Nonsense Mutation Research Articles

Loss-of-function mutations of the TIE1 receptor tyrosine kinase cause late-onset primary lymphedema.

Primary lymphedema (PL), characterized by tissue swelling, fat accumulation, and fibrosis, results from defects in lymphatic vessels or valves caused by mutations in genes involved in development, maturation, and function of the lymphatic vascular system. Pathogenic variants in various genes have been identified in about 30% of PL cases. By screening of a cohort of 755 individuals with PL, we identified two TIE1 (tyrosine kinase with immunoglobulin- and epidermal growth factor-like domains 1) missense variants and one truncating variant, all predicted to be pathogenic by bioinformatic algorithms. The TIE1 receptor, in complex with TIE2, binds angiopoietins to regulate the formation and remodeling of blood and lymphatic vessels. The premature stop codon mutant encoded an inactive truncated extracellular TIE1 fragment with decreased mRNA stability, and the amino acid substitutions led to decreased TIE1 signaling activity. By reproducing the two missense variants in mouse Tie1 via CRISPR/Cas9, we showed that both cause edema and are lethal in homozygous mice. Thus, our results indicate that TIE1 loss-of-function variants can cause lymphatic dysfunction in patients. Together with our earlier demonstration that ANGPT2 loss-of-function mutations can also cause PL, our results emphasize the important role of the ANGPT2/TIE1 pathway in lymphatic function.

Antisense oligonucleotides as a potential therapeutic strategy in hypertrophic cardiomyopathy caused by MYBPC3 variants

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Sociedad Española de Cardiología Background/Introduction Non-sense or frameshift mutations in the MYBPC3 gene encoding cardiac myosin-binding protein C (cMyBP-C) are a major cause for inherited forms of Hypertrophic Cardiomyopathy (HCM), the most frequent inherited cardiac disease, affecting 0.2% of the general population and represents the leading cause for sudden cardiac death in young athletes. During the last decade, RNA-based therapeutic approaches have been studied for neuromuscular disorders and recently became to be considered to envision causal therapies for cardiomyopathies, utilizing antisense oligonucleotide mediated exon skipping, which attempts to reframe mutated mybpc3 transcripts, resulting in shortened but functional protein. MYBPC3 gene has a total of 34 coding exons and, exons 2, 22, and 24-27 are ideal targets for skipping. Purpose We aim to design and evaluate antisense oligonucleotides (AONs) for efficient exon-skipping in targeted MYBPC3 exons. Methods We designed two specific AONs to mask the MYBPC3 exon 26 and confirmed successfully mediated exon skipping without disrupting the MYBPC3 reading frame. We chose a previously identified MYBPC3 c.2671insG/p.R891Afs*160 variant in exon 26, which causes a frameshift mutation leading to truncated MYBPC3 in HCM. Primary Human Cardiac Myocytes (PHCM) (Promocell) were transfected with the splice-site targeting AONs. Results In this study we designed two chemically modified AONs (AON1 and AON2) through the addition of a 2′-O-methyl RNA phosphorothioate backbone, targeting exonic splicing enhancers (ESEs) or splice sites (SSs) can induce skipping of MYBPC3 exon 26. We evaluated AON1 (i26-3SS) and AON2 (ESE)-treated Primary Human Cardiac Myocytes (PHCM) show 26-27 exons skipping in comparison to control samples. The identity of the exon 26-27 skipped bands detected by RT-PCR was confirmed by Sanger sequencing and preservation of both nucleotide sequences of MYBPC3 exons 25 and 28 were observed. These results indicate that AONs specifically for mutated MYBPC3 target exons, expanding the framework of future advancements in the therapeutic potential of antisense-mediated exon skipping MYBPC3-based HCM. In addition, skipping exons 26 and 27 we eliminate a total of 22 possible exonic mutations, which represent 7.2% of the total known MYBPC3 exonic mutations. Conclusion(s) These results demonstrate that disruption of the MYBPC3 reading frame due to a truncating HCM mutation can be restored by exon skipping in cardiomyocytes in vitro indicating RNA-based strategies as a potential treatment option for HCM.Exon skipping scheme

Zebrafish dspb-/- mutant as model for arrhythmogenic cardiomyopathy - impact of physical activity

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Instituto de Salud Carlos III Background Zebrafish has been reported as a model to study the Arrythmogenic cardiomyopathy, which is generally associated with desmosomal gene mutations. We have identified a founder non-sense human variant in patients from our region (DSP p.Q447*) associated with left ventricle Arrythmogenic cardiomyopathy and we have selected it for modelling in zebra fish by CRISPR/Cas9 method, choosing exon 10 as the region of homology to be mutated and obtaining the p.T449fs* variant that determines a premature stop codon. Purpose Our aim was the characterization of dspb-/- mutants, studying the impact of physical activity in development of the disease. Methods The characterization of phenotype was performed through viability assays, cardiac function measurements and gene expression assays in larvae (3 days post fertilization) and adult (8 months old) individuals. The impact of physical activity was determined through moderated training periods (1 month) and endurance assays using a swimming tunnel. Results Viability assays showed that dspb mutants suffer a premature death, with significant higher mortality rates than the wildtype group (survival WT 89.7% vs dspb-/- 61.3%, p<0.01). Larvae cardiac function showed dspb mutants had a higher heart rate (WT 106.8 ± 26.8 vs dspb-/- 141.0 ± 7.2, p<0.01) while echocardiography showed differences in heart rate (WT 127.2 ± 49,8 vs dspb-/- 219.2 ± 26.8, p<0.05), systolic (WT 26.9 ± 5.3 vs dspb-/- 18.5 ± 5.2, p<0.05) and diastolic (WT 37.6 ± 8.4 vs dspb-/- 23.7 ± 5.4, p<0.05) areas. Gene expression analysis in adult heart showed dspb expression drastically decreased (WT 0.9 ± 0.1 vs dspb-/- 0.4 ± 0.01, p<0.01), whilst pkp2 expression increased (WT 0.9 ± 0.1 vs dspb-/- 2.59 ±0.22, p<0.001) as well as dspa gene expression (wt 0.9 ± 0.1 vs dspb-/- 4.41 ± 0.18, p< 0.001) as a compensation mechanism. Moderated long term physical activity had a positive impact in heart rate (dspb-/-pre 219.2 ± 26.8 vs dspb-/-post 138 ± 7.5, p<0.01) and diastolic area (dspb-/-pre 24 ± 5 vs dspb-/-post 32 ± 2.1, p<0.05). Adult mutants showed a better endurance showing improvement in immobile cumulative duration (dspb-/-pre 849.3 ± 29.3 vs dspb-/-post 179.8 ± 29.7, p<0.01) and immobile frequency (dspb-/-pre 5399.5 ± 1127.8 vs dspb-/-post 1310 ± 481.9, p<0.01). Conclusion The dspb-/- zebrafish presented pathological cardiac phenotype at larvae and adult stages, and less endurance in intensity exercises. The moderate exercise has turned out to be a beneficial modulator of the phenotype.

Zebrafish dspb -/- tert +/- mutant as model for arrhythmogenic cardiomyopathy. Impact of aging in the development of the disease

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Instituto de Salud Carlos III Background/Objectives Zebrafish has been reported as model to study the Arrythmogenic Cardiomyopathy, which is generally associated with desmosomal gene mutations. We have identified a founder non-sense human variant in patients from our region (DSP p.Q447*) associated with left ventricle Arrythmogenic Cardiomyopathy and we have selected it for modelling in zebra fish by CRISPR/Cas9 method, choosing exon 10 as the region of homology to be mutated and obtaining the p.T449fs* variant that determines a premature stop codon. On the other hand, tert gene enables telomerase activity and telomere maintenance and mutations in this gene have been used for studying aging effects. After multiple crossings of our dspb model with tert mutants we have obtained dspb-/-tert+/- model line. Purpose Our aim was the characterization of dspb-/-tert+/- mutants, studying the impact of aging in the development of the disease. Methods The model was obtained by crossing dspb-/- and tert-/- mutants, crossing the heterozygous mutants and genotyping for the selection of the dspb-/-tert+/- mutants. The characterization of the phenotype was performed through viability assays, cardiac function measurements and gene expression assays in larvae (3 days post fertilization) young adult (8 months old) and old adult (3 years old) individuals. Results Viability assays showed that dspb-/-tert+/- mutants suffer a premature death, with much lower survival rates than the dspb-/- group (survival dspb-/- 61.3% vs dspb-/-tert+/- 35.6%, p<0.01). Larvae heart rate analysis showed no significant differences. Echocardiography in young adults showed no significant differences between dspb-/-tert+/- and dspb-/- mutants. In old adults, echocardiography showed differences in heart rate (dspb-/- 142.5 ± 15.5 vs dspb-/-tert+/ -97.5 ± 3.5, p<0.01), respiratory frequency (dspb-/- 288.7 ± 36.1 vs dspb-/-tert+/- 95 ± 49.4, p<0.01) and atrial contraction velocity (dspb-/-155.7 ± 26.09 vs dspb-/-tert+/ -227.2 ± 17.6, p<0.05). Gene expression analysis in larvae showed that dspb expression drastically decreased (dspb-/- 0.3 ± 0.1 vs dspb-/-tert+/ -0.01 ± 0.008, p<0.01). In adults, dspb expression drastically decreased in heart tissue (dspb-/- 0.4 ± 0.01 vs dspb-/-tert+/- 0.06 ± 0.004, p<0.01) and skin (dspb-/ -0.34 ± 0.02 vs dspb-/-tert+/- 0.25 ± 0.01, p<0.01). Conclusion The dspb-/-tert+/- zebrafish presented worse survival rates, lower dspb gene expression levels and worse pathological cardiac phenotype at adult stages than dspb-/- mutants, proving that age is an important factor in the development of the disease and proving that dspb-/-tert+/- zebrafish is an appropriate model to study the impact of aging.

Generation of an induced pluripotent stem cell line IGIBi18-A from an Indian patient with Rubinstein Taybi Syndrome

Rubinstein Taybi Syndrome (RSTS) is a rare genetic disorder which is caused by mutations in either CREBBP or EP300. RSTS with mutations in CREBBP is known as RSTS-1. We have generated an induced pluripotent stem cell (iPSC) line, IGIBi018-A from an Indian RSTS-patient using the episomal reprogramming method. The CREBBP gene in the patient harbours a nonsense mutation at position NM_004380.3(c.6876 del C). IGIBi018-A iPSC showed expression of pluripotent stem cell markers, has a normal karyotype and could be differentiated into three germ layers. This iPSC line will help to explore the role of CREBBP in RSTS associated developmental defects.

A novel PLS-DYW type PPR protein OsASL is essential for chloroplast development in rice

Oryza longistaminata (OL), an AA-genome African wild rice which can propagate clonally via rhizome, is an important germplasm for improvement of Asian cultivated rice, however recessive lethal alleles can hitchhike clonal propagation in heterozygous state. Selfing of OL is difficult due to its self-incompatibility, but simple selfing of hybrid progeny between OL and O. sativa is effective to disclose and eliminate recessive lethal alleles. Here, we identified an exhibited albino-lethal phenotype mutant, from an F2 population between OL and O. sativa, named it albino seedling-lethal (asl). The leaves of asl mutant showed abnormal chloroplast development. The albino characteristics of asl were determined to be governed by a set of recessive nuclear genes through genetic analysis. Map-based cloning experiments found that a single nucleotide variation (G to A) was detected in the exon of OsASL in OL, which causes a premature stop codon. OsASL encodes a PLS-type PPR protein with 12 pentratricopeptide repeat domains, and is translocalized to chloroplasts. Complementation and knockout transgenic experiments further confirmed that OsASL is responsible for the albino-lethal phenotype. Loss-of-function OsASL (i.e. osasl) resulted in devoid of intron splicing of chloroplast RNA atpF, ndhA, rpl2 and rps12, and also RNA editing of ndhB, but facilitates the RNA editing of rpl2 in the plastid. Transcriptome sequencing showed that OsASL was mainly involved in chlorophyll synthesis pathway. The expression of Chlorophyll-associated genes were significantly decreased in asl plants, especially PEP (plastid-encoded RNA polymerase)-mediated genes. Our results suggest that OsASL is crucial for RNA editing, RNA splicing of chloroplast RNA group II genes, and plays an essential role in chloroplast development during early leaf development in rice.

Descriptive Study of the Clinical and Molecular Features of Epidermolysis Bullosa Patients in a Romanian European Reference Network-Skin Affiliated Reference Center.

During the last 10 years, in Romania, progress has been made for the welfare of patients suffering from epidermolysis bullosa (EB). In five university hospitals, affiliated with the National Program for the Treatment of Rare Diseases, highly trained specialists diagnose and treat patients with this rare condition. Regarding diagnosis, limitations still exist as immunofluorescence mapping and molecular genetic analysis are not accessible, and generally not reimbursed. Our objective is to present the experience in diagnosing EB patients at Colentina Clinical Hospital, highlighting genotype-phenotype correlations observed in our cohort of patients. The records of the patients enrolled between 2012 and 2024 were analyzed considering clinical aspects, and, when available, immunofluorescence mapping, transmission electron microscopy, and genetic molecular analysis. Fifty-six patients were identified, of whom 31 cases were of dystrophic EB, threewere of junctional EB, and 11 were of simplex EB. For 11 cases, the EB type could not be determined. Regarding EB simplex, two patients with KRT5 mutations and three patients with KRT14 mutations with various clinical expressions, from mild phenotype to severe forms, were identified. Three severe junctional EB patients were registered in our database and for one of the patients, two previously unreported mutations in the LAMA3 gene were identified. Regarding dystrophic EB, 31 cases were identified, of which 25 were recessive dystrophic casesand six were dominant dystrophic cases. Molecular genetic testing was performed for 15 patients, and the most common variant was c.425A>G, identified in six cases. Two previously unreported mutations were identified, namely,COL7A1 c.5416G>C, a heterozygous missense variant in a patient with a mild phenotype, mainly with nail involvement, and COL7A1 c.5960del, a variant that generates a frameshift in exon 72 resulting in a premature stop codon; this variant was identified in two siblings with a severe recessive dystrophic. Important steps have been made in identifying the correct and complete diagnosis, as well as the characterization of EB patients addressing our reference center. The findings underscore the pivotal role of molecular genetic testing in confirming diagnoses and elucidating inheritance patterns, especially in cases with atypical presentations or de novo mutations.

Therapeutic targeting of TP53 nonsense mutations in cancer.

Mutations in the TP53 tumor suppressor gene occur with high prevalence in a wide range of human tumors. A significant fraction of these mutations (around 10%) are nonsense mutations, creating a premature termination codon (PTC) that leads to the expression of truncated inactive p53 protein. Induction of translational readthrough across a PTC in nonsense mutant TP53 allows the production of full-length protein and potentially restoration of normal p53 function. Aminoglycoside antibiotics and a number of novel compounds have been shown to induce full-length p53 in tumor cells carrying various TP53 nonsense mutations. Full-length p53 protein generated by translational readthrough retains the capacity to transactivate p53 target genes and trigger tumor cell death. These findings raise hopes for efficient therapy of TP53 nonsense mutant tumors in the future.

Mechanisms and Delivery of tRNA Therapeutics.

Transfer ribonucleic acid (tRNA) therapeutics will provide personalized and mutation specific medicines to treat human genetic diseases for which no cures currently exist. The tRNAs are a family of adaptor molecules that interpret the nucleic acid sequences in our genes into the amino acid sequences of proteins that dictate cell function. Humans encode more than 600 tRNA genes. Interestingly, even healthy individuals contain some mutant tRNAs that make mistakes. Missense suppressor tRNAs insert the wrong amino acid in proteins, and nonsense suppressor tRNAs read through premature stop signals to generate full length proteins. Mutations that underlie many human diseases, including neurodegenerative diseases, cancers, and diverse rare genetic disorders, result from missense or nonsense mutations. Thus, specific tRNA variants can be strategically deployed as therapeutic agents to correct genetic defects. We review the mechanisms of tRNA therapeutic activity, the nature of the therapeutic window for nonsense and missense suppression as well as wild-type tRNA supplementation. We discuss the challenges and promises of delivering tRNAs as synthetic RNAs or as gene therapies. Together, tRNA medicines will provide novel treatments for common and rare genetic diseases in humans.

Environment modulates protein heterogeneity through transcriptional and translational stop codon readthrough

Stop codon readthrough events give rise to longer proteins, which may alter the protein’s function, thereby generating short-lasting phenotypic variability from a single gene. In order to systematically assess the frequency and origin of stop codon readthrough events, we designed a library of reporters. We introduced premature stop codons into mScarlet, which enabled high-throughput quantification of protein synthesis termination errors in E. coli using fluorescent microscopy. We found that under stress conditions, stop codon readthrough may occur at rates as high as 80%, depending on the nucleotide context, suggesting that evolution frequently samples stop codon readthrough events. The analysis of selected reporters by mass spectrometry and RNA-seq showed that not only translation but also transcription errors contribute to stop codon readthrough. The RNA polymerase was more likely to misincorporate a nucleotide at premature stop codons. Proteome-wide detection of stop codon readthrough by mass spectrometry revealed that temperature regulated the expression of cryptic sequences generated by stop codon readthrough in E. coli. Overall, our findings suggest that the environment affects the accuracy of protein production, which increases protein heterogeneity when the organisms need to adapt to new conditions.

Retinoschisin Is Required for Pineal Gland Calcification and Cellular Communication in Pinealocytes of Rats and Mice

Retinoschisin (RS1) is a secretory protein specifically localized to the extracellular domains in both the lateral retina and the pineal gland (PG). However, the functions of RS1 in the pineal body are poorly understood. To address this knowledge gap, in this study, we undertook histochemical, ultrastructural, and Western blotting analyses of the PG in rats and RS1-knock-in transgenic. We found that RS1 plays a key role in pineal gland calcification (PGC) in mice through both extracellular and intracellular pathways. RS1 was clustered around the cell membrane or intracellularly in pinealocytes, actively participating in the exchange of calcium and thereby mediating PGC. Additionally, RS1 deposition is essential for maintaining PGC architecture in the intercellular space of the adult PG. In RS1-knock-in mice with a nonsense mutation (p.Y65X) in the Rs1-domain of RS1, the Rs1-domain is chaotically dispersed in pinealocytes and the intercellular region of the PG. This prevents RS1 from binding calcified spots and forming calcified nodules, ultimately leading to the accumulation of calcareous lamellae in microvesicles. Additionally, RS1 was observed to colocalize with connexin-36, thereby modulating intercellular communication in the PG of both rats and mice. Our study revealed for the first time that RS1 is essential for maintaining PGC architecture and that it colocalizes with connexin 36 to modulate intercellular communication in the PG. These findings provide novel insights into the function of the RS1 gene in the PG.

#2003 Genetic association in patients with chronic kidney disease of unknown etiology: an observational study

Abstract Background and Aims Chronic kidney disease (CKD), impacting over 850 million people worldwide, is a diverse group of inherited and acquired nephropathies. While only about 10% of adult CKD is hereditary, predominantly due to mutations in genes like PKD1, PKD2, and PKHD1, the etiology often remains unidentified, leading to challenges in management, especially in transplantation and genetic counseling contexts. This study used whole exome sequencing to investigate CKD of unknown origin, aiming to improve diagnostic yields and establish genetic associations. Our objective was to find the diagnostic yield of Whole exome sequencing in patients with CKD of unknown etiology and to establish genetic association for same. Method The study was conducted in the Department of Nephrology and the Institute of Genetics and Genomics at Sir Ganga Ram Hospital, New Delhi. It was an observational cross-sectional analysis. All patients, both male and female of all age groups with Chronic Kidney Disease of unknown etiology who were willing to participate. All patients, both male and female of all age groups with Chronic Kidney Disease of known etiology were excluded as following: Results In the investigation of 56 chronic kidney disease samples with unknown etiology, genetic testing yielded 45 mutated gene variants in 37 subjects. The variants comprised 32 missense mutations, 8 deletions, 3 nonsense mutations, and 1 splice site mutation. Variant zygosity was as follows: 34 heterozygous, 6 homozygous, and 2 hemizygous. Pathogenic variants were detected in 9 patients (20%), likely pathogenic in 6 (13.33%), and variants of uncertain significance in 29 (64.44%). Eighteen subjects (32.14%) demonstrated no genetic mutations. A genetic diagnosis was established in 15 patients, representing a diagnostic yield of 26.78%. Stratified by disorder, the yield was 40% for glomerular, 100% for tubular, 50% for renal calculi, and 18.42% for undetermined CKD. Genetic testing facilitated a novel diagnosis or an alteration of the existing diagnosis in 6 patients (10.71%). Conclusion The results affirm the efficacy of genetic testing in elucidating the etiologies of chronic kidney disease where traditional diagnostic methods do not yield definitive causation. Genetic alterations were identified in a significant proportion of the patient population, indicating the presence of underlying genetic contributors to the disease. The identification of pathogenic and likely pathogenic variants highlights the potential of genetic testing to impact clinical decision-making and patient care.

#662 Characterization of novel gene mutation sites associated with autosomal dominant polycystic kidney disease on 19 chromosomes

Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD) is the most common single-gene inherited kidney disorder, affecting approximately 1 in 500-1000 newborns and often leading to end-stage renal disease. It involves cyst formation in various organs, with renal manifestations including cysts, hematuria, kidney stones, proteinuria, and hypertension. Liver, pancreas, seminal vesicles, spleen, and intracranial aneurysms may also be affected. Genetic diagnosis, particularly for patients without a family history or with atypical imaging, is considered the gold standard. ADPKD is associated with several pathogenic genes, such as PKD1 and PKD2, as well as others like HNF-1B, GANAB, DNAJB11, SEC63, ALG8, ALG9, PRKCSH, LRP5, SEC61B, and SEC63. Understanding the genetic basis is crucial for early diagnosis and identifying high-risk individuals. This study, based on genetic data from patients with renal polycystic kidney disease at Zhong Da Hospital Southeast University, identified novel ADPKD gene mutations. It explored structural changes in proteins, predicting effects on phenotype, to contribute to the enrichment of the ADPKD gene database. Method In a single-center cross-sectional retrospective study, patients with renal multiple cysts diagnosed from January 2019 to February 2023 at the Zhong Da Hospital Southeast University were included. Genetic and clinical data of patients and their families were collected. Unreported novel gene mutation sites associated with ADPKD were identified, and AlphaFold v2.3.1 was employed to predict protein structures. Changes in protein structure before and after mutations were compared to explore genotype-phenotype correlations and enrich the ADPKD gene database. Results The study included 96 patients with renal multiple cysts and their relatives from 62 families. Twelve mutated genes associated with renal cysts were detected in 52 families. Nineteen novel gene mutation sites associated with ADPKD were identified (Table 1, Fig. 1) , including 17 mutations in the PKD1 gene (1 splicing mutation, 7 frameshift mutations, 4 nonsense mutations, 1 whole-codon insertion, and 4 missense mutations), 1 ALG9 missense mutation, and 1 chromosomal structural variation. Truncating mutations in the PKD1 gene were correlated with a more severe clinical phenotype, while non-truncating mutations were associated with greater clinical heterogeneity. Conclusion Numerous novel gene mutation sites associated with ADPKD remain unreported. It is essential to analyze the pathogenicity of these novel mutation sites, establish genotype-phenotype correlations, and enrich the ADPKD gene database.

Whole-exome sequencing combined with in silico protein analysis identifies rare truncating variants in the TYR gene associated with oculocutaneous albinism IA (OCA1A) in an Iranian patient

Oculocutaneous albinism type IA (OCA1A) is an autosomal recessive tyrosinase-negative condition caused by deleterious variants in the TYR gene. Using whole-exome sequencing (WES) followed by Sanger sequencing, we detected rare pathogenic variants (c.286dupA and c.763C > T) within the TYR gene in a 12-year-old Iranian female who met the clinical criteria proposed for OCA. Through comprehensive in silico protein analysis, we elucidated the functional impact of these two variants. c.286dupA induced a frameshift, leading to the loss of the entire copper-binding tyrosinase domain and the only transmembrane melanosome-bound tyrosinase helix. Similarly, c.763C > T introduced a premature stop codon, resulting in the loss of approximately two-thirds of the copper-binding tyrosinase domain and the transmembrane helix. These findings signified a substantial disturbance in the normal function of tyrosinase. Our study presents the first report of co-occurring pathogenic c.286dupA and c.763C > T variants, comprehensively investigating their functional impact using in silico protein tools.

#2498 PKD2 recurrent haplotype in ADPKD patients

Abstract Background and Aims Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a systemic renal ciliopathy characterized by progressive renal cyst expansion and extrarenal manifestations following loss-of-function mutations. ADPKD is primarily caused by PKD1 (72-75%) and PKD2 (15-18%), with a high level of allelic heterogeneity: it is rare to identify the same germ-line mutation in different families since mutations are “private”. In recent years the identification of several new genes has underlined the genetic heterogeneity of ADPKD. The Aim of this study is to describe a cohort of PKD2 gene-linked families with the same germ-line mutation. Method Patients (pts) with ADPKD referral to the Genetic Kidney Diseases clinic of the UOC of Nephrology of ULSS8 Berica (San Bortolo Hospital in Vicenza) were enrolled and followed prospectively. Diagnosis of ADPKD was made upon the revised Ravine's criteria. Complete clinical details were recorded, including family history and pedigree. We performed NGS with Sophia Genetic “Nephropathies Solution” Panel or Sanger sequencing to identify disease-causing mutations in the cohort. Furthermore, microsatellite analysis was performed in PKD2 gene pts with same germ-line nonsense mutation c.2533C>T (p.Arg845Ter), to identify disease haplotype. Results We identified a cohort of 30 ADPKD pts belonging to 13 families with same PKD2 germ-line nonsense mutation c.2533C>T (p.Arg845Ter). Regarding pedigree analysis, 8 families had their origin from the same geographical area and the remaining 5 were from an area 100 km away. In 4 families, we found a common individual. Concerning the microsatellite analysis, all 30 pts shared the same haplotype Chromosome 4 CEN – VG2 (TG)20, VG3 (TA + GA)32, VG4 (CA)13, D4S2929 (AC)26, D4S1563 (TG)20 – TEL indicating that those families may originate from a common ancestor. Conclusion We found the same PKD2 germline mutation in 30 ADPKD pts belonging to 13 families that share the same haplotype. Since, ADPKD mutations are “private” and Vicenza province counts almost 860000 inhabitants, our findings provide an evidence that a founder effect exists, maybe due to a founder effect in our region.

PSMC5 insufficiency and P320R mutation impair proteasome function.

The ubiquitin-proteasome system mediates the degradation of a wide variety of proteins. Proteasome dysfunction is associated with neurodegenerative diseases and neurodevelopmental disorders in humans. Here we identified mutations in PSMC5, an AAA ATPase subunit of the proteasome 19S regulatory particle, in individuals with neurodevelopmental disorders, which were initially considered as variants of unknown significance. We have now found heterozygotes with the following mutations: P320R (6 individuals), R325W, Q160A, and one nonsense mutation at Q69. We focused on understanding the functional consequence of PSMC5 insufficiency and the P320R mutation in cells and found that both impair proteasome function and activate apoptosis. Interestingly, the P320R mutation impairs proteasome function by weakening the association between the 19S regulatory particle and the 20S core particle. Our study supports that proteasome dysfunction is the pathogenic cause of neurodevelopmental disorders in individuals carrying PSMC5 variants.

Improving nitrofurantoin resistance prediction in Escherichia coli from whole-genome sequence by integrating NfsA/B enzyme assays.

Nitrofurantoin resistance in Escherichia coli is primarily caused by mutations damaging two enzymes, NfsA and NfsB. Studies based on small isolate collections with defined nitrofurantoin MICs have found significant random genetic drift in nfsA and nfsB, making it extremely difficult to predict nitrofurantoin resistance from whole-genome sequence (WGS) where both genes are not obviously disrupted by nonsense or frameshift mutations or insertional inactivation. Here, we report a WGS survey of 200 oqxAB-negative E. coli from community urine samples, of which 34 were nitrofurantoin resistant. We characterized individual non-synonymous mutations seen in nfsA and nfsB among this collection using complementation cloning and NfsA/B enzyme assays in cell extracts. We definitively identified R203C, H11Y, W212R, A112E, and A112T in NfsA and R121C, Q142H, F84S, P163H, W46R, K57E, and V191G in NfsB as amino acid substitutions that reduce enzyme activity sufficiently to cause resistance. In contrast, E58D, I117T, K141E, L157F, A172S, G187D, and A188V in NfsA and G66D, M75I, V93A, and A174E in NfsB are functionally silent in this context. We identified that 9/166 (5.4%) nitrofurantoin-susceptible isolates were "pre-resistant," defined as having loss of function mutations in nfsA or nfsB. Finally, using NfsA/B enzyme assays and proteomics, we demonstrated that 9/34 (26.5%) ribE wild-type nitrofurantoin-resistant isolates also carried functionally wild-type nfsB or nfsB/nfsA. In these cases, NfsA/B activity was reduced through downregulated gene expression. Our biological understanding of nitrofurantoin resistance is greatly improved by this analysis but is still insufficient to allow its reliable prediction from WGS data.

Generation of induced pluripotent stem cells from an individual with early onset and severe hypertrophic cardiomyopathy linked to MYBPC3: c.772G > A mutation

Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in the MYPBC3 gene, which encodes the cardiac myosin-binding protein C (cMyBP-C). Most pathogenic variants in MYPBC3 are either nonsense mutations or result in frameshifts, suggesting that the primary disease mechanism involves reduced functional cMyBP-C protein levels within sarcomeres. However, a subset of MYPBC3 variants are missense mutations, and the molecular mechanisms underlying their pathogenicity remain elusive. Upon in vitro differentiation into cardiomyocytes, induced pluripotent stem cells (iPSCs) derived from HCM patients represent a valuable resource for disease modeling. In this study, we generated two iPSC lines from peripheral blood mononuclear cells (PBMCs) of a female with early onset and severe HCM linked to the MYBPC3: c.772G > A variant. Although this variant was initially classified as a missense mutation, recent studies indicate that it interferes with splicing and results in a frameshift. The generated iPSC lines exhibit a normal karyotype and display hallmark characteristics of pluripotency, including the ability to undergo trilineage differentiation. These novel iPSCs expand the existing repertoire of MYPBC3-mutated cell lines, broadening the spectrum of resources for exploring how diverse mutations induce HCM. They additionally offer a platform to study potential secondary genetic elements contributing to the pronounced disease severity observed in this individual.

Exon-junction complex association with stalled ribosomes and slow translation-independent disassembly

Exon junction complexes are deposited at exon-exon junctions during splicing. They are primarily known to activate non-sense mediated degradation of transcripts harbouring premature stop codons before the last intron. According to a popular model, exon-junction complexes accompany mRNAs to the cytoplasm where the first translating ribosome pushes them out. However, they are also removed by uncharacterized, translation-independent mechanisms. Little is known about kinetic and transcript specificity of these processes. Here we tag core subunits of exon-junction complexes with complementary split nanoluciferase fragments to obtain sensitive and quantitative assays for complex formation. Unexpectedly, exon-junction complexes form large stable mRNPs containing stalled ribosomes. Complex assembly and disassembly rates are determined after an arrest in transcription and/or translation. 85% of newly deposited exon-junction complexes are disassembled by a translation-dependent mechanism. However as this process is much faster than the translation-independent one, only 30% of the exon-junction complexes present in cells at steady state require translation for disassembly. Deep RNA sequencing shows a bias of exon-junction complex bound transcripts towards microtubule and centrosome coding ones and demonstrate that the lifetimes of exon-junction complexes are transcript-specific. This study provides a dynamic vision of exon-junction complexes and uncovers their unexpected stable association with ribosomes.

P09 Next-generation sequencing: on-and off-target analysis of gene editing in fibroblasts from patients with recessive dystrophic epidermolysis bullosa

Abstract Introduction and aims Base editors (BEs) and prime editors (PEs) have proved to have advantages over traditional clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 in correcting pathogenic mutations. We previously demonstrated 94.6% efficiency using adenine base editor (ABE)8e in correcting nonsense mutation (c.5047 C>T) in fibroblasts, which causes recessive dystrophic epidermolysis bullosa (RDEB). However, safety profile and cell-type-specific efficiencies of gene editing techniques have not been characterized. The aim of our research is to establish corrections of c.5047 C>T pathogenic variant in fibroblasts and compare off-target effects and efficiency of CRISPR-Cas9, BE and PE systems. Methods Primary fibroblasts harbouring heterozygous C>T pathogenic variant on exon 54 will be transfected with CRISPR-Cas9, ABE8e and PE using electroporation or lipofectamine. Both Sanger sequencing and next-generation sequencing (NGS) will be conducted to confirm pathogenic variant corrections. Galaxy platform which incorporates different NGS analysis tools will be used to analyse the NGS data. Subsequently, whole-exome sequencing and targeted amplicon sequencing will be used for off-target analysis. Results On-target efficiency for ABE8e to correct c.5047 C>T pathogenic variant in patient primary fibroblasts was determined first by Sanger sequencing showing 100% editing in contrast to 84% determined by NGS. Moving forward, we plan to correct the same pathogenic variant with PEs. Initially experiments are under way with PEmax mRNA and engineered PE guide RNAs (epegRNA) to compare different PEs. Furthermore, we will investigate the off-target effects of CRISPR-Cas9, ABE8e and PEs to edit RDEB primary fibroblasts using NGS Methods and conduct functional studies. Conclusions Here, we showed that the c.5047 C>T pathogenic variant in RDEB fibroblasts could be successfully edited using ABE8e. We demonstrated that NGS is required to precisely determine the editing efficacy. Taken together, we have established a pipeline for base editing in RDEB and we aim to achieve the same for prime editing in the future.