The dystrophin protein encoding DMD gene is the longest human gene. The 2.2 Mb long human dystrophin transcript takes 16 hours to be transcribed and is co-transcriptionally spliced. It contains long introns (24 over 10kb long, 5 over 100kb long) and the heterogeneity in intron size makes it an ideal transcript to study different aspects of the human splicing process. Splicing is a complex process and much is unknown regarding the splicing of long introns in human genes.Here, we used ultra-deep transcript sequencing to characterize splicing of the dystrophin transcripts in 3 different human skeletal muscle cell lines, and explored the order of intron removal and multi-step splicing. Coverage and read pair analyses showed that around 40% of the introns were not always removed sequentially. Additionally, for the first time, we report that non-consecutive intron removal resulted in 3 or more joined exons which are flanked by unspliced introns and we defined these joined exons as an exon block. Lastly, computational and experimental data revealed that, for the majority of dystrophin introns, multistep splicing events are used to splice out a single intron.Overall, our data show for the first time in a human transcript, that multi-step intron removal is a general feature of mRNA splicing.

An accurate algorithm is essential for effective molecular diagnosis of hereditary colorectal cancer (CRC). Here, we have extended the analysis of 71 CRC cases suspected to be Lynch syndrome cases for MSH2, MLH1, MSH6, and PMS2 gene defects. All cases were screened for mutations in MSH2, MLH1, and MSH6, and all cases where tumors were available were screened for microsatellite instability (MSI) and expression of MSH2 and MLH1. Subsequently, mutation-negative cases were screened for MLH1 methylation and mutations in PMS2. Of the MSI-high (MSI-H) cases, 96% had a mismatch repair (MMR) gene defect, mostly involving MSH2 or MLH1; one PMS2 mutation, one MLH1 epimutation, and no MSH6 mutations were found. Four of the 28 MSI-H cases, including one Amsterdam criteria case, had biallelic tumor MLH1 methylation, indicating that sporadic cases can be admixed in with Lynch syndrome cases, even those meeting the strongest criteria for Lynch syndrome. MMR gene defects were found in similar frequency in cases where tumors were and were not available. One MLH1 and one MSH2 deletion mutation were found in MSIstable/low cases, indicating that MSI testing can exclude cases with pathogenic mutations. Our analysis supports a diagnostic algorithm where cases are selected for analysis based on clinical criteria or prediction models; isolated sporadic young-onset cases can be prescreened by tumor testing, whereas familial cases may be directly subjected to molecular analysis for mutations in MMR genes followed by MSI, protein expression, and DNA methylation analysis to aid in the resolution of mutation-negative cases.

Defects in human DNA mismatch repair have been reported to underlie a variety of hereditary and sporadic cancer cases. We characterized the structure of the MSH6 promoter region to examine the mechanisms of transcriptional regulation of the MSH6 gene. The 5-flanking region of the MSH6 gene was found to contain seven functional Sp1 transcription factor binding sites that each bind Sp1 and Sp3 and contribute to promoter activity. Transcription did not appear to require a TATA box and resulted in multiple start sites, including two major start sites and at least nine minor start sites. Three common polymorphisms were identified in the promoter region (؊557 T3G, ؊448 G3A, and ؊159 C3T): the latter two were always associated, and each of these functionally inactivated a different Sp1 site. The polymorphic allele ؊448 A ؊159 T was demonstrated to be a common Caucasian polymorphism found in 16% of Caucasians and resulted in a five-Sp1-site promoter that had 50% less promoter activity and was more sensitive to inactivation by DNA methylation than the more common seven Sp1 site promoter allele, which was only partially inactivated by DNA methylation. In cell lines, this five-Sp1-site polymorphism resulted in reduced MSH6 expression at both the mRNA and protein level. An additional 2% of Caucasians contained another polymorphism, ؊210 C3T, which inactivated a single Sp1 site that also contributes to promoter activity.The human DNA mismatch repair (MMR) system functions to repair mispaired bases in DNA that result from DNA replication errors and thereby prevents the accumulation of mutations due to such replication errors. Biochemical and genetic studies have identified a number of mismatch repair proteins involved in this system, including those encoded by the MSH2, MSH3, MSH6, MLH1, MLH3, PMS2, and EXO1 genes, as well as the replication proteins PCNA, RFC, RPA, and DNA polymerase delta (for reviews, see references 24 and 33). Loss of MMR function is associated with both inherited cancer susceptibility and the development of sporadic tumors. Inherited mutations in MSH2 and MLH1 are the most prevalent cause of hereditary nonpolyposis colorectal carcinoma (HNPCC) (for a review, see reference 52), and epigenetic silencing of MLH1 has been found to underlie most MMR defective sporadic cancer cases (14,25,31,46,47). Inherited mutations in MSH6 have been found in a small proportion (0 to 3%) of HNPCC families and appear to underlie a higher proportion for familial colorectal cancer cases that show later onset and a less pronounced family history than HNPCC (6,34,45,65,67,68). Mutations in PMS2 have been found in patients with Turcots syndrome but are only rarely found in patients with HNPCC (11, 39, 64, 66). Whether or not mutations in MLH3 or EXO1 underlie a significant proportion of HNPCC is unclear (2,29,38,69,70).In the human MMR system, two heterodimeric complexes-MSH2-MSH6 (MutS␣) MSH2-MSH3 (MutS␤)-function to recognize mispaired bases in DNA (1,19,21,49,61). MutS␣ appears to function in the repair of base-base and insert...

A loss-of-function mutation in DAG1 can result in Walker-Warburg syndrome and is not embryonic lethal.