The role of gene encoding collagen secretion protein (SERPINH1) in the pathogenesis of a hypermobile type of Ehlers-Danlos syndrome.

Introduction: Hypermobile (hEDS) Ehlers-Danlos syndrome (EDS) is a non-inflammatory, autosomal dominant connective tissue disorder. hEDS, unlike other types of EDS, has no known genetic aetiology, so diagnosis is conducted based on a person's medical history, a physical examination, and exclusion of other types of EDS after genetic tests. Aim: The present study was a sequencing analysis of the SERPINH1 gene and the evaluation of the potential impact of variants of this gene on their role in the aetiology of the hypermobile type of EDS. Material and methods: The study group included 100 hEDS patients of Polish origin. The SERPINH1 gene analysis was performed on genomic DNA (gDNA). In all patients, other types of EDS or other connective tissue disorders were excluded by testing them with NGS technology. Results: Among 100 tested patients, 4 different types of missense variants (heterozygote) were detected. All SERPINH1 alterations were classified as benign according to ACMG guidelines. Conclusions: Mutations in the SERPINH1 gene have been described in a rare type of OI but have never been analysed in hypermobile Ehlers-Danlos syndrome. In our investigation among 100 hEDS patients, we did not identify pathogenic or likely pathogenic variants. Though only benign variants were detected, which play no role in the pathogenesis of hEDS, we should take into account mechanisms other than gene structure alterations, which may have an impact on collagen and other ECM protein transport.

The Lack of Influence of Homozygous Long Allele of the 5-HTTLPR Gene on the Severity of Alcohol Craving During 6 Weeks of Rehab Hospitalisation in Comparison to Not Homozygous and Homozygous Short Alleles - Preliminary Report.

Purpose: The aim of this study was to assess changes in the severity of alcohol craving according to allelic variants of the 5-HTTLPR gene polymorphism during hospitalisation and their association with selected clinical variables in alcohol-dependent patients. Patients and Methods: The study is exploratory. Participants were investigated at the 2nd and 6th week of alcohol-dependence therapy in the addiction treatment unit. Recruitment was conducted among alcohol-dependent patients from several Polish drug treatment centres. The total sample size was 130 persons (12 females and 118 males). Study subjects' mean age was 43.0 years. Patients were investigated twice by using the Penn Alcohol Craving Scale (PACS) and Beck Depression Inventory (BDI), and once by using Short Alcohol Dependence Data Questionnaire (SADD) and taking a swab for genetic testing. The polymorphism of the gene encoding the serotonin transporter 5-HTTLPR (SLC6A4) was determined from isolated DNA and its homozygous variants of short/short or long/long alleles and heterozygous short/long alleles were analysed. Results: At 6th week of the follow-up, there was a decrease in the severity of alcohol craving in half of subjects with the short/short allele (p = 0.033) and in one-fifth of subjects with the long/short allele (p = 0.002) of the 5-HTTLPR gene. In subjects with long/long allele of the 5-HTTLPR gene, there was no change in the severity of alcohol craving between 2nd and 6th weeks of the study (p = 0.242). Conclusion: There was no statistical influence of the homozygous long allele of the 5-HTTLPR gene on severity of alcohol craving during 6 weeks of rehab hospitalisation in comparison to not homozygous and homozygous short alleles. The s-allele was associated with decrease of alcohol craving. It may point on the potential need for differentiated rehabilitation methods depending on the genetic diversity of addicted patients and its role in the severity of alcohol craving.

A novel mutation in collagen transport protein, MIA3 gene, detected in a patient with clinical symptoms of Ehlers-Danlos hypermobile syndrome.

BACKGROUND: Collagen, the most abundant human protein, is a significant component of the extracellular matrix (ECM) in tissues and organs like skin, bone, ligaments, and tendons. Collagen secretion is a complex, multistage process involving many molecules. A protein playing one of the main functions in this process is TANGO1 encoded by MIA3 gene. In the hypermobile type of Ehlers-Danlos syndrome (hEDS), one of the most common collagenopathies with no known genetic background, disrupted secretion of many molecules (including collagen) was observed. OBJECTIVES: The aim of this study was the evaluation of the MIA3 gene role in hEDS patients. MATERIAL AND METHODS: One hundred patients with clinically diagnosed hEDS and negative next-generation sequencing (NGS) testing for connective tissue disorder (e.g. Ehlers-Danlos syndrome, osteogenesis imperfect (OI), Marfan syndrome, and others) were tested for molecular changes in the MIA3 gene. RESULTS: Among the 100 tested patients, 14 single structural changes in the MIA3 gene were detected. Thirteen were missense benign or likely benign, while 1 variant (c.567dup, p.Leu1880ThrfsTer6) was truncating the TANGO1 protein. CONCLUSIONS: We suppose that the presence of truncating variant (c.5637dup) in the MIA3 gene and disrupted secretion of connective tissue protein may be one of the pathogenic mechanisms of clinical symptoms present in the tested patient, but these findings require a more comprehensive multidimensional investigation.

Next-Generation Sequencing of Connective Tissue Genes in Patients with Classical Ehlers-Danlos Syndrome.

BACKGROUND: Ehlers-Danlos syndrome (EDS) is a common non-inflammatory, congenital connective tissue disorder. Classical type (cEDS) EDS is one of the more common forms, typically caused by mutations in the COL5A1 and COL5A2 genes, though causative mutations in the COL1A1 gene have also been described. MATERIAL AND METHODS: The study group included 59 patients of Polish origin, diagnosed with cEDS. The analysis was performed on genomic DNA (gDNA) with NGS technology, using an Illumina sequencer. Thirty-five genes related to connective tissue were investigated. The pathogenicity of the detected variants was assessed by VarSome. RESULTS: The NGS of 35 genes revealed variants within the COL5A1, COL5A2, COL1A1, and COL1A2 genes for 30 of the 59 patients investigated. Our panel detected no sequence variations for the remaining 29 patients. DISCUSSION: Next-generation sequencing, with an appropriate multigene panel, showed great potential to assist in the diagnosis of EDS and other connective tissue disorders. Our data also show that not all causative genes giving rise to cEDS have been elucidated yet.

Clinical Significance of Gene Mutations and Polymorphic Variants and their Association with Prostate Cancer Risk in Polish Men.

Objectives: We tested the association of germline variants in BRCA1, BRCA2, CHEK2, CDKN2A, CYP1B1, HOXB13, MLH1, NBS1, NOD2 andPALB2 genes, as well as in 8q24 region, with prostate cancer (PC) risk and estimated their impact on disease clinical course, including overall survival time in Polish men with localized PC qualified for radical treatment.Materials and Methods: DNA of 110 patients with localized prostate cancer treated with radical prostatectomy (RP), from each age group and with different stages of the disease. DNA samples of the control group consisted of 111 men, volunteers, without PC (age-matched to study group). Sanger sequencing, AS-PCR, RFLP-PCR, and multiplex-PCR were used for  variants detection.Results: The percentage of men with ≥1 germline variant was higher in PC group (52.7%) than in healthy men (37.8%) (P = .03). The presence of ≥2 variants was associated with shorter survival than the presence of one or no variant in the PC group (P = .14, trend). The HOXB13 G84E was detected in 2.9% of PC men and in no healthy men (P = .19, trend, OR = 7.21). A CHEK2 truncating mutation (1100delC or IVS2+1G>A) was detected in 2/110 (1.8%) PC patients and in no healthy men (P = .29, OR=5.14). The NBS1 I171V was detected in 2/110 (1.8%) PC patients and in no men from the control group (OR=5.14, P = .29, NS).Conclusions: We conclude that the presence of more than 2 germline variants was probably associated with shorter survival of patients with localized prostate cancer qualified for radical treatment. The HOXB13 (G84E), CHEK2 (1100delC or IVS2+1G>A) truncating variants and NBS1 (I171V) are associated with PC and hereditary form of the disease. The HOXB13 (G84E) and NOD2 (3020insC) single variants are associated with shorter and CYP1B1 (48CC, 119GG) single genotypes with longer overall survival.

Initial Study on COMT and DRD2 Gene Polymorphisms as Well as the Influence of Temperament and Character Trait on the Severity of Alcohol Craving in Alcohol-Dependent Patients.

The main aim of this work was to determine the impact of COMT and DRD2 gene polymorphisms together with temperament and character traits on alcohol craving severity alcohol-dependent persons. The sample comprised of 89 men and 16 women (aged 38±7). For the sake of psychological assessment various analytic methods have been applied like the Short Alcohol Dependence Data Questionnaire (SADD), Penn Alcohol Craving Scale (PACS) or Temperament and Character Inventory (TCI) test. The SNP polymorphism of the analyzed genes was determined by Real Time PCR test. The results showed, that the COMT polymorphismmay have an indirected relationship with the intensity and changes in alcohol craving during abstinence. The DRD2 receptor gene polymorphisms are related with the intensity of alcohol craving. It seems that the character traits like "self-targeting", including "self-acceptance", are more closely related to the severity of alcohol craving and polymorphic changes in the DRD2 receptor than temperamental traits. Although this is a pilot study the obtained results appeared to be promising and clearly indicate the link betweengene polymorphisms alcohol craving and its severity.

Germline mutations among Polish patients with acute myeloid leukemia.

BACKGROUND: A small but important proportion of patients (4-10 %) with AML have germline mutations. They can cause the development of AML at an earlier age, confer a higher risk of relapse or predispose to secondary leukemias, including therapy-related leukemias. The analysis of germline mutations in a patient and his/her family is also critical for the selection of suitable family donors if the patient is a candidate for hematopoietic stem cell transplantation (HSCT). METHODS: 103 unrelated consecutive patients with de novo AML were enrolled in the study. Control group consisted of 103 persons from the general population. We performed NGS sequencing of bone marrow cells and buccal swabs DNA of six genes: CEBPA, DDX41, ETV6, TERT, GATA2, and IDH2 to detect germline pathogenic mutations. RESULTS: In the investigated group, 49 variants were detected in six genes. 26 of them were somatic and 23 germline. Germline variants were detected in all six tested genes. Eight pathogenic germline mutations were detected in 7 AML patients, in three genes: CEBPA, ETV6, and IDH2. One patient had two pathogenic germinal mutations, one in ETV6 and one in CEBPA gene. We identified one novel pathogenic germline mutation in CEBPA gene. The difference in frequency of all pathogenic germline mutations between the tested (7.77 %) and control groups (0.97 %) was statistically significant (p = 0.046). In the tested group, the median age at AML diagnosis was 11 years lower in patients with pathogenic germline mutations than in patients without them (p = 0.028). CONCLUSIONS: We showed higher frequency of CEBPA, ETV6, and IDH2 germline mutations in AML patients than in control group, which confirms the role of these mutations in the development of AML. We also showed that the median age at the onset of AML in patients with pathogenic germline mutations is significantly lower than in patients without them.

Searching for germline mutations in the RUNX1 gene among Polish patients with acute myeloid leukemia.

The aim of the study was the identification of constitutional RUNX1 mutations among AML patients. The study group included 100 patients of Polish origin, diagnosed with de novo AML. 14 out of 100 AML patients had together 17 RUNX1 mutations, three of which were found to be germline changes. The difference in germline mutation frequency between study and control groups was not statistically significant (p = 0.193), but the odds ratio was 7.215. In all patients with germline mutations, chromosome 7 aberrations were found. The difference in the frequency of chromosome 7 aberrations between the group of patients with and without germline mutations was statistically significant (p = 0.008, OR = 73.00). We showed a higher frequency of germline mutations of RUNX1 in AML patients than in the control group, which confirms the role of these mutations in the development of AML, and an association of germline mutations with aberrations of chromosome 7.

G84E germline mutation in HOXB13 gene is associated with increased prostate cancer risk in Polish men.

We tested the association between HOXB13 G84E (rs138213197) germline mutation and PC risk in Polish men. DNA from 103 consecutive, newly diagnosed patients hospitalised because of PC and DNA from 103 men: volunteers, healthy at the time of the study. The G84E mutation was genotyped using Sanger sequencing. The HOXB13 G84E germline mutation was detected in 2.9% of PC men (3/103) and not detected in any healthy man. Two mutation carriers originated from two of 25 families fulfilling hereditary prostate cancer criteria (HPC) and one mutation carrier from one family among 78 families without HPC (PC frequency: 8% vs. 1.3%, OR = 6.70, p = 0.13). In two of three mutation carriers, disease was detected above 60 years of age. There was a trend for a lower probability of 5-year survival in patients with G84E than in patients without it (66.7% vs. 94.0%, p = 0.08). The HOXB13 G84E germline mutation is associated with increased prostate cancer risk in Polish men, with hereditary form of the disease, and probably with older age at PC onset (> 60 years of age) and shorter survival. However, it is not associated with PSA level, or PC stage or grade at the time of diagnosis.

New variants in COL5A1 gene among Polish patients with Ehlers-Danlos syndrome: analysis of nine cases.

INTRODUCTION: The Ehlers-Danlos syndrome (EDS) is a non-inflammatory, heritable connective tissue disorder divided into 13 types according to the 2017 International Classification of the Ehlers-Danlos syndromes. One of the subtypes of EDS, classical (cEDS), is characterized by joint hypermobility, skin hyperextensibility and atrophic scars, which are major criteria of cEDS. AIM: In this study, the first in Central Eastern Europe, 44 patients were investigated. All of them were tested for COL5A1 mutations with direct DNA sequencing. MATERIAL AND METHODS: The study group included 44 patients of Polish origin, all of whom fulfilled criteria for the classical type of Ehlers-Danlos syndrome. Direct sequencing of the COL5A1, COL5A2 and COL1A1 c.934C>T genes was performed for all of them. Evaluation of potential pathogenicity of detected missense mutation was conducted using SIFT (Sorting Intolerant from Tolerant), PolyPhen-2, AlignGVGD (Align Grantham Variance/Grantham Difference). The effect of the splice site mutations was predicted by Human Splicing Finder and NetGene2 tools. RESULTS: Among all tested patients, nine mutations of COL5A1 gene were detected (8 missense mutations and 1 splice site). The alterations identified by us are new, hitherto not described in other reports. Evaluation of the mutations by in silico tools indicate their pathogenicity. CONCLUSIONS: Our study is the first COL5A1 gene molecular investigation conducted among cEDS patients from Central Eastern Europe. Besides new COL5A1 variant findings, we gained molecular confirmation of clinical diagnosis of cEDS. In some cases, specific and adequate evaluation and classification of EDS patients based only on clinical features, may be difficult.

Searching for new breast cancer-associated genes. ABRAXAS1 gene mutations in the group of BRCA1-negative patients.

In the present study, we analysed the association of mutations of a BRCA1-associated gene, ABRAXAS1, with the risk of development of breast cancer (BC) in BRCA1-negative women from North-Central Poland. A hundred women with consecutively diagnosed BC and 100 women belonging to the control group were screened for new mutations predisposing to breast cancer. The first step was a test carried out in order to find one of the three Polish founder mutations in the BRCA1 gene. In 96 BRCA1-negative patients two missense variants: c.422C>T and c.1042G>A as well as two intronic variants: IVS3-34G>A, IVS3-44T>C were detected in the ABRAXAS1 gene. The c.422C>T mutation was detected in one of 96 women diagnosed with breast cancer (1.04%); it was not associated with increased risk of disease in this group, compared to the controls (p = 0.49), but the odds ratio was 3.314; 95% CI: 0.122-75.352. IVS3-44T>C was found more frequently in the control group (15/93) than in the tested group (1/85), OR 0.062; 95% CI: 0.008-0.480, p = 0.007, which may suggest protective properties of this variant against tumorigenicity. The data obtained from the present study suggest the necessity for further research to be conducted on the ABRAXAS1 gene in relation to hereditary predisposition to breast cancer.

Genetical background of intelligence.

Intelligence as an ability to reason, think abstractly and adapt effectively to the environment is a subject of research in the field of psychology, neurobiology, and in the last twenty years genetics as well. Genetical testing of twins carried out from XX century indicated heritebility of intelligence, therefore confirmed an influence of genetic factor on cognitive processes. Studies on genetic background of intelligence focus on dopaminergic (DRD2, DRD4, COMT, SLC6A3, DAT1, CCKAR) and adrenergic system (ADRB2, CHRM2) genes as well as, neutrofins (BDNF) and oxidative stress genes (LTF, PRNP). Positive effect of investigated gene polymorphism was indicated by variation c.957C>T DRD2 gene (if in polymorphic site is thymine), polymorphism c.472G>A COMT gene (presence of adenine) and also gene ADRB2 c.46A->G (guanine), CHRM2 (thymine in place c.1890A>T) and BDNF (guanine in place c.472G>A) Obtained results indicate that intelligence is a feature dependent not only on genetic but also an environmental factor.

A risk of breast cancer in women - carriers of constitutional CHEK2 gene mutations, originating from the North - Central Poland.

BACKGROUND: Germline mutations of the CHEK2 gene have been reported to be associated with breast cancer. In this study, we analyzed the association of CHEK2 mutations with the risk of development of breast cancer in women of North-Central Poland. METHODS: 420 women with breast cancer and 435 controls were tested for three protein truncating (IVS2 + 1G > A, 1100delC, del5395) and one missense (I157T) CHEK2 mutation. IVS2 + 1G > A and I157T mutations were identified by RFLP-PCR, 1100delC variant was analyzed using an ASO-PCR and del5395 mutation by multiplex-PCR. The statistical tests: the odds ratio (OR) and Fisher's exact test were used. RESULTS: In 33 out of 420 (7.9%) women consecutively diagnosed with breast cancer, we detected one of four analyzed CHEK2 mutations: I157T, 1100delC, IVS2 + 1G > A or del5395. Together they were not associated with the increased risk of breast cancer (North-Central control group: OR = 1.6, p = 0.124; the general Polish population: OR = 1.4, p = 0.109). This association was only seen for IVS2 + 1G > A mutation (OR = 3.0; p = 0.039). One of the three truncating CHEK2 mutations (IVS2 + 1G > A, 1100delC, del5395) was present in 9 of 420 women diagnosed with breast cancer (2.1%) and in 4 of 121 women (3.3%) with a history of breast cancer in a first- and/or second- degree relatives. Together they were associated with the increased risk of disease in these groups, compared to the general Polish population (OR = 2.1, p = 0.053 and OR = 3.2; p = 0.044, respectively). I157T mutation was detected in 25 of 420 women diagnosed with breast cancer (6.0%) and in 8 of 121 women (6.6%) with a history of breast cancer in first- and/or second- degree relatives. The prevalance of I157T mutation was 4.1% (18/435) in North-Central control group and 4.8% (265/5.496) in the general Polish population. However it was not associated with an increased risk of breast cancer. CONCLUSION: Obtained results suggest that CHEK2 mutations could potentially contribute to the susceptibility to breast cancer. The germline mutations of CHEK2, especially the truncating ones confer low-penetrance breast cancer predisposition that contribute significantly to familial clustering of breast cancer at the population level.

Prevalence of the BRCA1 c.68_69delAG (BIC: 185delAG) mutation in women with breast cancer from north-central Poland and a review of the literature on other regions of the country.

AIM OF THE STUDY: Germline mutations in BRCA tumor suppressor genes are strongly associated with breast and ovarian cancer. The lifetime risk of these cancers in women with BRCA1 mutation is 84% and 27%, respectively. Studies on the prevalence of BRCA1 c.68_69delAG congenital mutation, the most frequent in Ashkenazi Jews, among women with breast cancer from north-central Poland and review of the literature on other regions of the country. Evaluation of the c.68_69delAG association with breast cancer risk, with respect to women's age at diagnosis and family history of cancer. MATERIAL AND METHODS: 252 women with breast cancer, without any of the mutations c.5266dupC, c.181T > G, or c.4034delA, regardless of histological type and family history of cancer. The mutation was detected using allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) assay and confirmed by sequence analysis. RESULTS: The c.68_69delAG mutation was disclosed in one out of the 252 women (0.4%), who had been diagnosed with breast cancer at age 43. Family investigations revealed the presence of c.68_69delAG also in the patient's mother, diagnosed with breast cancer at age 68. Sequence analysis confirmed the heterozygous status of the mutation, and family investigation its hereditary character. In the group of families with breast cancer history 1.4% frequency of c.68_69delAG was shown. CONCLUSIONS: Among families with breast cancer aggregation, originating from north-central Poland, c.68_69delAG is a rare BRCA1 alteration, similarly to other central regions of the country, investigated by other authors. However, in northern, north-western and south-western parts of Poland, it occurs 2-4 times more frequently than in our region.

A risk of essential thrombocythemia in carriers of constitutional CHEK2 gene mutations.

Germline mutations of the CHEK2 gene have been reported in some myeloid and lymphoid malignancies, but their impact on development of essential thrombocythemia has not been studied. In 16 out of 106 (15.1%) consecutive patients, newly diagnosed with essential thrombocythemia, we found one of four analyzed CHEK2 mutations: I157T, 1100delC, IVS2+1G>A or del5395. They were associated with the increased risk of disease (OR=3.8; P=0.002). The median age at ET diagnosis among CHEK2+/JAK2V617F+ patients was seven years lower than that among CHEK2-/JAK2V617F+ (52 vs. 59 years; P=0.04), whereas there was no difference in the medians of hematologic parameters between these groups. The results obtained suggest that CHEK2 mutations could potentially contribute to the susceptibility to essential thrombocythemia. The germline inactivation of CHEK2, as it seems, has no direct impact on the development of disease, but it could cause disruption of cell cycle checkpoints and initiate or support the cancerogenic process of essential thrombocythemia at a younger age.

Frequency of CYP1B1 homozygous genotype 355T/T in prostate cancer families from Poland.

A group of 98 families were analyzed for CYP1B1 gene 355T/T homozygous genotype frequency because of prostate cancer history. Molecular investigations were performed using the restriction fragment length polymorphism-PCR method. 355T/T genotype was detected in 14 of the 98 prostate cancer patients (14.3%). Among them, it was found in one man (7.1%) from a family suspected of hereditary prostate cancer (his age at prostate cancer diagnosis was 57 years) and in 13 men (92.9%) originating from families that did not strictly fulfill hereditary prostate cancer criteria (the median age at prostate cancer diagnosis was 60.1 years). Among 14 355T/T genotype-positive families, in 10 (71.4%) other types of cancers, for example, breast, uterus, stomach, colon, ovary, lung, larynx, bladder, pancreas and melanoma other than prostate cancer, were present, and in four (28.6%) only one cancer type, that is, prostate cancer, occurred. In the Polish population, the CYP1B1 355T/T genotype seems to be associated with prostate cancer; the frequency of this genotype was 5.9% higher in prostate cancer patients than in the general population (8.4%). However, it is not associated with prostate cancer family history.