Diagnostic Yield and Benefits of Whole Exome Sequencing in CAKUT Patients Diagnosed in the First Thousand Days of Life

Infancy; Reverse phenotyping; Whole exome sequencingInfancia; Fenotipado inverso; Secuenciación del exoma completoInfància; Fenotipat invers; Seqüenciació de l'exoma completIntroduction Congenital anomalies of the kidney and urinary tract (CAKUT) are the predominant cause of chronic kidney disease (CKD) and the need for kidney replacement therapy (KRT) in children. Although more than 60 genes are known to cause CAKUT if mutated, genetic etiology is detected, on average, in only 16% of unselected CAKUT cases, making genetic testing unproductive. Methods Whole exome sequencing (WES) was performed in 100 patients with CAKUT diagnosed in the first 1000 days of life with CKD stages 1 to 5D/T. Variants in 58 established CAKUT-associated genes were extracted, classified according to the American College of Medical Genetics and Genomics guidelines, and their translational value was assessed. Results In 25% of these mostly sporadic patients with CAKUT, a rare likely pathogenic or pathogenic variant was identified in 1 or 2 of 15 CAKUT-associated genes, including GATA3, HNF1B, LIFR, PAX2, SALL1, and TBC1D1. Of the 27 variants detected, 52% were loss-of-function and 18.5% de novo variants. The diagnostic yield was significantly higher in patients requiring KRT before 3 years of age (43%, odds ratio 2.95) and in patients with extrarenal features (41%, odds ratio 3.5) compared with patients lacking these criteria. Considering that all affected genes were previously associated with extrarenal complications, including treatable conditions, such as diabetes, hyperuricemia, hypomagnesemia, and hypoparathyroidism, the genetic diagnosis allowed preventive measures and/or early treatment in 25% of patients. Conclusion WES offers significant advantages for the diagnosis and management of patients with CAKUT diagnosed before 3 years of age, especially in patients who require KRT or have extrarenal anomalies.This work was supported by grants from the Else Kröner-Fresenius-Stiftung (2018_Kolleg.12, Clinician Scientist Program TITUS at Hannover Medical School to LW) and the Deutsche Forschungsgemeinschaft (MA 9606/1-1 to HM, and KO 5614/2-1 to AC and RGW)

Biological mechanisms and pharmacological inducibility of translational readthrough.

Die Termination der Translation wird initiiert, wenn die A-Stelle des Ribosoms ein Stopcodon erreicht und Terminationsfaktoren mit dem Stopcodon interagieren. Binden anstatt der Terminationsfaktoren eine fast komplementäre tRNA, wird das Stopcodon überlesen. Dieser Prozess wird als translationaler Readthrough bezeichnet. Endogener translationaler Readthrough führt zur Bildung neuer Protein-Isoformen. Neben dem endogenen translationalen Readthrough kann der Prozess auch durch Medikamente induziert werden, was ein möglicher Therapieansatz für Krankheiten, die durch Nonsense-Mutationen verursacht werden, darstellt.  Im ersten Teil dieser Arbeit wurde ein auf der Durchflusszytometrie basierender Dual-Reporter-Assay erfolgreich als Methode zur quantitativen Messung des translationalen Readthroughs etabliert.  Im zweiten Teil wurden peroxisomale Enzyme (Malatdehydrogenase 1 [MDH1] und Laktatdehydrogenase B [LDHB]), die einen besonders hohen endogenen Readthrough-Anteil zeigen, in Modellzellen von Nichtsäugetierzellen näher charakterisiert: Dabei war bekannt, dass die peroxisomale MDH1x und LDHBx über ein in den Readthrough-Erweiterungen verstecktes Zielsignal (peroxisomal targeting Signal, PTS) in das Peroxisom transportiert werden. In der Readthrough-Erweiterung der LDHBx ist bei Nichtsäugetieren bekanntermaßen kein PTS konserviert. In der vorliegenden Arbeit konnte mittels Immunfluoreszenzmikroskopie gezeigt werden, dass die LDHBx tatsächlich nur in 20 % bis 33 % der Fälle in Zellen von Nichtsäugetieren im Peroxisom lokalisiert ist. Hieraus ergeben sich Hinweise auf die Funktion und die Evolution der Laktatdehydrogenase B. Basierend auf zuvor erhobenen Daten der Göttinger neuropädiatrischen Arbeitsgruppe konnte zusätzlich festgestellt werden, dass die MDH1(x) in CEF-Zellen im Vergleich zur MDH1(x) in humanen HeLa-Zellen keinen signifikanten Unterschied bezüglich der Quantität des Readthrough zeigt. Im dritten Teil wurde der Einfluss der Nukleotide, die um das Stopcodon lokalisiert sind, auf den translationalen Readthrough analysiert. In-silico-Modellierungen sprachen für einen signifikanten Einfluss der Base sechs Positionen proximal des Stopcodons. Die Frage nach dem Einfluss der proximalen Basen auf den translationalen Readthrough wurde in der vorliegenden Arbeit mit einem neu etablierten dual reporter assay untersucht. Der Assay ermöglichte es, Readthrough-Werte der DNA-Sequenzen zwei verschiedener Proteine mit Mutationen sechs Nukleotide upstream des Stopcodons zu vergleichen. Mutationen sechs Positionen upstream des Stopcodons reduzierten den endogenen translationalen Readthrough signifikant. Die In-silico-Daten zum maßgeblichen Einfluss der Base sechs Positionen proximal des Stopcodons konnten erstmals in vivo im Experiment mit lebenden Zellen bestätigt werden. Eine frühere Studie deutete darauf hin, dass für das Protein VEGF-Ax die 63 Nukleotide downstream des Stopcodons für den translationalen Readthrough entscheidend sein könnten. Dies konnte in dieser Arbeit nicht bestätigt werden, was die Bedeutung der Nukleotide vor dem Stopcodon unterstreicht.  Im vierten Teil der Arbeit wurde der induzierte translationale Readthrough der Nonsense-Mutationen des Rett-Syndroms untersucht. Es wurde die Effizienz der Readthrough-Induktion durch das Aminoglykosid Geneticin bei den elf häufigsten Nonsense-Mutationen des Rett-Syndroms einzeln in HeLa-Zellen evaluiert. Es konnte ein deutlich signifikanter Unterschied in der Effizienz der Readthrough-Induzierbarkeit zwischen den Nonsense-Mutationen festgestellt werden. Für einige Mutationen ließ sich translationaler Readthrough sehr gut induzieren (z. B. RettpR270X), während andere, darunter auch die häufigste Mutation des Rett-Syndroms in Deutschland (RettpR168X), keinen induzierbaren Readthrough zeigten. Dies könnte klinisch relevant werden, weil auf der Basis dieser Ergebnisse Readthrough-induzierende und möglicherweise nebenwirkungsreiche Medikamente zielgerichtet eingesetzt werden könnten.Termination of translation is mediated when the A-site of the ribosome reaches a stop codon and release factors interact with the stop codon. Due to the competition between release factors and near-cognate tRNAs a stop codon can function as a sense codon. This process is called translational readthrough. Translational readthrough can occur endogenously which leads to the formation of new protein isoforms or can be induced by drugs which is a possible therapeutical approach for diseases caused by nonsense mutations.  In the first part of this thesis, a flow cytometry based dual reporter assay was successfully established as a method for quantitative measurement of translational readthrough.  In the second part, peroxisomal enzymes (malate dehydrogenase 1 [MDH1] and lactate dehydrogenase B [LDHB]) which show particularly high endogenous readthrough contents were further characterized in non-mammalian cells: It was known that peroxisomal MDH1x and LDHBx are transported to the peroxisome via a peroxisomal targeting signal (PTS) hidden in the readthrough extensions. In non-mammals no known PTS is conserved in the readthrough extension on the DNA of LDHBx. In this thesis, immunofluorescence microscopy demonstrated that LDHBx is indeed localized in the peroxisome in only 20% to 33% of cases in non-mammalian cells (chicken embryo fibroblast, CEF) providing insights about the function and the evolution of the lactate dehydrogenase B. Based on previously collected data from neuropediatric group in Göttingen, it was additionally found that MDH1(x) in CEF showed no significant difference in the readthrough quantity compared with MDH1(x) in human HeLa cells.  In the third part of this thesis the influence of nucleotides localized around the stop codon on translational readthrough was analyzed. In silico modeling suggested a significant influence of the nucleotide six positions upstream of the stop codon on translational readthrough. In this thesis the influence of the nucleotide six positions upstream of the stop codon was investigated using the newly established dual reporter assay. The assay allowed us to compare readthrough values of DNA sequences of two different proteins with mutations of the nucleotide six positions upstream of the stop codon. The mutations significantly reduced endogenous translational readthrough showing for the first time the influence of the nucleotide six positions upstream of the stop codon in experiments with living cells. A previous study indicated that the 63 nucleotides downstream of the stop codon are critical for translational readthrough in the protein VEGF-Ax. This could not be confirmed in this work which highlights the importance of the nucleotides upstream the stop codon.  In the fourth part of the thesis, the induced translational readthrough of nonsense mutations was investigated in Rett syndrome. The efficiency of readthrough induction by the aminoglycoside Geneticin was evaluated in eleven nonsense mutations of Rett syndrome in HeLa cells. A significant difference between the nonsense mutations was observed in the efficiency of readthrough inducibility. For some mutations translational readthrough could be induced very well (e.g. RettpR270X), while others, including the most common Rett syndrome mutation in Germany (RettpR168X), showed no inducible readthrough. This may be clinically relevant because readthrough-inducing drugs with potentially high side effects could be used depending on the mutation based on these results.2021-12-1

Systematic and quantitative analysis of stop codon readthrough in Rett syndrome nonsense mutations

Lebeda D, Fierenz A, Werfel L, Rosin-Arbesfeld R, Hofhuis J, Thoms S. Systematic and quantitative analysis of stop codon readthrough in Rett syndrome nonsense mutations. Journal of Molecular Medicine. 2024.Rett syndrome (RTT) is a neurodevelopmental disorder resulting from genetic mutations in the methyl CpG binding protein 2 (MeCP2) gene. Specifically, around 35% of RTT patients harbor premature termination codons (PTCs) within the MeCP2 gene due to nonsense mutations. A promising therapeutic avenue for these individuals involves the use of aminoglycosides, which stimulate translational readthrough (TR) by causing stop codons to be interpreted as sense codons. However, the effectiveness of this treatment depends on several factors, including the type of stop codon and the surrounding nucleotides, collectively referred to as the stop codon context (SCC). Here, we develop a high-content reporter system to precisely measure TR efficiency at different SCCs, assess the recovery of the full-length MeCP2 protein, and evaluate its subcellular localization. We have conducted a comprehensive investigation into the intricate relationship between SCC characteristics and TR induction, examining a total of 14 pathogenic MeCP2 nonsense mutations with the aim to advance the prospects of personalized therapy for individuals with RTT. Our results demonstrate that TR induction can successfully restore full-length MeCP2 protein, albeit to varying degrees, contingent upon the SCC and the specific position of the PTC within the MeCP2 mRNA. TR induction can lead to the re-establishment of nuclear localization of MeCP2, indicating the potential restoration of protein functionality. In summary, our findings underscore the significance of SCC-specific approaches in the development of tailored therapies for RTT. By unraveling the relationship between SCC and TR therapy, we pave the way for personalized, individualized treatment strategies that hold promise for improving the lives of individuals affected by this debilitating neurodevelopmental disorder. KEY MESSAGES: The efficiency of readthrough induction at MeCP2 premature termination codons strongly depends on the stop codon context. The position of the premature termination codon on the transcript influences the readthrough inducibility. A new high-content dual reporter assay facilitates the measurement and prediction of readthrough efficiency of specific nucleotide stop contexts. Readthrough induction results in the recovery of full-length MeCP2 and its re-localization to the nucleus. MeCP2 requires only one of its annotated nuclear localization signals. © 2024. The Author(s)