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BACKGROUND: Pathogenic PTEN germline variants cause PTEN Hamartoma Tumor Syndrome (PHTS), a rare disease with a variable genotype and phenotype. Knowledge about these spectra and genotype-phenotype associations could help diagnostics and potentially lead to personalized care. Therefore, we assessed the PHTS genotype and phenotype spectrum in a large cohort study. METHODS: Information was collected of 510 index patients with pathogenic or likely pathogenic (LP/P) PTEN variants (n = 467) or variants of uncertain significance. Genotype-phenotype associations were assessed using logistic regression analyses adjusted for sex and age. RESULTS: At time of genetic testing, the majority of children (n = 229) had macrocephaly (81%) or developmental delay (DD, 61%), and about half of the adults (n = 238) had cancer (51%), macrocephaly (61%), or cutaneous pathology (49%). Across PTEN, 268 LP/P variants were identified, with exon 5 as hotspot. Missense variants (n = 161) were mainly located in the phosphatase domain (PD, 90%) and truncating variants (n = 306) across all domains. A trend towards 2 times more often truncating variants was observed in adults (OR = 2.3, 95%CI = 1.5-3.4) and patients with cutaneous pathology (OR = 1.6, 95%CI = 1.1-2.5) or benign thyroid pathology (OR = 2.0, 95%CI = 1.1-3.5), with trends up to 2-4 times more variants in PD. Whereas patients with DD (OR = 0.5, 95%CI = 0.3-0.9) or macrocephaly (OR = 0.6, 95%CI = 0.4-0.9) had about 2 times less often truncating variants compared to missense variants. In DD patients these missense variants were often located in domain C2. CONCLUSION: The PHTS phenotypic diversity may partly be explained by the PTEN variant coding effect and the combination of coding effect and domain. PHTS patients with early-onset disease often had missense variants, and those with later-onset disease often truncating variants. ispartof: EUROPEAN JOURNAL OF MEDICAL GENETICS vol:65 issue:12 ispartof: location:Netherlands status: published

Asperger syndrome (AS) is a pervasive developmental disorder characterized by general impairment in socialization, stereotypical behavior, defective adaptation to the social context usually without intellectual disability, and some high functioning areas related to memory and mathematics. Clinical criteria are not well defined and the etiology is heterogeneous and mostly unknown. Like in typical autism spectrum disorders (ASD), the genetic background plays a crucial role in AS, and often an almost mendelian segregation can be observed in some families. We performed a whole exome sequencing (WES) in three relatives of a family with vertical transmission of AS-ASD to identify variants in candidate genes segregating with the phenotype. Variant p.(Cys834Ser) in the RADX gene was the only one segregating among all the affected family members. This gene encodes a single-strand DNA binding factor, which mediates the recruitment of genome maintenance proteins to sites of replication stress. Replication stress and genome instability have been reported recently in neural progenitor cells derived from ASD patients, leading to a disruption of long neural genes involved in cell–cell adhesion and migration. We propose RADX as a new gene that when mutated could represent a predisposing factor to AS-ASD.

Neural tube defects are a group of birth defects caused by failure of neural tube closure during development. The etiology of NTD, requiring a complex interaction between environmental and genetic factors, is not well understood. We performed whole-exome sequencing (WES) in six trios, with a single affected proband with spina bifida, to identify rare/novel variants as potential causes of the NTD. Our analysis identified four de novo and ten X-linked recessive variants in four of the six probands, all of them in genes previously never implicated in NTD. Among the 14 variants, we ruled out six of them, based on different criteria and pursued the evaluation of eight potential candidates in the following genes: RXRγ, DTX1, COL15A1, ARHGAP36, TKTL1, AMOT, GPR50, and NKRF. The de novo variants where located in the RXRγ, DTX1, and COL15A1 genes while ARHGAP36, TKTL1, AMOT, GPR50, and NKRF carry X-linked recessive variants. This analysis also revealed that four patients presented multiple variants, while we were unable to identify any significant variant in two patients. Our preliminary conclusion support a major role for the de novo variants with respect to the X-linked recessive variants where the X-linked could represent a contribution to the phenotype in an oligogenic model.

DNA methylation in the human genome is largely programmed and shaped by transcription factor binding and interaction between DNA methyltransferases and histone marks during gamete and embryo development. Normal methylation profiles can be modified at single or multiple loci, more frequently as consequences of genetic variants acting in cis or in trans, or in some cases stochastically or through interaction with environmental factors. For many developmental disorders, specific methylation patterns or signatures can be detected in blood DNA. The recent use of high-throughput assays investigating the whole genome has largely increased the number of diseases for which DNA methylation analysis provides information for their diagnosis. Here, we review the methylation abnormalities that have been associated with mono/oligogenic diseases, their relationship with genotype and phenotype and relevance for diagnosis, as well as the limitations in their use and interpretation of results.

To assess the involvement of microRNAs (miRNAs) in B-cell receptor (BCR) stimulation, we first evaluated miRNA profiling following IgM cross-linking in chronic lymphocytic leukemia (CLL) cells and in normal B lymphocytes. Second, we combined miRNA and gene expression data to identify putative miRNA functional networks. miRNA profiling showed distinctive patterns of regulation after stimulation in leukemic versus normal B lymphocytes and identified a differential responsiveness to BCR engagement in CLL subgroups according to the immunoglobulin heavy chain variable region mutational status and clinical outcome. The most significantly modulated miRNAs in stimulated CLL are miR-132 and miR-212. Notably, these miRNAs appeared regulated in progressive but not in stable CLL. Accordingly, gene profiling showed a significant transcriptional response to stimulation exclusively in progressive CLL. Based on these findings, we combined miRNA and gene expression data to investigate miR-132 and miR-212 candidate interactions in this CLL subgroup. Correlation analysis pointed to a link between these miRNAs and RB/E2F and TP53 cascades with proproliferative effects, as corroborated by functional analyses. Finally, basal levels of miR-132 and miR-212 were measured in an independent cohort of 20 unstimulated CLL cases and both showed lower expression in progressive compared to stable patients, suggesting an association between the expression of these molecules and disease prognosis. Overall, our results support a model involving miR-132 and miR-212 upregulation in sustaining disease progression in CLL. These miRNAs may therefore provide new valuable strategies for therapeutic intervention. © 2015 Wiley Periodicals, Inc.

1519 Background: DICER1 is a key endoribonuclease in the microRNA pathway that modulates gene expression. Germline loss of function variants in DICER1, first found in pleuropulmonary blastoma, have been subsequently linked to a variety of cancerous (and non) conditions referred to as DICER1 syndrome. In 2018, the Italian Society of Human Genetics launched an initiative aimed at establishing a national registry of DICER1 germline sequence variants. Methods: Centers involved in genetic testing for cancer predisposition were asked to report any identified DICER1 germline variants and related clinical information. Five University and/or research institutes filled-in the electronic survey. Informed consent was obtained from patients or their legal guardians prior to DNA testing by NGS and/or Sanger sequencing. Results: Six DICER1 sequence variants were identified in 11 individuals. Three missense variants are secondary results of NGS panels for cancer predisposition and lack definitive categorization in online databases. Three previously unreported variants are predicted to be protein truncating and, hence, likely pathogenic. Of these, DICER1 c.4844delA p.(Lys1615Argfs*5) and c.4886C > G p.(Ser1629*) result from ad hoc testing offered to probands based on a history of early onset follicular thyroid carcinoma and botryoid-type embryonal rhabdomyosarcoma of the cervix and of pleuropulmonary blastoma 2nd type, respectively. DICER1 c.4643T > A p.(Leu1548*), instead, results from whole exome sequencing in two siblings with malignant melanoma who tested non informative for alterations in the CDKN2A and CDK4 melanoma predisposing genes. Further investigation unearthed thyroid disease in the family and identified two other young carrier individuals, one unaffected and one thyroidectomized due to multinodular goiter. A DICER1 somatic hot spot sequence variant was detected in goiter specimens. Conclusions: Via the newly established national registry we uncovered novel DICER1 germline sequence variants and uncommon genotype-phenotype associations. Our joint effort will help us to refine our knowledge of the rare DICER1 syndrome , to inform research studies, and to improve testing and clinical management strategies.

// Monica Messina 1,* , Sabina Chiaretti 1,* , Jiguang Wang 2,* , Anna Lucia Fedullo 1 , Nadia Peragine 1 , Valentina Gianfelici 1 , Alfonso Piciocchi 3 , Fulvia Brugnoletti 1 , Filomena Di Giacomo 4 , Simona Pauselli 1 , Antony B. Holmes 5 , Maria Cristina Puzzolo 1 , Giulia Ceglie 1 , Valerio Apicella 1 , Marco Mancini 1 , Geertruy te Kronnie 6 , Anna Maria Testi 1 , Antonella Vitale 1 , Marco Vignetti 3 , Anna Guarini 1 , Raul Rabadan 2 and Robin Foa 1 1 Hematology, Department of Cellular Biotechnologies and Hematology, Sapienza University, Rome, Italy 2 Department of Systems Biology, Biomedical Informatics and Center for Computational Biology and Bioinformatics, Columbia University, New York, NY, USA 3 GIMEMA Data Center, Rome, Italy 4 Department of Molecular Biotechnology and Health Science, and Center for Experimental Research and Medical Studies (CeRMS), University of Torino, Torino, Italy 5 Institute for Cancer Genetics and The Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY, USA 6 Department of Women’s and Children’s Health, University of Padova, Padova, Italy * These authors have contributed equally to this work Correspondence to: Robin Foa, email: // Keywords : acute lymphoblastic leukemia, next generation sequencing, copy number aberrations, novel prognostic markers, genetic-driven targeted therapy Received : October 23, 2015 Accepted : January 28, 2016 Published : February 12, 2016 Abstract To shed light into the molecular bases of B-lineage acute lymphoblastic leukemia lacking known fusion transcripts, i.e. BCR-ABL1 , ETV6-RUNX1 , E2A-PBX1 , and MLL rearrangements (B-NEG ALL) and the differences between children, adolescents/young adults (AYA) and adults, we analyzed 168 B-NEG ALLs by genome-wide technologies. This approach showed that B-NEG cases carry 10.5 mutations and 9.1 copy-number aberrations/sample. The most frequently mutated druggable pathways were those pertaining to RAS/RTK (26.8%) and JAK/STAT (12.5%) signaling. In particular, FLT3 and JAK/STAT mutations were detected mainly in AYA and adults, while KRAS and NRAS mutations were more frequent in children. RAS/RTK mutations negatively affected the outcome of AYA and adults, but not that of children. Furthermore, adult B-NEG ALL carrying JAK/STAT mutations had a shorter survival. In vitro experiments showed that FLT3 inhibitors reduced significantly the proliferation of FLT3 -mutated primary B-NEG ALL cells. Likewise, PI3K/mTOR inhibitors reduced the proliferation of primary cells harboring RAS and IL7R mutations. These results refine the genetic landscape of B-NEG ALL and suggest that the different distribution of lesions and their prognostic impact might sustain the diverse outcome between children, adults and partly AYA - whose genomic scenario is similar to adults - and open the way to targeted therapeutic strategies.

Background A deregulated CRLF2 (d- CRLF2 ) expression was described in B-cell acute lymphoblastic leukemia without recurrent fusion genes (B-NEG ALL). While the role of d- CRLF2 in children has been extensively described, little is known about its role and impact in adult ALL. Methods Expression levels of CRLF2 were evaluated by quantitative real-time PCR in 102 newly-diagnosed adult B-NEG ALL and correlated with the clinico-biological characteristics and outcome. Incidence and clinical impact of the P2RY8/CRLF2 transcript was also assessed. Results High CRLF2 levels, as continuous variable, were significantly associated with hyperleucocytosis ( p = 0.0002) and thrombocytopenia ( p = 0.005); when a cut-point at ΔCt ≤ 8 was applied, 35 cases (34.3%), mostly males (80%), proved positive for CRLF2 expression. High CRLF2 levels, as continuous or categorical variable, were associated with a worse disease-free ( p = 0.003 and p = 0.015) and overall survival ( p = 0.017 and 0.0038). Furthermore, when CRLF2 was analyzed as a categorical variable, a high statistical association was found with IKZF1 deletion and mutations in the JAK/STAT pathway ( p = 0.001 and p P2RY8/CRLF2 transcript, identified in 8/102 patients (7.8%), was associated with a poor outcome. Conclusions In adult B-NEG ALL, high CRLF2 expression is associated with distinct clinico-biological features and an unfavourable prognosis in both univariate and multivariate analysis; similarly, P2RY8/CRLF2 positivity correlates with a poor outcome. The quantification of CRLF2 is an important prognostic marker in adult B-lineage ALL without known genetic lesions.

To assess the involvement of microRNAs (miRNAs) in B-cell receptor (BCR) stimulation, we first evaluated miRNA profiling following IgM cross-linking in chronic lymphocytic leukemia (CLL) cells and in normal B lymphocytes. Second, we combined miRNA and gene expression data to identify putative miRNA functional networks. miRNA profiling showed distinctive patterns of regulation after stimulation in leukemic versus normal B lymphocytes and identified a differential responsiveness to BCR engagement in CLL subgroups according to the immunoglobulin heavy chain variable region mutational status and clinical outcome. The most significantly modulated miRNAs in stimulated CLL are miR-132 and miR-212. Notably, these miRNAs appeared regulated in progressive but not in stable CLL. Accordingly, gene profiling showed a significant transcriptional response to stimulation exclusively in progressive CLL. Based on these findings, we combined miRNA and gene expression data to investigate miR-132 and miR-212 candidate interactions in this CLL subgroup. Correlation analysis pointed to a link between these miRNAs and RB/E2F and TP53 cascades with proproliferative effects, as corroborated by functional analyses. Finally, basal levels of miR-132 and miR-212 were measured in an independent cohort of 20 unstimulated CLL cases and both showed lower expression in progressive compared to stable patients, suggesting an association between the expression of these molecules and disease prognosis. Overall, our results support a model involving miR-132 and miR-212 upregulation in sustaining disease progression in CLL. These miRNAs may therefore provide new valuable strategies for therapeutic intervention.

Systemic and intrathecal methotrexate (MTX) are integral components of acute lymphoblastic leukemia (ALL) therapy, but can be associated with neurotoxicity. We describe here the case of an adolescent male with T-cell ALL who developed recurrent episodes of subacute neurotoxicity characterized by slurred speech, emotional lability, and hemiparesis after intrathecal MTX administration. Serial magnetic resonance imaging with diffusion-weighted imaging showed recurrent areas of restricted diffusion within cerebral hemispheric white matter, which correlated chronologically with the administration of intrathecal therapy and severity of clinical symptoms. Resolution of diffusion abnormalities did not preclude further toxicity and a large lesion could cause persisting symptoms.

Acute lymphoblastic leukemia (ALL) is a disease of either B-cell (80–85%) or T-cell (20–25%) derivation. Several molecular aberrations (i.e. BCR-ABL1, MLL/AFF1, SIL/TAL1 and E2A/PBX1 ) confer an overall poor outcome.[1][1],[2][2] However, a proportion of patients do not carry known genetic

We discuss an unusual clinical presentation of childhood Hodgkin lymphoma; a 16-year-old girl was referred for Coombs-positive severe anemia, thrombocytopenia, and asymptomatic anterior mediastinal mass. Bone marrow examination showed no evidence of neoplastic disease. Biopsy of the mass was possible only after administration of both intravenous immunoglobulins and steroids resulting in a prompt rise of the platelet count and partial hemoglobin level stabilization. The identification of this clinical picture as a possible complication of an underlying Hodgkin lymphoma presents difficulties in diagnosis and management of the primary condition.

Systemic and intrathecal methotrexate (MTX) are integral components of acute lymphoblastic leukemia (ALL) therapy, but can be associated with neurotoxicity. We describe here the case of an adolescent male with T-cell ALL who developed recurrent episodes of subacute neurotoxicity characterized by slurred speech, emotional lability, and hemiparesis after intrathecal MTX administration. Serial magnetic resonance imaging with diffusion-weighted imaging showed recurrent areas of restricted diffusion within cerebral hemispheric white matter, which correlated chronologically with the administration of intrathecal therapy and severity of clinical symptoms. Resolution of diffusion abnormalities did not preclude further toxicity and a large lesion could cause persisting symptoms.

This study was conducted to determine the feasibility, safety and efficacy of the FAND plus Campath-1H combination (FAND-Cam) given to 19 previously treated CLL patients with adverse prognostic features. The median age was 55 years (range 43–65), the median duration of CLL was 68 months; 89 % of patients were unmutated, 74% CD38+ and 75% ZAP-70+. A 17p− deletion was observed in 5/11 evaluable cases. The median number of prior treatments was 2 (range 1–6). Seventeen patients (89%) were refractory to prior therapy, which included fludarabine in 14 (fludarabine, n = 3; fludarabine-cyclophosphamide, n = 10; fludarabine-cyclophosphamide-rituximab, n = 1). The first 6 patients received 2 courses of FAND, for tumor debulking, followed by 2 FAND-Cam courses. However, a high infection rate was observed. In an attempt to reduce the risk of infection, the subsequent 13 patients received 2 courses of FAND-Cam without prior debulking with FAND. The treatment regimen of FAND was as follows: fludarabine, 25 mg/m2 intravenously (IV) administered daily on days 1–3 (0, 24 and 48 hours); Ara-C, 700 mg/m2 IV on days 1–3 (4, 28 and 52 hours); mitoxantrone, 10 mg/m2 IV on day 1 at 6 hours; dexamethasone, 20 mg IV on days 1–3. Before the first FAND-Cam course, Campath-1H was dose escalated from 3 mg 10 mg and then to 30 mg IV. After the FAND regimen, patients received Campath-1H, 30 mg IV on 3 consecutive days. G-CSF was used in the event of severe neutropenia. Infection prophylaxis consisted of trimethoprim-sulfamethoxazole, fluconazole, acyclovir 200 mg 3 times a day, for the first 9 patients, valacyclovir 2g 3 times a day for the last 10 patients. CMV viremia was performed weekly. To date, the response is evaluable in 18 patients. All 6 patients who received 2 courses of FAND for tumor debulking, followed by 2 courses of FAND-Cam, responded to treatment (complete response [CR], n = 1; cytometric CR, n = 4; partial response [PR], n = 1). However, infections were recorded in all cases (Nocardia pneumonia + CMV reactivation, n = 1; pneumonia, n = 1; pneumonia + CMV reactivation, n = 1; sinus infection, n = 1; CMV reactivation, n = 2). In the second group of patients who, without prior debulking, received only 2 courses of FAND-Cam, a response was observed in 10/12 (83%) evaluable cases (CR, n = 3; cytometric CR, n = 3; PR, n = 4; no response [NR], n = 2). An infection was recorded in 8/12 cases (Pseudomonas sepsis, n = 1; Pseudomonas sepsis + CMV reactivation, n = 1; Aspergillus, n = 1; Pneumonia, n = 3; CMV reactivation, n = 2). Prophylactic treatment with valacyclovir reduced the rate of CMV reactivation from 67% (6/9 pts) to 11% (1/9pts). During treatment there were no deaths related to infection. Six patients who achieved a response after Fand-Cam underwent allogeneic peripheral stem cell transplantation. CLL refractory patients with adverse prognostic factors respond poorly to therapy and show a high infection rate due to the severe immunodeficiency related to both refractory disease and treatment. In this study, FAND-Cam combination proved to be a feasible and effective salvage therapy in most patients. Infections were common. Careful surveillance and extended prophylaxis for infection are therefore required.

Chronic lymphocytic leukemia (CLL) is the most frequent form of leukemia in which familial aggregation has been observed. The affected relative is usually a sibling and less frequently a child. In the present study we evaluated whether CLL affecting parents and their children was associated with different clinical features, survival, immunoglobulin (Ig) VH genes repertoire and mutation status. The family history of 2% of the CLL patients diagnosed at our institution was characterized by the presence of a parent or a child affected by CLL. Other lymphoproliferative diseases, such as non-Hodgkin lymphoma, Hodgkin disease and acute lymphoid leukemia, affecting parents or children were also recorded in 3% of cases. Among the 17 identified parent/child CLL pairs there was a higher, though not significant, rate of gender concordant pairs than expected. Children were significantly younger at diagnosis than their parents. No significant differences in stage distribution, clinical course, survival and proportion of unmutated cases emerged between the two generations. In no case the parent and the respective child shared an identical IgVH repertoire. However, a significantly higher than expected usage of VH3-23 (p

T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy of the lymphoblasts committed to the T-cell lineage. Despite the therapeutic improvements witnessed over the years, ∼25% of children and ∼50% of adults still show a poor long-term outcome. While many recurrent oncogenic lesions have been identified through the characterization of chromosomal aberrations and candidate gene sequencing, several observations indicate that additional genetic alterations, not evident by conventional cytogenetics, might influence leukemogenesis and treatment outcome. Improvement of our knowledge in the identification and characterization of new oncogenic genome variations is expected to lead to a better prognostic classification and should also allow the design of tailored therapeutic strategies. To get further insights into the molecular pathogenesis of T-ALL and to identify novel markers for risk stratification and treatment improvement, we performed whole transcriptome sequencing (RNA-seq) on 18 refractory T-ALL cases sampled at diagnosis (median age 37.5 years, range 11-55). A pool of normal thymus cells was used as negative control. Next generation sequencing libraries were constructed from the mRNA fraction, followed by paired-end sequencing on a HiSeq2000 (Illumina). Sequence reads were aligned to the reference genome and were processed to identify gene expression levels, gene fusion transcripts and single nucleotide variations (SNVs). We first determined accurate gene expression levels from the RNA-seq data and used them to classify patients into T-ALL subtypes. Next, we applied the deFuse algorithm to detect fusion transcripts. Fusion transcripts detected also in normal thymus cells were filtered out, as well as fusions involving ribosomal genes. After applying these filters, we obtained 407 fusion transcripts (average: 22.6/sample, range: 0-84) predominantly involving genes localized on the same chromosome and mostly generated by deletion (306/407). Novel candidate fusion transcripts were confirmed by RT-PCR and Sanger sequencing. The SET-NUP214 fusion was identified in 2 cases, as well as 2 novel fusion transcripts involving the T-cell receptor (TCR) genes and not detected by conventional cytogenetics: the first fusion resulted in a chromosomal rearrangement between HOXA-AS4 and TRBC2 (also accompanied by overexpression of the HOXA genes) and the second between TRAC and SOX8 (associated with SOX8 overexpression). Interestingly, we also found out-of-frame fusion transcripts leading to the potential inactivation of tumor suppressor genes, such as PTEN-FAS and MAST3-C19orf10. Finally, we performed SNV calling on our dataset. After removing the most common polymorphisms, we obtained 1,483 protein-altering SNVs (missense, nonsense mutations and mutations affecting splicing), ranging between 30 and 131 per sample, with 85 genes that contain a protein-altering mutation in at least 3 of the 18 samples (i.e. 16% of cohort). Members or modulators of NOTCH and JAK/STAT pathways were the most recurrently mutated, each accounting for ∼38% of cases. In particular, 7/18 samples showed previously reported lesions in the NOTCH1 (n=5) and FBXW7 (n=1) genes but also in novel candidates as NOTCH2 (n=1), NOTCH3 (n=1) and SPEN (n=1). Interestingly, 1 patient showed 2 different mutations in the exon 26 of NOTCH1, while in 2 samples NOTCH1 mutation was associated with mutations in NOTCH2 or NOTCH3. Similarly, the JAK/STAT pathway was affected in 7/18 samples, including JAK1 (n=2), JAK3 (n=5), TYK2 (n=1) but also the novel candidates STAT5A (n=2) and STAT6 (n=1). Four of the 5 JAK3-positive patients showed also a mutation in another gene of the same pathway, such as JAK1 (n=1), STAT5A (n=2) and STAT6 (n=1). Thus, mutational screening of both the NOTCH and JAK/STAT pathway shows that mutations can occur simultaneously and suggests that more than one lesion is required for leukemic transformation. In conclusion, RNA-seq appears as a promising tool to dissect the heterogeneity of T-ALL and to identify targets that might be useful for tailored therapeutic interventions. Further investigations are ongoing to determine the recurrence and specificity of these lesions, and their potential in inducing a refractory phenotype. Finally, in vitro experiments will be carried out to investigate the transforming capability of specific lesions and the targettability of the recurrently impaired pathways. Disclosures: No relevant conflicts of interest to declare.