Cryptic Splice Site In Intron Research Articles

Three exonic variants in the PHEX gene cause aberrant splicing in a minigene assay.

Background: X-linked hypophosphatemia (XLH, OMIM 307800) is a rare phosphorus metabolism disorder caused by PHEX gene variants. Many variants simply classified as missense or nonsense variants were only analyzed at the DNA level. However, growing evidence indicates that some of these variants may alter pre-mRNA splicing, causing diseases. Therefore, this study aimed to use bioinformatics tools and a minigene assay to ascertain the effects of PHEX variations on pre-mRNA splicing. Methods: We analyzed 174 variants in the PHEX gene described as missense or nonsense variants. Finally, we selected eight candidate variants using bioinformatics tools to evaluate their effects on pre-mRNA splicing using a minigene assay system. The complementary DNA (cDNA) sequence for the PHEX gene (RefSeq NM_000444.6) serves as the basis for DNA variant numbering. Results: Of the eight candidate variants, three were found to cause abnormal splicing. Variants c.617T>G p.(Leu206Trp) and c.621T>A p.(Tyr207*) in exon 5 altered the splicing of pre-mRNA, owing to the activation of a cryptic splice site in exon 5, which produced an aberrant transcript lacking a part of exon 5, whereas variant c.1700G>C p.(Arg567Pro) in exon 16 led to the activation of a cryptic splice site in intron 16, resulting in a partial inclusion of intron 16. Conclusion: Our study employed a minigene system, which has a great degree of flexibility to assess abnormal splicing patterns under the circumstances of patient mRNA samples that are not available, to explore the impact of the exonic variants on pre-mRNA splicing. Based on the aforementioned experimental findings, we demonstrated the importance of analyzing exonic variants at the mRNA level.

XRCC4 rs28360071 intronic variant is associated with increased risk for infant acute lymphoblastic leukemia with KMT2A rearrangements.

Early age acute leukemia (EAL) shows a high frequency of KMT2A-rearrangements (KMT2A-r). Previous investigations highlighted double-strand breaks arising from maternal exposure to xenobiotics during pregnancy as a risk factor for EAL and KMT2A-r. In this case-control study, we investigated the relationship between EAL and genetic variants of the nonhomologous end-joining (XRCC6 rs5751129, XRCC4 rs6869366 and rs28360071), since they might affect DNA repair capacity, leading to KMT2A-r and leukemogenesis. Samples from 577 individuals (acute lymphoblastic leukemia-ALL, n=164; acute myeloid leukemia-AML, n=113; controls, n=300) were genotyped. No significant association was found for rs5751129 and rs6869366, whereas rs28360071 was associated with an increased risk for ALL with KMT2A-r (IIxID: OR - Odds ratio 2.23, CI 1.17-4.25, p=0.014). Bone marrow samples from ALL patients showed a higher expression of XRCC4 compared to AML patients (p=0.025). Human Splicing Finder 3.1 predicted that the deleted allele of rs28360071 is potentially associated with the activation of a 5’ cryptic splice site in intron 3 of XRCC4. The sequencing of cDNA did not show any differences on the splicing process for the rs28360071 genotypes. Our results suggest that the deleted allele for rs28360071 increases the risk for ALL with KMT2A-r, but not by modifying the XRCC4 expression levels or its structure.

P.133 - Identification of variants that affect severity of the spinal muscular atrophy phenotype within and outside of the SMN2 gene

SMA is caused by loss or mutation of the SMN1 gene and retention of SMN2. The SMN2 gene copy number correlates with phenotypic severity. However, there are exceptions, with type 3 cases having 2 copies of SMN2 or type 1 cases with 3 copies as well as discordant siblings with the same copy number. With effective therapeutics for SMA early introduction of therapy is important in achieving maximum benefit. Newborn screening is progressing towards implementation but precise prediction of phenotype from genotype is critical to optimize timing of treatment. It is often not known if the SMN2 gene is intact or contains variants which can affect the amount of SMN produced. In order to identify deletion junctions as well as intact SMN2 copies and variants within the gene that affect phenotypic severity we have sequenced the entire SMN2/SMN1gene present in 60 patients. In addition, we have employed genomic sequencing in a set of discordant siblings in comparison to concordant siblings in order to identify variants in the genome associated with the milder sibling. The entire SMN2/SMN1 gene along with the CFTR and PLS3 gene was captured and sequenced with an adapted Multiplexed direct genomic selection (MDiGS) technique. The sequences were aligned and variants were determined. To determine SMN2 copy number the SMN read count was split into bins and normalized to the CFTR and PLS3 reads. We defined a deletion occurring in 8 patients, the deletion occurred between two nearly identical repeats located in intron 6 and in sequence located 3 prime of SMN2. We detected a cryptic splice site in intron 6 of a patient with one copy of SMN1 and a missense mutation (A75T) in the SMN2 gene of a patient. We detected 3 variants associated with a milder phenotype in 3 copy SMN2 individuals. Lastly, we have performed genomic sequencing of select discordant and concordant families to identify modifiers of phenotype that act in trans.

SNX10 gene mutation leading to osteopetrosis with dysfunctional osteoclasts

Autosomal recessive osteopetrosis (ARO) is a heterogeneous disorder, characterized by defective osteoclastic resorption of bone that results in increased bone density. We have studied nine individuals with an intermediate form of ARO, from the county of Västerbotten in Northern Sweden. All afflicted individuals had an onset in early infancy with optic atrophy, and in four patients anemia was present at diagnosis. Tonsillar herniation, foramen magnum stenosis, and severe osteomyelitis of the jaw were common clinical features. Whole exome sequencing, verified by Sanger sequencing, identified a splice site mutation c.212 + 1 G > T in the SNX10 gene encoding sorting nexin 10. Sequence analysis of the SNX10 transcript in patients revealed activation of a cryptic splice site in intron 4 resulting in a frame shift and a premature stop (p.S66Nfs * 15). Haplotype analysis showed that all cases originated from a single mutational event, and the age of the mutation was estimated to be approximately 950 years. Functional analysis of osteoclast progenitors isolated from peripheral blood of patients revealed that stimulation with receptor activator of nuclear factor kappa-B ligand (RANKL) resulted in a robust formation of large, multinucleated osteoclasts which generated sealing zones; however these osteoclasts exhibited defective ruffled borders and were unable to resorb bone in vitro.

Splicing Defects in the AAAS Gene Leading to both Exon Skipping and Partial Intron Retention in a Tunisian Patient with Allgrove Syndrome

Background/Aims: Allgrove syndrome is a rare autosomal recessive disorder characterized by the triad of adrenal insufficiency, achalasia, and alacrima. This syndrome is caused by mutations in the AAAS gene. A major splice site mutation c.1331+1G>A was found previously in North African families affected by Allgrove syndrome. In this study, we analyzed in vivo and in silico the effect of this mutation on the splicing process. Methods: Using reverse transcriptase-polymerase chain reaction, sequencing and bioinformatics tools, we analyzed all transcripts produced by the AAAS gene containing this splice site mutation. Results: The altered splicing of mRNA produces two aberrant transcripts: one with exon 14 skipping, the other with concurrent exon 14 skipping and retention of 99 bp of intron 14, both outcomes resulting in frameshifts with a new stop codon generation in the untranslated region of the last exon. Using in silico bioinformatics tools, we demonstrated that this mutation abolishes the splice donor site of exon 14 and activates a new intronic cryptic splice site in intron 14. Conclusion: This study demonstrated that a single splicing mutation affects the AAAS transcripts and consequently the ALADIN protein structure and function.

A 3′ splice site mutation of IDS gene in a Chinese family with mucopolysaccharidosis type II

The purpose of this study was to identify the underlying genetic cause in a four generation Chinese family diagnosed with mucopolysaccharidosis type II. Peripheral blood samples were collected from family members and the iduronate-2-sulfatase (IDS) gene was analyzed by DNA sequencing. The impact of IDS mutations on mRNA transcription was determined by quantitative real-time RT-PCR (qRT-PCR) in both patients as well as in healthy control samples. In addition, RT-PCR was performed to confirm the characteristics of a found mutation located in non-canonical splicing site.A 3′ splice site mutation c.880-8A>G (IVS 6–8A>G) was identified in two members of the analyzed MPS II family and sequencing of RT-PCR products showed that this mutation activates an upstream cryptic splice-site in intron 6, leads to the 7 nucleotide insertion in exon 7, which in turn results in an exon 7 frameshift introducing a premature stop codon and shorter peptide chain. In addition, qRT-PCR products from the two patients showed a reduced IDS mRNA expression (43.9% and 71.2%, respectively), when compared with the average IDS mRNA expression in healthy control samples, possibly as a result of nonsense-mediated mRNA decay. In conclusion, in this study, we have identified an IDS gene splice mutation which is associated with clinically attenuated MPS II phenotype. In addition, our study accentuates the importance of cDNA analysis in the detection of intronic mutations, since in the studies examining only gDNA, the link between genotype and phenotype may have been misinterpreted.

The first founder DGUOK mutation associated with hepatocerebral mitochondrial DNA depletion syndrome

Deoxyguanosine kinase (dGK) deficiency is a frequent cause of mitochondrial DNA depletion associated with a hepatocerebral phenotype. In this study, we describe a new splice site mutation in the DGUOK gene and the clinical, radiologic, and genetic features of these DGUOK patients. This new DGUOK homozygous mutation (c.444-62C>A) was identified in three patients from two North-African consanguineous families with combined respiratory chain deficiencies and mitochondrial DNA depletion in the liver. Brain MRIs are normal in DGUOK patients in the literature. Interestingly, we found subtentorial abnormal myelination and moderate hyperintensity in the bilateral pallidi in our patients. This new mutation creates a cryptic splice site in intron 3 (in position −62) and is predicted to result in a larger protein with an in-frame insertion of 20 amino acids. In silico analysis of the putative impact of the insertion shows serious clashes in protein conformation: this insertion disrupts the α5 helix of the dGK kinase domain, rendering the protein unable to bind purine deoxyribonucleosides. In addition, a common haplotype that segregated with the disease in both families was detected by haplotype reconstruction with 10 markers (microsatellites and SNPs), which span 4.6 Mb of DNA covering the DGUOK locus. In conclusion, we report a new DGUOK splice site mutation that provide insight into a critical protein domain (dGK kinase domain) and the first founder mutation in a North-African population.

New splicing mutations in propionic acidemia

Propionic acidemia results from mutations in either of the two genes, PCCA or PCCB, that encode the two subunits of the propionyl-CoA carboxylase (PCC) enzyme. In this study, we report the identification and analysis of seven novel splicing mutations involving consensus donor and acceptor splice sites. Most of them were identified in patients with a Central Asian origin, and some present in several alleles, probably reflecting founder effects. The functional consequences of the splicing mutations were analyzed in patients' fibroblasts, as well as transcript quantification using real-time PCR methods. In the PCCA gene, two mutations were demonstrated to affect 5' splice sites (c.231+1G>C and c.1209+3A>G) and two 3' acceptor splice sites (c.1210delG and c.1430G>T), all causing skipping of the exons involved, with no detectable levels of normally spliced transcript. In the PCCB gene, all three mutations involved 5' donor splice sites-two affected exon 1 splicing (c.154_183+17del46 and c.183+2T>C), the latter activating a cryptic splice site in intron 1, and the remaining mutation (c.1498+2T>C) resulted in exon 14 skipping. The results highlight the necessity to perform transcript analysis in addition to genomic DNA sequencing to characterize the effect of splicing mutations and add relevant information on the genetic epidemiology of the disease.

Six novel mutations of the LDL receptor gene in FH kindred of Sicilian and Paraguayan descent

Familial hypercholesterolemia (FH) is an autosomal dominant inherited disease caused by mutations in the gene coding for the low density lipoprotein receptor (LDL-R). It is characterized by a high concentration of low density lipoprotein (LDL), which frequently gives rise to premature coronary artery disease. We studied the probands of five FH Sicilian families with 'definite' FH and one proband of Paraguayan descent with homozygous FH who has been treated with an effective living-donor liver transplantation. In order to seek the molecular defect in these six families, we used direct sequencing to define the molecular defects of the LDL-R gene responsible for the disease. We described three novel missense mutations (C100Y, C183Y and G440C), two frameshift mutations (g.1162delC in exon 8 and g.2051delC in exon 14) and one mutation (g.2390-1Gright curved arrow A) at splicing acceptor consensus sequences located in intron 16 of the LDL-R gene; the analysis of cDNA of this splicing mutation showed the activation of a cryptic splice site in intron 16 and the binding studies showed a reduction in internalisation of LDL-DIL in the proband's cultured fibroblasts. Moreover, a g.2051delC in exon 14 was identified in the proband of Paraguayan ancestry with clinical features of homozygous FH. The mutation identified in the South American patient represents the first description of a variant in South American patients other than Brazilian FH patients. The 5 mutations identified in the Sicilian patients confirm the heterogeneity of LDL-R gene mutations in Sicily.

Combined partial exon skipping and cryptic splice site activation as a new molecular mechanism for recessive type 1 von Willebrand disease

We describe the complex picture associated with a mutated splice junction in intron 13 of von Willebrand factor (VWF) gene. The proband, characterized by a marked decrease in plasma and platelet VWF and near normal multimer organization, was classified as recessive type 1 von Willebrand disease (VWD). Genetic analysis demonstrated that he was homozygous for the 1534-3C > A mutation in the consensus sequence of the acceptor splicing site of intron 13 of the VWF gene. Platelet mRNA analysis documented three VWF transcripts: a wild type generated by the correct recognition of the mutated splice site, a smaller transcript not containing exon 14, and a longer one that, in addition to exons 13 and 14, included a 62bp fragment corresponding to the end of intron 13. The small transcript derives from the skipping of exon 14, the long one from the activation of a cryptic splice site in intron 13; both show a premature stop codon in VWF propeptide, so the proband VWF derives entirely from the correct splice site recognition. Combined incomplete exon skipping and cryptic splice site activation are first recognized in VWD. Since the 1534-3C > A mutation does not abolish the normal processing of mRNA, it is unlikely to be found in type 3 VWD. This mutation therefore appears to be peculiar to type 1 VWD.

Mutation spectrum in patients with Rett syndrome in the German population: Evidence of hot spot regions.

Mutations in the MECP2 (Methyl-CpG-binding protein) gene recently have been reported to cause Rett syndrome (RTT), an X-linked dominant neurodevelopmental disease. We investigated 125 sporadic cases of Rett syndrome by direct sequencing. Thirty different mutations were found in 97 patients with Rett syndrome. Seventeen mutations have not been described previously. We provide evidence for the existence of several hot spot regions and of a deletion-prone region located at the 3' most region of the gene. This latter region most probably forms secondary structures in vitro. Similar structures in vivo could explain the high frequency of deletions in this region. Nine of 10 recurrent mutations were located in either the methyl CpG binding domain (MBD) or in the transcriptional repression domain (TRD), and all missense mutations were located in one of these functionally important domains. There was a high frequency of more than 60% of truncating mutations (nonsense mutations along with frameshift mutations). One patient with a mild form of the disease and a normal head growth carries a novel c.27-6C>A mutation that causes a cryptic splice site in intron I resulting in a frameshift transcript. The detection rate in our collective was 77.6%. Our findings show that the majority of German Rett patients carry mutations in the MECP2 gene confirming the suggested locus homogeneity for the disease.

Normolipidemia and hypercholesterolemia in persons heterozygous for the same 1592+5G→A splice site mutation in the low‐density lipoprotein receptor gene

In the present study, we have characterized a unique splice donor G to A substitution in the moderately conserved + 5 position in intron 10 of the low-density lipoprotein (LDL) receptor gene. In two Danish families, carriers of the 1592 + 5G --> A mutation display a clinical phenotype ranging from healthy normocholesterolemic persons to classical heterozygous familial hypercholesterolemia (FH) patients. Reverse transcription-polymerase chain reaction (RT-PCR) of RNA from Epstein Barr virus (EBV)-transformed lymphoblasts obtained from members of both families demonstrated abnormal splicing generating two aberrant mRNAs due to either alternative splicing and skipping of exon 10 or activation of a cryptic splice site in intron 10 inserting 66 intronic base pairs. These abnormally spliced mRNAs were predicted to encode two abnormal receptor proteins containing an in-frame deletion of 75 amino acids and an insertion of 22 novel amino acids, respectively. Results obtained by immunofluorescence staining, flow cytometry, and confocal microscopy of transfected Chang and COS-7 cells expressing normal and mutant LDL receptors were compatible with nearly complete retention of the mutant proteins in the endoplasmic reticulum. Quantitative measurements of LDL receptor mRNAs from EBV-transformed lymphoblasts, however, did not reveal any significant differences in variant mRNA contents between mutation carriers in the families that could be related to degree of hypercholesterolemia.

A Novel Alu-Like Element Rearranged in the Dystrophin Gene Causes a Splicing Mutation in a Family with X-Linked Dilated Cardiomyopathy

We have identified and characterized a genomic sequence with some features typical of Alu-like mobile elements rearranged into the dystrophin gene in a family affected by X-linked dilated cardiomyopathy. The Alu-like sequence rearrangement occurred 2.4 kb downstream from the 5' end of intron 11 of the dystrophin gene. This rearrangement activated one cryptic splice site in intron 11 and produced an alternative transcript containing the Alu-like sequence and part of the adjacent intron 11, spliced between exons 11 and 12. Translation of this alternative transcript is truncated because of the numerous stop codons present in every frame of the Alu-like sequence. Only the mutant mRNA was detected in the heart muscle, but in the skeletal muscle it coexisted with the normal one. This result is supported by the immunocytochemical findings, which failed to detect dystrophin in the patient's cardiac muscle but showed expression of a reduced level of protein in the skeletal muscle. Comparative analysis of the Alu-like sequence showed high homology with other repeated-element-containing regions and with several expressed sequence tags. We suggest that this Alu-like sequence could represent a novel class of repetitive elements, reiterated and clustered with some known mobile elements and capable of transposition. Our report underlines the complexity of the pathogenic mechanism leading to X-linked dilated cardiomyopathy but suggests that differences in tissue-specific expression of dystrophin mutations may be a common feature in this condition.

Three splicing defects, an insertion, and two missense mutations responsible for acute intermittent porphyria.

Three splicing defects (IVS1+3G-->T, 86A-->T, IVS13-2A-->G), an insertion (416insCA), and two missense mutations (664G-->A and 833T-->G) in the porphobilinogen deaminase (PBGD) gene were identified in six unrelated Finnish patients with acute intermittent porphyria (AIP). The IVS1+3G-->T substitution resulted in activation of a cryptic splice site in intron 1 and retention of a 67-bp fragment in the mutant transcript. The 86A-->T mutation at the end of exon 3 was predicted to cause an amino acid substitution (E29L). However, sequencing of the cDNA sample of the proband revealed exon 3 skipping from the mutant transcript. The IVS13-2A-->G substitution caused retention of intron 13 in the mutant transcript. In exon 8, 416insCA resulted in a frameshift. All three splicing defects and the CA insertion resulted in a truncated protein and thus, probably the loss of PBGD activity. The two novel missense mutations, 664G-->A in exon 12 and 833T-->C in exon 14 caused a single amino acid substitution (V222M and L278P). So far 25 different mutations have been characterized from 37 (93%) of a total of 40 unrelated Finnish AIP families, confirming the genetic heterogeneity of the disease even in a previously isolated area of Finland.

Activation of a cryptic splice-site in intron 24 leads to the formation of apolipoprotein B-27.6

Apo B expression is confined to the intestine and liver, and its secretion from these tissues is dependent on the expression of a lipid transfer protein, microsomal triglyceride transfer protein (MTP). Previously, we reported a model system for the study of apolipoprotein (apo B) biogenesis using heterologous expression in COS cells (Patel SB, Grundy SM. J. Lipid Res. 1995;36:2090–2103). We now report the characterization of the effects of a T→C transition in the splice-site at +2 of intron 24 previously reported by Talmud et al. (J. Lipid Res. 1994;35:468–77). Using our heterologous expression system, we show that the mutation led to aberrant processing of intron 24, but normal processing of intron 25. The resultant translation of this mutant mRNA produced a truncated apo B protein of the size of apo B-27.6. Reverse transcription, polymerase chain reaction and sequencing of the amplified products were used to show that a cryptic donor splice-site within intron 24 was utilized, resulting in the generation of a novel hydrophilic 29 amino acid carboxyl-terminal tail. Co-expression of apo B-27.6 with microsomal triglyceride transfer protein (MTP) showed that this protein could bind MTP and resulted in the secretion of a lipoprotein particle with a buoyant density in the range 1.16–1.25 g/ml. These results indicate that this splice-site mutation leads to an activation of a downstream cryptic splice-site within intron 24, causing an insertion of 40 bases of intron 24 sequences into the mature RNA. This leads to a frame-shift of translation resulting in addition of 29 new amino acids at the carboxyl-terminus, before an in-frame stop translation codon is encountered, truncating the apo B at B-27.6.