The overall prevalence of germline BRCA1/2 mutations is estimated between 11% and 15% of all ovarian cancers. Individuals with germline BRCA1/2 alterations treated with the PARP1 inhibitors (iPARP1) tend to respond better than patients with wild‐type BRCA1/2. Additionally, also somatic BRCA1/2 alterations induce the sensitivity to iPARP1. Therefore, the detection of both germline and somatic BRCA1/2 mutations is required for effective iPARP1 treatment. The aim of this study was to identify the frequency and spectrum of germline and somatic BRCA1/2 alterations in a group of Polish patients with ovarian serous carcinoma. In total, 100 formalin‐fixed paraffin‐embedded (FFPE) ovarian serous carcinoma tissues were enrolled to the study. Mutational analysis of BRCA1/2 genes was performed by using next‐generation sequencing. The presence of pathogenic variants was confirmed by Sanger sequencing. In addition, to confirm the germline or somatic status of the mutation, the nonneoplastic tissue was analyzed by bidirectional Sanger sequencing. In total, 27 (28% of patient samples) mutations (20 in BRCA1 and 7 in BRCA2) were identified. For 22 of 27 patients, nonneoplastic cells were available and sequencing revealed the somatic character of two BRCA1 (2/16; 12.5%) and two BRCA2 (2/6; 33%) mutations. Notably, we identified six novel frameshift or nonsense BRCA1/2 mutations. The heterogeneity of the detected mutations confirms the necessity of simultaneous analysis of BRCA1/2 genes in all patients diagnosed with serous ovarian carcinoma. Moreover, the use of tumor tissue for mutational analysis allowed the detection of both somatic and germline BRCA1/2 mutations.

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Autosomal dominant hypercholesterolemia (ADH) is caused by mutations in the genes coding for the low-density lipoprotein receptor (LDLR), apolipoprotein B-100 (APOB), or proprotein convertase subtilisin/kexin type 9 (PCSK9). In this study, a molecular analysis of LDLR and APOB was performed in a group of 378 unrelated ADH patients, to explore the mutation spectrum that causes hypercholesterolemia in Poland. All patients were clinically diagnosed with ADH according to a uniform protocol and internationally accepted WHO criteria. Mutational analysis included all exons, exon-intron boundaries and the promoter sequence of the LDLR, and a fragment of exon 26 of APOB. Additionally, the MLPA technique was applied to detect rearrangements within LDLR. In total, 100 sequence variations were identified in 234 (62%) patients. Within LDLR, 40 novel and 59 previously described sequence variations were detected. Of the 99 LDLR sequence variations, 71 may be pathogenic mutations. The most frequent LDLR alteration was a point mutation p.G592E detected in 38 (10%) patients, followed by duplication of exons 4-8 found in 16 individuals (4.2%). Twenty-five cases (6.6%) demonstrated the p.R3527Q mutation of APOB. Our findings imply that major rearrangements of the LDLR gene as well as 2 point mutations (p.G592E in LDLR and p.R3527Q in APOB) are frequent causes of ADH in Poland. However, the heterogeneity of LDLR mutations detected in the studied group confirms the requirement for complex molecular studies of Polish ADH patients.

Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify patients at risk of prolonged paralysis following the administration of neuromuscular blocking agents. The assay plays an important role in clinical chemistry as a good diagnostic marker for intoxication with pesticides and nerve agents. Furthermore, the assay is also commonly used for in vitro characterization of cholinesterases, their toxins and drugs. There is still lack of standardized procedure for measurement of BChE activity and many laboratories use different substrates at various concentrations. The purpose of this study was to validate the BChE activity assay to determine the best dilution of human serum and the most optimal concentration of substrates and inhibitors. Serum BChE activity was measured using modified Ellman’s method applicable for a microplate reader. We present our experience and new insights into the protocol for high-throughput routine assays of human plasma cholinesterase activities adapted to a microplate reader. During our routine assays used for the determination of BChE activity, we have observed that serum dilution factor influences the results obtained. We show that a 400-fold dilution of serum and 5mM S-butyrylthiocholine iodide can be successfully used for the accurate measurement of BChE activity in human serum. We also discuss usage of various concentrations of dibucaine and fluoride in BChE phenotyping. This study indicates that some factors of such a multicomponent clinical material like serum can influence kinetic parameters of the BChE. The observed inhibitory effect is dependent on serum dilution factor used in the assay.

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Approximately 25% of patients with ovarian cancer harbor a pathogenic BRCA1/2 mutation that has been associated with favorable responses for targeted therapy with poly (ADP-ribose) polymerase 1 (PARP1) inhibitors compared to wild-type individuals. The overall frequency of germline and somatic BRCA1/2 alterations is estimated at 13-15% and 3-10%, respectively. A high incidence of BRCA1/2 somatic variants significantly increases the number of patients eligible for treatment with PARP1 inhibitors. Here, we assessed circulating tumor DNA (ctDNA) from 121 patients with ovarian cancer for BRCA1/2 mutational analysis by next generation sequencing. A total number of patients carrying the pathogenic BRCA1/2 variants was 30/121 (24.8%), including 22 and 7 individuals with exclusively germline or somatic mutations, respectively and one patient with variants of both origin. Among this cohort, more than one known pathogenic BRCA1 and/or BRCA2 alterations were identified in 7/30 individuals. The most recurrent mutations were detected in the BRCA1 gene: c.5266dupC (p.Gln1756Profs*74) with the frequency of ~18%, followed by c.3756_3759del (p.Ser1253Argfs*10) and c.181T>G (p.Cys61Gly). In seven (5.8%) patients, coincidence of two or more BRCA1/2 pathogenic mutations have been identified. Our results clearly demonstrate that the detection of both germline and somatic BRCA1/2 mutations in ctDNA from ovarian cancer patients is feasible and may be a valuable complementary tool for identification of somatic alterations when the standard diagnostic procedures are insufficient. Finally, ctDNA can potentially allow to monitor the efficacy of PARP1 inhibitors and to detect a secondary reversion BRCA1/2 mutations.

While the life expectancy of the population has increased, Alzheimer’s disease (AD) has emerged as one of the greatest health problems of old age. AD is characterized by neuronal loss and cognitive decline. In the AD brain, there is a decrease in levels of acetylcholinesterase (AChE) and an increase in the levels of the related enzyme butyrylcholinesterase (BChE), that accumulate in plaques and tangles. Apolipoprotein E (ApoE) is a major cholesterol carrier and plays an important role in maintaining lipid homeostasis. APOE-ε4 constitutes the most important known genetic risk factor for late-onset AD. It has been proposed that the BCHE-K allele (Ala539Thr) acts in synergy with the APOE-ε4 allele to promote risk for AD. However, there is insufficient evidence to support a correlation. Most studies focused only on the coding regions of the genes. In this study, we analyzed sequence regions beyond the BCHE coding sequence. We found synergy between APOE-ε4 and SNPs localized in 5′UTR (rs1126680) and in intron 2 (rs55781031) of the BCHE-K allele (rs1803274) in 18% of patients with late-onset AD (n = 55). The results show that the coexistence of the APOE-ε4 allele and 3 SNPs in the BCHE gene is associated with a highly elevated risk of late-onset AD. SNP (rs1126680) in 5′UTR of the BCHE gene is located 32 nucleotides upstream of the 28 amino acid signal peptide. Mass spectrometry analysis of the BChE protein produced by SNP (rs1126680) showed that the mutation caused an in frame N-terminal extension of 41 amino acids of the BChE signal peptide. The resultant variant with a 69 amino acid signal peptide, designated N-BChE, may play a role in development of AD.