A deep intronic recurrent CHEK2 variant c.1009-118_1009-87delinsC affects pre-mRNA splicing and contributes to hereditary breast cancer predisposition.

Germline CHEK2 pathogenic variants confer an increased risk of female breast cancer (FBC). Here we describe a recurrent germline intronic variant c.1009-118_1009-87delinsC, which showed a splice acceptor shift in RNA analysis, introducing a premature stop codon (p.Tyr337PhefsTer37). The variant was found in 21/10,204 (0.21%) Czech FBC patients compared to 1/3250 (0.03%) controls (p = 0.04) and in 4/3639 (0.11%) FBC patients from an independent German dataset. In addition, we found this variant in 5/2966 (0.17%) Czech (but none of the 443 German) ovarian cancer patients, three of whom developed early-onset tumors. Based on these observations, we classified this variant as likely pathogenic.

Targeted long-read sequencing identified a causal structural variant in X-linked nephrogenic diabetes insipidus.

BACKGROUND: X-linked nephrogenic diabetes insipidus (NDI) is a rare genetic renal disease caused by pathogenic variants in the AVPR2 gene. Single nucleotide variants and small insertions/deletions in AVPR2 are reliably detected by routine clinical sequencing. Nevertheless, structural variants involving AVPR2 are challenging to identify accurately by conventional genetic testing. Here, we report a novel deletion of AVPR2 in a Czech family identified for the first time by targeted long-read sequencing (T-LRS). METHODS: A male proband with X-linked NDI underwent clinical sequencing of the AVPR2 gene that failed and thus indicated possible whole-gene deletion. Therefore, PCR mapping and subsequent targeted long-read sequencing (T-LRS) using a Pacific Biosciences sequencer were applied to search for the suspected deletion. To validate the deletion breakpoints and prove variant segregation in the family with X-linked NDI, Sanger sequencing of the deletion junction was performed. Quantitative real-time PCR was further carried out to confirm the carrier status of heterozygous females. RESULTS: By T-LRS, a novel 7.5 kb deletion of AVPR2 causing X-linked NDI in the proband was precisely identified. Sanger sequencing of the deletion junction confirmed the variant breakpoints and detected the deletion in the probands´ mother, maternal aunt, and maternal cousin with X-linked NDI. The carrier status in heterozygous females was further validated by quantitative real-time PCR. CONCLUSIONS: Identifying the 7.5 kb deletion gave a precise molecular diagnosis for the proband, enabled genetic counselling and genetic testing for the family, and further expanded the spectrum of structural variants causing X-linked NDI. Our results also show that T-LRS has significant potential for accurately identifying putative structural variants.

A recurrent synonymous L1CAM variant in a fetus with hydrocephalus.

We report the case of a hydrocephalic fetus in which clinical exome sequencing revealed a recurrent synonymous variant of unknown significance, c.453G>T, in the L1CAM gene. This report presents the second case of X-linked hydrocephalus in a fetus with this variant. Since we reproduced the RNA analysis, we were able to reclassify this variant as likely pathogenic. Our results stress the importance of not excluding synonymous variants during prioritization.

Lynch syndrome-associated upper tract urothelial carcinoma frequently occurs in patients older than 60 years: an opportunity to revisit urology clinical guidelines.

Upper tract urothelial carcinoma (UTUC) is the third most common malignancy associated with Lynch syndrome (LS). The current European urology guidelines recommend screening for LS in patients with UTUC up to the age of 60 years. In this study, we examined a cohort of patients with UTUC for potential association with LS in order to establish the sensitivity of current guidelines in detecting LS. A total of 180 patients with confirmed diagnosis of UTUC were enrolled in the study during a 12-year period (2010-2022). Loss of DNA-mismatch repair proteins (MMRp) expression was identified in 15/180 patients (8.3%). Germline analysis was eventually performed in 8 patients confirming LS in 5 patients (2.8%), including 4 germline mutations in MSH6 and 1 germline mutation in MSH2. LS-related UTUC included 3 females and 2 males, with a mean age of 66.2 years (median 71 years, range 46-75 years). Four of five LS patients (all with MSH6 mutation) were older than 65 years (mean age 71.3, median 72 years). Our findings indicate that LS-associated UTUCs can occur in patients with LS older than 60 years. In contrast to previous studies which used mainly highly pre-selected populations with already diagnosed LS, the most frequent mutation in our cohort involved MSH6 gene. All MSH6 mutation carriers were > 65 years, and UTUC was the first LS manifestation in 2/4 patients. Using current screening guidelines, a significant proportion of patients with LS-associated UTUC may be missed. We suggest universal immunohistochemical MMRp screening for all UTUCs, regardless of age and clinical history.

Germline multigene panel testing of patients with endometrial cancer.

Endometrial cancer (EC) is the most common gynecological malignancy in developed countries. The present study aimed to determine the frequency of germline pathogenic variants (PV) in patients with EC. In this multicenter retrospective cohort study, germline genetic testing (GGT) was performed in 527 patients with EC using a next generation sequencing panel targeting 226 genes, including 5 Lynch syndrome (LS) and 14 hereditary breast and ovarian cancer (HBOC) predisposition genes, and 207 candidate predisposition genes. Gene-level risks were calculated using 1,662 population-matched controls (PMCs). Patients were sub-categorized to fulfill GGT criteria for LS, HBOC, both or none. A total of 60 patients (11.4%) carried PV in LS (5.1%) and HBOC (6.6%) predisposition genes, including two carriers of double PV. PV in LS genes conferred a significantly higher EC risk [odds ratio (OR), 22.4; 95% CI, 7.8-64.3; P=1.8×10-17] than the most frequently altered HBOC genes BRCA1 (OR, 3.9; 95% CI, 1.6-9.5; P=0.001), BRCA2 (OR, 7.4; 95% CI, 1.9-28.9; P=0.002) and CHEK2 (OR, 3.2; 95% CI, 1.0-9.9; P=0.04). Furthermore, >6% of patients with EC not fulfilling LS or HBOC GGT indication criteria carried a PV in a clinically relevant gene. Carriers of PV in LS genes had a significantly lower age of EC onset than non-carriers (P=0.01). Another 11.0% of patients carried PV in a candidate gene (the most frequent were FANCA and MUTYH); however, their individual frequencies did not differ from PMCs (except for aggregated frequency of loss-of-function variants in POLE/POLD1; OR, 10.44; 95% CI, 1.1-100.5; P=0.012). The present study demonstrated the importance of GGT in patients with EC. The increased risk of EC of PV carriers in HBOC genes suggests that the diagnosis of EC should be included in the HBOC GGT criteria.

Post-mortem genetic testing in sudden cardiac death and genetic screening of relatives at risk: lessons learned from a Czech pilot multidisciplinary study.

Sudden cardiac death (SCD) might have an inherited cardiac condition background. Genetic testing supports post-mortem diagnosis and screening of relatives at risk. Our aim is to determine the feasibility of a Czech national collaboration group and to establish the clinical importance of molecular autopsy and family screening. From 2016 to 2021, we have evaluated 100 unrelated SCD cases (71.0% males, age: 33.3 (12.8) years). Genetic testing was performed by next-generation sequencing utilizing a panel of 100 genes related to inherited cardiac/aortic conditions and/or whole exome sequencing. According to autopsy, cases were divided into cardiomyopathies, sudden arrhythmic death syndrome, sudden unexplained death syndrome, and sudden aortic death. We identified pathogenic/likely pathogenic variants following ACMG/AMP recommendations in 22/100 (22.0%) of cases. Since poor DNA quality, we have performed indirect DNA testing in affected relatives or in healthy parents reaching a diagnostic genetic yield of 11/24 (45.8%) and 1/10 (10.0%), respectively. Cardiological and genetic screening disclose 83/301 (27.6%) relatives at risk of SCD. Genetic testing in affected relatives as starting material leads to a high diagnostic yield offering a valuable alternative when suitable material is not available. This is the first multidisciplinary/multicenter molecular autopsy study in the Czech Republic which supports the establishment of this type of diagnostic tests. A central coordinator and proper communication among centers are crucial for the success of a collaboration at a national level.

Prevalence of cancer in patients with superficial vein thrombosis and its clinical importance.

OBJECTIVE: The objective of the present study was to evaluate the prevalence of cancer in patients with superficial vein thrombosis (SVT) of the legs. Moreover, we evaluated the potential determinants of SVT complications by comparing a subgroup with isolated SVT and a subgroup of SVT complicated by concurrent deep vein thrombosis (DVT) and/or pulmonary embolism (PE) with respect to the presence of cancer and other clinical and laboratory characteristics. METHODS: The present single-center, retrospective study of prospectively collected data was conducted in a tertiary care setting. We included patients who had been treated in the thrombosis clinic from 2006 to 2018 for symptomatic SVT of the legs, either isolated SVT or SVT complicated by concurrent DVT/PE. We evaluated the prevalence and type of malignancy (diagnosed ≤12 months before SVT and/or ongoing therapy), demographics, and clinical and laboratory characteristics of the patients. For statistical evaluation, we used the Student t test, Kruskal-Wallis test, Fisher exact two-sided test, and logistic regression. RESULTS: Of 276 patients with SVT (mean age, 58.9 ± 14.7 years; 60.9% women), 191 had had isolated SVT and 85 had had SVT complicated by concurrent DVT/PE. The prevalence of malignancy was 8.7% in the whole group (mainly breast and urinary tract cancer), including 4.2% of those with isolated SVT and 18.8% of those with SVT and concurrent DVT/PE (P < .001). Between the two subgroups, no significant differences were present in the duration of leg symptoms, family or personal history of SVT and/or DVT, SVT location, and smoking. In logistic regression, several factors were significantly associated with the concurrent presence of DVT/PE: age (odds ratio [OR], 1.024; 95% confidence interval [CI], 1.004-1.044), female gender (OR, 0.545; 95% CI, 0.309-0.960), varicose vein SVT (OR, 0.42; 95% CI, 0.194-0.902), thrombophilia (OR, 1.939; 95% CI, 1.089-3.454), and cancer (OR, 4.727; 95% CI, 1.814-12.316). CONCLUSIONS: The prevalence of malignancy in the patients with SVT was 8.7%. Age, thrombophilia, male gender, nonvaricose vein SVT, and cancer were significantly associated with the presence of concurrent DVT/PE. Cancer was the strongest determinant of concurrent DVT/PE.

Cystic ovarian teratoma as a novel tumor and growth hormone deficiency as a new condition presenting in Multiple Endocrine Neoplasia type 2B: Case reports and review of the literature.

BACKGROUND: We describe early and typical nonendocrine symptoms of Multiple Endocrine Neoplasia type 2B (MEN2B) presented in our patients with de novo M918T mutation in the RET proto-oncogene in early childhood, however, the diagnosis of MEN2B and medullary thyroid carcinoma (MTC) was confirmed late, in the second decade of life. In this paper, we emphasize the possibility of growth retardation, growth hormone (GH) deficiency and ovarian teratoma as a new symptom of MEN2B. CASE REPORTS: Advanced MTC with palpable mass on the neck and nonendocrine symptoms such as marfanoid habitus, thickened lips, mucosal neuromas led to the diagnosis in case 1 at the age of 13 years and GH deficiency and nonendocrine symptoms in case 2 at the age of 11 years. The earliest feature of MEN2B was alacrima and constipation. Patient 1 was operated on for a slipped femoral capital epiphysis and for a cystic ovarian teratoma. CONCLUSIONS: Improved awareness of nonendocrine signs of MEN2B could lead to earlier diagnosis, when surgical cure of MTC is possible. Alacrima is the first sign of MEN2B. We confirmed the possibility of growth retardation and GH deficiency in MEN2B, which had been previously rarely described. We suggest that patients with MEN2B may develop cystic ovarian teratoma, to the best of our knowledge, which has never been described so far in the literature. The results of this study could be used to guide further diagnosing of MENB2 at the early stage for better clinical outcome. We emphasize that MEN2B carries a risk for development of cystic ovarian teratoma as a novel tumor in this disease.

Unique characteristics of informed consent in clinical genetics and genetic counselling.

Rapid development of clinical genetics was enabled by the advances of molecular genetic laboratory diagnostics. Genetic laboratory testing has unique characteristics, and results of germinal genome testing has consequences not only for the patient but also for his relatives. Genetic laboratory testing in the Czech Republic is governed by the act no. 373/2011, which explicitly states that the testing requires the completion of a written informed consent. This article explains in detail the process of obtaining an informed consent within a broader framework of genetic counselling. An informed consent with genetic laboratory testing not only informs the patient (this being its primary purpose), but can also serve as a lead for physicians of other clinical specialties intending to order genetic laboratory tests.

Late diagnosis of complete androgen insensitivity syndrome and transmission/carriers of the condition in a family with mutation c.2495G> T p.(Arg832Leu) in exon 7 of the androgen receptor gene: genetic, clinical and ethical aspects.

BACKGROUND: The complete androgen insensitivity syndrome (CAIS) is a rare genetic disorder causing insensitivity to androgens in a person with female phenotype and 46,XY karyotype due to a mutation in the androgen receptor gene located on chromosome X. These children are born with female external genitalia, and females are transmitters. CASE REPORT: We illustrate an unexpected diagnosis of CAIS in two siblings during examination for short stature, and describe transmission/carriers in the family along with ethical aspects. CONCLUSION: A genetic examination could have earlier revealed the transmission of c.2495G>Tp.(Arg832Leu) mutation in exon 7. Our experience highlights the possibility of prenatal testing for the management of pregnancy in a family with a history of CAIS. The implications of prenatal testing in relation to CAIS with clearer explication of ethical and clinical issues warrant further investigation.

First 2 cases with thiamine-responsive megaloblastic anemia in the Czech Republic, a rare form of monogenic diabetes mellitus: a novel mutation in the thiamine transporter SLC19A2 gene-intron 1 mutation c.204+2T>G.

Thiamine-responsive megaloblastic anemia (TRMA) is a rare autosomal recessive disorder caused by mutations in the SLC19A2 gene. To date at least 43 mutations have been reported for the gene encoding a plasma membrane thiamine transporter protein (THTR-1). TRMA has been reported in less than 80 cases worldwide. Here, we illustrate 2 female patients with TRMA first diagnosed in the Czech Republic and in central Europe being confirmed by sequencing of the THTR-1 gene SLC19A2. Both subjects are compound heterozygotes with 3 different mutations in the SLC19A2 gene. In case 2, the SLC19A2 intron 1 mutation c.204+2T>G has never been reported before. TRMA subjects are at risk of diabetic ketoacidosis during intercurrent disease and arrythmias. Thiamine supplementation has prevented hematological disorders over a few years in both pediatric subjects, and improved glycaemic control of diabetes mellitus. Patient 1 was suffering from hearing loss and rod-cone dystrophy at the time of diagnosis, however, she was unresponsive to thiamine substitution. Our patient 2 developed the hearing loss despite the early thiamine substitution, however no visual disorder had developed. The novel mutation described here extends the list of SLC19A2 mutations causing TRMA.

Thrombophilia testing results in patients with a first venous thromboembolic event: should the selection criteria for testing be revisited?

BACKGROUND: After the first episode of venous thromboembolism (VTE), the guidelines recommend selective thrombophilia testing and suggest not to test the patients older than 40 years with a provoked event and all patients above 60. METHODS: We compared thrombophilia workup results in 544 patients, meeting or not meeting the selection criteria. Homozygous factor V Leiden or prothrombin gene mutation, natural anticoagulant deficiencies, antiphospholipid syndrome or combination of ≥2 disorders were considered a strong thrombophilia. RESULTS: Thrombophilia was detected in 28.5% and strong thrombophilia in 6.6% of patients. In the subgroup aged 40-60 years, in men with unprovoked cases the prevalence of thrombophilia was 35.7% and that of strong thrombophilia 12.5%; in provoked cases it was 19.5% and 4.9%, respectively. In women aged 40-60 with unprovoked events, thrombophilia was found in 18.8%, in cases provoked solely by estrogens or pregnancy in 40.9%, and in those with another trigger in 9.1%. Comparing the patients above and under 60, thrombophilia was detected in 27.6% and 29.2%, respectively (P=0.67) and strong thrombophilia in 9.1% and 4.7%, respectively (P=0.041). Factors significantly associated with positive thrombophilia testing were family history of VTE (including superficial vein thrombosis) - OR 1.80; 95% CI 1.71-2.77 and proximal location of deep vein thrombosis - OR 1.94; 95% CI 1.25-3.02. CONCLUSIONS: Of VTE patients not meeting selection criteria for testing, the prevalence of thrombophilia and even strong thrombophilia was high in those older than 60 years. Selection criteria for testing should be respected but in some cases an individual approach might be considered.

First case report of rare congenital adrenal insufficiency caused by mutations in the CYP11A1 gene in the Czech Republic.

We characterized a case of congenital adrenal insufficiency caused by cholesterol side-chain cleavage enzyme (P450scc) deficiency. The patient presented after birth with cardiopulmonary instability, hyponatremia, hyperkalemia, hypoglycemia and metabolic acidosis. We confirmed primary adrenal insufficiency. There were no signs of the external genitalia virilism. The replacement therapy with glucocorticoids and mineralocorticoids led to normal laboratory results. At the age of 12 years, we confirmed hypergonadotropic hypogonadism, which revealed disorder of steroidogenesis in the adrenal glands and in the gonads. The enzymatic block was found at the beginning of steroidogenesis. The mutation was confirmed in the CYP11A1 gene. The patient is compound heterozygote for the novel CYP11A1 missense mutation c.412G>A (p.Gly138Arg) in exon 2 and frameshift mutation c.508_509delCT (p.Leu170Valfs*30) in exon 3. The CYP11A1: c.412G>A (p.Gly138Arg) was predicted as pathogenic by in silico analysis. So far, only 19 patients with CYP11A1 mutations causing P450scc deficiency have been reported worldwide. There are no related reports in the Czech Republic.

The translocation t(2;11)(p21;q23) without MLL gene rearrangement--a possible marker of good prognosis in myelodysplastic syndrome patients.

The translocation t(2;11)(p21;q23) is associated with de novo myelodysplastic syndromes (MDS) and has an overall frequency of approximately 1%. The outcome of MDS patients with this translocation is not clear until now, because most of the clinical data addressing the t(2;11)(p21;q23) has been collected without investigating the status of the mixed lineage leukemia (MLL) gene. In this report, we present seven new patients with MDS diagnosis and the t(2;11)(p21;q23) in bone marrow cells; all of them without MLL gene rearrangement. They were found in two databases consisting of 1185 patients of two Czech institutions. These patients tended to be younger and showed a strong male predominance. A cytological and histological assessment of bone marrow at diagnosis revealed only mild MDS with marked dysplasia in megakaryopoiesis. Similar to other primary abnormalities in MDS (e.g. deletion of 11q), the t(2;11)(p21;q23) was frequently associated with deletion of 5q. Our results stress the common clinicopathological features of this entity and indicate that the t(2;11)(p21;q23) may be associated with a good prognosis for MDS patients (median survival 72 months).

Recurrent pregnancy loss, plasminogen activator inhibitor-1 (-675) 4G/5G polymorphism and antiphospholipid antibodies in Czech women.

PROBLEM: This study compares the frequencies of plasminogen activator inhibitor-1 (-675) 4G/5G polymorphism and its relationship with eight antiphospholipid antibodies (aPLs) in serum of 157 patients with repeated pregnancy loss (RPL). METHOD OF STUDY: PAI-1 (-675) 4G/5G polymorphism was determined using standard PCR-RFLP method. Enzyme-linked immunosorbent assay was used for the detection of aPLs against ph-serine, ph-ethanolamine, ph-inositol, ph-DL-glycerol, phosphatidic acid, annexin V, cardiolipin, and beta2-GPI. Allelic frequency and distribution of genotypes were calculated. The prevalence of the risk conferring 4G allele and 4G/4G homozygous genotype in patients and controls was compared, and the correlation between aPLs positivity and PAI-1 4G/4G genotype was tested by chi-square test. RESULTS: Statistically highly significant correlation between RPL and PAI-1 (-675) 4G/4G genotype was found. No correlation between PAI-1 (-675) 4G/5G polymorphism and the presence of antiphospholipid antibodies in RPL patients was observed. CONCLUSIONS: PAI-1 (-675) 4G/4G homozygous genotype increases the risk of RPL independently from the aPLs positivity.

Six new gap junction beta 1 gene mutations and their phenotypic expression in Czech patients with Charcot-Marie-Tooth disease.

X-linked Charcot-Marie-Tooth (CMTX) disease is a hereditary motor and sensory neuropathy caused by mutations in the gap junction beta 1 gene (GJB1 codes for connexin 32). In this study we report six novel mutations p.Met1Arg, p.Leu9Phe, p.Ser17Tyr, p.Val63Phe, p.Val170Ile, and p.Leu212Phe in GJB1 and their phenotypic expression. These mutations affect both intracellular and extracellular parts of the GJB1 protein. The screened patients had previously excluded the duplication/deletion on 17p11.2 and the male-to-male transfer in the pedigree. Except p.Val170Ile, all reported mutations segregated with the CMT phenotype in the families and caused CMTX1 neuropathy. Mutations were not found in 200 control DNA samples. Additionally, we performed in silico analysis of the novel mutations with the program PANTHER. The PANTHER scored five mutations, all but p.Val170Ile, as likely deleterious and supported the pathogenicity of the found mutations. These results provided evidence that these five mutations are causative for CMTX1.

Urgent termination of pregnancy in pre-eclampsia and panel of antiphospholipid antibodies.

PROBLEM: The present work was undertaken to investigate the occurence of autoantibodies to eight various phospholipids in time of urgent termination of the pregnancy (sectio caesarea) in patients in reproductive age with severe preeclamptic symptoms. METHOD OF STUDY: Autoantibodies against annexin V, ph-serine, ph-ethanolamine, ph-inositol, ph-DL-glycerol, cardiolipin, beta2-glycoprotein I (beta2-GPI), and phosphatidic acid were studied by ELISA methods. RESULTS: Increased levels of IgA-beta2-glycoprotein I, IgG-beta2-glycoprotein I, IgG- anti-ph-serine, and IgG-anticardiolipin were found in sera of preeclamptic women in the time of urgent sectio caesarea when compared to the control group with physiological pregnancy. CONCLUSION: Supposed increase in various antiphospholipid antibodies (aPLs) levels due to the stress during the short time of admission and a need for a quick medical decision to terminate the pregnancy was not unambiguously proven, but our results are evidently influenced by the current urgent life-saving treatment.