Spectrum and frequency of CHEK2 variants in breast cancer affected and general population in the Baltic states region, initial results and literature review.

BACKGROUND: While BRCA1/2 gene mutational spectrum and clinical features are widely studied, there is limited data on breast cancer-predisposing non-BRCA pathogenic/likely pathogenic variants (PV/LPVs) in the Baltic states region. According to previous studies, CHEK2 is the most frequent moderate-risk breast cancer predisposition gene. The study aimed to analyse the frequency and mutational spectrum of CHEK2 PV/LPVs in the Baltic states region and perform a literature review on the subject. METHODS: The study includes two cohorts - population-based Estonian biobank (EstBB) (N-152 349) and breast cancer affected cases from Latvia (N-105). In the cohort from Latvia, CHEK2, BRCA1, BRCA2, PALB2 testing with next-generation sequencing (NGS) was carried out in selected breast cancer cases. In the EstBB, the full SNP genotyped dataset Global Screening Array (GSA) (N-152 349) was used to screen CHEK2 PV/LPVs and variants c.319+2T > A (p.(?)), c.444+1G>A (p.(?)), c.433C > T (p.Arg145Trp), c.283C > T (p.Arg95*) in CHEK2 are reported from this dataset. In addition, a subset of the EstBB (N-4776) underwent whole-genome sequencing (WGS, N-2420) and whole-exome sequencing (WES, N-2356) and founder variants c.470T > C (p.Ile157Thr), c.444+1G>A (p.(?)), c.1100delC (p.Thr367Metfs*15) in CHEK2 were reported from this dataset. Moreover, a literature overview was performed on April 1, 2021, using the PubMed search of keywords 'CHEK2', 'breast cancer', 'Estonia', 'Lithuania', 'Latvia', 'Poland', 'Belarus' and 'Russia'. RESULTS: In the breast cancer affected cohort from Latvia 6 CHEK2 variants, classified as PV/LPVs, were observed (6/105; 5.7%), including recurrent ones c.470T > C (p.Ile157Thr) (1.9%) and del5395(ex9-10del; (p.Met304Leufs*16)) (1.9%), as well as single ones - c.1100delC (p.Thr367Metfs*15) (1%) and c.444+1G>A (p.(?)) (1%). From EstBB NGS data (N-4776) CHEK2 variant c.470T > C (p.Ile157Thr) was detected in 8.6% of cases, c.1100delC (p.Thr367Metfs*15) in 0.6% and c.444+1G>A (p.(?)) in 0.2% of cases. In the EstBB full cohort of SNP array data (N-152 349) CHEK2 variant c.444+1G>A (p.(?)) was detected in 0.02% of cases, c.319+2T > A (p.(?)) in 0.09% of cases, c.433C > T (p.Arg145Trp) in 0.02% of cases and c.283C > T (p.Arg95*) in <0.001% of cases. For the literature review altogether, 49 PubMed articles were found, 23 of which were relevant, representing CHEK2 PV/LPVs in the population of interest. Ten publications are from Poland, eight from Russia, three from Latvia and two from Belarus. CONCLUSIONS: This study is the first combined report on complete CHEK2 PV/LPVs screening in selected breast cancer affected cases in Latvia and large-scale population screening in Estonia, providing insight into the CHEK2 mutational spectrum in the Baltic states region. The initial results are in line with other studies that CHEK2 PV/LPVs frequency is around 5-6% of selected breast cancer cases. Here we report three CHEK2 PV/LPV - c.319+2T > A (p.(?)), c.433C > T (p.Arg145Trp), c.283C > T (p.Arg95*), that are novel for the Baltic states region. This is also the first report on c.1100delC (p.Thr367Metfs*15) and c.444+1G>A (p.(?)) from the Baltic states. High population frequency of c.470T > C (p. Ile157Thr) (8.6%) continues to question the variant's pathogenicity in particular populations. Other findings are concordant with previous reports from Latvia and neighbouring populations.

Urinary organic acids spectra in children with altered gut microbiota composition and autistic spectrum disorder.

Introduction: Alteration of human gut microbiota is described in a number of neuro-developmental and cognitive disorders including autistic spectrum disorder (ASD). Along with the changes in the gut microbiota, children with ASD are also reported to have changes in urinary organic acid spectra implying these metabolites as potential biomarkers for gastrointestinal dysbiosis.Aim: Identify urinary metabolites that would indicate specific changes in the gut microbiota and could be useful as biomarkers.Methods: The study group consisted of 44 children with ASD. Urinary organic acids spectra and composition of gut microbiota were analysed.Results: Any significant deviation in quantified metabolites compared to the reference values were not confirmed. The main variations were detected in concentration of p-cresol and 3-(3-hydroxyphenyl)-3-hydroxypropionic acid (HPHPA), but we cannot confirm the presence of HPHPA in urine as a biomarker for Clostridium sp. overgrowth in the gut. The acquired results indicate higher relative abundance of Firmicutes phylum alone may be attributed to increased concentration of p-cresol in urine. Decreased Bacteroidetes/Firmicutes ratio was found in the group with the presence of HPHPA in urine.Conclusions: Metabolites of human urine can be used as biomarkers for alterations of gut microbiota with caution, guided treatment should be administrated only based on gut microbiota analysis results or in combination with urinary organic acid results, but not solely based on organic acid biomarkers.

Hypercalcemia and CYP24A1 Gene Mutation Diagnosed in the 2nd Trimester of a Twin Pregnancy: A Case Report.

BACKGROUND Loss-of-function mutations of the CYP24A1 gene cause a deficiency of the CYP24A1 enzyme, which is involved in the catabolism of 1,25-dihydroxyvitamin D3. Patients who are CYP24A1 enzyme deficient are at increased risk of developing hypercalcemia during pregnancy and should avoid additional vitamin D supplementation. This case report provides additional information for managing and diagnosing patients with a CYP24A1 gene mutation. CASE REPORT A primipara woman with a twin pregnancy was admitted to our hospital for frequent hypertensive crises. She had no history of hypercalcemia-associated signs and symptoms except nephrocalcinosis, and reported no other abnormalities or discomfort at presentation. Laboratory tests revealed that the parathyroid hormone level was suppressed and the serum calcium level was markedly increased. The 25-hydroxyvitamin D level was at the upper limit of the reference range while the 1,25-dihydroxyvitamin D3 level was elevated, suggesting a vitamin D catabolism disorder. A genetic test was performed and a homozygous likely pathogenic variant (based on the American College of Medical Genetics and Genomics guidelines) c.964G>A (p.Glu322Lys) was detected in the CYP24A1 gene (NM_000782.5). A cesarean section delivery was performed due to a single intrauterine demise at 33 weeks of gestation. The preterm newborn was diagnosed with transitional hypercalcemia and hyperphosphatemia; however, he was not treated, as he was asymptomatic. CONCLUSIONS Patients with a CYP24A1 gene mutation are at increased risk of hypercalcemia and fetal demise; therefore, 25-hydroxyvitamin D and calcium levels should be monitored in routine blood tests during pregnancy. Hypercalcemia in a newborn should be carefully evaluated and treated, as hypercalciuria can lead to nephrocalcinosis.

X-Linked Lymphoproliferative Disease in Latvia: A Report of Two Clinically Distinct Cases.

X-linked lymphoproliferative disease (XLP) is a rare primary immunodeficiency. Affected individuals usually present with the Epstein-Barr virus infection and have no apparent disease prior to presentation. The most common clinical manifestations are fulminant infectious mononucleosis, dysgammaglobulinaemia, and lymphoma (usually of B-cell origin). XLP is caused by mutations in the SH2D1A gene which encodes the intracellular adaptor molecule SAP (signalling lymphocyte activation molecule- (SLAM-) associated protein). SAP is predominantly expressed in T cells and NK cells and functions to regulate signal transduction pathways downstream of the SLAM family of surface receptors to control CD4+ T cell (and by extension B-cell), CD8+ T cell and NK cell function, and development of NKT cells. Thus, SAP mutations cause dysregulation of the immune system, with defects in both cellular and humoral immunity. Here we report two clinical cases of three patients who presented with different manifestations of XLP, namely, fulminant infectious mononucleosis, Burkitt lymphoma and hypogammaglobulinaemia.

Application of the UHPLC-DIA-HRMS Method for Determination of Cheese Peptides.

Until now, cheese peptidomics approaches have been criticised for their lower throughput. Namely, analytical gradients that are most commonly used for mass spectrometric detection are usually over 60 or even 120 min. We developed a cheese peptide mapping method using nano ultra-high-performance chromatography data-independent acquisition high-resolution mass spectrometry (nanoUHPLC-DIA-HRMS) with a chromatographic gradient of 40 min. The 40 min gradient did not show any sign of compromise in milk protein coverage compared to 60 and 120 min methods, providing the next step towards achieving higher-throughput analysis. Top 150 most abundant peptides passing selection criteria across all samples were cross-referenced with work from other publications and a good correlation between the results was found. To achieve even faster sample turnaround enhanced DIA methods should be considered for future peptidomics applications.

Pathogenic APC Variants in Latvian Familial Adenomatous Polyposis Patients.

Background and objectives: Familial adenomatous polyposis is one of the APC-associated polyposis conditions described as genetically predetermined colorectal polyposis syndrome with a variety of symptoms. The purpose of this study was to determine sequence variants of the APC gene in patients with familial adenomatous polyposis (FAP) phenotype and positive or negative family history. Materials and Methods: Eight families with defined criteria of adenomatous polyposis underwent molecular genetic testing. Coding regions and flanking intron regions of the APC gene were analyzed by Sanger sequencing. Results: Eight allelic variants of the APC gene coding sequence were detected. All allelic variants of the APC gene were predicted to be pathogenic based on criteria according to the "Joint Consensus Recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology" (2015), four of them c.1586_1587insAT, c.2336delT, c.3066_3067insGA, and c.4303_4304insC, were considered novel. Conclusions: The timely molecular genetic analysis of APC germline variants and standardized interpretation of the pathogenicity of novel allelic variants has a high impact on choice for treatment, cancer prevention, and family genetic counseling.

Allelic variants of breast cancer susceptibility genes PALB2 and RECQL in the Latvian population.

BACKGROUND: Large-scale case control studies revealed a number of moderate risk - low frequency breast cancer alleles of the PALB2 and RECQL genes. Some of these were reported as founder variants of Central and Eastern Europe. Based on highly similar founder variant spectra of the BRCA1 in Poland and Latvia, we decided to test the frequency of other common variants of moderate breast cancer risk - c.509_510delGA (rs515726124) and c.172_175delTTGT (rs180177143) of the PALB2 gene and c.1667_1667+3delAGTA variant of the RECQL gene in a breast cancer case-control series from Latvia to better understand the role of genes in susceptibility to breast cancer and their clinical significance. METHODS: The case-control study was performed based on an unselected breast cancer case group of 2480 women and a control group, including 1240 voluntary, to our knowledge unrelated, female donors without reported oncological disease. RESULTS: The calculated frequency for c.509_510delGA of the PALB2 gene in the case group is 0.35 and 0.00% in the control group, with respective relative risk (RR) 7.18 (CI 95% 0.37-138.75; p = 0.19). As for the PALB2 c.172_175delTTGT variant, the frequency in the case group of our study is 0.04%. In the control group of our study all individuals were homozygous for the wild-type allele, which lead to calculated RR = 1.50 (CI 95% 0.06-36.83; p-value = 0.80). There were no carriers of the RECQL variant c.1667_1667+3delAGTA identified in our case group and 2 heterozygotes were identified in the control group. The calculated RR = 0.26 (CI 95% 0.01-5.33; p-value = 0.38). CONCLUSION: Results obtained for the PALB2 gene variants are able to supplement evidence on the allele frequency in breast cancer patients from the region of Central and Eastern Europe. Based on our results we cannot confirm the contribution of the RECQL variant c.1667_1667+3delAGTA allele to breast cancer development.

An Effect of Culture Media on Epithelial Differentiation Markers in Breast Cancer Cell Lines MCF7, MDA-MB-436 and SkBr3.

Background and objectives: Cell culture is one of the mainstays in the research of breast cancer biology, although the extent to which this approach allows to preserve the original characteristics of originating tumor and implications of cell culture findings to real life situations have been widely debated in the literature. The aim of this study was to determine the role of three cell culture media on transcriptional expression of breast cancer markers in three breast cancer reference cell lines (MCF7, SkBr3 and MDA-MB-436). Materials and methods: Cell lines were conditioned in three studied media (all containing 5% fetal bovine serum (FBS) + hormones/growth factors; different composition of basal media) for four passages. Population growth was characterized by cumulative population doubling levels, average generation time, cell yield and viability at the fourth passage. Transcriptional expression of breast cancer differentiation markers and regulatory transcriptional programs was measured by qPCR. Results: Differences in the composition of growth media significantly influenced the growth of studied cell lines and the expression of mammary lineage governing transcriptional programs and luminal/basal markers. Effects of media on transcriptional expression were more pronounced in luminal cell lines (MCF7, SkBr3), than in the basal cell line (MDA-MB-436). Changes in growth media in terms of supplementation and basal medium delayed growth of cells, but improved cell yields. Conclusions: The expression of breast cancer cell differentiation phenotypic markers depends on the composition of cell growth medium, therefore cell culture as a tool in phenotypic studies should be used considering this effect. The findings of such studies should always be interpreted with caution. The formulation of cell growth media has greater effect on the expression of phenotypic markers in luminal, rather than basal cell lines. Media containing mitogens and higher vitamin content improved efficacy of cell culture in terms of cell yields, although greatly increased growth times.

Chromothripsis and progression-free survival in metastatic colorectal cancer.

Metastatic dissemination of the primary tumor is the major cause of death in colorectal cancer (CRC) patients. Multiple chromosomal breaks and chromothripsis, a phenomenon involving multiple chromosomal fragmentations occurring in a single catastrophic event, are associated with cancer genesis, progression and developing of metastases. The aim of this study was to evaluate the effect of chromothripsis and total breakpoint count (breakpoint instability index) on progression-free survival (PFS). A total of 19 patients with metastatic CRC (mCRC) receiving FOLFOX first-line palliative chemotherapy between August, 2011 and October, 2012 were selected for this study. The results indicated that the highest breakpoint count was observed in chromosomes 1, 2 and 6. Chromothripsis was detected in 52.6% of the study patients. Furthermore, chromothripsis was associated with an increased median PFS (mPFS; 14 vs. 8 months, respectively; P=0.03), but an association with overall survival was not identified. The present study demonstrated that chromothripsis affected CRC patient survival, suggesting a role for this event as a prognostic and predictive marker in mCRC treatment.