Molecular mechanisms of human overgrowth and use of omics in its diagnostics: chances and challenges.

Overgrowth disorders comprise a group of entities with a variable phenotypic spectrum ranging from tall stature to isolated or lateralized overgrowth of body parts and or organs. Depending on the underlying physiological pathway affected by pathogenic genetic alterations, overgrowth syndromes are associated with a broad spectrum of neoplasia predisposition, (cardio) vascular and neurodevelopmental anomalies, and dysmorphisms. Pathologic overgrowth may be of prenatal or postnatal onset. It either results from an increased number of cells (intrinsic cellular hyperplasia), hypertrophy of the normal number of cells, an increase in interstitial spaces, or from a combination of all of these. The underlying molecular causes comprise a growing number of genetic alterations affecting skeletal growth and Growth-relevant signaling cascades as major effectors, and they can affect the whole body or parts of it (mosaicism). Furthermore, epigenetic modifications play a critical role in the manifestation of some overgrowth diseases. The diagnosis of overgrowth syndromes as the prerequisite of a personalized clinical management can be challenging, due to their clinical and molecular heterogeneity. Physicians should consider molecular genetic testing as a first diagnostic step in overgrowth syndromes. In particular, the urgent need for a precise diagnosis in tumor predisposition syndromes has to be taken into account as the basis for an early monitoring and therapy. With the (future) implementation of next-generation sequencing approaches and further omic technologies, clinical diagnoses can not only be verified, but they also confirm the clinical and molecular spectrum of overgrowth disorders, including unexpected findings and identification of atypical cases. However, the limitations of the applied assays have to be considered, for each of the disorders of interest, the spectrum of possible types of genomic variants has to be considered as they might require different methodological strategies. Additionally, the integration of artificial intelligence (AI) in diagnostic workflows significantly contribute to the phenotype-driven selection and interpretation of molecular and physiological data.

Interindividual variability in cold-pressor pain sensitivity is not explained by peripheral vascular responding and generalizes to a C-nociceptor-specific pain phenotype.

ABSTRACT: Pain sensitivity of healthy subjects in the cold-pressor (CP) test was proposed to be dichotomously distributed and to represent a pain sensitivity trait. Still, it has not been systematically explored which factors influence this pain sensitivity readout. The aim of this study was to distinguish potential contributions of local tissue-related factors such as perfusion and thermoregulation or gain settings in nociceptive systems. Cold-pressor-sensitive and CP-insensitive students screened from a medical student laboratory course were recruited for a CP retest with additional cardiovascular and bilateral local vascular monitoring. In addition, comprehensive quantitative sensory testing according to Deutscher Forschungsverbund Neuropathischer Schmerz standards and a sustained pinch test were performed. Cold pressor was reproducible across sessions (Cohen kappa 0.61 ± 0.14, P < 0.005). At 30 seconds in ice water, CP-sensitive subjects exhibited not only more pain (78.6 ± 26.3 vs 29.5 ± 17.5, P < 0.0001) but also significantly stronger increases in mean arterial blood pressure (12.6 ± 9.3 vs 5.6 ± 8.1 mm Hg, P < 0.05) and heart rate (15.0 ± 8.2 vs 7.1 ± 6.2 bpm, P < 0.005), and lower baroreflex sensitivity, but not local or vasoconstrictor reflex-mediated microcirculatory responses. Cold-pressor-sensitive subjects exhibited significantly lower pain thresholds also for cold, heat, and blunt pressure, and enhanced pain summation, but no significant differences in Aδ-nociceptor-mediated punctate mechanical pain. In conclusion, differences in nociceptive signal processing drove systemic cardiovascular responses. Baroreceptor activation suppressed pain and cardiovascular responses more efficiently in CP-insensitive subjects. Cold-pressor sensitivity generalized to a pain trait of C-fiber-mediated nociceptive channels, which was independent of local thermal and vascular changes in the ice-water-exposed hand. Thus, the C-fiber pain trait reflects gain setting of the nociceptive system.

Further understanding of paternal uniparental disomy in Beckwith-Wiedemann syndrome.

INTRODUCTION: Paternal uniparental disomy of chromosome 11 (upd(11)pat) accounts for up to 20% of molecularly confirmed Beckwith-Wiedemann spectrum (BWSp) cases. It belongs to the BWSp subgroup with the second highest tumor risk, and therefore needs particular awareness in research, diagnostics and clinical management. AREAS COVERED: We overview the contribution of paternal (mosaic) uniparental disomy of chromosome 11 (UPD, upd(11)pat) and mosaic paternal uniparental diploidy in patients with Beckwith-Wiedemann features. The review comprises the current knowledge on their formation and their molecular and clinical consequences. Accordingly, the consequences for diagnostic testing and clinical monitoring are compiled. EXPERT OPINION: The necessity to diagnostically identify and thus discriminate genome-wide paternal uniparental disomy, and upd(11)pat becomes obvious, due to the differences in the clinical course, disease prognosis, and treatment. In particular, monitoring of tumor development by liquid biopsy might be a promising option in the future. From the research point of view, it should be addressed why 11p is prone to mitotic recombination and thus also provide to the role of upd(11) as second hit in tumorigenesis.

Comprehensive genetic testing approaches as the basis for personalized management of growth disturbances: current status and perspectives.

The implementation of high-throughput and deep sequencing methods in routine genetic diagnostics has significantly improved the diagnostic yield in patient cohorts with growth disturbances and becomes increasingly important as the prerequisite of personalized medicine. They provide considerable chances to identify even rare and unexpected situations; nevertheless, we must be aware of their limitations. A simple genetic test in the beginning of a testing cascade might also help to identify the genetic cause of specific growth disorders. However, the clinical picture of genetically caused growth disturbance phenotypes can vary widely, and there is a broad clinical overlap between different growth disturbance disorders. As a consequence, the clinical diagnosis and therewith connected the decision on the appropriate genetic test is often a challenge. In fact, the clinician asking for genetic testing has to weigh different aspects in this decision process, including appropriateness (single gene test, stepwise procedure, comprehensive testing), turnaround time as the basis for rapid intervention, and economic considerations. Therefore, a frequent question in that context is 'what to test when'. In this review, we aim to review genetic testing strategies and their strengths and limitations and to raise awareness for the future implementation of interdisciplinary genome medicine in diagnoses, treatment, and counselling of growth disturbances.

Molecular Basis of Beckwith-Wiedemann Syndrome Spectrum with Associated Tumors and Consequences for Clinical Practice.

Beckwith-Wiedemann syndrome (BWS, OMIM 130650) is a congenital imprinting condition with a heterogenous clinical presentation of overgrowth and an increased childhood cancer risk (mainly nephroblastoma, hepatoblastoma or neuroblastoma). Due to the varying clinical presentation encompassing classical, clinical BWS without a molecular diagnosis and BWS-related phenotypes with an 11p15.5 molecular anomaly, the syndromic entity was extended to the Beckwith-Wiedemann spectrum (BWSp). The tumor risk of up to 30% depends on the molecular subtype of BWSp with causative genetic or epigenetic alterations in the chromosomal region 11p15.5. The molecular diagnosis of BWSp can be challenging for several reasons, including the range of causative molecular mechanisms which are frequently mosaic. The molecular basis of tumor formation appears to relate to stalled cellular differentiation in certain organs that predisposes persisting embryonic cells to accumulate additional molecular defects, which then results in a range of embryonal tumors. The molecular subtype of BWSp not only influences the overall risk of neoplasia, but also the likelihood of specific embryonal tumors.

Hypermethylation of RAD9A intron 2 in childhood cancer patients, leukemia and tumor cell lines suggest a role for oncogenic transformation.

Most childhood cancers occur sporadically and cannot be explained by an inherited mutation or an unhealthy lifestyle. However, risk factors might trigger the oncogenic transformation of cells. Among other regulatory signals, hypermethylation of RAD9A intron 2 is responsible for the increased expression of RAD9A protein, which may play a role in oncogenic transformation. Here, we analyzed the RAD9A intron 2 methylation in primary fibroblasts of 20 patients with primary cancer in childhood and second primary cancer (2N) later in life, 20 matched patients with only one primary cancer in childhood (1N) and 20 matched cancer-free controls (0N), using bisulfite pyrosequencing and deep bisulfite sequencing (DBS). Four 1N patients and one 2N patient displayed elevated mean methylation levels (≥ 10 %) of RAD9A. DBS revealed ≥ 2 % hypermethylated alleles of RAD9A, indicative for constitutive mosaic epimutations. Bone marrow samples of NHL and AML tumor patients (n=74), EBV (Epstein Barr Virus) lymphoblasts (n=6), tumor cell lines (n=5) and FaDu subclones (n=13) were analyzed to substantiate our findings. We find a broad spectrum of tumor entities with an aberrant methylation of RAD9A. We detected a significant difference in mean methylation of RAD9A for NHL versus AML patients (p ≤0.025). Molecular karyotyping of AML samples during therapy with hypermethylated RAD9A showed an evolving duplication of 1.8 kb on Chr16p13.3 including the PKD1 gene. Radiation, colony formation assays, cell proliferation, PCR and molecular karyotyping SNP-array experiments using generated FaDu subclones suggest that hypermethylation of RAD9A intron 2 is associated with genomic imbalances in regions with tumor-relevant genes and survival of the cells. In conclusion, this is the very first study of RAD9A intron 2 methylation in childhood cancer and Leukemia. RAD9A epimutations may have an impact on leukemia and tumorigenesis and can potentially serve as a biomarker.

Correction to: Molecular karyotyping and gene expression analysis in childhood cancer patients.

The correct Author names are presented in this paper.

Genetic testing in inherited endocrine disorders: joint position paper of the European reference network on rare endocrine conditions (Endo-ERN).

BACKGROUND: With the development of molecular high-throughput assays (i.e. next generation sequencing), the knowledge on the contribution of genetic and epigenetic alterations to the etiology of inherited endocrine disorders has massively expanded. However, the rapid implementation of these new molecular tools in the diagnostic settings makes the interpretation of diagnostic data increasingly complex. MAIN BODY: This joint paper of the ENDO-ERN members aims to overview chances, challenges, limitations and relevance of comprehensive genetic diagnostic testing in rare endocrine conditions in order to achieve an early molecular diagnosis. This early diagnosis of a genetically based endocrine disorder contributes to a precise management and helps the patients and their families in their self-determined planning of life. Furthermore, the identification of a causative (epi)genetic alteration allows an accurate prognosis of recurrence risks for family planning as the basis of genetic counselling. Asymptomatic carriers of pathogenic variants can be identified, and prenatal testing might be offered, where appropriate. CONCLUSIONS: The decision on genetic testing in the diagnostic workup of endocrine disorders should be based on their appropriateness to reliably detect the disease-causing and -modifying mutation, their informational value, and cost-effectiveness. The future assessment of data from different omic approaches should be embedded in interdisciplinary discussions using all available clinical and molecular data.

Cancer incidence and spectrum among children with genetically confirmed Beckwith-Wiedemann spectrum in Germany: a retrospective cohort study.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) is a cancer predisposition syndrome caused by defects on chromosome 11p15.5. The quantitative cancer risks in BWS patients depend on the underlying (epi)genotype but have not yet been assessed in a population-based manner. METHODS: We identified a group of 321 individuals with a molecularly confirmed diagnosis of BWS and analysed the cancer incidence up to age 15 years and cancer spectrum by matching their data with the German Childhood Cancer Registry. RESULTS: We observed 13 cases of cancer in the entire BWS cohort vs 0.4 expected. This corresponds to a 33-fold increased risk (standardised incidence ratio (SIR) = 32.6; 95% confidence interval = 17.3-55.7). The specific cancers included hepatoblastoma (n = 6); nephroblastoma (n = 4); astrocytoma (n = 1); neuroblastoma (n = 1) and adrenocortical carcinoma (n = 1). The cancer SIR was highest in patients with a paternal uniparental disomy of 11p15.5 (UPDpat). A high cancer risk remained when cases of cancer diagnosed prior to the BWS diagnosis were excluded. CONCLUSIONS: This study confirms an increased cancer risk in children with BWS. Our findings suggest that the highest cancer risk is associated with UPDpat. We were unable to confirm an excessive cancer risk in patients with IC1 gain of methylation (IC1-GOM) and this finding requires further investigation.

In renal cell carcinoma the PTEN splice variant PTEN-Δ shows similar function as the tumor suppressor PTEN itself.

BACKGROUND: Loss of PTEN is involved in tumor progression of several tumor entities including renal cell carcinoma (RCC). During the translation process PTEN generates a number of splice variants, including PTEN-Δ. We analyzed the impact of PTEN-Δ in RCC progression. METHODS: In specimens of RCC patients the expression of PTEN-Δ and PTEN was quantified. The PTEN expressing RCC cell line A498 and the PTEN deficient 786-O cell line were stably transfected with the PTEN-Δ or PTEN transcript. In Caki-1 cells that highly express PTEN-Δ, this isoform was knocked down by siRNA. Cell migration, adhesion, apoptosis and signaling pathways activities were consequently analyzed in vitro. RESULTS: Patients with a higher PTEN-Δ expression had a longer lymph node metastasis free and overall survival. In RCC specimens, the PTEN-Δ expression correlated with the PTEN expression. PTEN-Δ as well as PTEN induced a reduced migration when using extracellular matrix (ECM) compounds as chemotaxins. This effect was confirmed by knockdown of PTEN-Δ, inducing an enhanced migration. Likewise a decreased adhesion on these ECM components could be shown in PTEN-Δ and PTEN transfected cells. The apoptosis rate was slightly increased by PTEN-Δ. In a phospho-kinase array and Western blot analyses a consequently reduced activity of AKT, p38 and JNK could be shown. CONCLUSIONS: We could show that the PTEN splice variant PTEN-Δ acts similar to PTEN in a tumor suppressive manner, suggesting synergistic effects of the two isoforms. The impact of PTEN-Δ in context of tumor progression should thus be taken into account when generating new therapeutic options targeting PTEN signaling in RCC.

Calcium-sensing receptor (CaSR) promotes development of bone metastasis in renal cell carcinoma.

Bone metastasis is an important prognostic factor in renal cell carcinoma (RCC). The calcium-sensing receptor (CaSR) has been associated with bone metastasis in several different malignancies. We analyzed the impact of CaSR in bone metastasis in RCC in vitro and in vivo. The RCC cell line 786-O was stably transfected with the CaSR gene and treated with calcium alone or in combination with the CaSR antagonist NPS2143. Afterwards migration, adhesion, proliferation and prominent signaling molecules were analyzed. Calcium treated CaSR-transfected 768-O cells showed an increased adhesion to endothelial cells and the extracellular matrix components fibronectin and collagen I, but not to collagen IV. The chemotactic cell migration and proliferation was also induced by calcium. The activity of SHC, AKT, ERK, P90RSK and JNK were enhanced after calcium treatment of CaSR-transfected cells. These effects were abolished by NPS2143. Development of bone metastasis was evaluated in vivo in a mouse model. Intracardiac injection of CaSR-transfected 768-O cells showed an increased rate of bone metastasis. The results indicate CaSR as an important component in the mechanism of bone metastasis in RCC. Therefore, targeting CaSR might be beneficial in patients with bone metastatic RCC with a high CaSR expression.

[Beckwith-Wiedemann Syndrome (BWS) Current Status of Diagnosis and Clinical Management: Summary of the First International Consensus Statement]. / Beckwith-Wiedemann-Syndrom (BWS): Aktueller Stand der Diagnostik und des klinischen Managements.

Beckwith-Wiedemann syndrome (BWS) belongs to the group of imprinting disorders and is characterized by variable clinical features, including overgrowth, macroglossia, abdominal wall defect, neonatal hypoglycemia, body asymmetry and an increased risk for embryonal tumors. In the majority of cases, molecular alterations of the Imprinting Center (IC) regions in the chromosomal region 11p15.5 can be detected, and a correlation of single clinical features with specific genomic and epigenetic changes is obvious. Therefore, the detailed molecular diagnosis is a prerequisite for a precise prediction of the tumor risk and the tumor spectrum. Furthermore, it is the basis for a well-directed genetic counselling of the families. Despite a huge number of comprehensive studies based on a large number of cases, standardized diagnostic criteria and advices for therapeutic management were missing. In the following, the recently published first international consensus guidelines drafted by 41 experts in the field of BWS from 11 European countries and the USA are summarized. Patients support groups had been included as well. In total, 72 consented recommendations for clinical and molecular diagnosis as well as for the clinical management of BWS have been published. They refer both to patients with the classical BWS phenotype and to those with "atypical" phenotypes which are summarized as BWS spectrum (BWSp). A modified clinical scoring system is now suggested, which represents the basis to initiate molecular diagnostics. Therapeutic recommendations comprise the major clinical questions in BWS/BWSp, i. e. early monitoring of an increased tumor risk, treatment of the macroglossia and the abdominal wall defects, and therapeutic interventions for hypoglycemia. However, though there was a broad consensus on the majority of therapeutic interventions, discussions on tumor monitoring are foreseeable. Thus, prospective studies to evaluate the consensus guidelines and their use are planned.

Expert consensus document: Clinical and molecular diagnosis, screening and management of Beckwith-Wiedemann syndrome: an international consensus statement.

Beckwith-Wiedemann syndrome (BWS), a human genomic imprinting disorder, is characterized by phenotypic variability that might include overgrowth, macroglossia, abdominal wall defects, neonatal hypoglycaemia, lateralized overgrowth and predisposition to embryonal tumours. Delineation of the molecular defects within the imprinted 11p15.5 region can predict familial recurrence risks and the risk (and type) of embryonal tumour. Despite recent advances in knowledge, there is marked heterogeneity in clinical diagnostic criteria and care. As detailed in this Consensus Statement, an international consensus group agreed upon 72 recommendations for the clinical and molecular diagnosis and management of BWS, including comprehensive protocols for the molecular investigation, care and treatment of patients from the prenatal period to adulthood. The consensus recommendations apply to patients with Beckwith-Wiedemann spectrum (BWSp), covering classical BWS without a molecular diagnosis and BWS-related phenotypes with an 11p15.5 molecular anomaly. Although the consensus group recommends a tumour surveillance programme targeted by molecular subgroups, surveillance might differ according to the local health-care system (for example, in the United States), and the results of targeted and universal surveillance should be evaluated prospectively. International collaboration, including a prospective audit of the results of implementing these consensus recommendations, is required to expand the evidence base for the design of optimum care pathways.

Recommendations for a nomenclature system for reporting methylation aberrations in imprinted domains.

The analysis of DNA methylation has become routine in the pipeline for diagnosis of imprinting disorders, with many publications reporting aberrant methylation associated with imprinted differentially methylated regions (DMRs). However, comparisons between these studies are routinely hampered by the lack of consistency in reporting sites of methylation evaluated. To avoid confusion surrounding nomenclature, special care is needed to communicate results accurately, especially between scientists and other health care professionals. Within the European Network for Human Congenital Imprinting Disorders we have discussed these issues and designed a nomenclature for naming imprinted DMRs as well as for reporting methylation values. We apply these recommendations for imprinted DMRs that are commonly assayed in clinical laboratories and show how they support standardized database submission. The recommendations are in line with existing recommendations, most importantly the Human Genome Variation Society nomenclature, and should facilitate accurate reporting and data exchange among laboratories and thereby help to avoid future confusion.

Overriding TKI resistance of renal cell carcinoma by combination therapy with IL-6 receptor blockade.

Metastatic renal cell carcinoma (RCC) is a tumor entity with poor prognosis due to limited therapy options. Tyrosine kinase inhibitors (TKI) represent the standard of care for RCCs, however a significant proportion of RCC patients develop resistance to this therapy. Interleukin-6 (IL-6) is considered to be associated with poor prognosis in RCCs. We therefore hypothesized that TKI resistance and IL-6 secretion are causally connected. We first analyzed IL-6 expression after TKI treatment in RCC cells and RCC tumor specimens. Cell proliferation and signal transduction activity were then quantified after co-treatment with tocilizumab, an IL-6R inhibitor, in vitro and in vivo. 786-O RCC cells secrete high IL-6 levels after low dose stimulation with the TKIs sorafenib, sunitinib and pazopanib, inducing activation of AKT-mTOR pathway, NFκB, HIF-2α and VEGF expression. Tocilizumab neutralizes the AKT-mTOR pathway activation and results in reduced proliferation. Using a mouse xenograft model we can show that a combination therapy with tocilizumab and low dosage of sorafenib suppresses 786-O tumor growth, reduces AKT-mTOR pathway and inhibits angiogenesis in vivo more efficient than sorafenib alone. Furthermore FDG-PET imaging detected early decrease of maximum standardized uptake values prior to extended central necrosis. Our findings suggest that a combination therapy of IL-6R inhibitors and TKIs may represent a novel therapeutic approach for RCC treatment.

Integrin α5 triggers the metastatic potential in renal cell carcinoma.

The therapy of advanced renal cell carcinoma (RCC) is still a major challenge. To intervene therapeutically a deeper comprehension of the particular steps of metastasis is necessary. In this context membrane bound receptors like integrins play a decisive role. We analyzed the integrin α5 expression in 141 clear cell RCC patients by Western blot. Patients with RCC expressed a significant higher level of integrin α5 in tumor than in normal tissue. The integrin α5 expression correlated with tumor grade, the development of distant metastases within five years after tumor nephrectomy and reduced survival. The RCC cell lines Caki-1 and CCF-RC1, which highly express integrin α5, were treated with fibronectin in combination with or without an inhibiting anti-integrin α5 antibody. Afterwards the migration, adhesion, viability and prominent signaling molecules were analyzed. Both cell lines showed a significant reduced migration potential as well as a decreased adhesion potential to fibronectin after treatment with an integrin α5 blocking antibody. A contribution of the AKT and ERK1/2 signaling pathways could be demonstrated. The results indicate integrin α5 as a potent marker to discriminate patients' tumor prognosis. Consequently the integrin subunit α5 can be considered as a target for individual therapy of advanced RCC.

Personalized therapy: CNS HGNET-BCOR responsiveness to arsenic trioxide combined with radiotherapy.

High-grade neuroepithelial tumor of the central nervous system with BCOR alteration (HGNET-BCOR) is a rare, highly malignant tumor. At the time of this publication, no standard protocol exists to treat this tumor entity. In this work, we tested the responsiveness of the primary culture PhKh1 derived from tumor tissue from a pediatric HGNET-BCOR patient (P1) to inhibitors of the Sonic hedgehog pathway combined with radiation. The SMO inhibitors vismodegib and itraconazole had low effect on the proliferation of the PhKh1 cells. However, the GLI inhibitor arsenic trioxide reduced the expression of GLI target genes in the PhKh1 cells and in combination with radiotherapy significantly decreased their clonogenic potential. PhKh1 cells resistant to arsenic trioxide were characterized by the overexpression of molecular chaperones. We combined arsenic trioxide and radiation in the relapse therapy protocol of P1, achieving complete remission after seven weeks. Clinical remission lasted for six months, when P1 developed systemic metastases. Meanwhile, an increase in the concentration of circulating tumor DNA carrying a BCOR internal tandem duplication was observed. Molecular characterization of a second patient (P2) was also performed. In P2, we detected a larger tandem duplication and greater activation of the Sonic hedgehog pathway than in P1. These findings suggest that combining arsenic trioxide with radiotherapy may represent a new therapeutic approach. Moreover, peripheral blood analysis for circulating tumor DNA could help in the early detection of systemic metastases.

Novel deletion in 11p15.5 imprinting center region 1 in a patient with Beckwith-Wiedemann syndrome provides insight into distal enhancer regulation and tumorigenesis.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) is an early-onset overgrowth disorder with a high risk for embryonal tumors. It is mainly caused by dysregulation of imprinted genes on chromosome 11p15.5; however, the driving forces in the development of tumors are not fully understood. PROCEDURE: We report on a female patient presenting with macrosomia, macroglossia, organomegaly and extensive bilateral nephroblastomatosis. Adjuvant chemotherapy was initiated; however, the patient developed hepatoblastoma and Wilms tumor at 5 and 12 months of age, respectively. Subsequent radiofrequency ablation of the liver tumor and partial nephrectomy followed by consolidation therapy achieved complete remission. RESULTS: Molecular genetic analysis revealed a maternally derived large deletion of the complete H19-differentially methylated region (H19-DMR; imprinting control region-1 [ICR1]), the whole H19 gene itself as well as large parts of the distal enhancer region within the imprinting cluster-1 (IC1). Extended analysis showed highly elevated insulin-like growth factor 2 (IGF2) expression, possibly explaining at least in part the distinct BWS features and tumor manifestations. CONCLUSIONS: This study of a large maternal deletion encompassing the H19 gene and complete ICR1 is the first to demonstrate transcriptional consequences on IGF2 in addition to methylation effects resulting in severe overgrowth and occurrence of multiple tumors in a BWS patient. Studying this deletion helps to clarify the complex molecular processes involved in BWS and provides further insight into tumorigenesis.

Next-generation sequencing reveals germline mutations in an infant with synchronous occurrence of nephro- and neuroblastoma.

Although neuro- and nephroblastoma are common solid tumors in children, the simultaneous occurrence is very rare and is often associated with syndromes. Here, we present a unique case of synchronous occurrence of neuro- and nephroblastoma in an infant with no signs of congenital anomalies or a syndrome. We performed genetic testing for possible candidate genes as underlying mutation using the next-generation sequencing (NGS) approach to target 94 genes and 284 single-nucleotide polymorphisms (SNPs) involved in cancer. We uncovered a novel heterozygous germline missense mutation p.F58L (c.172T→C) in the anaplastic lymphoma kinase (ALK) gene and one novel heterozygous rearrangement Q418Hfs(*)11 (c.1254_1264delins TTACTTAGTACAAGAACTG) in the Fanconi anemia gene FANCD2 leading to a truncated protein. Besides, several SNPs associated with the occurrence of neuroblastoma and/or nephroblastoma or multiple primary tumors were identified. The next-generation sequencing approach might in the future be useful not only in understanding tumor etiology but also in recognizing new genetic markers and targets for future personalized therapy.

Phenotypic spectrum and extent of DNA methylation defects associated with multilocus imprinting disturbances.

AIM: To characterize the genotypic and phenotypic extent of multilocus imprinting disturbances (MLID). MATERIALS & METHODS: We analyzed 37 patients with imprinting disorders (explorative cohort) for DNA methylation changes using the Infinium HumanMethylation450 BeadChip. For validation, three independent cohorts with imprinting disorders or cardinal features thereof were analyzed (84 patients with imprinting disorders, 52 with growth disorder, 81 with developmental delay). RESULTS: In the explorative cohort 21 individuals showed array-based MLID with each one displaying an Angelman or Temple syndrome phenotype, respectively. Epimutations in ZDBF2 and FAM50B were associated with severe MLID regarding number of affected regions. By targeted analysis we identified methylation changes of ZDBF2 and FAM50B also in the three validation cohorts. CONCLUSION: We corroborate epimutations in ZDBF2 and FAM50B as frequent changes in MLID whereas these rarely occur in other patients with cardinal features of imprinting disorders. Moreover, we show cell lineage specific differences in the genomic extent of FAM50B epimutation.