Search Results (17)

Calcium-dependent protein kinases (CDPKs) are crucial regulators in calcium-mediated signal transduction pathways, playing a pivotal role in plant response to abiotic stresses. However, there is still limited knowledge regarding the genes of the Populus tomentosa CDPK family and their underlying functions in response to arsenic (As) stress and arbuscular mycorrhizal fungi (AMF) colonization. In our study, 20 PtCDPKs were identified in the P. tomentosa genome. Phylogenetic analysis categorized these PtCDPK genes into four subgroups based on sequence homology. Motif analysis revealed that PtCDPK genes within the same group share a similar exon–intron structure, conserved domains, and composition. The promoters of PtCDPK genes were found to contain a multitude of cis-acting elements, including light-response elements, phytohormone-response elements, and stress-response elements. The analysis of genes provided insights into the evolutionary dynamics and expansion of the PtCDPK gene family within P. tomentosa. The PtCDPK genes exhibited a strong collinear relationship with the CDPK genes of two model plants, namely, Arabidopsis thaliana and Oryza sativa L. Specifically, 10 gene pairs showed collinearity with Arabidopsis; in contrast, 14 gene pairs were collinear with rice. Transcriptome analysis of gene expression levels in P. tomentosa roots under both As stress and arbuscular mycorrhizal fungi (AMF) colonization conditions revealed that 20 PtCDPK genes had differential expression patterns. Under As stress, AMF inoculation led to the upregulation of 11 PtCDPK genes (PtCDPKSK5, X2, 1-3, 20-1, 24, 26-X1-1, 26-X1-2, 29-1, 29-2, 32, and 32-X1) and the downregulation of 8 PtCDPK genes, including PtCDPK1-1, 1-2, 8-X1, 10-X4, 13, 20-2, 26-X2, and 26-X3. The RT-qPCR results for 10 PtCDPK genes were consistent with the transcriptome data, indicating that AMF symbiosis plays a regulatory role in modulating the expression of PtCDPK genes in response to As stress. The principal findings of this study were that PtCDPK genes showed differential expression patterns under As stress and AMF colonization, with AMF regulating PtCDPK gene expression in response to As stress. Our study contributes to developing a deeper understanding of the function of PtCDPKs in the Ca²⁺ signaling pathway of P. tomentosa under As stress and AMF inoculation, which is pivotal for elucidating the molecular mechanisms underlying As tolerance in AMF-inoculated P. tomentosa. Full article

Tree peony (Paeonia ostii T. Hong et J. X. Zhang) is an important medicinal and ornamental plant. It would be useful to propagate this plant in tissue culture, but it is difficult to induce root formation. Auxin plays a pivotal role in adventitious root formation, and ABCB transporter proteins are involved in auxin transport. To elucidate the function of the ABCB transporter family in P. ostii, we identified members of the ABCB gene family in the P. ostii genome and analyzed the functional characteristics of the putative proteins. In total, 29 ABCB genes were identified in P.ostii, distributed on five chromosomes. In a phylogenetic analysis, the PoABCBs were grouped into four subfamilies, with the largest being Subfamily I, characterized by their MDR structure. PoABCB genes in the same subfamily exhibited similar intron/exon arrangements and motif composition. The promoters of PoABCBs contained cis-acting elements associated with the photoresponse and hormone signaling. qRT-PCR analyses showed that, after treatment of tissue-cultured P. ostii seedlings with auxin, five PoABCB gene family members (PoABCB6, PoABCB10, PoABCB11, PoABCB12, and PoABCB16) were significantly upregulated during adventitious root development. These genes may play roles in the auxin response and adventitious root development of P. ostii in vitro. Full article

Spinal muscular atrophy (SMA) is a degenerative neuromuscular condition resulting from a homozygous deletion of the survival motor neuron 1 (SMN1) gene in 95% of patients. A timely diagnosis via newborn screening (NBS) and initiating treatment before the onset of symptoms are critical for improving health outcomes in affected individuals. We carried out a screening test by quantitative PCR (qPCR) to amplify the exon seven of SMN1 using dried blood spot (DBS) samples. From October 2021 to August 2024, a total of 31,560 samples were tested in the Valencian Community (Spain) and 4 of them were positive for SMA, indicating an incidence of 1/7890. Genetic confirmation was performed using multiplex ligation-dependent probe amplification (MLPA) and AmplideX PCR/CE SMN1/2 Plus kit, in parallel obtaining concordant results in survival motor neuron 2 (SMN2) gene copy number. Within the first few weeks of their lives, two of the four patients detected by NBS showed signs of severe hypotonia, becoming ineligible for treatment. The other two patients were the first presymptomatic patients with two copies of SMN2 to receive treatment with Risdiplam in Spain. In order to treat positive cases in their early stages, we conclude that the official deployment of SMA newborn screening is necessary. Full article

In newborn screening (NBS), it is important to consider the availability of multiplex assays or other tests that can be integrated into existing systems when attempting to implement NBS for new target diseases. Recent developments in innovative testing technology have made it possible to simultaneously screen for severe primary immunodeficiency (PID) and spinal muscular atrophy (SMA) using quantitative real-time polymerase chain reaction (qPCR) assays. We describe our experience of optional NBS for severe PID and SMA in Osaka, Japan. A multiplex TaqMan qPCR assay was used for the optional NBS program. The assay was able to quantify the levels of T-cell receptor excision circles and kappa-deleting recombination excision circles, which is useful for severe combined immunodeficiency and B-cell deficiency screening, and can simultaneously detect the homozygous deletion of SMN1 exon 7, which is useful for NBS for SMA. In total, 105,419 newborns were eligible for the optional NBS program between 1 August 2020 and 31 August 2023. A case each of X-linked agammaglobulinemia and SMA were diagnosed through the optional NBS and treated at early stages (before symptoms appeared). Our results show how multiplex PCR-based NBS can benefit large-scale NBS implementation projects for new target diseases. Full article

Spinal muscular atrophy 5q (SMA) is one of the most common neuromuscular inherited diseases and is the most common genetic cause of infant mortality. SMA is associated with homozygous deletion of exon 7 in the SMN1 gene. Recently developed drugs can improve the motor functions of infants with SMA when they are treated in the pre-symptomatic stage. With aim of providing an early diagnosis, newborn screening (NBS) for SMA using a real-time PCR assay with dried blood spots (DBS) was performed from January 2022 through November 2022 in Saint Petersburg, which is a representative Russian megapolis. Here, 36,140 newborns were screened by the GenomeX real-time PCR-based screening test, and three genotypes were identified: homozygous deletion carriers (4 newborns), heterozygous carriers (772 newborns), and wild-type individuals (35,364 newborns). The disease status of all four newborns that screened positive for the homozygous SMN1 deletion was confirmed by alternate methods. Two of the newborns had two copies of SMN2, and two of the newborns had three copies. We determined the incidence of spinal muscular atrophy in Saint Petersburg to be 1 in 9035 and the SMA carrier frequency to be 1 in 47. In conclusion, providing timely information regarding SMN1, confirmation of disease status, and SMN2 copy number as part of the SMA newborn-screening algorithm can significantly improve clinical follow-up, testing of family members, and treatment of patients with SMA. Full article

Background and Objectives: Hereditary multiple exostoses (HME) is a disease characterized by cartilage-capped bony protuberances at the site of growth plates of long bones. Functional mutations in the exostosin genes (EXT1 and EXT2) are reported to affect the hedgehog signalling pathways leading to multiple enchondromatosis. However, the exact role of each EXT protein in the regulation of heparan sulphate (HS) chain elongation is still an enigma. In this study, a Pakistani family with HME is investigated to find out the genetic basis of the disease. Materials and Methods: Genotyping of eight members of the family by amplifying microsatellite markers, tightly linked to the EXT1 and EXT2 genes. Results: The study revealed linkage of the HME family to the EXT1 locus 8q24.1. Sanger sequencing identified a heterozygous deletion (c.247Cdel) in exon 1 of EXT1, segregating with the disease phenotype in the family. In silico analysis predicted a shift in the frame causing an early stop codon (p.R83GfsX52). The predicted dwarf protein constituting 134 amino acids was functionally aberrant with a complete loss of the catalytic domain at the C-terminus. Interestingly, an alternative open reading frame 3 (ORF3) caused by the frame shift is predicted to encode a protein sequence, identical to the wild type and containing the catalytic domain, but lacking the first 100 amino acids of the wild-type EXT1 protein. Conclusion: Consequently, haploinsufficiency could be the cause of HME in the investigated family as the mutated copy of EXT1 is ineffective for EXT-1/2 complex formation. The predicted ORF3 protein could be of great significance in understanding several aspects of HME pathogenesis. Full article

Mitogen-activated protein kinase (MAPK) is an important component of the signal transduction pathway, which plays important roles in regulating plant growth and development, and abiotic stress. Potato (Solanum tuberosum L.) is one of the most popular tuber crops in the world. Genome-wide identification and analysis of the MAPK and MAPKK gene family in potato is not clear. A total of 20 MAPK genes and 8 MAPKK genes were identified in the potato genome. A conservative motif analysis showed that the MAPK protein contained a typical TxY phosphorylation site, and the MAPKK protein contained a conservative characteristic motif S/T-x5-S/T. Phylogenetic analysis showed that potato MAPK (mitogen-activated protein kinase) and MAPKK (mitogen-activated protein kinase kinase) were similar to Arabidopsis, including four groups of members A, B, C and D. Gene structure and promoter sequence analysis showed that all 28 gene family members of potato Solanum tuberosum MAPK (StMAPK) and StMAPKK have coding regions (CDS), and family members in the same group have similar intron and exon compositions, and that most cis-acting elements upstream of gene promoters elements have related to stress response. Chromosome location analysis found that MAPKs were unevenly distributed on 11 chromosomes, while MAPKKs were only distributed on chromosomes Chr. 03 and Chr. 12. Collinearity analysis showed that StMAPKK3 and StMAPKK6 have the same common ancestors among potato, pepper, and tomato. qRT-PCR results showed that the relative expressions of StMAPK14 and StMAPKK2 were significantly upregulated under low-temperature stress. These results could provide new insights into the characteristics and evolution of the StMAPK and StMAPKK gene family and facilitate further exploration of the molecular mechanism responsible for potato abiotic stress responses. Full article

Objectives: Several new cancer therapies targeting signaling pathways involved in the growth and progression of cancer cells were developed as personalized medicine. Our study aimed to identify epidermal growth factor receptor (EGFR) mutations for TKI treatment in non-small-cell lung cancer (NSCLC) Tunisian patients. Methods: Analysis of the TKI sensitivity mutations in exons 18 to 21 of the EGFR gene and exon 15 of the B-raf gene was performed in 79 formalin fixed-paraffin embedded (FFPE) NSCLC samples using pyrosequencing. Results: EGFR mutations were detected in 34 cases among 79 (43%), with the predominance of the L861Q in exon 21 found in 35.3% of the cases (12 out of 34). Deletions in exon 19 were found in 8 cases (23.5%), and only one young male patient had the T790M mutation. Three patients harbored composite EGFR mutations (p.E746_A750del/p.L861R, p.E746_S752>V/p.S768I, and p.G719A/p.L861Q). Furthermore, the EGFR mutated status was significantly more frequent in female patients (p = 0.019), in non-smoker patients (p = 0.008), and in patients with metastasis (p = 0.044). Moreover, the B-raf V600E was identified in 5 EGFR negative patients among 39 analyzed samples (13.15%). Conclusion: The p.L861Q localized in exon 21 of the EGFR gene was the most common mutation identified in our patients (35.3%), whereas the “classic” EGFR mutations such as Del19 and p.L858R were found in 23.5% and 11.7% of the cases, respectively. Interestingly, most of p.L861X mutation-carrying patients showed good response to TKI treatment. Altogether, our findings suggest a particular distribution of the EGFR-TKIs sensitivity mutations in Tunisian NSCLC patients. Full article

Solitary fibrous tumor is a rare subtype of soft-tissue sarcoma with a wide spectrum of histopathological features and clinical behaviors, ranging from mildly to highly aggressive tumors. The defining genetic driver alteration is the gene fusion NAB2–STAT6, resulting from a paracentric inversion within chromosome 12q, and involving several different exons in each gene. STAT6 (signal transducer and activator of transcription 6) nuclear immunostaining and/or the identification of NAB2–STAT6 gene fusion is required for the diagnostic confirmation of solitary fibrous tumor. In the present study, a new gene fusion consisting of Nuclear Factor I X (NFIX), mapping to 19p13.2 and STAT6, mapping to 12q13.3 was identified by targeted RNA-Seq in a 74-year-old female patient diagnosed with a deep-seated solitary fibrous tumor in the pelvis. Histopathologically, the neoplasm did not display nuclear pleomorphism or tumor necrosis and had a low proliferative index. A total of 378 unique reads spanning the NFIXexon8–STAT6exon2 breakpoint with 55 different start sites were detected in the bioinformatic analysis, which represented 59.5% of the reads intersecting the genomic location on either side of the breakpoint. Targeted RNA-Seq results were validated by RT-PCR/ Sanger sequencing. The identification of a new gene fusion partner for STAT6 in solitary fibrous tumor opens intriguing new hypotheses to refine the role of STAT6 in the sarcomatogenesis of this entity. Full article

In this study, the yak’s TLR4 gene alternative spliceosomes were investigated using PCR amplification and cloning to improve disease-resistance in yak and promote efficient utilization of yak’s resources. qRT-PCR was used to determine the expression levels of two alternatively spliced transcripts of the TLR4 gene in seven distinct tissues. To predict the function of proteins expressed by each TLR4 spliceosome, bioinformatic analysis of yak’s TLR4 protein structure and function was performed, which led to the identification of two alternative spliceosomes of yak’s TLR4 gene. The TLR4-X1 sequence length was 2526 bp, and it encoded full-length TLR4 protein (841 amino acids). The sequence length of the exon-2 deleted TLR4-X2 sequence was 1926 bp, and it encoded truncated TLR4 protein (641 amino acids). TLR4-X2 sequence was consistent with the predicted sequence of the TLR4 gene in GenBank. Each tissue showed significantly different expression levels of these two alternative spliceosomes. As per the bioinformatic analysis of the structure and function of TLR4 protein, deletion of exon-2 in the TLR4 gene resulted in frameshift mutations of the reading frame in the corresponding protein, which altered its ligand-binding and active sites. Besides, biological property such as substrate specificity of truncated TLR4 protein was also altered, leading to altered protein function. This study has laid a theoretical foundation for exploring the role of two variants of the TLR4 gene in yak’s disease resistance. Besides, this study’s data could be analyzed further to explore the molecular mechanism associated with disease-resistance in the yak. Full article

This article reports a case of a 7-year-old girl with Turner syndrome, treated with growth hormone (GH), who developed ovarian dysgerminoma. The patient karyotype was mosaic for chromosome Xq deletion: 46,X,del(X)(q22)/45,X. No Y chromosome sequences were present. Molecular studies revealed the presence of a driving mutation in exon 17 of the KIT gene in the neoplastic tissue, as well as Sonic-hedgehog (SHH) pathway activation at the protein level. The patient responded well to chemotherapy and remained in complete remission. This is the first case of dysgerminoma in a Turner syndrome patient with such oncogenic pathway. Full article

Approximately 10% of the epidermal growth factor receptor (EGFR) mutations in non-small-cell lung cancer (NSCLC) are uncommon EGFR mutations. Although the efficacy of second (2G) or third generation (3G) EGFR tyrosine kinase inhibitors (EGFR-TKIs) in the patients with uncommon EGFR mutation has been proven, further studies are warranted to define the optimal treatment approach for uncommon EGFR mutation-positive NSCLC. This study retrospectively investigated the treatment patterns and outcomes of patients with uncommon EGFR mutation-positive NSCLC from January 2011 to December 2019 at the Samsung Medical Center, Seoul, Korea. During the study, 2121 patients with EGFR mutation-positive NSCLC received first-generation (1G, gefitinib or erlotinib) or 2G EGFR-TKI (afatinib) as the first-line (1L) systemic therapy. Of this, 135 (6.4%) patients harbored uncommon EGFR mutations. Of 135, 54 (40%, 54/135) patients had overlapping mutations with major EGFR mutations. The objective response rate (ORR) for the 1L EGFR-TKI was 63.3%. The median progression-free survivals (PFSs) were 8.6 months (95% CI: 3.8–13.5), 11.7 months (95% CI: 6.6–16.7), 7.7 months (95% CI: 4.9–17.4), and 5.0 months (95% CI: 3.7–6.1) for major uncommon EGFR mutation (G719X, L861Q), compound mutation with major EGFR mutation (Del 19 or EGFR exon 21 p.L858R), other compound mutation, and other uncommon mutations, respectively. The median overall survivals (OSs) were 25.6 months (16.9–34.2), 28.8 (95% CI: 24.4–33.4), 13.5 months (95% CI: 7.4–27.8), and 9.4 months (95% CI: 3.4–10.5) for major uncommon EGFR mutation (G719X), compound mutation with major EGFR mutation (Del 19 or EGFR exon 21 p.L858R), other compound mutation, and other uncommon mutations, respectively. The response rate, median PFS, and OS were 63.3%, 16.3 months (95% CI: 15.6–16.9), and 37.5 months (95% CI: 35.4–39.6) for common EGFR mutation-positive NSCLC. After failing 1L EGFR-TKI, repeated tissue or liquid biopsy were carried out on 44.9% (35/78) of patients with T790M detected in 10/35 (28.6%) patients. With subsequent 3G EGFR-TKI after failing the first-line EGFR-TKI, the ORR and PFS for 3G EGFR-TKI were 80% and 8.9 months (95% CI: 8.0–9.8). These patients showed a median OS of 34.6 months (95% CI: 29.8–39.4). The ORR, PFS and OS were poorer in patients with uncommon (especially other compound and other uncommon mutation) than those with common EGFR mutations. T790M was detected in 28.6% of the uncommon EGFR mutation-positive patients for whom prior 1G/2G EGFR-TKIs failed and underwent repeat biopsy at the time of progression. Full article

Niemann Pick disease type C (NPC) is a neurovisceral disorder due to mutations in NPC1 or NPC2. This review focuses on poorly characterized clinical and molecular features of early infantile form of NPC (EIF) and identified 89 cases caused by NPC1 (NPC1) and 16 by NPC2 (NPC2) mutations. Extra-neuronal features were common; visceromegaly reported in 80/89 NPC1 and in 15/16 NPC2, prolonged jaundice in 30/89 NPC1 and 7/16 NPC2. Early lung involvement was present in 12/16 NPC2 cases. Median age of neurological onset was 12 (0–24) and 7.5 (0–24) months in NPC1 and NPC2 groups, respectively. Developmental delay and hypotonia were the commonest first detected neurological symptoms reported in 39/89 and 18/89 NPC1, and in 8/16 and 10/16 NPC2, respectively. Additional neurological symptoms included vertical supranuclear gaze palsy, dysarthria, cataplexy, dysphagia, seizures, dystonia, and spasticity. The following mutations in homozygous state conferred EIF: deletion of exon 1+promoter, c.3578_3591 + 9del, c.385delT, p.C63fsX75, IVS21-2delATGC, c. 2740T>A (p.C914S), c.3584G>T (p.G1195V), c.3478-6T>A, c.960_961dup (p.A321Gfs*16) in NPC1 and c.434T>A (p.V145E), c.199T>C (p.S67P), c.133C>T (p.Q45X), c.141C>A (p.C47X) in NPC2. This comprehensive analysis of the EIF type of NPC will benefit clinical patient management, genetic counselling, and assist design of novel therapy trials. Full article

The olive fruit fly (olf) Bactrocera oleae is the most damaging olive pest. The intensive use of organophosphates (OPs) to control it, led to an increase in resistance in field populations. This study assesses the presence and distribution of three mutations at the ace gene related to target site insensitivity to OPs in Spain. Samples from other Mediterranean countries were included as external references. Resistance-conferring alleles (from exons IV and VII of the ace gene) reached almost an 80% frequency in olf Spanish populations. In total, 62% of them were homozygous (RR/RR), this being more common in eastern mainland Spain. High frequencies of RR/RR individuals were also found in North Mediterranean samples. Conversely, in Tunisia, only sensitive alleles were detected. Finally, the exon X mutation ∆Q3 had an extremely low frequency in all samples. The high frequency of genotype RR/RR in Spain indicates high fitness in an agroecosystem treated with pesticides, in contrast to ∆Q3. At exon IV all flies carried the same haplotype for the allele conferring resistance. The sequence analysis at this exon suggests a unique origin and fast expansion of the resistant allele. These results provide evidence that OPs appropriate use is needed and prompt the search for alternative methods for olf pest control. Full article

Adipogenesis is a complicated but precisely orchestrated process mediated by a series of transcription factors. Our previous study has identified a novel long noncoding RNA (lncRNA) that was differentially expressed during bovine adipocyte differentiation. Because this lncRNA overlaps with miR-221 in the genome, it was named miR-221 host gene (MIR221HG). The purpose of this study was to clone the full length of MIR221HG, detect its subcellular localization, and determine the effects of MIR221HG on bovine adipocyte differentiation. The 5′ rapid amplification of cDNA ends (RACE) and 3′ RACE analyses demonstrated that MIR221HG is a transcript of 1064 nucleotides, is located on the bovine X chromosome, and contains a single exon. Bioinformatics analyses suggested that MIR221HG is an lncRNA and the promoter of MIR221HG includes the binding consensus sequences of the forkhead box C1 (FOXC1) and krüppel-like factor5 (KLF5). The semi-quantitative PCR and quantitative real-time PCR (qRT-PCR) of nuclear and cytoplasmic fractions revealed that MIR221HG mainly resides in the nucleus. Inhibition of MIR221HG significantly increased adipocyte differentiation, as indicated by a dramatic increment in the number of mature adipocytes and in the expression of the respective adipogenic markers, peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), and fatty acid binding protein 4 (FABP4). Our results provide a basis for elucidating the mechanism by which MIR221HG regulates adipocyte differentiation. Full article