Background Mutations in PIK3CA, which encodes p110 subunit of PI3K class IA enzyme, are highly frequent in breast cancer. Here, we aimed to probe mutations in exon 9 of PIK3CA and computationally simulate their function. Method PCR/HRM and PCR/sequencing were used for mutation detection in 40 breast cancer specimens. The identified mutations were queried via in silico algorithms to check the pathogenicity. The molecular dynamics (MD) simulations were utilized to assess the function of mutant proteins. Result Three samples were found to harbor at least one of the E542K, E545K and L551Q mutations of which L511Q has not been reported previously. All mutations were confirmed to be pathogenic and MD simulations revealed their impact on protein function and regulation. The novel L551Q mutant dynamics was similar to that of previously found carcinogenic mutants, E542K and E545K. A functional role for the helical domain was also suggested by which the inhibitory signal of p85α is conducted to kinase domain via helical domain. Helical domain mutations lead to impairment of kinase domain allosteric regulation. Interestingly, our results show that p110α substrate binding pocket of helical domain in mutants may have differential affinity for enzyme substrates, including anit-p110α drugs. Conclusion The novel p110α L551Q mutation could has carcinogenic feature similar to previously known mutations.
One of the most common somatic mutations in breast cancer is found in PIK3CA with a prevalence rate of 18-45%. Different variants of this gene are considered as resistance markers for treatment with HER2-targeted medicines. Conventional molecular methods such as Sanger sequencing are not able to detect mutations with low abundance in a mixture of wild-type DNA, especially in the early stages of cancer development. In this study, two methods of co-amplification at lower denaturation temperature PCR (COLD-PCR) and high-resolution melting (HRM) were combined for detection of mutations in exon 9 of PIK3CA; DNA, therefore, was extracted from MCF-7 and BT-474 as mutant and wild-type cell lines respectively. Thereafter, serial dilutions of extracted DNA were used to determine sensitivity of full-COLD PCR/HRM in comparison with conventional PCR sequencing as the gold standard method. Cell line experiments resulted in almost 30 fold increase in sensitivity by use of full-COLD PCR/HRM. In addition, 40 patients with primary breast cancer were investigated with the mentioned methods. As a result of this part of study, four mutations were detected by conventional PCR sequencing including E542K and E545K mutations in three and one samples respectively. Whereas, full-COLD PCR/HRM was able to detect one E542K mutation more than gold standard method which caused the percentage of sensitivity to get improved by 2.5% (10 to 12.5%). Our results clearly demonstrated that full-COLD PCR/HRM could detect lower levels of mutations in wild-type background as a sensitive method with simple and cost-effective procedure; therefore, it can prospectively be used in screening of patients with early-stage breast cancers.
Background Limb‐girdle muscular dystrophy (LGMD) is a non‐syndromic muscular dystrophy caused by variations in the genes involved in muscle structure, function and repair. The heterogeneity in the severity, progression, age of onset, and causative genes makes next‐generation sequencing (NGS) a necessary approach for the proper diagnosis of LGMD. Methods In this article, 26 Iranian patients with LGMD criteria were diagnosed with disease variants in the genes encoding calpain3, dysferlin, sarcoglycans and Laminin α‐2. Patients were referred to the hospital with variable distribution of muscle wasting and progressive weakness in the body. The symptoms along with biochemical and EMG tests were suggestive of LGMD; thus the genomic DNA of patients were investigated by whole‐exome sequencing including flanking intronic regions. The target genes were explored for the disease‐causing variants. Moreover, the consequence of the amino acid alterations on proteins' secondary structure and function was investigated for a better understanding of the pathogenicity of variants. Variants were sorted based on the genomic region, type and clinical significance. Results In a comprehensive investigation of previous clinical records, 6 variations were determined as novel, including c.1354–2 A > T and c.3169_3172dupCGGC in DYSF , c.568 G > T in SGCD , c.7243 C > T, c.8662_8663 insT and c. 4397G > C in LAMA2 . Some of the detected variants were located in functional domains and/or near to the post‐translational modification sites, altering or removing highly conserved regions of amino acid sequence.
Background Mutations in PIK3CA, which encodes p110 subunit of PI3K class IA enzyme, are highly frequent in breast cancer. Here, we aimed to probe mutations in exon 9 of PIK3CA and computationally simulate their function. Method PCR/HRM and PCR/sequencing were used for mutation detection in 40 breast cancer specimens. The identified mutations were queried via in silico algorithms to check the pathogenicity. The molecular dynamics (MD) simulations were utilized to assess the function of mutant proteins. Result Three samples were found to harbor at least one of the E542K, E545K and L551Q mutations of which L511Q has not been reported previously. All mutations were confirmed to be pathogenic and MD simulations revealed their impact on protein function and regulation. The novel L551Q mutant dynamics was similar to that of previously found carcinogenic mutants, E542K and E545K. A functional role for the helical domain was also suggested by which the inhibitory signal of p85α is conducted to kinase domain via helical domain. Helical domain mutations lead to impairment of kinase domain allosteric regulation. Interestingly, our results show that p110α substrate binding pocket of helical domain in mutants may have differential affinity for enzyme substrates, including anit-p110α drugs. Conclusion The novel p110α L551Q mutation could has carcinogenic feature similar to previously known mutations.
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