New Mutations Associated with Rasopathies in a Central European Population and Genotype-Phenotype Correlations.

We performed the genetic analysis of Rasopathy syndromes in patients from Central European by direct sequencing followed by next generation sequencing of genes associated with Rasopathies. All 51 patients harboured the typical features of Rasopathy syndromes. Thirty-five mutations were identified in the examined patients (22 in PTPN11, two in SOS1, one in RIT1, one in SHOC2, two in HRAS, three in BRAF, two in MAP2K1 and two in the NF1 gene). Two of them (p.Gly392Glu in the BRAF gene and p.Gln164Lys in the MAP2K1 gene) were novel with a potentially pathogenic effect on the structure of these proteins. Statistically significant differences in the presence of pulmonary stenosis (63.64% vs. 23.81%, P = 0.013897) and cryptorchidism (76.47% vs. 30%, P = 0.040224) were identified as the result of comparison of the prevalence of phenotypic features in patients with the phenotype of Noonan syndrome and mutation in the PTPN11 gene, with the prevalence of the same features in patients without PTPN11 mutation. Cryptorchidism as a statistically significant feature in our patients with PTPN11 mutation was not reported as significant in other European countries (Germany, Italy and Greece). The majority of mutations were clustered in exons 3 (45.45%), 8 (22.73%), and 13 (22.73%) of the PTPN11 gene.

Guidelines for surveillance of individuals with constitutional mismatch repair-deficiency proposed by the European Consortium "Care for CMMR-D" (C4CMMR-D).

Lynch syndrome (LS) is an autosomal dominant disorder caused by a defect in one of the DNA mismatch repair genes: MLH1, MSH2, MSH6 and PMS2. In the last 15 years, an increasing number of patients have been described with biallelic mismatch repair gene mutations causing a syndrome referred to as 'constitutional mismatch repair-deficiency' (CMMR-D). The spectrum of cancers observed in this syndrome differs from that found in LS, as about half develop brain tumours, around half develop digestive tract cancers and a third develop haematological malignancies. Brain tumours and haematological malignancies are mainly diagnosed in the first decade of life, and colorectal cancer (CRC) and small bowel cancer in the second and third decades of life. Surveillance for CRC in patients with LS is very effective. Therefore, an important question is whether surveillance for the most common CMMR-D-associated cancers will also be effective. Recently, a new European consortium was established with the aim of improving care for patients with CMMR-D. At a workshop of this group held in Paris in June 2013, one of the issues addressed was the development of surveillance guidelines. In 1968, criteria were proposed by WHO that should be met prior to the implementation of screening programmes. These criteria were used to assess surveillance in CMMR-D. The evaluation showed that surveillance for CRC is the only part of the programme that largely complies with the WHO criteria. The values of all other suggested screening protocols are unknown. In particular, it is questionable whether surveillance for haematological malignancies improves the already favourable outcome for patients with these tumours. Based on the available knowledge and the discussions at the workshop, the European consortium proposed a surveillance protocol. Prospective collection of all results of the surveillance is needed to evaluate the effectiveness of the programme.

Late onset of familial neurogenic diabetes insipidus in monozygotic twins.

OBJECTIVE: Autosomal dominant familial diabetes insipidus (FNDI) is a rare disease characterized by polydipsia and polyuria due to deficiency of the antidiuretic hormone, arginine vasopressin (AVP). We report the first Slovak family with the disease. Noteworthy is the concordantly belated debut of the disease symptoms in two monozygotic twin proband girls in the age of 17 years. Because of inconclusive results of water deprivation test consistent with partial diabetes insipidus (DI), missing "bright spot" of posterior pituitary gland in T1-weighted magnetic resonance imaging and family occurrence of polyuria and polydipsia on anamnestic evaluation. METHODS: Molecular genetic testing of the AVP gene was proceeded, because of the inconclusive results of water deprivation test consistent with partial diabetes insipidus, missing "bright spot" of posterior pituitary gland in T1-weighted magnetic resonance imaging and family occurrence of polyuria and polydipsia on anamnestic evaluation. RESULTS: Genetic analysis revealed a heterozygous g.279G>A substitution that predicts a p.Ala19Thr substitution in the signal peptide of the AVP prohormone. The wide intrafamiliar variations (3 to 17 years) in disease onset together with the concordantly delayed debut of polyuria in two monozygotic twin girls suggest that individual differences in genetic influences family environmental factors may modify the penetrance of the mutation of the AVP gene. CONCLUSIONS: The present paper supports the notion that molecular genetic evaluation should be performed in all patients with familial occurrence of DI regardless of the clinical results.

Rasopathies - dysmorphic syndromes with short stature and risk of malignancy.

OBJECTIVES: The term ´Rasopathies´ represents a group of five neurodevelopmental syndromes (Noonan, LEOPARD, Costello, Cardio-facio-cutaneous, and Neurofibromatose-Noonan syndrome) caused by germline mutation in genes encoding proteins involved in RAS/MAPK (rat sarcoma/mitogen-activated protein kinase) signaling pathway. The RAS/MAPK signaling pathway participates in regulation of cell determination, proliferation, differentiation, migration, and senescence and dysregulation of this pathway can lead to the risk of tumorigenesis. In this review, we aim to summarize the current clinical and molecular genetic knowledge on Rasopathies with special attention for the risk of cancer. We propose also clinical and therapeutic approach for patients with malignancy. METHODS: We are reviewing the clinical and molecular basis of Rasopathies based on recent studies, clinical examination, and molecular diagnostics (mutation analysis of causal genes for Rasopathies) in Slovak pediatric patients. RESULTS: Some clinical features, such as short stature, a specific facial dysmorphology and cardiac abnormalities are common to all of Rasopathy syndromes. However, there are unique signs by which the syndromes can differ from each other, especially multiple lentigo in LEOPARD syndrome, increased risk of malignancy in Costello syndrome, dry hyperkeratotic skin in patients with cardio-facio-cutaneous syndrome, and neurofibromas and cafe-au-lait spots in neurofibromatosis-Noonan syndrome. CONCLUSION: Despite the overlapping clinical features, Rasopathy syndromes exhibit unique fenotypical features and the precise molecular diagnostics may lead to confirmation of each syndrome. The molecular diagnostics may allow the detection of pathogenic mutation associated with tumorigenesis.

Clustering of mutations in the 5' tertile of the NF1 gene in Slovakia patients with optic pathway glioma.

Optic pathway gliomas (OPG) occur in 15% of patients with neurofibromatosis type 1 (NF1; OMIM 162200). Genotype-phenotype correlations in patients with NF1 may help to determine the risk group for developing complications such as OPG in coincidence with other NF1.features. We evaluated 52 patients with NF1 (25 with OPG and 27 without OPG). All subjects underwent a clinical examination focused on neurofibromatosis type 1 and molecular diagnostics of NF1 gene using protocol based on RNA analysis confirming the diagnosis of NF1. In the group with OPG patients, there was a significantly higher incidence of freckling (P=0.017), neurofibromatosis bright objects (NBO) (P=0.0038), compared to the group without OPG. The differences between the groups with respect to Lisch nodules were on the borderline of statistical significance (P=0.088). The frequency of neurofibromas in the group with OPG was not significant (P=0.9). From all patients with the mutation localized in the first tertile of the NF1 gene majority (71%) had optic glioma compared to individuals who didn't have the OPG 29% (P=0.0049). Our results present the clustering of mutations in the 5'tertile of NF1 gene in patients with optic nerve glioma and suggest higher incidence of freckling and neurofibromatosis brain objects in these patients. Molecular analysis of NF1 gene is important part in complex management of NF1 patients and contributes to a better understanding of clinical picture of NF1 patients. .

[Constitutional mismatch repair-deficiency syndrome (CMMR-D) - a case report of a family with biallelic MSH6 mutation]. / Syndróm konstitucného deficitu mismec opravného systému (CMMR-D) - kazuistika rodiny s bialelickou MSH6 mutáciou.

This work gives comprehensive information about new recessively inherited syndrome characterized by development of childhood malignancies. Behind this new described syndrome, called Constitutional mismatch repair-deficiency syndrome (CMMR-D), there are biallelic mutations in genes, which cause adult cancer syndrom termed Lynch syndrom (Hereditary non-polyposis cancer syndrom-HNPCC) if they are heterozygous mutations. Biallelic germline mutations of genes MLH1, MSH2, MSH6 and PMS2 in CMMR-D are characterized by increased risk of hematological malignancies, atypical brain tumors and early onset of colorectal cancers. An accompanying manifestation of the disease are skin spots with diffuse margins and irregular pigmentation reminiscent of Café au lait spots of NF1. This paper reports a case of a family with CMMR-D caused by novel homozygous MSH6 mutations leading to gliomatosis cerebri, T-ALL in an 11-year-old female and glioblastoma multiforme in her 10-year-old brother, both with rapid progression of the diseases. A literature review of brain tumors in CMMR-D families shows that they are treatment-resistant and lead to early death. Therefore, this work highlights the importance of early identification of patients with CMMR-D syndrome - in terms of initiation of a screening program for early detection of malignancies as well as early surgical intervention.

[Clinical dysmorphic syndromes with tumorigenesis]. / Klinické dysmorfické syndrómy s tumorigenézou.

Genetic alterations cause predisposition to malignancy by increased cancer risk related to constitutional mutations in growth-regulating or DNA repair genes. Some pediatric malignancies are associated with dysmorphic features in several body areas. Through physical examination, we recognise characteristic signs of genetic dysmorphic disorders, such as somatic overgrowth, undergrowth, macrocephaly, microcephaly and dysmorphic changes of the face, eyes, mouth and lips, heart, gastrointestinal tract, urinary tract, genitalia and skeleton. Recognition of a cancer-associated dysmorphic syndrome allows intensive cancer screening and genetic counseling. Therefore, it is recommended that every child with cancer should be examined by a clinical geneticist. Molecular diagnostics of germinal mutations may very effectively detect families at high risk of malignancy and help provide primary prevention. This work presents clinical syndromes with genetic background and cancer screening recommendations for 18 syndromes with increased cancer risk.

Recurring multifocal leiomyosarcoma of the urinary bladder 22 years after therapy for bilateral (hereditary) retinoblastoma: a case report and review of the literature.

We report on a case of urinary bladder leiomyosarcoma in a 23-year-old woman, 22 years after therapy for bilateral retinoblastoma. The tumor presented with dysuria and macroscopic haematuria. Cystoscopy revealed a tumor localized in the trigonum covered by an ulcerated urothelium. The patient underwent a transvesical tumor resection. Eight months later, a second leiomyosarcoma developed in the vertex, at a site different from the previous one. A cystoscopic trans-urethral tumor resection was performed, followed by combined chemotherapy. One year later another recurrence occurred at the site of the primary resection. Open laparotomic resection of the involved bladder wall was performed. The patient remains both recurrence and metastases free after twenty months of follow-up. Molecular analysis of the peripheral blood showed rare germline point mutation in the intron 24 of the RB1 gene. FISH analysis of the tumor tissue revealed polyploid cells with relative loss of normal RB1 gene locus, indicating deletion and second hit loss of the second RB1 allele function. Along with the ten previously reported cases, this report suggests a non-random association between the hereditary retinoblastoma and urinary bladder leiomyosarcoma. Therapy with cyclophosphamide seems to be an important risk factor. Life-long surveillance for second malignancies, including bladder leiomyosarcoma is therefore mandatory in these patients.

[Identification of molecular markers in children with acute myeloid leukemia (AML)]. / Identifikácia molekulárnych markerov u detí s akútnou myeloblastovou leukémiou (AML).

BACKGROUNDS: AML is an aggressive, phenotypically and genetically heterogenous clonal disease of hematopoietic progenitor cells with a great molecular variability. New WHO classification 2008 divides de novo AML according to cytogenetic and molecular prognostic and predictive markers. Recently, it is increasingly possible to identify a subgroup of poorer prognosis patients among those with normal karyotype AML. The aim of our study was to identify prognostically important molecular markers in children with AML, to stratify patients with normal karyotype and to monitor the disease according the genetic findings. MATERIAL AND METHODS: In 2008-2010, we analyzed bone marrow and peripheral blood samples of 20 children with de novo AML by conventional cytogenetic analysis, fluorescence in situ hybridisation and molecular diagnostics. The molecular analysis was performed on the cDNA level, with the restriction analysis of PCR products (FLT3-TKD), conventional PCR (MLL-PTD, NPM1mut, FLT3-ITD) and quantification RT-PCR method (expression of fusion transcripts, BAALC, WT1). RESULTS: Samples from 20 children with AML were analyzed using the conventional cytogenetics, FISH and molecular methods. Abnormal karyotype was identified in 13 patients (65%). Further analysis revealed FLT3-ITD in 5/20 (25%), FLT3-TKD in 3/20 (15%), NPM1mut in 2/20 (10%) and MLL-PTD in 1/20 (5%), overexpression of WT1 gene in 15/20 (75%) and overexpression of BAALC in 13/20 (65%) patients. CONCLUSION: Wide cytogenetic and molecular screening helped to find at least one genetic marker in all 20 patients for later follow-up and risk stratification. 4/20 (20%) patients died of the disease progression.

Burkitt lymphoma with unusual granulomatous reaction. A case report.

Formation of epithelioid histiocytic cell granulomas has been described in the post in various neoplasms, hematologic malignancies included. Among lymphoproliferative disorders such changes are commonly found in Hodgkin lymphoma and T-cell non-Hodgkin lymphomas (NHL), but are rarely described in B-NHL, like Burkitt lymphoma. This report presents a case of sporadic Burkitt lymphoma accompanied by a sarcoid-like reaction without any clinical, laboratory or histological evidence of microorganisms nor sarcoidosis. Using in situ hybridization and polymerase chain reaction the presence of the Epstein-Barr virus (EBV) was detected in the analyzed lymphoma cells. EBV demonstrated latency I phenotype as defined by the lack of immunohistochemical positivity of latent membrane protein 1 (LMP1). Cytogenetic investigation using fluorescence in situ hybridization uncovered c-MYC mutation and provided indirect indication for the MYC/IgL fusion gene. The lack of EBV positivity in histiocytes indicated the reactive character of the granulomatous reaction in relation to the neoplasm. The role of the granulomatous reaction in the biology and prognosis of Burkitt lymphoma and the function of EBV infection in its development remain to be established.

Minimal residual disease detection using real-time quantitative PCR analysis of immunoglobulin and T-cell receptor gene rearrangements in the non-MRD-based ALL IC-BFM 2002 protocol for childhood ALL: Slovak experience.

Acute lymphoblastic leukemia is the most common form of cancer in children. The 10-year event-free survival ranged from 77 to 85% after having achieved complete remission rates of 93% or higher. The main cause of treatment failure is relapse arising from outgrowth of residual leukemic cells that are refractory to therapy. An intense effort has been made to develop methods to determine the degree of minimal residual leukemia cells present in patients considered to be in morphological remission. Because of the strong correlation between minimal residual disease (MRD) levels and risk of relapse, monitoring of MRD provides unique information regarding treatment response. The MRD monitoring based on real-time quantitative PCR detection of patient-specific immunoglobulin and T-cell receptor (Ig/TCR) gene rearrangements is currently considered to be the most reliable tool for MRD-based diagnosis in ALL. Because the significance of MRD monitoring has been strongly supported by several studies and because it has been implemented in the latest protocols, there has been a significant effort to develop MRD monitoring in the Slovak Republic since 2005. Between October 2006 and December 2009, 50 children with ALL who were treated at three Slovak centers were included in the RQ PCR MRD pilot project. A total of 40 patients with BCP-ALL ( B cell precursor ALL) and 4 patients with T ALL were analyzed for Ig/TCR rearrangement. We identified 106 different rearrangements in the 44 ALL patients analyzed. Based on MRD stratification, we identified 26 patients who were stratified into the HRG ( high risk group) (n = 3; 11.5%), IRG ( intermediate risk group) (n = 14; 54%) and SRG ) standard risk group) (n = 9; 34.5%). Morphology-based risk stratification allows the identification of most HRG patients identified also by MRD-based stratification, but fails to discriminate the IRG assigned to therapy reduction. Patients in the SRG and the IRG could profit from MRD-based risk assignment

[Acute myeloblastic leukaemia with alternations of MLL proto-oncogene protein (11q23/MLL+ AML)]. / Akútna myeloblastová leukémia s alteráciami MLL protoonkogénu (11q23/MLL+ AML).

One of the most common chromosomal breakpoint regions in acute myeloid leukaemia is the chromosome band 11q23. The analysis of this region led to the discovery of the extremely promiscuous MLL gene, in which more than 60 MLL translocation partner genes have been described. Among the most frequent are t(9;11)(p21-22;q23)/MLL-AF9, t(10; 11)(p13; q23)/MLL-AF10, t(11;19)(q23;p13)/MLL-ELL, ENL and t(6;11)(q27;q23)/MLL-AF6. The presented work provides an overview of the molecular mechanisms by means of which MLL proto-oncogene can be converted into oncogene. Genetic alternations of the MLL Proto-Oncogene Protein besides translocation are also represented by complex chromosomal rearrangements, deletions, insertions, partial tandem duplications, amplifications and gains. These genetic alterations are described in the work from the diagnostic and prognostic point of view. Abnormalities of the MLL ProtoOncogene Protein are usually connected with bad prognosis. For that reason, in oncological practice, particular attention is paid to introducing new genetic methods for their identification. The above work gives well arranged information about different types of genetic tests and their outcomes, which can help oncologists in predicting the prognosis, in minimal residual disease monitoring and in modifying oncological patient treatment.

New insights to the MLL recombinome of acute leukemias.

Chromosomal rearrangements of the human MLL gene are associated with high-risk pediatric, adult and therapy-associated acute leukemias. These patients need to be identified, treated appropriately and minimal residual disease was monitored by quantitative PCR techniques. Genomic DNA was isolated from individual acute leukemia patients to identify and characterize chromosomal rearrangements involving the human MLL gene. A total of 760 MLL-rearranged biopsy samples obtained from 384 pediatric and 376 adult leukemia patients were characterized at the molecular level. The distribution of MLL breakpoints for clinical subtypes (acute lymphoblastic leukemia, acute myeloid leukemia, pediatric and adult) and fused translocation partner genes (TPGs) will be presented, including novel MLL fusion genes. Combined data of our study and recently published data revealed 104 different MLL rearrangements of which 64 TPGs are now characterized on the molecular level. Nine TPGs seem to be predominantly involved in genetic recombinations of MLL: AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, MLLT4/AF6, ELL, EPS15/AF1P, MLLT6/AF17 and SEPT6, respectively. Moreover, we describe for the first time the genetic network of reciprocal MLL gene fusions deriving from complex rearrangements.

The spectrum and incidence of BRCA1 pathogenic mutations in Slovak breast/ovarian cancer families.

Pathogenic germline mutations in BRCA1 and BRCA2 account for the majority of hereditary breast/ovarian cancer cases. The analysis of BRCA1 gene was carried out in 156 breast/ovarian cancer families: 82 families with strong family history and 59 families with medium family history. Generally, 31 families and 71 cases with BRCA1 pathologic mutations (14 different types) were identified in this study by combination of SSCP and direct sequencing techniques. Using approved systematic nomenclature numbering, c.5266dupC (8 families, 21 cases), c.181T>G (5 families, 11 cases), c.68_69delAG (3 families, 5 samples) and c.843_846del4 (3 families, 4 samples) were the most frequently found mutations in BRCA1 gene. Altogether these 4 mutations accounted for 61.3% of all detected pathogenic mutations in BRCA1. One novel mutation c.1166delG was detected in one family (4 cases). Frame-shift mutations were found in 21 families (46 cases), nonsense mutations in 4 families (8 cases) and missense mutations in 6 families (17 cases). Even though the 4 most frequent mutations account for 61.3% of all detected pathogenic mutations, screening of the whole BRCA1 coding region is necessary, due to the large scale of low frequency disease causing mutations in breast/ovarian cancer families in Slovakia.

The novel exon 11 mutation of BRCA1 gene in a high-risk family.

Germline mutations in the BRCA1 and BRCA2 genes are required for the initiation of the development of hereditary forms of breast and ovarian cancer, which represent 10-15% of all cases. The course of the disease varies from case to case that can be due even to the possibility of multiple genetic changes including inactivation of other tumor suppressor genes--TP53 and APC genes or activation of oncogenes, especially K-ras oncogene. The combination of these changes results in an early expression of the broad variety of malignancies. The analyzed proband (II-5) comes from a high-risk family, in which various types of cancer were observed. The novel BRCA1 mutation in exon 11 (2057delCAGTGAAGAG) was detected by SSCP, HDA techniques and confirmed by automatic sequencing. The same deletion was observed in DNA sample of her first daughter (III-1), but DNA of her second one was without any mutational changes (III-2). Due to the occurrence of different types of cancer in this family, the incidental mutations in the APC; resp. TP53 tumor supressor genes and K-ras oncogene were searched as well. Any mutation was found after sequencing of SSCP interesting exons of these genes. The reasons for such strong malignant manifestation in this high risk family are discussed.

Approaches to identification of HNPCC suspected patients in Slovak population.

Patients with hereditary non-polyposis colorectal cancer (HNPCC) have a DNA mismatch repair defect (MMR) in their tumor tissue that results in instability of microsatellite DNA sequences (MSI). Thus, MSI analysis may effectively indicate this form of cancer that should be then proved by analysis of germline mutations in MMR genes. The aim of this study was to identify HNPCC suspected patients in the Slovak population by investigating microsatellite instability in colorectal tumor tissues. MSI was studied at 5-11 loci in matched tumor and normal DNA using radioactively labeled PCR products separated on sequencing gels. High microsatellite instability (MSI-H) was present only in patients younger than 50 years, in 100% of patients having two affected relatives by colorectal cancer and in 67% of patients with only one affected relative. In both groups of patients colorectal cancer was present in two successive generations. No MSI-H was found in the group of patients older than 50 years, even if they had positive family history for colorectal cancer. Among all markers used, the BAT26 mononucleotide repeat (100%), DI0S197 and D13S175 (62.5%) dinucleotide repeats were the most frequently altered in the tumor tissues. Retrospective analysis revealed that some of the patients having MSI-H tumors have had clinicopathological characteristics frequently reported to HNPCC. The family members of those patients with MSI-H are enrolled in preventive health care program until mutational analyses will enable to select carriers from non-carriers of mutated MMR genes.

A double germline mutations in the APC and p53 genes.

Germline mutation in the APC gene is required for the initiation of the development of familial adenomatous polyposis (FAP). According to Fearon and Vogelstein model, further somatic mutations in the K-ras oncogene, DCC gene and p53 tumor suppressor gene are prerequisite for development of colon carcinoma. We have found that the germline mutations in the DNA isolated from lymphocytes of an 18 years old girl with extraordinary expressive phenotype in codons 1060-1061 of the APC gene result in truncation of the APC protein. The mutation in codons 12 and 13 of the K-ras oncogene was not detected, but another germline mutation was found in codon 210 of the p53 gene. Furthermore, no one of these germline mutations was detected in the DNA of peripheral blood lymphocytes of the patient's 21 years old healthy sister. Until now, there has been no evidence about the expressive phenotype due to mutation in codons 1060-1061 of the APC gene; the role of germline missense mutation in codon 210 of the p53 gene in the FAP malignant process remains to be elucidated too. The effect of the combination of germline mutation in two different tumor suppressor genes in the progress of disease is discussed.