中国汉族人ATM基因的单核苷酸多态与点突变

目的研究中国汉族人共济失调性毛细血管扩张症(ataxiatelangiectasiamutated,ATM)基因的单核苷酸多态和点突变。方法首先用PCR扩增ATM基因第39、61和63外显子的靶片段,然后用单链构象多态性(singlestrandconformationpolymorphism,SSCP)技术进行筛选,选择典型带型经全自动DNA测序证实。结果在ATM基因第39外显子以及第61和63内含子发现6个新的单核苷酸多态,它们分别是第39外显子第5689位和第5691位的A/T多态,第61内含子第 69位的T/G多态、第 94位的A/G多态和第 99位的T/G多态,第63内含子第 17位的G/C多态。在ATM基因第61外显子、第62内含子和第63外显子发现5个新的点突变,它们分别是第61外显子第8618位的T/G颠换、第62内含子第-13位的T/G颠换、第63外显子第8793位的T/G颠换、第8816位和第8848位的G/A转换。证实了ATM基因第39外显子第5557位G/A、第61内含子第 104位T/C和第62内含子第-55位T/C多态在中国汉族人中的存在。结论中国汉族人ATM基因的单核苷酸多态与白人存在较大差异。     

Desmin splice variants causing cardiac and skeletal myopathy

Desmin myopathy is a hereditary or sporadic cardiac and skeletal myopathy characterised by intracytoplasmic accumulation of desmin reactive deposits in muscle cells. We have characterised novel splice site mutations in the gene desmin resulting in deletion of the entire exon 3 during the pre-mRNA splicing. Sequencing of cDNA and genomic DNA identified a heterozygous de novo A to G change at the +3 position of the splice donor site of intron 3 (IVS3+3A→G) in a patient with sporadic skeletal and cardiac myopathy. A G to A transition at the highly conserved -1 nucleotide position of intron 2 affecting the splice acceptor site (IVS2-1G→A) was found in an unrelated patient with a similar phenotype. Expression of genomic DNA fragments carrying the IVS3+3A→G and IVS2-1G→A mutations confirmed that these mutations cause exon 3 deletion. Aberrant splicing leads to an in frame deletion of 32 complete codons and is predicted to result in mutant desmin lacking 32 amino acids from the 1B segment of the alpha helical rod. Functional analysis of the mutant desmin in SW13 (vim-) cells showed aggregation of abnormal coarse clumps of desmin positive material dispersed throughout the cytoplasm. This is the first report on the pathogenic potentials of splice site mutations in the desmin gene.


Keywords: cardiac and skeletal myopathy; desmin splice site mutations; expression study; genotype-phenotype correlation     

Fibrillin gene (FBN1) mutations in Japanese patients with Marfan syndrome

Marfan syndrome (MFS; MIM #154700) is a connective tissue disorder characterized by cardiovascular, skeletal, and ocular abnormalities. The fibrillin-1 gene (FBN1; MIM no. 134797) on chromosome 15 was revealed to be the cause of Marfan syndrome. To date over 137 types of FBN1 mutations have been reported. In this study, two novel mutations and a recurrent de-novo mutation were identified in patients with MFS by means of single-strand conformational polymorphism (SSCP) analysis. The two novel mutations are a 4-bp deletion at nucleotide 2820-2823 and a G-to-T transversion at nucleotide 1421 (C474F), located on exon 23 and exon 11, respectively. A previously reported mutation at the splicing donor site of intron 2 (IVS2 G + 1A), which is predicted to cause exon skipping, was identified in a sporadic patient with classical MFS. Received: November 1, 1999 / Accepted: November 9, 1999     

Mutations causing defective splicing in the human hprt gene.

Ten intron mutations and one exon mutation giving rise to defective splicing in the human gene for hypoxanthine phosphoribosyl transferase (hprt) in T-lymphocytes have been characterized. The splicing mutants were detected by PCR amplification of hprt cDNA and direct sequencing. Nine of the mutants showed skipping of whole exons or parts of exons in the cDNA, one mutant had an inclusion of an intron sequence into the cDNA, and one mutant showed both inclusion of an intron sequence and skipping of exons as well as a normal cDNA. Genomic PCR and direct sequencing of the splice sites involved showed one deletion of three base pairs and 10 different single base alterations to be responsible for these splice alterations. One mutation in the last base pair of exon 6 causing skipping of the entire exon 6 was found, whereas an identical mutation in the last base pair of exon 2 caused no aberrant splicing. It was also found that a deletion mutation in the pyrimidine rich stretch of the acceptor site of intron 7 caused skipping of the entire exon 8, whereas a base substitution in the last base of intron 7 caused exclusion of only the first 21 base pairs of exon 8 as a result of the activation of a cryptic acceptor site in exon 8. The results show that many different types of mutations at several different sites can cause splicing errors in the hprt gene and that the sequence differences between the splice sites influence the possible spectrum of mutations in each site.     

Newly recognized exons induced by a splicing abnormality from an intronic mutation of the dystrophin gene resulting in Duchenne muscular dystrophy. Mutations in brief no. 213. Online

A boy with the clinical phenotype of Duchenne muscular dystrophy had no detectable deletion or duplication in the dystrophin gene by the routine multiplex PCR method. In mRNA extracted from his muscle biopsy, newly recognized extra-exons of 172 bp and 202 bp were present between exon 25 and 26 suggesting a splicing abnormality. Genomic DNA of the intron 25 including the above insertions were amplified and sequenced. There was one nucleotide substitution of A-to-G at 2 Kb downstream from the 5' end of intron 25 which formed consensus dinucleotide 'GT' motif for 5' splice site resulting in aberrant splicing. This is the first patient who had a mutation at the central part of an intron of the dystrophin gene instead of at the exon-intron border.     

Molecular diagnosis of inherited coagulation disorders--sequence analysis of hemophilia B patients with anti-factor IX antibodies

Seven hemophilia B patients with anti-factor IX antibodies are studied with molecular means. A total factor IX gene deletion was detected in four patients from three distinct families. Three other patients without detectable gene arrangements were investigated at the DNA sequence level. Enzymatic amplification of the factor IX gene and subsequent DNA sequencing revealed four novel nucleotide mutations in these patients. Patient HB 5 had two point mutations in his factor IX gene. One is located at nucleotide -793 from the translation start (G-A) and the other (C-T) was found in the codon for 191-Gln changing to a termination codon. Patient HB6 had a point mutation (G-A) in the splicing junction of intron g. A2 bp nucleotide deletion was detected in the third exon of the patient HB7 yielding 8 frameshifted amino acids and a stop codon. These results suggested that not only a large deletion of the factor IX gene but also point mutations or small deletion of the gene which may cause the substantial loss of the coding information for the mature protein are involved in the development of anti-factor IX antibodies in hemophilia B patients.     

Deletion of nonconserved helices near the 3' end of the rRNA intron of Tetrahymena thermophila alters self-splicing but not core catalytic activity

The self-splicing rRNA intron of Tetrahymena thermophila contains two stem-loop structures (P9.1 and P9.2) near its 3' end that are not conserved among group I introns. As a step toward deriving the smallest active self-splicing RNA, 78 nucleotides encompassing P9.1 and P9.2 have been deleted. This deletion has no effect on the core catalytic activity of the intron, as judged by its ability to catalyze poly(C) polymerization and other related reactions. In contrast, reactions at the 3' splice site of the rRNA precursor--exon ligation and intermolecular exon ligation--take place with reduced efficiency, and exon ligation becomes rate-limiting for self-splicing. Moreover, intermolecular exon ligation with pentaribocytidylic acid is inaccurate, occurring primarily at a cryptic site in the 3' exon. A deletion of 79 nucleotides that disrupts P9, as well as removing P9.1 and P9.2, has more severe effects on both the first and second steps of splicing. P9, a conserved helix at the 5' edge of the deletion point, can form stable alternative structures in the deletion mutants. This aberrant folding may be responsible for the reduced activity and accuracy of reactions with mutant precursors. Analysis of the cryptic site suggests that choice of the 3' splice site may not only depend on sequence but also on proximity to P9. In the course of these studies, evidence has been obtained for an alternative 5' exon-binding site distinct from the normal site in the internal guide sequence.     

Spectrum of splicing errors caused by CHRNE mutations affecting introns and intron/exon boundaries

Background: Mutations in CHRNE, the gene encoding the muscle nicotinic acetylcholine receptor ε subunit, cause congenital myasthenic syndromes. Only three of the eight intronic splice site mutations of CHRNE reported to date have had their splicing consequences characterised. Methods: We analysed four previously reported and five novel splicing mutations in CHRNE by introducing the entire normal and mutant genomic CHRNEs into COS cells. Results and conclusions: We found that short introns (82–109 nucleotides) favour intron retention, whereas medium to long introns (306–1210 nucleotides) flanking either or both sides of an exon favour exon skipping. Two mutations are of particular interest. Firstly, a G→T substitution at the 3'' end of exon 8 predicts an R286M missense mutation, but instead results in skipping of exon 8. In human genes, a mismatch of the last exonic nucleotide to U1 snRNP is frequently compensated by a matching nucleotide at intron position +6. CHRNE intron 8 has a mismatch at position +6, and accordingly fails to compensate for the exonic mutation at position –1. Secondly, a 16 bp duplication, giving rise to two 3'' splice sites (g.IVS10-9_c.1167dup16), results in silencing of the downstream 3'' splice site. This conforms to the scanning model of recognition of the 3'' splice site, which predicts that the first "ag" occurring after the branch point is selected for splicing.     

Compound heterozygous patient with glycogen storage disease type III: identification of two novel AGL mutations, a donor splice site mutation of Chinese origin and a 1-bp deletion of Japanese origin

Glycogen storage disease type III (GSD III) is an autosomal recessive disorder caused by deficiency of glycogen-debranching enzyme (AGL). We studied a 2-year-old GSD III patient whose parents were from different ethnic groups. Nucleotide sequence analysis of the patient showed two novel mutations: a single cytosine deletion at nucleotide 2399 (2399delC) in exon 16, and a G-to-A transition at the +5 position at the donor splice site of intron 33 (IVS33+5G>A). Analysis of the mRNA produced by IVS33+5G>A showed aberrant splicing: skipping of exon 33 and activation of a cryptic splice site in exon 34. Mutational analysis of the family revealed that the 2399delC was inherited from her father, who is of Japanese origin, and the IVS33+5G>A from her mother, who is of Chinese descent, establishing that the patient was a compound heterozygote. To our knowledge, this is the first report of a mutation identified in a GSD III patient from the Chinese population.     

Germinal HPRT splice donor site mutation results in multiple RNA splicing products in T-lymphocyte cultures

We have used peripheral blood T-lymphocyte cultures to analyze the hprt mutation in two Lesch-Nyhan syndrome males who are cousins and to confirm the carrier status of female members of the family. Both cDNA and genomic DNA sequencing studies show that this patient carries a hitherto undescribed single base deletion in the exon 5 donor splice site sequence (15:+1, δG, base number 31635). The largest cDNA product contained all nine hprt exons plus an insertion of 66 bases of intron 5, consistent with the use of a cryptic splice site in intron 5 (aag₆₇/gtaagc). This splicing error would result in a chain terminating codon immediately after exon 5 (15:2–4, taa) and predicts a polypeptide of 133 amino acids. This loss of the normal splice donor site also results in multiple hprt mRNA species, combining the use of the cryptic splice site in intron 5 and splicing errors involving exons 2–6. In addition to defining a new Lesch-Nyhan mutation (hprt_Henryville), these results provide insight into aberrant splicing of hprt mRNA in T-lymphocytes.     

Donor splice site mutation in keratin 5 causes in-frame removal of 22 amino acids of H1 and 1A rod domains in Dowling-Meara epidermolysis bullosa simplex

Epidermolysis bullosa simplex (EBS) arises from mutations within the keratin 5 and 14 (K5 and K14) genes which alter the integrity of basal keratinocytes cytoskeleton. The majority of these defects are missense mutations in the rod domain, whose locations influence the disease severity. We investigated a large family dominantly affected with the Dowling-Meara form of EBS (EBS-DM). Sequencing of amplified and cloned K5 cDNA from cultured keratinocytes revealed a 66 nucleotide deletion in one allele corresponding to the last 22 amino acid residues encoded by exon 1 (Val164 to Lys185). Sequencing of amplified genomic DNA spanning the mutant region revealed a heterozygous G-to-A transition at +1 position of the consensus GT donor splice site of intron 1 of K5. This mutation leads to the use of an exonic GT cryptic donor splice site, located 66 nucleotides upstream from the normal donor splice site of intron 1. The corresponding peptide deletion includes the last five amino acids of the H1 head domain and the first 17 amino acids of the conserved amino terminal end of the 1A rod domain, including the first two heptad repeats and the helix initiation peptide. The shortened polypeptide is expressed in cultured keratinocytes at levels which are comparable to the normal K5 protein. This is the first splice site mutation to be reported as a cause of EBS-DM. Owing to the functional importance of the removed region, our data strongly suggest that shortened keratin polypeptide can impair keratin filament assembly in a dominant manner and causes EBS-DM.