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PubMed1993

The human gene encoding tryptophanyl-tRNA synthetase: interferon-response elements and exon-intron organization.

Recently, we cloned and sequenced the cDNA encoding human tryptophanyl-tRNA synthetase (hWRS) [Frolova et al., Gene 109 (1991) 291-296]. Independently, it has been shown that this protein is induced by interferons (IFN) gamma and alpha [Fleckner et al., Proc. Natl. Acad. Sci. USA 88 (1991) 11520-11524; Rubin et al., J. Biol. Chem. 266 (1991) 24245-24248]. This unusual feature of a housekeeping enzyme raises the problem of how the gene is regulated. Since at present the genomic structure of hWRS is unknown, this issue remains unsolved. Here, the exon-intron organization of hWRS has been deciphered. This gene consists of at least 12 exons that span more than 35 kb of DNA. At least two alternative noncoding exons precede ten coding exons. Upstream from the first exon, two GGAAAN(N/-)GAAA sequences, which are considered to be IFN-stimulating response elements (ISRE), have been revealed. The same consensus was also found in the intron region in close vicinity to the 5' end of the second exon. Thus, the IFN-stimulated synthesis of hWRS is presumably due to gene activation at the transcriptional level. Alignment of hWRS amino acid sequences has shown that exons V to XI of hWRS encode regions of structural similarity with bacterial WRS, whereas the N-terminal portion of the protein encoded by exons II to IV exhibits no homology with bacterial WRS.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2006

An alternative intron-exon pairing scheme implied by unexpected in vitro activities of group II intron RmInt1 from Sinorhizobium meliloti.

RmInt1 is a mobile group II intron which interrupts ISRm2011-2, another mobile element from the bacterium Sinorhizobium meliloti. Ribozyme constructs derived from intron RmInt1 self-splice in vitro when incubated under permissive conditions, but the excised intron and ligated exons are largely replaced by unconventional products. These include a slightly shorter, 5'-end truncated 3' exon, truncated variants of the linear and lariat forms of the intron-3' exon reaction intermediate, as well as presumably circular molecules derived from the latter. Two factors explain the abundance of these products: (i) nucleotides 5-11 of the 3' exon (IBS1*) provide a better match to the EBS1 5'-exon-binding site than the authentic IBS1 sequence in the 5' exon; (ii) exon ligation is unusually inefficient, and especially so when the 5' exon is truncated close to the second (IBS2) intron-binding site. We propose that reactions at the IBS1* site play a part in the regulation of the intron ISRm2011-2 host in vivo.

Alternative Splicing↗

PubMed2006

TGF-β1 regulation of human AT1 receptor mRNA splice variants harboring exon 2.

At least four alternatively spliced mRNAs can be synthesized from the human AT(1)R (hAT(1)R) gene that differ only in the inclusion or exclusion of exon 2 and/or 3. RT-PCR experiments demonstrate that splice variants harboring exon 2 accounts for at least 30% of all the hAT(1)R mRNA transcripts expressed in the human tissues investigated. Since exon 2 contains two upstream AUGs or open reading frames (uORFs), we hypothesized that these AUGs would inhibit the translation of the downstream hAT(1)R protein ORF harbored in exon 4. This study demonstrates that the inclusion of exon 2 in hAT(1)R mRNA transcripts dramatically reduces hAT(1)R protein levels (nine-fold) and significantly attenuates Ang II responsiveness ( approximately four-fold). Interestingly, only when both AUGs were mutated in combination were the hAT(1)R density and Ang II signaling levels comparable with those values obtained using mRNA splice variants that did not include exon 2. This observation is consistent with a model where the majority of the ribosomes likely translate uORF#1 and are then unable to reinitiate at the downstream hAT(1)R ORF, in part due to the presence of AUG#2 and to the short intercistronic spacing. Importantly, TGF-beta(1) treatment (4ng/ml for 4h) of fibroblasts up-regulated hAT(1)R mRNA splice variants, which harbored exon 2, six-fold. Since AT(1)R activation is closely associated with cardiovascular disease, the inclusion of exon 2 by alternative splicing represents a novel mechanism to reduce the overall production of the hAT(1)R protein and possibly limit the potential pathological effects of AT(1)R activation.

Alternative Splicing↗

PubMed2002

Detection of polymorphisms at exons 3 (Tyr113-->His) and 4 (His139-->Arg) of the microsomal epoxide hydrolase gene using fluorescence PCR method combined with melting curves analysis.

An association between exon 3 polymorphisms of the gene encoding microsomal epoxide hydrolase (mEH) and susceptibility to the development of chronic obstructive pulmonary disease (COPD) has been described. We have developed two methods for detecting polymorphisms at exons 3 (Tyr113-->His) and 4 (His139-->Arg) of the mEH gene based on different melting temperatures (T(m)) of fluorescent-labeled oligonucleotide hybridization probes using single-step assays that combine fluorescence PCR and melting curve analysis (LightCycler methodology). DNA was extracted from blood in 79 COPD patients and 146 healthy controls. Results were compared with those obtained by restriction fragment length polymorphism (RFLP) analysis to detect Tyr113His variants and a single-strand conformation polymorphism (SSCP) assay for His139Arg detection. The T(m) of the exon 3 polymorphisms were 61.3 degrees C for Tyr113 (wild type) and 67.5 degrees C for His113 (mutant). The T(m) values of the exon 4 polymorphisms were 67.5 degrees C for His139 (wild type) and 59.2 degrees C for Arg139 (mutant). The within- and between-run melting peaks for the same allele differed by less than 0.5 degrees C for both the exon 3 and the exon 4 polymorphisms. Thus, melting analysis allowed easy and unambiguous assignment of genotyping by means of the respective melting curves. The proportion of individuals who were homozygous mutant for exon 3 was significantly higher in the COPD group than in the control group (p=0.004). LightCycler fluorescence genotyping of exon 4 polymorphisms correlated perfectly with SSCP results. RFLP assay classified 2 patients as homozygous mutant while LightCycler analysis genotyped them as heterozygous. DNA analysis by PCR and sequencing confirmed the LightCycler result. These high-speed (about 40 min for 32 samples), highly sensitive, and specific small-volume assays with low labor requirements hold great promise as tools for rapid detection of COPD susceptibility.

Adult↗

PubMed1998

Identification of two additional exons at the 3' end of the amelogenin gene.

Alternative splicing of the amelogenin gene generates a family of proteins secreted by ameloblasts that are primarily responsible for tooth enamel formation. Here the presence of two additional exons, downstream of exon 7, which are followed by an alternate polyadenylation site, is reported. Long polymerase chain reaction was used with a rat genomic amelogenin template to amplify sequences downstream from the primary polyadenylation site. Within the amplified sequences, two exons of 45 bp (exon 8) and 110 bp (exon 9) were identified. The presence of these additional exons in the human and mouse genome was indicated by Southern blot analysis. Antibodies raised against a synthetic peptide corresponding to a sequence encoded by exon 9 positively stained ameloblasts in sections of developing murine and porcine unerupted teeth. These results suggest that an amelogenin protein(s) that includes sequences derived from exons 8 and 9 is synthesized by the ameloblasts.

Alternative Splicing↗

PubMed1994

The first analysis of exon 1 (the transactivation domain) of the androgen receptor gene in infertile men with oligospermia or azoospermia.

OBJECTIVE: To examine the role of the androgen receptor (AR) gene in spermatogenesis by evaluating infertile men with idiopathic oligospermia or azoospermia, with special emphasis on the transactivation domain (exon 1) of AR gene because it has not been studied in this population previously. STUDY DESIGN: A molecular study of the AR gene. Deoxyribonucleic acid samples were screened for possible AR gene mutations using polymerase chain reactions (PCR). SETTING: The offices and laboratories of the Medical College of Georgia. PARTICIPANTS: Infertile men with oligospermia or azoospermia and an otherwise negative laboratory evaluation. Controls consisted of healthy fertile men. MAIN OUTCOME MEASURES: Each exon (2 to 8) and each of five overlapping exon segments for exon 1 of the AR gene was amplified using PCR for each participant's DNA sample. The PCR products were evaluated by size using electrophoresis and a DNA size marker. RESULTS: Sixteen idiopathic oligospermic or azoospermic men entered the study. All seven exons and the five overlapping segments of exon 1 were amplified and were of the appropriate size on electrophoresis when compared with controls, the DNA size marker, and the exon sequence. CONCLUSIONS: Preliminary protein studies on AR suggested that up to 40% of infertile men may have AR abnormalities. Since the availability of molecular analysis, no studies to date have evaluated the transcriptional activation domain (exon 1) of the AR gene in this population of infertile men. Our study found no gross AR mutations in the individuals studied. These results emphasize the importance of further studies needed to understand the regulation of spermatogenesis.

DNA↗

PubMed1995

CD44 variant exon epitopes in primary breast cancer and length of survival.

CD44 designates a group of closely related cell-surface proteins generated by alternative splicing. We have previously shown that splice variants carrying sequences encoded by exon v6 are preferentially expressed in metastatic animal cancer cell lines and that they confer metastatic behaviour on non-metastatic animal tumour cell lines. In this study we set out to assess the expression of CD44 epitopes specific for variant exon sequences in human breast cancer and their potential for determining prognosis. We used affinity-purified polyclonal sera and four monoclonal antibodies raised against the human homologues of CD44 variant exon sequences to investigate the presence of CD44 on 100 primary invasive breast tumours, 12 local recurrences, 18 lymph node metastases, and normal tissue controls. Whereas normal mammary ductal epithelial cells and cells derived from hyperplastic lesions do not express CD44 variant exons, expression of v3, v5, and v6 epitopes was found in most tumour samples. The DIII (exon v6) epitope was present in 84% of the primary tumours and in 100% of axillary lymph node metastases and local recurrences. The presence of these CD44 epitopes is correlated with poor overall survival. 15 patients with exon-v6-negative tumours had good survival compared with 76 patients with exon-v6-positive tumours (p = 0.005; log rank test). Multivariate analysis showed that the CD44 epitope encoded by exon v6 was a good marker for prognosis independent of progesterone receptor, lymph node status, tumour size, and grade.

Adult↗

PubMed2000

Regulation of SRp20 exon 4 splicing.

SR proteins are essential splicing factors involved in the use of both constitutive and alternative exons. We previously showed that the SR proteins SRp20 and ASF/SF2 have antagonistic activities on SRp20 pre-mRNA splicing. SRp20 activates exon 4 recognition in its pre-mRNA, whereas ASF/SF2 inhibits this recognition. In experiments aimed at testing the specificity of SRp20 and ASF/SF2 for exon 4 splicing regulation, we show here that this specificity lies in the RNA binding domains of SRp20 and ASF/SF2 and not in the RS domains. Surprisingly, a deletion of 14 amino acids at the end of ASF/SF2-RBD2 converts ASF/SF2 from an inhibitor to an activator of exon 4 splicing. We found that ASF3 also inhibits exon 4 recognition, thus acting similarly to ASF/SF2, while SC35 activates a cryptic 5' splice site downstream of exon 3 and, in doing so, represses exon 4 use. In contrast, Tra2 and the SR proteins 9G8 and SRp40 do not appear to affect exon 4 splicing.

Alternative Splicing↗

PubMed2001

The point mutation in the promoter region and the single nucleotide polymorphism in exon 1 of the cytokeratin 19 gene in human lung cancer cell lines.

The CYFRA 21-1 assay which detects the cytokeratin 19 (CK19) fragment is widely used as a tumor marker for lung cancer. We previously suggested that the failure of PCR amplification of exon 1 is closely related to the inability of the expression of mRNA for CK19, and hypothesized that point mutations might exist within exon 1. In order to prove this, sequence analysis of the promoter region and exon 1 was performed in 14 human lung cancer cell lines. Among the 14 lung cancer cell lines evaluated, point mutations within the promoter region (at -99, G-->C) of the CK19 gene were demonstrated in two cell lines (Lu135 and HI1017). In addition, point mutations within exon 1 (at 90, T-->C, Ala-->Ala and at 179, G-->C, Gly-->Ala) were also demonstrated in three cell lines (LU135, HI1017, and LC2/AD). Point mutations within the promoter region of CK19 (at -99) and within exon 1 (at 179) were confirmed by analysis of digestion by specific restriction enzymes. Since the same point mutation within exon 1 (at 179) was observed in genomes of normal volunteers, this mutation was considered as a single nucleotide polymorphism. In contrast, there were no mutations within the promoter region of exon 1 in genomes of normal volunteers. After a computer search, it was demonstrated that several transcription factors bind to the sense primer sequence which was designed for amplification of exon 1. In addition, after point mutations within the promoter region occurred (at -99), new sequences appeared to which known transcription factors (AP2) bind. In conclusion, analysis of genomic DNA for CK19 suggested that expression of mRNA for CK19 was regulated by several transcription factors which bound to the specific sequence with the promoter region of the CK19 gene. It was also suggested that the mutation in the promoter region of the CK19 gene down-regulated the expression of mRNA for CK19.

Antigens, Neoplasm↗

PubMed1998

The unique exon 10 of the human luteinizing hormone receptor is necessary for expression of the receptor protein at the plasma membrane in the human luteinizing hormone receptor, but deleterious when inserted into the human follicle-stimulating hormone receptor.

The LH receptor (LHR) is a member of the family of G protein-coupled seven-times plasma membrane transversing receptors. Its gene consists of 11 exons, the last one encoding the transmembrane and intracellular domains of the receptor. The FSHR, and its gene, resemble structurally those of the LHR, with the exception that the sequences corresponding to exon 10 in LHR are missing in FSHR, which is thus encoded by a total of ten exons. Our recent studies on the marmoset monkey testis LHR cDNA indicated that an 81 bp nucleotide sequence, encoding the complete exon 10 of the LHR gene in other mammalian species, is absent in this species without affecting the LHR function. To study further the role of the exon 10 encoded sequences of the LHR in the gonadotropin receptor function, a deletion of exon 10 from the human LHR (hLHdeltaexon10R), and a chimeric hFSHR with exon 10 from hLHR inserted (hFSHLHexon10R), were constructed in expression vectors. The results presented here demonstrate that 293 cells transfected with the hLHdeltaexon10R display a decrease in the proportion of the receptor binding at the cell surface, compared with cells transfected with wild-type hLHR. However, the cells expressing hLHdeltaexon10R showed similar high affinity binding of [125I]iodo-hCG as those transfected with wild-type hLHR, in either intact cells or their detergent extracts. In addition, cells expressing the hLHdeltaexon10R and wild-type hLHR displayed similar dose-response of cAMP production to hCG stimulation. Cells transfected with chimeric hFSHLHexon10R showed barely detectable [125I]iodo-FSH binding in intact cells compared with those transfected with wild-type hFSHR. The FSH binding detected in cellular detergent extracts displayed 10-fold lower binding activity than wild-type receptors, in spite of similar level of immunoreactive FSHR protein expression in the transfected cells. The hFSHLHexon10R had a modest 5-fold lower binding affinity for FSH as compared with wild-type hFSHR. In conclusion, the present study indicates that the sequences encoding exon 10 of the hLHR are essential for the LHR expression at the plasma membrane, but deleterious for function if inserted into the hFSHR.

Animals↗

PubMed2000

The human cytochrome P450 3A locus. Gene evolution by capture of downstream exons.

Using a bacterial artificial chromosome (BAC) clone, we have mapped the human cytochrome P450 3A (CYP3A) locus containing the genes encoding for CYP3A4, CYP3A5 and CYP3A7. The genes lie in a head-to-tail orientation in the order of 3A4, 3A7 and 3A5. In both intergenic regions (3A4-3A7 and 3A7-3A5), we have detected several additional cytochrome P450 3A exons, forming two CYP3A pseudogenes. These pseudogenes have the same orientation as the CYP3A genes. To our surprise, a 3A7 mRNA species has been detected in which the exons 2 and 13 of one of the pseudogenes (the one that is downstream of 3A7) are spliced after the 3A7 terminal exon. This results in an mRNA molecule that consists of the 13 3A7 exons and two additional exons at the 3' end. The additional two exons originating from the pseudogene are in an altered reading frame and consequently have the capability to code a completely different amino acid sequence than the canonical CYP3A exons 2 and 13. These findings may represent a generalized evolutionary process with genes having the potential to capture neighboring sequences and use them as functional exons.

Alternative Splicing↗

PubMed2003

Expression of estrogen receptor beta exon-deleted variant mRNAs in ovary and uterine endometrium.

Various estrogen receptor beta exon-deleted variant (ER-beta EDV) mRNAs were expressed in human ovary and uterine endometrium. Estrogen receptor beta (ER-beta) completely or partially deleted exon n is expressed as ER-beta EnDV or En'DV, respectively. The mRNAs for ER-beta single exon-deleted variant (EDV), ER-beta E2DV, E4DV, E5DV and E6DV; for ER-beta double exon-deleted variants, ER-beta E1'+2DV, E4+5DV and E5+6DV; and for ER-beta triple exon-deleted variants, ER-beta E2'+3+4DV and E4+5+6DV were detected. In ER-beta E2DV, E4+5DV, E5DV and E6DV mRNAs, the new stop codon is made in the exon following the deleted exon(s), and the new proteins may lack the corresponding domains. In ER-beta E1'+2DV, E2'+3+4DV, E4DV, E4+5+6DV and E5+6DV mRNAs, the original stop codon is still present, and the new proteins may conserve the new short amino acid sequences surrounding the deleted exons. ER-beta E1'+2DV, E2DV, E2'+3+4DV, E4DV, E4+5DV and E4+5+6 are unlikely to work as a transcription factors. On the other hand, ER-beta E5DV, E6DV and E5+6DV lack only the ligand-binding domain, and might work as dominant positive or negative factors. Therefore, ER-beta E5DV, E6DV and E5+6DV, constitutively expressed in human ovary and uterine endometrium might, in part regulate estrogen responsiveness.

Adult↗

PubMed1996

Aromatase gene expression and its exon I usage in human breast tumors. Detection of aromatase messenger RNA by reverse transcription-polymerase chain reaction.

The expression of aromatase in human breast tumors has been studied by the reverse-transcription polymerase chain reaction (RT-PCR) method on 70 breast tissue specimens. An RT-PCR analysis using two oligonucleotide primers derived from the exon II of the human aromatase gene revealed that aromatase mRNA was detected in all but three tissue specimens. Furthermore, primer-directed RT-PCR was performed to determine the exon I usage in aromatase mRNA in these breast tumor specimens. The analysis has revealed that exons I.3 and PII are the two major exon Is present in aromatase mRNA isolated from breast tumors, suggesting that promoters I.3 and II are the major promoters driving aromatase expression in breast cancer and surrounding adipose stromal cells. The RT-PCR analysis also detected two products, I.3A (334 bp in length) and I.3B (222 bp in length), when it was carried out using a primer derived from exon I.3 and a reverse primer derived from exon II. The nucleotide sequences of these products have been determined and indicate that I.3A contains a region which was previously thought to be an intron. In addition, RT-PCR analyses of RNA isolated from eight pairs of breast tumor and neighboring normal tissue specimens were performed to evaluate the exon I usage and the distribution of I.3A- and I.3B-containing aromatase RNA messages in breast tumor and neighboring normal tissues. The results suggest that I.3B- and I.3A-containing messages are mainly present in breast tumor and neighboring normal tissues, respectively. Finally, the exon I/promoter usage for aromatase expression in eight cell lines (skin fibroblast, MCF-7, MDA-MB-231, T-47D, SK-BR-3, JAR, OVCAR-3, and human adipose stromal cells) was examined by primer-directed RT-PCR analyses. These studies provide a basis for further evaluation of the control mechanism of aromatase expression and estrogen biosynthesis in breast tumors.

Adipose Tissue↗

PubMed2002

RNAi-mediated PTB depletion leads to enhanced exon definition.

Mutually exclusive use of exons IIIb or IIIc in FGF-R2 transcripts requires the silencing of exon IIIb. This repression is mediated by silencer elements upstream and downstream of the exon. Both silencers bind the polypyrimidine tract binding protein (PTB) and PTB binding sites within these elements are required for efficient silencing of exon IIIb. Recruitment of MS2-PTB fusion proteins upstream or downstream of exon IIIb causes repression of this exon. Depletion of endogenous PTB using RNAi increases exon IIIb inclusion in transcripts derived from minigenes and from the endogenous FGF-R2 gene. These data demonstrate that PTB is a negative regulator of exon definition in vivo.

Blotting, Western↗

PubMed2004

Mechanism of gamma-secretase cleavage activation: is gamma-secretase regulated through autoinhibition involving the presenilin-1 exon 9 loop?

Maturation of gamma-secretase requires an endoproteolytic cleavage in presenilin-1 (PS1) within a peptide loop encoded by exon 9 of the corresponding gene. Deletion of the loop has been demonstrated to cause familial Alzheimer's disease. A synthetic peptide corresponding to the loop sequence was found to inhibit gamma-secretase in a cell-free enzymatic assay with an IC(50) of 2.1 microM, a value similar to the K(m) (3.5 microM) for the substrate C100. Truncation at either end, single amino acid substitutions at certain residues, sequence reversal, or randomization reduced its potency. Similar results were also observed in a cell-based assay using HEK293 cells expressing APP. In contrast to small-molecule gamma-secretase inhibitors, kinetic inhibition studies demonstrated competitive inhibition of gamma-secretase by the exon 9 peptide. Consistent with this finding, inhibitor cross-competition kinetics indicated noncompetitive binding between the exon 9 peptide and L685458, a transition-state analogue presumably binding at the catalytic site, and ligand competition binding experiments revealed no competition between L685458 and the exon 9 peptide. These data are consistent with the proposed gamma-secretase mechanism involving separate substrate-binding and catalytic sites and binding of the exon 9 peptide at the substrate-binding site, but not the catalytic site of gamma-secretase. NMR analyses demonstrated the presence of a loop structure with a beta-turn in the middle of the exon 9 peptide and a loose alpha-helical conformation for the rest of the peptide. Such a structure supports the hypothesis that this exon 9 peptide can adopt a distinct conformation, one that is compact enough to occupy the putative substrate-binding site without necessarily interfering with binding of small molecule inhibitors at other sites on gamma-secretase. We hypothesize that gamma-secretase cleavage activation may be a result of a cleavage-induced conformational change that relieves the inhibitory effect of the intact exon 9 loop occupying the substrate-binding site on the immature enzyme. It is possible that the DeltaE9 mutation causes Alzheimer's disease because cleavage activation of gamma-secretase is no longer necessary, alleviating constraints on Abeta formation.

Alzheimer Disease↗

PubMed2001

Novel exon 11 skipping mutation in a patient with glycogen storage disease type IIId.

We report the molecular genetic abnormalities of a patient with GSD IIId presenting with progressive myopathy and cardiopathy leading to a fatal outcome. We identified two independent deletions including a 4 bp deletion (117-1120) and a 98 bp deletion (1135-1232) in cDNA. Sequencing of the genomic DNA of the corresponding region revealed a 4 bp deletion in exon 10; however, the other 98 bp deletion corresponding to exon 11, which was deleted in cDNA, was present in genomic DNA. We therefore concluded that skipping of exon 11 occurred in the cDNA of the patient. Intron/exon boundary analysis of the skipped exon 11 revealed no mutation in the consensus splice-site sequence. If normal splicing had occurred, a stop codon would have appeared within exon II due to frameshift mutation. The mechanism of exon skipping observed in our patient is as yet unknown, and it is still not clear whether intraexonal mutation of the preceding exon can influence splice-site selection. It is possible that a unique exon skipping occurred, preventing the appearance of a stop codon in our patient.

Adult↗

PubMed2002

Differential expression of mutually exclusive exons of the fast skeletal muscle troponin T gene in the chicken wing and leg muscles.

To determine the physiological significance of developmentally regulated and muscle tissue-specific expression of troponin T (TnT) isoforms and also to elucidate mechanisms of alternative splicing, we investigated splicing patterns of fast skeletal muscle TnT by sequencing cDNAs obtained from the chicken wing and leg muscles. The chicken fast skeletal muscle TnT gene has 27 exons, and produces potentially 2(15) transcripts if alternative exons are randomly spliced. For the fourteen alternative exons in the 5'-region of the gene, we found 7 and 12 splice variants in the wing biceps brachii and leg gastrocnemius, respectively. Out of 19, 12 variants were newly found in this study. Twenty two different splice variants were reported by the previous studies, but this number has now increased to 34 in the chicken TnT gene, suggesting that alternative splicing is not random, but strictly regulated. We also investigated splicing patterns of mutually exclusive exons 16 and 17 in the 3'-region of the gene. Approximately equal amounts of cDNAs containing exons 16 and 17 were detected in proximal regions of adult biceps brachii and gastrocnemius, respectively, whereas cDNA with exon 17 dominated in distal regions. Only cDNA containing exon 17 was found in both proximal and distal regions of 5-day-old gastrocnemius. The inclusion of exon 16 into cDNAs was detected in the proximal region of 15-day-old gastrocnemius and increased during development.

Alternative Splicing↗

PubMed2000

SMA carrier testing--validation of hemizygous SMN exon 7 deletion test for the identification of proximal spinal muscular atrophy carriers and patients with a single allele deletion.

To facilitate the detection of carriers of a hemizygous survival motor neuron (SMN) exon 7 deletion we have modified the quantitative SMN exon 7 assay described by McAndrew et al (1997). The major changes include quantitative analysis of the amount of SMN exon 7-specific fluorescently-labelled PCR product on an automated sequencer, and the monitoring of the completeness of a DraI digestion necessary to distinguish the PCR products of exons 7 of SMN and its copy gene. In our method the amount of SMN exon 7 PCR product is compared with the amount of a co-amplified PCR product of the retinoblastoma (RB1) exon containing a DraI restriction site. By co-amplification using the same primers of plasmids included in the reaction as internal standards containing SMN exon 7 with a 36-nucleotide deletion and RB1 exon 13 with a 19-nucleotide deletion, respectively, the relative amplification efficacy can be monitored. The assay has been validated in 63 ascertained carriers and 28 ascertained non-carriers. The sensitivity of the test is approximately 97%, the specificity approaches 100%. In four out of six SMA patients without a homozygous deletion we detected a hemizygous deletion. The implications of the use of this assay for carrier testing and for confirmation of the clinical diagnosis of SMA are discussed.

Cyclic AMP Response Element-Binding Protein↗