A G-->A transition creates a branch point sequence and activation of a cryptic exon, resulting in the hereditary disorder neurofibromatosis 2

We describe a G-->A transition within intron 5 of the NF2 gene. This mutation creates a consensus splice branch point sequence. To our knowledge this is the first report of a mutation that creates a functional branch point sequence in a human hereditary disorder. The new branch point sequence is located 18 bp upstream of a consensus splice acceptor site. A consensus splice donor site is found 106 bp 3' of the acceptor site. Asa consequence the G-->A transition results in an alternatively spliced mRNA containing an additional exon 5a of 106 bp derived from intron sequences. We cloned the mutant cDNA and show that due to an in-frame stop codon the cDNA codes for a truncated NF2 protein. The mutation was observed in three affected members of an NF2 family. In a tumour of one of the family members both alternatively spliced and wild-type mRNA were found, although the wild-type allele of the gene is absent due to an interstitial deletion on chromosome 22. We also show that immunoprecipitations reveal the presence of full-length wild-type NF2 protein in the tumour lysate. These data support the hypothesis that some degree of normal splicing of the mutant precursor RNA is taking place. It is therefore likely that this residual activity of the mutant allele explains the relatively mild phenotype in the family. These data also indicate that complete inactivation of the gene is not required for tumour formation.      

神经母细胞瘤SK-N-SH细胞内DHRS4L2基因一种新选择性剪接亚型S4的克隆和亚细胞定位

目的 神经母细胞瘤SK-N-SH细胞系脱氢酶/还原酶(SDR家族)成员4类2[dehydrogenase/reductase(SDR family)member 4 like 2,DHRS4L2]基因的一种新的选择性剪接亚型克隆、生物信息学分析及其亚细胞定位.方法 以SK-N-SH细胞cDNA为模板,PCR扩增DHRS4基因簇Ea1转录本.将PCR产物A-T克隆至pGEMT-Easy质粒,对质粒进行DNA Sanger测序.将测序所得序列用NCBI ORF finder分析其编码区,用Motif Scan分析预测蛋白氨基酸序列.用Clustal Omega进行蛋白序列比对分析.将新亚型完整编码框cDNA以及删除偶核定位信号的编码框分别插入pEGFP-C1质粒,所得质粒和空质粒分别转染SK-N-SH细胞,在荧光显微镜下观察转染表达蛋白亚细胞定位.结果 用RT-PCR和Sanger测序方法发现,SK-N-SH表达DHRS4L2 Ea1转录本,未检测到其表达脱氢酶/还原酶(SDR家族)成员4类1[dehydrogenase/reductase(SDR family)member 4 like 1,DHRS4L1]的Ea2转录本.DHRS4L2 Ea1表达一个新的选择性剪接亚型DHRS4L2-S4(KU141377),由AY616183基础上在Ea1与E2外显子之间插入新外显子Ej形成,外显子Ej含有新亚型翻译起始密码子ATG.转录本KU141377预测蛋白羧基端具有偶核定位信号(bipartite nuclear localization signal,NLS),提示其可能定位于细胞核.绿色荧光蛋白融合蛋白实验显示,在SK-N-SH细胞该蛋白定位于细胞核.该蛋白还含有一个甘氨酸密集区(glycine-rich region)和阿片样生长因子受体重复(opioid growth factor receptor repeat)序列.结论 研究发现SK-N-SH细胞表达的一种DHRS4L2新选择性剪接亚型KU141377,其预测编码蛋白含有细胞偶核定位信号,融合荧光蛋白实验显示该新亚型定位于细胞核,这为后续研究DHRS4L2在神经母细胞瘤中的潜在功能奠定基础.     

Characterization of four mutations in the neurofibromatosis type 1 gene by denaturing gradient gel electrophoresis (DGGE)

Neurofibromatosis type 1 (NF1) Is one of the most common Inheriteddisorders. The gene responsible for the disease has a very highmutation rate, approximately fifty per cent of NF1 patientsappear to have a de novo mutation. The search for mutationsis hampered by the large size of the NF1 gene and up to date,relatively few mutations have been characterized. In the presentwork, we report the results of screening seventy unrelated NF1patients for mutations in NF1 exons 29 and 31 by using an experimentalapproach that combines the polymerase chain reaction (PCR) withdenaturing gradient gel electrophoresis (DGGE). Four mutationswere Identified and characterized. Three of them consist ofC-T transitions resulting in nonsense mutations, two In exon29, C5242T and C5260T, and one In exon 31, C5839T. The fourthmutation consists of a two-base pair deletion In exon 31, 5843delAA,also resulting in a premature stop codon. The finding In ourpatients of mutation C5839T, previously reported In three Independentstudies, supoports that thisposition is a hotspot within theNF1 gene.     

Dysregulation of human brain microtubule-associated tau mRNA maturation in myotonic dystrophy type 1

Intraneuronal aggregates of hyperphosphorylated tau proteins, referred to as pathological tau, are found in brain areas of demented patients affected by numerous different neurodegenerative disorders. We previously described a particular biochemical profile of pathological tau proteins in myotonic dystrophy type 1 (DM1). This multisystemic disorder is characterized by an unstable CTG repeat expansion in the 3'-untranslated region of the DM protein kinase gene. In the human central nervous system, tau proteins consist of six isoforms that differ by the presence or absence of the alternatively spliced exons 2, 3 and 10. Here we show that the pattern of tau isoforms aggregated in DM1 brain lesions is characteristic. It consists mainly of the aggregation of the shortest human tau isoform. A disruption in normal tau isoform expression consisting of a reduced expression of tau isoforms containing the exon 2 was observed at both the mRNA and protein levels. Large expanded CTG repeats were detected and showed marked somatic heterogeneity between DM1 cases and in cortical brains regions analysed. Our data suggest a relationship between the CTG repeat expansion and the alteration of tau expression showing that DM1 is a peculiar tauopathy.     

A novel dystrophin isoform is required for normal retinal electrophysiology

Dystrophin is present in the outer plexiform layer of the retinaand is required for normal retinal function as measured by electroretinography.We describe the identification of a novel isoform of dystrophln(Dp260) present in the mouse retina. The unIque 5' terminusof the mRNA originates from a newly identified exon and is splicedin frame to exon 30 of the Duchenne muscular dystrophy (DMD)gene. The retinal isoform of dystrophln has 13 novel amino acidsas its N-terminus followed by most of the dystrophin rod domainand the cysteine-rich C-terminal domains. Analysis of mousetissues indicated this isoform of dystrophin Is expressed inretina, brain and cardiac tissue. Comparison of retinal electrophysiologyin mdx and mdx^cv3 mouse suggests that Dp260 is required fornormal retinal function.     

Three alternatively spliced variants of the gene coding for the human bone morphogenetic protein-1

 The human bone morphogenetic protein-1 was originally identified as a protein with the capacity to stimulate bone and cartilage growth in vitro. Its gene sequence identified it as an alternatively spliced human homolog of the Drosophila dorsal-ventral patterning tolloid gene and suggested that it activates transforming growth factor-β-like molecules by proteolytic cleavage. Its expression pattern and its recently identified activity as a procollagen C proteinase, however, suggest that it has a more general function in the early stages of embryogenesis. This view is strengthened by the previous observation of a third alternatively spliced isoform of the gene, called bone morphogenetic protein 1/His. We now show that the gene is expressed in three additional variants, leading to shorter and slightly modified C-termini. The three variants are preferentially expressed in placenta but show individual differences in their expression profiles in other soft tissues. Received: 27 January 1997 / Accepted: 14 October 1997     

Novel alleles, hemizygosity and deletions at an Alu-repeat within the neurofibromatosis type 1 (NF1) gene

Neurofibromatosis type 1 (NF1) (von Recklinghausen) is a commonautosomal dominant disorder, characterised by the presence ofperipheral neurofibromas, café-au-lait spots and Lischnodules of the iris. Due to the high mutation rate at the NF1locus, most patients are expected to have different mutations,limiting molecular analysis and genetic counseling to the identificationof the mutation in each patient or family, or to the use ofDNA polymorphisms. We have analysed an Alu-repeat polymorphicsequence (AAAT), located in intron 27 of the NF1 gene, in 70NF1 and 40 CEPH families and we have detected several geneticand molecular abnormalities. In two families the NF1 individualswere hemizygous at the AAAT-repeat and/or at the CA-repeat ofintron 27 of NF1, due to interstitial deletions, which includeintron 27 to exon 37 of the NF1 gene. A 71-bp deletion at theAlu sequence was detected in non-NF1 chromosomes of membersof three NF1 families. New alleles at the AAAT-repeat were foundin one NF1 family and in three CEPH families giving a mutationrate for this AAAT-repeat of 0.36% per allele, which is oneof the highest detected for a microsatellite locus.     

Slow troponin T mRNA in striated muscles is expressed in both cell type and developmental stage specific manner

We have cloned cDNA sequences of both rat and mouse slow troponin T gene. These sequences share a high level of homology with each other and with the human slow troponin T gene although we were unable to detect an alternatively spliced exon present at 3 end of human slow troponin T cDNA in either mouse or rat cDNAs. Northern blot analysis detected a high level expression of slow troponin T in adult mouse Soleus with a lower level expression in mixed postnatal skeletal muscles. Unlike late fetal and postnatal skeletal muscles in which slow troponin T expression is restricted to slow muscle fibre rich regions only, in situ hybridisation analysis detected this isoform to be highly expressed in somitic myotome and all muscle masses at 10–14 days of gestation after which its expression was rapidly downregulated. The unexpected expression of slow troponin T mRNA in fetal heart was apparent by both northern blotting and in situ hybridisation analyses. Slow troponin T mRNA in fetal heart was first detected at 10 day in utero reaching maximum levels of expression at 12–15 days gestation. The slow troponin T in the heart was mainly expressed in the ventral ventricles until day 15 after which low level expression was also observed in both atria. Slow troponin T mRNA in both atrium and ventricle was mainly expressed in outer wall of the myocardium although it was also expressed in interventricular septum. This study therefore shows that in addition to being a cell type specific marker during later fetal and postnatal skeletal muscle development, slow troponin T represented one of the major developmental isoforms expressed in embryonic and fetal skeletal muscle as well as in the cardiac muscle.     

Tissue-Specific Expression of Splice Variants of Human IL-4 and IL-6 Gene mRNA

The spectrum of alternatively spliced IL-4 and IL-6 gene mRNA was studied in peripheral blood mononuclears from healthy donors and in human fetal tissues. It was found that the expression of alternatively spliced IL-4 and IL-6 gene mRNA in fetal tissues is tissue specific and that hemopoiesis- and immunopoiesis-related tissues differ by the amount of IL-4 and IL-4δ2 mRNA. An mRNA variant IL-4alt3 carrying partial exon 3 deletion was for the first time identified in human mononuclear cells.