any protein synthesized.     

Novel Y283C mutation of the A subunit for coagulation factor XIII: molecular modelling predicts its impaired protein folding and dimer formation

In an Italian patient with severe factor XIII deficiency, a novel mutation, Y283C (TAT to TGT), was identified heterozygously by nucleotide sequencing analysis in exon VII of the gene for the A subunit. The presence of this mutation was confirmed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis in the proband and his brother. Molecular modelling predicts that the mutant molecule would be misfolded. It is probable that the impaired folding of the mutant Y283C A subunit led to its instability, which is at least in part responsible for the factor XIII deficiency of this patient.     

A complex heterozygous mutation of His373Leu and Asp487-Ser488-Phe489 deletion in human cytochrome P450c17 causes 17alpha-hydroxylase/17,20-lyase deficiency in three Chinese sisters

Cytochrome P450c17 deficiency is one of the rare forms of enzyme disorders in steroid biosynthesis, resulting from defects in 17alpha-hydroxylase and 17,20-lyase activities. The disease is caused by the mutations in CYP17 gene, inherited in an autosomal recessive pattern. We reported a Chinese family with three sisters suffering from P450c17 deficiency based on their clinical features and molecular genetics. The patients were found to be compound heterozygotes with two different mutations. Screened by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), a heterozygous point mutation His373Leu was detected in the exon 6 of CYP17 gene which was proved to be derived from paternal allele. The other allele contained nine-base pair deletion, located in exon 8, eliminating codons 487-489 (Asp-Ser-Phe) near the carboxy-terminus of P450c17. The mother and the brother have been demonstrated to be carriers of deletion mutation through restriction enzyme analysis. Both mutations have been reported previously in Asia. This is the first report of the molecular genetic study of 17alpha-hydroxylase/17,20-lyase deficiency in mainland China with a novel compound heterozygous mutation.     

Novel point mutations in the αIIb subunit (Phe289 → Ser, Glu324 → Lys and Gln747 → Pro) causing thrombasthenic phenotypes in four Japanese patients

We analysed the molecular basis of Glanzmann thrombasthenia (GT) in four Japanese patients with type I or type II disease. Polymerase chain reaction (PCR) and subsequent direct sequencing of platelet RNA and genomic DNA revealed three single nucleotide substitutions of the αIIb gene, which were confirmed by allele-specific PCR or restriction analysis. One patient with type I GT had a T to C base substitution in exon 11 resulting in a Phe (TTT)-289 to Ser (TCT) mutation (F289S) of the subunit. Another type I patient had a G to A base substitution in exon 12 resulting in a Glu (GAA)-324 to Lys (AAA) mutation (E324K). Interestingly, two unrelated patients with type II GT shared an A to C base substitution in exon 23, a region previously not associated with GT, resulting in a Gln (CAA)-747 to Pro (CCA) mutation (Q747P). To analyse the effects of these mutations on αIIbβ3 surface expression, the wild-type αIIb cDNA or mutant αIIb cDNAs were transfected into Chinese hamster ovary (CHO) cells together with a wild-type β3 cDNA. Flow cytometric analysis using an anti-αIIbβ3 complex antibody revealed that 50.6% of CHO cells with wild-type αIIbβ3 expressed complexes, whereas only 1.6%, 7.7% and 31.3% of cells, with αIIb(F289S)β3, αIIb(E324K)β3 and αIIb(Q747P)β3 expressed complexes, respectively. Our data indicate that these three novel point mutations in the αIIb subunit may hamper surface expression of the αIIbβ3 complex, thus resulting in the quantitative GT phenotypes of platelets from these patients.     

A complete deficiency of coagulation factor XIII A-subunit due to a novel compound heterozygote of Ser 413 Leu missense and an nt 389 (ins G) frameshift mutation

Coagulation factor XIII consists of two A- and two B-subunits, and either gene mutation can cause a complete deficiency. In a newborn patient with persistent bleeding from the umbilical cord stump, the plasma A-subunit protein was not detectable. Direct PCR sequencing revealed an nt 389 (ins G) frameshift mutation in exon 4 resulting in a new stop codon and a Ser 413 Leu missense mutation in exon 10 in either allele. His mother and father were heterozygous for the nt 389 (ins G) and the Ser 413 Leu, respectively, with about 50% reduction of the plasma A-subunit proteins. In all family members examined only those with either mutation showed the reduced subunit A protein levels. Thus, this complete deficiency of factor XIII was due to a novel compound heterozygous mutation in the A-subunit gene.     

Molecular characterization of five Italian families with inherited severe factor XIII deficiency

Summary.  Factor XIII (FXIII) deficiency is a very rare (1:2 000 000) severe autosomal recessive bleeding disorder, mostly due to mutations in the coagulation FXIII A-subunit gene. We have studied the molecular basis of FXIII deficiency in five unrelated Italian families. The coding region, intron–exon boundaries and 5'- and 3'-untranslated regions of the FXIII gene encoding the A subunit were amplified and directly sequenced. Candidate mutations were identified in all the patients. Three novel mutations occurred in three patients. These include a novel homozygous deletion of two base pairs (bp) in exon 14 (c.2002–2003 DelCT). This deletion causes a frameshift from Leu667 and the formation of a stop codon at amino acid position 681. The second patient presents a novel homozygous (c.2126 G>A) transition in exon 15, predicting a Ser708Asn in Barrel 2 domain. The third patient is compound heterozygote for two missense mutations, a previously reported Arg260His substitution, and a novel transition in exon 4 (c.560 C>T) predicting a Pro186Leu in the core domain. The remaining two patients have two previously reported mutations: a 4-bp homozygous deletion in exon 11 (c.1392–1395 Del AATT), previously reported to occur in the Vicenza Area, and a homozygous nonsense mutation in exon 8 (c.979 C>T) predicting an Arg326X in the core domain. The novel mutations occurred at amino acid residues highly conserved among different species (pig, monkey, mouse and dog) and were not detected in 110 normal alleles. Structural analysis shows that Pro186Leu mutation leads to the replacement of the rigid proline pyrrolidine ring by the larger and more flexible leucine side chain and Ser708Asn may probably disrupt the hydrogen bond with Ala291.     

Identification of a new mutation (Gly420Ser), distal to the active site, that leads to factor XIII deficiency

The molecular defects of the factor XIII A subunit gene were studied in a patient with factor XIII deficiency. Mutation analysis was performed on amplified DNA from each exon of this gene by single-strand conformation polymorphism (SSCP) and DNA sequencing techniques. A substitution of guanine by adenine at nucleotide 1258 in exon 10 of the coagulation factor XIII A subunit gene has been identified in the patient. The mutation results in the replacement of Gly420 by Ser in the core domain of the enzyme. Restriction enzyme analysis of amplified exon 10 DNA confirmed that the patient was homozygous for this mutation. A family study revealed that the mutation was inherited from both parents, who were first cousins. The potential effects of the mutation were predicted by molecular modeling of the amino acid substitution within the coordinates of the crystal structure. The substitution occurred within the core domain of the enzyme at a residue completely conserved among all known members of the transglutaminase family. The model of the mutant protein suggests that although the substitution of Gly420 by Ser causes only minor readjustment of the residues and does not appear to be particularly deleterious in terms of structure, the mutation is, however, likely to decrease the molecule's ability to undergo the conformational change that is thought to be required for full transglutaminase activity. Our data strongly support the previously published information about the functional significance of the residues surrounding, but not forming, the catalytic pocket in the A subunit of factor XIII.     

Congenital factor XIII deficiency in Pakistan: characterization of seven families and identification of four novel mutations

Deficiency of coagulation factor XIII (FXIII) belongs to the rare bleeding disorders and its incidence is higher in populations with consanguineous marriages. The aims of this study were to characterize patients and relatives from seven families with suspected FXIII deficiency from Pakistan and to identify the underlying mutations. As a first indicator of FXIII deficiency, a 5M urea clot solubility test was used. Plasma FXIII A‐ and B‐subunit antigen levels were determined by ELISA. FXIII activity was measured with an incorporation assay. Sequencing of all exons and intron/exon boundaries of F13A was performed, and a novel splice site defect was confirmed by RT‐PCR analysis. Genetic analysis revealed six different mutations in the F13A gene. Two splice site mutations were detected, a novel c.1460+1G>A mutation in the first nucleotide of intron 11 and a previously reported c.2045G>A mutation in the last nucleotide of exon 14. Neither of them was expressed at protein level. A novel nonsense mutation in exon 4, c.567T>A, p.Cys188X, was identified, leading in homozygous form to severe FXIII deficiency. Two novel missense mutations were found in exons 8 and 9, c.1040C>A, p.Ala346Asp and c.1126T>C, p.Trp375Arg, and a previously reported missense mutation in exon 10, c.1241C>T, p.Ser413Leu. All patients homozygous for these missense mutations presented with severe FXIII deficiency. We have analysed a cohort of 27 individuals and reported four novel mutations leading to congenital FXIII deficiency.     

重型遗传性凝血因子Ⅶ缺陷症家系分子遗传学分析

目的 探讨遗传性凝血因子Ⅶ(FⅦ)缺陷症家系基因突变的类型。方法 检测凝血和止血指标以明确诊断;用DNA直接测序法对先证者及其家庭成员FⅦ基因的全部外显子及其侧翼、5’和3’非翻译区进行分析;将含杂合缺失突变外显子克隆入pMD18-TTA克隆载体中,对来自父母的两条染色体的相应序列分别测序。应用限制性内切酶StuI、MspI进行酶切分析。结果 先证者FⅦ的6号、8号外显子分别发现了1个错义杂合突变：9482位G→T,导致Arg(CG-A)152被Leu(CU-G)替代;11348位C→T突变,导致Arg(C-GG)304Trp(U-GG)突变;克隆的pMD18-TTA克隆载体测序结果显示一个杂合缺失：在11487～9位(CCC)缺失一个C,引起框移突变(已证实,后两种杂合突变均来自母亲),使351位His变为Thr,在其后编码与正常氨基酸序列完全不同的6个氨基酸后出现了翻译终止密码。其中,先证者Arg152 Leu杂合突变来自父亲,其祖父具有相同的杂合突变,父亲还含有Arg353Gln杂合多态性;其祖母为Arg304Trp杂合突变且含有Arg353Gln杂合多态性;姑姑和大伯分别为Arg353Gln杂合多态性和Arg304Trp杂合突变。PCR辅助限制性酶切证实了先证者及其家系成员的两种错义突变。结论 在该例家系中发现了Arg304Trp、Arg152Leu、11487～9位(CCC)缺失一个C三种杂合性突变,其中后两种突变为国际首次报道。     

Identification of a novel point mutation (Leu72Pro) in the NADH-cytochrome b5 reductase gene of a patient with hereditary methaemoglobinaemia type I

Utilizing polymerase chain reaction (PCR) related technology, we investigated the b5R gene of a Chinese patient with hereditary methaemoglobinaemia type I and found a novel missense mutation (CTC-CCC) at codon 72 in exon 3 of the gene. As the mutation generates an Apa I recognition site, homozygosity for the mutation was confirmed by restriction analysis of PCR-amplified fragments from the patient's genomic DNA. We predicted that the residue replacement of Leu with Pro of the mutant enzyme would account for the b5R deficiency in the patient. The results further confirm the genetic polymorphism of b5R gene mutations found in the RCM type I.     

X-linked sideroblastic anemia: identification of the mutation in the erythroid-specific delta-aminolevulinate synthase gene (ALAS2) in the original family described by Cooley

In 1945, Thomas Cooley described the first cases of X-linked sideroblastic anemia (XLSA) in two brothers from a large family in which the inheritance of the disease was documented through six generations. Almost 40 years later the enzymatic defect in XLSA was identified as the deficient activity of the erythroid-specific form of delta-aminolevulinate synthase (ALAS2), the first enzyme in the heme biosynthetic pathway. To determine the nature of the mutation in the ALAS2 gene causing XLSA in Cooley's original family, genomic DNAs were isolated from two affected hemizygotes, and each ALAS2 exon was PCR amplified and sequenced. A single transversion (A to C) was identified in exon 5. The mutation predicted the substitution of leucine for phenylalanine at residue 165 (F165L) in the first highly conserved domain of the ALAS2 catalytic core shared by all species. No other nucleotide changes were found by sequencing each of the 11 exons, including intron/exon boundaries, 1 kb of 5'-flanking and 350 nucleotides of 3'-flanking sequence. The mutation introduced an Mse I site and restriction analysis of PCR-amplified genomic DNA confirmed the presence of the lesion in the two affected brothers and in three obligate heterozygotes from three generations of this family. Carrier diagnosis of additional family members identified the mutation in one of the proband's sisters. After prokaryotic expression and affinity purification of both mutant and normal ALAS2 fusion proteins, the specific activity of the F165L mutant enzyme was about 26% of normal. The cofactor, pyridoxal 5'-phosphate, activated and/or stabilized the purified mutant recombinant enzyme in vitro, consistent with the pyridoxine-responsive anemia in affected hemizygotes from this family.     

Mutations in coagulation factor XIII A gene in three Turkish patients: two novel mutations and a known insertion

Molecular analysis of factor XIII A gene on three unrelated Turkish families identified two novel and one known mutations. One novel mutation is a substitution of cytidine by guanine at codon 541 in exon 12, beta barrel 1 domain of the coagulation factor XIII A subunit gene resulting in the conversion of asparagine to lysine. The mutation alters the restriction site of the enzyme MboII. The second novel mutation, a 4 bp (-CAAA) deletion located in a direct repetitive sequence (CAAACAAA) between codons 466-469, results in premature termination of translation at codon 474. The third mutation is a previously reported single nucleotide (cytidine) insertion at codon 400 in exon 9 of the factor XIII gene.     

A novel mutation that leads to a congenital factor XI deficiency in a Japanese family

We have identified a novel mutation leading to a congenital deficiency of the coagulation factor XI (FXI) in a Japanese family. A propositus was a 42-year-old female patient without bleeding tendency. Coagulant activity and the antigen level of FXI in her plasma were below the detectable range. The nucleotide sequences of the FXI gene of this patient were determined by a direct sequence method established in this study. A novel nonsense mutation (CAA; Gly263 --> TAA; stop) was identified in exon 8 of the FXI gene. Her parents are first cousins, and a polymerase chain reaction-restriction-fragment length polymorphism analysis revealed that her parents were heterozygous at this nucleotide position. This patient inherited mutant alleles from her parents and is homozygous at this nucleotide position. The nonsense mutation in the FXI gene is responsible for her deficiency of FXI.     

A novel mutation at the donor splice site of intron 3 of the GH-I gene in a patient with isolated growth hormone deficiency

A G - C transversion at the fifth nucleotide of intron 3 of GH-I gene was identified in a sporadic case of isolated growth hormone deficiency (IGHD). The mutation was absent in both of the parents, indicating that the mutation occurred de novo. An abnormal hGH mRNA lacking a region encoded by exon 3 was spliced when the mutant GH-I gene was expressed in cultured cells. Since skipping of exon 3 is a common feature for four different mutant GH-I genes identified in patients with autosomal dominantly inherited IGHD, we conclude that the mutation causes IGHD in this case.     

Congenital plasminogen deficiency caused by a Ser572 to Pro mutation

We used a polymerase chain reaction (PCR) strategy and restriction fragment polymorphism analysis to evaluate all 19 exons of the plasminogen (PLG) gene in a Japanese patient with congenital PLG deficiency and her family members. She presented with cerebral infarction. Sequence analysis following amplification of each exon and its flanking regions showed a single T to C transition in exon 14, which changed a Ser572 codon (TCC) to Pro572 codon (CCC). Since this mutation generates a new Fok I site, the Fok I digestion pattern of the PCR-amplified exon 14 fragments from each family member was analyzed. In all cases, the patterns were consistent with the activities and antigen levels of plasma PLG in those members. Furthermore, all PCR- amplified exon 14 fragments from 15 normal individuals were not restricted with Fok I endonuclease. We conclude that a T to C transition in exon 14 identified in the propositus is responsible for PLG deficiency inherited in this Japanese family with thrombotic episodes.     

A new mutation of the arginine vasopressin-neurophysin II gene in a family with autosomal dominant neurohypophyseal diabetes insipidus.

Familial neurohypophyseal diabetes insipidus (FNDI) is an autosomally dominant inherited disorder with a typical onset at one to six years of age. The genetic locus of FNDI is the arginine vasopressin-neurophysin II (AVP-NPII) gene. The gene encoding the precursor hormone (prepro-AVP-neurophysin II) is located in the chromosomal region 20p13 and contains three exons. Mutations that cause FNDI have been found to occur within the signal peptide of the prepro-AVP-neurophysin II precursor, within the coding sequence for neurophysin II and the vasopressin-coding sequence. A family (four members with FNDI, two without FNDI) in three consecutive generations was investigated. Index case was a now 22-year old man with a history of severe polyuria (18 L/day) and polydipsia first recognized at about 4-5 months of age. The arginine vasopressin-neurophysin II gene was investigated by direct sequencing of PCR products amplified from each exon. Subsequently, a restriction analysis was performed to verify the sequencing results. The affected individuals were found to have a missense mutation in exon 2 at nucleotide position 1887 (G to C) of the AVP-NPII gene. Using both restriction enzyme digestion and sequence analysis, the mutation was found in all affected family members, but not in the unaffected members studied. This mutation (1887 G to C) represents a novel mutation of the AVP-NPII gene.     

Identification of a novel mutation in the arginine vasopressin-neurophysin II gene in familial central diabetes insipidus.

Familial central diabetes insipidus is an inherited disease of predominant autosomal dominant trait characterized by a deficiency of arginine vasopressin. The arginine vasopressin-neurophysin II ( AVP-NPII) gene consists of three exons and is located on chromosome 20p13 encoding for the precursor protein of AVP. We investigated two Caucasian families with a typical autosomal dominant trait of familial central diabetes insipidus, defined by deficiency of arginine vasopressin. After PCR amplification of exon 1 and exon 2/3, fragments were pooled and purified. Nucleotide sequencing was performed with the Taq DyeDeoxy-terminator cycle sequencing method using nested primers. Two mutations in the coding region of NPII were identified. In family C we found a heterozygous G ==> C missense mutation (AA61) in exon 2 leading to the substitution of cystein with serine. In family D a novel heterozygous nonsense mutation in exon 3 (AA 83, GAG ==> TAG) was indentified, leading to a stop codon instead of glutamine. Both mutations were confirmed by restriction analysis and were found in all affected but not in healthy family members or control subjects. We therefore have identified a missense mutation of the AVP-NPII gene and a novel mutation predicting a truncated protein.     

Novel mutations of the thyroid peroxidase gene in patients with permanent congenital hypothyroidism.

OBJECTIVE: It is suggested that iodide organification defects account for 10% of all cases with congenital hypothyroidism (CH). One candidate gene for these defects is the thyroid peroxidase (TPO) gene. DESIGN: Exons 2, 8-10 and 14 of the TPO gene were examined in 30 patients with permanent CH without a family history of CH. This group was characterized by the presence of an orthotopic thyroid gland and elevated TSH levels. METHODS: The mutational screening was performed by single-strand conformational polymorphism followed by sequence analysis of fragments with abnormal migration patterns and by restriction enzyme analysis. RESULTS: In four patients we were able to identify mutations on both alleles which have not been described so far. One patient was a carrier of a new homozygous point mutation in exon 9 resulting in an exchange from Leu to Pro at codon 458. Another patient was found to be compound heterozygous for two mutations, a 20 bp duplication in exon 2 and a new mutation in exon 9 (Arg491His). Two brothers of consanguineous parents showed a homozygous T deletion in exon 14 at position 2512. CONCLUSIONS: Our findings confirm the genetic heterogeneity of TPO defects and support the suggested prevalence of organification defects.