Premature Termination Codon Mutations Research Articles

Readthrough-induced misincorporated amino acid ratios guide mutant-specific therapeutic approaches for two CFTR nonsense mutations.

Cystic fibrosis (CF) is a monogenic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Premature termination codons (PTCs) represent ∼9% of CF mutations that typically cause severe expression defects of the CFTR anion channel. Despite the prevalence of PTCs as the underlying cause of genetic diseases, understanding the therapeutic susceptibilities of their molecular defects, both at the transcript and protein levels remains partially elucidated. Given that the molecular pathologies depend on the PTC positions in CF, multiple pharmacological interventions are required to suppress the accelerated nonsense-mediated mRNA decay (NMD), to correct the CFTR conformational defect caused by misincorporated amino acids, and to enhance the inefficient stop codon readthrough. The G418-induced readthrough outcome was previously investigated only in reporter models that mimic the impact of the local sequence context on PTC mutations in CFTR. To identify the misincorporated amino acids and their ratios for PTCs in the context of full-length CFTR readthrough, we developed an affinity purification (AP)-tandem mass spectrometry (AP-MS/MS) pipeline. We confirmed the incorporation of Cys, Arg, and Trp residues at the UGA stop codons of G542X, R1162X, and S1196X in CFTR. Notably, we observed that the Cys and Arg incorporation was favored over that of Trp into these CFTR PTCs, suggesting that the transcript sequence beyond the proximity of PTCs and/or other factors can impact the amino acid incorporation and full-length CFTR functional expression. Additionally, establishing the misincorporated amino acid ratios in the readthrough CFTR PTCs aided in maximizing the functional rescue efficiency of PTCs by optimizing CFTR modulator combinations. Collectively, our findings contribute to the understanding of molecular defects underlying various CFTR nonsense mutations and provide a foundation to refine mutation-dependent therapeutic strategies for various CF-causing nonsense mutations.

Induction of Translational Readthrough on Protein Tyrosine Phosphatases Targeted by Premature Termination Codon Mutations in Human Disease.

Nonsense mutations generating premature termination codons (PTCs) in various genes are frequently associated with somatic cancer and hereditary human diseases since PTCs commonly generate truncated proteins with defective or altered function. Induced translational readthrough during protein biosynthesis facilitates the incorporation of an amino acid at the position of a PTC, allowing the synthesis of a complete protein. This may evade the pathological effect of the PTC mutation and provide new therapeutic opportunities. Several protein tyrosine phosphatases (PTPs) genes are targeted by PTC in human disease, the tumor suppressor PTEN being the more prominent paradigm. Here, using PTEN and laforin as examples, two PTPs from the dual-specificity phosphatase subfamily, we describe methodologies to analyze in silico the distribution and frequency of pathogenic PTC in PTP genes. We also summarize laboratory protocols and technical notes to study the induced translational readthrough reconstitution of the synthesis of PTP targeted by PTC in association with disease in cellular models.

CSF biomarker analysis of ABCA7 mutation carriers suggests altered APP processing and reduced inflammatory response

BackgroundThe Alzheimer’s disease (AD) risk gene ABCA7 has suggested functions in lipid metabolism and the immune system. Rare premature termination codon (PTC) mutations and an expansion of a variable number of tandem repeats (VNTR) polymorphism in the gene, both likely cause a lower ABCA7 expression and hereby increased risk for AD. However, the exact mechanism of action remains unclear. By studying CSF biomarkers reflecting different types of AD-related pathological processes, we aim to get a better insight in those processes and establish a biomarker profile of mutation carriers.MethodsThe study population consisted of 229 AD patients for whom CSF was available and ABCA7 sequencing and VNTR genotyping had been performed. This included 28 PTC mutation and 16 pathogenic expansion carriers. CSF levels of Aβ1–42, Aβ1–40, P-tau181, T-tau, sAPPα, sAPPβ, YKL-40, and hFABP were determined using ELISA and Meso Scale Discovery assays. We compared differences in levels of these biomarkers and the Aβ ratio between AD patients with or without an ABCA7 PTC mutation or expansion using linear regression on INT-transformed data with APOE-status, age and sex as covariates.ResultsCarriers of ABCA7 expansion mutations had significantly lower Aβ1–42 levels (P = 0.022) compared with non-carrier patients. The effect of the presence of ABCA7 mutations on CSF levels was especially pronounced in APOE ε4-negative carriers. In addition, VNTR expansion carriers had reduced Aβ1–40 (P = 0.023), sAPPα (P = 0.047), sAPPβ (P = 0.016), and YKL-40 (P = 0.0036) levels.ConclusionsOur results are suggestive for an effect on APP processing by repeat expansions given the changes in the amyloid-related CSF biomarkers that were found in carriers. The decrease in YKL-40 levels in expansion carriers moreover suggests that these patients potentially have a reduced inflammatory response to AD damage. Moreover, our findings suggest the existence of a mechanism, independent of lowered expression, affecting neuropathology in expansion carriers.

249 Pharmacological rescue of CFTR premature termination codon mutations by novel small molecules

249 Pharmacological rescue of CFTR premature termination codon mutations by novel small molecules

Myotonic Dystrophy Type 1 - An Atypical Presentation Having Symmetric Parkinsonism and Early Proximal Muscle Involvement.

Myotonic Dystrophy Type 1 - An Atypical Presentation Having Symmetric Parkinsonism and Early Proximal Muscle Involvement.

Genetic compensation response could exist in colorectal cancer: UPF3A upregulates the oncogenic homologue gene SRSF3 expression corresponding to SRSF6 to promote colorectal cancer metastasis.

Genetic compensation response (GCR) is a mechanism that maintains the robustness of functional genes, which has been recently identified. Whether GCR exists in tumors and its effects on tumor progression remains unknown. Whole exome sequencing was performed to identify premature termination codon (PTC) gene mutations in colorectal cancer (CRC) tissues. RNA sequencing, Cancer Cell Line Encyclopedia database analysis, and high-throughput output of homologous genes using the Ensemble genome database were performed to further identify homologous genes of target PTC gene mutations. Serine and arginine-rich splicing factor 3 (SRSF3) increased the invasion ability in CRC cells and could be the target gene of up-frameshift 3A (UPF3A). The deletion of the 660th base A in the coding sequence region of SRSF6 caused a frameshift mutation of serine at position 220 (s220fs), which contributed to a PTC UAA termination of translation in HCT116 cells. We further found that SRSF3 was the only homologue of SRSF6 with a frameshift mutation. The transfection of s220fs of SRSF6 into HCT116 cells led to upregulation of its corresponding oncogenic homologue gene SRSF3 expression to promote CRC metastasis. SRSF3 was highly expressed in CRC liver metastases and was positively correlated with UPF3A expression and contributed to poor prognosis. GCR may exist in CRC and exert effects on the progression of CRC. Targeted inhibition of UPF3A could reduce the GCR effects and suppress the expression of oncogenic homologue genes corresponding to PTC mutations, indicating a novel therapeutic strategy for treatment of CRC metastasis.

Novel read through agent: ZKN-0013 demonstrates efficacy in APCmin model of familial adenomatous polyposis.

Familial adenomatous polyposis (FAP) is a precancerous, colorectal disease characterized by hundreds to thousands of adenomatous polyps caused by mutations in the tumor suppressor gene adenomatous polyposis coli (APC). Approximately 30% of these mutations are premature termination codons (PTC), resulting in the production of a truncated, dysfunctional APC protein. Consequently, the β-catenin degradation complex fails to form in the cytoplasm, leading to elevated nuclear levels of β-catenin and unregulated β-catenin/wnt-pathway signaling. We present in vitro and in vivo data demonstrating that the novel macrolide, ZKN-0013, promotes read through of premature stop codons, leading to functional restoration of full-length APC protein. Human colorectal carcinoma SW403 and SW1417 cells harboring PTC mutations in the APC gene showed reduced levels of nuclear β-catenin and c-myc upon treatment with ZKN-0013, indicating that the macrolide-mediated read through of premature stop codons produced bioactive APC protein and inhibited the β-catenin/wnt-pathway. In a mouse model of adenomatous polyposis coli, treatment of APCmin mice with ZKN-0013 caused a significant decrease in intestinal polyps, adenomas, and associated anemia, resulting in increased survival. Immunohistochemistry revealed decreased nuclear β-catenin staining in the epithelial cells of the polyps in ZKN-0013-treated APCmin mice, confirming the impact on the β-catenin/wnt-pathway. These results indicate that ZKN-0013 may have therapeutic potential for the treatment of FAP caused by nonsense mutations in the APC gene. KEY MESSAGES: • ZKN-0013 inhibited the growth of human colon carcinoma cells with APC nonsense mutations. • ZKN-0013 promoted read through of premature stop codons in the APC gene. • In APCmin mice, ZKN-0013 treatment reduced intestinal polyps and their progression to adenomas. • ZKN-0013 treatment in APCmin mice resulted in reduced anemia and increased survival.

STR-based feature extraction and selection for genetic feature discovery in neurological disease genes

Gene expression, often determined by single nucleotide polymorphisms, short repeated sequences known as short tandem repeats (STRs), structural variants, and environmental factors, provides means for an organism to produce gene products necessary to live. Variation in expression levels, sometimes known as enrichment patterns, has been associated with disease progression. Thus, the STR enrichment patterns have recently gained interest as potential genetic markers for disease progression. However, to the best of our knowledge, we are unaware of any study that evaluates and explores STRs, particularly trinucleotide sequences, as machine learning features for classifying neurological disease genes for the purpose of discovering genetic features. Thus, in this paper, we proposed a new metric and a novel feature extraction and selection algorithm based on statistically significant STR-based features and their respective enrichment patterns to create a statistically significant feature set. The proposed new metric has shown that the neurological disease family genes have a non-random AA, AT, TA, TG, and TT enrichment pattern. This is an important result, as it supports prior research that has established that certain trinucleotides, such as AAT, ATA, ATT, TAT, and TTA, are favored during protein misfolding. In contrast, trinucleotides, such as TAA, TAG, and TGA, are favored during premature termination codon mutations as they are stop codons. This suggests that the metric has the potential to identify patterns that may be genetic features in a sample of neurological genes. Moreover, the practical performance and high prediction results of the statistically significant STR-based feature set indicate that variations in STR enrichment patterns can distinguish neurological disease genes. In conclusion, the proposed approach may have the potential to discover differential genetic features for other diseases.

Homology-directed repair of an MYBPC3 gene mutation in a rat model of hypertrophic cardiomyopathy.

Variants in myosin-binding protein C3 (MYBPC3) gene are a main cause of hypertrophic cardiomyopathy (HCM), accounting for 30% to 40% of the total number of HCM mutations. Gene editing represents a potential permanent cure for HCM. The aim of this study was to investigate whether genome editing of MYBPC3 using the CRISPR/Cas9 system in vivo could rescue the phenotype of rats with HCM. We generated a rat model of HCM ("1098hom") that carried an Mybpc3 premature termination codon mutation (p.W1098x) discovered in a human HCM pedigree. On postnatal day 3, the CRISPR/Cas9 system was introduced into rat pups by a single dose of AAV9 particles to correct the variant using homology-directed repair (HDR). Analysis was performed 6 months after AAV9 injection. The 1098hom rats didn't express MYBPC3 protein and developed an HCM phenotype with increased ventricular wall thickness and diminished cardiac function. Importantly, CRISPR HDR genome editing corrected 3.56% of total mutations, restored MYBPC3 protein expression by 2.12%, and normalized the HCM phenotype of 1098hom rats. Our work demonstrates that the HDR strategy is a promising approach for treating HCM associated with MYBPC3 mutation, and that CRISPR technology has great potential for treating hereditary heart diseases.

ABCA7‐reducing mutation carriers have reduced CSF amyloid biomarker levels, suggestive of increased amyloid deposition

AbstractBackgroundThe Alzheimer’s disease (AD) risk gene ABCA7 has suggested functions in lipid metabolism and the immune system. Rare premature termination codon (PTC) mutations and an expansion of a variable number of tandem repeats (VNTR) polymorphism in the gene, both likely cause a lower ABCA7 expression and hereby increased risk for AD. However, the exact mechanism‐of‐action remains unclear. By studying CSF biomarkers reflecting different types of AD‐related pathological processes, we aim to get a better insight in those processes and establish a biomarker profile of mutation carriers.MethodThe study population consisted of 228 AD patients (27 PTC mutation and 16 pathogenic expansion carriers) for whom CSF was available and ABCA7 sequencing and VNTR genotyping had been performed. CSF levels of amyloid β (Aβ) 40 and 42, sAPP‐α and ‐β, YKL‐40 and hFABP were determined using ELISA and Meso Scale Discovery assays. We compared differences in levels of these biomarkers AD patients with or without an ABCA7 PTC mutation or expansion, and further stratified on APOE e4 status.ResultCarriers of ABCA7 mutations/expansions had significantly lower Aβ42 levels (p= 0.0031) and amyloid ratio (p=0.020) compared to non‐carrier patients, and were threefold more likely to have abnormal Aβ42 values. The effect on amyloid biomarkers remained significant when considering PTC mutations or VNTR expansions separately. APOE e4 carriers had significantly lower Aβ42 (p=0.0013) and amyloid ratio (p<0.001) compared to non‐APOE e4 carriers. When combined, both APOE e4 allele and ABCA7 carrier status influenced amyloid ratio and Aβ42 levels. Remarkably, within non‐APOE e4 carriers, ABCA7 mutation/expansion carriers did have significantly reduced Aβ42 levels (mean=540 pg/mL) compared to ABCA7 non‐carriers (812 pg/mL; p=0.0051), attaining lower levels as e4 carriers (612 pg/mL). Finally, in carriers of the pathogenic expansion we observed lower YKL40 levels (p=0.011), suggesting reduced neuroinflammation.ConclusionOur results highlight that carriers of ABCA7 PTC mutations and repeat expansions show more pronounced abnormalities in CSF amyloid beta markers than non‐carrier patients, especially in non‐APOE e4 carriers. Expansion carriers also had a decrease in neuroinflammation marker YKL40. Our results suggest that reduced ABCA7 expression leads to increase in amyloid pathology and potentially a reduced inflammatory response to damage.

Investigating the use of translational readthrough inducing drugs in keratocytes from a mouse model of brittle cornea syndrome

AbstractPurpose: Brittle Cornea Syndrome (BCS) is a rare recessive condition characterized by extreme thinning of the cornea. The collagen‐rich corneal stroma accounts for 90% of corneal thickness in humans, and is a major determinant of visual acuity. BCS results from homozygous loss‐of‐function mutations in ZNF469 (BCS1) or PRDM5 (BCS2). Our mouse model of BCS1, caused by a premature termination codon (PTC) mutation in Zfp469, showed that the stroma is thinner due to of decreased type 1 collagen expression1. To elucidate disease mechanisms and test potential therapies, we established a primary keratocyte disease‐in‐a‐dish model of BCS1. Translational readthrough inducing drugs (TRIDs) can restore full‐length proteins truncated by PTC and are a potential treatment for 20% of BCS patients with PTC mutations. Primary keratocytes from our BCS1 mouse model were treated with TRIDs to assess their efficacy in improving the BCS phenotype in vitro.Methods: Zfp469BCS1/BCS1 keratocytes were treated with TRIDs PTC124 (ataluren), amlexanox, gentamicin, geneticin or vehicle‐only, for 2 or 5 days. Following treatment, Zfp469 and Col1a1 mRNA levels were evaluated by RT‐qPCR. Changes to keratocyte cell‐derived matrices were assessed using immunostaining or the collagen dye CNA35.Results: Zfp469BCS1/BCS1 keratocytes express 40% more Zfp469 mRNA than wildtype cells; this was unchanged by treatment with amlexanox, geneticin or gentamicin. PTC124 decreased expression of Zfp469 by 50%. Expression of Col1a1, a key component of the stromal matrix, was significantly decreased by PTC124 treatment (<65% relative to vehicle‐only Zfp469BCS1/BCS1 keratocytes). Collagen deposition, visualized using CNA35 dyes, was decreased by PTC124 treatment in BCS and wildtype keratocytes.Conclusions: BCS is a devastating condition. Extreme thinning of the corneal stroma, as a result of impaired collagen production by keratocytes, makes the cornea prone to rupture. This causes irreversible sight loss. There is currently no treatment for BCS. Our initial work suggests that TRIDs may not restore functional full‐length Zfp469 in primary keratocytes and are unlikely to be of therapeutic benefit in BCS.Reference1. Stanton CM et al. A mouse model of brittle cornea syndrome caused by mutation in Zfp469. Dis Model Mech. 2021;14(9):dmm049175.

Belgian Carriers of Rare ABCA7 Mutations Present with Pronounced Cerebral Amyloid Angiopathy and Alzheimer’s Disease

AbstractBackground ABCA7 is a major risk gene for Alzheimer’s disease (AD) and rare premature termination codon (PTC) and missense mutations are enriched in AD patients. In preliminary studies we obtained that ABCA7 PTC and missense mutation carriers present with a classic AD phenotype, but severe levels of cerebral amyloid angiopathy (CAA) were also found (table 1, figures 1‐2). We aim to delineate the clinicopathological phenotype of rare ABCA7 mutation carriers in Belgian CAA patients.MethodsGenetic screening of ABCA7 in a Belgian CAA cohort (n = 83), with genotype‐phenotype comparison of the ABCA7 carriers using demographics and clinicopathology data.ResultsIn 21.4% (18/84) of CAA patients a rare ABCA7 mutation was identified. Seven patients carried a PTC mutation, 10 a rare missense variant, and in one CAA patient we found a deletion. The mean onset age was 66.4±12.8 (range 47‐84) years, while the mean age at death was 68.0±10.3 (range 48‐92) years. In 87.5% (7/8) of patients a positive family history of disease was mentioned. Cognitive decline was present in 52.9% (9/17). Nine patients were diagnosed as probable CAA, and four as possible CAA. In six patients postmortem examination showed moderate‐to‐severe levels of CAA in all but one patient (83.3%, 5/6) and AD neuropathological hallmarks (100%, 6/6). Extensive levels of CAA were present in both the meningeal and capillary blood vessels, and moderate to high levels of CAA in the parenchymal blood vessels (Figure 3).ConclusionsOur data suggests that rare ABCA7 mutations are frequently present in CAA patients. Carriers show signs of severe levels of CAA, as well as AD neuropathological hallmarks. The findings of this study have important implications for future research and clinical practice.

Genotype and phenotype correlations in 441 patients with epidermolysis bullosa from China.

Epidermolysis bullosa (EB) is a heterogeneous group of rare and incurable genetic blistering disorders. The objective was to analyse the genotype-phenotype correlation in EB among Chinese individuals. Next-generation sequencing and Sanger sequencing were performed to genetically confirm clinically diagnosed EB. Reverse transcription-PCR and splice-site analysis were used to evaluate the consequences of splicing mutations. A total of 441 cases (413 families) across 11 genes were included. EB simplex (EBS), junctional EB (JEB), dystrophic EB (DEB), Kindler EB, simplex and junctional compound EB accounted for 23.4%, 12.7%, 61.5%, 1.1% and 0.2%, respectively. In 16 probands with presumptive recessive EB, failed to find the second allele, COL7A1 (10), COL17A1 (4), LAMB3 (1) and ITGB4 (1). De novo mutations are common in dominant EB (63.8% in EBS, 27.5% in DEB) but extremely rare in recessive DEB (RDEB; 0.74%). Mosaicism is more common than presumed, with 5.4% of dominant EBS. In JEB, only 45.0% of patients with biallelic premature termination codon (PTC) mutations in laminin 332 genes died within 24 months, with a longer average survival age of 11.1months. In JEB, unusual phenotypes are frequently observed, notably urinary tract involvement, duodenal atresia and EB nevi. In RDEB, 48.8% of cases with biallelic PTC mutations in COL7A1 exhibited a relatively mild phenotype; they are likely to develop a severe phenotype at 0-4 years old, and the PTC mutations position closer to the N-terminal, leading to earlier onset. Glycine substitution mutations in DEB have complex genotypic and phenotypic heterogeneity. The rare subtype, dominant and recessive compound DEB, consists of 1.8% of the total DEB. This study reveals the general rules governing genotype-phenotype correlations, rare phenotypes and complex genotypes. Collectively, mutation analysis in different forms of EB provides the basis for improved subclassification with accurate genetic counselling and for prenatal diagnosis.

454 Genotype-phenotype associations in recessive dystrophic epidermolysis bullosa (RDEB)

RDEB is a rare bullous genodermatosis caused by mutations in COL7A1. Clinical features range from severe wounds to esophageal strictures and anemia. Prior work has identified associations of biallelic COL7A1 premature termination codon (PTC) mutations with more severe disease, likely from absent or severely truncated type VII collagen (C7), but genotype-phenotype associations for other mutations including splice site (SP) or missense (MS) mutations remain unexplored. We analyzed data from 83 RDEB patients for clinical characteristics and functional genotypes as defined by a mutation’s impact on C7 function using available literature and in silico predictions, classifying genotypes by degree of deleterious effect on C7 function as “severe” (PTC/PTC, n=40; PTC/SP, n=12), “moderate” (PTC/MS, n=20; SP/SP, n=2) and “mild” (SP/MS, n=4; MS/MS, n=5).

Pharmacological Responses of the G542X-CFTR to CFTR Modulators.

Cystic fibrosis (CF) is a lethal hereditary disease caused by loss-of-function mutations of the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR). With the development of small-molecule CFTR modulators, including correctors that facilitate protein folding and expression and potentiators that promote channel activity, about 90% of the CF patients are now receiving efficacious target therapies. G542X-CFTR, a premature termination codon (PTC) mutation, is the most common disease-associated mutation found in the remaining 10% of patients that await effective drugs to rectify the fundamental defects caused by PTC. In this study, we employed biophysical and biochemical techniques to characterize the pharmacological responses of the translational products of G542X-CFTR to a range of new CFTR modulators. Specifically, we identified two different proteins translated from the G542X-CFTR cDNA using western blotting: the C-terminus truncated protein that responds to the C1 corrector which binds to the N-terminal part of the protein and a full-length CFTR protein through the read-through process. Electrophysiological data suggest that the read-through protein, but not the C-terminus truncated one, is functional and responds well to CFTR potentiators despite a lower open probability compared to wild-type CFTR. As the expression of the read-through products can be increased synergistically with the read-through reagent G418 and C1 corrector, but not with combinations of different types of correctors, we concluded that an efficacious read-through reagent is a prerequisite for mitigating the deficits of G542X-CFTR. Moreover, the CFTR potentiators may help improve the effectiveness of future combinational therapy for patients carrying PTCs such as G542X.

Rare missense mutations in ABCA7 might increase Alzheimer\u2019s disease risk by plasma membrane exclusion

The adenosine triphosphate–binding cassette subfamily A member 7 gene (ABCA7) is associated with Alzheimer’s disease (AD) in large genome-wide association studies. Targeted sequencing of ABCA7 suggests a role for rare premature termination codon (PTC) mutations in AD, with haploinsufficiency through nonsense-mediated mRNA decay as a plausible pathogenic mechanism. Since other classes of rare variants in ABCA7 are poorly understood, we investigated the contribution and pathogenicity of rare missense, indel and splice variants in ABCA7 in Belgian AD patient and control cohorts. We identified 8.36% rare variants in the patient cohort versus 6.05% in the control cohort. For 10 missense mutations identified in the Belgian cohort we analyzed the pathogenetic effect on protein localization in vitro using immunocytochemistry. Our results demonstrate that rare ABCA7 missense mutations can contribute to AD by inducing protein mislocalization, resulting in a lack of functional protein at the plasma membrane. In one pedigree, a mislocalization-inducing missense mutation in ABCA7 (p.G1820S) co-segregated with AD in an autosomal dominant inheritance pattern. Brain autopsy of six patient missense mutation carriers showed typical AD neuropathological characteristics including cerebral amyloid angiopathy type 1. Also, among the rare ABCA7 missense mutations, we observed mutations that affect amino acid residues that are conserved in ABCA1 and ABCA4, of which some correspond to established ABCA1 or ABCA4 disease-causing mutations involved in Tangier or Stargardt disease.

Topical Gentamicin 0.1% Promotes Collagen 7 Expression in Recessive Dystrophic Epidermolysis Bullosa.

Background:Currently, there is no cure for epidermolysis bullosa (EB) but few studies have explored the role of aminoglycosides in promoting collagen 7 expression in recessive dystrophic EB (RDEB).Materials and Methods:Consecutive patients aged >1 year with a confirmed diagnosis of dystrophic EB (DEB) were advised to apply 0.1% w/w gentamicin cream in a collagen base (Derbriment G™) twice daily on a representative area on right lower limb (RLL) and paraffin gauze dressings on the corresponding opposite side on the left lower limb (LLL). Skin lesions were evaluated clinically during the 12-week treatment period at the end of which a repeat skin biopsy was sent for immunofluorescence antigen mapping (IFM).Results:Twelve patients with DEB were recruited but only eight completed the study and were analyzed. The mean fluorescence intensity (MFI) of the study cohort increased from 2765 ± 1732.07 (263–4845) at baseline to 5412.75 ± 3937.64 (2100–13536) at 12 weeks; a 95.75% (range 5.34%–775.14%) increase in the MFI of collagen 7 from baseline (P = 0.06). Among patients with a known termination codon mutation (n = 3), the percentage increase in MFI was greater among patients with known premature termination codon (PTC) mutations compared to those with unknown mutations. The clinical severity did not change significantly in terms of the mean number of blisters, erosions, and scarring during the study period. None of the parents reported any adverse effect.Conclusions:Topical gentamicin 0.1% w/w is a safe and effective way to promote the expression of COL7A1 in DEB patients, especially those carrying PTC mutations.

Two Novel Mutations in LAMC2 Gene in Iranian Families Affected by Junctional Epidermolysis Bullosa.

Junctional epidermolysis bullosa (JEB) is an autosomal recessive skin disorder with defective adhesion of dermal- epidermal within the lamina lucida region of the basement membrane zone. The main characterization of JEB is blistering and fragile skin and mucous membrane. Laminins are noncollagenous part of basement membrane and classified as a family of extracellular matrix glycoprotein. Laminins contain three chains: Laminin α, Laminin β and Laminin γ. LAMC2 (laminin subunit gamma 2) gene encodes γ subunit of laminin and its mutation contributes to JEB. Here, we report a disease-causing nonsense mutation and a large deletion mutation in LAMC2 gene in two families affected by JEB. Whole exome sequencing (WES) was carried out on the mother of patient in family I and the patient himself in family II to detect the underlying mutations. Then, sanger sequencing was performed to confirm the identified mutations. Next generation sequencing (NGS) data analysis of the first family showed a novel, nonsense mutation in LAMC2 gene (LAMC2: NM_005562: exon14:c.C2143T: p.R715X). The heterozygous state of the mutation was confirmed by sanger sequencing in the parents and unaffected brother. In Family II, NGS data had no coverage in the large area of LAMC2 gene. Thus, to confirm the possible deletion sanger sequencing was done and blasting of sequence showed the deleted region of 9.4 kb (exon10-17) in LAMC2 gene. In summary, current study reported a novel disease-causing premature termination codon (PTC) mutation in LAMC2 gene and a large deletion mutation in patients affected by JEB.

Rare missense mutations and compound heterozygous mutations in ABCA7 contribute to Alzheimer's disease in Belgian patients

Initially, ABCA7 was associated with Alzheimer's disease (AD) in large genome-wide association studies. Targeted resequencing of ABCA7 suggested a role for rare premature termination codon (PTC) mutations. We observed loss of ABCA7 in PTC carriers, although with high variability due to differences in transcript rescue. Additionally, rare missense mutations are present in ABCA7 with unknown effect on the protein. We aimed to investigate the contribution of rare (MAF≤1%) mutations or compound heterozygous mutations in ABCA7 in Belgian AD patient and control cohorts, as well as the effect of rare missense mutations on the subcellular localization of the protein. Targeted resequencing or whole exome sequencing of ABCA7 exons and splice sites in 1375 Belgian AD patients and 976 controls. We validated all rare, non-synonymous coding and splice mutations. Allele-specific PCR, haplotype sharing analysis and long-read sequencing to determine cis/trans configuration of compound heterozygous mutations. In vitro mutagenesis to introduce missense mutations of interest in an entry vector. Gateway cloning to generate wild type and mutant ABCA7 pCR3 expression vectors with a C-terminal EmGFP-tag. Transfection of HeLa and HEK293 cells for immunofluorescence microscopy to localize wild type versus mutant ABCA7. We identified rare mutations in 12.7% (175/1375) of the patients and in 7.8% (76/976) of the controls. We observed co-segregation of p.G1820S with AD in a family with apparent autosomal dominant inheritance. We observed compound heterozygous mutations in 10 patients (0.73%) and five controls (0.51%). We confirmed trans configuration in four of the patients and cis configuration in four other patients. In the remaining two patients, configuration remains unknown. In the five controls, we confirmed trans configuration in one and cis configuration in the other four. We selected eight missense mutations present in Belgian AD patients (Figure) to introduce in an entry vector. Mutant and wild type entry clones were subcloned in the expression vector to transfect and generate immunofluorescence localization data. We observed an enrichment in patients of rare single mutations and compound heterozygous mutations. In addition, trans compound heterozygous mutations might modify penetrance and pathogenicity. Functional studies of missense mutations are necessary to determine their potential pathogenic effect.

Integrity and Stability of PTC Bearing CFTR mRNA and Relevance to Future Modulator Therapies in Cystic Fibrosis.

Major advances have recently been made in the development and application of CFTR (cystic fibrosis transmembrane conductance regulator) mutation class-specific modulator therapies, but to date, there are no approved modulators for Class I mutations, i.e., those introducing a premature termination codon (PTC) into the CFTR mRNA. Such mutations induce nonsense-mediated decay (NMD), a cellular quality control mechanism that reduces the quantity of PTC bearing mRNAs, presumably to avoid translation of potentially deleterious truncated CFTR proteins. The NMD-mediated reduction of PTC-CFTR mRNA molecules reduces the efficacy of one of the most promising approaches to treatment of such mutations, namely, PTC readthrough therapy, using molecules that induce the incorporation of near-cognate amino acids at the PTC codon, thereby enabling translation of a full-length protein. In this study, we measure the effect of three different PTC mutations on the abundance, integrity, and stability of respective CFTR mRNAs, using CFTR specific RT-qPCR-based assays. Altogether, our data suggest that optimized rescue of PTC mutations has to take into account (1) the different steady-state levels of the CFTR mRNA associated with each specific PTC mutation; (2) differences in abundance between the 3′ and 5′ regions of CFTR mRNA, even following PTC readthrough or NMD inhibition; and (3) variable effects on CFTR mRNA stability for each specific PTC mutation.