Mutations in the bone morphogenetic protein 15 gene causing defective protein secretion in Cholistani infertile sheep

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.

Resumo 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.

Selective type II TRK inhibitors overcome xDFG mutation mediated acquired resistance to the second-generation inhibitors selitrectinib and repotrectinib

Neurotrophic receptor kinase (NTRK) fusions are actionable oncogenic drivers of multiple pediatric and adult solid tumors, and tropomyosin receptor kinase (TRK) has been considered as an attractive therapeutic target for "pan-cancer" harboring these fusions. Currently, two generations TRK inhibitors have been developed. The representative second-generation inhibitors selitrectinib and repotrectinib were designed to overcome clinic acquired resistance of the first-generation inhibitors larotrectinib or entrectinib resulted from solvent-front and gatekeeper on-target mutations. However, xDFG (TRKAG667C/A/S, homologous TRKCG696C/A/S) and some double mutations still confer resistance to selitrectinib and repotrectinib, and overcoming these resistances represents a major unmet clinical need. In this review, we summarize the acquired resistance mechanism of the first- and second-generation TRK inhibitors, and firstly put forward the emerging selective type II TRK inhibitors to overcome xDFG mutations mediated resistance. Additionally, we concluded our perspectives on new challenges and future directions in this field.

Mutations in the bone morphogenetic protein 15 gene causing defective protein secretion in Cholistani infertile sheep / Mutações no gene da proteína morfogenética óssea 15 causadoras de defeitos na secreção proteica em ovelhas inférteis Cholistani

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.

Research Analysis of SLC12A3 Gene Mutation and Molecular Mechanism in Gitelman Syndrome / 罕见病研究

ObjectiveThis study aimed at conducting retrospective analysis of the clinical symptoms and genetic mutations in 20 children with Gitelman syndrome treated at the Affiliated Children′s Hospital of Nanjing Medical University from August 2015 to November 2022 and also explored the molecular mechanism of the pathogenic high-frequency mutation D486N in the Chinese population.MethodsWe collected the clinical manifestations, growth and development status, laboratory examination results, and SLC12A3 gene variations of the patients. We distinguished the wild-type and mutant SLC12A3 genes overexpressed in human embryonic kidney 293T cells (HEK293T). We used protein immunoblotting to detect the expression level of NCC, and used immunofluorescence techniques to examine the subcellular localization of NCC. In addition, we investigated the impact of the high-frequency SLC12A3 gene mutation D486N on NCC protein expression and localization.ResultsIn the 20 patients with Gitelman syndrome, all of them had hypokalemia. We indemnified twenty-six SLC12A3 gene mutations, 13 of which are missense mutation, 1 of which synonymous mutation, 1 nonsense mutation, 4 frameshift mutation, and 7 splicing site mutation. Among them, four mutations (p.T235K, c.1096-1G > A, p.A464A, and c.2660+1_2660+2insT) were novel mutations.ConclusionsWe found the preliminary evidence that the high-frequency mutation D486N in the Chinese population affected the expression of total and membrane-bound NCC protein and influenced the membrane localization of NCC protein. The findings of this study provides experimental evidence for genetic counseling, diagnosis, and treatment of Gitelman syndrome.

Resistencia transmitida de VIH-1 en pacientes sin exposición a tratamiento antirretroviral: Guatemala / Transmitted HIV-1 resistance in patients without exposure to antiretroviral treatment: Guatemala / Resistência transmitida ao HIV-1 em pacientes sem exposição ao tratamento antirretroviral: Guatemala

El objetivo del estudio fue describir los niveles de resistencia transmitida de VIH-1 en adultos atendidos en Unidades de Atención Integral de Guatemala. El estudio incluyó registros de 185 pacientes adultos VIH-1 positivo, de reciente diagnóstico sin antecedente de uso de TAR, de noviembre del 2019 a noviembre del 2020. El análisis se realizó en el software DeepChek® v2.0, para la clasificación de la resistencia se siguió el algoritmo de Stanford HIVdb (v9.4 - 07/12/2022). Se encontró 18.4% (IC 95% 13.1 - 24.7%) de resistencia general a alguna familia de ARVs. Se evidenció 15.1% (IC 95% 10.3 - 21.1%) de resistencia individual a la familia de INNTR afectando principalmente a NVP y EFV; 2.2% (IC 95% 0.6 - 5.4%) de resistencia a INTR, mayormente a FTC/3TC; y 2.7% (IC 95% 0.9 - 6.2%) de resistencia intermedia y baja los IP NFV y LPV/r. Tres casos presentaron resistencia múltiple a los INTR + INNTR. Las mutaciones más frecuentemente encontradas fueron K103N (41.2%), M184V/I (8.8%) y M46I (5.9%). La elevada resistencia transmitida del VIH-1 en pacientes atendidos en distintas Unidades de Atención Integral del VIH, demuestra la importancia de analizar periódicamente la tendencia de la resistencia en personas que no han estado expuestas a ARVs, lo cual a su vez es un marcador indirecto de presencia de resistencia adquirida en el país, datos que evidencian la necesidad de acciones e intervenciones prontas y efectivas dado su impacto en la salud pública.

The objective of this study was to describe the levels of transmitted HIV-1 resistance in patients with a recent HIV diagnosis before starting ART, treated in Comprehensive Care Units in Guatemala during the years 2019 and 2020. The study included records of 185 HIV-positive adult patients, recently diagnosed with HIV without a history of ART use. The analysis was carried out in the DeepChek® v2.0 software, the Stanford HIVdb algorithm (v9.4 - 07/12/2022) was followed to classify resistance. 18.4% (95% CI 13.1 - 24.7%) of general resistance to some family of ARVs was found. There was evidence of 15.1% (95% CI 10.3 - 21.1%) of individual resistance to the NNRTI family, mainly affecting NVP and EFV; 2.2% (95% CI 0.6 - 5.4%) resistance to INTR, mostly to FTC/3TC; and 2.7% (95% CI 0.9 - 6.2%) of intermediate and low resistance IP NFV and LPV/r. Three cases presented multiple resistance to NRTIs + NNRTIs. The most frequently found mutations were K103N (41.2%), M184V/I (8.8%) and M46I (5.9%). The high transmitted resistance of HIV-1 in patients treated in different Comprehensive HIV Care Units demonstrates the importance of periodically analyzing the trend of resistance in people who have not been exposed to ARVs, which in turn is an indirect marker. of the presence of acquired resistance in the country, data that demonstrate the need for prompt and effective actions and interventions given its impact on public health.

O objetivo deste estudo foi descrever os níveis de resistência transmitida ao HIV-1 em adultos tratados em Unidades de Cuidados Integrais na Guatemala. O estudo incluiu prontuários de 185 pacientes adultos HIV-1 positivos, recentemente diagnosticados sem histórico de uso de TARV, no período de novembro de 2019 a novembro de 2020. A análise foi realizada no software DeepChek® v2.0, para classificação da resistência, O algoritmo Stanford HIVdb (v9.4 - 07/12/2022) foi seguido. Foi encontrada 18.4% (IC 95% 13.1 - 24.7%) de resistência geral a alguma família de ARVs. Houve evidência de 15.1% (IC 95% 10.3 - 21.1%) de resistência individual à família de NNRTI, afetando principalmente NVP e EFV; 2.2% (IC 95% 0.6 - 5.4%) resistência ao INTR, principalmente ao FTC/3TC; e 2.7% (IC 95% 0.9 - 6.2%) de resistência intermediária e baixa ao IP NFV e LPV/r. Três casos apresentaram resistência múltipla a NRTIs + NNRTIs. As mutações mais frequentemente encontradas foram K103N (41.2%), M184V/I (8.8%) e M46I (5.9%). A elevada resistência transmitida do HIV-1 em pacientes atendidos em diferentes Unidades de Cuidados Integrados ao HIV demonstra a importância de analisar periodicamente a tendência de resistência em pessoas que não foram expostas aos ARVs, o que por sua vez é um marcador indireto da presença de ARVs adquiridos. resistência no país, dados que demonstram a necessidade de ações e intervenções rápidas e eficazes dado o seu impacto na saúde pública.

Clinicopathological and molecular analyses of uterine carcinosarcomas using next-generation sequencing: A single-center experience

Background: Uterine carcinosarcomas (UCS) constitute 3–4% of all uterine malignancies and 16% of deaths caused due to uterine neoplasms. Aim: In this study, we aimed to perform DNA-based mutation analysis in 12 genes (KRAS, NRAS, EGFR, C-KIT, BRAF, PDGFRA, ALK, ERBB2, ERBB3, ESR1, RAF1, PIK3CA) to determine the molecular subtypes of UCS using next-generation sequencing (NGS) in patients with aggressive UCS and poor prognosis. We aimed to compare the results of our analysis with clinicopathological data to contribute to the development of targeted therapy approaches related to the molecular changes of UCS. Materials and Methods: In this study, we included 12 cases diagnosed with uterine carcinosarcomas and examined the changes in oncogenes that play a role in UCS pathogenesis. For the analysis of mutation, the clinicopathological data were compared with the variations in the DNA-based gene panel consisting of 12 genes and 1237 variants in the UCS using the NGS method. Results: EGFR mutation was found in 91.7% of the cases, mutation in 41.7%, PDGFRA mutation in 25%, KRAS and PIK3CA mutation in 16.7%, and C-KIT mutation in 8.3% of the cases. Although no statistical significance was found between the detected mutation and clinicopathological data, it was concluded that PDGFRA mutation might be associated with advanced-stage disease development. Conclusion: This study's findings regarding different molecular types of UCS and information on oncogenesis of UCS can provide inferences for targeted therapies in the future by identifying targetable mutations representing early oncogenic events and thereby contribute toward further studies on this subject.

Relación estructura-función de la enzima uroporfirinógeno descarboxilasa: implicancias para la comprensión y el tratamiento de la porfiria cutánea tarda / Structure-function relationship of the enzyme uroporphyrinogen decarboxylase: implications for the understanding and treatment of porphyria cutanea tarda / Relação estrutura-função da enzima uroporfirinogênio descarbioxilase: implicações para a compreensão e tratamento da porfiria cutânea tardia / Reconocimiento a la trayectoria del Prof. Dr. Juan Miguel Castagnino†

Resumen La uroporfirinógeno descarboxilasa humana (UROD-h) es la quinta enzima del camino biosintético del hemo y su actividad deficiente, relacionada a mutaciones en su gen, se encuentra asociada a un subgrupo de porfirias. El objetivo de este trabajo fue estudiar la relación entre la dimerización de la enzima y su actividad enzimática y comprobar si la dimerización de UROD-h es imprescindible tanto para la primera etapa de la reacción (urogen→heptagen), como para la segunda etapa (heptagen→coprogen). Con ese objetivo, se expresó y purificó la UROD-h hasta homogeneidad, se analizó el comportamiento dímero-monómero bajo distintas condiciones que pudieran desplazar el equilibrio de dimerización y se evaluó la actividad enzimática en dichas condiciones. Los resultados obtenidos sugieren que la especie activa para la primera etapa de la reacción es el homodímero y que tanto el dímero como el monómero se comportan como especies activas para la segunda etapa de la reacción. Se propone que mutaciones clínicas como la Y311C, existentes en pacientes con porfiria cutánea tarda, podrían afectar la estabilidad del dímero y podrían ser el blanco para futuras terapias génicas.

Abstract Human uroporphyrinogen decarboxylase (UROD-h) is the fifth enzyme in the heme biosynthetic pathway and its deficient activity, related to mutations in its gene, is associated with a subset of porphyrias. The objective of this work was to study the relationship between the dimerisation of the enzyme and its enzymatic activity and to verify if the dimerisation of UROD-h is essential both for the first stage of the reaction (urogen→heptagen), and for the second stage (heptagen→ coprogen). With this objective, the UROD-h was expressed and purified to homogeneity, the dimer- monomer behaviour was analysed under different conditions, which could shift the dimerisation equilibrium, and the enzymatic activity was evaluated under these conditions. The results obtained suggest that the active species for the first stage of the reaction is the homodimer, and both the dimer and the monomer behaved as active species for the second stage of the reaction. It is proposed that clinical mutations such as Y311C, existing in porphyria cutanea tarda patients, could affect dimer stability and could be the target of future gene therapies.

Resumo A enzima uroporfirinogênio descarboxilase humana (UROD-h) é a quinta enzima da via biossintética do heme e sua atividade deficiente, relacionada com mutações em seu gene, está associada a um subgrupo de porfirias. O objetivo deste trabalho foi estudar a relação entre a dimerização da enzima e sua atividade enzimática e comprovar se a dimerização da UROD-h é imprescindível tanto para a primeira etapa da reação (urogênio→heptagênio), quanto para a segunda etapa (heptagênio→coprogênio). Com esse objetivo, a UROD-h foi expressa e purificada até a homogeneidade, o comportamento de dímero-monômero foi analisado sob diversas condições, que puderam deslocar o equilíbrio de dimerização, e a atividade enzimática foi avaliada em tais condições. Os resultados obtidos sugerem que a espécie ativa para a primeira etapa da reação é o homodímero, e tanto o dímero quanto o monômero se comportam como espécies ativas para a segunda etapa da reação. Propõe-se que mutações clínicas como Y311C, existentes em pacientes com porfiria cutânea tardia, poderiam afetar a estabilidade do dímero e poderiam ser o alvo de futuras terapias gênicas em porfiria cutânea tardia.

Current status of insecticide resistance and its underlying mechanisms in Aedes aegypti (L.) in Punjab, Pakistan

Aedes aegypti (Linnaeus) plays an important role as a vector of different deadly diseases particularly dengue fever. Insecticides are used as a primary tool to control Ae. aegypti. However, due to the excessive use of insecticides on agricultural, public health, and industrial levels, mosquitoes have developed resistance. In this study, the current susceptibility status of Ae. aegypti mosquitoes against different insecticides (Temephos, DDT, dieldrin, Malathion, Bendiocarb, Permethrin, Cypermethrin, and Lambda-cyhalothrin) was evaluated in district Lahore and district Muzaffargarh of Punjab, Pakistan. For this purpose, WHO bioassays and biochemical assays were performed on Ae. aegypti population from Lahore (APLa) and Aedes population from Muzaffargarh (APMg). Results of APLa and APMg showed high levels of resistance against the larvicide Temephos. Resistance against all adulticides was also observed in APLa and APMg (% mortality < 98%). The biochemical assays indicated statistically significant elevated levels of detoxification enzymes in APLa and APMg. APLa showed slightly higher levels as compared to APMg. Mosquitoes were also screened for the presence of kdr mutations. The results revealed no mutation in domain II while the presence of mutation F1534C in domain III was found in both field populations. The results showed the presence of moderate to high grade resistance against all insecticides in Ae. aegypti in district Lahore and district Muzaffargarh of Punjab, Pakistan

Predicting and overcoming resistance to CDK9 inhibitors for cancer therapy

Abnormally activated CDK9 participates in the super-enhancer mediated transcription of short-lived proteins required for cancer cell survival. Targeting CDK9 has shown potent anti-tumor activity in clinical trials among different cancers. However, the study and knowledge on drug resistance to CDK9 inhibitors are very limited. In this study, we established an AML cell line with acquired resistance to a highly selective CDK9 inhibitor BAY1251152. Through genomic sequencing, we identified in the kinase domain of CDK9 a mutation L156F, which is also a coding SNP in the CDK9 gene. By knocking in L156F into cancer cells using CRISPR/Cas9, we found that single CDK9 L156F could drive the resistance to CDK9 inhibitors, not only ATP competitive inhibitor but also PROTAC degrader. Mechanistically, CDK9 L156F disrupts the binding with inhibitors due to steric hindrance, further, the mutation affects the thermal stability and catalytic activity of CDK9 protein. To overcome the drug resistance mediated by the CDK9-L156F mutation, we discovered a compound, IHMT-CDK9-36 which showed potent inhibition activity both for CDK9 WT and L156F mutant. Together, we report a novel resistance mechanism for CDK9 inhibitors and provide a novel chemical scaffold for the future development of CDK9 inhibitors.

TCOF1 Gene variation in Treacher Collins syndrome and evaluation of speech rehabilitation after bone bridge surgery / 临床耳鼻咽喉头颈外科杂志

Objective:By analyzing the clinical phenotypic characteristics and gene sequences of two patients with Treacher Collins syndrome（TCS）, the biological causes of the disease were determined. Then discuss the therapeutic effect of hearing intervention after bone bridge implantation. Methods:All clinical data of the two family members were collected, and the patients signed the informed consent. The peripheral blood of the proband and family members was extracted, DNA was extracted for whole exome sequencing, and Sanger sequencing was performed on the family members for the mutation site.TCOF1genetic mutations analysis was performed on the paitents. Then, the hearing threshold and speech recognition rate of family 2 proband were evaluated and compared under the sound field between bare ear and wearing bone bridge. Results:In the two pedigrees, the probands of both families presented with auricle deformity, zygomatic and mandibular hypoplasia, micrognathia, hypotropia of the eye fissure, and hypoplasia of the medial eyelashes. The proband of Family 1 also presents with specific features including right-sided narrow anterior nasal aperture and dental hypoplasia, which were consistent with the clinical diagnosis of Treacher Collins syndrome. Genetic testing was conducted on both families, and two heterozygous mutations were identified in the TCOF1 gene: c. 1350_1351dupGG（p. A451Gfs*43） and c. 4362_4366del（p. K1457Efs*12）, resulting in frameshift mutations in the amino acid sequence. Sanger sequencing validation of the TCOF1 gene in the parents of the proband in Family 1 did not detect any mutations. Proband 1 TCOF1 c. 1350_1351dupGG heterozygous variants have not been reported previously. The postoperative monosyllabic speech recognition rate of family 2 proband was 76%, the Categories of Auditory Performance（CAP） score was 6, and the Speech Intelligibility Rating（SIR） score was 4. Assessment using the Meaningful Auditory Integration Scale（MAIS） showed notable improvement in the patient's auditory perception, comprehension, and usage of hearing aids. Evaluation using the Glasgow Children's Benefit Inventory and quality of life assessment revealed significant improvements in the child's self care abilities, daily living and learning, social interactions, and psychological well being, as perceived by the parents. Conclusion:This study has elucidated the biological cause of Treacher Collins syndrome, enriched the spectrum of TCOF1 gene mutations in the Chinese population, and demonstrated that bone bridge implantation can improve the auditory and speech recognition rates in TCS patients.

Analysis of the ocular clinical features and pathogenic genes in patients with Alstr?m syndrome / 中华眼底病杂志

Objective:To observe and analyze the ocular clinical features and pathogenic genes of Alstr?m syndrome (ALMS).Methods:A retrospective clinical study. From October 2020 to July 2022, 3 patients and 5 normal family members from 2 families affected with ALMS who visited in the Ophthalmology Department of Henan Children's Hospital were enrolled in the study. These 2 families were without blood relationship. The medical history and family history were inquired. Best corrected visual acuity (BCVA), fundus color photography, full-field electroretinogram (ERG), frequency domain optical coherence tomography (OCT) and systemic examination were performed. 3 ml peripheral venous blood of patients and their family members were collected, and the whole genomic DNA was extracted. The second generation sequencing analysis was performed on these members. The suspected pathogenic mutation sites were verified by Sanger, and the pathogenicity of the gene mutation sites were determined by bioinformatics analysis.Results:Three patients from two families all developed nystagmus and photophobia in infancy. In the family 1, the BCVA of both eyes of the proband was no light perception. The fundus examination revealed vascular attenuation and retinal pigment abnormality. OCT showed retinal thinning, loss of photoreceptor layer and atrophy of the retinal pigment epithelium layer. ERG examination showed extinguished. The BCVA of the proband’s younger brother was 0.04 in the right eye and 0.02 in the left eye. The fundus examination revealed vascular attenuation but the pigment distribution was roughly normal. OCT showed blurred photoreceptor layers in both eyes. ERG examination showed extinguished. Two patients developed sensorineural deafness, obesity, acanthosis nigricans, insulin resistance/diabetes, and abnormal liver function. In addition, the proband also had left heart enlargement, hyperlipidemia and abnormal kidney function. The results of genetic testing showed that the proband and his younger brother had compound heterozygous mutations in exon 8 (c.1894C>T/p.Gln632*, M1) and exon 10 (c.9148_9149delCT/p.Leu 3050 Leufs*9, M2) of ALMS1, which were both known mutations. The father of the proband was a carrier of M1 and the mother of the proband was a carrier of M2. The proband of the family 2 had a normal fundus at 23 months old. The amplitude of ERG b wave under the stimulation of the dark adaptation 0.01 and a, b wave under the stimulation of dark adaptation 0.3 were all mild reduced. The amplitude of ERG a, b wave under the stimulation of the light adaptation 0.3 was severity decreased. At 4 years old, the BCVA was 0.01 in the right eye and 0.05 in the left eye. The fundus examination revealed vascular attenuation and bilateral blunted foveal reflex. In addition to severely diminished of a, b wave under the stimulation of dark adaptation 0.3, the rest showed extinguished. There were no systemic abnormalities. The results of genetic testing showed that the proband had compound heterozygous mutations in exon 11 (c.9627delT/p.Pro3210Glnfs*22, M3) and exon 5 (c.1089delT/p.Asp364Ilefs*13, M4) of ALMS1, which were both novel mutations. The father of the proband was a carrier of M3 and the mother of the proband was a carrier of M4. Conclusions:Nystagmus and photophobia are often the first clinical manifestations of ALMS. In the early stage, the fundus can be basically normal. As the disease progresses, the fundus examination reveals vascular attenuation and retinal pigment abnormality, and the reflection of the fovea is unclear. OCT shows the photoreceptor cell layers are blurred or even lost. The final ERG is extinguished. M1, M2, and M3, M4 compound heterozygous mutations may be the pathogeny for family 1 and family 2, respectively.

The efficacy and safety comparison of radium-223 in the treatment of mCRPC patients with and without homologous recombination repair gene mutation / 中华泌尿外科杂志

Objective:To compare the efficacy and safety of radium-223 in the treatment of metastatic castration-resistant prostate cancer (mCRPC) patients with and without homologous recombination repair (HRR) gene mutation.Methods:The clinical data of 27 patients with mCRPC bone metastases who received radium-223 therapy from April 2021 to November 2022 in Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine were retrospectively analyzed. Among the 27 mCRPC patients, 18 patients carrying HRR gene mutations belonged to the HRD(+ ) group, and 9 patients without HRR gene mutation belonged to the HRD(-) group. The age of patients in HRD(+ ) group was 69.5 (63.8, 77.0) years old, alkaline phosphatase (ALP) was 243.0 (82.8, 301.3) U/L, prostate specific antigen (PSA) was 71.6 (7.3, 329.8) ng/ml, pain score was 3.0 (1.0, 5.0) points. Eastern Cooperative Oncology Group (ECOG) score ranged from 0 to 1 points in 7 cases, and 2 points in 11 cases. In the HRD(-) group, the median age was 72.0 (64.5, 76.5) years old, ALP was 88.0 (67.5, 260.6) U/L, PSA was 19.1 (1.1, 117.8) ng/ml, and pain score was 2.0 (0, 4.5) points. The ECOG score ranged from 0 to 1 in 4 cases, and 2 in 5 cases in the HRD(-) group. There was no significant difference in the above general data between the two groups ( P>0.05). All patients received radium-223 treatment every 4 weeks, no more than 6 times. The changes of ALP, PSA, pain score and hematological adverse reactions were compared between the two groups. Results:In the HRD(+ ) group, the median number of radium-223 treatment was 4.5 (3.0, 5.3) couses, 4 patients (22.2%) completed 6 courses, and 6 patients died of prostate cancer during follow-up. In the HRD(-) group, the median number of radium treatment was 4.0 (2.5, 6.0) couses, 3 patients (33.3%) completed 6 courses, and 1 patient died of prostate cancer during follow-up. There was no significant difference in the number of radium treatment courses between the two groups ( P=0.320). ALP in HRD(+ ) group was 101.8 (61.3, 147.0) U/L after radium-223 treatment, which was significantly lower than that before treatment ( P=0.002). ALP in HRD(-) group was 73.0 (64.0, 113.5) U/L after radium-223 treatment, and it was not significantly different from that before treatment ( P=0.327). The rate of ALP response (ALP decrease >10%) in HRD(+ ) group was significantly higher than that in HRD(-) group [83.3% (15/18) vs. 44.4% (4/9), P=0.037]. PSA was 105.9(5.2, 798.4) ng/ml in HRD (+ ) group after radium-223 treatment, and was 25.6(0.8, 1 031.0) ng/ml in HRD(-) group, and they were not significantly different from that before treatment ( P=0.145, P=0.386). There were no significant differences in the rate of PSA response (PSA decrease>10%) between HRD(+ ) group and HRD(-) group [38.9% (7/18) vs. 22.2% (2/9), P=0.386]. The median pain score of HRD(+ ) group was 3.0 (0, 4.0) points after treatment, which was significantly lower than that before treatment ( P=0.028). The pain score of HRD(-) group was 1.0(0, 3.0) points after treatment, and it was not significantly different from that before treatment ( P=0.129). There was no significant difference in pain relief rate between HRD(+ ) group and HRD(-) group [66.7% (12/18) vs. 44.4% (4/9), P=0.411]. The incidence of at least one hematological adverse event during radium-223 treatment in the HRD(+ ) group was higher than that in the HRD(-) group [77.8% (14/18) vs. 33.3% (3/9), P=0.039]. There was no significant difference in the incidence of grade 1-2 hematological adverse events between the two groups [72.2%(13/18) vs. 33.3%(3/9), P=0.097]. Only 1 patient in the HRD(+ ) group experienced grade 3 anemia during treatment which was recovered after blood transfusion. Conclusions:Compared to mCRPC patients without HRR gene mutation, patients with HRR gene mutations had better ALP response and bone pain relief after radium-223 treatment. The overall incidence of adverse events in the HRD(+ ) group is higher than that in HRD(-) group, and there was no significant difference in grade 1-2 hematological adverse events between the two goups. It is necessary to expand the sample size to further verify the conclusion.

Progress on the impact of pathogenic gene mutations in epilepsy on treatment choices / 国际儿科学杂志

In the past decade, significant progress has been made in the study of epilepsy-causing genetic mutations and the molecular mechanisms of epilepsy clinical manifestations.A growing number of studies have shown that the mechanism of action of pathogenic genes related to clinical symptoms shows significant correlation.In the selection of antiepileptic drugs for patients with different gene mutation, early identification of pathogenic genes has guiding significance for the selection of antiepileptic drugs.This review summairzes common epilepsy pathogenic genes, including ion channels genes, cellular metabolism related genes and cell signaling pathway related genes, and research progress on therapeutic targets corresponding to pathogenic genes in recent years.As research deepens, specific gene defects and their machanisms of action provide a basis for studying new treatment methods.

Construction of a High-Throughput Mutation Library of Mycobacterium Rifampin Resistance Gene rpoB / 中山大学学报(医学科学版)

ObjectiveTo establish a mutation library of rifampicin resistance gene rpoB. MethodsThe ΔrpoB attB：：rpoB strain of Mycobacterium smegmatis （M. smegmatis） be constructed by homologous recombination and L5 attB phage integration site exchange. Based on the L5 attB plasmid exchange system and resistance selection medium， 48 clones are selected to verify plasmid replacement efficiency. Degenerate primers are designed every 3 bases in the rifampicin resistance determining region （RRDR）， and a full-coverage mutation library of 81 bases in RRDR region is obtained by PCR amplification. The library fragments are seamlessly cloned into the vector and transformed into Escherichia coli （E. coli）to form an E. coli mutation library. Based on the principle of plasmid exchange， the mutant plasmid library is transformed into the M. smegmatis strain ΔrpoB attB：：rpoB， and the original L5 attB site plasmid is replaced to form the M. smegmatis mutant library. The genotype of the library are determined by genome extraction， library construction and high-throughput sequencing. ResultsCompared with the wild-type rpoB gene （5 600 bp）， the amplified fragment of the rpoB knockout strain is 2 200 bp， which proved that the ΔrpoB attB：：rpoB conditional knockout strain of M. smegmatis is successfully constructed. The success rate of plasmid replacement is 100%. There were 540 kinds of single amino acid mutations in both E. coli library and M. smegmatis library， 5 301 kinds of multi-point mutations in E. coli library， and 853 kinds of multi-point mutations in M. smegmatis library. The correlation coefficient between E. coli library and M. smegmatis library is 0.84. ConclusionsWe have developed a strategy to construct a library of mutants targeting the essential mycobacterial gene rpoB， and successfully established a mutant library of rifampicin resistance gene rpoB.

Human 8-cell embryos enable efficient induction of disease-preventive mutations without off-target effect by cytosine base editor

Approximately 140 million people worldwide are homozygous carriers of APOE4 (ε4), a strong genetic risk factor for late onset familial and sporadic Alzheimer's disease (AD), 91% of whom will develop AD at earlier age than heterozygous carriers and noncarriers. Susceptibility to AD could be reduced by targeted editing of APOE4, but a technical basis for controlling the off-target effects of base editors is necessary to develop low-risk personalized gene therapies. Here, we first screened eight cytosine base editor variants at four injection stages (from 1- to 8-cell stage), and found that FNLS-YE1 variant in 8-cell embryos achieved the comparable base conversion rate (up to 100%) with the lowest bystander effects. In particular, 80% of AD-susceptible ε4 allele copies were converted to the AD-neutral ε3 allele in human ε4-carrying embryos. Stringent control measures combined with targeted deep sequencing, whole genome sequencing, and RNA sequencing showed no DNA or RNA off-target events in FNLS-YE1-treated human embryos or their derived stem cells. Furthermore, base editing with FNLS-YE1 showed no effects on embryo development to the blastocyst stage. Finally, we also demonstrated FNLS-YE1 could introduce known protective variants in human embryos to potentially reduce human susceptivity to systemic lupus erythematosus and familial hypercholesterolemia. Our study therefore suggests that base editing with FNLS-YE1 can efficiently and safely introduce known preventive variants in 8-cell human embryos, a potential approach for reducing human susceptibility to AD or other genetic diseases.

Chinese Expert Consensus on Non-small Cell Lung Cancer with EGFR Exon 20 Insertion Mutations (2023 Edition) / 中国肺癌杂志

With the development of precision diagnosis and treatment for non-small cell lung cancer (NSCLC), the epidermal growth factor receptor (EGFR) exon 20 insertion (ex20ins) mutations, as a rare subset of EGFR mutaions, have gradually attracted attention recently. The heterogeneity of EGFR ex20ins mutations is very high, different variants have different clinical benefits, and the prognosis is extremely poor. The available traditional treatment outcomes are poor in patients with EGFR ex20ins positive NSCLC and polymerase chain reaction (PCR) tests would miss aprocimately 50% of the variants. Therefore, high attention should be paid to EGFR ex20ins positive NSCLC during the clinical practice. The expert panel has formed a consensus on the standardized clinical diagnosis and treatment of EGFR ex20ins mutation NSCLC through reference to literature and clinical data, and combined with the experts' own clinical experience, the consensus recommendations including clinicopathologic characteristics, therapies, testing methods and recent relevant clinical trials for NSCLC patients with EGFR ex20ins mutation, in order to provide medication reference for clinical physicians at all levels.

Research Progress on Gene Mutation and CHIP in Pathogenesis of MDS --Review / 中国实验血液学杂志

With the development of molecular biology techniques, the people's understanding of myelodysplastic syndromes (MDS) has greatly improved, a heterogeneous hematopoietic pre-malignant disorder of the stem cells. Gene mutations include RNA splicing, DNA methylation, chromosome modification, transcription factors, signal transduction kinases, RAS pathways, cohesion complexes, DNA repair, etc. Gene mutation is the determinant of diagnostic typing and therapeutic efficacy of MDS. The new concepts of CHIP and ICUS have aroused people's attention to the elderly patients with clonal hematopoiesis and non-clonal cytopenia but without MDS characteristics, who have the possibility of high-risk transformation to MDS and leukemia. In order to better understand the pathogenesis of MDS, the significance of gene mutations, CHIP and ICUS in the diagnosis and prognosis of MDS were reviewed in this paper.

Clinical and gene mutation characteristics of patients with hereditary ellipsocytosis: nine cases report and literature review / 中华血液学杂志

Objective: To report gene mutations in nine patients with hereditary elliptocytosis (HE) and analyze the characteristics of pathogenic gene mutations in HE. Methods: The clinical and gene mutations of nine patients clinically diagnosed with HE at Institute of Hematology & Blood Diseases Hospital from June 2018 to February 2022 were reported and verified by next-generation sequencing to analyze the relationship between gene mutations and clinical phenotypes. Results: Erythrocyte membrane protein gene mutations were detected among nine patients with HE, including six with SPTA1 mutation, one with SPTB mutation, one with EPB41 mutation, and one with chromosome 20 copy deletion. A total of 11 gene mutation sites were involved, including 6 known mutations and 5 novel mutations. The five novel mutations included SPTA1: c.1247A>C (p. K416T) in exon 9, c.1891delG (p. A631fs*17) in exon 15, E6-E12 Del; SPTB: c.154C>T (p. R52W) ; and EPB41: c.1636A>G (p. I546V) . Three of the six patients with the SPTA1 mutation were SPTA1 exon 9 mutation. Conclusion: SPTA1 is the most common mutant gene in patients with HE.

Prevalence of Somatic BRCA1 and BRCA2 mutations in ovarian cancer among Filipinos using next generation sequencing

INTRODUCTION@#Ovarian cancer is one of the leading causes of mortality in women. In 2020, 5,395 (6.2%) of diagnosed malignancies in females were ovarian in origin. It also ranked second among gynecologic malignancies after cervical cancer. The prevalence in Asian /Pacific women is 9.2 per 100,000 population. Increased mortality and poor prognosis in ovarian cancer are caused by asymptomatic growth and delayed or absent symptoms for which about 70% of women have an advanced stage (III/IV) by the time of diagnosis. The most associated gene mutations are Breast Cancer gene 1 (BRCA1) which is identified in chromosome 17q21 and Breast Cancer gene 2 (BRCA2) identified in chromosome 13. Both proteins function in the double-strand DNA break repair pathway especially in the large framework repair molecules. Olaparib is a first-line drug used in the management of ovarian cancer. It targets affected cells by inhibition of poly (ADP-ribose) polymerase (PARP) activity which induces synthetic lethality in mutated BRCA1/2 cancers by selectively targeting tumor cells that fail to repair DNA double-strand breaks (DSBs).@*OBJECTIVE@#The study aims to determine the prevalence of pathogenic somatic mutations in BRCA1 and BRCA2 among patients diagnosed of having ovarian cancer, to characterize the identified variants into benign/ no pathogenic variant identified, variant of uncertain significance (VUS), and pathogenic, and to determine the relationship of specific mutations detected with histomorphologic findings and clinical attributes.@*METHODOLOGY@#Ovarian cancer tissues available at the St. Luke’s Medical Center Human Cancer Biobank and formalin-fixed paraffin-embedded (FFPE) tissue blocks diagnosed as ovarian cancer from the year 2016 to 2020 were included. Determination of the prevalence of somatic BRCA1 and BRCA2 mutations using Next Generation Sequencing (NGS).@*RESULTS@#A total of 60 samples were processed, and three samples were excluded from the analysis due to an inadequate number of cells. In the remaining 57 samples diagnosed ovarian tumors, pathogenic BRCA1/2 variants were identified in 10 (17.5%) samples. Among the BRCA1/2 positive samples, 3 (5.3%) BRCA1 and 7 (12.3%) BRCA2 somatic mutations were identified.@*CONCLUSION@#Identification of specific BRCA1/2 mutations in FFPE samples with NGS plays a big role in the management of ovarian cancer, particularly with the use of targeted therapies such as Olaparib. The use of this drug could provide a longer disease-free survival for these patients. Furthermore, we recommend that women diagnosed with ovarian cancer should be subjected to genetic testing regardless of the histologic subtypes or clinical features. Lastly, genetic testing should be done along with proper genetic counseling, especially for patients who are susceptible to these mutations.

Antitumor activity of aumolertinib, a third-generation EGFR tyrosine kinase inhibitor, in non-small-cell lung cancer harboring uncommon EGFR mutations

Uncommon epidermal growth factor receptor (EGFR) mutations account for 10%-20% of all EGFR mutations in non-small-cell lung cancer (NSCLC). The uncommon EGFR-mutated NSCLC is associated with poor clinical outcomes and generally achieved unsatisfactory effects to the current therapies using standard EGFR-tyrosine kinase inhibitors (TKIs), including afatinib and osimertinib. Therefore, it is necessary to develop more novel EGFR-TKIs to treat uncommon EGFR-mutated NSCLC. Aumolertinib is a third-generation EGFR-TKI approved in China for treating advanced NSCLC with common EGFR mutations. However, it remains unclear whether aumolertinib is effective in uncommon EGFR-mutated NSCLC. In this work, the in vitro anticancer activity of aumolertinib was investigated in engineered Ba/F3 cells and patient-derived cells bearing diverse uncommon EGFR mutations. Aumolertinib was shown to be more potent in inhibiting the viability of various uncommon EGFR-mutated cell lines than those with wild-type EGFR. And in vivo, aumolertinib could also significantly inhibit tumor growth in two mouse allograft models (V769-D770insASV and L861Q mutations) and a patient-derived xenografts model (H773-V774insNPH mutation). Importantly, aumolertinib exerts responses against tumors in advanced NSCLC patients with uncommon EGFR mutations. These results suggest that aumolertinib has the potential as a promising therapeutic candidate for the treatment of uncommon EGFR-mutated NSCLC.