Auswahl der wissenschaftlichen Literatur zum Thema „Nonsense alteration“

Sixty-three cases of benign and malignant canine mammary tumors were analyzed to define the alteration of exons 5–8 for the p53 tumor suppressor gene using polymerase chain reaction direct sequence analysis with paraffin-embedded tissues. Four missense mutations were found in 38 benign mammary tumors (11%), and five missense (one tumor had two missense mutations) and one nonsense mutations were found in 25 mammary carcinomas (20%). These data suggest that the p53 gene alterations might be initiated at an early stage of canine mammary carcinogenesis and p53 mutations might be associated with malignancy. However, there was no evidence of any relationship between the p53 alterations and the histologic types of tumors or breeds of dogs.

This study aimed to clarify the genetic difference between Korean triple-negative breast cancer (TNBC) and other breast cancer (BC) subtypes. TNBC was defined as the absence of hormonal receptors and human epidermal growth factor receptor 2 (HER2) amplification. DNA panel of the Ion Torrent Oncomine Comprehensive Assay (OCA) v3 was performed to identify somatic alteration in 48 specimens. In a total of 102 alterations (37 nonsense, 35 missense, 8 frameshift and 22 amplifications), 30 nucleotide alterations (24 nonsense, 1 missense, and 5 frameshift) were newly identified. The eight most commonly altered genes were PIK3CA, TP53, ERBB2, BRCA2, FANCD2, AKT1, BRCA1, and FANCA. TNBC had significantly lower mutation frequency in PIK3CA (TNBC vs. hormone receptor-positive and HER2-negative BC [HRPBC], p = 0.009), but higher mutation frequency in TP53 (TNBC vs. HRPBC, p = 0.036; TNBC vs. hormone receptor-positive and HER2- positive BC [HHPBC], p = 0.004). TNBC showed frequently higher Ki-67 expression than any positive BC (p = 0.004) due to HRPBC (p < 0.001). TNBC with high Ki-67/unmutated PIK3CA/mutated TP53 appears at a younger age (52.2 ± 7.6 years), compared to other subtypes (63.7 ± 11.0 years). TNBC with high Ki-67/unmutated PIK3CA/mutated TP53 may be related to relatively early onset BC. These findings demonstrate the genomic heterogeneity between TNBC and other BC subtypes and could present a new approach for molecular targeted therapy in TNBC patients.

275 Background: Circulating tumor-derived DNA (ctDNA) is an accessible method for characterizing somatic alterations. We report longitudinal ctDNA screenings of mCRPC patients (pts) who have had germline testing. Methods: Patients with both germline testing and ctDNA assessment were included. Germline testing was performed with a multi-gene cancer panel from Invitae (50-83 genes) and somatic alterations in ctDNA were identified by testing with Guardant 360 (70-83 genes). ctDNA alterations were characterized as deletions, frameshift, missense, nonsense, and other mutations. A total of 177 patients in various stages of therapy had both ctDNA and germline DNA tested. Results: From 2015-2021, 177 mCRPC patients were included and had an average of 3 ctDNA tests. 11.3% (20/177) had pathogenic or likely-pathogenic germline mutations. The common pathogenic germline mutations were in BRCA2 (25%; 5/20), ATM (10%; 2/20), and MSH2 (10%; 2/20). In ctDNA, missense mutations were the most prevalent type of gene alteration in germline negative (n = 539/790, 68%) and germline positive (n = 124/218, 57%) followed by frameshift mutations at 22% (n = 48/218) in germline positive and 10% (n = 80/790) in germline negative patients. Germline positive patients were more likely to have somatic frameshift mutations (OR = 2.09, 95% C.I. (1.3792, 3.1618), p = 0.001) and less likely to have missense mutations (OR = 0.61, 95% C.I. (0.4519, 0.8351), p = 0.002). Other alterations including deletions, nonsense, and other mutations were not significantly different. Of the germline positive pts, BRCA2 mutation was associated with the highest number of somatic alterations. Conclusions: Germline positive CRPC patients were more likely to have frameshift mutations and less likely to missense mutations compared to germline negative CRPC patients. Patients with germline mutations in BRCA2 and TP53 had the highest number of somatic alterations detected in ctDNA over the course of ctDNA evaluation.

e20548 Background: SMARCA4 gene is one of the catalytic subunits of the SWI/SNF chromosomal remodeling complex, which can regulate important cellular processes and functions and is closely associated to tumors. The clinical features, therapeutic efficacy, prognosis and pathological features of lung adenocarcinoma with this genetic variation are still unknown and controversial. Methods: The study recruited 274 patients (pts) with lung adenocarcinoma whose samples were sent to perform parallel hybridization-based next-generation sequencing. Two categories of SMARCA4 mutations were divided into Type1 mutations (frameshift mutations, nonsense mutations, splice-3 mutations, copy number amplification) and Type2 mutations (missense mutations and copy number loss) based on whether the mutation may result in defective protein. Furthermore, comparative analysis by using the clinical outcome data, the genomic and pathological characteristics were be performed in SMARCA4 Type 1 alterations corhort and Type 2 alterations corhort. Results: Among 274 pts were recruited, the mutation rate of SMARCA4 gene in lung adenocarcinoma was 9.1%. Furthermore, the presence of SMARCA4 alteration was associated with smoking (P＜0.05). Missense, nonsense, frameshift and splice were the most common types of mutations (92%). The pts with SMARCA4 Type1 alterations which probably lead to defective protein expression, had a worse prognosis compared with pts with SMARCA4 Type1 alterations (The role leading to defective protein expression is uncertain) and SMARCA4 Wild groups (P＜0.05). In addition, EGFR alterations were strongly associated with SMARCA4 Wild corhort compared to SMARCA4 Type1 alterations corhort (67% vs. 31% ), and SMARCA4 Type1 alterations was more associated with the absence of TP53, RB1, and Robo3 alterations. GO enrichment analysis suggested that the differentiated mutated genes between SMARCA4 Type1 alterations corhort and SMARCA4 Wild corhort were mainly enriched in cell cycle regulation. Pathologically, The SMARCA4 Type1 alterations was mostly poorly or moderately differentiated and strongly accompanied by the loss of expression of TTF-1(83.3%) and BRG1(80%) in immunohistochemistry. Conclusions: SMARCA4 Type1 alterations which probably lead to abnormality of protein was associated with poor prognosis and having different the genomic and pathological characteristics.

Abstract In an attempt to decipher their role in the life history and senescence process of the filamentous fungus Podospora anserina, we have cloned the su1 and su2 genes, previously identified as implicated in cytosolic translation fidelity. We show that these genes are the equivalents of the SUP35 and SUP45 genes of Saccharomyces cerevisiae, which encode the cytosolic translation termination factors eRF3 and eRF1, respectively. Mutations in these genes that suppress nonsense mutations may lead to drastic mycelium morphology changes and sexual impairment but have little effect on life span. Deletion of su1, coding for the P. anserina eRF3, is lethal. Diminution of its expression leads to a nonsense suppressor phenotype whereas its overexpression leads to an antisuppressor phenotype. P. anserina eRF3 presents an N-terminal region structurally related to the yeast eRF3 one. Deletion of the N-terminal region of P. anserina eRF3 does not cause any vegetative alteration; especially life span is not changed. However, it promotes a reproductive impairment. Contrary to what happens in S. cerevisiae, deletion of the N terminus of the protein promotes a nonsense suppressor phenotype. Genetic analysis suggests that this domain of eRF3 acts in P. anserina as a cis-activator of the C-terminal portion and is required for proper reproduction.

The existence of a neurofilament-deficient mutant of Japanese quail was recently documented (Yamasaki, H., C. Itakura, and M. Mizutani. 1991. Acta Neuropathol. 82:427-434), but the genetic events leading to the neurofilament deficiency have yet to be determined. Our molecular biological analyses revealed that the expression of neurofilament-L (NF-L) gene was specifically repressed in neurons of this mutant. To search for mutation(s) responsible for the shutdown of this gene expression, we cloned and sequenced the NF-L genes in the wild-type and mutant quails. It is eventually found that the NF-L gene in the mutant includes a nonsense mutation at the deduced amino acid residue 114, indicating that the mutant is incapable of producing even a trace amount of polymerization-competent NF-L protein at any situation. The identification of this nonsense mutation provides us with a solid basis on which molecular mechanisms underlying the alteration in the neuronal cytoskeletal architecture in the mutant should be interpreted.

Abstract Abstract 4121 The activation of the JAK/STAT pathway caused by the JAK2 gene mutations is an important pathogenetic mechanism of myeloproliferative neoplasm(MPN). Recently, many evidences suggest that there are factors besides the mutations of JAK2 gene participate in the pathogenesis of MPN. Suppressors of cytokine signaling (SOCS) proteins are potent inhibitors of JAK/STAT pathway, therefore we hypothesized that the down regulation of SOCS protein system may be a possible pathogenetic mechanism of MPN through the activation of the JAK/STAT pathway. In order to testify our hypothesis, we investigated mutated points, the expression and methylation status of the SOCS1, SOCS2 and SOCS3 gene in 100 MPN patients(44 polycythemia vera (PV), 38 essential thrombocythemia (ET) and 18 idiopathic myelofibrosis(MF)). We obtained some interesting results: (1) By using DNA sequence analysis, two mutations of SOCS3 were identified with in the coding region in 1 of PV patients and 1 of ET patients (2%), respectively, and both of these 2 patients are with JAK2V617F mutation.(wide type ACG, coding Threonine, alterering to mutant type AAG, coding Lysine). Furthermore, three types of nonsense mutations were identified in SOCS3:Firstly,38 (38%) mutations of SOCS3 were identified with in the coding region in 19 of PV patients,17 of ET patients and 2 of MF respectively, (wide type CCC, coding Proline, alterering to mutant type CCA, coding Proline); Secondly, 44 (44%) mutations of SOCS3 were identified with in the coding region in 21 of PV patients,18 of ET patients and 5 of MF respectively, (wide type GTA, coding Valine, alterering to mutant type GTG, coding Valine); At last, 35 (35%) mutations of SOCS3 were identified with in the coding region in 13 of PV patients,20 of ET patients and 2 of MF respectively, (wide type GAT, coding Aspartic acid, alterering to mutant type GAC, coding Aspartic acid).Five nonsense mutations were found in SOCS2: 2 of PV patients,3 of ET patients, (wide type AAT, coding Asparagine, alterering to mutant type AAC, coding Asparagine). On the contrary, the presence of JAK2V617F mutation did not affect the nonsense mutations of SOCS2 or SOCS3. (2) By using Methylation Specific PCR (MSP), SOCS1 hypermethylation was identified in 27 patients. Hypermethylation of the SOCS2 promoter was identified in 9 of 100 (9%) patients. Hypermethylation of the SOCS3 promoter was identified in 35 of 100 (35%) patients. There was no hypermethylation of the SOCS1, SOCS2 and SOCS3 gene in 173 normal controls. (3) By using semi-quantitative PCR, the RNA expression levels of SOCS1, SOCS2 and SOCS3 were also investigated. We observed hypermethylated patients had lower SOCS1 or SOCS3 mRNA levels than unmethylated MPN samples, also observed that among patients with unmethylated SOCS1 and SOCS3, mRNA expression was higher from patients carrying the JAK2V617F mutation as compared with JAK2 wild type patients. On the contrary, the presence of JAK2V617F mutation did not affect the expression of SOCS1 or SOCS3 mRNA in methylated patients. Moreover, SOCS3 transcript levels were highest in patients with polycythemia vera and other JAK2 V617F negative myeloproliferative neoplasm. (4) According to SOCS1, SOCS3 methylation was not significantly correlated with survival or other clinical variables. In conclusion, SOCS1 and SOCS3 hypermethylation can activate the JAK/STATsignaling pathway in alternative or together with JAK2 mutations. These alterations might represent a potential therapeutic target. Disclosures: No relevant conflicts of interest to declare.

Abstract Introduction/Objective Melanoma, a highly invasive form of skin cancer associated with high mortality rates, poses significant challenges in diagnosis and treatment. RAC1, a member of the RHO family of GTPases, plays a crucial role as a molecular switch in cellular processes. A specific mutation in RAC1, caused by C &gt; T transition induced by UV radiation, leads to P29S alteration, activating the SRF/MRTF pathway and triggering a phenotypic switch towards a mesenchymal phenotype. Methods/Case Report We present a case of a 74-year-old male patient who presented with a six-week history of right neck swelling. Ultrasonography revealed an enlarged lymph node in the right posterior triangle of the neck. PET scan showed a well- defined, moderately metabolically active lesion measuring 1.4x1.7cm. Initial biopsy indicated a diagnosis of malignant metastatic sarcomatous neoplasm. To establish a definitive diagnosis and determine the appropriate treatment plan, excision of the lymph node was performed. Histological examination revealed lymph node involvement by large, highly atypical cells exhibiting prominent nucleoli and frequent mitotic figures. Immunohistochemical analysis demonstrated positive staining for SOX10 and S100, while Melan-A and HMB45 were negative. Epithelial markers were also negative, although focal keratin positivity was observed. Further investigations were conducted to identify a primary or extranodal site. Routine dermatology visits had previously shown no evidence of primary cutaneous melanoma. Results (if a Case Study enter NA) Genomic profiling through next-generation sequencing (NGS) identified multiple alterations, including BRAF (G469R- exon 11, missense alteration), TP53 (R248W, missense alteration in TP53), RAC1 (P29S, missense alteration), TERT (Promoter Site Variant; chr5:g.1295228G&gt;A c.-124C&gt;T), and NF1 (Q1815, nonsense alteration). The tumor exhibited a high tumor mutational burden (TMB) and an ultraviolet (UV) signature characterized by C &gt; T transitions. Conclusion Diagnosing melanoma with RAC1 mutation poses significant challenges due to its potential to induce phenotypic switching. Understanding the role of RAC1 mutations in driving melanoma progression can help improve diagnostic accuracy and guide targeted therapeutic strategies.

4522 Background: Fumarate hydratase-deficient renal cell carcinoma (FH-deficient RCC) is a rare yet highly lethal kidney cancer. To deepen our understanding of FH-deficient RCC, we conducted a comprehensive integrated genomic study. Methods: A total of 126 treatment naïve FH-deficient RCC tissue samples were extracted from our multi-center database. Whole-exome, RNA-seq, and DNA-methylation sequencing were performed. All cases were confirmed with both FH/2SC immunohistochemical staining and FH alterations analysis. Results: Through whole-exome sequencing, we found FH alteration patterns were associated with both tumor behavior and patient survival outcomes. Tumors harboring FH truncating alteration (including nonsense, frameshift and splice site mutations) and mutations near hinge regions (such as p.S419L), were predominantly associated with more aggressive tumor behavior and poorer prognosis. We further developed a CpG sites-specific methylation signature for precise identification of FH-deficient RCC. More importantly, transcriptomic analysis unveiled three distinctive molecular subtypes characterized by enrichment of immune/Angiogenic/Stromal (C1), WNT/Notch/MAPK (C2), and proliferation/stemness (C3) pathways, respectively. Tumors in C1 derived the most substantial survival benefit from a combination of immune checkpoint blockade (ICB) and anti-angiogenic therapy. Tumors in C2 displayed moderate response to this therapeutic approach. In contrast, tumors in C3 exhibited an unfavorable response to anti-angiogenic monotherapy and its combination with ICB. Conclusions: These findings contribute to a profound understanding of the aggressive nature of FH-deficient RCC, offering insights into potential precision medicine approaches for disease management.

La maladie de Charcot-Marie-Tooth (CMT) est la neuropathie périphérique héréditaire la plus fréquente chez l’humain. Elle touche les motoneurones (MN) et les cellules de Schwann (CS). La majorité des gènes impliqués, dont SH3TC2 et GDAP1, peuvent être affectés par des mutations non-sens. En 2021, peu de modèles cellulaires humains existaient, et aucun traitement curatif n'était disponible pour les patients. Les travaux de cette thèse se centre sur SH3TC2, responsable de la forme démyélinisante autosomique récessive la plus fréquente des CMT, nommée CMT4C ou AR-CMTde-SH3TC2 et sur GDAP1 notamment responsable d’une forme axonale AR-CMTax-GDAP1. Dans un premier temps, nous avons analysé une cohorte de 103 patients mutés sur SH3TC2 et montré que plus de 80 % des patients possédaient au moins un allèle avec une mutation non-sens, associé à une gravité clinique accrue. Nous avons également identifié 22 nouvelles mutations pathogènes sur ce gène. La seconde partie de ce travail a consisté à créer les premiers modèles cellulaires neuronaux humains pour SH3TC2. À partir de cellules souches pluripotentes induites (hiPSCs) issues d’un individu contrôle, nous avons utilisé la technologie CRISPR-Cas9 pour produire, avec plus de 90% d’efficacité, deux modèles humains in vitro contenant des mutations non-sens induisant un codon stop prématuré (PTC) : un modèle homozygote p.(Arg954*) (PTC de type UGA) et un modèle homozygote p.(Gln71*) (PTC de type UAG). Ces hiPSCs contrôle et mutées ont ensuite été différenciées en CS. Nous avons mis en évidence une expression précoce de SH3TC2 dans les CS contrôle. Dans les modèles CS AR-CMTde-SH3TC2, une expression réduite de SH3TC2, un retard de maturation, une capacité réduite à soutenir les MN en coculture, et des anomalies dans le recyclage des récepteurs à la transferrine ont été observées. Enfin, nous avons testé plusieurs molécules thérapeutiques ciblant les mutations non-sens, des agents de translecture et des inhibiteurs du mécanisme de surveillance des ARN non-sens (NMDi). Sur un modèle de progéniteurs neuronaux dérivés d’hiPSCs portant la mutation homozygote non-sens p.(Ser194*) (UGA) sur GDAP1, nous avons testé une de ces molécules et montré qu’elle stabilisait l'ARNm muté GDAP1, restaurait son expression protéique et corrigeait la morphologie mitochondriale. Dans les modèles CS créés dans cette thèse pour SH3TC2, nos premiers résultats suggèrent l’effet positif de deux de ces molécules sur la réexpression de la protéine pour les deux types de codons UGA et UAG. Dans la quatrième partie de ce travail, nous avons développé un modèle 3D de coculture CS/MN permettant d’induire la myélinisation, étape ultime pour étudier les maladies démyélinisantes comme l’AR-CMTde-SH3TC2. Les molécules thérapeutiques identifiées pourront être testées sur ces modèles cellulaires de coculture et potentiellement in vivo pour évaluer leur capacité à induire une remyélinisation. Ce travail de thèse souligne l'importance des modèles cellulaires adaptés pour comprendre les mécanismes physiopathologiques de la CMT et ouvre des perspectives prometteuses pour de nouvelles approches thérapeutiques
Charcot-Marie-Tooth disease (CMT) is the most common hereditary peripheral neuropathy in humans. It affects motor neurons (MNs) and Schwann cells (SCs). Most of the genes involved, such as SH3TC2 and GDAP1, can be affected by nonsense mutations. As of 2021, few human cellular models existed, and no curative treatment was available for patients. This thesis primarily focuses on SH3TC2, responsible for the most common autosomal recessive demyelinating form of CMT, known as CMT4C or AR-CMTde-SH3TC2, and on GDAP1, notably responsible for an axonal form, AR-CMTax-GDAP1. In the first part of this work, we analyzed a cohort of 103 patients with SH3TC2 mutations and demonstrated that more than 80% of the patients carried at least one allele with a nonsense mutation, associated with increased clinical severity. We also identified 22 new pathogenic mutations in this gene. The second part of my work involved creating the first human neuronal cell models for SH3TC2. Using induced pluripotent stem cells (hiPSCs) derived from a control individual, we employed CRISPR-Cas9 technology to generate, with over 90% efficiency, two in vitro human models containing nonsense mutations inducing a premature stop codon (PTC): a homozygous p.(Arg954*) model (UGA-type PTC) and a homozygous p.(Gln71*) model (UAG-type PTC). These controls and mutated hiPSCs were then differentiated into Schwann cells (SCs). We observed early SH3TC2 expression in control SCs. In AR-CMTde-SH3TC2 SC models, reduced SH3TC2 expression, delayed maturation, impaired ability to support MNs in co-culture, and abnormalities in transferrin receptor recycling were noted. Finally, we tested several therapeutic molecules targeting nonsense mutations, including readthrough agents and inhibitors of nonsense-mediated mRNA decay (NMDi). In a model of neuronal progenitors derived from hiPSCs carrying the homozygous nonsense mutation p.(Ser194*) (UGA) on GDAP1, we tested one of these molecules and demonstrated that it stabilizes the mutated GDAP1 mRNA, restores its protein expression, and corrects mitochondrial morphology. In the SC models created in this thesis for SH3TC2, our early results suggest a positive effect of two of these molecules on protein re-expression for both UGA and UAG codons. In the fourth part of this work, we developed a 3D co-culture model of SCs/MNs that enables myelination, the ultimate step to studying demyelinating diseases such as AR-CMTde-SH3TC2. The identified therapeutic molecules can be tested on these co-culture cellular models and potentially in vivo to evaluate their capacity to induce remyelination. This thesis highlights the importance of appropriate cellular models to understand the pathophysiological mechanisms of CMT and opens promising perspectives for new therapeutic approaches

Aujourd’hui, le défi majeur en génétique médicale est l’interprétation clinique des variations détectées dans le génome des patients. Dans un contexte de maladie monogénique, l’identification de la variation pathogène permet d’optimiser la prise en charge des patients et de leurs apparentés. Les variations non-sens ainsi que celles localisées au niveau des sites canoniques d’épissage (IVS±1/2) sont généralement considérées causales. Toutefois, il est possible que certaines d’entre elles induisent des anomalies d’épissage en phase permettant potentiellement la production d’une protéine fonctionnelle. Afin de tester cette hypothèse, nous avons choisi comme modèles d’étude deux gènes majeurs de prédisposition aux cancers : BRCA2, pour le syndrome sein-ovaire et MSH2, pour le syndrome de Lynch. Des approches expérimentales complémentaires ont été mises à profit afin de caractériser l’impact de ce type de variations non seulement au niveau de l’épissage mais également au niveau de la protéine. Notre étude portant sur des variations de BRCA2 a démontré que certaines variations IVS±1/2 et non-sens (i) induisent un saut en phase de l’exon 12 du fait de l’altération de sites d’épissage ou d’éléments de régulation et (ii) sont de ce fait responsables du maintien de l’activité BRCA2, tout en étant hypomorphes. Ces travaux montrent, pour la première fois dans un gène de prédisposition au cancer, que certaines variations présumées nulles peuvent contourner la perte totale de fonction du fait de leur impact sur l’épissage. Nos données remettent ainsi en question le caractère pathogène de ces variations. Dans le cadre de notre étude portant sur des variations IVS±1/2 de MSH2, nous avons caractérisé trois biotypes d’anomalies d’épissage en phase : (i) des sauts d’exon, (ii) des délétions de portions exoniques et (iii) des rétentions introniques, avec pour conséquence au niveau protéique des délétions ou insertions de plus ou moins grande taille dans différents domaines fonctionnels. Toutes les isoformes protéiques MSH2 résultant de ces variations splicéogéniques se sont avérées inactives, confirmant ainsi leur caractère pathogène. L’ensemble de ces travaux souligne l’importance d’exercer une certaine prudence dans l’interprétation des variations considérées a priori pathogènes mais susceptibles de générer des anomalies d’épissage en phase. Dans ce contexte, la combinaison d’approches expérimentales complémentaires pour appréhender l’impact biologique au niveau de l’ARN et de la protéine est essentielle pour une interprétation fiable de ce type de variations. Ces données mettent également en exergue la problématique de l’interprétation clinique des variations hypomorphes dans les gènes de prédisposition aux cancers
Today, the major challenge in medical genetics is the clinical interpretation of nucleotide variants detected in a patient’s genome. In the context of a monogenic disease, the identification of the pathogenic variant allows the optimization of the medical care of patients and their relatives. Nonsense variations as well as those located at the canonical splice sites (IVS±1/2) are generally considered pathogenic. However, it is possible that a fraction of them induce in-frame splicing anomalies that can potentially result in the production of a functional protein. To test this hypothesis, we used as model systems two major cancer-predisposition genes: BRCA2, implicated in hereditary breast and ovarian cancer syndrome and MSH2, involved in Lynch syndrome. We took advantage of complementary experimental approaches to characterize the impact of this type of variants not only in RNA splicing but also at the protein level. Our study on BRCA2 demonstrated that a subset of IVS±1/2 and nonsense variants (i) induce in-frame skipping of exon 12 via the modification of splice sites or of splicing regulatory elements and (ii) are able to maintain BRCA2 activity though being hypomorphic. These data provide the first evidence, in a cancer-predisposition gene, that certain presumed null variants can bypass total loss of function due to their impact on splicing. Thus our findings call into question the pathogenic nature of these variants. In our study on MSH2 IVS±1/2 variants, we characterized three biotypes of in-frame splicing defects responsible for deletions or insertions at the protein level in different MSH2 functional domains: (i) exon skipping, (ii) deletions of exonic portions and (iii) intron retentions. All the MSH2 protein isoforms resulting from these spliceogenic variants were found to be inactive, thus confirming their pathogenic classification. Altogether, our findings highlight the need to exercise caution in the interpretation of putative pathogenic variants susceptible to induce in-frame splicing modifications. In this context, the combination of complementary experimental approaches assessing the biological impact at the RNA and protein level is essential for a reliable interpretation of this type of variants. Furthermore, our results stress the problem of the clinical interpretation of hypomorphic variants in cancer-predisposition genes

A detailed, step-by-step guide to successfully altering and repairing ready-made apparel that will help you achieve the perfect fit and extend the life of your clothing. Whether you are interested in tailoring your wardrobe, starting a business, or learning a skill that will save you money and the planet, you'll find what you need through illustrated step-by-step projects and no-nonsense videos. You'll learn to make alterations to your ready-made clothing, including a variety of hemming techniques and taking in/ letting out seams, and repair methods to fix zippers, tears, and holes. There are also detailed guidelines on more complex techniques, including adjusting suit jacket sleeves, reshaping necklines and even fixing backpacks, tents and bags.

Abstract The detection and characterization of point mutations in genes responsible for inherited disorders has become one of the most common practices in human molecular genetics. Several protocols are nowadays available: single strand conformation polymorphism (SSCP) (1), RNase protection (2), hydroxylamine and osmium tetroxide (HOT) chemical cleavage (3), and denaturing gradient gel electrophoresis (DGGE) (4, 5). These techniques are generally aimed at the identification of single base substitutions, insertions, or deletions in relatively small DNA fragments (50-500 bp). However, when dealing with large genes, it is desirable to analyse larger fragments of the coding region to speed up the analysis and pin-point the location of the alteration prior to the nucleotide sequence determination. To this aim, mRNA-based mutation detection protocols are often employed. Moreover, if the spectrum and the type (i.e. missense, nonsense, or frame-shift) of the prevalent mutations at a given diseasecausing gene has already been established, one would prefer to employ a protocol ‘targeted’ for the detection of the most frequent type of mutations.

Basal cell nevoid carcinoma syndrome (BCNCS) is a rare autosomal dominant genetic syndrome that is predisposed to cancer. It is characterized by the presence of multiple basal cell carcinomas (BCCs) on the skin, as well as numerous maxillary odontogenic keratocyst (QTOs), palmoplantar punctate depressions (pits), skeletal abnormalities, and other developmental defects. The genetic basis of this syndrome lies in causal mutations in the PTCH1 gene, a tumor suppressor gene located on chromosome 9. The present study aimed to review recent literature concerning SCNBC, addressing aspects such as clinical manifestations, diagnostic criteria, genetic etiology, and molecular tests used. A total of 88 articles were included, most of which were clinical cases. Among the clinical manifestations, QTOs were the most frequently mentioned major diagnostic criteria, followed by calcification of the cerebral sickle. Ocular anomalies, on the other hand, were the alterations belonging to the most prevalent minor criteria. A total of 18 clinical cases underwent molecular testing for mutations in the PTCH1 gene. The most used methods were genetic sequencing and the mutations frequently found were frameshift and nonsense mutation, which occurred in exons 2, 3, 6, 8, 11, 12, 18, and 21. Despite the existence of several mutations in the PTCH1 gene that are attributable to the etiology of SCNBC, the performance of diagnostic molecular tests were not performed in many of the studies analyzed, and even those that did not identify a correlation with the patient's phenotype or prognosis, and these are indicated only in some particular cases.

Hemophilia A is a common X linked hereditary disorder of blood coagulation due to deficiency of factor 8. The gene for factor 8 has been cloned and characterized (Nature 312:326-342, 1984). It is divided into 26 exons and 25 introns and spans 186 kb of DNA. The CGNA is 9 kb and codes for 2351 amino acids. The first 19 amino acids comprise the secretory leader peptide and the mature excreted polypeptide consists of 2332 amino acids. The nucleotide sequence of the exons and the exon-intron junctions is known and the complete amino acid sequence has been deducedSeveral laboratories have used cloned factor 8 DNA sequences as probes to characterized mutations that are responsible for hemophilia A in certain pedigrees. These mutations have been characterized by restriction analysis, oligonucleotide hybridization, cloning and sequencing of DNA from appropriate patientsIn about 500 patients with hemophilia A examined, the molecular defect has been recognized in 39. Both gross alterations (mainly deletions) and point mutations of the factor 8 gene have been found.A total of 19 different deletions have been observed. No two unrelated pedigrees share the same exact deletion.The size of the deleted DNA varies from 1.5 kb to more than 210 kb. All but one of these deletions are associated with severe hemophilia A. A deletion of 6 kb that contains exon 22 only is associated with moderate hemophilia. Some deletions are present in patients with inhibitors to factor 8. No correlation of the size or the position of the deletions can be found with the presence of inhibitors to factor 8.A total of 20 point mutations have been characterized. All are recognized by restriction analysis and involve Taq I sites. All are mutations of CpG dinucleotides and generate nonsense or missence codons. Unrelated pedigrees have the same single nucleotide change because of independent origin of the same mutation. In many instances de novo occurrence of a point mutation has been observed. CpG dinucleotides are hot spots for mutation to TG or CA presumably because of spontaneous deamination of methylcytosine. Some point mutations are present in patients with inhibitors but no correlation of the site of mutation and inhibitor formation has been found. The nonsense mutations are present in patients with severe hemophilia A. A missense mutation (Arg Gin) in exon 26 was found in a patient with mild hemophilia while another Arg Gin mutation in exon 24 has been observed in a patient with severe disease. The creation of a donor splice site in IVS 4 of factor 8 gene has been observed in a patient with mild hemophilia.Few DNA polymorphisms within the factor 8 gene and two other closely linked polymorphisms have been used for carrier detection and prenatal diagnosis of hemophilia A. These DNA markers are useful in more than 90% of families at risk for hemophilia A.The author thanks Drs. Gitschier, Din, Olek, Pirastou, Lawn for communication of their data prior to publication.The hemophilia project at Johns Hopkins was supported by an Institutional grant and NIH grant to S.S.A. and Haig H. Kazazian, Jr.