Mutation analysis in the iduronate-2-sulphatase gene in 43 Japanese patients with mucopolysaccharidosis type II (Hunter disease)

Our series of studies on Hunter disease in Japanese patients showed allelic heterogeneity of IDS gene mutations, genotype/phenotype correlation and racial differences in distribution of mutations. Twenty-five different small mutations have been characterized. Small mutations in the Japanese population are widely distributed through the IDS gene, although some mutations were unevenly concentrated on exon 5 (28%) and on exon 9 (24%). Mutations were seen at the same codon 468 in exon 9 in 5 patients. These findings are in good agreement with data on other ethnic groups. Two unique mutations linked to a severe phenotype were apparently associated with aberrant splicings; one was a point mutation within exon 3 (P86L), partially activating a cryptic splice acceptor site at 28 bp downstream from the mutation site within exon 3 and producing a 44-base truncated mRNA, and the other was a point mutation at the consensus sequence of the splice donor site of intron 2, causing exon 2 skipping.     

Molecular basis and haematological characterization of β-thalassaemia major in Taiwan, with a mutation of IVS-1 3'end TAG → GAG in a Chinese patient

Summary We studied 41 patients with β-thalassaemia major and their parents by using a combination of polymerase chain reaction (PCR) amplification, slot-blot hybridization of allele-specific oligonucleotide (ASO), and direct genomic sequencing. Eight different point mutations were characterized. C to T substitution at nucleotide (nt) 654 of intervening sequences (IVS) 2, accounting for 46.3% of mutant β-globin genes, is the most common mutation in Taiwan, followed by frameshift codons 41/42 with four nucleotides (TCTT) deletion for 31.7%, A to G substitution at position ?28 of promotor area for 8.5%, A to T substitution at codon 17 for 6.1%, frameshift codons 27/28 (insertion of C) for 2.4%, G to T substitution at nucleotide 1 of IVS-1 for 2.4%, frameshift codons 71/72 (insertion of A) and IVS-1 3’end TAG→GAG for 1.2%. The former four mutations showed no obvious difference between two major ethnic groups in Taiwan. As to mutations in each individual of β-thalassaemia major, the incidence of compound heterozygotes of two different mutations is much higher than homozygotes of single mutation, 78.0%v 22.0%. Compound heterozygotes of C to T substitution at nt 654 of IVS-2 and frameshift codons 41/42 with four nucleotides deletion is the most common pattern of β-thalassaemia mutations in each individual (41.5%). The results are somewhat different from other documented reports concerning the mutations of β-thalassaemia in southern China. This is the first report of mutation of IVS-1 3’end TAG→GAG which causes consensus change in Chinese people. Patients with heterozygotes of β° and –28 β⁺-thalassaemia mutations would have a greater delay in initial transfusion in comparison to patients with homozygotes of both β°-thalassaemia mutation, but their initial clinical manifestation might be aggravated when combined with a glucose-6-phosphate dehydrogenase (G-6-PD) deficiency and an insult such as exposure to infection and certain drugs.     

Novel <Emphasis Type="Italic">hMSH</Emphasis>2, <Emphasis Type="Italic">hMSH</Emphasis>6 and <Emphasis Type="Italic">hMLH</Emphasis>1 gene mutations and microsatellite instability in sporadic colorectal cancer

Purpose To detect the hMSH2, hMSH6 and hMLH1 DNA mismatch repair gene mutations and microsatellite instability in somatic colorectal cancer.Patients and methods The mutations of hMSH2, hMSH6, and hMLH1 genes, including microsatellite instability of BAT-26, BAT-40, D2S123, D5S346 and D17S250 were analyzed in 31 patients with colorectal.Results The results revealed that eight cases (25.8%) harbored mutations in DNA mismatch repair genes. Of these, five novel mutations including I237V in exon 4 of hMSH2, ins T at codon 1196 in exon 7 of hMSH6, and ins G at codon 154 in exon 6, N158H in exon 6, and del A at codon 257 in exon 9 of hMLH1 were identified. Moreover, several intronic polymorphisms, including c–g transversion at IVS-1 nt211 + 9 of hMSH2, del T in poly T track at IVS-6 nt3559-5, ATCT duplicate in IVS-7 nt 3642 + 35 and t–g transversion at IVS-10 nt4080 + 185 of hMSH6 were demonstrated in these patients. In addition, seven cases (22.5%) exhibited microsatellite instability (MSI).Conclusion These results suggested that the inactivation of DNA mismatch repair genes and microsatellite instability may play a minor role in somatic colorectal cancer development.     

In vitro splicing of influenza viral NS1 mRNA and NS1-beta-globin chimeras: possible mechanisms for the control of viral mRNA splicing.

In influenza virus-infected cells, the splicing of the viral NS1 mRNA catalyzed by host nuclear enzymes is controlled so that the steady-state amount of the spliced NS2 mRNA is only 5-10% of that of the unspliced NS1 mRNA. Here we examine the splicing of NS1 mRNA in vitro, using nuclear extracts from HeLa cells. We show that in addition to its consensus 5' and 3' splice sites, NS1 mRNA has an intron branch-point adenosine residue that was functional in lariat formation. Nonetheless, this RNA was not detectably spliced in vitro under conditions in which a human beta-globin precursor was efficiently spliced. Using chimeric RNA precursors containing both NS1 and beta-globin sequences, we show that the NS1 5' splice site was effectively utilized by the beta-globin branch-point sequence and 3' splice site to form a spliced RNA, whereas the NS1 3' splice site did not function in detectable splicing in vitro, even in the presence of the beta-globin branch-point sequence or in the presence of both the branch-point sequence and 5' exon and splice site from beta-globin. With the chimeric precursors that were not detectably spliced, as with NS1 mRNA itself, a low level of a lariat structure containing only intron and not 3' exon sequences was formed. The inability of the consensus 3' splice site of NS1 mRNA to function effectively in in vitro splicing suggests that this site is structurally inaccessible to components of the splicing machinery. Based on these results, we propose two mechanisms whereby NS1 mRNA splicing in infected cells is controlled via the accessibility of its 3' splice site.     

Exon sequences at the splice junctions affect splicing fidelity and alternative splicing

Identification of splice sites is essential for the expression of most eukaryotic genes, allowing accurate splicing of pre-mRNAs. The splice sites are recognized by the splicing machinery based on sequences within the pre-mRNA. Here, we show that the exon sequences at the splice junctions play a significant, previously unrecognized role in the selection of 3′ splice sites during the second step of splicing. The influence of the exon sequences was enhanced by the Prp18 mutant Prp18ΔCR, and the strength of an exon sequence in Prp18ΔCR splicing predicted its effect in wild-type splicing. Analysis of the kinetics of splicing in vitro demonstrated that 3′ splice sites were chosen competitively during the second step, likely at the same time as exon ligation. In wild-type yeast, splice site selection for two genes studied was altered by point mutations in their exon bases, affecting splicing fidelity and alternative splicing. Finally, we note that the degeneracy of the genetic code allows competing 3′ splice sites to be eliminated from coding regions, and we suggest that the evolution of the splicing signals and the genetic code are connected.     

Splice site mutations are a common cause of X-linked chronic granulomatous disease.

Chronic granulomatous disease (CGD) is characterized by the absence of a respiratory burst in activated phagocytes. Defects in at least four different genes lead to CGD. Patients with the X-linked form of CGD have mutations in the gene for the beta-subunit of cytochrome b558 (gp91-phox). We studied the molecular defect in four patients with X-linked CGD. In a fifth family, we studied the mother of a patient with X-linked CGD who had died before our investigations. Gp91-phox messenger RNA (mRNA) was reverse transcribed into cDNA and the coding region was amplified by polymerase chain reaction into three fragments. Sequence analysis showed the absence of the exon 7, 5, 3, and 2 sequences in patients 1, 2, 3, and 4, respectively. In carrier 5, we found both normal cDNA and cDNA that lacked 57 3'-nucleotides of exon 6. We analyzed the splice sites of the flanking introns of the missing exons. In patients 1, 2, and 3, we found single nucleotide substitutions within the first five positions of the down-stream 5' donor splice sites. In patient 4, a similar substitution was found at position -1 of the 3' acceptor splice site of intron 1. In carrier 5, no mutation was found in the exon 6-intron 6 boundary sequence. Instead, a single substitution was observed in exon 6 (C----A at nucleotide 633) that created a new donor splice site. Apparently, mRNA splicing occurs preferentially at this newly created splice site. We conclude that the absence of the exon sequences in the gp91-phox mRNA of these patients is due to splicing errors. Of 30 European X-linked CGD patients studied by us so far, five appear to be caused by mutations that affect correct mRNA splicing. Thus, such mutations appear to be a common cause of X-linked CGD.     

Molecular characterization of the PK-LR gene in sixteen pyruvate kinase-deficient patients

We studied the PK-LR gene in 16 unrelated patients with congenital haemolytic anaemia associated with erythrocyte pyruvate kinase deficiency. Fifteen different mutations were detected among the 28 mutated alleles identified: two deletions (del 1010G, del 1042--1044); one four nucleotide duplication (nt 1515--1518, GGTC); one splice site [IVS6(-2)t]; nine missense (991A, 1003A, 1151T, 1160G, 1181T, 1181A, 1456T, 1483A, 1529A); and two nonsense (721T, 1675T) mutations. Eight of them [del 1010G, del 1042--1044, dupl 1515--1518, IVS6(-2)t, 1003A, 1160G, 1181T, 1181A] were novel. The deletion 1042-1044 causes the loss of Lys 348. Deletion 1010G and duplication 1515-1518 determine a frameshift and the creation of a stop codon at nucleotides 1019 and 1554 respectively. Mutation IVS6(-2)t leads to an alteration of the 5' and 3' splice site consensus sequence; the cDNA analysis shows a 67-bp deletion in the first part of exon 11 (del 1437--1503). All the four new missense mutations involve highly conserved amino acids. The most frequent mutation in Italy would appear to be 1456T. Correlation was made between mutations, biochemical characteristics of the enzyme and clinical course of the disease.     

Factor XIIIA calgary: a candidate missense mutation (Leu667Pro) in the beta barrel 2 domain of the factor XIIIA subunit

Summary. Molecular analysis performed on a Canadian family with congenital factor XIII deficiency revealed a homozygous missense mutation (Leu667Pro) in exon 14 of the A subunit gene in three affected siblings. The mutation results from a T-to-C transition at nucleotide position 2087 and generates a new Msp1 restriction site. Digestion of an amplified fragment containing exon 14 with this restriction enzyme enabled the heterozygous allele to be identified in both parents (who were third cousins) and three other family members. SSCP analysis detected no additional mutations in the coding or consensus splice sequences of the A subunit gene. The mutant nucleotide substitution was absent in 60 normal alleles and 10 unrelated patients with XIII_A deficiency. Leu667 is located in the carboxyl terminal beta barrel 2 domain of the A subunit molecule. Computer modelling based on 3D crystallographic data predicts that the mutant protein has aberrant folding and is likely to be rapidly degraded following translation.     

Characterization of two novel splice site mutations in human factor VII gene causing severe plasma factor VII deficiency and bleeding diathesis

The molecular basis of severe type I factor (F)VII deficiency was investigated in two Algerian patients. One patient, a 13-year-old-girl who has suffered from severe bleeding since birth, was homozygous for a 7-bp deletion (nt 7774-7780) and a 251-bp insertion (nt 7773-7781) of mitochondrial origin, in IVS 4 acceptor splice site. The other patient, an infant who died from massive intracranial haemorrhage, was homozygous for a transversion in the IVS 7 donor splice site (T9726+2-->G) and a missense mutation in exon 8 (G10588-->A; Arg224-->Gln). In both cases, the deleterious mutations are probably the splice site junction abnormalities impairing mRNA processing. These three lesions have not yet been reported.     

A new mutation in IVS-1 of the human beta globin gene causing beta thalassemia due to abnormal splicing

A G to T transversion at the fifth nucleotide of the first intervening sequence (IVS-1) of the beta-globin gene has been identified in cloned beta-thalassemia genes of two unrelated individuals, one of Mediterranean and the other of Anglo Saxon ancestry. In each patient the mutation was present in a different beta globin gene framework, defined by intragenic restriction site polymorphisms, thereby suggesting the occurrence of independent mutations. The study of the RNA products of one of these cloned genes, after transfer and transient expression in HeLa cells, showed partial inactivation of the normal donor splice site of IVS-1 and activation of two major and one minor cryptic splice sites. Only one of the two major cryptic sites was utilized in a cell-free splicing extract. The effects of this mutation on messenger RNA (mRNA) splicing are similar to that of another beta thalassemia gene with a G to C transition at the same position.     

The spectrum of β-thalassaemia mutations in Sicily

To characterize beta-thalassaemia genes among the Sicilian population we have previously determined the DNA haplotypes in the beta-globin gene cluster of 99 beta-thal chromosomes. We found seven haplotypes, although 95 of 99 beta-thal chromosomes contained framework 1 and framework 3 beta genes. We have now determined the mutation in all 99 of these beta-thal genes by the use of oligonucleotide hybridization. PCR-amplification and direct genomic sequencing, and direct restriction analysis. Our results indicate that (1) the beta (0)-39 mutation is most frequent (35%); (2) beta(0)-39, IVS-1 nt 110 and IVS-1 nt 6 mutations account for 90% of beta-thal genes: (3) the IVS-1 nt 6 mutation is more frequent in thalassaemia intermedia (77%) than in Cooley's disease (34%): (4) the association between haplotypes and specific mutations is imperfect, but mutation spread has occurred within haplotypes containing the same beta-gene framework: (5) the beta(0)-39 and the IVS-1 nt 6 mutations, with a mutation spread to two major haplotypes, may be older than the IVS-1 nt 110 mutation: (6) these data make possible first-trimester prenatal diagnosis in many families (85%) in Sicily using only three pairs of oligonucleotides. In addition, a new mutation, a frameshift at codon 76 due to loss of a C residue, was found in a single beta-thal chromosome.     

Inverse splicing of a group II intron.

I describe the self-splicing of an RNA that consists of exon sequences flanked by group II intron sequences. I find that this RNA undergoes accurate splicing in vitro, yielding an excised exon circle. This splicing reaction involves the joining of the 5' splice site at the end of an exon to the 3' splice site at the beginning of the same exon; thus, I term it inverse splicing. Inverse splicing provides a potential mechanism for exon scrambling, for exon deletion in alternative splicing pathways, and for exon shuffling in gene evolution.