An apoptosis directed multi-target approach for therapeutic siRNA oligonucleotides

Die Entdeckung des RNA interference Mechanismus als natürliches Phänomen in eukaryotischen Zellen führte zu einer Reihe neuer Möglichkeiten in Krebsforschung und – therapie. Dabei spielen siRNAs, kurze RNA Sequenzen (19-23bp), eine große Rolle indem sie die Expression spezifischer Gensequenzen herunterregulieren und im Rahmen des RNAi – Mechanismus als sogenannte „gene – silencing“ Substanzen wirken. Auf dem Gebiet von RNA-interference-basierenden Studien ist dieser siRNA – vermittelte knockdown von spezifischen apoptose- und krebsrelevanten Genen eine viel versprechende Anwendung. Weiterführend zum knockdown eines Gens mittels spezifischer siRNA – Sequenz wurden in dieser Arbeit mehrere synthetisch hergestellte siRNAs, deren Zielsequenzen verschiedene Karzinom – Biomarker sind, gemeinsam in Krebszelllinien transfiziert, um mögliche Synergieeffekte im Ausmass der Downregulierung hervorzurufen. Die dabei verwendeten siRNA – Zielsequenzen waren Gene aus verschiedensten metabolischen Pathways, um eine möglichst weitreichende Abdeckung zu erzielen. Mittels Proliferationstests wurden sechs mögliche siRNA – Ziele ermittelt: galectin-1, sphingosin kinase 1, BCL-2, Her2/Neu, ß-catenin und EpCAM. Für eine weitere Validierung dieser Ergebnisse wurden zusätzlich Apoptosetests und realtime PCRs durchgeführt. Als Selektivitätskontrolle für somatisches Endothelgewebe wurden HUVEC – Zellen (human umbilical vein endothelial cells) verwendet. Mit Ausnahme von zwei Gentargets wurde die Herunterregulierung von mRNA mittels qPCR verifiziert. Die siRNAs zeigten auch in Kombination keinen Wirkungsverlust. Die Induktion von Apoptose und die Proliferationsraten waren stark abhängig von der verwendeten Zelllinie, Synergieeffekte der siRNA-Kombinationen konnten jedoch in keiner der Krebszelllinien nachgewiesen werden.The discovery of RNA interference (RNAi) as a naturally occuring phenomenon in eukaryotic cells has risen an entire new field of possibilities in cancer research and therapy. siRNAs, small RNA structures (19-23bp), are gene-silencing substances which downregulate the expression of specific gene sequences by using this cell-innate RNAi pathway. The siRNA-mediated knockdown of apoptosis- and cancer-relevant genes in carcinoma cell lines appeared to be a promising application in the area of RNA-interference-based therapeutical studies. As continuative attempt to a single siRNA knockdown of the same gene, the aim of this study was to find possible synergistic functional effects in the extend of gene – downregulation by combining synthetical siRNAs targeted at different cancer biomarkers in order to reduce proliferation and increase apoptosis of the target cell lines. For this multi – target approach, siRNAs were transfected as single agents and in various combinations of two to three sequences into carcinoma cell lines (607B, HT-29, MCF-7). Gene targets were chosen due to their role in preferably different apoptosis- and cancer relevant pathways for a wide range of potential effects. Using proliferation assays the most promising siRNAs, in combination, appeared to be galectin-1, sphingosin kinase 1, BCL-2, Her2/Neu, ß-catenin and EpCAM. For further validation, apoptosis assays and realtime PCR was performed. In order to find targets with selectivity on cancer cells, HUVECs (human umbilical vein endothelial cells) were used as control for somatic endothelial tissue. Successful downregulation of mRNAs were verified by qPCR-based quantification for all but two of the targets. siRNAs did not lose their potency when applied in combinations. Effects on proliferation rates and apoptosis induction were found to be dependent on the cell line. No synergistic effect was identified for all of the cancer cells used for any siRNA combination

The current benefit of genome sequencing compared to exome sequencing in patients with developmental or epileptic encephalopathies

Background: As the technology of next generation sequencing rapidly develops and costs are constantly reduced, the clinical availability of whole genome sequencing (WGS) increases. Thereby, it remains unclear what exact advantage WGS offers in comparison to whole exome sequencing (WES) for the diagnosis of genetic diseases using current technologies. Methods: Trio-WGS was conducted for 20 patients with developmental or epileptic encephalopathies who remained undiagnosed after WES and chromosomal microarray analysis. Results: A diagnosis was reached for four patients (20%). However, retrospectively all pathogenic variants could have been detected in a WES analysis conducted with today's methods and knowledge. Conclusion: The additional diagnostic yield of WGS versus WES is currently largely explained by new scientific insights and the general technological progress. Nevertheless, it is noteworthy that whole genome sequencing has greater potential for the analysis of small copy number and copy number neutral variants not seen with WES as well as variants in noncoding regions, especially as potentially more knowledge of the function of noncoding regions arises. We, therefore, conclude that even though today the added value of WGS versus WES seems to be limited, it may increase substantially in the future

Assessing clinical utility of preconception expanded carrier screening regarding residual risk for neurodevelopmental disorders

The magnitude of clinical utility of preconception expanded carrier screening (ECS) concerning its potential to reduce the risk of affected offspring is unknown. Since neurodevelopmental disorders (NDDs) in their offspring is a major concern of parents-to-be, we addressed the question of residual risk by assessing the risk-reduction potential for NDDs in a retrospective study investigating ECS with different criteria for gene selection and definition of pathogenicity. We used exome sequencing data from 700 parents of children with NDDs and blindly screened for carrier-alleles in up to 3046 recessive/X-linked genes. Depending on variant pathogenicity thresholds and gene content, NDD-risk-reduction potential was up to 43.5% in consanguineous, and 5.1% in nonconsanguineous couples. The risk-reduction-potential was compromised by underestimation of pathogenicity of missense variants (false-negative-rate 4.6%), inherited copy-number variants and compound heterozygosity of one inherited and one de novo variant (0.9% each). Adherence to the ACMG recommendations of restricting ECS to high-frequency genes in nonconsanguineous couples would more than halve the detectable inherited NDD-risk. Thus, for optimized clinical utility of ECS, screening in recessive/X-linked genes regardless of their frequency (ACMG Tier-4) and sensible pathogenicity thresholds should be considered for all couples seeking ECS

Deleterious ZNRF3 germline variants cause neurodevelopmental disorders with mirror brain phenotypes via domain-specific effects on Wnt/β-catenin signaling

Zinc and RING finger 3 (ZNRF3) is a negative-feedback regulator of Wnt/β-catenin signaling, which plays an important role in human brain development. Although somatically frequently mutated in cancer, germline variants in ZNRF3 have not been established as causative for neurodevelopmental disorders (NDDs). We identified 12 individuals with ZNRF3 variants and various phenotypes via GeneMatcher/Decipher and evaluated genotype-phenotype correlation. We performed structural modeling and representative deleterious and control variants were assessed using in vitro transcriptional reporter assays with and without Wnt-ligand Wnt3a and/or Wnt-potentiator R-spondin (RSPO). Eight individuals harbored de novo missense variants and presented with NDD. We found missense variants associated with macrocephalic NDD to cluster in the RING ligase domain. Structural modeling predicted disruption of the ubiquitin ligase function likely compromising Wnt receptor turnover. Accordingly, the functional assays showed enhanced Wnt/β-catenin signaling for these variants in a dominant negative manner. Contrarily, an individual with microcephalic NDD harbored a missense variant in the RSPO-binding domain predicted to disrupt binding affinity to RSPO and showed attenuated Wnt/β-catenin signaling in the same assays. Additionally, four individuals harbored de novo truncating or de novo or inherited large in-frame deletion variants with non-NDD phenotypes, including heart, adrenal, or nephrotic problems. In contrast to NDD-associated missense variants, the effects on Wnt/β-catenin signaling were comparable between the truncating variant and the empty vector and between benign variants and the wild type. In summary, we provide evidence for mirror brain size phenotypes caused by distinct pathomechanisms in Wnt/β-catenin signaling through protein domain-specific deleterious ZNRF3 germline missense variants

Elucidation of the phenotypic spectrum and genetic landscape in primary and secondary microcephaly

Purpose: Microcephaly is a sign of many genetic conditions but has been rarely systematically evaluated. We therefore comprehensively studied the clinical and genetic landscape of an unselected cohort of patients with microcephaly. Methods: We performed clinical assessment, high-resolution chromosomal microarray analysis, exome sequencing, and functional studies in 62 patients (58% with primary microcephaly [PM], 27% with secondary microcephaly [SM], and 15% of unknown onset). Results: We found severity of developmental delay/intellectual disability correlating with severity of microcephaly in PM, but not SM. We detected causative variants in 48.4% of patients and found divergent inheritance and variant pattern for PM (mainly recessive and likely gene-disrupting [LGD]) versus SM (all dominant de novo and evenly LGD or missense). While centrosome-related pathways were solely identified in PM, transcriptional regulation was the most frequently affected pathway in both SM and PM. Unexpectedly, we found causative variants in different mitochondria-related genes accounting for ~5% of patients, which emphasizes their role even in syndromic PM. Additionally, we delineated novel candidate genes involved in centrosome-related pathway (SPAG5, TEDC1), Wnt signaling (VPS26A, ZNRF3), and RNA trafficking (DDX1). Conclusion: Our findings enable improved evaluation and genetic counseling of PM and SM patients and further elucidate microcephaly pathways

Deleterious ZNRF3 germline variants cause neurodevelopmental disorders with mirror brain phenotypes via domain-specific effects on Wnt/β-catenin signaling

Zinc and RING finger 3 (ZNRF3) is a negative-feedback regulator of Wnt/β-catenin signaling, which plays an important role in human brain development. Although somatically frequently mutated in cancer, germline variants in ZNRF3 have not been established as causative for neurodevelopmental disorders (NDDs). We identified 12 individuals with ZNRF3 variants and various phenotypes via GeneMatcher/Decipher and evaluated genotype-phenotype correlation. We performed structural modeling and representative deleterious and control variants were assessed using in vitro transcriptional reporter assays with and without Wnt-ligand Wnt3a and/or Wnt-potentiator R-spondin (RSPO). Eight individuals harbored de novo missense variants and presented with NDD. We found missense variants associated with macrocephalic NDD to cluster in the RING ligase domain. Structural modeling predicted disruption of the ubiquitin ligase function likely compromising Wnt receptor turnover. Accordingly, the functional assays showed enhanced Wnt/β-catenin signaling for these variants in a dominant negative manner. Contrarily, an individual with microcephalic NDD harbored a missense variant in the RSPO-binding domain predicted to disrupt binding affinity to RSPO and showed attenuated Wnt/β-catenin signaling in the same assays. Additionally, four individuals harbored de novo truncating or de novo or inherited large in-frame deletion variants with non-NDD phenotypes, including heart, adrenal, or nephrotic problems. In contrast to NDD-associated missense variants, the effects on Wnt/β-catenin signaling were comparable between the truncating variant and the empty vector and between benign variants and the wild type. In summary, we provide evidence for mirror brain size phenotypes caused by distinct pathomechanisms in Wnt/β-catenin signaling through protein domain-specific deleterious ZNRF3 germline missense variants

The current benefit of genome sequencing compared to exome sequencing in patients with developmental or epileptic encephalopathies

Abstract Background As the technology of next generation sequencing rapidly develops and costs are constantly reduced, the clinical availability of whole genome sequencing (WGS) increases. Thereby, it remains unclear what exact advantage WGS offers in comparison to whole exome sequencing (WES) for the diagnosis of genetic diseases using current technologies. Methods Trio‐WGS was conducted for 20 patients with developmental or epileptic encephalopathies who remained undiagnosed after WES and chromosomal microarray analysis. Results A diagnosis was reached for four patients (20%). However, retrospectively all pathogenic variants could have been detected in a WES analysis conducted with today's methods and knowledge. Conclusion The additional diagnostic yield of WGS versus WES is currently largely explained by new scientific insights and the general technological progress. Nevertheless, it is noteworthy that whole genome sequencing has greater potential for the analysis of small copy number and copy number neutral variants not seen with WES as well as variants in noncoding regions, especially as potentially more knowledge of the function of noncoding regions arises. We, therefore, conclude that even though today the added value of WGS versus WES seems to be limited, it may increase substantially in the future

Clinical and experimental evidence suggest a link between KIF7 and C5orf42-related ciliopathies through Sonic Hedgehog signaling

Acrocallosal syndrome (ACLS) is an autosomal recessive neurodevelopmental disorder caused by KIF7 defects and belongs to the heterogeneous group of ciliopathies related to Joubert syndrome (JBTS). While ACLS is characterized by macrocephaly, prominent forehead, depressed nasal bridge, and hypertelorism, facial dysmorphism has not been emphasized in JBTS cohorts with molecular diagnosis. To evaluate the specificity and etiology of ACLS craniofacial features, we performed whole exome or targeted Sanger sequencing in patients with the aforementioned overlapping craniofacial appearance but variable additional ciliopathy features followed by functional studies. We found (likely) pathogenic variants of KIF7 in 5 out of 9 families, including the original ACLS patients, and delineated 1000 to 4000-year-old Swiss founder alleles. Three of the remaining families had (likely) pathogenic variants in the JBTS gene C5orf42, and one patient had a novel de novo frameshift variant in SHH known to cause autosomal dominant holoprosencephaly. In accordance with the patients' craniofacial anomalies, we showed facial midline widening after silencing of C5orf42 in chicken embryos. We further supported the link between KIF7, SHH, and C5orf42 by demonstrating abnormal primary cilia and diminished response to a SHH agonist in fibroblasts of C5orf42-mutated patients, as well as axonal pathfinding errors in C5orf42-silenced chicken embryos similar to those observed after perturbation of Shh signaling. Our findings, therefore, suggest that beside the neurodevelopmental features, macrocephaly and facial widening are likely more general signs of disturbed SHH signaling. Nevertheless, long-term follow-up revealed that C5orf42-mutated patients showed catch-up development and fainting of facial features contrary to KIF7-mutated patients

Elucidation of the phenotypic spectrum and genetic landscape in primary and secondary microcephaly

PURPOSE Microcephaly is a sign of many genetic conditions but has been rarely systematically evaluated. We therefore comprehensively studied the clinical and genetic landscape of an unselected cohort of patients with microcephaly. METHODS We performed clinical assessment, high-resolution chromosomal microarray analysis, exome sequencing, and functional studies in 62 patients (58% with primary microcephaly [PM], 27% with secondary microcephaly [SM], and 15% of unknown onset). RESULTS We found severity of developmental delay/intellectual disability correlating with severity of microcephaly in PM, but not SM. We detected causative variants in 48.4% of patients and found divergent inheritance and variant pattern for PM (mainly recessive and likely gene-disrupting [LGD]) versus SM (all dominant de novo and evenly LGD or missense). While centrosome-related pathways were solely identified in PM, transcriptional regulation was the most frequently affected pathway in both SM and PM. Unexpectedly, we found causative variants in different mitochondria-related genes accounting for ~5% of patients, which emphasizes their role even in syndromic PM. Additionally, we delineated novel candidate genes involved in centrosome-related pathway (SPAG5, TEDC1), Wnt signaling (VPS26A, ZNRF3), and RNA trafficking (DDX1). CONCLUSION Our findings enable improved evaluation and genetic counseling of PM and SM patients and further elucidate microcephaly pathways

Total hip arthroplasty with acetabular reconstruction using a bulk autograft for patients with developmental dysplasia of the hip results in high loosening rates at mid-term follow-up

PURPOSE: Managing a deficient acetabulum in patients with developmental dysplasia of the hip (DDH) can be challenging. The purpose of the study was to determine the mid-term results of total hip arthroplasty (THA) using a bulk structural autograft for reconstruction of the acetabular roof in patients with DDH. METHODS: Between 1982 and 1999, 112 patients underwent THA with acetabular roof-plasty using a bulk structural autograft for secondary osteoarthritis related to DDH. A total of 106 patients (115 hips) met inclusion criteria and were followed for an average of 11.6 years (seven to 24 years). The mean age was 52.5 years at the index operation. Clinical and radiological evaluations were performed according to the methods of Merle d'Aubigne and Postel, Johnston et al. and DeLee and Charnley. RESULTS: The overall Merle d'Aubigne hip score significantly improved (3.7 vs 10.4, p < 0.01). The limb length discrepancy decreased from 30 to 6 mm (p < 0.01). The average distance that the hip centre was distalised was 22.3 mm (0-56 mm). However, radiolucent lines were observed in 27 % of patients at final follow-up, and the overall rate of revision for aseptic loosening was 16 %. Further, Kaplan-Meier survivorship curves predicted a rapid increase in the failure rate at 15 years. CONCLUSIONS: The mid-term functional outcome of THA with an acetabular roof-plasty using a bulk autograft is satisfactory; however, the long-term results are questionable