Splice Site Consensus Research Articles

A birth of bipartite exon by intragenic deletion.

BackgroundDisease‐causing mutations that activate transposon‐derived exons without creating a new splice‐site consensus have been reported rarely, but they provided unique insights into our understanding of structural motifs required for inclusion of intronic sequences in mature transcripts.MethodsWe employ a combination of experimental and computational techniques to characterize the first de novo bipartite exon activation in genetic disease.ResultsThe exon originated from two separate introns as a result of an in‐frame COL4A5 deletion associated with a typical Alport syndrome. The deletion encompassed exons 38 through 41 and activated a cryptic 3′ and 5′ splice site that were derived from intron 37 and intron 41, respectively. The deletion breakpoint was in the middle of the new exon, with considerable complementarity between the two exonic parts, potentially bringing the cryptic 3′ and 5′ splice site into proximity. The 3′ splice site, polypyrimidine tract and the branch site of the new exon were derived from an inactive, 5′ truncated LINE‐1 retrotransposon. This ancient LINE‐1 copy sustained a series of mutations that created the highly conserved AG dinucleotide at the 3′ splice site early in primate development. The exon was fully included in mature transcripts and introduced a stop codon in the shortened COL4A5 mRNA, illustrating pitfalls of inferring disease severity from DNA mutation alone.ConclusionThese results expand the repertoire of mutational mechanisms that alter RNA processing in genetic disease and illustrate the extraordinary versatility of transposed elements in shaping the new exon‐intron structure and the phenotypic variability.

Characterization of aberrant splicing of von Willebrand factor in von Willebrand disease: an underrecognized mechanism

AbstractApproximately 10% of von Willebrand factor (VWF) gene mutations are thought to alter messenger RNA (mRNA) splicing through disruption of consensus splice sites. This mechanism is likely underrecognized and affected by mutations outside consensus splice sites. During VWF synthesis, splicing abnormalities lead to qualitative defects or quantitative deficiencies in VWF. This study investigated the pathologic mechanism acting in 3 von Willebrand disease (VWD) families with putative splicing mutations using patient-derived blood outgrowth endothelial cells (BOECs) and a heterologous human embryonic kidney (HEK 293(T)) cell model. The exonic mutation c.3538G>A causes 3 in-frame splicing variants (23del, 26del, and 23/26del) which cannot bind platelets, blood coagulation factor VIII, or collagen, causing VWD through dominant-negative intracellular retention of coexpressed wild-type (WT) VWF, and increased trafficking to lysosomes. Individuals heterozygous for the c.5842+1G>C mutation produce exon 33 skipping, exons 33-34 skipping, and WT VWF transcripts. Pathogenic intracellular retention of VWF lacking exons 33-34 causes their VWD. The branch site mutation c.6599-20A>T causes type 1 VWD through mRNA degradation of exon 38 skipping transcripts. Splicing ratios of aberrant transcripts and coexpressed WT were altered in the BOECs with exposure to shear stress. This study provides evidence of mutations outside consensus splice sites disrupting splicing and introduces the concept that VWF splicing is affected by shear stress on endothelial cells.

Evaluation of Bioinformatic Programmes for the Analysis of Variants within Splice Site Consensus Regions.

The increasing diagnostic use of gene sequencing has led to an expanding dataset of novel variants that lie within consensus splice junctions. The challenge for diagnostic laboratories is the evaluation of these variants in order to determine if they affect splicing or are merely benign. A common evaluation strategy is to use in silico analysis, and it is here that a number of programmes are available online; however, currently, there are no consensus guidelines on the selection of programmes or protocols to interpret the prediction results. Using a collection of 222 pathogenic mutations and 50 benign polymorphisms, we evaluated the sensitivity and specificity of four in silico programmes in predicting the effect of each variant on splicing. The programmes comprised Human Splice Finder (HSF), Max Entropy Scan (MES), NNSplice, and ASSP. The MES and ASSP programmes gave the highest performance based on Receiver Operator Curve analysis, with an optimal cut-off of score reduction of 10%. The study also showed that the sensitivity of prediction is affected by the level of conservation of individual positions, with in silico predictions for variants at positions −4 and +7 within consensus splice sites being largely uninformative.

A Novel Splice Site Mutation in the 5'UTR of GATA1 resulting in Abnormal Hematopoiesis

Diamond-Blackfan anemia (DBA) is a rare disorder of impaired erythropoiesis belonging to a broader family of inherited and acquired anemias known as pure red cell aplasias (PRCA). The cardinal feature of DBA is aregenerative anemia accompanied by macrocytosis, reticulocytopenia and decreased or absent erythroid precursors in an otherwise normocellular marrow. Classic DBA is associated with mutations of genes encoding ribosome subunits. Advancements in molecular techniques have prompted discussion regarding the genetic basis of the disease and its prognostic implications as non-ribosomal genes are beginning to be implicated in DBA.Acquired mutations within the X-linked transcription factor GATA1 have been described in Down syndrome-related transient myeloproliferative disorder (TMD) and acute megakaryoblastic leukemia (AKML), while germline GATA1 mutations affecting both isoforms are associated with congenital thrombocytopenia. Normal alternative splicing of GATA1 produces a full-length GATA1 isoform (flGATA1) as well as a shortened GATA1 isoform (GATA1s), which lacks the N-terminal domain that activates GATA1-driven erythropoiesis program and recruits flGATA1 to a subset of megakaryocyte and erythroblast genes. Rare germline defects known as "GATA1s mutations" are characterized by an unbalanced production of the GATA1s isoform at the expense of a flGATA1 transcript due to disrupting exon 2 splice sites or the ATG initiation codon. GATA1s mutations had been reported to phenocopy Diamond-Blackfan anemia by causing isolated red blood cell aplasia in some patients, although thrombocytopenia with structural platelet abnormalities and dyserythropoiesis as well as DBA-like picture progressing into MDS had been described in other GATA1s families. Therefore, while GATA1s mutations uniformly disrupt erythropoiesis, the impact of defective flGATA production on megakaryopoiesis in non-trisomy 21 individuals is less clear.Here we describe a child with dyserthropoietic anemia, marked megakaryocyte dysplasia, peripheral thrombocytosis and platelet dysfunction due to a novel disease-causing mutation within the 5' UTR of GATA1. DBA was initially suspected due to progressive macrocytic anemia beginning in infancy. Bone marrow analysis revealed not only a paucity of erythroid precursor cells and dyserythropoiesis, but also prominent megakaryocytosis and megakaryocyte dysplasia, not typically associated with classic DBA. Ribosome mutational analysis was unremarkable, as were cytogenetics and MDS-FISH analysis. Given aregenerative anemia mimicking DBA as well as megakaryocyte dysplasia, the question was raised whether the findings could be associated with a germline mutation in GATA1. Indeed, sequencing revealed a novel, pathogenic mutation in the 5'UTR of GATA1 (c.-21 A>G). We explored the impact of this novel mutation in silico, found it to disrupt a consensus splice site, and further demonstrated that this mutation conferred a striking predominance of the GATA1s isoform at the transcript and protein level. Two-color immunohistochemistry confirmed loss of full-length GATA1 protein in the patient's bone marrow and showed that full-length GATA1 expression is restricted to megakaryocytes and erythroblasts in healthy bone marrow, consistent with dysmegakaryopoiesis and decreased erythropoiesis in our patient.Our findings support a role for the N-terminus of GATA1 in both erythroblast and megakaryocyte maturation and function in vivo. Furthermore, the findings of prominent megakaryocytes in the bone marrow and resultant functional platelet abnormalities may provide subtle clinical clues to differentiate Diamond-Blackfan anemia due to ribosomopathy from dyserythropoiesis secondary to GATA1s mutations. We conclude that GATA1 sequencing, including non-coding GATA1 regions, should be considered in males with congenital multi-lineage dysplasia and/or DBA-like clinical presentation. DisclosuresNo relevant conflicts of interest to declare.

A novel splicing mutation in the albumin gene (c.270+1G>T) causes analbuminaemia in a German infant.

Congenital analbuminaemia is a rare autosomal recessive disorder manifested by the presence of a very low amount of circulating serum albumin. The clinical diagnosis may be challenging because of the absence of unambiguous symptoms and because hypoalbuminemia may have many causes different from a genetic lack of the protein. We describe the clinical and molecular characterization of a new case of congenital analbuminaemia in an infant of apparently non-consanguineous parents from Treves, Germany. For molecular diagnosis, we used our strategy, based on the screening of the albumin gene by single-strand conformation polymorphism, heteroduplex analysis and direct DNA sequencing, which revealed that the proband is homozygous and both parents are heterozygous, for a novel G > T transversion at nucleotide c.270+ 1, the first base of intron 3. The mutation inactivates the strongly conserved GT dinucleotide at the 5' splice site consensus sequence of this intron. In conclusion, we report the clinical findings and the molecular defect of this case, which contributes to a better understanding of the biological mechanism of congenital analbuminaemia.

Functional and splicing defect analysis of 23 ACVRL1 mutations in a cohort of patients affected by Hereditary Hemorrhagic Telangiectasia.

Hereditary Hemorrhagic Telangiectasia syndrome (HHT) or Rendu-Osler-Weber (ROW) syndrome is an autosomal dominant vascular disorder. Two most common forms of HHT, HHT1 and HHT2, have been linked to mutations in the endoglin (ENG) and activin receptor-like kinase 1 (ACVRL1or ALK1) genes respectively. This work was designed to examine the pathogenicity of 23 nucleotide variations in ACVRL1 gene detected in more than 400 patients. Among them, 14 missense mutations and one intronic variant were novels, and 8 missense mutations were previously identified with questionable implication in HHT2. The functionality of missense mutations was analyzed in response to BMP9 (specific ligand of ALK1), the maturation of the protein products and their localization were analyzed by western blot and fluorescence microscopy. The splicing impairment of the intronic and of two missense mutations was examined by minigene assay. Functional analysis showed that 18 out of 22 missense mutations were defective. Splicing analysis revealed that one missense mutation (c.733A>G, p.Ile245Val) affects the splicing of the harboring exon 6. Similarly, the intronic mutation outside the consensus splicing sites (c.1048+5G>A in intron 7) was seen pathogenic by splicing study. Both mutations induce a frame shift creating a premature stop codon likely resulting in mRNA degradation by NMD surveillance mechanism. Our results confirm the haploinsufficiency model proposed for HHT2. The affected allele of ACVRL1 induces mRNA degradation or the synthesis of a protein lacking the receptor activity. Furthermore, our data demonstrate that functional and splicing analyses together, represent two robust diagnostic tools to be used by geneticists confronted with novel or conflicted ACVRL1 mutations.

Dinoflagellate Gene Structure and Intron Splice Sites in a Genomic Tandem Array.

Dinoflagellates are one of the last major lineages of eukaryotes for which little is known about genome structure and organization. We report here the sequence and gene structure of a clone isolated from a cosmid library which, to our knowledge, represents the largest contiguously sequenced, dinoflagellate genomic, tandem gene array. These data, combined with information from a large transcriptomic library, allowed a high level of confidence of every base pair call. This degree of confidence is not possible with PCR‐based contigs. The sequence contains an intron‐rich set of five highly expressed gene repeats arranged in tandem. One of the tandem repeat gene members contains an intron 26,372 bp long. This study characterizes a splice site consensus sequence for dinoflagellate introns. Two to nine base pairs around the 3′ splice site are repeated by an identical two to nine base pairs around the 5′ splice site. The 5′ and 3′ splice sites are in the same locations within each repeat so that the repeat is found only once in the mature mRNA. This identically repeated intron boundary sequence might be useful in gene modeling and annotation of genomes.

Splicing analysis for exonic and intronic mismatch repair gene variants associated with Lynch syndrome confirms high concordance between minigene assays and patient RNA analyses.

A subset of DNA variants causes genetic disease through aberrant splicing. Experimental splicing assays, either RT-PCR analyses of patient RNA or functional splicing reporter minigene assays, are required to evaluate the molecular nature of the splice defect. Here, we present minigene assays performed for 17 variants in the consensus splice site regions, 14 exonic variants outside these regions, and two deep intronic variants, all in the DNA mismatch-repair (MMR) genes MLH1, MSH2, MSH6, and PMS2, associated with Lynch syndrome. We also included two deep intronic variants in APC and PKD2. For one variant (MLH1 c.122A>G), our minigene assay and patient RNA analysis could not confirm the previously reported aberrant splicing. The aim of our study was to further investigate the concordance between minigene splicing assays and patient RNA analyses. For 30 variants results from patient RNA analyses were available, either performed by our laboratory or presented in literature. Some variants were deliberately included in this study because they resulted in multiple aberrant transcripts in patient RNA analysis, or caused a splice effect other than the prevalent exon skip. While both methods were completely concordant in the assessment of splice effects, four variants exhibited major differences in aberrant splice patterns. Based on the present and earlier studies, together showing an almost 100% concordance of minigene assays with patient RNA analyses, we discuss the weight given to minigene splicing assays in the current criteria proposed by InSiGHT for clinical classification of MMR variants.

The low information content of Neurospora splicing signals: implications for RNA splicing and intron origin.

When we expressed a small (0.9 kb) nonprotein-coding transcript derived from the mitochondrial VS plasmid in the nucleus of Neurospora we found that it was efficiently spliced at one or more of eight 5′ splice sites and ten 3′ splice sites, which are present apparently by chance in the sequence. Further experimental and bioinformatic analyses of other mitochondrial plasmids, random sequences, and natural nuclear genes in Neurospora and other fungi indicate that fungal spliceosomes recognize a wide range of 5′ splice site and branchpoint sequences and predict introns to be present at high frequency in random sequence. In contrast, analysis of intronless fungal nuclear genes indicates that branchpoint, 5′ splice site and 3′ splice site consensus sequences are underrepresented compared with random sequences. This underrepresentation of splicing signals is sufficient to deplete the nuclear genome of splice sites at locations that do not comprise biologically relevant introns. Thus, the splicing machinery can recognize a wide range of splicing signal sequences, but splicing still occurs with great accuracy, not because the splicing machinery distinguishes correct from incorrect introns, but because incorrect introns are substantially depleted from the genome.

Extensive functional analyses of RHD splice site variants: Insights into the potential role of splicing in the physiology of Rh.

Among more than 300 mutated alleles identified so far within the RHD gene, almost 40 are assumed to alter cellular splicing and therefore may have a direct effect on Rh phenotype both at the quantitative and at the qualitative levels. Functional data are, however, mostly unavailable to assess the direct involvement of splicing defect in the underlying physiology. We generated plasmid constructs to carry out an exhaustive investigation of 38 RHD variants located within or in the vicinity of exon-intron junctions by a minigene splicing assay, further characterized the transcript structures by sequencing, and identified cryptic sites activated by the genetic defect. Bioinformatics predictions were carried out in parallel and compared with the functional data. For the first time we demonstrate that a product including the full-length Exon 9 is transcribed in the presence of the c.1227G>A substitution frequently carried by Asians with DEL phenotype and confirmed that splicing is altered in the RHD*weak D Type 2 allele, a rare variant most commonly found in Caucasians. Overall we 1) show significant correlation between functional analyses, bioinformatics predictions, and phenotypes, when available, especially for variants in close proximity of the consensus splice sites; 2) classify the variations as splicing or nonsplicing variants; and 3) provide functional data to further improve bioinformatics splicing tools. Conversely assessment of seven silent exonic variants was mainly inconclusive.

Finding the unexpected--how we identified a second class of introns and the U12-dependent spliceosome.

Finding the unexpected--how we identified a second class of introns and the U12-dependent spliceosome.

Splicing: still so much to learn.

Splicing: still so much to learn.

Splicing abnormality of integrin β4 gene (ITGB4) due to nucleotide substitutions far from splice site underlies pyloric atresia-junctional epidermolysis bullosa syndrome

BackgroundPyloric atresia-junctional epidermolysis bullosa syndrome (PA-JEB) is a rare subgroup of epidermolysis bullosa, which is inherited disorder characterized by skin fragile. PA-JEB is caused by mutation of ITGB4 or ITGA6, which encodes integrin β4 or α6, respectively. ObjectiveTo clarify the molecular basis of PA-JEB and to expand the mutational database, we carried out the mutational analysis of a 29-year-old Japanese PA-JEB patient. MethodsStandard methods were used to prepare, PCR-amplify, and sequence DNA or mRNA in peripheral blood or skin samples, respectively. ResultsSequence analysis revealed two novel mutations in ITGB4, c.264+2TtoA and c.1762−25TtoA. The paternal c.264+2TtoA resided within a splice site consensus region and generated two splice variants resulting in a premature termination codon (PTC). The maternal c.1762−25TtoA was a unique mutation because of its location, 25bp away from the splice site, and resided in branch-point consensus sequence. This c.1762−25TtoA substitution resulted in generation of two abnormal transcripts each with a PTC. Genotype–phenotype correlation in this case was also unique because the proband showed a non-lethal phenotype regardless of both mutations resulted in only abnormal transcripts with a PTC. ConclusionThe present case expands the mutational database and further elucidates the genotype–phenotype correlation for this rare disease, PA-JEB.

De novo mutations in moderate or severe intellectual disability.

Genetics is believed to have an important role in intellectual disability (ID). Recent studies have emphasized the involvement of de novo mutations (DNMs) in ID but the extent to which they contribute to its pathogenesis and the identity of the corresponding genes remain largely unknown. Here, we report a screen for DNMs in subjects with moderate or severe ID. We sequenced the exomes of 41 probands and their parents, and confirmed 81 DNMs affecting the coding sequence or consensus splice sites (1.98 DNMs/proband). We observed a significant excess of de novo single nucleotide substitutions and loss-of-function mutations in these cases compared to control subjects, suggesting that at least a subset of these variations are pathogenic. A total of 12 likely pathogenic DNMs were identified in genes previously associated with ID (ARID1B, CHD2, FOXG1, GABRB3, GATAD2B, GRIN2B, MBD5, MED13L, SETBP1, TBR1, TCF4, WDR45), resulting in a diagnostic yield of ∼29%. We also identified 12 possibly pathogenic DNMs in genes (HNRNPU, WAC, RYR2, SET, EGR1, MYH10, EIF2C1, COL4A3BP, CHMP2A, PPP1CB, VPS4A, PPP2R2B) that have not previously been causally linked to ID. Interestingly, no case was explained by inherited mutations. Protein network analysis indicated that the products of many of these known and candidate genes interact with each other or with products of other ID-associated genes further supporting their involvement in ID. We conclude that DNMs represent a major cause of moderate or severe ID.

Compound heterozygous mutations in RIPPLY2 associated with vertebral segmentation defects.

Segmentation defects of the vertebrae (SDV) are caused by aberrant somite formation during embryogenesis and result in irregular formation of the vertebrae and ribs. The Notch signal transduction pathway plays a critical role in somite formation and patterning in model vertebrates. In humans, mutations in several genes involved in the Notch pathway are associated with SDV, with both autosomal recessive (MESP2, DLL3, LFNG, HES7) and autosomal dominant (TBX6) inheritance. However, many individuals with SDV do not carry mutations in these genes. Using whole-exome capture and massive parallel sequencing, we identified compound heterozygous mutations in RIPPLY2 in two brothers with multiple regional SDV, with appropriate familial segregation. One novel mutation (c.A238T:p.Arg80*) introduces a premature stop codon. In transiently transfected C2C12 mouse myoblasts, the RIPPLY2 mutant protein demonstrated impaired transcriptional repression activity compared with wild-type RIPPLY2 despite similar levels of expression. The other mutation (c.240-4T>G), with minor allele frequency <0.002, lies in the highly conserved splice site consensus sequence 5' to the terminal exon. Ripply2 has a well-established role in somitogenesis and vertebral column formation, interacting at both gene and protein levels with SDV-associated Mesp2 and Tbx6. We conclude that compound heterozygous mutations in RIPPLY2 are associated with SDV, a new gene for this condition.

First steps in exploring prospective exome sequencing of consanguineous couples

Consanguinity is one of the most frequent risk factors for congenital disorders. In theory, prospective exome sequencing of consanguineous couples could identify couples who both are carriers of autosomal recessive diseases, and empower such couples to make informed reproductive decisions. To investigate this, we sent blood samples to our laboratory of four pairs of consanguineous parents having one or more children affected by an autosomal recessive disorder, without revealing any diagnostic information. The study was restricted to find identical, previously described, or evidently pathogenic mutations in both parents of each couple, in over 400 genes known to result in severe autosomal recessive disorders. Out of the six autosomal recessive disorders known to the four couples studied, two were correctly identified. Carrier status of one not previously known autosomal recessive disorder was discovered. As expected, given the pipeline used, large deletions, mutations in genes not present in the gene list, mutations outside the exons and consensus splice sites, and mutations that were not evidently pathogenic and previously not reported, were not identified. The restriction to detecting only couples with identical mutations diminishes the risk of revealing unsolicited findings and shortens the time needed for analysis, but also results in missing couples with different mutations in the same gene. In addition to the proposed pipeline, couples should be offered testing for carrier status of frequent disorders that can present themselves by large deletions, non-exonic mutations or compound heterozygous mutations (e.g. thalassemia, spinal muscular atrophy, cystic fibrosis). Even though sensitivity is reduced, offering exome sequencing prospectively will increase reproductive options for consanguineous couples.

T.P.30: Recurrent episodes of myoglobinuria, mental retardation and epilepsy but no haemolysis in two brothers with phosphoglycerate kinase deficiency

We report two brothers, aged 14 and 16, with a mild intellectual deficiency. Both brothers presented recurrent episodes of generalized weakness following effort since the age of 4. At the age of 8, after spending twenty minutes playing in a swimming pool, the youngest brother presented an episode of weakness, together with a painful swelling of the left thigh and myoglobinuria (max CK 540,000 UI/l). He was hospitalized in an intensive care unit and treated by intravenous fluids and loop diuretics. 4 months later, a muscle biopsy disclosed abnormal vacuolisation with slightly increased muscle lipid content. An extensive workup excluded a mitochondrial disease, a defect of fatty acid oxidation and Mac Ardle disease. At the age of 13, the youngest brother presented a refractory status epilepticus, complicated by rhabdomyolysis and acute renal failure (max CK 710,000 UI/l). Physical examination of both brothers, between acute episodes, was completely normal and CK level was approximately 500 UI/l ( N PGK1 gene sequencing revealed a splice site consensus sequence change that co-segregates with decreased PGK activity. Its putative functional consequences are currently under investigation. Conclusion: Phosphoglycerate kinase (PGK) deficiency, a rare defect in glycolysis pathway, has its place in the differential diagnosis of metabolic myopathies. The association of exercise intolerance and recurrent episodes of myoglobinuria with haemolytic anaemia, intellectual deficiency or epilepsy is highly suggestive of the disease.

Influenza viruses and mRNA splicing: doing more with less.

ABSTRACTDuring their nuclear replication stage, influenza viruses hijack the host splicing machinery to process some of their RNA segments, the M and NS segments. In this review, we provide an overview of the current knowledge gathered on this interplay between influenza viruses and the cellular spliceosome, with a particular focus on influenza A viruses (IAV). These viruses have developed accurate regulation mechanisms to reassign the host spliceosome to alter host cellular expression and enable an optimal expression of specific spliced viral products throughout infection. Moreover, IAV segments undergoing splicing display high levels of similarity with human consensus splice sites and their viral transcripts show noteworthy secondary structures. Sequence alignments and consensus analyses, along with recently published studies, suggest both conservation and evolution of viral splice site sequences and structure for improved adaptation to the host. Altogether, these results emphasize the ability of IAV to be well adapted to the host’s splicing machinery, and further investigations may contribute to a better understanding of splicing regulation with regard to viral replication, host range, and pathogenesis.

Targeted Suppression of a Dystrophin Pseudo-exon using Antisense Oligonucleotides

Antisense oligonucleotide induced exon skipping is showing great promise as a potential therapy to restore functional dystrophin expression in Duchenne muscular dystrophy. Redirecting mRNA processing can excise one or more exons flanking frame-shifting deletions or duplications so that the reading frame is restored and a Becker muscular dystrophy-like transcript is induced. Here, we report the application of exon skipping to excise a diseasecausing 72 bp pseudo-exon from the mature dystrophin mRNA. This pseudo-exon arises from a single T>G base change in intron 47 that creates an acceptor site with a strong consensus splice site score. Although in-frame, this pseudo-exon contains several stop codons and its inclusion is consistent with a diagnosis of Duchenne muscular dystrophy. The 21 year old patient is no longer ambulant but can stand and sit unassisted, indicating leaky premRNA processing and the generation of some normal transcripts has delayed the muscle wasting to some extent. In cases where the DMD lesion activates one or more cryptic splice sites and results in the retention of intronic sequences in that mature mRNA, exon skipping has the potential to generate a normal gene transcript. Antisense oligonucleotides targeted to the mutant splice site were able to remove the pseudo-exon from the mature transcript and restore full-length dystrophin expression. This is in contrast to most exon-skipping strategies for Duchenne muscular dystrophy in which antisense oligonucleotides are used to restore expression but the induced Becker muscular dystrophy-like transcript is shorter and potentially less functional than the full-length mRNA. As such, pseudo-exons may be particularly amenable to exon skipping, especially when basal levels of expression have raised endogenous dystrophin levels, reducing disease severity and should mitigate immune responses to the induced normal isoform.

Congenital analbuminemia caused by a novel aberrant splicing in the albumin gene

Introduction:Congenital analbuminemia is a rare autosomal recessive disorder manifested by the presence of a very low amount of circulating serum albumin. It is an allelic heterogeneous defect, caused by variety of mutations within the albumin gene in homozygous or compound heterozygous state. Herein we report the clinical and molecular characterization of a new case of congenital analbuminemia diagnosed in a female newborn of consanguineous (first degree cousins) parents from Ankara, Turkey, who presented with a low albumin concentration (< 8 g/L) and severe clinical symptoms.Materials and methods:The albumin gene of the index case was screened by single-strand conformation polymorphism, heteroduplex analysis, and direct DNA sequencing. The effect of the splicing mutation was evaluated by examining the cDNA obtained by reverse transcriptase - polymerase chain reaction (RT-PCR) from the albumin mRNA extracted from proband’s leukocytes.Results:DNA sequencing revealed that the proband is homozygous, and both parents are heterozygous, for a novel G>A transition at position c.1652+1, the first base of intron 12, which inactivates the strongly conserved GT dinucleotide at the 5′ splice site consensus sequence of this intron. The splicing defect results in the complete skipping of the preceding exon (exon 12) and in a frame-shift within exon 13 with a premature stop codon after the translation of three mutant amino acid residues.Conclusions:Our results confirm the clinical diagnosis of congenital analbuminemia in the proband and the inheritance of the trait and contribute to shed light on the molecular genetics of analbuminemia.