Splice-shifting oligonucleotide (SSO) mediated blocking of an exonic splicing enhancer (ESE) created by the prevalent c.903+469T>C MTRR mutation corrects splicing and restores enzyme activity in patient cells

The prevalent c.903+469T>C mutation in MTRR causes the cblE type of homocystinuria by strengthening an SRSF1 binding site in an ESE leading to activation of a pseudoexon. We hypothesized that other splicing regulatory elements (SREs) are also critical for MTRR pseudoexon inclusion. We demonstrate that the MTRR pseudoexon is on the verge of being recognized and is therefore vulnerable to several point mutations that disrupt a fine-tuned balance between the different SREs. Normally, pseudoexon inclusion is suppressed by a hnRNP A1 binding exonic splicing silencer (ESS). When the c.903+469T>C mutation is present two ESEs abrogate the activity of the ESS and promote pseudoexon inclusion. Blocking the 3′splice site or the ESEs by SSOs is effective in restoring normal splicing of minigenes and endogenous MTRR transcripts in patient cells. By employing an SSO complementary to both ESEs, we were able to rescue MTRR enzymatic activity in patient cells to approximately 50% of that in controls. We show that several point mutations, individually, can activate a pseudoexon, illustrating that this mechanism can occur more frequently than previously expected. Moreover, we demonstrate that SSO blocking of critical ESEs is a promising strategy to treat the increasing number of activated pseudoexon

Topoisomerase 1 inhibits MYC promoter activity by inducing G-quadruplex formation

We have investigated the function of human topoisomerase 1 (TOP1) in regulation of G-quadruplex (G4) formation in the Pu27 region of the MYC P1 promoter. Pu27 is among the best characterized G4 forming sequences in the human genome and it is well known that promoter activity is inhibited upon G4 formation in this region. We found that TOP1 downregulation stimulated transcription from a promoter with wildtype Pu27 but not if the G4 motif in Pu27 was interrupted by mutation(s). The effect was not specific to the MYC promoter and similar results were obtained for the G4 forming promoter element WT21. The other major DNA topoisomerases with relaxation activity, topoisomerases 2α and β, on the other hand, did not affect G4 dependent promoter activity. The cellular studies were supported by in vitro investigations demonstrating a high affinity of TOP1 for wildtype Pu27 but not for mutant sequences unable to form G4. Moreover, TOP1 was able to induce G4 formation in Pu27 inserted in double stranded plasmid DNA in vitro. This is the first time TOP1 has been demonstrated capable of inducing G4 formation in double stranded DNA and of influencing G4 formation in cells

Splicing of phenylalanine hydroxylase (PAH) exon 11 is vulnerable: molecular pathology of mutations in PAH exon 11

In about 20-30% of phenylketonuria (PKU) patients, phenylalanine (Phe) levels can be controlled by cofactor 6R-tetrahydrobiopterin (BH(4)) administration. The phenylalanine hydroxylase (PAH) genotype has a predictive value concerning BH(4)-response and therefore a correct assessment of the mutation molecular pathology is important. Mutations that disturb the splicing of exons (e.g. interplay between splice site strength and regulatory sequences like exon splicing enhancers (ESEs)/exon splicing silencers (ESSs)) may cause different severity of PKU. In this study, we identified PAH exon 11 as a vulnerable exon and used patient derived lymphoblast cell lines and PAH minigenes to study the molecular defect that impacted pre-mRNA processing. We showed that the c.1144T>C and c.1066-3C>T mutations cause exon 11 skipping, while the c.1139C>T mutation is neutral or slightly beneficial. The c.1144T>C mutation resides in a putative splicing enhancer motif and binding by splicing factors SF2/ASF, SRp20 and SRp40 is disturbed. Additional mutations in potential splicing factor binding sites contributed to elucidate the pathogenesis of mutations in PAH exon 11. We suggest that PAH exon 11 is vulnerable due to a weak 3' splice site and that this makes exon 11 inclusion dependent on an ESE spanning position c.1144. Importantly, this implies that other mutations in exon 11 may affect splicing, since splicing is often determined by a fine balance between several positive and negative splicing regulatory elements distributed throughout the exon. Finally, we identified a pseudoexon in intron 11, which would have pathogenic consequences if activated by mutations or improved splicing conditions. Exonic mutations that disrupt splicing are unlikely to facilitate response to BH(4) and may lead to inconsistent genotype-phenotype correlations. Therefore, recognizing such mutations enhances our ability to predict the BH(4)-response

Absence of an Intron Splicing Silencer in Porcine Smn1 Intron 7 Confers Immunity to the Exon Skipping Mutation in Human SMN2

Spinal Muscular Atrophy is caused by homozygous loss of SMN1. All patients retain at least one copy of SMN2 which produces an identical protein but at lower levels due to a silent mutation in exon 7 which results in predominant exclusion of the exon. Therapies targeting the splicing of SMN2 exon 7 have been in development for several years, and their efficacy has been measured using either in vitro cellular assays or in vivo small animal models such as mice. In this study we evaluated the potential for constructing a mini-pig animal model by introducing minimal changes in the endogenous porcine Smn1 gene to maintain the native genomic structure and regulation. We found that while a Smn2-like mutation can be introduced in the porcine Smn1 gene and can diminish the function of the ESE, it would not recapitulate the splicing pattern seen in human SMN2 due to absence of a functional ISS immediately downstream of exon 7. We investigated the ISS region and show here that the porcine ISS is inactive due to disruption of a proximal hnRNP A1 binding site, while a distal hnRNP A1 binding site remains functional but is unable to maintain the functionality of the ISS as a whole

Identification of Six Novel PTH1R Mutations in Families with a History of Primary Failure of Tooth Eruption

Primary Failure of tooth Eruption (PFE) is a non-syndromic disorder which can be caused by mutations in the parathyroid hormone receptor 1 gene (PTH1R). Traditionally, the disorder has been identified clinically based on post-emergent failure of eruption of permanent molars. However, patients with PTH1R mutations will not benefit from surgical and/or orthodontic treatment and it is therefore clinically important to establish whether a given failure of tooth eruption is caused by a PTH1R defect or not. We analyzed the PTH1R gene in six patients clinically diagnosed with PFE, all of which had undergone surgical and/or orthodontic interventions, and identified novel PTH1R mutations in all. Four of the six mutations were predicted to abolish correct mRNA maturation either through introduction of premature stop codons (c.947C>A and c.1082G>A), or by altering correct mRNA splicing (c.544-26_544-23del and c.989G>T). The latter was validated by transfection of minigenes. The six novel mutations expand the mutation spectrum for PFE from eight to 14 pathogenic mutations. Loss-of-function mutations in PTH1R are also associated with recessively inherited Blomstrand chondrodysplasia. We compiled all published PTH1R mutations and identified a mutational overlap between Blomstrand chondrodysplasia and PFE. The results suggest that a genetic approach to preclinical diagnosis will have important implication for surgical and orthodontic treatment of patients with failure of tooth eruption

Use of Molecular Genetic Analyses in Danish Routine Newborn Screening

Historically, the analyses used for newborn screening (NBS) were biochemical, but increasingly, molecular genetic analyses are being introduced in the workflow. We describe the application of molecular genetic analyses in the Danish NBS programme and show that second-tier molecular genetic testing is useful to reduce the false positive rate while simultaneously providing information about the precise molecular genetic variant and thus informing therapeutic strategy and easing providing information to parents. When molecular genetic analyses are applied as second-tier testing, valuable functional data from biochemical methods are available and in our view, such targeted NGS technology should be implemented when possible in the NBS workflow. First-tier NGS technology may be a promising future possibility for disorders without a reliable biomarker and as a general approach to increase the adaptability of NBS for a broader range of genetic diseases, which is important in the current landscape of quickly evolving new therapeutic possibilities. However, studies on feasibility, sensitivity, and specificity are needed as well as more insight into what views the general population has towards using genetic analyses in NBS. This may be sensitive to some and could have potentially negative consequences for the NBS programme

Normal levels of plasma free carnitine and acylcarnitines in follow-up samples from a presymptomatic case of carnitine palmitoyl transferase 1 (CPT1) deficiency detected through newborn screening in Denmark

Carnitine palmitoyl transferase (CPT) 1 A deficiency is a rare disorder of hepatic long-chain fatty acid oxidation. CPT1 deficiency is included in newborn screening programs in a number of countries to allow presymptomatic detection and early treatment of affected patients. We present a case of presymptomatic CPT1A deficiency detected through newborn screening in Denmark with diagnostic levels of carnitine and acylcarnitines in the initial dried blood spot. Levels of plasma-free carnitine and acylcarnitines in follow-up samples were normal, but reverted to diagnostic levels when the patient developed clinical symptoms at the age of 8 months. At that time, a diagnosis of CPT1A deficiency was confirmed by sequence analysis of the CPT1A gene revealing homozygosity for a novel c.167C>T variation in exon 3. Enzyme activity measurements showed a relatively mild enzyme defect with a decreased residual enzyme activity of 17–25%. We conclude that CPT1A gene testing and/or enzyme assay is mandatory to confirm an abnormal newborn screen suggesting CPT1A deficiency to avoid delayed diagnoses