In this study, we used deletions at 22q13, which represent a substantial source of human pathology (Phelan/McDermid syndrome), as a model for investigating the molecular mechanisms of terminal deletions that are currently poorly understood. We characterized at the molecular level the genomic rearrangement in 44 unrelated patients with 22q13 monosomy resulting from simple terminal deletions (72%), ring chromosomes (14%), and unbalanced translocations (7%). We also discovered interstitial deletions between 17–74 kb in 9% of the patients. Haploinsufficiency of the SHANK3 gene, confirmed in all rearrangements, is very likely the cause of the major neurological features associated with PMS. SHANK3 mutations can also result in language and/or social interaction disabilities. We determined the breakpoint junctions in 29 cases, providing a realistic snapshot of the variety of mechanisms driving non-recurrent deletion and repair at chromosome ends. De novo telomere synthesis and telomere capture are used to repair terminal deletions; non-homologous end-joining or microhomology-mediated break-induced replication is probably involved in ring 22 formation and translocations; non-homologous end-joining and fork stalling and template switching prevail in cases with interstitial 22q13.3. For the first time, we also demonstrated that distinct stabilizing events of the same terminal deletion can occur in different early embryonic cells, proving that terminal deletions can be repaired by multistep healing events and supporting the recent hypothesis that rare pathogenic germline rearrangements may have mitotic origin. Finally, the progressive clinical deterioration observed throughout the longitudinal medical history of three subjects over forty years supports the hypothesis of a role for SHANK3 haploinsufficiency in neurological deterioration, in addition to its involvement in the neurobehavioral phenotype of PMS.

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In-frame missense and splicing mutations (resulting in a 2 amino acid insertion or a 34 amino acid deletion) dispersed through the MAP3K1 gene tilt the balance from the male to female sex-determining pathway, resulting in 46,XY disorder of sex development. These MAP3K1 mutations mediate this balance by enhancing WNT/β-catenin/FOXL2 expression and β-catenin activity and by reducing SOX9/FGF9/FGFR2/SRY expression. These effects are mediated at multiple levels involving MAP3K1 interaction with protein co-factors and phosphorylation of downstream targets. In transformed B-lymphoblastoid cell lines and NT2/D1 cells transfected with wild-type or mutant MAP3K1 cDNAs under control of the constitutive CMV promoter, these mutations increased binding of RHOA, MAP3K4, FRAT1 and AXIN1 and increased phosphorylation of p38 and ERK1/2. Overexpressing RHOA or reducing expression of MAP3K4 in NT2/D1 cells produced phenocopies of the MAP3K1 mutations. Using siRNA knockdown of RHOA or overexpressing MAP3K4 in NT2/D1 cells produced anti-phenocopies. Interestingly, the effects of the MAP3K1 mutations were rescued by co-transfection with wild-type MAP3K4. Although MAP3K1 is not usually required for testis determination, mutations in this gene can disrupt normal development through the gains of function demonstrated in this study.

Objective We surveyed the datasheets of 29 laboratories concerning prenatal diagnosis of de novo apparently balanced chromosome rearrangements to assess the involvement of specific chromosomes, the breakpoints distribution and the impact on the pregnancy outcome.Method By means of a questionnaire, data on 269.371 analyses performed from 1983 to 2006 on amniotic fluid, chorionic villus and fetal blood samples were collected.Results A total of 246 balanced anomalies were detected at frequencies of 72% for reciprocal translocations, 18% for Robertsonian translocations, 7% for inversions and 3% for complex chromosome rearrangements. The total frequencies of balanced rearrangements were 0.09%, 0.08% and 0.05% on amniotic fluid, chorionic villus and fetal blood samples.Conclusion A preferential involvement of chromosomes 22, 7, 21, 3, 9 and 11 and a less involvement of chromosomes X, 19, 12, 6 and 1 was observed. A nonrandom distribution of the breakpoints across chromosomes was noticed. Association in the location of recurrent breakpoints and fragile sites was observed for chromosomes 11, 7, 10 and 22, while it was not recorded for chromosome 3. The rate of pregnancy termination was about 20%, with frequencies decreasing from complex chromosomal rearrangements (33%), reciprocal translocations (24%) to inversions (11%) and Robertsonian translocations (3%).

IntroductionChronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder induced by a chimeric gene that results from the fusion of the ABL gene on chromosome 9 with the BCR gene on chromosome 22, leading to the formation of a new leukemiaspecific fusion gene that codes for constitutionally activated protein tyrosine kinases (PTK) of different molecular weight (p210, rarely p230 or p185/190). The oncogenic PTK, which is located in the cytoplasm, is responsible of the leukemic phenotype through the constitutive activation of several downstream pathways. [1][2][3] In more than 95% of cases, the translocation between chromosome 9 and 22 is balanced and results in the formation of a small chromosome 22 that was identified originally as Philadelphia (Ph). Rare variant translocations may be masked and detected by fluorescence in situ hybridization of interphase nuclei (I-FISH). [2][3][4][5] The BCR-ABL mRNA is detected by reverse transcriptase polymerase chain reaction (PCR) and may be quantified by real-time quantitative PCR (RT-Q-PCR). [6][7][8] After the introduction of interferon-␣ and the PTK inhibitor imatinib mesylate (IM), it has become more and more important to monitor cytogenetically the response to treatment and the course of the disease. 9-13 On the basis of chromosome banding analysis (CBA) of marrow cell metaphases, the cytogenetic response (CgR) is classified as none, minimal, minor, partial, or complete according to the percentage of Ph ϩ metaphases (95%, 95%-66%, 65%-36%, 35%-1%, and none). 14 The achievement and the maintenance of a complete cytogenetic response (CCgR) are of particular importance because a CCgR is the most solid, confirmed, early surrogate marker of progression-free survival and overall survival. [11][12][13][14][15][16] The definition of CgR by CBA requires marrow cells, which cannot be always sampled, and an adequate number of banded metaphases, which cannot be always obtained. For these reasons, I-FISH is sometimes used, with increasing frequency, as a substitute for CBA, but although there is a fairly good relationship between I-FISH and CBA data, [17][18][19][20][21][22][23][24][25][26][27][28] there are no controlled and shared For personal use only. on May 12, 2018. by guest www.bloodjournal.org From definitions of CgR by I-FISH. This study was designed with the purpose of comparing CBA and I-FISH data for the definition of CCgR. Methods PatientsAll the patients, at least 18 years of age, were required to have a Ph ϩ and BCR-ABL ϩ CML in early chronic phase (CP). They were enrolled in 3 prospective and concurrent studies: CML/021 (ClinTrials.gov no. NCT00514488), a phase 2 trial exploring IM 800 mg daily in intermediate Sokal risk patients with CP CML; CML/022 (ClinTrials.gov no. NCT00510926), a phase 3 trial comparing IM 400 versus 800 mg daily in high Sokal risk patients with CP CML; and CML/023, an observational study of IM 400 mg daily in patients with CP CML. These studies were promoted, sponsored, and operated by the CML Working Party of GIMEMA (previous...

Imatinib mesylate (IM) is the first line therapy against Chronic Myeloid Leukemia, effectively prolonging overall survival. Because discontinuation of treatment is associated with relapse, IM is required indefinitely to maintain operational cure. To assess minimal residual disease, cytogenetic analysis is insensitive in a high background of normal lymphocytes. The qRT-PCR provides highly sensitive detection of BCR-ABL1 transcripts, but mRNA levels are not directly related to the number of leukemic cells, and undetectable results are difficult to interpret. We developed a sensitive approach to detect the number of leukemic cells by a genomic DNA (gDNA) Q-PCR assay based on the break-point sequence, with a formula to calculate the number of Ph-positive cells. We monitored 8 CML patients treated with IM for more than 8 years. We tested each samples by patient specific gDNA Q-PCR in parallel by the conventional techniques. In all samples positive for chimeric transcripts we showed corresponding chimeric gDNA by Q-PCR, and in 32.8% (42/128) of samples with undetectable levels of mRNA we detected the persistence of leukemic cells.The gDNA Q-PCR assay could be a new diagnostic tool used in parallel to conventional techniques to support the clinician's decision to vary or to STOP IM therapy.

Human bone marrow mesenchymal stromal cells (BM-MSC) represent one of the most investigated "advanced therapeutic medicinal products".1 Recent safety concerns have focused attention on the possible malignant transformation due to mutations acquired during their large-scale in vitro expansion.2 Indeed, spontaneous oncogenic transformation has been described for murine MSC 3 although not for human cells, 4,5 with the exception of a few studies, 6which were subsequently retracted when it was realized that this was due to cross-contamination by a tumor cell line. 7,8 One single report has described the in vitro outgrowth of a transformed subpopulation from a normal BM sample.9 Furthermore, genetic aberrations of MSC have been very occasionally observed after long-term cultures 4,10,11 but interpreted to be related to senescence. 5In order to investigate the frequency of cytogenetic alterations in a broad "collection" of clinical-grade BM-MSC products, we performed cytogenetic analysis of 92 preparations expanded under Good Manufacturing Practice conditions.12 More precisely, 67 expansions were performed from 33 healthy donors, 4 β−thalassemia patients and 21 multiple sclerosis patients (Table 1). MSC were expanded from BM washouts or aspirates using human platelet lysate as previously described.12,13 Metaphases were prepared according to standard procedures 12 and analyzed by QFQ-banding. At least 20 metaphases per sample were analyzed. Karyotype was described according to the International System for Human Cytogenetic Nomenclature. Furthermore, p53 gene mutations were analyzed by deep sequencing of exons 5 to 11.Chromosomal abnormalities were detected in 17 of 86 expansions (19.8%). In all cases, the genetic lesions were spontaneous abnormalities.2 In 14 cases they were nonclonal: in 8 they involved one metaphase (MSC46, MSC70, MSC74, MSC79, MSC82, MSC87, MSC121, MSC126); in 5 two different chromosome abnormalities in two metaphases (MSC52, MSC55, MSC66 MSC100, MSC116). Only in one case were three different alterations in three metaphases found (MSC80). "Clonal chromosome changes" 2 were detected in 3 cases: in MSC114 monosomy of chromosome X was found in three metaphases, while in MSC119 and MSC122 inversion of chromosome 1 and a translocation involving chromosomes 9 and 4, respectively, were found in two metaphases. We also examined the results of multiple expansions from the same donors. Chromosomal anomalies were observed for 7 out of 13 donors (ns. 18, 20, 23, 32, 37, 50, 63), but these lesions were not recurrent and present only in some of the expansions performed. This suggests that chromosome aberrations do not associate with specific donors. In 6 cases, cytogenetic evaluation could not be performed on the final fresh P2 products due to lack of metaphases (Table 1) but in 5 of these the analyses could be repeated using a frozen P2 aliquot and in 4 cases karyotypes were normal. Similarly, when spontaneous and non-clonal abnormalities were detected, the karyotype analysis was repeated using a frozen P2 aliqu...

CD56 is expressed in 15–20% of acute myeloid leukaemias (AML) and is associated with extramedullary diffusion, multidrug resistance and poor prognosis. We describe the establishment and characterisation of a novel disseminated model of AML (AML-NS8), generated by injection into mice of leukaemic blasts freshly isolated from a patient with an aggressive CD56+ monoblastic AML (M5a). The model reproduced typical manifestations of this leukaemia, including presence of extramedullary masses and central nervous system involvement, and the original phenotype, karyotype and genotype of leukaemic cells were retained in vivo. Recently Polo-Like Kinase 1 (PLK1) has emerged as a new candidate drug target in AML. We therefore tested our PLK1 inhibitor NMS-P937 in this model either in the engraftment or in the established disease settings. Both schedules showed good efficacy compared to standard therapies, with a significant increase in median survival time (MST) expecially in the established disease setting (MST = 28, 36, 62 days for vehicle, cytarabine and NMS-P937, respectively). Importantly, we could also demonstrate that NMS-P937 induced specific biomarker modulation in extramedullary tissues. This new in vivo model of CD56+ AML that recapitulates the human tumour lends support for the therapeutic use of PLK1 inhibitors in AML.