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Purpose: In many children with cancer and characteristics suggestive of a genetic predisposition syndrome, the genetic cause is still unknown. We studied the yield of pathogenic mutations by applying whole-exome sequencing on a selected cohort of children with cancer.Experimental Design: To identify mutations in known and novel cancer-predisposing genes, we performed trio-based whole-exome sequencing on germline DNA of 40 selected children and their parents. These children were diagnosed with cancer and had at least one of the following features: (1) intellectual disability and/or congenital anomalies, (2) multiple malignancies, (3) family history of cancer, or (4) an adult type of cancer. We first analyzed the sequence data for germline mutations in 146 known cancer-predisposing genes. If no causative mutation was found, the analysis was extended to the whole exome.Results: Four patients carried causative mutations in a known cancer-predisposing gene: TP53 and DICER1 (n = 3). In another 4 patients, exome sequencing revealed mutations causing syndromes that might have contributed to the malignancy (EP300-based Rubinstein–Taybi syndrome, ARID1A-based Coffin–Siris syndrome, ACTB-based Baraitser–Winter syndrome, and EZH2-based Weaver syndrome). In addition, we identified two genes, KDM3B and TYK2, which are possibly involved in genetic cancer predisposition.Conclusions: In our selected cohort of patients, pathogenic germline mutations causative or likely causative of the cancer phenotype were found in 8 patients, and two possible novel cancer-predisposing genes were identified. Therewith, our study shows the added value of sequencing beyond a cancer gene panel in selected patients, to recognize childhood cancer predisposition. Clin Cancer Res; 24(7); 1594–603. ©2018 AACR.

Supplementary Table S1 - Cancer gene panel Supplementary Table S2 - Patients with multiple malignancies Supplementary Table S3 - Tumor types of index patients Supplementary Table S4 - Candidate genes Supplementary Table S5 - Overview of clinical features and mutations found in category 1 (ID and/or congenital anomalies) Supplementary Table S6 - Overview of clinical features and mutations found in category 2 (cancer twice) Supplementary Table S7 - Overview of clinical features and mutations found in category 3 (family history) Supplementary Table S8 - Overview of clinical features and mutations found in category 4 (adult type of cancer) Supplementary Table S9 - Overview of clinical features and mutations found in category 5 (multiple reasons for inclusion) Supplementary Figure S1 - Detection of mosaic mutation in ARID1A Supplementary Figure S2 - Family tree of case #04 Supplementary Figure S3 - Family tree of case #05 Supplementary Figure S4 - Family tree of case #07 Supplementary Figure S5 - Family tree of case #10 Supplementary Figure S6 - Family tree of case #12 Supplementary Figure S7 - Family tree of case #17 Supplementary Figure S8 - Family tree of case #21 Supplementary Figure S9 - Family tree of case #22 Supplementary Figure S10 - Family tree of case #24 Supplementary Figure S11 - Family tree of case #27 Supplementary Figure S12 - Family tree of case #28 Supplementary Figure S13 - Family tree of case #29 Supplementary Figure S14 - Family tree of case #34 Supplementary Figure S15 - Family tree of case #36 Supplementary Figure S16 - Family tree of case #39

This file contains all mutations identified in all patients.

Introduction: In children, chronic pancreatitis (CP) is usually associated with anatomical anomalies of the pancreas and biliary tract or is genetically determined. Autosomal dominant polycystic kidney disease (ADPKD) may present with extrarenal cyst formation, sometimes involving the pancreas. Large enough, these cysts may cause pancreatitis in ADPKD patients. Case presentation: Herein, we present a case of a 12-year-old Caucasian girl with recurrent pancreatitis with no identifiable traumatic, metabolic, infectious, drug, or immunologic causes. Structural anomalies of the pancreas, including cysts, were ruled out by imaging. However, bilateral cystic kidneys were found as an incidental finding. Her family history was negative for pancreatitis, but positive for polycystic kidney disease. Molecular analysis of ADPKD-causing mutations revealed a novel c.9659C>A (p.Ser3220*) mutation in the PKD1 gene confirming the clinical suspicion of ADPKD. Although CP may rarely occur as an extrarenal manifestation of ADPKD with pancreatic cysts, it is unusual in their absence. Thus, molecular analysis of pancreatitis susceptibility genes was performed and a homozygous pathologic c.180C>T (p.G60=) variant of the CTRC gene, known to increase the risk of CP, was confirmed. Conclusion: This is the first reported case of a pediatric patient with coincidence of genetically determined CP and ADPKD. Occurrence of pancreatitis in children with ADPKD without pancreatic cysts warrants further investigation of CP causing mutations. Resumen: Introducción: En niños, la pancreatitis crónica (CP, por sus siglas en inglés) generalmente se asocia con anomalías anatómicas del páncreas y el tracto biliar, o está genéticamente determinada. La enfermedad renal poliquística autosómica dominante (ADPKD, por sus siglas en inglés) puede presentarse con la formación de quistes extrarrenales, que a veces afecta al páncreas. Suficientemente grandes, estos quistes pueden causar pancreatitis en pacientes con ADPKD. Presentación del caso: Presentamos el caso de una niña caucásica de 12 años con pancreatitis recurrente sin causas identificables traumáticas, metabólicas, infecciosas, farmacológicas o inmunológicas. Las anomalías estructurales del páncreas, incluidos los quistes, se descartaron mediante imágenes. Sin embargo, los riñones quísticos bilaterales se encontraron como un hallazgo accidental. Su historia familiar fue negativa para la pancreatitis, pero positiva para la enfermedad renal poliquística. El análisis molecular de las mutaciones causantes de ADPKD reveló una nueva mutación c.9659C>A (p.Ser3220*) en el gen PKD1 que confirma la sospecha clínica de ADPKD. Aunque la CP rara vez ocurre como una manifestación extrarrenal de ADPKD con quistes pancreáticos es inusual. Por lo tanto, se realizó el análisis molecular de los genes de susceptibilidad a pancreatitis y se confirmó una variante homocigótica patológica c.180C>T (p.G60=) del gen CTRC, que se sabe que aumenta el riesgo de CP. Conclusión: Este es el primer caso reportado de un paciente pediátrico con coincidencia de CP y ADPKD genéticamente determinados. La aparición de pancreatitis en niños con ADPKD sin quistes pancreáticos justifica una mayor investigación de CP que causan mutaciones.

Tetrasomy 18p is an extremely rare disorder with estimated incidence 1:140,000-180,000. The most common clinical presentations include developmental delay, intellectual disability, and several dysmorphic features such as the triangular face, low-set ears, depressed nasal bridge or smooth philtrum. The disease's hallmark is a small supernumerary isochromosome 18p consisting of two copies of the same arm of chromosome 18. Males and females are affected equally. Here, we present the case of 20 months old girl with tetrasomy 18p, the only affected child out of the dizygotic twins born to healthy parents. Routine cytogenetic testing revealed the presence of the marker chromosome in the girl's sample. Combination of Multicolor Banding fluorescent in situ hybridization and array-based Comparative Genomic Hybridization confirmed the isochromosome 18p in this girl. Quantitative Fluorescence PCR determined the maternal origin of the isochromosome 18p. The presented case of tetrasomy18p in one of the non-identical twins supports the hypothesis that tetrasomy 18p arises de novo and are of maternal origin. We conclude that the formation of isochromosome 18p is a random event that can occur in pregnancies of cytogenetically normal parents and maternal age is probably the main risk factor in etiology of the tetrasomy 18p.

Autosomal dominant disorder Legius syndrome (NF1- like syndrome) shows phenotype features that overlap with neurofibromatosis type 1 (NF1), such as CALMs, freckling, macrocephaly and learning disability. Mutation analysis provides an important tool in order to distinguish two entities that have different clinical implications. We analyzed SPRED1 gene by cDNA and/or gDNA sequencing in a cohort of 46 Slovak patients in whom previously NF1 mutation was excluded. In one case we identified a nonsense mutation c.46C>T (p.Arg16*) in exon 2 of SPRED1 gene, confirming diagnosis of Legius syndrome. This mutation was reported previously.

Graf, Norbert/0000-0002-2248-323X; Reis, Andre/0000-0002-6301-6363; Diets, Illja/0000-0001-7603-8898; Popp, Bernt/0000-0002-3679-1081; Thiel, Christian T/0000-0003-3817-7277; Roeleveld, Nel/0000-0002-3390-4466; Reis, AlessanRSS/0000-0001-8486-7469; Vasileiou, Georgia/0000-0002-1993-1134; Gessler, Manfred/0000-0002-7915-6045 WOS:000472571300008 PubMed ID: 30694527 Two percent of patients with Wilms tumors have a positive family history. In many of these cases the genetic cause remains unresolved. By applying germline exome sequencing in two families with two affected individuals with Wilms tumors, we identified truncating mutations in TRIM28. Subsequent mutational screening of germline and tumor DNA of 269 children affected by Wilms tumor was performed, and revealed seven additional individuals with germline truncating mutations, and one individual with a somatic truncating mutation in TRIM28. TRIM28 encodes a complex scaffold protein involved in many different processes, including gene silencing, DNA repair and maintenance of genomic integrity. Expression studies on mRNA and protein level showed reduction of TRIM28, confirming a loss-of-function effect of the mutations identified. The tumors showed an epithelial-type histology that stained negative for TRIM28 by immunohistochemistry. The tumors were bilateral in six patients, and 10/11 tumors are accompanied by perilobar nephrogenic rests. Exome sequencing on eight tumor DNA samples from six individuals showed loss-of-heterozygosity (LOH) of the TRIM28-locus by mitotic recombination in seven tumors, suggesting that TRIM28 functions as a tumor suppressor gene in Wilms tumor development. Additionally, the tumors showed very few mutations in known Wilms tumor driver genes, suggesting that loss of TRIM28 is the main driver of tumorigenesis. In conclusion, we identified heterozygous germline truncating mutations in TRIM28 in 11 children with mainly epithelial-type Wilms tumors, which become homozygous in tumor tissue. These data establish TRIM28 as a novel Wilms tumor predisposition gene, acting as a tumor suppressor gene by LOH. Dutch Cancer SocietyKWF Kankerbestrijding [KUN2012-5366]; KiKa Foundation [127] Grant sponsor: Dutch Cancer Society; Grant number: KUN2012-5366; Grant sponsor: The KiKa Foundation (project 127)

Purpose: In many children with cancer and characteristics suggestive of a genetic predisposition syndrome, the genetic cause is still unknown. We studied the yield of pathogenic mutations by applying whole-exome sequencing on a selected cohort of children with cancer. Experimental Design: To identify mutations in known and novel cancer-predisposing genes, we performed trio-based whole-exome sequencing on germline DNA of 40 selected children and their parents. These children were diagnosed with cancer and had at least one of the following features: (1) intellectual disability and/or congenital anomalies, (2) multiple malignancies, (3) family history of cancer, or (4) an adult type of cancer. We first analyzed the sequence data for germline mutations in 146 known cancer-predisposing genes. If no causative mutation was found, the analysis was extended to the whole exome. Results: Four patients carried causative mutations in a known cancer-predisposing gene: TP53 and DICER1 (n = 3). In another 4 patients, exome sequencing revealed mutations causing syndromes that might have contributed to the malignancy (EP300-based Rubinstein–Taybi syndrome, ARID1A-based Coffin–Siris syndrome, ACTB-based Baraitser–Winter syndrome, and EZH2-based Weaver syndrome). In addition, we identified two genes, KDM3B and TYK2, which are possibly involved in genetic cancer predisposition. Conclusions: In our selected cohort of patients, pathogenic germline mutations causative or likely causative of the cancer phenotype were found in 8 patients, and two possible novel cancer-predisposing genes were identified. Therewith, our study shows the added value of sequencing beyond a cancer gene panel in selected patients, to recognize childhood cancer predisposition. Clin Cancer Res; 24(7); 1594–603. ©2018 AACR.

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.

Over the past decades, a pleiotropic spectrum of B-cell intrinsic defects leading to early onset agammaglobulinemia and absent B cells has been described. Herein we report terminal 14q32.33 deletion as a novel cause of agammaglobulinemia. We describe a 20-year old man with a 1 MB terminal 14q32.33 deletion resulting in a loss of the entire Immunoglobulin heavy chain gene region of chromosome 14. The patient presented with absent serum immunoglobulin levels and absent circulating B cells since age 2. The clinical picture was dominated by severe episodes of recurrent upper respiratory tract infections. In the literature, the most prevalent features of terminal 14q32.33 deletions include mental disability, facial malformation, hypotonia, seizures, and visual problems with retinal abnormalities. Neither increased susceptibility to infections nor agammaglobulinemia have been described as a manifestation of terminal 14q32.33 deletion. Thus, our findings expand the known clinical spectrum of terminal 14q32.33 deletion to include susceptibility to infections.

Constitutional mismatch repair deficiency (CMMRD) syndrome is a distinct childhood cancer predisposition syndrome that results from biallelic germline mutations in one of the four MMR genes, MLH1, MSH2, MSH6 or PMS2. The tumour spectrum is very broad, including mainly haematological, brain and intestinal tract tumours. Patients show a variety of non-malignant features that are indicative of CMMRD. However, currently no criteria that should entail diagnostic evaluation of CMMRD exist. We present a three-point scoring system for the suspected diagnosis CMMRD in a paediatric/young adult cancer patient. Tumours highly specific for CMMRD syndrome are assigned three points, malignancies overrepresented in CMMRD two points and all other malignancies one point. According to their specificity for CMMRD and their frequency in the general population, additional features are weighted with 1-2 points. They include multiple hyperpigmented and hypopigmented skin areas, brain malformations, pilomatricomas, a second childhood malignancy, a Lynch syndrome (LS)-associated tumour in a relative and parental consanguinity. According to the scoring system, CMMRD should be suspected in any cancer patient who reaches a minimum of three points by adding the points of the malignancy and the additional features. The diagnostic steps to confirm or refute the suspected diagnosis are outlined. We expect that application of the suggested strategy for CMMRD diagnosis will increase the number of patients being identified at the time when they develop their first tumour. This will allow adjustment of the treatment modalities, offering surveillance strategies for second malignancies and appropriate counselling of the entire family.

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. .

Summary We performed a complex analysis of the neurofibromatosis type 1 (NF1) gene in Slovakia based on direct cDNA sequencing supplemented by multiple ligation dependent probe amplification (MLPA) analysis. All 108 patients had cafe-au-lait spots, 85% had axilary and/or inguinal freckling, 61% neurofibromas, 36% Lisch nodules of the iris and 31% optic pathway glioma, 5% suffered from typical skeletal disorders, and 51% of patients had family members with NF1. In 78 of the 86 (90.7%) index patients our analysis revealed the presence of NF1 mutations, 68 of which were small changes (87.2%), including 39 (50%) novel. Among the identified mutations the most prevalent were small deletions and insertions causing frameshift (42.3%), followed by nonsense (14.1%), missense (12.8%), and typical splicing (11.5%) mutations. Type 1 NF1 deletions and intragenic deletions/duplication were identified in five cases each (6.4%). Interestingly, in five other cases nontypical splicing variants were found, whose real effect on NF1 transcript would have remained undetected if using a DNA-based method alone, thus underlying the advantage of using the cDNA-based sequencing. We show that Slovak NF1 patients have a similar repertoire of NF1 germline mutations compared to other populations, with some prevalence of small deletions/insertions and a decreased proportion of nonsense mutations.

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.

Biallelic germline mutations of Constitutional mismatch repair-deficiency syndrome (CMMR-D) genes, MLH1, MSH2, MSH6, and PMS2 are characterized by increased risk of childhood malignancy. We report a case with CMMR-D caused by novel homozygous MSH6 mutations leading to gliomatosis cerebri and 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 on brain tumors in CMMR-D families shows that they are treatment-resistant and lead to early death. Identification of patients with CMMR-D is critical, and specific cancer screening programs with early surgery are recommended.

Background and Objective: Chemotherapy for advanced non-small-cell lung cancer (NSCLC) remains marginally effective, with a 5-year overall survival rate of approximately 5%. Recently, the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib was approved in Slovakia for the treatment of metastatic NSCLC. Gefitinib is a selective EGFR inhibitor that binds to the adenosine triphosphate binding pocket of the kinase domain and blocks downstream signaling pathways. Mutations of the EGFR gene, particularly an in-frame 15 bp deletion (delE746_A750) in exon 19 and the L858R mutation in exon 21, correlate with enhanced clinical responsiveness to EGFR tyrosine kinase inhibitors. However, the detection of these mutations and thereby prediction of the therapy outcome is sometimes unreliable due to the low sensitivity of direct sequencing if the proportion of tumor cells in the tissue is less than 25%. Therefore we decided to test the applicability of other methods, particularly high-resolution melting analysis (HRMA), for detection of these mutations in clinical samples. Methods: We analyzed 53 archival cytologic specimens for the presence of EGFR mutations, using the HRMA method. Results were verified by direct sequencing. For samples containing less than 25% tumor cells, we used mutant-enriched PCR before sequencing. We also performed a titration assay to establish the lower limit of the proportion of tumor cells for detection of EGFR mutations. Results: EGFR mutations were detected in 13 cases (24%). In-frame deletions in exon 19 were detected in eight cases (15%) and the L858R mutation in exon 21 was detected in five cases (9%). The positive results of the HRMA were confirmed by direct sequencing only in five of 13 cases. In the remaining eight positive samples, HRMA results were confirmed by sequencing analysis after mutant-DNA enrichment. The titration assay established that the lower limit for detection of EGFR mutations by HMRA was 1% tumor cells in the clinical sample. Conclusion: Our results indicated that HRMA in combination with mutant-enriched PCR represents a sensitive method for detection of EGFR mutations from cytologic specimens. When properly executed, this protocol allows identification of EGFR mutations in specimens containing a minimal percentage of tumor cells.

MIRAGE syndrome caused by mutations in SAMD9 is associated with potential loss of chromosome 7 (-7/7q-) and an increased risk to develop myelodysplastic syndrome (MDS). We report a case of MIRAGE syndrome, caused by a novel SAMD9 mutation p.Leu641Pro, leading to characteristic clinical features as well as to the coexistence of cells with monosomy 7 (20%) and with uniparental disomy of long arm of chromosome 7 (UPD7q). In contrast to previously reported MIRAGE patients with -7/7q- developing MDS, our patient achieved complete cytogenetic remission of monosomy 7. As UPD7q remained unchanged, it seems to be a protective factor against MDS.

Voltage-gated sodium channels are essential for generation and propagation of the action potential mainly in nerve and muscle cells. Causative variants in SCN1A gene which codes the main, pore-forming subunit of the channel expressed in central nervous system are associated predominantly with Dravet syndrome (DS), as well as with generalized epilepsy with febrile seizures plus (GEFS+) making it one of the most significant epilepsy gene. Our goal was to determine whether SCN1A screening is relevant in patients with a broad range of epileptic syndromes. 52 patients diagnosed with DS, generalized epilepsy with GEFS+ or similar types of epileptic syndromes were included. Sequencing of the protein coding parts of the gene complemented with MLPA analysis was carried out. One already described nonsense variant, four novel protein truncating variants and a deletion encompassing the whole SCN1A gene were revealed, all in heterozygous state. All identified variants were found in DS patients with 85.7% sensitivity, thus supporting the role of profound SCN1A gene variants in etiology of DS phenotype. No causative variants were identified in any of non-DS epileptic patients in our cohort, suggesting a minor, but not irrelevant role for SCN1A in patients with other types of childhood epilepsy.

BACKGROUND: Germ-line mutations in mismatch repair genes are associated with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome, which is characterized by susceptibility to cancer of the colon, endometrium, small bowel or urothelium at an unusually young age and with a high degree of penetration in all generations. MATERIAL AND METHODS: One hundred and nine individuals from 46 Austrian families who fulfilled the Amsterdam criteria (n = 29) or at least one of the Bethesda guidelines (n= 17) were analyzed for mutations in MLH1 and MSH2. Microsatellite instability was determined in the tumors of index persons and affected relatives. RESULTS AND CONCLUSION: High-grade instability was present in 60.6% of the tumor samples from index patients. Twenty-three germ-line DNA sequence variants in 24/46 families and four somatic mutations in three tumors were detected in MLH1 and MSH2. Fifteen mutations are novel. None of the newly identified germ-line variants was found in 100 alleles of healthy control individuals. We were able to characterize two intronic variants (MLH1 c.589-10T > A; MSH2 c.367-1G > A) with regard to their effect on mRNA. Both created new splice sites that replaced the regular ones. Germ-line mutations occurred in 44.8% of the families fulfilling the Amsterdam criteria and in 35.3% of the Bethesda patients. The detection of a pathogenic mutation was strongly correlated with microsatellite instability in the tumor DNA (p = 0.007). This study is the first comprehensive report of mutations in mismatch repair genes in Austrian patients with HNPCC.

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant disorders with mainly mild cutaneous manifestations. Some patients with NF1, however, develop severe complications such as progressive optic pathway glioma, plexiform neurofibroma or malignant peripheral nerve sheath tumour. Due to potentially progressive and asymptomatic course of the disease, patients with NF1 require a regular multidisciplinary follow-up in coordination with various specialties and early intervention. In this article, we summarise our long-term experience with multidisciplinary follow-up of NF1 patients in the Centre for Neurofibromatosis Type 1 patients at the Children's University Hospital in Bratislava.

Although most of the pertinent data on the sequence-directed processes leading to genome rearrangements (GRs) have come from studies on somatic tissues, little is known about GRs in the germ line of patients with hereditary disorders. This study aims at identifying DNA motifs and higher order structures of genome architecture, which can result in losses and gains of genetic material in the germ line. We first identified candidate motifs by studying 112 pathogenic germ-line GRs in hereditary colorectal cancer patients, and subsequently created an algorithm, termed recombination type ratio, which correctly predicts the propensity of rearrangements with respect to homologous versus nonhomologous recombination events.

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.

In this study we performed the CHEK2 c.1100delC mutation analysis in 1046 breast cancer patients and 730 unaffected control individuals. The mutated allele was found in 3 out of 688 unselected sporadic breast cancer patients and in 1 out of 358 familial/early onset breast cancer patients. Two mutations were identified in a cohort of 730 controls. Our results support the finding that frequency of CHEK2 c.1100delC mutation varies among different populations. Based on our results, genotyping of CHEK2 c.1100delC mutation in clinical settings in the Czech Republic could not be recommended.

Mutation analysis of the epidermal growth factor receptor (EGFR) gene is an essential part of the diagnostic algorithm in patients with metastatic or recurrent non-small cell lung cancer (NSCLC). Small biopsies or cytology specimens represent80% of the available diagnostic material. EGFR mutation analyses were realized on 835 samples (675 cytology specimens, 151 formalin-fixed paraffin-embedded blocks, 5 tumors, and 4 pleural effusions). EGFR mutation analysis was performed by high-resolution melting analysis in combination with mutant-enriched polymerase chain reaction and sequencing analysis. Because of increased risk of inaccuracy in histology diagnosis of small specimens, all subtypes of NSCLC were analyzed. EGFR mutations were detected in 83 cases (10%). EGFR mutation testing failed in 5% (42/835) and was associated with poor cellularity, low percentage of tumor cells, and bad quality of DNA. Although 281 samples were evaluated as insufficient material (poor cellularity and/or unrepresentative tumor content), mutation rates were 7%. Although only adenocarcinomas or NSCLC-not otherwise specified are recommended for EGFR mutation testing, EGFR mutations in 11% of the large cell carcinomas and 4% of the squamous cell carcinomas were observed. Our results indicate that defined algorithm for EGFR testing of small diagnostic samples is sensitive, fast, and suitable even for samples with poor cellularity. The results of this testing should be evaluated depending on tumor content and DNA quality for each sample individually. At the conclusion of our results, we recommend to realize EGFR mutation analysis of small diagnostic samples regardless of the histologic subtypes of NSCLC.

Acute myeloid leukemia (AML) is characterized by the malignant transformation of myeloid cells from myeloblasts to a pathological cell clone. These pathological cell clones lose their ability to differentiate and mature, are no longer subject to regulatory mechanisms and suppress other components of normal hemopoiesis. AML does not fall under a single nosological entity. The heterogeneity of AML is reflected by differences in morphology and immunophenotype, as well as cytogenetic and molecular genetic abnormalities. It includes a number of subtypes, which can be further classified according to the FAB and World Health Organization (WHO) criteria.

We performed a complex analysis of the neurofibromatosis type 1 (NF1) gene in Slovakia based on direct cDNA sequencing supplemented by multiple ligation dependent probe amplification (MLPA) analysis. All 108 patients had café-au-lait spots, 85% had axilary and/or inguinal freckling, 61% neurofibromas, 36% Lisch nodules of the iris and 31% optic pathway glioma, 5% suffered from typical skeletal disorders, and 51% of patients had family members with NF1. In 78 of the 86 (90.7%) index patients our analysis revealed the presence of NF1 mutations, 68 of which were small changes (87.2%), including 39 (50%) novel. Among the identified mutations the most prevalent were small deletions and insertions causing frameshift (42.3%), followed by nonsense (14.1%), missense (12.8%), and typical splicing (11.5%) mutations. Type 1 NF1 deletions and intragenic deletions/duplication were identified in five cases each (6.4%). Interestingly, in five other cases nontypical splicing variants were found, whose real effect on NF1 transcript would have remained undetected if using a DNA-based method alone, thus underlying the advantage of using the cDNA-based sequencing. We show that Slovak NF1 patients have a similar repertoire of NF1 germline mutations compared to other populations, with some prevalence of small deletions/insertions and a decreased proportion of nonsense mutations.

Ig class-switch recombination (Ig-CSR) deficiencies are rare primary immunodeficiencies characterized by defective switched isotype (IgG/IgA/IgE) production. Depending on the molecular defect, defective Ig-CSR may also be associated with impaired somatic hypermutation (SHM) of the Ig V regions. Although the mechanisms underlying Ig-CSR and SHM in humans have been revealed (at least in part) by studying natural mutants, the role of mismatch repair in this process has not been fully elucidated. We studied in vivo and in vitro Ab maturation in eight MSH6-deficient patients. The skewed SHM pattern strongly suggests that MSH6 is involved in the human SHM process. Ig-CSR was found to be partially defective in vivo and markedly impaired in vitro. The resolution of γH2AX foci following irradiation of MSH6-deficient B cell lines was also found to be impaired. These data suggest that in human CSR, MSH6 is involved in both the induction and repair of DNA double-strand breaks in switch regions.

Biallelic germline mutations of Constitutional mismatch repair-deficiency syndrome (CMMR-D) genes, MLH1, MSH2, MSH6, and PMS2 are characterized by increased risk of childhood malignancy. We report a case with CMMR-D caused by novel homozygous MSH6 mutations leading to gliomatosis cerebri and 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 on brain tumors in CMMR-D families shows that they are treatment-resistant and lead to early death. Identification of patients with CMMR-D is critical, and specific cancer screening programs with early surgery are recommended.

Germline mutations in the BRCA1/2 genes account for the majority of hereditary breast ovarian cancer (HBOC). Identification of causal mutations may have significant impact on clinical management of such families. Despite high mutation detection rate, many HBOC cases remain without identified cause. These cases warrant use of several analysis methods, such as those for large genomic rearrangements and DNA copy number changes, or analysis other genes, shown to be associated with increased HBOC risk. We assessed 585 Slovak HBOC for the presence of mutations in BRCA genes. Sequencing revealed mutations in 100 families, representing 17.1% (88 and 12% of mutations were located in BRCA1 and BRCA2, respectively). Four of the mutations, c.80+4del4, c.1938_1947del10 and c.1166delG in BRCA1 and c.6589delA in BRCA2 gene have been described only in Slovak population. Using MLPA analysis, we detected two large genomic rearrangements in three families, a deletion of exons 21 and 22, and a rare deletion of a whole BRCA1 gene. Twenty-seven different variants of uncertain clinical effect (four novel) and 14 distinct SNP BRCA1 haplotypes were detected. Their potential effect was considered using the prediction software packages Align-GVGD, Pmut and Polyphen. We observed that the best clinical criterion for the initiation of BRCA1 analysis is the presence of breast cancer at 40 years of age in the association with the presence of ovarian cancer diagnosed around the age of 50. Conversely, the best clinical criterion for starting with BRCA2 analysis is the presence of breast cancer diagnosed in older age (above 50), or the presence of breast cancer in conjunction with carcinomas at different sites e.g., prostate, colorectum, ovary and uterus. Finally we have seen that the analyses of other HBOC risk gene TP53 and specific mutation in CHEK2*c.1100delC in Slovak HBOC families were not efficient since no mutations were found in these genes.

Background Depending on the population studied, large genomic rearrangements (LGRs) of the mismatch repair (MMR) genes constitute various proportions of the germline mutations that predispose to hereditary non-polyposis colorectal cancer (HNPCC). It has been reported that loss of heterozygosity (LOH) at the LGR region occurs through a gene conversion mechanism in tumors from MLH1/MSH2 deletion carriers; however, the converted tracts were delineated only by extragenic microsatellite markers. We sought to determine the frequency of LGRs in Slovak HNPCC patients and to study LOH in tumors from LGR carriers at the LGR region, as well as at other heterozygous markers within the gene to more precisely define conversion tracts. Methods The main MMR genes responsible for HNPCC, MLH1, MSH2, MSH6, and PMS2, were analyzed by MLPA (multiplex ligation-dependent probe amplification) in a total of 37 unrelated HNPCC-suspected patients whose MLH1/MSH2 genes gave negative results in previous sequencing experiments. An LOH study was performed on six tumors from LGR carriers by combining MLPA to assess LOH at LGR regions and sequencing to examine LOH at 28 SNP markers from the MLH1 and MSH2 genes. Results We found six rearrangements in the MSH2 gene (five deletions and dup5-6), and one aberration in the MLH1 gene (del5-6). The MSH2 deletions were of three types (del1, del1-3, del1-7). We detected LOH at the LGR region in the single MLH1 case, which was determined in a previous study to be LOH-negative in the intragenic D3S1611 marker. Three tumors displayed LOH of at least one SNP marker, including two cases that were LOH-negative at the LGR region. Conclusion LGRs accounted for 25% of germline MMR mutations identified in 28 Slovakian HNPCC families. A high frequency of LGRs among the MSH2 mutations provides a rationale for a MLPA screening of the Slovakian HNPCC families prior scanning by DNA sequencing. LOH at part of the informative loci confined to the MLH1 or MSH2 gene (heterozygous LGR region, SNP, or microsatellite) is a novel finding and can be regarded as a partial LOH. The conversion begins within the gene, and the details of conversion tracts are discussed for each case.

Background: In the workup of patients with suspected hereditary nonpolyposis colorectal cancer (HNPCC), detection of loss of heterozygosity (LOH) could help pinpoint the mismatch-repair (MMR) gene carrying the germline mutation, but analysis of microsatellite markers has proved unreliable for this purpose. We developed a simple, low-cost method based on single-nucleotide polymorphism (SNP) genotyping and capillary electrophoresis for the assessment of LOH at 2 MMR loci simultaneously. Methods: We used the Applied Biosystems SNaPshot® Multiplex Kit with meticulously selected primers to assess 14 common SNPs in MLH1 [mutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli)] and MSH2 [mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli)] and optimized the protocol for DNA isolated from peripheral blood and fresh/frozen or archival microsatellite-unstable tumors from patients with confirmed (n = 42) or suspected (n = 25) HNPCC. The 42 tumors from patients with confirmed MLH1 or MSH2 germline mutations were used to validate the method’s diagnostic accuracy against results obtained with DNA sequencing or multiplex ligation-dependent probe amplification. Results: The SNaPshot assay provided better detection of certain SNPs than DNA sequencing. The MLH1 and MSH2 SNP marker sets were informative in 82% and 76% of the 67 cases analyzed, respectively. The new assay displayed 100% specificity for detecting LOH and predicted the location of the germline mutation in 40% of the cases (54% of those involving MLH1, 22% in MSH2). Conclusions: Our SNP-based method for detecting LOH in MLH1 and MSH2 is simple to perform with instruments available in most clinical genetics laboratories. It can be a valuable addition to protocols now used to guide mutational screening of patients with suspected HNPCC.

Background Germline mutations in the adenomatous polyposis gene (APC) result in familial adenomatous polyposis (FAP). FAP is an autosomal dominantly inherited disorder predisposing to colorectal cancer. Typical FAP is characterized by hundreds to thousands of colorectal adenomatous polyps and by several extracolonic manifestations. An attenuated form of polyposis (AFAP) is characterized by less than 100 adenomas and later onset of the disease. Methods Here, we analyzed the APC gene for germline mutations in 59 Czech and 15 Slovak FAP patients. In addition, 50 apparently APC mutation negative Czech probands and 3 probands of Slovak origin were screened for large deletions encompassing the APC gene. Mutation screening was performed using denaturing gradient gel electrophoresis and/or protein truncation test. DNA fragments showing an aberrant electrophoretic banding pattern were sequenced. Screening for large deletions was performed by multiplex ligation dependent probe amplification. The extent of deletions was analyzed using following microsatellite markers: D5S299, D5S82, D5S134 and D5S346. Results In the set of Czech and Slovak patients, we identified 46 germline mutations among 74 unrelated probands. Total mutation capture is 62,2% including large deletions. Thirty seven mutations were detected in 49 patients presenting a classical FAP phenotype (75,5%) and 9 mutations in 25 patients with attenuated FAP (36%). We report 20 novel germline APC mutations and 3 large deletions (6%) encompassing the whole-gene deletions and/or exon 14 deletion. In the patients with novel mutations, correlations of the mutation localization are discussed in context of the classical and/or attenuated phenotype of the disease. Conclusion The results of the molecular genetic testing are used both in the establishment of the predictive diagnosis and in the clinical management of patients. In some cases this study has also shown the difficulty to classify clinically between the classical and the attenuated form of FAP according to the established criteria. Interfamilial and/or intrafamilial phenotype variability was also confirmed in some cases which did not fit well with predicted genotype-phenotype correlation. All these findings have to be taken into consideration both in the genetic counselling and in the patient care.

Hereditary nonpolyposis colorectal cancer (HNPCC) is a dominantly-inherited cancer predisposition syndrome, in which the susceptibility to cancer of the colon, endometrium and ovary is linked to germline mutations in DNA mismatch repair (MMR) genes. We have recently initiated a cancer prevention program in suspected HNPCC families in the Slovak Republic. The first ten families fulfilling Amsterdam criteria or Bethesda guidelines were screened for germline mutations in MLH1 and MSH2, two MMR genes most frequently mutated in HNPCC families. Six mutations were identified, five of which have not been reported previously. Two of the three new mutations in MLH1 (c.380+2TA; c.307-2AC) were absent from 100 chromosomes of healthy controls and probably cause a splicing defect, while the third was a 1 bp deletion (c.1261delA). In the MSH2 gene, one new nonsense (c.1030CT [p.Q344X]) and one missense (c.524TC [p.L175P]) mutation were identified. This latter variant was not found in 104 alleles of healthy control individuals. Moreover, a previously-reported pathogenic mutation (c.677GT [p.R226L]) was found in one kindred. The clinical data and the genotypic and phenotypic evaluation of the tumors indicate that all the new alterations are pathogenic HNPCC mutations.

Hereditary nonpolyposis colorectal cancer (HNPCC) is a dominantly-inherited cancer predisposition syndrome, in which the susceptibility to cancer of the colon, endometrium and ovary is linked to germline mutations in DNA mismatch repair (MMR) genes. We have recently initiated a cancer prevention program in suspected HNPCC families in the Slovak Republic. The first ten families fulfilling Amsterdam criteria or Bethesda guidelines were screened for germline mutations in MLH1 and MSH2, two MMR genes most frequently mutated in HNPCC families. Six mutations were identified, five of which have not been reported previously. Two of the three new mutations in MLH1 (c.380+2T>A; c.307-2A>C) were absent from 100 chromosomes of healthy controls and probably cause a splicing defect, while the third was a 1 bp deletion (c.1261delA). In the MSH2 gene, one new nonsense (c.1030C>T [p.Q344X]) and one missense (c.524T>C [p.L175P]) mutation were identified. This latter variant was not found in 104 alleles of healthy control individuals. Moreover, a previously-reported pathogenic mutation (c.677G>T [p.R226L]) was found in one kindred. The clinical data and the genotypic and phenotypic evaluation of the tumors indicate that all the new alterations are pathogenic HNPCC mutations.