Assessment of hOGG1 Genetic Polymorphism (rs1052133) and DNA Damage in Radiation-Exposed Workers.

OBJECTIVE: The aim of this study was to assess the effect of radiation exposure, human 8-oxoguanine DNA N-glycosylase-1 (hOGG1) exon 7 genetic polymorphism and confounding factors on DNA damage response. METHODS: Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and alkaline Comet assay method were applied to determine the hOGG1 genetic polymorphisms and DNA damage response. A total of 80 participants were enrolled in this study, consisting of 40 radiation-exposed workers as a case group and 40 non-radiation workers as a control group. RESULT: The genotypes frequencies for controls were Ser/Ser (35%), Ser/Cys (32.5%), and Cys/Cys (32.5%), with frequencies of alleles being 326Ser (0.52) and 326Cys (0.48), whereas the genotypes frequencies for radiation-exposed workers (cases group) were Ser/Ser (17.5%), Ser/Cys (57.5%), and Cys/Cys (25%), with frequencies of alleles being 326Ser (0.46) and 326Cys (0.54). The results indicated that DNA damage response were not significantly higher in the exposed workers than in controls (22.55 ± 6.02 versus 21.72 ± 7.14; P=0.58). The time of exposure has a significantly negative correlation with comet tail length value among radiation workers. In addition, it was found that the DNA damage response was strongly associated with age and time of exposure with a decrease of 0.6 percent (P-value: 0.008) and 0.58 percent (P-value: 0.009), respectively. Whereas gender, smoking habit, and equivalent dose were not correlated with DNA damage. CONCLUSION: The single-nucleotide polymorphism of hOGG1 exon 7 (rs1052133) demonstrated no association with the extent of DNA damage in radiation-exposed workers.

Efficacy of erlotinib in NSCLC harboring rare EGFR extracellular domain mutation (T263P) and common mutations: Case report and literature review.

The epidermal growth factor receptor (EGFR) typically contains an extracellular domain (ECD), a transmembrane (TM) domain, and an intracellular kinase (KD) domain. ECD mutations of EGFR in NSCLC may affect its normal function and intrinsic resistance to tyrosine kinase inhibitors (TKIs) and the effectiveness of drugs for these patients is unsatisfactory. Recently, we found an EGFR T263P mutation located at the ECD, which has never been reported in Chinese non-small cell lung cancer (NSCLC). Hence, we reported that a patient with advanced lung adenocarcinoma harboring the EGFR T263P mutation, L858R mutation and MET amplification was resistant to osimertinib but significantly benefited from erlotinib and capmatinib treatment. This patient achieved a partial response and had progression-free survival (PFS) for more than 19 months. In summary, we are the first researchers to report in detail on a Chinese patient carrying the T263P mutation and summarize all the ECD mutations in NSCLC. We believe this finding will enlighten us to treat patients with EGFR ECD mutations and more patients deserve further study.

NOVA1 promotes SMN2 exon 7 splicing by binding the UCAC motif and increases SMN protein expression.

Spinal muscular atrophy (SMA) is a rare hereditary neuromuscular disease with a high lethality rate in infants. Variants in the homologous genes survival of motor neuron (SMN)1 and SMN2 have been reported to be SMA pathogenic factors. Previous studies showed that a high inclusion rate of SMN2 exon 7 increased SMN expression, which in turn reduced the severity of SMA. The inclusion rate of SMN2 exon 7 was higher in neural tissues than in non-neural tissues. Neuro-oncological ventral antigen (NOVA) is a splicing factor that is specifically and highly expressed in neurons. It plays a key role in nervous system development and in the induction of nervous system diseases. However, it remains unclear whether this splicing factor affects SMA. In this study, we analyzed the inclusion of SMN2 exon 7 in different tissues in a mouse model of SMA (genotype smn-/-SMN22tg/0) and littermate controls (genotype smn+/-SMN22tg/0). We found that inclusion level of SMN2 exon 7 was high in the brain and spinal cord tissue, and that NOVA1 was also highly expressed in nervous system tissues. In addition, SMN2 exon 7 and NOVA1 were expressed synchronously in the central nervous system. We further investigated the effects of NOVA1 on disease and found that the number of neurons in the anterior horn of spinal cord decreased in the mouse model of SMA during postnatal days 1-7, and that NOVA1 expression levels in motor neurons decreased simultaneously as spinal muscular atrophy developed. We also found that in vitro expression of NOVA1 increased the inclusion of SMN2 exon 7 and expression of the SMN2 protein in the U87MG cell line, whereas the opposite was observed when NOVA1 was knocked down. Finally, point mutation and RNA pull-down showed that the UCAC motif in SMN2 exon 7 plays a critical role in NOVA1 binding and promoting the inclusion of exon 7. Moreover, CA was more essential for the inclusion of exon 7 than the order of Y residues in the motif. Collectively, these findings indicate that NOVA1 interacts with the UCAC motif in exon 7 of SMN2, thereby enhancing inclusion of exon 7 in SMN2, which in turn increases expression of the SMN protein.

Activated macrophages of CD 163 gene edited pigs generated by direct cytoplasmic microinjection with CRISPR gRNA/Cas9 mRNA are resistant to PRRS virus assault.

Porcine reproductive and respiratory syndrome virus (PRRSV) infects placental and lung macrophages, causing a global epidemic with economic loss. Attempts to develop an effective vaccine to control the disease have not been effective. Currently, developing PRRSV disease-resistant pigs via a gene editing (GE) strategy to mutate the PRRSV receptor or to delete the binding domain on the macrophage appears promising. In this study, we used the strategy of Edinburg University to construct two guide RNAs (gRNAs) located on the proximal front and post sites of exon 7. Directive microinjection of two gRNAs and Cas9 mRNA into the cytoplasm of pronuclear zygotes efficiently generated four piglets confirmed as CD163 knockout (KO) and/or CD163 exon 7 deleted (CD163ΔE7). In four GE piglets, three pigs carried two chromosome CD163 KO or ΔE7. Peripheral blood mononuclear cells (PBMCs) from three GE and wild-type (WT) pigs were activated into macrophages for in vitro transfection. The results showed that the activated macrophages from all GE pigs were significantly more viable than those from WT pig. Current results suggest that we have successfully generated PRRSV-resistant pigs, although in vivo challenge is needed to validate that the pigs are PRRSV resistant.

The clinical outcomes of chronic myeloid leukemia patients harboring alternatively spliced BCR-ABL variants.

Objectives and importance: Tyrosine kinase inhibitors (TKIs) are indispensable for the treatment of chronic myeloid leukemia (CML). However, alternative splicing variants have been recently proposed as mechanisms of TKI resistance, although the clinical significance of these mutations remains controversial. We here present the long-term clinical courses of three CML patients harboring such unique mutations and try to assess their clinical significances. Moreover, the exon 6 frameshift presented here has been rarely reported, which may provide important information on this rare mutation. Clinical presentation: We report three cases of CML harboring an exon 7 deletion, insertion of 35 intronic nucleotides and an exon 6 frameshift, respectively. Remarkably, all patients obtained better than molecular response4.0 following administration of TKIs. Discussion and conclusion: Three CML cases highlighted an association between such splicing variants and clinical outcomes. The premature termination in the kinase domain due to these mutations likely causes conformational changes and inhibits TKI binding, but it also results in abrogating kinase activities of CML cells. Thus, the above-mentioned mutants might less affect outcomes of treatment. Noteworthy, clinically available International Scale RT-PCR system cannot distinguish kinase-active mutants from kinase-inactive mutants, which may possibly influence upon interpretation of the treatment efficacy. Clonal quantification on respective mutants could more precisely evaluate CML status in these patients. Therefore, one should realize these important splicing variants and accumulate further experiences.

Hepatocellular adenoma: Classification, variants and clinical relevance.

Hepatocellular adenomas are benign tumors with two major complications, bleeding and malignant transformation. The overall narrative of hepatocellular adenoma has evolved over time. Solitary or multiple hepatocellular developing in the normal liver of women of child bearing age exposed to oral contraceptives still represents the most frequent clinical context, however, new associations are being recognized. Hepatocellular adenoma is discovered on a background of liver diseases such as non-alcoholic steatohepatitis, vascular diseases, and alcoholic cirrhosis. Hepatocellular adenoma is also reported in men, young or older adults, and even in infants. On the morpho-molecular side, the great leap forward was the discovery that hepatocellular adenoma was not a single entity and that at least 3 different subtypes exist, with specific underlying gene mutations. These mutations affect the HNF1A gene, several genes leading to JAK/STAT3 pathway activation and the CTNNB1 gene. All of them are associated with more or less specific histopathological characteristics and can be recognized using immunohistochemistry either with specific antibodies or with surrogate markers. Liver pathologists and radiologists are the key actors in the identification of the different subtypes of hepatocellular adenoma by the recognition of their specific morphological features. The major impact of the classification of hepatocellular adenoma is to identify subjects who are at higher risk of malignant transformation. With the development of new molecular technologies, there is hope for a better understanding of the natural history of the different subtypes, and, particularly for their mechanisms of malignant transformation.

Synthesis of a norcantharidin-tethered guanosine: Protein phosphatase-1 inhibitors that change alternative splicing.

Phosphorylation and dephosphorylation of splicing factors play a key role in pre-mRNA splicing events, and cantharidin and norcantharidin analogs inhibit protein phosphatase-1 (PP1) and change alternative pre-mRNA splicing. Targeted inhibitors capable of selectively inhibiting PP-1 could promote exon 7 inclusion in the survival-of-motorneuron-2 gene (SMN2) and shift the proportion of SMN2 protein from a dysfunctional to a functional form. As a prelude to the development of norcantharidin-tethered oligonucleotide inhibitors, the synthesis a norcantharidin-tethered guanosine was developed in which a suitable tether prevented the undesired cyclization of norcantharidin monoamides to imides and possessed a secondary amine terminus suited to the synthesis of oligonucleotides analogs. Application of this methodology led to the synthesis of a diastereomeric mixture of norcantharidin-tethered guanosines, namely bisammonium (1R,2S,3R,4S)- and (1S,2R,3S,4R)-3-((4-(2-(((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-2-(hydroxymethyl)-4-methoxytetrahydrofuran-3-yl)oxy)oxidophosphoryl)oxy)ethyl)-phenethyl)(methyl)carbamoyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylate, which showed activity in an assay for SMN2 pre-mRNA splicing.

Treatment of lipoid proteinosis with acitretin in two patients from two unrelated Chinese families with novel nonsense mutations of the ECM1 gene.

Lipoid proteinosis is a rare recessive genetic disorder caused by loss-of-function mutations to chromosome 1 at 1q21, the extracellular matrix protein 1 (ECM1) gene. Two children with lipoid proteinosis were reported from two unrelated Chinese families, both manifesting with a typical hoarse voice, white acne-like atrophic lesions and scarring on the skin, and beaded papules around the eyelids. The diagnosis had been confirmed by laboratory tests, skin biopsy and laryngoscope examination. Genomic DNA sequencing was performed for both children and their family members. The two children were treated with acitretin for 6 months and followed up for 1 year. Genomic DNA sequencing of the ECM1 gene showed a novel homozygous nonsense mutation of C1522>T (p.R508X) at exon 10 in one patient, and a novel compound heterozygote for a nonsense/frame-shift combination of mutations of R281X/1596delG at exons 7 and 10 in the other patient. The symptom of hoarse voice was improved by 6-month treatment with acitretin, while there was no improvement in the skin lesions. These results demonstrated that acitretin treatment may have efficacy for some of patients with lipoid proteinosis, with superior effect on laryngeal symptoms than skin lesions. However, the conclusive therapeutic effect and underlying mechanisms remain to be further investigated.

PSF contacts exon 7 of SMN2 pre-mRNA to promote exon 7 inclusion.

Spinal muscular atrophy (SMA) is an autosomal recessive genetic disease and a leading cause of infant mortality. Deletions or mutations of SMN1 cause SMA, a gene that encodes a SMN protein. SMN is important for the assembly of Sm proteins onto UsnRNA to UsnRNP. SMN has also been suggested to direct axonal transport of ß-actin mRNA in neurons. Humans contain a second SMN gene called SMN2 thus SMA patients produce some SMN but not with sufficient levels. The majority of SMN2 mRNA does not include exon 7. Here we show that increased expression of PSF promotes inclusion of exon 7 in the SMN2 whereas reduced expression of PSF promotes exon 7 skipping. In addition, we present evidence showing that PSF interacts with the GAAGGA enhancer in exon 7. We also demonstrate that a mutation in this enhancer abolishes the effects of PSF on exon 7 splicing. Furthermore we show that the RNA target sequences of PSF and tra2ß in exon 7 are partially overlapped. These results lead us to conclude that PSF interacts with an enhancer in exon 7 to promote exon 7 splicing of SMN2 pre-mRNA.

hnRNP M facilitates exon 7 inclusion of SMN2 pre-mRNA in spinal muscular atrophy by targeting an enhancer on exon 7.

Spinal muscular atrophy (SMA) is an autosomal recessive genetic disease, which causes death of motor neurons in the anterior horn of the spinal cord. Genetic cause of SMA is the deletion or mutation of SMN1 gene, which encodes the SMN protein. Although SMA patients include SMN2 gene, a duplicate of SMN1 gene, predominant production of exon 7 skipped isoform from SMN2 pre-mRNA, fails to rescue SMA patients. Here we show that hnRNP M, a member of hnRNP protein family, when knocked down, promotes exon 7 skipping of both SMN2 and SMN1 pre-mRNA. By contrast, overexpression of hnRNP M promotes exon 7 inclusion of both SMN2 and SMN1 pre-mRNA. Significantly, hnRNP M promotes exon 7 inclusion in SMA patient cells. Thus, we conclude that hnRNP M promotes exon 7 inclusion of both SMN1 and SMN2 pre-mRNA. We also demonstrate that hnRNP M contacts an enhancer on exon 7, which was previously shown to provide binding site for tra2ß. We present evidence that hnRNP M and tra2ß contact overlapped sequence on exon 7 but with slightly different RNA sequence requirements. In addition, hnRNP M promotes U2AF65 recruitment on the flanking intron of exon 7. We conclude that hnRNP M promotes exon 7 inclusion of SMN1 and SMN2 pre-mRNA through targeting an enhancer on exon 7 through recruiting U2AF65. Our results provide a clue that hnRNP M is a potential therapeutic target for SMA.

Clinical and molecular study of the extracellular matrix protein 1 gene in a spanish family with lipoid proteinosis.

BACKGROUND: Lipoid proteinosis (LP) is a rare autosomal recessive disorder characterized by a hoarse voice, variable scarring, and infiltration of the skin and mucosa. This disease is associated with mutations of the gene encoding extracellular matrix protein 1 (ECM1). CASE REPORT: This was a clinical and molecular study of a new case of LP with a severe phenotype. A 35-year-old female born to nonconsanguineous parents developed dermatological and extracutaneous symptoms in her 9th month of life. The neurological abnormalities of the disease began to appear at the age of 19 years. Computed tomography revealed cranial calcifications. CONCLUSIONS: The diagnosis of LP was confirmed by histopathological findings and direct sequencing of ECM1. A new homozygous nonsense mutation was identified in exon 7 of ECM1, c.1076G>A (p.Trp359(*)). This mutation was not detected in 106 chromosomes of healthy individuals with a similar demographic origin. Microsatellite markers around ECM1 were used to construct the haplotype in both the parents and the patient. Reports on genotype-phenotype correlations in LP point to a milder phenotype in carriers of missense mutations in the Ecm1a isoform, whereas mutations in the Ecm1b isoform are thought to be associated with more severe phenotypes. The present findings in a Spanish patient carrying a truncating mutation in exon 7 revealed complete dermatological and neurological manifestations.

Clinical and Molecular Study of the Extracellular Matrix Protein 1 Gene in a Spanish Family with Lipoid Proteinosis

BACKGROUND: Lipoid proteinosis (LP) is a rare autosomal recessive disorder characterized by a hoarse voice, variable scarring, and infiltration of the skin and mucosa. This disease is associated with mutations of the gene encoding extracellular matrix protein 1 (ECM1). CASE REPORT: This was a clinical and molecular study of a new case of LP with a severe phenotype. A 35-year-old female born to nonconsanguineous parents developed dermatological and extracutaneous symptoms in her 9th month of life. The neurological abnormalities of the disease began to appear at the age of 19 years. Computed tomography revealed cranial calcifications. CONCLUSIONS: The diagnosis of LP was confirmed by histopathological findings and direct sequencing of ECM1. A new homozygous nonsense mutation was identified in exon 7 of ECM1, c.1076G>A (p.Trp359*). This mutation was not detected in 106 chromosomes of healthy individuals with a similar demographic origin. Microsatellite markers around ECM1 were used to construct the haplotype in both the parents and the patient. Reports on genotype-phenotype correlations in LP point to a milder phenotype in carriers of missense mutations in the Ecm1a isoform, whereas mutations in the Ecm1b isoform are thought to be associated with more severe phenotypes. The present findings in a Spanish patient carrying a truncating mutation in exon 7 revealed complete dermatological and neurological manifestations.

A Case of Exon 7 and 8 Deletion of Survival Motor Neuron Gene in Spinal Muscular Atrophy / 대한소아신경학회지

Spinal muscular atrophy(SMA) is a genetic disorder of the motor neurons that cause muscular weakness and muscular atrophy due to anterior horn cell degeneration. Classic spinal muscular atrophy patient is caused by mutation in the chromosome 5(q11.2-q13.3), and the majority of the patient shows homozygous deletion of the telomeric survival motor neuron(SMN) gene in the chromosome 5. Deletion of exon 7 and 8 of the SMN gene and deletion of exon 4 and 5 of the neuronal apoptosis inhibitory protein(NAIP) are typically observed in SMA patients. The SMN protein plays a role in an essential cell metabolism process, the splicing of pre mRNA in the spliceosomes. We report a 7 month old male with SMA. He showed rapidly aggrdvatial muscular weakness and died at 7 months. His DNA analysis proved deletion of exon 7 and 8 of the telomeric copy of the SMN gene.

Genetic Polymorphism of Cytochrome P4501A1 Exon 7 and Glutathione S-Transferase M1 in the Head and Neck Squamous Cell Carcinoma Patients / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: An individual difference in susceptibility to chemically induced carcinomas is in part ascribed to genetic differences of metabolic activity of environmental procarcinogens. The cytochrome P450 family (CYPs) and glutathione S-transferase (GST) have been reported to be associated with human cancers related with smoking. The purpose of this study was to determine the frequencies of the genotypes of CYP1A1 and GSTM1 genes in healthy control of Koreans and to identify the high-risk genotypes of these metabolic genes in head and neck cancer patients. MATERIALS AND METHOD: The genetic polymorphism of CYP1A1 exon 7 and GSTM1 genes were analysed in a group of 115 healthy Koreans and 107 head and neck squamous cell carcinoma patients using allelic-specific polymerase chain reaction. RESULTS: The genotypes of CYP1A1 exon 7 (Ile/Ile, Ile/Val and Val/Val) were 59.1%, 36.5% and 4.4%, respectively, in the healthy control group, and 57.0%, 31.8% and 11.2%, respectively, in the cancer patients . The distributions of GSTM1 [GSTM1 (-), GSTM1 (+)] in healthy control group were 46.1%, 53.9% respectively, and 53.3%, 46.7%, respectively, in the cancer patients. The relative risk (odds ratio) for combination of CYP1A1 Val/Val and GSTM1 (-) genotype was estimated to be 5.17, taking the risk of combined genotype Ile/Ile and GSTM1 (+) as a reference in cancer patients. CONCLUSION: These results suggest that the genetic polymorphisms of CYP1A1 exon 7 and GSTM1 were an important major factor in determining the individual susceptibility to head and neck squamous cell carcinoma in Koreans.