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We investigated 52 cases of de novo unbalanced translocations, consisting in a terminally deleted or inverted-duplicated deleted (inv-dup del) 46th chromosome to which the distal portion of another chromosome or its opposite end was transposed. Array CGH, whole-genome sequencing, qPCR, FISH, and trio genotyping were applied. A biparental origin of the deletion and duplication was detected in 6 cases, whereas in 46, both imbalances have the same parental origin. Moreover, the duplicated region was of maternal origin in more than half of the cases, with 25% of them showing two maternal and one paternal haplotype. In all these cases, maternal age was increased. These findings indicate that the primary driver for the occurrence of the de novo unbalanced translocations is a maternal meiotic non-disjunction, followed by partial trisomy rescue of the supernumerary chromosome present in the trisomic zygote. In contrast, asymmetric breakage of a dicentric chromosome, originated either at the meiosis or postzygotically, in which the two resulting chromosomes, one being deleted and the other one inv-dup del, are repaired by telomere capture, appears at the basis of all inv-dup del translocations. Notably, this mechanism also fits with the origin of some simple translocations in which the duplicated region was of paternal origin. In all cases, the signature at the translocation junctions was that of non-homologous end joining (NHEJ) rather than non-allelic homologous recombination (NAHR). Our data imply that there is no risk of recurrence in the following pregnancies for any of the de novo unbalanced translocations we discuss here.

It is widely accepted that the therapeutic potential of stem cells can be largely mediated by paracrine factors, also included into exosomes. Thus, stem cell-derived exosomes represent a major therapeutic option in regenerative medicine avoiding, if compared to stem cells graft, abnormal differentiation and tumor formation. Exosomes derived from mesenchymal stem cells (MSC) induce damaged tissue repair, and can also exert immunomodulatory effects on the differentiation, activation and function of different lymphocytes. Therefore, MSC exosomes can be considered as a potential treatment for inflammatory diseases and also an ideal candidate for allogeneic therapy due to their low immunogenicity. Amniotic fluid stem cells (AFSCs) are broadly multipotent, can be expanded in culture, and can be easily cryopreserved in cellular banks. In this study, morphology, phenotype, and protein content of exosomes released into amniotic fluid in vivo and from AFSC during in vitro culture (conditioned medium) were examined. We found that AFSC-derived exosomes present different molecules than amniotic fluid ones, some of them involved in immunomodulation, such transforming growth factor beta and hepatic growth factors. The immunomodulatory effect of AFSC's exosomes on peripheral blood mononuclear cells stimulated with phytohemagglutinin was compared to that of the supernatant produced by such conditioned media deprived of exosomes. We present evidence that the principal effect of AFSC conditioned media (without exosomes) is the induction of apoptosis in lymphocytes, whereas exposure to AFSC-derived exosomes decreases the lymphocyte's proliferation, supporting the hypothesis that the entire secretome of stem cells differently affects immune-response. © 2017 BioFactors, 44(2):158-167, 2018.

Whole exome sequencing (WES) has made the identification of causative SNVs/InDels associated with rare Mendelian conditions increasingly accessible. Incorporation of softwares allowing CNVs detection into the WES bioinformatics pipelines may increase the diagnostic yield. However, no standard protocols for this analysis are so far available and CNVs in non-coding regions are totally missed by WES, in spite of their possible role in the regulation of the flanking genes expression. So, in a number of cases the diagnostic workflow contemplates an initial investigation by genomic arrays followed, in the negative cases, by WES. The opposite workflow may also be applied, according to the familial segregation of the disease. We show preliminary results for a diagnostic application of a single next generation sequencing panel permitting the concurrent detection of LOH and variations in sequences and copy number. This approach allowed us to highlight compound heterozygosity for a CNV and a sequence variant in a number of cases, the duplication of a non-coding region responsible for sex reversal, and a whole-chromosome isodisomy causing reduction to homozygosity for a WFS1 variant. Moreover, the panel enabled us to detect deletions, duplications, and amplifications with sensitivity comparable to that of the most widely used array-CGH platforms.

Background aims Current procedures for collection of human amniotic fluid stem cells (hAFSCs) indicate that cells cultured in a flask for 2 weeks can then be used for research. However, hAFSCs can be retrieved directly from a small amount of amniotic fluid that can be obtained at the time of diagnostic amniocentesis. The aim of this study was to determine whether direct freezing of amniotic fluid cells is able to maintain or improve the potential of a sub-population of stem cells. Methods We compared the potential of the hAFSCs regarding timing of freezing, cells obtained directly from amniotic fluid aspiration (D samples) and cells cultured in a flask before freezing (C samples). Colony-forming-unit ability, proliferation, morphology, stemness-related marker expression, senescence, apoptosis and differentiation potential of C and D samples were compared. Results hAFSCs isolated from D samples expressed mesenchymal stem cells markers until later passages, had a good proliferation rate and exhibited differentiation capacity similar to hAFSCs of C samples. Interestingly, direct freezing induced a higher concentration of cells positive for pluripotency stem cell markers, without teratoma formation in vivo . Conclusions This study suggests that minimal processing may be adequate for the banking of amniotic fluid cells, avoiding in vitro passages before the storage and exposure to high oxygen concentration, which affect stem cell properties. This technique might be a cost-effective and reasonable approach to the process of Good Manufacturing Process accreditation for stem-cell banks.

To date, 5 cases of 17p13.1 microduplications have been described in the literature. Intellectual disability was reported as the core feature, together with minor facial dysmorphisms and obesity, but a characteristic phenotype for 17p13.1 microduplication has not been delineated. Here, we describe a patient with a 1.56-Mb de novo duplication in 17p13.1, affected by mild intellectual disability, facial dysmorphisms, obesity, and diabetes. By comparing the different phenotypes of currently described cases, we delineated the main clinical features of 17p13.1 microduplication syndrome. All patients described to date had variable facial dysmorphisms; therefore, it was difficult to define a common facial gestalt. Furthermore, we stress endocrinological abnormalities as important features and the need to monitor these over time.

Gorlin syndrome or nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant condition mainly characterized by the development of mandibular keratocysts which often have their onset during the second decade of life and/or multiple basal cell carcinoma (BCC) normally arising during the third decade. Cardiac and ovarian fibromas can be found. Patients with NBCCS develop the childhood brain malignancy medulloblastoma (now often called primitive neuro-ectodermal tumor [PNET]) in 5% of cases. The risk of other malignant neoplasms is not clearly increased, although lymphoma and meningioma can occur in this condition. Wilms tumor has been mentioned in the literature four times. We describe a patient with a 10.9 Mb 9q22.3 deletion spanning 9q22.2 through 9q31.1 that includes the entire codifying sequence of the gene PTCH1, with Wilms tumor, multiple neoplasms (lung, liver, mesenteric, gastric and renal leiomyomas, lung typical carcinoid tumor, adenomatoid tumor of the pleura) and a severe clinical presentation. We propose including leiomyomas among minor criteria of the NBCCS. © 2013 Wiley Periodicals, Inc.

Traditional approaches for the classification of Small Supernumerary Marker Chromosomes (sSMC), mostly based on FISH techniques, are time-consuming and not always sufficient to fully understand the true complexity of this class of rearrangements. We describe four supernumerary marker chromosomes that, after array-CGH, were interpreted rather differently in respect to the early classification made by conventional cytogenetics and FISH investigations, reporting two types of complex markers which DNA content was overlooked by conventional approaches: 1. the sSMC contains non-contiguous regions of the same chromosome and, 2. the sSMC, initially interpreted as a supernumerary del(15), turns out to be a derivative 15 to which the portion of another chromosome was attached. All are likely derived from partial trisomy rescue events, bringing further demonstration that germline chromosomal imbalances are submitted to intense reshuffling during the embryogenesis, leading to unexpected complexity and changing the present ideas on the composition of supernumerary marker chromosomes.

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%). Copyright © 2009 John Wiley & Sons, Ltd.

22q11.2 deletion syndrome is mainly characterized by conotruncal congenital heart defects, velopharyngeal insufficiency, hypocalcemia and a characteristic craniofacial appearance. The etiology in the majority of patients is a 3-Mb recurrent deletion in region 22q11.2. Nevertheless, recently some cases of infrequent deletions with various sizes have been reported with a different phenotype. We report on a patient with congenital heart disease (truncus arteriosus type 2) in whom a de novo 1.3-Mb 22q11.2 deletion was detected by array comparative genomic hybridization. The deletion described corresponds to an atypical and distal deletion which spans low copy repeat (LCR) 4 and is associated with breakpoint sites that do not correspond to known LCRs of 22q11.2. We examine the clinical phenotype of our case and compare our findings with those published in the literature. The most prevalent clinical features in this type of deletion are a history of prematurity, pre-natal and post-natal growth retardation, slight facial dysmorphic features, microcephaly and developmental delay, with a speech defect in particular. These are clearly different from those found in the classic 22q11.2 deletion syndrome, and we believe that the main differential diagnosis should be with Silver-Russel syndrome. In our case we observe the cardiac phenotype with truncus arteriosus communis usually seen in the classic 22q11.2 deletion syndrome, and so far associated with the TBX1 gene. Significantly, however, TBX1 is not included in our patient’s deletion. The possible roles of a position effect or other genes are discussed.

A child with acute myelogenous leukemia who relapsed three months after an allogeneic bone marrow transplant received intermediate-dose cytarabine followed by interleukin 2 (IL-2). Complete remission was achieved after the first cycle of IL-2. Five more combined cycles of cytarabine and IL-2 were given over the next year, during which remission has persisted. IL-2 therapy affected serum tumor necrosis factor (TNF), interferon gamma (IFNγ) and soluble IL-2 receptor (sIL-2r) levels. In vitro cytotoxicity against leukemia cell lines and recipient leukemia cells was also increased.

Subtelomeric rearrangements are a common cause of idiopathic mental retardation (MR) accounting for 6.3-10.2% of moderate to severe cases and less than 1% of mildly retarded patients. We report on a cohort of 219 patients with idiopathic MR and normal 400-550 band karyotype screened for subtelomeric rearrangements by multiprobe Fluorescence in situ hybridization (FISH) in three Italian Genetics Centers. Twelve positive cases (5.5%) were found. Six were de novo deletions (1p, 7p, 9p, 9q, 20p, 22q) and four unbalanced translocations [a der(6)t(6q; 18p) and a der(18)t(8p; 18q) both de novo, a der(12)t(12p; 17q)mat and a der(2)t(2q; 17q) of unknown origin]. The remaining two cases were apparently balanced reciprocal translocations [a t(4p; 18q) and a t(1p; 16p)] of undetermined origin whose role in the pathogenesis of the clinical phenotype is doubtful. Dysmorphic features were present in all unbalanced patients, whilst a family history of MR was present in only four of them. The proposition that subtelomeric rearrangements are a significant cause of idiopathic MR is supported by our survey. Collection of the clinical data of positive patients will help to delineate the phenotype associated with the various subtelomeric abnormalities, to tailor healthcare services to the needs of these patients and their families and to determine the appropriate use of the test.

PURPOSE: We evaluated the experiences of 19 Italian laboratories concerning 241 small supernumerary marker chromosomes (sSMCs) with the aim of answering questions arising from their origin from any chromosome, their variable size and genetic content, and their impact on the carrier's phenotype. METHODS: Conventional protocols were used to set up the cultures and chromosome preparations. Both commercial and homemade probes were used for the fluorescent in situ hybridization analyses. RESULTS: A total of 113 of the 241 sSMCs were detected antenatally, and 128 were detected postnatally. There were 52 inherited and 172 de novo cases. Abnormal phenotype was present in 137 cases (57%), 38 of which were antenatally diagnosed. A mosaic condition was observed in 87 cases (36%). In terms of morphology, monocentric and dicentric bisatellited marker chromosomes were the most common, followed by monocentric rings and short-arm isochromosomes. The chromosomes generating the sSMCs were acrocentric in 132 cases (69%) and non-acrocentric chromosomes in 60 cases (31%); a neocentromere was hypothesized in three cases involving chromosomes 6, 8, and 15. CONCLUSION: The presented and published data still do not allow any definite conclusions to be drawn concerning karyotype-phenotype correlations. Only concerted efforts to characterize molecularly the sSMCs associated or not with a clinical phenotype can yield results suitable for addressing karyotype-phenotype correlations in support of genetic counseling.

Abstract 4977 Introduction Lenalidomide (Len) is an immunomodulatory drug that has a selective inhibitory effect on the del(5q) clone. The exact mechanism of action has not been defined: Len is known to have multiple biologic activities and an impressive effect on myelodysplasia (MDS) with deletion of the long arm of chromosome 5, leading to transfusion-independence, a complete erythroid and cytogenetic response in over 65% of cases. Similar responses were also seen in patients with MDS and a complex karyotype including del(5q), which usually have a poor prognosis. Lenalidomide leads to an important myelotoxicity in 50% of patients, so the optimal schedule merits discussion. In this report we describe unexpected erythroid and cytogenetic responses in two elderly patients with del(5q) MDS treated with Len for less than 12 wks. Case Reports Since November 2008 we have been treating two female patients with a diagnosis of MDS and a deletion of the long arm of chromosome 5 [del(5q)] with Len. Both patients had a low-int-1 IPSS risk. At the time of diagnosis severe cardio-pulmonary co-pathologies were present, therefore we observed a very bad tolerance to anaemia. The monthly transfusion requirement before starting treatment was 6 packed RBC units. Len was started at a full dose (10 mg/d for 21 days every 4 wks). Patient 1 received a diagnosis of “5q- syndrome” (Fig. 1). She stopped therapy after only 10 days due to severe agitation and panic attacks; however, one month later we observed a progressive reduction of transfusion need. Two months later, the patient was transfusion-free and the response had been ongoing for 6 months; she obtained a very good partial cytogenetic response. After 6 months the anaemia worsened again; we started Len 10 mg/d, but after 15 days the therapy was stopped due to the same side effects. Forty days later, we observed transfusion-independence for another 6 months, and a very good partial cytogenetic response. When the patient's condition worsened again, she was treated with Len 10 mg on alternative days with a better tolerance. After the first cycle, she presented the same side effects. Again the karyotype analysis showed a very good partial cytogenetic response. In Patient 2 the morphologic analysis was compatible with RAEB-1 and the cytogenetic analysis showed a [del(16)(q22)] together with del (5q) (Fig. 2). Len was stopped after 15 days due to renal impairment, cardiopulmonary and cardiac failure; she was then given only palliative therapy, but four wks later she achieved a complete erythroid response and remained transfusion-free for one year. In January 2010 the patient worsened and started Len again (10 mg/d for 21 dd every 4 wks) with good tolerance, a complete hematologic response and a very good partial cytogenetic response. Hematologic responses are illustrated in Tab. 1 and Tab. 2. In these two patients, after a very short treatment with Len we observed a complete erythroid response and a very good partial cytogenetic response. The median time to achieve the response was 6 wks (range 4–8 weeks). Transfusion-independence was durable (24 and 38 weeks respectively). Discussion Giagounidis et al (Ann. Hematol, 2007) describe two similar cases in which short-time Len treatment was given and a good response was obtained. One of our patients experienced grade III hematologic toxicity during the first cycle of therapy; despite this, we did not observe severe infectious or hemorrhagic complications. Despite the very short duration of Len treatment in our two patients, in both cases we achieved a very good partial cytogenetic response. Conclusions We observed unexpected effects of Len in myelodysplastic patients with del(5q) and a low-int1 IPSS risk who were treated for a very short period; additionally, this response was observed again at least twice when treatment was begun again: although these patients obviously represent only selected cases, the good erythroid and cytogenetic responses suggest that some patients with MDS and del(5q) may benefit from Len treatment even if this must be discontinued. Disclosures: No relevant conflicts of interest to declare.