Daniela Bellotti

De novo balanced chromosome rearrangements in prenatal diagnosis

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.

Cytogenetic and molecular evaluation of 241 small supernumerary marker chromosomes: cooperative study of 19 Italian laboratories

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 carriers 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.

MATERNAL SERUM SCREENING AND TRISOMY 16 CONFINED TO THE PLACENTA

Five cases of trisomy 16 confined to the placenta have been detected by invasive procedures (amniocentesis and chorionic villus sampling) after high‐risk results for Down syndrome and neural tube defects in a maternal serum screening programme of 6614 consecutive cases. All five pregnancies displayed unusually elevated levels of human chorionic gonadotropin and four out of five also had raised alpha‐fetoprotein values. No structural malformation was present but all five pregnancies were complicated by fetal growth retardation, and one by intrauterine death. From our results, we suggest that both amniocentesis and chorionic villus sampling should be considered in the management of cases with high mid‐trimester levels of these analytes.

‘Dual Positivity’ for Neural Tube Defects and Down Syndrome at Maternal Serum Screening: Gestational Outcome

Objective: To evaluate the gestational outcome of pregnancies screen-positive for both neural tube defects (NTD) and Down syndrome (DS) (‘dual positivity’). Methods: Among 10,667 mid-trimester women screened for DS and NTD with α-fetoprotein (AFP), unconjugated estriol (uE3), and human chorionic gonadotropin (hCG), delivered up to July 1996, we have selected cases with both an unexplained AFP value ≥2.5 multiples of median (MoM) and a DS risk ≥1:250. All these pregnant women were managed with amniocentesis and/or CVS, ultrasound scans, and Doppler velocimetry. We have collected all data about the gestations with ‘dual positivity’ and no obvious explanation for these findings (cases with fetal malformations related to raised AFP). Results: Twelve women (1.1:1,000) showed unexplained ‘dual positivity’. Abnormal karyotypes were found in 3 fetuses, and pregnancies were terminated: there were 2 triploidies with partial hydatiform mola, and 1 DS. In 9 cases the fetal karyotype was normal, but a confined placental trisomy 16 was found in 4. Of the 9 continuing gestations, 8 displayed fetal growth retardation (FGR). One gestation ended with fetal death at 27 weeks. All 9 fetuses were morphologically normal, and 8 were small for gestational age. Conclusions: ‘Dual positivity’ at NTD/DS screening may anticipate pregnancy complications. The finding of trisomy 16 confined to the placenta and FGR in 4 cases suggests that at least some fetuses with growth restriction may suffer from a distinct placental disease. Maternal serum screening may have implications different from DS and NTD, as demonstrated by the 2 cases with triploidy and incomplete hydatiform mola, the 4 cases with placental trisomy 16, and the 4 cases of FGR of the 5 fetuses without chromosome abnormalities. As the pathologic outcome of these pregnancies is more important than the mere serum screening results, we feel that these cases need a strict work-up, including CVS, amniocentesis and ultrasound studies to better address the obstetrical management.

BDNF Val66Met polymorphism and protein levels in Amniotic Fluid

BackgroundBrain-Derived Neurotrophic Factor (BDNF) is a neurotrophin which plays survival- and growth-promoting activity in neuronal cells and it is involved in cellular plasticity mechanisms as it controls activity dependent synaptic transmission. A functional polymorphism (Val66Met) in the pro-region of BDNF, which affects the intracellular trafficking of proBDNF has been associated with memory and cognitive deficits as well as to an increased susceptibility for several psychiatric disorders especially those with a neurodevelopmental origin. To date, no study has evaluated the influence of the Val66Met polymorphism on BDNF levels in a peripheral system that may reflect fetal neurodevelopment. Therefore we investigated in amniotic fluids (AF) obtained from 139 healthy women during 15-17 week of pregnancy, BDNF protein levels in correlation with the Val66Met polymorphism.ResultsInterestingly we found a significant BDNF protein levels reduction in 55 Met carriers (Val/Met and Met/Met) (p = 0.002) as compared to 84 non carriers (Val/Val), and no effect of fetus gender, maternal age or gestation week on BDNF levels has been observed.ConclusionThese results, although explorative, indicate that during fetal life the Val66Met genotype might influences BDNF protein levels in AF supporting the involvement of this polymorphism in behavioral and functional brain individual differences in the adulthood.

Dissection of partial 21q monosomy in different phenotypes: clinical and molecular characterization of five cases and review of the literature

BackgroundPartial deletion of chromosome 21q is a very rare chromosomal abnormality associated with highly variable phenotypes, such as facial dysmorphic features, heart defects, seizures, psychomotor delay, and severe to mild intellectual disability, depending on the location and size of deletions. So far, three broad deletion regions of 21q have been correlated with the clinical phenotype.ResultsWe described the clinical and genetic features of three family members (father and two siblings) and other two unrelated patients with very wide range in age of diagnosis. All of them showed intellectual disability with very variable symptoms, from mild to severe, and carried 21q interstitial deletions with different sizes and position, as detected by conventional karyotype and array-CGH.ConclusionsOur study provided additional cases of partial 21q deletions, allowing to better delineate the genotype-phenotype correlations. In contrast to previous observations, we showed that deletions of the 21q proximal region are not necessarily associated with severe phenotypes and, therefore, that mild phenotypes are not exclusively related to distal deletions. To the best of our knowledge, this is the first report showing 21q deletions in adult patients associated with mild phenotypes, mainly consisting of neurobehavioral abnormalities, such as obsessive-compulsive disorders, poor social interactions and vulnerability to psychosis.

Involvement of a member of the histone cluster 1 at 6p21 in NUP98-positive MDS/AML

HIST1H1T, one of the eleven members of the histone H1 gene family (H1A, H1B, H1C, H1D, H1E, H1T, H1F0, H1FNT, H1FOO, HILS1 and H1FX), is considered a germinal variant as it is specifically expressed in early meiotic spermatocytes until late spermatids in mammals [1]. Information on the involvement of this family in malignancy are still scarce. Members of the histone cluster 1 have never been reported in myeloid malignancies, so far, though HIST1H1B, C, D and E mutations were observed in chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) [2]. A deregulation of HIST1H1T was found in carcinomas of prostate, head and neck, bladder and endometrium [2]. In addition, a pathogenetic role has been attributed to the rs2051542 HIST1H1T polymorphism because of its association with lung carcinoma [3]. We here provide the first evidence of HIST1H1T gene involvement in human myeloid leukemia as a new partner of the promiscuous NUP98 gene. In 2012, a 38-year-old Jamaican woman with adult T-cell leukaemia lymphoma (ATLL) HTLVþ was treated with combination chemotherapy (VCAP-AMO-VECP, six cycles), obtaining complete remission. Maintenance therapy was a-interferon (3MU) and zidovudine (600mg/day) for 10 months. Three years later, in April 2015, the patient developed secondary myelodysplastic syndrome (MDS), with trilinear cytopenia and macrocytosis (WBC 2.67 10/l, PMN 18%, Hb 93 g/l, PLT 80 10/l, MCV 121 fl). Bone marrow aspirate showed RAEB1 with trilinear dysplasia and 7% blasts. Karyotype was 46,XX,t(6;11)(p21;p15)[10]/46,XX[12] (Figure 1(A)). No therapy was administered. In August 2015, patient developed frank AML with 48% bone marrow blasts CD34 , CD117, CD33þ, CD13þ, CD14 , CD16 and HLA-DR. Karyotypic evolution was observed: 47,XX,t(6;11)(p21;p15),þ8[13]/ 46,XX,t(6;11)(p21;p15) [7]. Mutational analysis (primers in Supplementary Table 2) showed FLT3 mutation (D835H) at the AML diagnosis, but not at the MDS phase. No other mutations were found in ASXL1, RUNX1, SETBP1, SRSF2, TET2, EZH2, DNMT3A, IDH1-2, SF3B1, JAK2, HRAS, WT1, KIT, NRAS and KRAS genes. Induction therapy used idarubicin 12mg/m/day, 3 days, cytosine arabinoside 100mg/m/dayX2, 7 days, VP16 100mg/m/day, 5 days. Consolidation therapy was idarubicin (10mg/m/day, 2 days), cytosine arabinoside (100mg/m/day 2, 7 days) followed by cytarabine (1 g/m/day 2, 4 days). Complete remission was obtained and in February 2016 the patient underwent successful syngeneic peripheral blood stemcell transplantation from her HLA-identical twin after conditioning with busulphan and fludarabine. To characterize the t(6;11)(p21;p15) chromosomal translocation, FISH was performed on bone marrow cells. At least 200 interphase nuclei and/or seven abnormal metaphases were analyzed in each experiment. A breakapart FISH assay with RP11-348A20 (NUP98 exons 1–27) and CTD-3234F16 (NUP98 exons 13–32) indicated the 11p15 breakpoint fell within NUP98 exons 1–13 in 75% of cells at AML diagnosis, with RP11-348A20 showing three hybridization signals: on normal 11, on derivative 11 and on derivative 6. Homebrew BAC and PAC clones (Supplementary Table 1) investigated the 6p breakpoint, which was identified within clone RP1-221C16 at 6p22.2. Figure 1(B) shows the reciprocal translocation in a double fusion experiment. RP1-221C16 clone encompasses the 30-end of the hemochromatosis gene (HFE) and H4C, H1T, H2BC, H2AC, H1E, H2BD, H2BE and H4D genes, members of histone gene cluster 1, on chromosome 6p21. The hybridization pattern indicated that the breakpoint localized to the most telomeric RP1-221C16 region, where two putative partner genes, HIST1H1T and HIST1H2BC, were mapped with appropriate centromere–telomere orientation. Total RNA was extracted by Trizol reagent

Lenalidomide in patients with red blood cell transfusion-dependent myelodysplastic syndrome and del(5q): a single-centre “real-world” experience

“Real life” data are needed to complement published trials on the efficacy of lenalidomide in patients with myelodysplastic syndrome (MDS) and del(5q) and on the risk of inducing acute myeloid leukemia (AML) progression. Here, we present results of lenalidomide treatment in a consecutive, population-based series of 21 red blood cell (RBC) transfusion-dependent elderly patients with multiple comorbidities. Of 18 evaluable patients (median follow-up: 22 months), 17 achieved an erythroid hematologic response (HI-E) and 16 an RBC transfusion independence. Cytogenetic response (CyR) rate was 80%, median overall survival was 48 months (range 3–164), and 5-year leukemia-free survival was 84%. Three patients progressed to AML; one, with baseline TP53 mutation, achieved HI-E, partial CyR, and did not progress to AML. Eighteen patients experienced hematological adverse events. Overall, lenalidomide was very effective and well tolerated even in unselected elderly patients with multiple comorbidities and did not appear to increase the risk of AML.