DAZ基因外显子与无精子症的关系

目的探讨Y染色体无精子缺失(DAZ)基因外显子与无精子症的关系。方法应用多重聚合酶链反应(PCR)技术对97例无精子症病人和60例正常生育男性DAZ基因外显子进行检测。结果 97例无精子症病人DAZ基因外显子缺失4例,缺失率4.12%,正常生育男性DAZ外显子未检测到缺失。结论 DAZ基因外显子缺失可导致无精子症;检测不育病人是否有Y染色体微缺失,对于通过辅助生殖技术(ART)将这种基因缺陷遗传给下一代的可能性评估具有重要意义。     

A deep intronic mutation in FGB creates a consensus exonic splicing enhancer motif that results in afibrinogenemia caused by aberrant mRNA splicing, which can be corrected in vitro with antisense oligonucleotide treatment

We previously described a novel homozygous point mutation (FGB c.115-600A>G) located deep within intron 1 of the fibrinogen beta gene (FGB), as a likely cause of afibrinogenemia. While this was the only mutation detected, its pathological mechanism was unclear. Here we show the mutation causes the inclusion of a 50-bp cryptic exon by creating a consensus heptad motif recognized by the spliceosome recruiting protein pre-mRNA splicing factor (SF2)/arginine/serine-rich alternative splicing factor (ASF) splicing factor 2/alternative splicing factor (SF2/ASF). Translation of the aberrant mRNA would result in truncation of the Bbeta chain, preventing fibrinogen synthesis. Selective introduction of a second mutation into the enhancer motif abolished the SF2/ASF binding motif and re-established normal pre-mRNA splicing. Subsequent introduction of antisense phosphorodiamidate morpholino oligonucleotides (PMOs) into transfected cells containing the mutant construct blocked the protein-RNA interaction and successfully restored normal splicing ( approximately 50% at 2 microM and approximately 90% at 10 microM). The molecular characterization of this case has revealed a unique disease mechanism, shown the importance of screening for deep intronic mutations, and provided evidence that antisense gene therapy is potentially practical for the treatment of diseases caused by this class of mutation.     

Coevolutionary networks of splicing cis-regulatory elements

Accurate and efficient splicing of eukaryotic pre-mRNAs requires recognition by trans-acting factors of a complex array of cis-acting RNA elements. Here, we developed a generalized Bayesian network to model the coevolution of splicing cis elements in diverse eukaryotic taxa. Cross-exon but not cross-intron compensatory interactions between the 5' splice site (5'ss) and 3' splice site (3'ss) were observed in human/mouse, indicating that the exon is the primary evolutionary unit in mammals. Studied plants, fungi, and invertebrates exhibited exclusively cross-intron interactions, suggesting that intron definition drives evolution in these organisms. In mammals, 5'ss strength and the strength of several classes of exonic splicing silencers (ESSs) evolved in a correlated way, whereas specific exonic splicing enhancers (ESEs), including motifs associated with hTra2, SRp55, and SRp20, evolved in a compensatory manner relative to the 5'ss and 3'ss. Interactions between specific ESS or ESE motifs were not observed, suggesting that elements bound by different factors are not commonly interchangeable. Thus, the splicing elements defining exons coevolve in a way that preserves overall exon strength, allowing specific elements to substitute for loss or weakening of others.     

Regulation of enterocyte apoptosis by acyl-CoA synthetase 5 splicing

BACKGROUND AND AIMS: The constant renewal of enterocytes along the crypt-villus axis (CVA) of human small intestine is due to cell-inherent changes resulting in the apoptotic cell death of senescent enterocytes. The aim of the present study was to examine underlying molecular mechanisms of the cell death at the villus tip. METHODS: Characterization of human acyl-coenzyme A (CoA) synthetase 5 (ACSL5) was performed by cloning, recombinant protein expression, biochemical approaches, and several functional and in situ analyses. RESULTS: Our data show that different amounts of acyl-CoA synthetase 5-full length (ACSL5-fl) and a so far unknown splice variant lacking exon 20 (ACSL5-Delta 20) are found in human enterocytes. In contrast with the splice variant ACSL5-Delta 20, recombinant and purified ACSL5-fl protein is active at a highly alkaline pH. Over expression of ACSL5-fl protein is associated with a decrease of the anti-apoptotic FLIP protein in a ceramide-dependent manner and an increased cell-surface expression of the death receptor TRAIL-R1. Expression analyses revealed that the ACSL5-fl/ACSL5-Delta 20 ratio increases along the CVA, thereby sensitizing ACSL5-fl-dominated cells at the villus tip to the death ligand TRAIL, which is corroborated by functional studies with human small intestinal mucosal samples and an immortalized human small intestinal cell line. CONCLUSIONS: Our results suggest an ACSL5-dependent regulatory mechanism that contributes to the cellular renewal along the CVA in human small intestine. Deregulation of the ACSL5-fl/ACSL5-Delta 20 homeostasis in the maturation and shedding of cells along the CVA might also be of relevance for the development of intestinal neoplasia.     

Clinical utility of routine MPL exon 10 analysis in the diagnosis of essential thrombocythaemia and primary myelofibrosis

Approximately 50% of essential thrombocythaemia and primary myelo‐fibrosis patients do not have a JAK2 V617F mutation. Up to 5% of these are reported to have a MPL exon 10 mutation but testing for MPL is not routine as there are multiple mutation types. The ability to routinely assess both JAK2 and MPL mutations would be beneficial in the differential diagnosis of unexplained thrombocytosis or myelofibrosis. We developed and applied a high resolution melt (HRM) assay, capable of detecting all known MPL mutations in a single analysis, for the detection of MPL exon 10 mutations. We assessed 175 ET and PMF patients, including 67 that were JAK2 V617F‐negative by real time polymerase chain reaction (PCR). Overall, 19/175 (11%) patients had a MPL exon 10 mutation, of whom 16 were JAK2 V617F‐negative (16/67; 24%). MPL mutation types were W515L (11), W515K (4), W515R (2) and W515A (1). One patient had both W515L and S505N MPL mutations and these were present in the same haemopoietic colonies. Real time PCR for JAK2 V617F analysis and HRM for MPL exon 10 status identified one or more clonal marker in 71% of patients. This combined genetic approach increases the sensitivity of meeting the World Health Organization diagnostic criteria for these myeloproliferative neoplasms.     

Focal β‐catenin mutation identified on formalin‐fixed and paraffin‐embedded inflammatory hepatocellular adenomas

The identification of hepatocellular adenoma (HCA) with mutation in exon 3 of the CTNNB1 gene encoding for β‐catenin is clinically relevant due to a higher risk of malignant transformation. Inflammatory HCA (IHCA) can exhibit β‐catenin activation (β‐IHCA). We report two cases with multiple IHCA in which focal β‐catenin activation has been found in one of the IHCA. In both cases, the diagnosis of IHCA was confirmed on the resected nodules by routine stains, immunohistochemical detection of C‐reactive protein (CRP) and molecular biology on frozen material. An additional molecular analysis was performed on formalin‐fixed paraffin‐embedded (FFPE) material that showed focal glutamine synthetase (GS) staining, the surrogate marker of β‐catenin activation. In case 1, it was a 1.8‐cm area within the 7.5 cm IHCA, and in case 2 a small 0.3‐cm area within a 1.8 cm resected IHCA located close to a larger IHCA, negative for GS. In both cases, nuclear β‐catenin expression and decreased reticulin network were observed in the GS expressing foci, together with cholestasis and diffuse CD34 expression in case 1. Molecular analysis by pyrosequencing on FFPE material using the GS‐stained slides as reference to select areas with/without positive staining revealed a CTNNB1 exon 3 mutation restricted to the areas exhibiting both positive GS and CRP expression, whereas wild‐type CTNNB1 was found in areas showing only CRP staining. These two cases illustrate focal β‐catenin activation that can occur within IHCAs. Additional data are needed to determine if β‐catenin mutation is a secondary event in IHCA.     

乳腺增生病p53基因第6外显子突变检测

目的：探讨p53基因在乳腺癌发生早期的作用及早期诊断乳腺癌的分子病理指标。方法：用PCR－SSCP检测36例乳腺单纯性增生、31例不典型增生、30例乳腺癌中p53基因第6外显子突变，用DNA直接测序技术确定突变的碱基及其所在的密码子。结果：乳腺单纯性增生、不典型增生、乳腺癌中p53基因第6外显子的突变率分别为0、6．5％（2／31）、13．3％（4／30）。6个点突变均为碱基替换，其中4个发生于第192密码子（CAG→TAG），2个发生于第213密码子（CGA→TGA），两者均导致多肽链合成提前终止。结论：乳腺癌不典型增生中存在p53基因第6外显子突变，该突变可能在乳腺不典型增生发展到乳腺癌过程中起重要作用，可作为早期诊断乳腺癌的辅助指标。