Showing papers on "Exon published in 2001"

Aberrant regulation of insulin receptor alternative splicing is associated with insulin resistance in myotonic dystrophy

Abstract: Myotonic dystrophy type 1 (DM1) is caused by a CTG trinucleotide expansion in the 3' untranslated region of the DM protein kinase gene. People with DM1 have an unusual form of insulin resistance caused by a defect in skeletal muscle. Here we demonstrate that alternative splicing of the insulin receptor (IR) pre-mRNA is aberrantly regulated in DM1 skeletal muscle tissue, resulting in predominant expression of the lower-signaling nonmuscle isoform (IR-A). IR-A also predominates in DM1 skeletal muscle cultures, which exhibit a decreased metabolic response to insulin relative to cultures from normal controls. Steady-state levels of CUG-BP, a regulator of pre-mRNA splicing proposed to mediate some aspects of DM1 pathogenesis, are increased in DM1 skeletal muscle; overexpression of CUG-BP in normal cells induces a switch to IR-A. The CUG-BP protein mediates this switch through an intronic element located upstream of the alternatively spliced exon 11, and specifically binds within this element in vitro. These results support a model in which increased expression of a splicing regulator contributes to insulin resistance in DM1 by affecting IR alternative splicing.

Frequency of single nucleotide polymorphisms in the P-glycoprotein drug transporter MDR1 gene in white subjects.

Abstract: Background P-glycoprotein, the gene product of MDR1, confers multidrug resistance against antineoplastic agents but also plays an important role in the bioavailability of common drugs in medical treatment. Various polymorphisms in the MDR1 gene were recently identified. A silent mutation in exon 26 (C3435T) was correlated with intestinal P-glycoprotein expression and oral bioavailability of digoxin. Objective We wanted to establish easy-to-use and cost-effective genotyping assays for the major known MDR1 single nucleotide polymorphisms and study the allelic frequency distribution of the single nucleotide polymorphisms in a large sample of volunteers. Methods In this study, the distribution of the major MDR1 alleles was determined in 461 white volunteers with the use of polymerase chain reaction and restriction fragment length polymorphism. Results Five amino acid exchanges were found with allelic frequencies of 11.2% for Asn21Asp and 5.5% for Ser400Asn. Strikingly, in exon 21 three variants were discovered at the same locus: 2677G (56.4%), 2677T (41.6%), and 2677A (1.9%), coding for 893Ala, Ser, or Thr. A novel missense Gln1107Pro mutation was found in two cases (0.2%). The highest frequencies were observed for intronic and silent polymorphisms; C3435T occurred in 53.9% of the subjects heterozygously, and 28.6% of individuals were homozygous carriers of 3435T/T with functionally restrained P-glycoprotein. Conclusion This study provides the first analysis of MDR1 variant genotype distribution in a large sample of white subjects. It gives a basis for large-scale clinical investigations on the functional role of MDR1 allelic variants for bioavailability of a substantial number of drugs. Clinical Pharmacology & Therapeutics (2001) 69, 169–174; doi: 10.1067/mcp.2001.114164

Nuclear factor TDP‐43 and SR proteins promote in vitro and in vivo CFTR exon 9 skipping

Abstract: Alternative splicing of human cystic fibrosis transmembrane conductance regulator (CFTR) exon 9 is regulated by a combination of cis-acting elements distributed through the exon and both flanking introns (IVS8 and IVS9). Several studies have identified in the IVS8 intron 3' splice site a regulatory element that is composed of a polymorphic (TG)m(T)n repeated sequence. At present, no cellular factors have been identified that recognize this element. We have identified TDP-43, a nuclear protein not previously described to bind RNA, as the factor binding specifically to the (TG)m sequence. Transient TDP-43 overexpression in Hep3B cells results in an increase in exon 9 skipping. This effect is more pronounced with concomitant overexpression of SR proteins. Antisense inhibition of endogenous TDP-43 expression results in increased inclusion of exon 9, providing a new therapeutic target to correct aberrant splicing of exon 9 in CF patients. The clinical and biological relevance of this finding in vivo is demonstrated by our characterization of a CF patient carrying a TG10T9(DeltaF508)/TG13T3(wt) genotype leading to a disease-causing high proportion of exon 9 skipping.

A gene encoding a putative GTPase regulator is mutated in familial amyotrophic lateral sclerosis 2.

Abstract: Amyotrophic lateral sclerosis 2 (ALS2) is an autosomal recessive form of juvenile ALS and has been mapped to human chromosome 2q33. Here we report the identification of two independent deletion mutations linked to ALS2 in the coding exons of the new gene ALS2. These deletion mutations result in frameshifts that generate premature stop codons. ALS2 is expressed in various tissues and cells, including neurons throughout the brain and spinal cord, and encodes a protein containing multiple domains that have homology to RanGEF as well as RhoGEF. Deletion mutations are predicted to cause a loss of protein function, providing strong evidence that ALS2 is the causative gene underlying this form of ALS.

Extensive genetic polymorphism in the human CYP2B6 gene with impact on expression and function in human liver.

Abstract: The human cytochrome P450, CYP2B6, is involved in the metabolism of several therapeutically important drugs and environmental or abused toxicants. In this study, we present the first systematic investigation of genetic polymorphism in the CYP2B6 gene on chromosome 19. A specific direct sequencing strategy was developed based on CYP2B6 and CYP2B7 genomic sequence information and DNA from 35 subjects was completely analysed for mutations throughout all nine exons and their exon-intron boundaries. A total of nine novel point mutations were identified, of which five result in amino acid substitutions in exon 1 (C64T, Arg22Cys), exon 4 (G516T, Gln172His), exon 5 (C777A, Ser259Arg and A785G, Lys262Arg) and exon 9 (C1459T, Arg487Cys) and four are silent mutations (C78T, G216C, G714A and C732T). Polymerase chain reaction-restriction fragment length polymorphism tests were developed to detect each of the five nonsynonymous mutations in genomic DNA. By screening a population of 215 subjects the C64T, G516T, C777A, A785G and C1459T mutations were found at frequencies of 5.3%, 28.6%, 0.5%, 32.6% and 14.0%, respectively. Haplotype analysis revealed six different mutant alleles termed CYP2B6*2 (C64T), *3 (C777A), *4 (A785G), *5 (C1459T), *6 (G516T and A785G) and *7 (G516T, A785G and C1459T). By analysing a large number of human liver samples, significantly reduced CYP2B6 protein expression and S-mephenytoin N-demethylase activity were found in carriers of the C1459T (R487C) mutation (alleles *5 and *7). These data demonstrate that the extensive interindividual variability of CYP2B6 expression and function is not only due to regulatory phenomena, but also caused by a common genetic polymorphism.

Tissue-specific expression of a splicing mutation in the IKBKAP gene causes familial dysautonomia.

Abstract: Familial dysautonomia (FD; also known as "Riley-Day syndrome"), an Ashkenazi Jewish disorder, is the best known and most frequent of a group of congenital sensory neuropathies and is characterized by widespread sensory and variable autonomic dysfunction. Previously, we had mapped the FD gene, DYS, to a 0.5-cM region on chromosome 9q31 and had shown that the ethnic bias is due to a founder effect, with >99.5% of disease alleles sharing a common ancestral haplotype. To investigate the molecular basis of FD, we sequenced the minimal candidate region and cloned and characterized its five genes. One of these, IKBKAP, harbors two mutations that can cause FD. The major haplotype mutation is located in the donor splice site of intron 20. This mutation can result in skipping of exon 20 in the mRNA of patients with FD, although they continue to express varying levels of wild-type message in a tissue-specific manner. RNA isolated from lymphoblasts of patients is primarily wild-type, whereas only the deleted message is seen in RNA isolated from brain. The mutation associated with the minor haplotype in four patients is a missense (R696P) mutation in exon 19, which is predicted to disrupt a potential phosphorylation site. Our findings indicate that almost all cases of FD are caused by an unusual splice defect that displays tissue-specific expression; and they also provide the basis for rapid carrier screening in the Ashkenazi Jewish population.

Expression of P-glycoprotein in human placenta : relation to genetic polymorphism of the multidrug resistance (MDR)-1 gene

Abstract: To evaluate whether mutations in the human multidrug resistance (MDR)-1 gene correlate with placental P-glycoprotein (PGP) expression, we sequenced the MDR-1 cDNA and measured PGP expression by Western blotting in 100 placentas obtained from Japanese women. Nine single nucleotide polymorphisms (SNPs) were observed with an allelic frequency of 0.005 to 0.420. Of these SNPs, G2677A (allelic frequency = 0.18) and G2677T (0.39) in exon 21 were associated with an amino acid conversion from Ala to Thr and to Ser, respectively. Sixty-one of 65 samples (93.8%), which had a C3435T allele, also had a mutant G2677(A,T) allele, suggesting an association between the two SNPs. Correlations of mutations with expression levels were observed; individuals having the G2677(A,T) and/or T-129C (p < 0.05) allele had less placental PGP. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-based genotyping tests were developed for the detection of these SNPs. The PCR, in which genomic DNAs obtained from healthy subjects (n = 48) are used as samples, was successful. The frequency of mutations in placental cDNA was identical with that in genomic DNA. When genotype results were compared between Caucasians and Japanese, ethnic differences in the frequency of polymorphism in the MDR-1 gene were suspected. Although it remains to be determined whether these SNPs influence the pharmacokinetic and dynamic properties of clinically useful drugs that are substrates of PGP, the polymorphism of the MDR-1 gene presented here may provide useful information in in vivo study of these issues.

The der(17)t(x;17)(p11;q25) of human alveolar soft part sarcoma fuses the TFE3 transcription factor gene to ASPL, a novel gene at 17q25

Abstract: Alveolar soft part sarcoma (ASPS) is an unusual tumor with highly characteristic histopathology and ultrastructure, controversial histogenesis, and enigmatic clinical behavior. Recent cytogenetic studies have identified a recurrent der(17) due to a non-reciprocal t(X;17)(p11.2;q25) in this sarcoma. To define the interval containing the Xp11.2 break, we first performed FISH on ASPS cases using YAC probes for OATL1 (Xp11.23) and OATL2 (Xp11.21), and cosmid probes from the intervening genomic region. This localized the breakpoint to a 160 kb interval. The prime candidate within this previously fully sequenced region was TFE3, a transcription factor gene known to be fused to translocation partners on 1 and X in some papillary renal cell carcinomas. Southern blotting using a TFE3 genomic probe identified non-germline bands in several ASPS cases, consistent with rearrangement and possible fusion of TFE3 with a gene on 17q25. Amplification of the 5' portion of cDNAs containing the 3' portion of TFE3 in two different ASPS cases identified a novel sequence, designated ASPL, fused in-frame to TFE3 exon 4 (type 1 fusion) or exon 3 (type 2 fusion). Reverse transcriptase PCR using a forward primer from ASPL and a TFE3 exon 4 reverse primer detected an ASPL-TFE3 fusion transcript in all ASPS cases (12/12: 9 type 1, 3 type 2), establishing the utility of this assay in the diagnosis of ASPS. Using appropriate primers, the reciprocal fusion transcript, TFE3-ASPL, was detected in only one of 12 cases, consistent with the non-reciprocal nature of the translocation in most cases, and supporting ASPL-TFE3 as its oncogenically significant fusion product. ASPL maps to chromosome 17, is ubiquitously expressed, and matches numerous ESTs (Unigene cluster Hs.84128) but no named genes. The ASPL cDNA open reading frame encodes a predicted protein of 476 amino acids that contains within its carboxy-terminal portion of a UBX-like domain that shows significant similarity to predicted proteins of unknown function in several model organisms. The ASPL-TFE3 fusion replaces the N-terminal portion of TFE3 by the fused ASPL sequences, while retaining the TFE3 DNA-binding domain, implicating transcriptional deregulation in the pathogenesis of this tumor, consistent with the biology of several other translocation-associated sarcomas. Oncogene (2001) 20, 48 - 57.

Lewy bodies and parkinsonism in families with parkin mutations.

Abstract: Previous work has established that compound mutations and homozygous loss of function of the parkin gene cause early-onset, autosomal recessive parkinsonism. Classically, this disease has been associated with loss of dopaminergic neurons in the substantia nigra pars compacta and locus ceruleus, without Lewy body pathology. We have sequenced the parkin gene of 38 patients with early-onset Parkinson's disease (<41 years). Two probands with mutations were followed up. Clinical evaluation of their families was performed, blinded to both genetic and pathological findings. Chromosome 6q25.2-27 haplotype analysis was carried out independently of the trait; parkin gene expression was examined at both the RNA and protein levels. Haplotype analysis of these families revealed a common chromosome 6, with a novel 40 bp exon 3 deletion that cosegregated with disease. In the proband of the smaller kindred, an exon 7 R275W substitution was identified in addition to the exon 3 deletion; RNA analysis demonstrated that the mutations were on alternate transcripts. However, Lewy body pathology typical of idiopathic Parkinson's disease was found at autopsy in the proband from the smaller kindred. These data suggest that compound heterozygous parkin mutations and loss of parkin protein may lead to early-onset parkinsonism with Lewy body pathology, while a hemizygous mutation may confer increased susceptibility to typical Parkinson's disease.

A mechanism for exon skipping caused by nonsense or missense mutations in BRCA1 and other genes

Abstract: Point mutations can generate defective and sometimes harmful proteins. The nonsense-mediated mRNA decay (NMD) pathway minimizes the potential damage caused by nonsense mutations. In-frame nonsense codons located at a minimum distance upstream of the last exon-exon junction are recognized as premature termination codons (PTCs), targeting the mRNA for degradation. Some nonsense mutations cause skipping of one or more exons, presumably during pre-mRNA splicing in the nucleus; this phenomenon is termed nonsense-mediated altered splicing (NAS), and its underlying mechanism is unclear. By analyzing NAS in BRCA1, we show here that inappropriate exon skipping can be reproduced in vitro, and results from disruption of a splicing enhancer in the coding sequence. Enhancers can be disrupted by single nonsense, missense and translationally silent point mutations, without recognition of an open reading frame as such. These results argue against a nuclear reading-frame scanning mechanism for NAS. Coding-region single-nucleotide polymorphisms (cSNPs) within exonic splicing enhancers or silencers may affect the patterns or efficiency of mRNA splicing, which may in turn cause phenotypic variability and variable penetrance of mutations elsewhere in a gene.

The CELF family of RNA binding proteins is implicated in cell-specific and developmentally regulated alternative splicing.

Abstract: Alternative splicing of cardiac troponin T (cTNT) exon 5 undergoes a developmentally regulated switch such that exon inclusion predominates in embryonic, but not adult, striated muscle. We previously described four muscle-specific splicing enhancers (MSEs) within introns flanking exon 5 in chicken cTNT that are both necessary and sufficient for exon inclusion in embryonic muscle. We also demonstrated that CUG-binding protein (CUG-BP) binds a conserved CUG motif within a human cTNT MSE and positively regulates MSE-dependent exon inclusion. Here we report that CUG-BP is one of a novel family of developmentally regulated RNA binding proteins that includes embryonically lethal abnormal vision-type RNA binding protein 3 (ETR-3). This family, which we call CELF proteins for CUG-BP- and ETR-3-like factors, specifically bound MSE-containing RNAs in vitro and activated MSE-dependent exon inclusion of cTNT minigenes in vivo. The expression of two CELF proteins is highly restricted to brain. CUG-BP, ETR-3, and CELF4 are more broadly expressed, and expression is developmentally regulated in striated muscle and brain. Changes in the level of expression and isoforms of ETR-3 in two different developmental systems correlated with regulated changes in cTNT splicing. A switch from cTNT exon skipping to inclusion tightly correlated with induction of ETR-3 protein expression during differentiation of C2C12 myoblasts. During heart development, the switch in cTNT splicing correlated with a transition in ETR-3 protein isoforms. We propose that ETR-3 is a major regulator of cTNT alternative splicing and that the CELF family plays an important regulatory role in cell-specific alternative splicing during normal development and disease.

Gene structure prediction and alternative splicing analysis using genomically aligned ESTs.

Abstract: With the availability of a nearly complete sequence of the human genome, aligning expressed sequence tags (EST) to the genomic sequence has become a practical and powerful strategy for gene prediction. Elucidating gene structure is a complex problem requiring the identification of splice junctions, gene boundaries, and alternative splicing variants. We have developed a software tool, Transcript Assembly Program (TAP), to delineate gene structures using genomically aligned EST sequences. TAP assembles the joint gene structure of the entire genomic region from individual splice junction pairs, using a novel algorithm that uses the EST-encoded connectivity and redundancy information to sort out the complex alternative splicing patterns. A method called polyadenylation site scan (PASS) has been developed to detect poly-A sites in the genome. TAP uses these predictions to identify gene boundaries by segmenting the joint gene structure at polyadenylated terminal exons. Reconstructing 1007 known transcripts, TAP scored a sensitivity (Sn) of 60% and a specificity (Sp) of 92% at the exon level. The gene boundary identification process was found to be accurate 78% of the time. also reports alternative splicing patterns in EST alignments. An analysis of alternative splicing in 1124 genic regions suggested that more than half of human genes undergo alternative splicing. Surprisingly, we saw an absolute majority of the detected alternative splicing events affect the coding region. Furthermore, the evolutionary conservation of alternative splicing between human and mouse was analyzed using an EST-based approach. (See http://stl.wustl.edu/~zkan/TAP/)

MDR1 pharmacogenetics: frequency of the C3435T mutation in exon 26 is significantly influenced by ethnicity.

Abstract: P-glycoprotein (PGP), the product of the multidrug resistance gene (MDR1), acts as an energy-dependent efflux pump that exports its substrates out of the cell. PGP expression is an important factor regulating absorption of a wide variety of medications. It has also been associated with intrinsic and acquired cross resistance to a number of structurally unrelated anticancer drugs. A single nucleotide polymorphism (SNP) in exon 26 of the MDR1 gene, C3435T, was recently correlated with PGP protein levels and substrate uptake. Individuals homozygous for the T allele have more than four-fold lower PGP expression compared with CC individuals. As overexpression of PGP has been associated with altered drug absorption, therapy-resistant malignancies, and lower concentrations of HIV-1 protease inhibitors, this SNP may provide a useful approach to individualize therapy. To facilitate clinical application throughout the world, 1280 subjects from 10 different ethnic groups were evaluated for this SNP using the polymerase chain reaction-restriction fragment length polymorphism assay and the genotype and allele frequency for each group were ascertained. Marked differences in genotype and allele frequency were apparent between the African populations and the Caucasian/Asian populations (P < 0.0001). The Ghanaian, Kenyan, African American and Sudanese populations studied had frequencies of 83%, 83%, 84% and 73%, respectively, for the C allele. The British Caucasian, Portuguese, South-west Asian, Chinese, Filipino and Saudi populations had lower frequencies of the C allele compared to the African group (48%, 43%, 34%, 53%, 59%, and 55%, respectively). The high frequency of the C allele in the African group implies overexpression of PGP and may have important therapeutic and prognostic implications for use of PGP dependent drugs in individuals of African origin.

Familial dysautonomia is caused by mutations of the IKAP gene.

Abstract: The defective gene DYS, which is responsible for familial dysautonomia (FD) and has been mapped to a 0.5-cM region on chromosome 9q31, has eluded identification. We identified and characterized the RNAs encoded by this region of chromosome 9 in cell lines derived from individuals homozygous for the major FD haplotype, and we observed that the RNA encoding the IκB kinase complex–associated protein (IKAP) lacks exon 20 and, as a result of a frameshift, encodes a truncated protein. Sequence analysis reveals a T→C transition in the donor splice site of intron 20. In individuals bearing a minor FD haplotype, a missense mutation in exon 19 disrupts a consensus serine/threonine kinase phosphorylation site. This mutation results in defective phosphorylation of IKAP. These mutations were observed to be present in a random sample of Ashkenazi Jewish individuals, at approximately the predicted carrier frequency of FD. These findings demonstrate that mutations in the gene encoding IKAP are responsible for FD.

Identification of novel FLT-3 Asp835 mutations in adult acute myeloid leukaemia.

Abstract: Genomic DNA from 97 cases of adult de novo acute myeloid leukaemia (AML) was screened using polymerase chain reaction (PCR) and conformation-sensitive gel electrophoresis (CSGE) for FLT3 exon 20 mutations. Initial sequencing of four cases, representing the spectrum of CSGE abnormalities, revealed changes affecting codon Asp835 in three cases and also an intron 20 A to G change. In order to identify all possible Asp835 alterations, as well as the frequency of the intronic change nucleotide 2541 + 57 AG, the patient PCR products were digested with EcoRV and NlaIII respectively. Seven cases (7·2%) possessed a mutation affecting Asp835; these were identified, following DNA sequencing, as Asp835Tyr (n = 5), Asp835His (n = 1) and Asp835del (n = 1). Alterations affecting Asp835 were not found in 80 normal control DNA samples. In contrast, the nucleotide 2541 + 57 AG change was shown to be a polymorphism, with an allelic frequency of 0·24 for the G and 0·76 for the A allele. This study reports, for the first time, point mutations in the human FLT3 gene that, because of their homology with other class III receptor tyrosine kinase mutations, probably result in constitutive activation of the receptor.

Polypyrimidine Tract Binding Protein Antagonizes Exon Definition

Abstract: PTB appears to be a global repressor of weak or regulated exons. We propose here that PTB multimerization sequesters these exons to prevent exon definition. This is likely critical not only to prevent inclusion of pseudo-exons but also to set up cell-type-specific exon definition. What remains unclear about PTB can probably be broken down into two basic questions. First, what is the precise mechanism of repression? Second, how is this mechanism circumvented? Most of the research to resolve the first question has focused primarily on identifying instances of PTB repression but has done little to understand how that repression is achieved. Recently, both in vivo and in vitro assays for PTB repression have been developed (8, 65); thus, a detailed structure-function analysis can be done. Information from this approach may address mechanistic questions such as if PTB multimerization is required for repression or if there are PTB cofactors. Understanding how this repression is lifted will probably be a more complicated issue. Overwhelming PTB may occur by numerous mechanisms, such as strengthening weak splice sites via activators such as TIA-1 (18), causing the enhancement of inclusion via a tissue-specific expression of antagonizing RNA-binding proteins, or simply by modulating the expression of a PTB cofactor.

A computational analysis of sequence features involved in recognition of short introns

Abstract: Splicing of short introns by the nuclear pre-mRNA splicing machinery is thought to proceed via an "intron definition" mechanism, in which the 5' and 3' splice sites (5'ss, 3'ss, respectively) are initially recognized and paired across the intron. Here, we describe a computational analysis of sequence features involved in recognition of short introns by using available transcript data from five eukaryotes with complete or nearly complete genomic sequences. The information content of five different transcript features was measured by using methods from information theory, and Monte Carlo simulations were used to determine the amount of information required for accurate recognition of short introns in each organism. We conclude: (i) that short introns in Drosophila melanogaster and Caenorhabditis elegans contain essentially all of the information for their recognition by the splicing machinery, and computer programs that simulate splicing specificity can predict the exact boundaries of approximately 95% of short introns in both organisms; (ii) that in yeast, the 5'ss, branch signal, and 3'ss can accurately identify intron locations but do not precisely determine the location of 3' cleavage in every intron; and (iii) that the 5'ss, branch signal, and 3'ss are not sufficient to accurately identify short introns in plant and human transcripts, but that specific subsets of candidate intronic enhancer motifs can be identified in both human and Arabidopsis that contribute dramatically to the accuracy of splicing simulators.

Mutations in SPTLC1 , encoding serine palmitoyltransferase, long chain base subunit-1, cause hereditary sensory neuropathy type I

Abstract: Hereditary sensory neuropathy type I (HSN1) is the most common hereditary disorder of peripheral sensory neurons. HSN1 is an autosomal dominant progressive degeneration of dorsal root ganglia and motor neurons with onset in the second or third decades. Initial symptoms are sensory loss in the feet followed by distal muscle wasting and weakness. Loss of pain sensation leads to chronic skin ulcers and distal amputations. The HSN1 locus has been mapped to chromosome 9q22.1-22.3 (refs. 3,4). Here we map the gene SPTLC1, encoding serine palmitoyltransferase, long chain base subunit-1, to this locus. Mutation screening revealed 3 different missense mutations resulting in changes to 2 amino acids in all affected members of 11 HSN1 families. We found two mutations to be located in exon 5 (C133Y and C133W) and one mutation to be located in exon 6 of SPTLC1 (V144D). All families showing definite or probable linkage to chromosome 9 had mutations in these two exons. These mutations are associated with increased de novo glucosyl ceramide synthesis in lymphoblast cell lines in affected individuals. Increased de novo ceramide synthesis triggers apoptosis and is associated with massive cell death during neural tube closure, raising the possibility that neural degeneration in HSN1 is due to ceramide-induced apoptotic cell death.

Antisense-induced exon skipping restores dystrophin expression in DMD patient derived muscle cells

Abstract: Due to frame-shifting mutations in the DMD gene that cause dystrophin deficiency, Duchenne muscular dystrophy (DMD) patients suffer from lethal muscle degeneration. In contrast, mutations in the allelic Becker muscular dystrophy (BMD) do not disrupt the translational reading frame, resulting in a less severe phenotype. In this study, we explored a genetic therapy aimed at restoring the reading frame in muscle cells from DMD patients through targeted modulation of dystrophin pre-mRNA splicing. Considering that exon 45 is the single most frequently deleted exon in DMD, whereas exon (45+46) deletions cause only a mild form of BMD, we set up an antisense-based system to induce exon 46 skipping from the transcript in cultured myotubes of both mouse and human origin. In myotube cultures from two unrelated DMD patients carrying an exon 45 deletion, the induced skipping of exon 46 in only approximately 15% of the mRNA led to normal amounts of properly localized dystrophin in at least 75% of myotubes. Our results provide first evidence of highly effective restoration of dystrophin expression from the endogenous gene in DMD patient-derived muscle cells. This strategy may be applicable to not only >65% of DMD mutations, but also many other genetic diseases.

The IC-SNURF–SNRPN transcript serves as a host for multiple small nucleolar RNA species and as an antisense RNA for UBE3A

Abstract: The imprinted domain on human chromosome 15 consists of two oppositely imprinted gene clusters, which are under the coordinated control of an imprinting center (IC) at the 5' end of the SNURF-SNRPN gene. One gene cluster spans the centromeric part of this domain and contains several genes that are transcribed from the paternal chromosome only (MKRN3, MAGEL2, NDN, SNURF-SNRPN, HBII-13, HBII-85 and HBII-52). Apart from the HBII small nucleolar RNA (snoRNA) genes, each of these genes is associated with a 5' differentially methylated region (DMR). The second gene cluster maps to the telomeric part of the imprinted domain and contains two genes (UBE3A and ATP10C), which in some tissues are preferentially expressed from the maternal chromosome. So far, no DMR has been identified at these loci. Instead, maternal-only expression of UBE3A may be regulated indirectly through a paternally expressed antisense transcript. We report here that a processed antisense transcript of UBE3A starts at the IC. The SNURF-SNRPN sense/UBE3A antisense transcription unit spans more than 460 kb and contains at least 148 exons, including the previously identified IPW exons. It serves as the host for the previously identified HBII-13, HBII-85 and HBII-52 snoRNAs as well as for four additional snoRNAs (HBII-436, HBII-437, HBII-438A and HBII-438B), newly identified in this study. Almost all of those snoRNAs are encoded within introns of this large transcript. Northern blot analysis indicates that most if not all of these snoRNAs are indeed expressed by processing from these introns. As we have not obtained any evidence for other genes in this region, which, from the mouse data appears to be critical for the neonatal Prader-Willi syndrome phenotype, a lack of these snoRNAs may be causally involved in this disease.

Mutation spectrum and splicing variants in the OPA1 gene.

Abstract: Optic atrophy type 1 (OPA1, MIM 165500) is a dominantly inherited optic neuropathy that features low visual acuity leading in many cases to legal blindness. We have recently shown, with others, that mutations in the OPA1 gene encoding a dynamin-related mitochondrial protein, underlie the dominant form of optic atrophy. Here we report that OPA1 has eight mRNA isoforms as a result of the alternative splicing of exon 4 and two novel exons named 4b and 5b. In addition, we screened a cohort of 19 unrelated patients with dominant optic atrophy by direct sequencing of the 30 OPA1 exons (including exons 4b and 5b) and found mutations in 17 (89%) of them of which 8 were novel. A majority of these mutations were truncative (65%) and located in exons 8 to 28, but a number of them were amino acid changes predominantly found in the GTPase domain (exons 8 to 15). We hypothesize that at least two modifications of OPA1 may lead to dominant optic atrophy, that is alteration in GTPase activity and loss of the last seven C-terminal amino acids that putatively interact with other proteins.

Mutations of CD40 gene cause an autosomal recessive form of immunodeficiency with hyper IgM

Abstract: CD40 is a member of the tumor necrosis factor receptor superfamily, expressed on a wide range of cell types including B cells, macrophages, and dendritic cells. CD40 is the receptor for CD40 ligand (CD40L), a molecule predominantly expressed by activated CD4(+) T cells. CD40/CD40L interaction induces the formation of memory B lymphocytes and promotes Ig isotype switching, as demonstrated in mice knocked-out for either CD40L or CD40 gene, and in patients with X-linked hyper IgM syndrome, a disease caused by CD40L/TNFSF5 gene mutations. In the present study, we have identified three patients with an autosomal recessive form of hyper IgM who fail to express CD40 on the cell surface. Sequence analysis of CD40 genomic DNA showed that one patient carried a homozygous silent mutation at the fifth base pair position of exon 5, involving an exonic splicing enhancer and leading to exon skipping and premature termination; the other two patients showed a homozygous point mutation in exon 3, resulting in a cysteine to arginine substitution. These findings show that mutations of the CD40 gene cause an autosomal recessive form of hyper IgM, which is immunologically and clinically undistinguishable from the X-linked form.

CTLA-4 gene expression is influenced by promoter and exon 1 polymorphisms.

Abstract: CTLA-4, expressed mainly on activated T cells, helps maintain, through its inhibitory function, immune-system homeostasis. Polymorphisms in the CTLA-4 gene (CTLA4) are known to be important in several autoimmune diseases, including multiple sclerosis (MS). Here, we have performed genotyping for CTLA4 polymorphisms, and investigated expression by peripheral blood mononuclear cells of CTLA-4 mRNA and protein, in patients with MS and myasthenia gravis and in healthy controls. Expression levels for mRNA and protein were similar in the patient and control groups; however, there was a clear relationship between genotype and CTLA-4 expression. Specifically, individuals carrying thymine at position -318 of the CTLA4 promoter (T(-318)) and homozygous for adenine at position 49 in exon 1 showed significantly increased expression both of cell-surface CTLA-4 after cellular stimulation and of CTLA-4 mRNA in non-stimulated cells. The association was seen most clearly for unsorted CD3(+) cells and was absent in the CD8(+) subset. The T(-318) allele has been shown to be negatively associated with susceptibility to MS in an earlier study by our group. Thus, we propose that the susceptibility-influencing role of CTLA4 in MS may be related to genotypically conditioned promoter function, whereby high gene expression may decrease the risk of disease.