Identification of a novel splice-site mutation in the CYP1A2 gene.

AIMS: To identify the molecular basis for a low CYP1A2 metabolic status, as determined by a caffeine phenotyping test, in a 71-year-old, nonsmoking, Caucasian woman who presented with very high clozapine concentrations despite being administered a standard dose of the drug. METHODS: The nucleotide sequence of the 7 exons, exon-intron boundaries and 5'-flanking region of the CYP1A2 gene was analysed by direct sequencing. RESULTS: Only one heterozygous point mutation was identified in the donor splice site of intron 6 (3534G > A) of CYP1A2. This mutation could cause abnormal RNA splicing and therefore lead to a truncated nonfunctional enzyme. No other carrier of this mutation was identified in a population of 100 unrelated healthy Caucasians. CONCLUSIONS: This is the first report of a splice-site mutation affecting the CYP1A2 gene. This polymorphism is a likely explanation for the low CYP1A2 activity associated with high clozapine concentrations in this patient.

Utilização do Intron Splice Site primer EI-1 na discriminação de leveduras contaminantes do processo de fermentação alcoólica

Neste trabalho, utilizamos o Intron Splice Site primer EI-1 para a análise do perfil de amplificação de diferentes espécies de leveduras consideradas contaminantes no processo de fermentação alcoólica, originadas de uma destilaria no Estado da Paraíba na safra 2004/2005. Foram realizadas as etapas analíticas para discriminação molecular das leveduras a partir da extração do DNA total, amplificação por PCR e análise do perfil genético. Os resultados obtidos indicam que o Intron Splice Site primer EI-1é muito eficaz na discriminação das diferentes espécies de Saccharomyces e não Saccharomyces, evidenciando padrões de bandas específicos para as leveduras analisadas. Este primer, por ser complementar a uma região muito conservada do genoma das leveduras, mostrou-se incapaz de uma discriminação intraespecífica. Isto demonstra a utilidade deste marcador no auxílio à taxonomia de leveduras.

Growth hormone (GH)-releasing hormone increases the expression of the dominant-negative GH isoform in cases of isolated GH deficiency due to GH splice-site mutations

An autosomal dominant form of isolated GH deficiency (IGHD II) can result from heterozygous splice site mutations that weaken recognition of exon 3 leading to aberrant splicing of GH-1 transcripts and production of a dominant-negative 17.5-kDa GH isoform. Previous studies suggested that the extent of missplicing varies with different mutations and the level of GH expression and/or secretion. To study this, wt-hGH and/or different hGH-splice site mutants (GH-IVS+2, GH-IVS+6, GH-ISE+28) were transfected in rat pituitary cells expressing human GHRH receptor (GC-GHRHR). Upon GHRH stimulation, GC-GHRHR cells coexpressing wt-hGH and each of the mutants displayed reduced hGH secretion and intracellular GH content when compared with cells expressing only wt-hGH, confirming the dominant-negative effect of 17.5-kDa isoform on the secretion of 22-kDa GH. Furthermore, increased amount of 17.5-kDa isoform produced after GHRH stimulation in cells expressing GH-splice site mutants reduced production of endogenous rat GH, which was not observed after GHRH-induced increase in wt-hGH. In conclusion, our results support the hypothesis that after GHRH stimulation, the severity of IGHD II depends on the position of splice site mutation leading to the production of increasing amounts of 17.5-kDa protein, which reduces the storage and secretion of wt-GH in the most severely affected cases. Due to the absence of GH and IGF-I-negative feedback in IGHD II, a chronic up-regulation of GHRH would lead to an increased stimulatory drive to somatotrophs to produce more 17.5-kDa GH from the severest mutant alleles, thereby accelerating autodestruction of somatotrophs in a vicious cycle.

Isolated autosomal dominant growth hormone deficiency: stimulating mutant GH-1 gene expression drives GH-1 splice-site selection, cell proliferation, and apoptosis

The majority of mutations that cause isolated GH deficiency type II (IGHD II) affect splicing of GH-1 transcripts and produce a dominant-negative GH isoform lacking exon 3 resulting in a 17.5-kDa isoform, which further leads to disruption of the GH secretory pathway. A clinical variability in the severity of the IGHD II phenotype depending on the GH-1 gene alteration has been reported, and in vitro and transgenic animal data suggest that the onset and severity of the phenotype relates to the proportion of 17.5-kDa produced. The removal of GH in IGHD creates a positive feedback loop driving more GH expression, which may itself increase 17.5-kDa isoform productions from alternate splice sites in the mutated GH-1 allele. In this study, we aimed to test this idea by comparing the impact of stimulated expression by glucocorticoids on the production of different GH isoforms from wild-type (wt) and mutant GH-1 genes, relying on the glucocorticoid regulatory element within intron 1 in the GH-1 gene. AtT-20 cells were transfected with wt-GH or mutated GH-1 variants (5'IVS-3 + 2-bp T->C; 5'IVS-3 + 6 bp T->C; ISEm1: IVS-3 + 28 G->A) known to cause clinical IGHD II of varying severity. Cells were stimulated with 1 and 10 mum dexamethasone (DEX) for 24 h, after which the relative amounts of GH-1 splice variants were determined by semiquantitative and quantitative (TaqMan) RT-PCR. In the absence of DEX, only around 1% wt-GH-1 transcripts were the 17.5-kDa isoform, whereas the three mutant GH-1 variants produced 29, 39, and 78% of the 17.5-kDa isoform. DEX stimulated total GH-1 gene transcription from all constructs. Notably, however, DEX increased the amount of 17.5-kDa GH isoform relative to the 22- and 20-kDa isoforms produced from the mutated GH-1 variants, but not from wt-GH-1. This DEX-induced enhancement of 17.5-kDa GH isoform production, up to 100% in the most severe case, was completely blocked by the addition of RU486. In other studies, we measured cell proliferation rates, annexin V staining, and DNA fragmentation in cells transfected with the same GH-1 constructs. The results showed that that the 5'IVS-3 + 2-bp GH-1 gene mutation had a more severe impact on those measures than the splice site mutations within 5'IVS-3 + 6 bp or ISE +28, in line with the clinical severity observed with these mutations. Our findings that the proportion of 17.5-kDa produced from mutant GH-1 alleles increases with increased drive for gene expression may help to explain the variable onset progression, and severity observed in IGHD II.

Sensitivity of splice sites to antisense oligonucleotides in vivo.

A series of HeLa cell lines which stably express beta-globin pre-mRNAs carrying point mutations at nt 654, 705, or 745 of intron 2 has been developed. The mutations generate aberrant 5' splice sites and activate a common 3' cryptic splice site upstream leading to aberrantly spliced beta-globin mRNA. Antisense oligonucleotides, which in vivo blocked aberrant splice sites and restored correct splicing of the pre-mRNA, revealed major differences in the sensitivity of these sites to antisense probes. Although the targeted pre-mRNAs differed only by single point mutations, the effective concentrations of the oligonucleotides required for correction of splicing varied up to 750-fold. The differences among the aberrant 5' splice sites affected sensitivity of both the 5' and 3' splice sites; in particular, sensitivity of both splice sites was severely reduced by modification of the aberrant 5' splice sites to the consensus sequence. These results suggest large differences in splicing of very similar pre-mRNAs in vivo. They also indicate that antisense oligonucleotides may provide useful tools for studying the interactions of splicing machinery with pre-mRNA.

Role of adenine functional groups in the recognition of the 3′-splice-site AG during the second step of pre-mRNA splicing

The AG dinucleotide at the 3′ splice sites of metazoan nuclear pre-mRNAs plays a critical role in catalytic step II of the splicing reaction. Previous studies have shown that replacement of the guanine by adenine in the AG (AG → GG) inhibits this step. We find that the second step was even more severely inhibited by cytosine (AG → CG) or uracil (AG → UG) substitutions at this position. By contrast, a relatively moderate inhibition was observed with a hypoxanthine substitution (AG → HG). When adenine was replaced by a purine base (AG → PG) or by 7-deazaadenine (AG → c7AG), little effect on the second step was observed, suggesting that the 6-NH2 and N7 groups do not play a critical role in adenine recognition. Finally, replacement of adenine by 2-aminopurine (AG → 2-APG) had no effect on the second step. Taken together, our results suggest that the N1 group of adenine functions as an essential determinant in adenine recognition during the second step of pre-mRNA splicing.

Conserved stem–loop structures in the HIV-1 RNA region containing the A3 3′ splice site and its cis-regulatory element: possible involvement in RNA splicing

The HIV-1 transcript is alternatively spliced to over 30 different mRNAs. Whether RNA secondary structure can influence HIV-1 RNA alternative splicing has not previously been examined. Here we have determined the secondary structure of the HIV-1/BRU RNA segment, containing the alternative A3, A4a, A4b, A4c and A5 3′ splice sites. Site A3, required for tat mRNA production, is contained in the terminal loop of a stem–loop structure (SLS2), which is highly conserved in HIV-1 and related SIVcpz strains. The exon splicing silencer (ESS2) acting on site A3 is located in a long irregular stem–loop structure (SLS3). Two SLS3 domains were protected by nuclear components under splicing condition assays. One contains the A4c branch points and a putative SR protein binding site. The other one is adjacent to ESS2. Unexpectedly, only the 3′ A residue of ESS2 was protected. The suboptimal A3 polypyrimidine tract (PPT) is base paired. Using site-directed mutagenesis and transfection of a mini-HIV-1 cDNA into HeLa cells, we found that, in a wild-type PPT context, a mutation of the A3 downstream sequence that reinforced SLS2 stability decreased site A3 utilization. This was not the case with an optimized PPT. Hence, sequence and secondary structure of the PPT may cooperate in limiting site A3 utilization.

GeneSplicer: a new computational method for splice site prediction

GeneSplicer is a new, flexible system for detecting splice sites in the genomic DNA of various eukaryotes. The system has been tested successfully using DNA from two reference organisms: the model plant Arabidopsis thaliana and human. It was compared to six programs representing the leading splice site detectors for each of these species: NetPlantGene, NetGene2, HSPL, NNSplice, GENIO and SpliceView. In each case GeneSplicer performed comparably to the best alternative, in terms of both accuracy and computational efficiency.

Modulation of 5' splice site choice in pre-messenger RNA by two distinct steps.

Ser/Arg-rich proteins (SR proteins) are essential splicing factors that commit pre-messenger RNAs to splicing and also modulate 5' splice site choice in the presence or absence of functional U1 small nuclear ribonucleoproteins (snRNPs). Here, we perturbed the U1 snRNP in HeLa cell nuclear extract by detaching the U1-specific A protein using a 2'-O-methyl oligonucleotide (L2) complementary to its binding site in U1 RNA. In this extract, the standard adenovirus substrate is spliced normally, but excess amounts of SR proteins do not exclusively switch splicing from the normal 5' splice site to a proximal site (site 125 within the adenovirus intron), suggesting that modulation of 5' splice site choice exerted by SR proteins requires integrity of the U1 snRNP. The observation that splicing does not necessarily follow U1 binding indicates that interactions between the U1 snRNP and components assembled on the 3' splice site via SR proteins may also be critical for 5' splice site selection. Accordingly, we found that SR proteins promote the binding of the U2 snRNP to the branch site and stabilize the complex formed on a 3'-half substrate in the presence or absence of functional U1 snRNPs. A novel U2/U6/3'-half substrate crosslink was also detected and promoted by SR proteins. Our results suggest that SR proteins in collaboration with the U1 snRNP function in two distinct steps to modulate 5' splice site selection.

Lethal chondrodysplasia in a family of Holstein cattle is associated with a de novo splice site variant of COL2A1

Background Lethal chondrodysplasia (bulldog syndrome) is a well-known congenital syndrome in cattle and occurs sporadically in many breeds. In 2015, it was noticed that about 12 % of the offspring of the phenotypically normal Danish Holstein sire VH Cadiz Captivo showed chondrodysplasia resembling previously reported bulldog calves. Pedigree analysis of affected calves did not display obvious inbreeding to a common ancestor, suggesting the causative allele was not a rare recessive. The normal phenotype of the sire suggested a dominant inheritance with incomplete penetrance or a mosaic mutation. Results Three malformed calves were examined by necropsy, histopathology, radiology, and computed tomography scanning. These calves were morphologically similar and displayed severe disproportionate dwarfism and reduced body weight. The syndrome was characterized by shortening and compression of the body due to reduced length of the spine and the long bones of the limbs. The vicerocranium had severe dysplasia and palatoschisis. The bones had small irregular diaphyses and enlarged epiphyses consisting only of chondroid tissue. The sire and a total of four affected half-sib offspring and their dams were genotyped with the BovineHD SNP array to map the defect in the genome. Significant genetic linkage was obtained for several regions of the bovine genome including chromosome 5 where whole genome sequencing of an affected calf revealed a COL2A1 point mutation (g.32473300 G > A). This private sequence variant was predicted to affect splicing as it altered the conserved splice donor sequence GT at the 5’-end of COL2A1 intron 36, which was changed to AT. All five available cases carried the mutant allele in heterozygous state and all five dams were homozygous wild type. The sire VH Cadiz Captivo was shown to be a gonadal and somatic mosaic as assessed by the presence of the mutant allele at levels of about 5 % in peripheral blood and 15 % in semen. Conclusions The phenotypic and genetic findings are comparable to a previously reported COL2A1 missense mutation underlying lethal chondrodysplasia in the offspring of a mosaic French Holstein sire (Igale Masc). The identified independent spontaneous splice site variant in COL2A1 most likely caused chondrodysplasia and must have occurred during the early foetal development of the sire. This study provides a first example of a dominant COL2A1 splice site variant as candidate causal mutation of a severe lethal chondrodysplasia phenotype. Germline mosaicism is a relatively frequent mechanism in the origin of genetic disorders and explains the prevalence of a certain fraction of affected offspring. Paternal dominant de novo mutations are a risk in cattle breeding, especially because the ratio of defective offspring may be very high and be associated with significant animal welfare problems.

Molecular characterization and cancer risk associated with BRCA1 and BRCA2 splice site variants identified in multiple-case breast cancer families

Genetic screening of women from multiple-case breast cancer families and other research-based endeavors have identified an extensive collection of germline variations of BRCA1 and BRCA2 that can be classified as deleterious and have clinical relevance. For some variants, such as those in the conserved intronic splice site regions which are highly likely to alter splicing, it is not possible to classify them based on the identified DNA sequence variation alone. We studied 11 multiple-case breast cancer families carrying seven distinct splice site region genetic alterations in BRCA1 or BRCA2 (BRCA1, c.IVS6-2delA, c.IVS9-2A>C, c.IVS4-1G>T, c.IVS20+1G>A and BRCA2, c.IVS17-1G>C, c.IVS20+1G>A, c.IVS7-1G>A) and applied SpliceSiteFinder to predict possible changes in efficiency of splice donor and acceptor sites, characterized the transcripts, and estimated the average age-specific cumulative risk (penetrance) using a modified segregation analysis. SpliceSiteFinder predicted and we identified transcipts that illustrated that all variants caused exon skipping, and all but two led to frameshifts. The risks of breast cancer to age 70 yrs, averaged over all variants, over BRCA1 variants alone, and over BRCA2 variants alone, were 73% (95% confidence interval 47-93), 64% (95%CI 28-96) and 79% (95%CI 48-98) respectively (all P

No evidence for mutations of CTCFL/BORIS in Silver-Russell syndrome patients with IGF2/H19 imprinting control region 1 hypomethylation.

BACKGROUND: Silver-Russell syndrome (SRS) is a genetically and clinically heterogeneous disease. Although no protein coding gene defects have been reported in SRS patients, approximately 50% of SRS patients carry epimutations (hypomethylation) at the IGF2/H19 imprinting control region 1 (ICR1). Proper methylation at ICR1 is crucial for the imprinted expression of IGF2, a fetal growth factor. CTCFL, a testis-specific protein, has recently been proposed to play a role in the establishment of DNA methylation at the murine equivalent of ICR1. A screen was undertaken to assess whether CTCFL is mutated in SRS patients with hypomethylation, to explore a link between the observed epimutations and a genetic cause of the disease. METHODOLOGY/PRINCIPAL FINDINGS: DNA was obtained from 36 SRS patients with hypomethylation at ICR1. All CTCFL coding exons were sequenced and analyzed for duplications/deletions using both multiplex ligation-dependent probe amplification, with a custom CTCFL probe set, and genomic qPCR. Novel SNP alleles were analyzed for potential differential splicing in vitro utilizing a splicing assay. Neither mutations of CTCFL nor duplications/deletions were observed. Five novel SNPs were identified and have been submitted to dbSNP. In silico splice prediction suggested one novel SNP, IVS2-66A>C, activated a cryptic splice site, resulting in aberrant splicing and premature termination. In vitro splicing assays did not confirm predicted aberrant splicing. CONCLUSIONS/SIGNIFICANCE: As no mutations were detected at CTCFL in the patients examined, we conclude that genetic alterations of CTCFL are not responsible for the SRS hypomethylation. We suggest that analysis of other genes involved in the establishment of DNA methylation at imprinted genes, such as DNMT3A and DNMT3L, may provide insight into the genetic cause of hypomethylation in SRS patients.

Prevalence and nonrandom distribution of exonic mutations in interferon regulatory factor 6 in 307 families with Van der Woude syndrome and 37 families with popliteal pterygium syndrome

Purpose: Interferon regulatory factor 6 encodes a member of the IRF family of transcription factors. Mutations in interferon regulatory factor 6 cause Van der Woude and popliteal pterygium syndrome, two related orofacial clefting disorders. Here, we compared and contrasted the frequency and distribution of exonic Mutations in interferon regulatory factor 6 between two large geographically distinct collections of families with Van der Woude and between one collection of families with popliteal pterygium syndrome. Methods: We performed direct sequence analysis of interferon regulatory factor 6 exons oil samples from three collections, two with Van der Woude and one with popliteal pterygium syndrome. Results: We identified mutations in interferon regulatory factor 6 exons in 68% of families in both Van der Woude collections and in 97% of families with popliteal pterygium syndrome. In sum, 106 novel disease-causing variants were found. The distribution of mutations in the interferon regulatory factor 6 exons in each collection was not random; exons 3, 4, 7, and 9 accounted for 80%. In the Van der Woude collections, the mutations were evenly divided between protein truncation and missense, whereas most mutations identified in the popliteal pterygium syndrome collection were missense. Further, the missense mutations associated with popliteal pterygium syndrome were localized significantly to exon 4, at residues that are predicted to bind directly to DNA. Conclusion: The nonrandom distribution of mutations in the interferon regulatory factor 6 exons suggests a two-tier approach for efficient mutation screens for interferon regulatory factor 6. The type and distribution of mutations are consistent with the hypothesis that Van der Woude is caused by haploinsufficiency of interferon regulatory factor 6. Oil the other hand, the distribution of popliteal pterygium syndrome-associated mutations suggests a different, though not mutually exclusive, effect oil interferon regulatory factor 6 function. Genet Med 2009:11(4):241-247.