Mutations in MAP3K1 tilt the balance from SOX9/FGF9 to WNT/ß-catenin signaling.

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/ß-catenin/FOXL2 expression and ß-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.

Loss-of-Function FANCL Mutations Associate with Severe Fanconi Anemia Overlapping the VACTERL Association.

The diagnosis of VACTERL syndrome can be elusive, especially in the prenatal life, due to the presence of malformations that overlap those present in other genetic conditions, including the Fanconi anemia (FA). We report on three VACTERL cases within two families, where the two who arrived to be born died shortly after birth due to severe organs' malformations. The suspicion of VACTERL association was based on prenatal ultrasound assessment and postnatal features. Subsequent chromosome breakage analysis suggested the diagnosis of FA. Finally, by next-generation sequencing based on the analysis of the exome in one family and of a panel of Fanconi genes in the second one, we identified novel FANCL truncating mutations in both families. We used ectopic expression of wild-type FANCL to functionally correct the cellular FA phenotype for both mutations. Our study emphasizes that the diagnosis of FA should be considered when VACTERL association is suspected. Furthermore, we show that loss-of-function mutations in FANCL result in a severe clinical phenotype characterized by early postnatal death.

Molecular mechanisms generating and stabilizing terminal 22q13 deletions in 44 subjects with Phelan/McDermid syndrome.

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.

Variant Philadelphia translocations: molecular-cytogenetic characterization and prognostic influence on frontline imatinib therapy, a GIMEMA Working Party on CML analysis.

Variant Philadelphia (Ph) chromosome translocations have been reported in 5%-10% of patients with newly diagnosed chronic myeloid leukemia (CML). Variant translocations may involve one or more chromosomes in addition to 9 and 22, and can be generated by 2 different mechanisms, 1-step and 2-step rearrangements, as revealed by fluorescence in situ hybridization. The prognostic significance of the occurrence of variant translocations has been discussed in previous studies. The European LeukemiaNet recommendations do not provide a "warning" for patients with variant translocations, but there is limited information about their outcome after therapy with tyrosine kinase inhibitors. To identify the role of variant translocations in early chronic phase (CP) CML patients treated with imatinib mesylate, we performed an analysis in a large series of 559 patients enrolled in 3 prospective imatinib trials of the Gruppo Italiano Malattie EMatologiche dell'Adulto (GIMEMA) Working Party on CML. Variant translocations occurred in 30 patients (5%). Our data show that the presence of variant translocations has no impact on the cytogenetic and molecular response or on outcome, regardless of the involvement of different mechanisms, the number of involved chromosomes, or the presence of deletions. Therefore, we suggest that patients with variant translocations do not constitute a "warning" category in the imatinib era. This study is registered at www.clinicaltrials.gov as NCT00514488 and NCT00510926.

A novel, highly sensitive and rapid allele-specific loop-mediated amplification assay for the detection of the JAK2V617F mutation in chronic myeloproliferative neoplasms.

BACKGROUND: The identification of the JAK2V617F mutation is mandatory in the diagnostic work-up of Philadelphia chromosome-negative myeloproliferative neoplasms. Several molecular techniques to detect this mutation are currently available, but each of them has some limits. DESIGN AND METHODS: We set up a novel molecular method for the identification of the JAK2V617F mutation based on an allele-specific loop-mediated amplification, not polymerase chain reaction analysis. This innovative technique amplifies DNA targets under isothermal conditions with high specificity, efficiency and rapidity. The method does not require either a thermal cycler or gel separation and the DNA amplification reaction is visible to the naked eye and can be monitored by turbidimetry. This method was validated on DNA from cell lines as well as from patients with myeloproliferative neoplasms. The results were compared with those obtained by conventional polymerase chain reaction methods. RESULTS: This assay detects, within 1 hour, the JAK2V617F mutation down to an allele burden of 0.1-0.01%. All samples positive by polymerase chain reaction (n=146) proved positive when tested by allele-specific loop-mediated amplification and none of the 80 negative controls gave false positive results. In addition, six patients with essential thrombocythemia previously diagnosed as being JAK2V617F negative by polymerase chain reaction analysis were found to be positive (at a low level) by allele-specific loop-mediated amplification. Furthermore, this assay discriminated the amount of JAK2V617F tumor allele within intervals of positivity, above 50%, between 50% and 10% and below 10%. CONCLUSIONS: Allele-specific loop-mediated amplification is a simple, robust and easily applicable method for the molecular diagnosis and monitoring of JAK2V617F mutation in patients with chronic myeloproliferative neoplasms.

Chronic myeloid leukemia: a prospective comparison of interphase fluorescence in situ hybridization and chromosome banding analysis for the definition of complete cytogenetic response: a study of the GIMEMA CML WP.

In chronic myeloid leukemia, different methods are available to monitor the response to therapy: chromosome banding analysis (CBA), interphase fluorescence in situ hybridization (I-FISH), and real-time quantitative polymerase chain reaction (RT-Q-PCR). The GIMEMA CML WP (Gruppo Italiano Malattie Ematologiche Adulto Chronic Myeloid Leukemia Working Party) has performed a prospective study to compare CBA and I-FISH for the definition of complete cytogenetic response (CCgR). Samples (n = 664) were evaluated simultaneously by CBA and I-FISH. Of 537 cases in CCgR, the number of positive nuclei by I-FISH was less than 1% in 444 cases (82.7%). Of 451 cases with less than 1% positive nuclei by I-FISH, 444 (98.4%) were classified as CCgR by CBA. The major molecular response rate was significantly greater in cases with I-FISH less than 1% than in those with I-FISH 1% to 5% (66.8% vs 51.6%, P < .001) and in cases with CCgR and I-FISH less than 1% than in cases with CCgR and I-FISH 1% to 5% (66.1% vs 49.4%, P = .004). I-FISH is more sensitive than CBA and can be used to monitor CCgR. With appropriate probes, the cutoff value of I-FISH may be established at 1%. These trials are registered at http://www.clinicaltrials.gov as NCT00514488 and NCT00510926.

Immature Immunoglobulin Gene Rearrangements Are Recurrent in B Precursor Adult Acute Lymphoblastic Leukemia Carrying TP53 Molecular Alterations.

Here, we describe the immunoglobulin and T cell receptor (Ig/TCR) molecular rearrangements identified as a leukemic clone hallmark for minimal residual disease assessment in relation to TP53 mutational status in 171 Ph-negative Acute Lymphoblastic Leukemia (ALL) adult patients at diagnosis. The presence of a TP53 alterations, which represents a marker of poor prognosis, was strictly correlated with an immature DH/JH rearrangement of the immunoglobulin receptor (p < 0.0001). Furthermore, TP53-mutated patients were classified as pro-B ALL more frequently than their wild-type counterpart (46% vs. 25%, p = 0.05). Although the reasons for the co-presence of immature Ig rearrangements and TP53 mutation need to be clarified, this can suggest that the alteration in TP53 is acquired at an early stage of B-cell maturation or even at the level of pre-leukemic transformation.

Double homozygosity in CEP57 and DYNC2H1 genes detected by WES: Composite or expanded phenotype?

BACKGROUND: In the last few years trio-whole exome sequencing (WES) analysis has demonstrated its potential in obtaining genetic diagnoses even in nonspecific clinical pictures and in atypical presentations of known diseases. Moreover WES allows the detection of variants in multiple genes causing different genetic conditions in a single patient, in about 5% of cases. The resulting phenotype may be clinically discerned as variability in the expression of a known phenotype, or as a new unreported syndromic condition. METHODS: Trio-WES was performed on a 4-month-old baby with a complex clinical presentation characterized by skeletal anomalies, congenital heart malformation, congenital hypothyroidism, generalized venous and arterial hypoplasia, and recurrent infections. RESULTS: WES detected two different homozygous variants, one in CEP57, the gene responsible for mosaic variegated aneuploidy syndrome 2, the other in DYNC2H1, the main gene associated with short-rib thoracic dysplasia. CONCLUSION: The contribution of these two different genetic causes in determining the phenotype of our patient is discussed, including some clinical signs not explained by the detected variants. The report then highlights the role of WES in providing complete and fast diagnosis in patients with complex presentations of rare genetic syndromes, with important implications in the assessment of recurrence risk.

Insertional translocation involving an additional nonchromothriptic chromosome in constitutional chromothripsis: Rule or exception?

BACKGROUND: Chromothripsis, which is the local massive shattering of one or more chromosomes and their reassembly in a disordered array with frequent loss of some fragments, has been mainly reported in association with abnormal phenotypes. We report three unrelated healthy persons, two of which parenting a child with some degree of intellectual disability, carrying a chromothripsis involving respectively one, two, and three chromosomes, which was detected only after whole-genome sequencing. Unexpectedly, in all three cases a fragment from one of the chromothripsed chromosomes resulted to be inserted within a nonchromothripsed one. METHODS: Conventional cytogenetic techniques, paired-end whole-genome sequencing, polymerase chain reaction, and Sanger sequencing were used to characterize complex rearrangements, copy-number variations, and breakpoint sequences in all three families. RESULTS: In two families, one parent was carrier of a balanced chromothripsis causing in the index case a deletion and a noncontiguous duplication at 3q in case 1, and a t(6;14) translocation associated with interstitial 14q deletion in case 2. In the third family, an unbalanced chromothripsis involving chromosomes 6, 7, and 15 was inherited to the proband by the mosaic parent. In all three parents, the chromothripsis was concurrent with an insertional translocation of a portion of one of the chromothriptic chromosomes within a further chromosome that was not involved in the chromothripsis event. CONCLUSION: Our findings show that (a) both simple and complex unbalanced rearrangements may result by the recombination of a cryptic parental balanced chromothripsis and that (b) insertional translocations are the spy of more complex rearrangements and not simply a three-breakpoint event.

Feasibility and outcome of tandem stem cell autotransplants in multiple myeloma.

BACKGROUND AND OBJECTIVES: Clinical trials have shown that high dose chemotherapy (HDT) with peripheral stem cell autotransplantation is presently the best treatment for patients with symptomatic multiple myeloma (MM). In the context of an outcomes research project, we analyzed the feasibility of this strategy in clinical practice in a large cohort of consecutive, unselected patients with newly diagnosed MM and looked at the major determinants of response of patients enrolled in a HDT with tandem autotransplantation (Total Therapy I, TTI) program. DESIGN AND METHODS: Two hundred and fourteen patients were treated outside of a clinical trial and regularly followed-up at our Center for symptomatic MM. Ninety-seven patients (45%) received conventional chemo-radiotherapy regimens, 110 (51%) entered the TTI program and the remaining 7 patients (3.3%) were enrolled in other programs involving HDT with autotransplantation. RESULTS: Patients enrolled in HDT with tandem autotransplantation programs were 14 years younger and less likely to have co-morbidities than patients treated with conventional therapy. Median overall survivals of the two groups were 60 and 33 months, respectively. Thirteen percent of the patients enrolled in the TTI program did not receive the first HDT with autotransplantation, mostly because of disease progression, and another 16% did not proceed to the second HDT with autotransplantation mainly because of infections or drug-related complications. Most patients achieved complete remission after the second autotransplantation, with acceptable toxicity. However, only patients with a major reduction of the myeloma burden at the end of induction therapy enjoyed significantly prolonged event-free and overall survivals. INTERPRETATION AND CONCLUSIONS: Approximately one third of patients with newly diagnosed symptomatic MM completed the TTI program. These data suggest the need to improve the induction therapy in order to increase both the number of patients able to proceed to autotransplantation programs and to enhance the rate of early response.

Prolonged administration of all-trans retinoic acid in combination with intensive chemotherapy and G-CSF for adult acute myelogenous leukemia: single-centre pilot study in different risk groups.

INTRODUCTION: An exploratory trial was conducted to evaluate toxicity and potential therapeutic role of all trans-retinoic acid (ATRA) given long-term together with chemotherapy and G-CSF to adult patients with acute myelogenous leukemia (AML). MATERIALS AND METHODS: ATRA was administered orally at 45 mg/m(2)/day on days 1-14 and 25 mg/m(2)/day on days 15-28 of two standard cycles (idarubicin, etoposide, cytarabine, G-CSF) and of up to three high-dose courses (cytarabine, G-CSF). The results obtained in 19 patients enrolled in the ATRA trial were compared with those from 29 comparable cases treated with the same schedule without ATRA, according to patient risk class and an in vitro study. RESULTS: ATRA was administered for a median of 52 days to the patients selected for study who achieved a remission. ATRA-related toxicity was mostly non-severe apart from high incidence of headache in conjunction with high-dose cytarabine. Complete remission (CR) rate after cycle 1 (54%), kinetics of hematological recovery, postremission treatment realization, disease-free survival (DFS 37.5% at three years) and overall survival (30% at three years) were not different between ATRA-treated and untreated patients. The only significant prognostic factor was the patient risk class, as defined by cytogenetics and other clinical criteria: DFS rate was 57% at three years in standard-risk cases compared to only 19% in the high-risk group, with no influx by ATRA in either category. The in vitro study, in patients with a definite clinical response, failed to document any inhibitory or pro-apoptotic effect of ATRA on AML blast cells. CONCLUSION: As a consequence to these results, the pilot ATRA phase was closed. This study does not suggest a significant role for the present ATRA schedule as an adjunct to standard antileukemic therapy in adult AML.

Genomic quantitative real-time PCR proves residual disease positivity in more than 30% samples with negative mRNA-based qRT-PCR in Chronic Myeloid Leukemia.

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.

The Polo-Like Kinase 1 (PLK1) inhibitor NMS-P937 is effective in a new model of disseminated primary CD56+ acute monoblastic leukaemia.

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.

De novo balanced chromosome rearrangements in prenatal diagnosis.

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%).