Nonsense Mutation Research Articles

Role of IL3RA in a Family with Lumbar Spinal Stenosis.

Lumbar spinal stenosis (LSS) is a degenerative spinal condition characterized by the narrowing of the spinal canal, resulting in low back pain (LBP) and limited leg mobility. Twin and family studies have suggested that genetics contributes to disease progression. However, the genetic causes of familial LSS remain unclear. We performed whole-exome and direct sequencing on seven female patients from a Han Chinese family with LBP, among whom four developed LSS. Based on our genetic findings, we performed gene knockdown studies in human chondrocytes to study possible pathological mechanisms underlying LSS. We found a novel nonsense mutation, c.417C > G (NM_002183, p.Y139X), in IL3RA, shared by all the LBP/LSS cases. Knockdown of IL3RA led to a reduction in the total collagen content of 81.6% in female chondrocytes and 21% in male chondrocytes. The expression of MMP-1, -3, and/or -10 significantly increased, with a more pronounced effect observed in females than in males. Furthermore, EsRb expression significantly decreased following IL3RA knockdown. Moreover, the knockdown of EsRb resulted in increased MMP-1 and -10 expression in chondrocytes from females. We speculate that IL3RA deficiency could lead to a reduction in collagen content and intervertebral disk (IVD) strength, particularly in females, thereby accelerating IVD degeneration and promoting LSS occurrence. Our results illustrate, for the first time, the association between IL3RA and estrogen receptor beta, highlighting their importance and impact on MMPs and collagen in degenerative spines in women.

Hidden evolutionary constraints dictate the retention of coronavirus accessory genes.

Coronaviruses exhibit many mechanisms of genetic innovation, including the acquisition of accessory genes that originate by capture of cellular genes or through duplication of existing viral genes. Accessory genes influence viral host range and cellular tropism, but little is known about how selection acts on these variable regions of virus genomes. We used experimental evolution of mouse hepatitis virus (MHV) encoding a cellular AKAP7 phosphodiesterase and an inactive native phosphodiesterase, NS2 to model the evolutionary fate of accessory genes. After courses of serial infection, the gene encoding inactive NS2, ORF2, unexpectedly remained intact, suggesting it is under cryptic constraint uncoupled from the function of NS2. In contrast, AKAP7 was retained under strong selection but rapidly lost under relaxed selection. Experimental evolution also led to altered viral replication in a cell type-specific manner and changed the relative proportions of subgenomic viral RNA in plaque-purified viral isolates, revealing additional mechanisms of adaptation. Guided by the retention of ORF2 and similar patterns in related betacoronaviruses, we analyzed ORF8 of SARS-CoV-2, which arose via gene duplication and contains premature stop codons in several globally successful lineages. As with MHV ORF2, the coding-defective SARS-CoV-2 ORF8 gene remains largely intact, mirroring patterns observed during MHV experimental evolution, challenging assumptions on the dynamics of gene loss in virus genomes and extending these findings to viruses currently adapting to humans.

Clinical and genetic spectrum of factor XII deficiency in the Han population of East China

BackgroundFactor XII (FXII or F12) deficiency is a rare inherited disorder, typically lacking haemorrhagic symptoms. There is limited literature exists on FXII deficiency and mutations within the Chinese population. This study aimed to characterize the spectrum of F12 gene mutations in a Chinese cohort and to investigate the relationship between FXII mutations and clinical phenotypes.MethodsGenetic and clinical data from 51 unrelated probands with FXII deficiency, along with their families, were meticulously collected and analysed.ResultsGenetic analysis revealed that 94.1% of probands carried genetic defects, with 29 mutations pinpointed in the F12 gene. Of these, 18 mutations were previously reported for the first time by our research group, including c.303_304delCA, c.1078G > A, c.1285 C > T, among others. Of the mutations, 17 are missense, constituting 58.6% of the total. Additionally, 11 are deletions or insertions, of which 8 result in frameshifts, while the remaining one is a nonsense mutation. These mutations were predominantly concentrated in two crucial regions: the catalytic domain and the kringle domain. The most frequently observed mutations were c.1681G > A, closely followed by c.1561G > A and c.1078G > A, indicating a dominance among these mutations. Additionally, a prevalent polymorphism at position 46 was observed in the majority of probands, with 47.1% having the 46T/T genotype and 13.7% having the 46 C/T genotype, which may potentially impact FXII activity. The broad spectrum of asymptomatic FXII deficiency observed within the Han population of East China.ConclusionsWe speculate on the potential impact of recurrent mutations on the efficacy of new drugs being developed to target FXII for thrombosis prevention and treatment. Furthermore, it is important to explore their influence on FXII-related pathways beyond the activation of the contact pathway in the coagulation cascade.

Intrinsic cardiomyopathy in pediatric Marfan syndrome: predictive factors and risk assessments.

Marfan syndrome (MFS) is associated with cardiovascular complications, particularly valvulopathies; however, its association with primary cardiomyopathy remains unclear. This retrospective cohort study examined the cardiomyopathy characteristics (CMCs) in pediatric patients with MFS. CMCs were defined as meeting at least one of the following echocardiography or clinical parameters: (1) cardiac index (CI) too low for patient's age, (2) ejection fraction (EF) <50%, and (3) diastolic dysfunction. The predictive factors for CMCs were determined using a multivariable logistic regression model. Among 83 patients with MFS (age, median [range], 12.5 [0.4-22.3] years), 39.8% exhibited CMCs. Only 4 patients (5%) showed heart failure symptoms (NYHA > 1). Independent predictors for CMCs included a systemic score of ≥7 (revised Ghent criteria) and likely pathogenic or pathogenic variants in FBN1, including variants that introduce a premature stop codon, splice site variants, and missense variants involving cysteine. A multivariable score was constructed with an AUC of 0.733. This study offers valuable insights into the prevalence and predictors of CMC in pediatric patients with MFS and presents potential strategies for risk assessment of cardiomyopathy. The objective of this study was to elucidate the contentious issue of intrinsic cardiomyopathy in Marfan syndrome and demonstrate its notable occurrence even in pediatric patients who do not exhibit heart failure symptoms or valvular complications. We highlighted the importance of specific FBN1 variants and higher systemic scores in identifying the potential for intrinsic cardiomyopathy in pediatric patients with Marfan syndrome.

Genetic analysis of antithrombin deficiency among Thai patients with thrombosis

Hereditary antithrombin (AT) deficiency, primarily caused by mutations of SERPINC1 mutations, is a strong risk factor for occurrences and recurrences of venous thrombosis. While AT activity level measurements may be interfered by several factors, genetic analysis is more definitive for diagnosis. This study aims to address the knowledge gap of genetic basis of AT deficiency among Thai patients. Samples were collected from patients with thrombosis and low AT activities. Cases with acquired causes of low AT were excluded. All 7 exons of the SERPINC1 gene were PCR amplified and subjected to Sanger sequencing. The study included 15 patients and 6 controls with normal AT activity levels. The results revealed the presence of 6 benign single nucleotide polymorphisms (SNPs) located in intron 1, 2, exon 5 and intron 5. Two of these SNPs in intron 2 were first described here. Notably, a novel SNP in exon 3, SERPINC1 c.570 C&gt;G (p.Y190X), was discovered. This is likely pathogenic because the premature stop codon led to a non-functional protein. The results suggest that pathogenic SERPINC1 variants detectable by DNA sequencing are uncommon among Thai patients with low AT activity levels underscoring the role of genetic analysis for definitive diagnosis. Further studies involving larger numbers of patients are required.

8696 Whole Exome Sequence Analysis in a Naïve Cohort of Congenital Hypopituitarism in a Single Center

Abstract Disclosure: F.Q. Aratani: None. D.P. Di Matteo: None. I.P. de Magalhães: None. P. Frudit: None. L.S. Motomia: None. S.K. Beeby: None. L.R. Barros: None. D.D. Bissegatto: None. L.R. Carvalho: None. Background: Deficiency of one or more pituitary hormones is defined as hypopituitarism. In the literature, molecular diagnosis (MD) is defined in 12% by the Sanger sequencing and large scale sequencing increased this number to 15%. Aim: To perform whole exome sequencing (WES) in a cohort of patients with congenital hypopituitarism (CH) naïve to MD approach. Patients: Nine patients with CH followed in a single Brazilian tertiary center were included. Methods: Libraries were prepared with kits from Agilent and sequenced on Hi-Seq (Illumina). The variants were called following GATK best practices, using the Haplotypecaller and genome version hg38. They were classified in the Franklin by genoox (https://franklin.genoox.com/clinical-db/home). WES that did not pass the platform's initial quality control and variants that did not reach an adequate confidence level (low/medium confidence: VAF &amp;lt; 50%) were excluded. The remaining variants were classified according to ACMG criteria. Results: Three among 9 patients presented allelic variants and aredescribed here. Case 1: A 22-year-old female presented height of 143 cm (-3,13 SDS) at the first visit without signs of puberty, had delayed bone age (DBA), hypoplastic adenohypophysis, interrupted pituitary stalk (IPS) and ectopic neurohypophysis (ENH) at the magnetic resonance imaging (MRI). Hormonal profile showed low E2, GH, FSH and LH. Twoheterozygous missense variants in the ALMS1 gene (c.5450G&amp;gt;A : p.Arg1817GIn; and ALMS1:c.4225G&amp;gt;C: p.Ala1409Pro) were seen in the exome; both classified as VUS according to ACMG criteria. This allelic variant is related to Alstrom Syndrome. Case 2: A 7-year-old female presented with short stature and was diagnosed with GH followed by LH/FSH, TSH and ACTH deficiencies. At MRI presented IPS and ENH. A heterozygous variant in the PROK2 gene c.364C&amp;gt;T p.R122 with a premature stop codon and truncated protein was present and classified as probably pathogenic according to ACMG criteria. This variant was already described with hypogonadotropic hypogonadism (HH) with or without anosmia. Case3: A 15-year-old male was referred with growth hormone deficiency (GHD), DBA, and HH. Growth hormone replacement was started followed by testosterone. Despite delayed puberty, the testes were normal in size. WES showed a variant c.629G&amp;gt;A:p.Trp210*, in hemizygosity in IGSF1 gene and was classified as probably pathogenic according to ACMG criteria. Loss-of-function mutations in IGSF1 may cause an X-linked syndrome of central hypothyroidism, macroorchidism and delayed puberty (delayed rise of testosterone, but normal timing of testicular growth). Conclusion: WES in a cohort of patients with congenital hypopituitarism naïve to molecular diagnosis revealed 33% (3/9) with positive genetic findings, two of them with clinical significance and family segregation according to the genetic inheritance. Presentation: 6/3/2024

6864 Genetic Profile And Identification Of A Novel Mutation In PHEX In X-Linked Hypophosphatemia (XLH) Patients At A Brazilian Research Center: Insights And Implications

Abstract Disclosure: M. Monseff Rodrigues da silva: None. I.M. de Araújo: None. F.J. de Paula: None. Background: X-Linked Hypophosphatemia (XLH) is a rare genetic disorder characterized by low phosphate levels in the blood, primarily caused by mutations in the PHEX gene, leading to bone skeletal deformities and reduced quality of life. XLH genetics is extensively studied with over 500 pathogenic mutations in the PHEXgene having been identified. Common mutations include missense, nonsense, and splicing, accounting for approximately 50% of cases. However, genotype-phenotype correlations remain unclear. Methods: We conducted a cross-sectional study using a convenience sample selected from the osteometabolism an outpatient clinic in Brazil. Genetic analysis was performed using next-generation sequencing (NGS; Ilumina) in collaboration with Mendelics Laboratory. Results: In our study, 9 out of 10 patients showed previously known and reported mutations, with 60% exhibiting common mutation types. The mutations occur in different codons; however, the most prevalent consequence was the substitution of glycine (G) with adenine (A), present in 40% of the patients. One patient, however, showed a unique missense mutation in PHEX gene (arginine to glutamate at codon 266), which leads to a premature stop codon, a mutation absent in over 183,000 X chromosomes from XLH individuals. Even though this mutation was novel, no significant diferences in the phenotype of this patient were seen in comparision with the rest of the cohort. Discussion: The findings of our study underscore the genetic heterogeneity characteristic of XLH, as evidenced by the fact that 90% of our patient cohort exhibited mutations previously identified in the literature, with a significant 60% showing common mutation types. This highlights the recurring genetic patterns in XLH, which can be instrumental for diagnostic and - perhaps in the future - therapeutic strategies. Particularly notable is the predominant mutation involving the substitution of glycine with adenine, found in 40% of our patients, underscoring its potential role as a key mutation in the pathogenesis of XLH. Furthermore, the discovery of a novel missense mutation in one patient, resulting in a premature stop codon, enriches the mutation spectrum of XLH and suggests a deeper complexity in the genetic landscape of the disease. This mutation, not previously documented in the extensive database of over 183,000 X chromosomes from XLH individuals, opens new avenues for research into the disease's molecular mechanisms and potential genotype-phenotype correlations. Conclusion: This study underlines the genetic diversity within XLH, emphasizing the need for comprehensive genetic analysis to understand the full spectrum of the disease. More studies are yet necessary to elucidate if there is a genotype-phenotype correlation. Presentation: 6/1/2024

7438 DAX1/NR0B1 Related Adrenal Hypoplasia Congenita Without Hypogonadotropic Hypogonadism

Abstract Disclosure: R.L. Figueiredo: None. J.B. Drummond: None. A. Meireles: None. J.L. Machado: None. G. Vidigal: None. B.S. Rocha: None. Background: X-linked congenital adrenal hypoplasia (AHC) is a rare disease caused by genetic mutations in DXA1/NRB01 gene, which encodes a nuclear hormone receptor expressed in the adrenal cortex, gonads, hypothalamus and anterior pituitary gland, occurring in less than 1:12,500 live births (1,2). AHC is characterized by primary adrenal insufficiency (PAI) associated with hypogonadotropic hypogonadism (HH) and impaired spermatogenesis (oligospermia or azoospermia). Although DAX1/NR0B1 mutations can manifest with different phenotypes, HH is mostly present, which makes this present case a diagnostic challenge. Clinical case: A 23-year-old man presented with typical symptoms of adrenal insufficiency at the age of 7 (abdominal pain, dehydration, nausea, vomiting and cutaneous hyperpigmentation). Initial testing was consistent with primary adrenal failure: elevated ACTH levels (1250pg/mL, RR&amp;lt;46pg/mL) and very low serum cortisol (0.8mcg/dL, RR 3-20mcg/dL). Etiological investigation excluded autoimmune etiology (negative anti-adrenal and anti-21-hydroxylase antibodies) as well as adrenoleukodystrophy (normal very long chain fatty acid levels). Physical exam revealed normal virilization, testicular volume and penile length. The patient reported normal libido and sexual function. Hormonal evaluation showed normal total testosterone (424 and 713ng/dL, RR 165 to 753ng/dL) as well as calculated free testosterone (7.2 and 11.4ng/mL, RR&amp;gt;3.8ng/mL) levels. Abdominal CT showed signs of bilateral adrenal hypoplasia. We then proceeded to genetic testing that revealed a pathological variant c.625C&amp;gt;T:p (Gln 209*) in hemizygosity in exon 1 of the DAX 1/NR0B1 gene. This is a nonsense mutation that introduces a premature stop codon. This variant is not present on the global and Brazilian genetic databases (gnomAD and ABraOM) and has not been previously described in the literature. The patient is currently being treated for adrenal insufficiency with the lowest tolerated dose of prednisone (7.5mg/day) and fludrocortisone 0.1mg/day. Conclusion: Etiological investigation of patients with PAI is essential to understand the evolution of the disease, monitoring extra-adrenal manifestations and the possibility of genetic counseling for the family. An intact hypothalamic-pituitary gonadal axis should not preclude genetic evaluation for a DAX/NR0B1 mutation in patients with PAI and suspected AHC. Further studies will clarify if this specific mutation carries a genotype-phenotype correlation in patients with AHC without HH.

7694 Characterizing NF1 Germline Pathogenic Variants In A Cohort Of Patients With Pheochromocytomas And Neurofibromatosis Type 1

Abstract Disclosure: T. Hristova: None. S. Parisien-La Salle: None. S. Lapointe: None. D. Tremblay: None. N. Dumas: None. M. Labidi: None. Z. El Haffaf: None. I. Bourdeau: None. Introduction: Pheochromocytomas and paragangliomas (PPGLs) are the tumors with the highest heritability in adult patients, with identification of a germline mutation in more than 30% of cases. Up to 20 susceptibility genes for PPGLs are known, including NF1. Neurofibromatosis type 1 (NF1) is an inherited disease affecting approximately 1 in 3000 people that predisposes to the development of tumors, such as pheochromocytomas (PHEO). Up to recently, the diagnosis of NF1 was mainly based on clinical manifestations and NF1 genetic characterization was not systematically performed. Objectives: To characterize germline pathogenic variants in the NF1 gene in patients with NF1 developing and PHEO. Methods: We reviewed the charts of patients with a pathology-proven diagnosis of PHEOs that were investigated at Centre hospitalier de l’Université de Montréal (CHUM) between 2000 and May 2023. Genetic analysis included gene sequencing by Sanger method or multigene sequencing by NGS with a panel (Invitae, CA) Results: In our cohort of 220 PHEOs, 15 patients (6.8%) (Males: 6, Females: 9) had a diagnosis of NF1. Mean age at diagnosis of PHEO in NF1 patients was 48 ± 13.6 years, contrasting to the mean age of 38,7 ± 15.2 years in patients carrying germline mutations in non-NF1 genes, most likely reflecting lack of systematic biochemically screening of PHEO in NF1. Urinary metanephrines were elevated in 9/15 patients. 2/15 (15.4%) NF1 patients had bilateral PHEO and 1/15 (7,7%) metastatic disease. Mean tumour diameter was 4.64cm (min-max 1.5 – 12.5 cm). Eight out of 15 NF1 patients underwent NF1 genetic analysis. Heterozygous NF1 germline pathogenic variants were found in all of them: 3 deletions (c.7379delG, c.5844_5845delAA and one encompassing the entire gene), 3 premature stop codons leading to truncated proteins or loss of function (c.7549C&amp;gt;T, c.3916C&amp;gt;T, c.1246C&amp;gt;T), and 2 splicing defects (c.4269+1 G&amp;gt;A, c.1885G&amp;gt;A). Conclusions: We report a large cohort of patients with NF1 and PHEO. The older age at diagnosis of PHEO in NF1 is expected based on lack of systematic biochemical screening for PHEO in NF1 clinical guidelines. We report 8 germline NF1 mutations associated with PHEOs. Further studies are required to fully understand the impact of genetic pathogenic variants in this population and to unravel a possible genotype-phenotype correlation. Presentation: 6/2/2024

A Novel Deep Learning Approach for Analyzing Glomerular Basement Membrane Lesions in a Mouse Model of X-Linked Alport Syndrome

Alport syndrome is a rare kidney disease typically more severe in males due to its X-linked inheritance. However, female patients with heterozygous X-linked Alport syndrome (XLAS) can develop renal failure over time, necessitating accurate pathological assessment for effective therapy. A key pathological finding in female XLAS patients is the mosaic pattern of partial loss of α5 chains of type IV collagen. This study, using a mouse model of XLAS with a nonsense mutation (R471*) in the Col4a5 gene, analogous to human XLAS, aimed to examine the consistency of this pattern with the glomerular basement membrane (GBM) structure. A modified periodic acid-methenamine silver (PAMS) staining method was developed for clearer GBM visualization. The integrated images from COL4α5-stained fluorescence, PAMS, and low-vacuum scanning electron microscopy (LVSEM) into a single-slide section and applied supervised deep learning to predict GBM lesions. Results showed significant individual variability in urinary protein levels and histological lesions. Pathological parameters, including crescent formation, focal segmental glomerulosclerosis, and the COL4α5/α2 ratio, correlated with clinical parameters like urinary protein and plasma creatinine levels. Integrated LVSEM analysis revealed dense GBM regions corresponded to areas where COL4α5 was preserved, while coarse GBM (basket-weave lesions) occurred in COL4α5-deficient regions. These advanced techniques can enhance biopsy-based diagnosis of Alport syndrome and aid in developing AI diagnostic tools for diseases involving basement membrane lesions.

680P Developing effective RNA editing approaches to target the nonsense mutation in the key functional region of DMD

680P Developing effective RNA editing approaches to target the nonsense mutation in the key functional region of DMD

376P Ataluren in patients with nonsense mutation DMD (nmDMD): retrospective evaluation from a tertiary referral center in Turkey

376P Ataluren in patients with nonsense mutation DMD (nmDMD): retrospective evaluation from a tertiary referral center in Turkey

Spastin accumulation and motor neuron defects caused by a novel SPAST splice site mutation

BackgroundHereditary spastic paraplegia (HSP) is a rare genetically heterogeneous neurodegenerative disorder. The most common type of HSP is caused by pathogenic variants in the SPAST gene. Various hypotheses regarding the pathogenic mechanisms of HSP-SPAST have been proposed. However, a single hypothesis may not be sufficient to explain HSP-SPAST.ObjectiveTo determine the causative gene of autosomal dominant HSP-SPAST in a pure pedigree and to study its underlying pathogenic mechanism.MethodsA four-generation Chinese family was investigated. Genetic testing was performed for the causative gene, and a splice site variant was identified. In vivo and in vitro experiments were conducted separately. Western blotting and immunofluorescence were performed after transient transfection of cells with the wild-type (WT) or mutated plasmid. The developmental expression pattern of zebrafish spasts was assessed via whole-mount in situ hybridization. The designed guide RNA (gRNA) and an antisense oligo spast-MO were microinjected into Tg(hb9:GFP) zebrafish embryos, spinal cord motor neurons were observed, and a swimming behavioral analysis was conducted.ResultsA novel heterozygous intron variant, c.1004 + 5G > A, was identified in a pure HSP-SPAST pedigree and shown to cosegregate with the disease phenotypes. This intron splice site variant skipped exon 6, causing a frameshift mutation that resulted in a premature termination codon. In vitro, the truncated protein was evenly distributed throughout the cytoplasm, formed filamentous accumulations around the nucleus, and colocalized with microtubules. Truncated proteins diffusing in the cytoplasm appeared denser. No abnormal microtubule structures were observed, and the expression levels of α-tubulin remained unchanged. In vivo, zebrafish larvae with this mutation displayed axon pathfinding defects, impaired outgrowth, and axon loss. Furthermore, spast-MO larvae exhibited unusual behavioral preferences and increased acceleration.ConclusionThe adverse effects of premature stop codon mutations in SPAST result in insufficient levels of functional protein, and the potential toxicity arising from the intracellular accumulation of spastin serves as a contributing factor to HSP-SPAST.

Tyrp1 is the mendelian determinant of the Axolotl (Ambystoma mexicanum) copper mutant

Several dozen Mendelian mutants have been discovered in axolotl (Ambystoma mexicanum) populations, including several that affect pigmentation. Four recessive mutants have been described in the scientific literature and genes for three of these have been identified. Here we describe and genetically dissect copper, a mutant with an albino-like phenotype known only from the pet trade. We performed a cross segregating copper and wildtype color phenotypes and used bulked segregant RNA-Seq to identify a region on chromosome 6 that was enriched for single-nucleotide polymorphisms (SNPs) between the color phenotypes. This region included Tyrosinase-like Protein 1 (Tyrp1), a melanin synthesis protein that when mutated, is associated with lighter than black melanin coloration in animal models and oculocutaneous albinism in humans. Inspection of RNA-Seq reads identified a single nucleotide deletion that is predicted to change the coding frame, introduce a premature stop codon in exon 6 and yield a truncated Tyrp1 protein in copper individuals. Using CRISPR-Cas9 editing, we show that wildtype Tyrp1 crispants exhibit copper pigmentation, thus confirming Tyrp1 as the copper locus. Our results suggest that commercial and hobbyist axolotl populations may harbor useful mutants for biological research.

The evolution and functional significance of the programmed ribosomal frameshift in prfB.

Release Factor 2 (RF2) is one of two peptide release factors that terminate translation in bacteria. In Escherichia coli, the gene encoding RF2, prfB, contains an in-frame premature RF2-specific stop codon. Therefore, a programmed ribosomal frameshift is required to translate full-length RF2. Here, we investigate the diversity of prfB frameshifting through bioinformatic analyses of >12,000 genomes. We present evidence that prfB frameshifting autoregulates RF2 levels throughout the bacterial domain since (i) the prfB in-frame stop codon is always TGA or TAA, both of which are recognized by RF2, and never the RF1-specific TAG stop codon, and (ii) species that lack the autoregulatory programmed frameshift likely need higher RF2 levels since, on average, they have significantly higher RF2-specific stop codon usage. Overexpression of prfB without the autoregulatory frameshift motif is toxic to Bacillus subtilis, an organism with intermediate RF2-specific stop codon usage. We did not detect the programmed frameshift in any Actinobacteriota. Consistent with this finding, we observed very low frameshift efficiency at the prfB frameshift motif in the Actinobacterium Mycobacterium smegmatis. Our work provides a more complete picture of the evolution of the RF2 programmed frameshifting motif, and its usage to prevent toxic overexpression of RF2.

Amphiphilic shuttle peptide delivers base editor ribonucleoprotein to correct the CFTR R553X mutation in well-differentiated airway epithelial cells.

Base editing could correct nonsense mutations that cause cystic fibrosis (CF), but clinical development is limited by the lack of delivery methods that efficiently breach the barriers presented by airway epithelia. Here, we present a novel amphiphilic shuttle peptide based on the previously reported S10 peptide that substantially improved base editor ribonucleoprotein (RNP) delivery. Studies of the S10 secondary structure revealed that the alpha-helix formed by the endosomal leakage domain (ELD), but not the cell penetrating peptide (CPP), was functionally important for delivery. By isolating and extending the ELD, we created a novel shuttle peptide, termed S237. While S237 achieved lower delivery of green fluorescent protein, it outperformed S10 at Cas9 RNP delivery to cultured human airway epithelial cells and to pig airway epithelia in vivo, possibly due to its lower net charge. In well-differentiated primary human airway epithelial cell cultures, S237 achieved a 4.6-fold increase in base editor RNP delivery, correcting up to 9.4% of the cystic fibrosis transmembrane conductance regulator (CFTR) R553X allele and restoring CFTR channel function close to non-CF levels. These findings deepen the understanding of peptide-mediated delivery and offer a translational approach for base editor RNP delivery for CF airway disease.

Primary adrenal insufficiency and hypogonadotropic hypogonadism associated with NR0B1 (DAX1) gene nonsense mutation (p.Gln155*) in a patient with empty sellae syndrome.

Primary adrenal insufficiency and hypogonadotropic hypogonadism associated with NR0B1 (DAX1) gene nonsense mutation (p.Gln155*) in a patient with empty sellae syndrome.

Microphthalmia and disrupted retinal development due to a LacZ knock-in/knock-out allele at the Vsx2 locus.

Visual System Homeobox 2 (Vsx2) is a transcription factor expressed in the developing retina that regulates tissue identity, growth, and fate determination. Several mutations in the Vsx2 gene exist in mice, including a spontaneous nonsense mutation and two targeted missense mutations originally identified in humans. Here, we expand the genetic repertoire to include a LacZ reporter allele (Vsx2 LacZ ) designed to express beta-Galactosidase (b-GAL) and simultaneously disrupt Vsx2 function (knock-in/knock-out). The retinal expression pattern of b-GAL is concordant with VSX2, and the mutant allele is recessive. Vsx2 LacZ homozygous mice have congenital bilateral microphthalmia accompanied by defects in retinal development including ectopic expression of non-retinal genes, reduced proliferation, delayed neurogenesis, aberrant tissue morphology, and an absence of bipolar interneurons - all hallmarks of Vsx2 loss-of-function. Unexpectedly, the mutant VSX2 protein is stably expressed, and there are subtle differences in eye size and early retinal neurogenesis when compared to the null mutant, ocular retardation J. The perdurance of the mutant VSX2 protein combined with subtle deviations from the null phenotype leaves open the possibility that Vsx2 LacZ allele is not a complete knock-out. The Vsx2 LacZ allele exhibits loss-of-function characteristics and adds to the genetic toolkit for understanding Vsx2 function.

Sanger Sequencing Reveals Novel Variants in GLO-1, ACE, and CBR1 Genes in Patients of Early and Severe Diabetic Nephropathy.

Background and Objectives: Diabetes is a global health issue, with approximately 50% of patients developing diabetic nephropathy (DN) and 25% experiencing early and severe forms of the disease. The genetic factors contributing to rapid disease progression in a subset of these patients are unclear. This study investigates genetic variations in the GLO-1, CBR-1, and ACE genes associated with early and severe DN. Materials and Methods: Sanger DNA sequencing of the exons of CBR1, GLO1, and ACE genes was conducted in 113 patients with early and severe DN (defined as occurring within 10 years of the diagnosis of diabetes and with eGFR < 45 mL/min/1.73 m2) and 100 controls. The impact of identified genetic variations was analyzed using computational protein models created in silico with SWISS-Model and SWISS-Dock for ligand binding interactions. Results: In GLO1, two heterozygous missense mutations, c.102G>T and c.147C>G, and one heterozygous nonsense mutation, c.148G>T, were identified in patients. The SNP rs1049346 (G>A) at location 6:38703061 (GRCh38) was clinically significant. The c.147C>G mutation (C19S) was associated with ligand binding disruption in the GLO1 protein, while the nonsense mutation resulted in a truncated, non-functional protein. In CBR1, two heterozygous variations, one missense c.358G>A, and one silent mutation c.311G>C were observed, with the former (D120N) affecting the active site. No significant changes were noted in ACE gene variants concerning protein structure or function. Conclusions: The study identifies four novel and five recurrent mutations/polymorphisms in GLO1, ACE, and CBR1 genes associated with severe DN in Pakistani patients. Notably, a nonsense mutation in GLO1 led to a truncated, non-functional protein, while missense mutations in GLO1 and CBR1 potentially disrupt enzyme function, possibly accelerating DN progression.

Positive experience in treating patients with Duchenne muscular dystrophy caused by a nonsense mutation: family clinical case

Duchenne muscular dystrophy is a severe genetic disease caused by mutations in the dystrophin gene (DMD), which lead to a significant decrease or complete absence of the protein of the same name in muscle fibers. The disease manifests itself in boys from the age of 2–5 years in the form of progressive muscular weakness, cardiomyopathy and respiratory failure. As a result of progression, patients lose the ability to move by adolescence and die in the third decade from cardiopulmonary complications. However, timely pathogenetic therapy for Duchenne muscular dystrophy can prolong the life of patients and improve its quality. In this regard, early diagnosis of the disease and early initiation of pathogenetic therapy are very important.The article describes two clinical cases of the disease in siblings with onset at 2 years of age and different ages of diagnosis. Pathogenetic therapy in the first case made it possible to prolong the patient’s outpatient stay and improve the quality of life, and early diagnosis and initiation of pathogenetic therapy in the second case avoided disease progression and contributed to the development of new motor skills. The presented clinical case demonstrates the need for early diagnosis of the disease even before the onset of clinical manifestations.