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1.
Braz. j. biol ; 84: e255235, 2024. tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1355897

ABSTRACT

Abstract In soybean breeding program, continuous selection pressure on traits response to yield created a genetic bottleneck for improvements of soybean through hybridization breeding technique. Therefore an initiative was taken to developed high yielding soybean variety applying mutation breeding techniques at Plant Breeding Division, Bangladesh Institute of Nuclear Agriculture (BINA), Bangladesh. Locally available popular cultivar BARI Soybean-5 was used as a parent material and subjected to five different doses of Gamma ray using Co60. In respect to seed yield and yield attributing characters, twelve true breed mutants were selected from M4 generation. High values of heritability and genetic advance with high genotypic coefficient of variance (GCV) for plant height, branch number and pod number were considered as favorable attributes for soybean improvement that ensure expected yield. The mutant SBM-18 obtained from 250Gy provided stable yield performance at diversified environments. It provided maximum seed yield of 3056 kg ha-1 with highest number of pods plant-1 (56). The National Seed Board of Bangladesh (NSB) eventually approved SBM-18 and registered it as a new soybean variety named 'Binasoybean-5' for large-scale planting because of its superior stability in various agro-ecological zones and consistent yield performance.


Resumo No programa de melhoramento da soja, a pressão pela seleção contínua para a resposta das características de rendimento criou um gargalo genético para melhorias da soja por meio da técnica de melhoramento por hibridação. Portanto, foi desenvolvida uma variedade de soja de alto rendimento, aplicando técnicas de reprodução por mutação, na Divisão de Melhoramento de Plantas, no Instituto de Agricultura Nuclear de Bangladesh (BINA), em Bangladesh. A cultivar popular BARI Soybean-5, disponível localmente, foi usada como material original e submetida a cinco doses diferentes de raios gama usando Co60. Em relação ao rendimento de sementes e às características de atribuição de rendimento, 12 mutantes genuínos foram selecionados a partir da geração M4. Altos valores de herdabilidade e avanço genético com alto coeficiente de variância genotípico (GCV) para altura da planta, número de ramos e número de vagens foram considerados atributos favoráveis ​​ao melhoramento da soja, garantindo, assim, a produtividade esperada. O mutante SBM-18, obtido a partir de 250Gy, proporcionou desempenho de rendimento estável em ambientes diversificados e produtividade máxima de sementes de 3.056 kg ha-1 com o maior número de vagens planta-1 (56). O Conselho Nacional de Sementes de Bangladesh (NSB) finalmente aprovou o SBM-18 e o registrou como uma nova variedade de soja, chamada 'Binasoybean-5', para plantio em larga escala por causa de sua estabilidade superior em várias zonas agroecológicas e desempenho de rendimento consistente.


Subject(s)
Soybeans/growth & development , Soybeans/genetics , Phenotype , Bangladesh , Plant Breeding , Genotype , Mutation
2.
Braz. j. biol ; 84: e256923, 2024. tab, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1360219

ABSTRACT

Naturally occurring mutations in morphogenetic protein 15 (BMP15) are associated with decreased ovulation rate (OR), litter size (LS), and sterility. It is of a great interest to elucidate BMP15 gene in Cholistani sheep breed to uplift socio-economic status and the knowledge of Cholistani sheep breeding in Southern Punjab, Pakistan. In our study, a total of 50 infertile Cholistani sheep aged between 2-6 years and having no blood relation were screened for BMP15 mutations. For this purpose, a high-quality DNA was extracted from the blood of sheep followed by primer designing, Polymerase Chain Reaction (PCR) amplification, DNA sequencing, and in silico analyses. Out of total 50 samples, 9 samples including case 1 (T3), case 2 (T8), case 3 (T17), case 4 (T22), case 5 (T25), case 6 (T33), case 7 (T40), case 8 (T44), and case 9 (T47) were found positive for a variety of already reported and novel BMP15 mutations. Further in silico analyses of the observed mutations have shown the functional impact of these mutations on different characteristics (molecular weight, theoretical PI, estimated half-life, instability index, sub-cellular localization, and 3D confirmation) of the encoded proteins, possibly altering the normal functionality. In a nutshell, findings of this study have confirmed the possible essential role of the BMP15 mutations in the infertility of the Cholistani sheep.


Mutações de ocorrência natural na proteína morfogenética 15 (BMP15) estão associadas à diminuição da taxa de ovulação (TO), tamanho da ninhada (TN) e esterilidade. Estudar a BMP15 na raça Cholistani para elevar o status socioeconômico e o conhecimento da criação de ovinos Cholistani no sul de Punjab, Paquistão. Em nosso estudo, 50 ovelhas Cholistani inférteis sem parentesco sanguíneo foram rastreadas para mutações BMP15. Para tanto, um DNA de alta qualidade foi extraído do sangue dessas ovelhas, seguido de concepção do primer, amplificação da reação em cadeia da polimerase (PCR), sequenciamento de DNA e análises in silico. Do total de 50 amostras, 9, incluindo caso 1 (T3), caso 2 (T8), caso 3 (T17), caso 4 (T22), caso 5 (T25), caso 6 (T33), caso 7 (T40), caso 8 (T44) e caso 9 (T47), foram consideradas positivas para uma variedade de mutações BMP15 novas e já relatadas. Mais análises in silico das mutações observadas mostraram o impacto funcional dessas mutações em diferentes características (peso molecular, PI teórico, meia-vida estimada, índice de instabilidade, localização subcelular e confirmação 3D) das proteínas codificadas, possivelmente alterando a funcionalidade normal. Nossos achados confirmaram o possível papel essencial das mutações BMP15 na infertilidade de ovelhas Cholistani.


Subject(s)
Animals , Sheep , Infertility , Mutation/genetics
3.
Braz. j. biol ; 83: e246040, 2023. tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1285610

ABSTRACT

Abstract Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by a congenitally reduced head circumference (-3 to -5 SD) and non-progressive intellectual disability. The objective of the study was to evaluate pathogenic mutations in the ASPM gene to understand etiology and molecular mechanism of primary microcephaly. Blood samples were collected from various families across different remote areas of Pakistan from February 2017 to May 2019 who were identified to be affected with primary microcephaly. DNA extraction was performed using the salting-out method; the quality and quantity of DNA were evaluated using spectrophotometry and 1% agarose gel electrophoresis, respectively in University of the Punjab. Mutation analysis was performed by whole exome sequencing from the Cologne Center for Genomics, University of Cologne. Sanger sequencing was done in University of the Punjab to confirm the pathogenic nature of mutation. A novel 4-bp deletion mutation c.3877_3880delGAGA was detected in exon 17 of the ASPM gene in two primary microcephaly affected families (A and B), which resulted in a frame shift mutation in the gene followed by truncated protein synthesis (p.Glu1293Lysfs*10), as well as the loss of the calmodulin-binding IQ domain and the Armadillo-like domain in the ASPM protein. Using the in-silico tools Mutation Taster, PROVEAN, and PolyPhen, the pathogenic effect of this novel mutation was tested; it was predicted to be "disease causing," with high pathogenicity scores. One previously reported mutation in exon 24 (c.9730C>T) of the ASPM gene resulting in protein truncation (p.Arg3244*) was also observed in family C. Mutations in the ASPM gene are the most common cause of MCPH in most cases. Therefore, enrolling additional affected families from remote areas of Pakistan would help in identifying or mapping novel mutations in the ASPM gene of primary microcephaly.


Resumo Microcefalia primária autossômica recessiva (MCPH) é um distúrbio do neurodesenvolvimento caracterizado por uma redução congênita do perímetro cefálico (-3 a -5 DP) e deficiência intelectual não progressiva. O objetivo do estudo foi avaliar mutações patogênicas no gene ASPM a fim de compreender a etiologia e o mecanismo molecular da microcefalia primária. Amostras de sangue foram coletadas de várias famílias em diferentes áreas remotas do Paquistão de fevereiro de 2017 a maio de 2019, que foram identificadas como afetadas com microcefalia primária. A extração do DNA foi realizada pelo método salting-out; a qualidade e a quantidade de DNA foram avaliadas por espectrofotometria e eletroforese em gel de agarose a 1%, respectivamente, na Universidade de Punjab. A análise de mutação foi realizada por sequenciamento completo do exoma do Cologne Center for Genomics, University of Cologne. O sequenciamento de Sanger foi feito na Universidade do Punjab para confirmar a natureza patogênica da mutação. Uma nova mutação de deleção de 4 bp c.3877_3880delGAGA foi detectada no exon 17 do gene ASPM em duas famílias afetadas por microcefalia primária (A e B), que resultou em uma mutação de frame shift no gene seguida por síntese de proteína truncada (pGlu1293Lysfs * 10), bem como a perda do domínio IQ de ligação à calmodulina e o domínio do tipo Armadillo na proteína ASPM. Usando as ferramentas in-silico Mutation Taster, PROVEAN e PolyPhen, o efeito patogênico dessa nova mutação foi testado; foi previsto ser "causador de doenças", com altos escores de patogenicidade. Uma mutação relatada anteriormente no exon 24 (c.9730C > T) do gene ASPM, resultando em truncamento de proteína (p.Arg3244 *) também foi observada na família C. Mutações no gene ASPM são a causa mais comum de MCPH na maioria dos casos . Portanto, a inscrição de famílias afetadas adicionais de áreas remotas do Paquistão ajudaria a identificar ou mapear novas mutações no gene ASPM da microcefalia primária.


Subject(s)
Humans , Microcephaly/genetics , Nerve Tissue Proteins/genetics , Pakistan , Consanguinity , Mutation/genetics
5.
Methods Mol Biol ; 2564: 75-97, 2023.
Article in English | MEDLINE | ID: mdl-36107338

ABSTRACT

Directed evolution has revolutionized the way scientists create new biomolecules not found in nature. Error-prone polymerase chain reaction (PCR) introduces random mutations and was used to evolve jellyfish and coral fluorescent proteins in bacteria. We describe a novel method for the directed evolution of a far-red fluorescent protein in E. coli. The new method used genes to produce fluorophores inside E. coli and allowed changing the native fluorophore, phycocyanobilin, for a second small-molecule fluorophore, biliverdin. The directed evolution blueshifted the fluorescence, which enhanced the quantum yield to produce a brighter fluorescent protein. Finally, the evolution selected fluorescent proteins that expressed in large quantities in E. coli. The evolved fluorescent protein was named the small ultra-red fluorescent protein (smURFP) and was biophysically as bright as the enhanced green fluorescent protein (EGFP). This chapter describes the materials and methods used to evolve a far-red fluorescent protein in bacteria. While the focus is a fluorescent protein, the protocol is adaptable for the evolution of other biomolecules in bacteria when using a proper selection strategy.


Subject(s)
Anthozoa , Escherichia coli , Animals , Anthozoa/genetics , Anthozoa/metabolism , Biliverdine/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Fluorescence , Mutation
6.
Horm Metab Res ; 54(2): 67-75, 2022 Feb.
Article in English | MEDLINE | ID: mdl-35130567

ABSTRACT

Resistance to thyroid hormone syndrome (RTHS) is defined as increased thyroxine and triiodothyronine associated with normal or increased thyrotropin. This is usually due to a pathogenic variant of the gene coding for thyroid hormone receptor B (THRB). THRB is a rare genetic disorder characterized by an altered response of target tissue to the thyroid hormone action. Retrospective cross-sectional observational study with diagnosis of RTHS evaluated in secondary and tertiary hospitals for 6 years, from 2014 to 2020, in order to describe variables including age, sex, anthropometric data, clinical and biochemical characteristics of patients, who were divided according to age, in a pediatric group from 0 to 14 years (index cases), and an adult group composed of adult relatives of index cases. A molecular analysis of the THRB gene was performed. The total retrospective cohort included 7 pediatric patients and 15 adults. We found 22 cases with a clear male predominance (14/22). Mean age is 24.8 years old (22 days-70 years). Patients were referred because of symptoms 18.2% (4/22), analysis results 22.7% (5/22), or familial study 59.1% (13/22). About 31.8% (7/22) cases show goiter, 31.8% (7/22) sympathetic symptoms and 13.6% (3/22) abnormalities in behavior. In most cases, 77.3%, (17/22) show familial background of thyroid abnormalities. It is important to remark that 18.2% (4/22) relatives received previous incorrect treatments such as thyroidectomy, because of wrong diagnosis. In conclusion, a better understanding of RTHS, its prompt molecular diagnosis and genetic counseling, could avoid unnecessary tests and inappropriate treatments.


Subject(s)
Thyroid Hormone Resistance Syndrome , Adolescent , Adult , Child , Child, Preschool , Cross-Sectional Studies , Humans , Infant , Infant, Newborn , Male , Mutation , Retrospective Studies , Thyroid Hormone Receptors beta/genetics , Thyroid Hormone Resistance Syndrome/diagnosis , Thyroid Hormone Resistance Syndrome/genetics , Thyrotropin , Thyroxine , Triiodothyronine , Young Adult
7.
Clin. transl. oncol. (Print) ; 24(10): 1975–1985, octubre 2022. graf
Article in English | IBECS | ID: ibc-207953

ABSTRACT

Purpose: Lung cancer is one of the most common carcinomas with the highest mortality in the world. Non-small cell lung carcinoma has a large proportion of epidermal growth factor receptor (EGFR) mutations, of which rare EGFR mutations account for about 10%–20%. Currently, tyrosine kinase inhibitors (TKIs) therapy is a standard treatment for patients with non-small cell lung carcinoma with EGFR mutations. To date, the toxicological effects of the EGFR L861Q variant (less than 2%) have been rarely reported, so further investigation of its sensitivity to six first-in-class TKIs is of great clinical interest.MethodsIn this study, two EGFR L861Q variants cell lines (EGFR L861Q variant and EGFR L861Q + exon 19 deletion variant) were established by CRISPR-Cas9 gene-editing technology. The steady-state plasma concentrations of six TKIs (gefitinib/erlotinib/icotinib, the first generation; dacomitinib/afatinib, the second generation; and osimertinib, the third generation) were tested, respectively. The change of cell viability, proliferation, cloning ability, mitochondrial membrane potential and apoptosis were detected by MTT assay, EdU staining assay, colony formation assay, mitochondrial membrane potential and apoptosis test. TUNEL and Annexin V / PI staining were used to detect cell apoptosis, and flow cytometry was employed to explore the sensitivity of two variants to six TKIs.ResultsOur study indicated that the six TKIs inhibited the viability of the two cell lines in a time-dependent manner, and the inhibitory time of six TKIs on proliferation was different between the two cell lines. The proliferation and cloning ability of two cell lines were inhibited by six TKIs. The cytoskeleton morphology, microfilament structure and distribution of the two cell lines were changed by six TKIs. (AU)


Subject(s)
Humans , Carcinoma, Non-Small-Cell Lung , ErbB Receptors , Erlotinib Hydrochloride , Gefitinib , Lung Neoplasms , Mutation
8.
Clin. transl. oncol. (Print) ; 24(10): 1986–1997, octubre 2022. graf
Article in English | IBECS | ID: ibc-207954

ABSTRACT

Purpose: Prostate adenocarcinoma (PRAD) is a high incidence of malignant tumor of the urinary system and the second most common male cancer in the world. Immune checkpoint inhibitor (ICIS) therapy is becoming a new hope for cancer treatment.MethodsTo realize the possibility of PRAD patients benefiting from ICIS treatment, we analyzed the mutation spectrum of all PRAD patients, calculated the TMB of each PRAD patient, and divided the patients into high TMB group and low TMB group. Differentially expressed genes (DEGs) between the two groups were identified and path analysis was carried out. The immune cell infiltration of each PRAD patient was evaluated and survival analysis was performed to explore the effect of immune cell infiltration on the prognosis.ResultsWe found that high TMB was associated with better survival outcomes, with higher TMB scores in young patients, T2 and N0 patients. 28 hub genes were screened by the overlap between 229 DEGs and immune-related genes. T cells CD8 and CD4 memory activated in the high TMB group were higher than those in the low TMB group, while Mast cells resting in the low TMB group were higher than that in the high TMB group. High neutrophil infiltration is associated with poor prognosis in patients with PRAD. Finally, from the immune genes used to construct the prognostic risk model of TMB, it is found that CHP2 and NRG1 are independent prognostic factors of PRAD.ConclusionsThis study provides new insights into the immune microenvironment and potential immunotherapy of PRAD. (AU)


Subject(s)
Humans , Biomarkers, Tumor , Immunotherapy , Prostatic Neoplasms , Tumor Microenvironment , Prognosis , Mutation
9.
Clin. transl. oncol. (Print) ; 24(9): 1785–1799, septiembre 2022.
Article in English | IBECS | ID: ibc-206264

ABSTRACT

PurposeAnaplastic lymphoma kinase (ALK) is an endorsed molecular target in ALK-rearranged carcinomas, including lung adenocarcinoma. However, the clinical advantage of targeting ALK using druggable inhibitors is almost universally restricted by the development of drug resistance. Therefore, a strategy for combating ALK overexpression remains paramount for ALK-driven cancer.MethodsWe systemically analyzed the overexpression pattern of ALK and its clinical consequences, genetic alterations, and their significance in cancer hallmark genes, and correlation using integrated multidimensional approaches. The LwCas13a RNA molecular scissors was used to downregulate ALK-rearrangement by leveraging two target guide RNAs in lung adenocarcinoma (LUAD) cells. Immunocytochemistry, immunoblotting, and MTT assays were conducted to validate the downregulation.ResultsWe found elevated levels of ALK in several malignancies, including LUAD, than in normal tissues. Higher expression of ALK was significantly associated with worse or shorter survival than patients with lower expression. We identified numerous genetic alterations in ALK, which potentially alter the cancer hallmark genes, including STAT1 and CTSL, in patients with LUAD. Next, we observed that the LwCas13a molecular scissors robustly downregulated both phosphorylated and total ALK chimera protein expression in LUAD cells compared to the control. Furthermore, we found that downregulation of ALK chimera protein substantially inhibited cell viability and induced cell death, including apoptosis.ConclusionOur findings suggest a basis for ALK as a prognostic biomarker and the LwCas13a molecular scissors successfully downregulated the onco-driver ALK-rearrangement protein, which will potentially pave the way toward the development of novel therapeutic strategies for ALK-driven cancer. (AU)


Subject(s)
Humans , Adenocarcinoma of Lung/drug therapy , Adenocarcinoma of Lung/genetics , Lung Neoplasms/pathology , Mutation , RNA
12.
Gan To Kagaku Ryoho ; 49(8): 801-808, 2022 Aug.
Article in Japanese | MEDLINE | ID: mdl-36046960

ABSTRACT

Anti-EGFR antibodies(cetuximab and panitumumab)are molecular targeted agents that exert their antitumor effects by binding directly to the epidermal growth factor receptor(EGFR)and inhibiting its downstream signaling. Anti-EGFR antibodies have become one of the most important agents in the treatment of metastatic colorectal cancer(CRC), showing efficacy in combination with cytotoxic chemotherapeutic agents and as single agents in first-line and second-line treatment or later. Molecular targeted agents show more potent therapeutic effects than conventional chemotherapeutic agents, but their cost-effectiveness is often an issue due to their high drug costs. Predictive biomarkers of therapeutic efficacy that enable appropriate patient selection have an important role not only in improving the cost-effectiveness of molecular targeted agents, but also from the perspective of avoiding side effects in invalid patients. In addition to RAS and BRAF genotypes as molecular biological factors, anatomical factors such as the site of primary tumor(sidedness)are described in guidelines as important biomarkers for anti-EGFR treatment in the treatment of metastatic CRC. Recently, an association between HER2 gene amplification and anti-EGFR antibody resistance in RAS/BRAF wild-type metastatic CRC was reported. Furthermore, an epigenetic factor, DNA methylation status, was reported to be associated with the therapeutic effect of anti-EGFR treatments, and our subsequent studies have suggested that DNA methylation status is predictive of therapeutic efficacy of anti- EGFR antibodies regardless of the site of the primary tumor. Novel biomarkers will continue to be developed from a variety of approaches to provide more optimal cancer treatment for each individual patient.


Subject(s)
Antineoplastic Agents , Colonic Neoplasms , Colorectal Neoplasms , Antibodies, Monoclonal/therapeutic use , Antineoplastic Agents/therapeutic use , Biomarkers , Biomarkers, Tumor/genetics , Cetuximab/therapeutic use , Colonic Neoplasms/drug therapy , Colorectal Neoplasms/pathology , Humans , Mutation , Proto-Oncogene Proteins B-raf/genetics , ras Proteins/genetics
13.
Commun Biol ; 5(1): 965, 2022 Sep 15.
Article in English | MEDLINE | ID: mdl-36109659

ABSTRACT

Spotted hyenas are an exception in the animal kingdom not only due to female dominance over males, but also because of the strict female linear hierarchy which determines priority of access to resources and produces considerable female reproductive skew. This special social system raises a question: what would become of a beneficial mutation if it occurred in a low-ranking female? We used several simulation models in order to address this question. Our modeling results indicate that such a social system may inhibit the establishment of a beneficial mutation. However, this negative effect may be counteracted by random choice of mates by females.


Subject(s)
Reproduction , Social Dominance , Animals , Female , Male , Mutation , Reproduction/genetics
14.
Orphanet J Rare Dis ; 17(1): 359, 2022 Sep 15.
Article in English | MEDLINE | ID: mdl-36109815

ABSTRACT

OBJECTIVE: Alström syndrome (ALMS) is a rare autosomal recessive genetic disorder that is caused by homozygous or compound heterozygous mutation in the ALMS1 gene. Dilated cardiomyopathy (DCM) is one of the well-recognized features of the syndrome ranging from sudden-onset infantile DCM to adult-onset cardiomyopathy, sometimes of the restrictive hypertrophic form with a poor prognosis. We aimed to evaluate severe cardiomyopathy in Alström syndrome in infancy and display susceptible specific mutations of the disease, which may be linked to severe DCM. Secondarily we reviewed published mutations in ALMS1 with cardiomyopathies in the literature. METHOD: We represent new mutagenic alleles related to severe cardiomyopathy and cardiac outcome in this patient cohort. We evaluated echocardiographic studies of nine Turkish patients diagnosed with Alström syndrome (between 2014 and 2020, at age two weeks to twenty years). Thus, we examined the cardiac manifestations of a single-centre prospective series of nine children with specific ALMS mutations and multisystem involvement. All patients underwent genetic and biochemical testing, electrocardiograms, and echocardiographic imaging to evaluate systolic strain with speckle tracking. RESULTS: Four of the patients died from cardiomyopathy. Three patients (including three of the four fatalities) with the same mutation (c.7911dupC [p.Asn2638Glnfs*24]) had cardiomyopathy with intra-familial variability in the severity of cardiomyopathy. Global longitudinal strain, a measure of systolic contractile function, was abnormal in all patients that can be measured. CONCLUSION: Cardiac function in ALMS patients with infantile cardiomyopathy appears to have different clinical spectrums depending on the mutagenic allele. The c.7911dupC (p. Asn2638Glnfs*24) mutation can be related to severe cardiomyopathy. Parents can be informed and consulted about the progression of severe cardiomyopathy in a child carrying this mutagenic allele.


Subject(s)
Alstrom Syndrome , Cardiomyopathies , Cardiomyopathy, Dilated , Adult , Alstrom Syndrome/diagnosis , Alstrom Syndrome/genetics , Cardiomyopathies/genetics , Cardiomyopathy, Dilated/diagnosis , Cardiomyopathy, Dilated/genetics , Child , Homozygote , Humans , Mutation/genetics
15.
Biochem Biophys Res Commun ; 628: 49-56, 2022 Nov 05.
Article in English | MEDLINE | ID: mdl-36081278

ABSTRACT

The coagulation factor 9 gene (FIX) point mutation contributes to most hemophilia B cases, providing ideal gene correction models. Here we identified the frequent mutation G20519A (R226Q) in FIX, which resulted in many severe and moderate hemophilia B patients. This study aimed to investigate the effect of HDR and base editing in correcting FIX mutant. We first constructed HEK293 and liver-derived cell lines Huh7 cells stabling carrying mutated FIX containing G20519A (HEK293-FIXmut and Huh7-FIXmut). Then, CRISPR/Cas9-based homology-directed repair (HDR) and base editing were used for the correction of this mutated point. We used Cas9 nickase (nCas9) mediated HDR and the advanced base editor ABE8e to correct G20519A and then measured the concentration and activity of FIX. Furthermore, we used the star-shaped poly(lysine) gene nanocarriers to deliver the ABE8e correction systems into HEK293-FIXmut and Huh7-FIXmut stem cells to correct mutated FIX. As a result, we found that gRNAs directed inefficient HDR in correcting G20519A. The ABE8e corrected the mutation efficiently in both HEK293-FIXmut and Huh7-FIXmut stem cells. In addition, the star-shaped poly(lysine) carriers delivered non-viral vectors into stem cells efficiently. The nanocarriers-delivered ABE8e system corrected mutated FIX in stem cells, and the stem cells secreted active FIX in high concentration. In conclusion, our study provides a potential alternative for correcting mutated FIX in hemophilia B patients.


Subject(s)
Hemophilia A , Hemophilia B , Blood Coagulation Factors/genetics , Blood Coagulation Factors/metabolism , CRISPR-Cas Systems/genetics , Deoxyribonuclease I/metabolism , Gene Editing/methods , HEK293 Cells , Hemophilia A/genetics , Hemophilia A/metabolism , Hemophilia B/genetics , Hemophilia B/therapy , Humans , Mutation , Mutation, Missense , Polylysine , Stem Cells/metabolism
16.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 39(9): 969-973, 2022 Sep 10.
Article in Chinese | MEDLINE | ID: mdl-36082567

ABSTRACT

OBJECTIVE: To analyze the clinical and genetic characteristics of a Chinese pedigree affected with developmental and epileptic encephalopathy 9. METHODS: N048: epilepsy full version gene detection panel-V2 and genome wide copy number variation analysis were carried out on the genomic DNA extracted from the peripheral blood samples. Amniotic fluid was also sampled for single nucleoticle polymorphism array (SNP-array) analysis. RESULTS: Both the mother and her daughter were found to have loss of heterozygosity at Xq21.31q22.1, with which exons of protocadherin 19 (PCDH19) gene was deleted. SNP-array showed the fetus to be a female and had arr[hg19]Xq21.31q22.1 (89 558 626-99 701 006)x1. The mother, daughter and fetus of this family all had developmental and epileptic encephalopathy 9. CONCLUSION: Variant of the PCDH19 gene probably underlay the Developmental and epileptic encephalopathy 9 in this pedigree.


Subject(s)
Epilepsy, Generalized , Epilepsy , Cadherins/genetics , China , DNA Copy Number Variations , Epilepsy/diagnosis , Epilepsy/genetics , Female , Humans , Mutation , Pedigree , Protocadherins
17.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 39(9): 979-982, 2022 Sep 10.
Article in Chinese | MEDLINE | ID: mdl-36082569

ABSTRACT

OBJECTIVE: To explore the genetic basis for a neonate featuring developmental delay. METHODS: Clinical examination and laboratory tests were carried out for the patient. Peripheral venous blood samples of the proband and his parents were extracted and subjected to target capture next generation sequencing. Candidate variant was verified by Sanger sequencing. RESULTS: The patient, a four-month-old male, has presented with developmental delay and weakness of limbs. Genetic testing revealed that he had harbored a novel c.1432C>T variant of the TNPO3 gene, which was inherited from his mother. The nonsense variant has resulted in premature termination of protein translation and was predicted to be pathogenic by bioinformatics analysis. CONCLUSION: The heterozygous c.1432C>T variant of the TNPO3 gene probably underlay the limb-girdle muscular dystrophies form 1F in this patient. Above finding has enriched the variation spectrum of the TNPO3 gene.


Subject(s)
Muscular Dystrophies, Limb-Girdle , Genetic Testing , Heterozygote , High-Throughput Nucleotide Sequencing , Humans , Infant , Male , Muscular Dystrophies, Limb-Girdle/genetics , Mutation , Phenotype , beta Karyopherins/genetics
18.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 39(9): 988-991, 2022 Sep 10.
Article in Chinese | MEDLINE | ID: mdl-36082571

ABSTRACT

OBJECTIVE: To explore the genetic etiology of a patient with glycogen storage diseases. METHODS: Clinical data of child and his parents were collected. The genes associated with glycogen storage diseases were subjected to high-throughput sequencing to screen the variants. Candidate variant was validated by Sanger sequencing. Pathogenicity of the variant was predicted by bioinformatic analysis. RESULTS: High-throughput sequencing results showed that the boy has carried a hemizygous c.749C>T (p.S250L) variant of the PHKA2 gene. Sanger sequencing verified the results and confirmed that it was inherited from his mother. This variant was unreported previously and predicted to be pathogenic by bioinformatic analysis. CONCLUSION: The patient was diagnosed with glycogen storage disease type IXa due to a novel c.749C>T (p.S250L) hemizygous variant of the PHKA2 gene. High-throughput sequencing can facilitate timely and accurate differential diagnosis of glycogen storage disease type IXa.


Subject(s)
Glycogen Storage Disease , Child , Family , Genetic Testing , Glycogen Storage Disease/diagnosis , Glycogen Storage Disease/genetics , Glycogen Storage Disease/pathology , High-Throughput Nucleotide Sequencing/methods , Humans , Male , Mutation , Phosphorylase Kinase/genetics
19.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 39(9): 1001-1004, 2022 Sep 10.
Article in Chinese | MEDLINE | ID: mdl-36082574

ABSTRACT

OBJECTIVE: To analyze the clinical characteristics and genetic etiology of a child with Helsmoortel-Van der Aa syndrome (HVDAS). METHODS: Genetic testing was carried out for the child and his parents, and the clinical phenotypes and genetic variants of reported cases were summarized through literature review. RESULTS: The child has featured peculiar facies, accompanied by autism spectrum disorder, intellectual disability and motor retardation, and curving of the second toes, which was unreported previously. Genetic testing revealed that the child has harbored a heterozygous c.2157C>G (p.Tyr719*) variant of the ADNP gene, which was not found in either parent. Based on the guidelines of the American College of Medical Genetics and Genomics, this variant was rated as pathogenic. Among 80 HVDAS cases described in the literature, most had various degrees of behavioral abnormalities, intellectual disability, language retardation and motor retardation, with common features involving the nervous system, gastrointestinal system and eye. Variants of the ADNP gene mainly included frameshift variants and nonsense variants, with the hotspot variants including p.Tyr719*, p.Asn832lysfs*81 and p.Arg730*. CONCLUSION: The clinical phenotype of the child is closely correlated with the heterozygous variant of the ADNP gene, which expanded the phenotypic spectrum of HVDAS. As HVDAS may involve multiple systems and have high phenotypic heterogeneity, genetic testing technology can facilitate accurately diagnose.


Subject(s)
Abnormalities, Multiple , Autism Spectrum Disorder , Autistic Disorder , Intellectual Disability , Abnormalities, Multiple/genetics , Autism Spectrum Disorder/genetics , Autistic Disorder/genetics , Homeodomain Proteins/genetics , Humans , Intellectual Disability/complications , Intellectual Disability/genetics , Mutation , Nerve Tissue Proteins/genetics , Rare Diseases/complications
20.
Methods Mol Biol ; 2569: 343-359, 2022.
Article in English | MEDLINE | ID: mdl-36083457

ABSTRACT

Effective population size (Ne) determines the amount of genetic diversity and the fate of genetic variants in a species and thus is an essential parameter in evolutionary genetics. There are standard approaches to determine the Ne of evolving species. For example, the long-term Ne of an extant species is calculated based on its unbiased global mutation rate and the neutral genetic diversity of the species. However, approaches for inferring Ne of ancestral lineages are less known. Here, we introduce an evolutionary genetic statistic and an analytical procedure to assess the efficiency of natural selection for deep nodes by calculating rates of nonsynonymous nucleotide substitutions leading to radical (dR) and conservative (dC) amino acid replacements, respectively. Given that radical variants are more likely to be deleterious than conservative ones, an elevated dR/dC ratio in gene families across the genome means an accelerated genome-wide accumulation of the more deleterious type of mutations (i.e., radical variants), which indicates that natural selection is less efficient and genetic drift becomes more powerful. Earlier approaches that calculate dR/dC do not consider the impact of nucleotide composition (G+C content) on the dR/dC result, which is partially accounted for in more recent methods. Here, we use these methods to demonstrate that genetic drift may have driven the early evolution of Prochlorococcus, the most abundant carbon-fixing photosynthetic bacteria in the ocean.


Subject(s)
Genetic Drift , Selection, Genetic , Evolution, Molecular , Genetic Variation , Genome , Models, Genetic , Mutation , Nucleotides
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