Genetic risk of prediabetes and diabetes development in chronic myeloid leukemia patients treated with nilotinib.

Impaired fasting glucose and type 2 diabetes represent adverse events in patients with chronic myeloid leukemia (CML) treated with the second generation tyrosine kinase inhibitor nilotinib. An unweighted genetic risk score (uGRS) for the prediction of insulin resistance, consisting of 10 multiple single-nucleotide polymorphisms, has been proposed. We evaluated uGRS predictivity in 61 CML patients treated with nilotinib. Patients were genotyped for IRS1, GRB14, ARL15, PPARG, PEPD, ANKRD55/MAP3K1, PDGFC, LYPLAL1, RSPO3, and FAM13A1 genes. The uGRS was based on the sum of the risk alleles within the set of selected single-nucleotide polymorphisms. Molecular response (MR)3.0 and MR4.0 were achieved in 90% and 79% of patients, respectively. Before treatment, none of the patients had abnormal blood glucose. During treatment and subsequent follow-up at 80.2 months (range: 1-298), seven patients (11.5%) had developed diabetes that required oral treatment, a median of 14 months (range: 3-98) after starting nilotinib treatment. Twelve patients (19.7%) had developed prediabetes. Prediabetes/diabetes-free survival was significantly higher in patients with a uGRS <10 than in those with higher scores (100% vs. 22.8 ± 12.4%, p <0.001). Each increment of one unit in the uGRS caused a 42% increase in the prediabetes/diabetes risk (hazard ratio = 1.42, confidence interval: 1.04-1.94, p = 0.026). The presence of more than 10 allelic variants associated with insulin secretion, processing, sensitivity, and clearance is predictive of prediabetes/diabetes development in CML patients treated with nilotinib. In clinical practice, uGRS could help tailor the best tyrosine kinase inhibitor therapy.

NFIX mutations affecting the DNA-binding domain cause a peculiar overgrowth syndrome (Malan syndrome): a new patients series.

The Nuclear Factor I-X (NFIX) is a member of the nuclear factor I (NFI) protein family and is deleted or mutated in a subset of patients with a peculiar overgrowth condition resembling Sotos Syndrome as well as in patients with Marshall-Smith syndrome. We identified three additional patients with this phenotype each carrying a different new mutation affecting the DNA-binding/dimerization domain of the NFIX protein. The present report further adds weight to the hypothesis that mutations in DNA-binding/dimerization domain are likely to cause haploinsufficiency of the NFIX protein and confirms that NFIX is the second gene that should be tested in individuals with overgrowth conditions resembling Sotos syndrome, previously tested negative for NSD1 mutations. We then propose to consider this overgrowth syndrome (namely Malan syndrome) and Marshall-Smith syndrome NFIX-related diseases.

Update on epidemiology of HCV in Italy: focus on the Calabria Region.

The epidemiological profile of HCV infection is evolving in Europe, as well as in Italy. We have previously showed genotype distributions and their dynamics in 2,153 HCV RNA positive patients living in Calabria, Southern Italy, over 11 years. In this study, we extend and update this information by evaluating a hospital-based cohort of 945 HCV RNA positive patients attending five hospitals in the Calabria Region from January 2011 to August 2013. We assessed rates of HCV genotypes according to age and gender and the dynamics of HCV genotype distribution over the 3-year period studied. Data showed that genotype 1b is the most prevalent, followed by subtypes 2a/2c and genotype 3. Genotype 4 exhibited an increase between 2011 and 2013. Also, we found a significant decrease in the median age of subjects infected with HCV genotype 3 and 4 during the period studied. Since HCV genotypes are important in epidemiology, pathogenesis and response to antiviral therapy, a continuous epidemiological surveillance is needed.

Lenalidomide in International Prognostic Scoring System Low and Intermediate-1 risk myelodysplastic syndromes with del(5q): an Italian phase II trial of health-related quality of life, safety and efficacy.

In lower-risk myelodysplastic syndromes (MDS) with del(5q), lenalidomide induces erythroid responses associated with better survival. In a phase II, single-arm trial, 45 patients with anemia and lower-risk del(5q) MDS received lenalidomide 10 mg/day to evaluate quality of life (QoL) changes, measured by QOL-E, safety, responses and survival. Lenalidomide was well tolerated, with 80% completing ≥ 24 weeks of treatment. Earlier study discontinuation was related to disease progression (n = 5), death (n = 1) and withdrawal of consent (n = 3). Within 24 weeks, 82% obtained erythroid responses, durable in 69% at 52 weeks. Cytogenetic responses occurred in 29 patients (64%), with 10 patients achieving a complete cytogenetic response. QoL-E scores correlated with hemoglobin levels and improved in erythroid responders. Erythroid responders had an 86% reduced risk of disease progression and an 80% reduction in mortality risk compared with non-responders. These findings corroborate earlier studies and give further support to the use of lenalidomide in lower-risk MDS and del(5q).

Biological activity of lenalidomide in myelodysplastic syndromes with del5q: results of gene expression profiling from a multicenter phase II study.

In vitro studies suggest that haploinsufficiency is involved in the pathogenesis of myelodysplastic syndromes (MDS). In patients with del5q cytogenetic abnormality, RPS-14 and microRNAs (miRNAs) play a major role. In a multicenter phase II single-arm trial with lenalidomide in anemic primary del5q MDS patients with low- or int-1 risk IPSS, biological changes from baseline were investigated. Gene expression profiling of selected genes was performed (TaqMan® Low Density Array Fluidic card, Applied Biosystems PRISM® 7900HT) and normalized against the expression of the 18S housekeeping gene from a pool of healthy subjects. Thirty-two patients were evaluated at baseline and after 3 and 6 months of treatment. RPS-14, miR-145, and miR-146 were downregulated at baseline and significantly increased during treatment. Nuclear factor kappa B, IL-6, interferon regulatory factor-1, IFNγ-R2, IL-2, and many genes in the apoptotic pathways (TNF, IL-1B, and IL-10) were upregulated at baseline and significantly downregulated during lenalidomide treatment, while forkhead box P3, FAS, IFNγ, IL-12A, and IL-12B were downregulated at baseline and progressively upregulated during treatment. The crucial role of aberrant immunological pathways and haploinsufficiency in the pathogenesis of del5q MDS is confirmed in the present patient setting. Our results indicate that lenalidomide may act through defined immunological pathways in this condition.

A peculiar mutation in the DNA-binding/dimerization domain of NFIX causes Sotos-like overgrowth syndrome: a new case.

The Nuclear Factor I-X (NFIX) is a member of the nuclear factor I (NFI) family proteins, which are implicated as site-specific DNA-binding proteins and is deleted or mutated in a subset of patients with Sotos-like overgrowth syndrome and in patients with Marshall-Smith syndrome. We evaluated an additional patient with clinical features of Sotos-like syndrome by sequencing analysis of the NFIX gene and identified a 21 nucleotide in frame deletion predicting loss of 7 amino acids in the DNA-binding/dimerization domain of the NFIX protein. The deleted residues are all evolutionally conserved amino acids. The present report further confirms that mutations in DNA-binding/dimerization domain cause haploinsufficiency of the NFIX protein and strongly suggests that in individuals with Sotos-like features unrelated to NSD1 changes genetic testing of NFIX should be considered.

Absence of deletion and duplication of MLL2 and KDM6A genes in a large cohort of patients with Kabuki syndrome.

Kabuki syndrome is a rare, multiple congenital anomaly/mental retardation syndrome caused by MLL2 point mutations and KDM6A microdeletions. We screened a large cohort of MLL2 mutation-negative patients for MLL2 and KDM6A exon(s) microdeletion and microduplication. Our assays failed to detect such rearrangements in MLL2 as well as in KDM6A gene. These results show that these genomic events are extremely rare in the Kabuki syndrome, substantiating its genetic heterogeneity and the search for additional causative gene(s).

Changes in RPS14 expression levels during lenalidomide treatment in Low- and Intermediate-1-risk myelodysplastic syndromes with chromosome 5q deletion.

BACKGROUND: Haploinsufficiency of the ribosomal protein S14 RPS14 gene, located in the common deleted region of chromosome 5q, is a potential causal factor of 5q- syndrome. Lenalidomide elicits high response rates and morphological improvements in myelodysplastic syndrome (MDS) patients with chromosome 5q deletion [del(5q)]. METHODS: To further evaluate the role of RPS14, its transcription was tested in bone marrow cells from 17 patients with International Prognostic Scoring System defined Low- or Intermediate-1-risk MDS with del(5q) as a single or additional cytogenetic abnormality receiving treatment with lenalidomide. RESULTS: After 12 wk of lenalidomide treatment, erythroid responses were observed in all cases with an increase in hemoglobin levels of 2.7 +/- 2.5 g/dL (up to a mean 11.8 +/- 1.9 g/dL; P = 0.001). Before treatment, RPS14 expression levels were under-expressed in 15 patients with respect to normal controls. After 12 wk of lenalidomide treatment, all patients had an erythroid response. There was a significant increase in median RPS14 expression from baseline 0.01 (IQR 0.05-0.31) to 12 wk 204.71-fold (2.86-446.32; P < 0.0001). CONCLUSIONS: These observations in the patient setting support the importance of RPS14 in the pathogenesis of MDS with del(5q).

Prevalence of prothrombotic polymorphisms in a selected cohort of cryptogenic and noncryptogenic ischemic stroke patients.

Ischemic stroke is a complex multifactorial disease and approximately 30%, especially in the young, are cryptogenic. In some of the patients with cryptogenic ischemic stroke the underlying risk factor may be a prothrombotic state. We studied 101 patients with ischemic stroke under 55 years of age. All the patients underwent an extensive diagnostic evaluation to determine the cause of stroke. Common variations in the genes encoding factor V, prothrombin, 5,10-methylenetetrahydrofolate reductase, plasminogen activator inhibitor-1, and human platelet alloantigens-1 were evaluated. Of the 101 patients with ischemic stroke, 28 patients had cryptogenic ischemic stroke. At least one of the different genetic polymorphisms investigated was present in 44% patients in the total group and in 48% of patients with cryptogenic ischemic stroke. In this study population under 55 years of age there was no significant difference in the prevalence of various genetic polymorphisms, factor V, prothrombin, 5,10-methylenetetrahydrofolate reductase, plasminogen activator inhibitor-1, and human platelet alloantigens) in patients with cryptogenic ischemic stroke and in patients with ischemic stroke of determined cause.

The incidence of JAK2 V617F mutation in bcr/abl-negative chronic myeloproliferative disorders: assessment by two different detection methods.

A recurrent specific JAK2 V617F mutation has been reported in bcr/abl-negative chronic myeloproliferative diseases (cMPD), including polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). The mutation is detectable in a variable proportion of neoplastic clones, depending on the molecular methods employed. In this study, we attempted to establish the JAK2 V617F mutation frequency in two partially overlapping cMPD patient series by two different PCR-based techniques. Using an allele-specific polymerase chain reaction assay (AS-PCR), the JAK2 V617F mutation was detected in 124/158 (78.5%) cMPD patients; in particular, 90.2, 72.1, 63.2 and 50% of PV, ET, IMF and unclassified (U)-MPD cases, respectively, showed the mutation. Employing a semiquantitative 5' fluorogenic TaqMan assay, the JAK2 V617F mutation was identified in a much larger percentage of cMPDs patients (118/127: 92.9%). Rates of mutation in PV, ET, IMF and U-MPD cases were 95.9, 85.7, 91.7 and 92.9%, respectively. Furthermore, a statistically higher percentage of JAK2 mutated alleles was detected by TaqMan assay in PV (68+/-3.5, mean value+/-SEM) and IMF (64+/-9.3) cases as compared with ET (35+/-5.4). Finally, a significant correlation between JAK2 V617F mutational status and hematocrit (Ht), white blood cell and platelet counts in PV patients, and Ht values in ET cases, was observed by AS-PCR. Overall, these data indicate that TaqMan technology significantly improved sensitivity in detecting the JAK2 mutation in cMPD patients and may be worth of further evaluations as a clinically useful tool for detection of small amounts of mutated clones.

MS-MLPA is a specific and sensitive technique for detecting all chromosome 11p15.5 imprinting defects of BWS and SRS in a single-tube experiment.

Human chromosome 11p15.5 harbours a large cluster of imprinted genes. Different epigenetic defects at this locus have been associated with both Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS). Multiple techniques (Southern blotting, COBRA and microsatellite analysis) have been used so far to detect various DNA methylation abnormalities, uniparental disomies and copy number variations, which are characteristics of these two diseases. We have now evaluated a methylation-specific multiplex-ligation-dependent probe amplification assay (MS-MLPA) for the molecular diagnosis of BWS and SRS. Seventy-three samples derived from BWS- and SRS-affected individuals and 20 controls were analysed by conventional tests and MS-MLPA in blind. All cases that were found positive with conventional methods were also identified by MS-MLPA. These included cases with paternal UPD11, hyper- or hypo-methylation at the Imprinting Centre 1 or Imprinting Centre 2 and rare 11p15.5 duplications. In summary, this MS-MLPA assay can detect both copy number variations and methylation defects of the 11p15.5 critical region within one single experiment and represents an easy, low cost and reliable system for the molecular diagnostics of BWS and SRS.

Utility of the clinical practice of administering thrombophilic screening and antithrombotic prophylaxis with low-molecular-weight heparin to healthy donors treated with G-CSF for mobilization of peripheral blood stem cells.

The aim of the study was to verify the utility of the clinical practice of administering thrombophilic screening and antithrombotic prophylaxis with low-molecular-weight heparin to healthy donors receiving granulocyte colony-stimulating factor to mobilize peripheral blood stem cells. Thrombophilia screening comprised of testing for factor V Leiden G1691A, prothrombin G20210A, the thermolabile variant (C677T) of the methylene tetrahydrofolate reductase gene, protein C, protein S, factor VIII and homocysteine plasmatic levels, antithrombin III activity, and acquired activated protein C resistance. We investigated prospectively 72 white Italian healthy donors, 39 men and 33 women, with a median age of 42 years (range, 18-65). Five donors (6.9%) were heterozygous carriers of Factor V Leiden G1691A; two healthy donors had the heterozygous prothrombin G20210A gene mutation; C677T mutation in the methylene tetrahydrofolate reductase gene was present in 34 (47.2%) donors in heterozygous and in 7 donors (9.7%) in homozygous. Acquired activated protein C resistance was revealed in 8 donors of the study (11.1%). The protein C plasmatic level was decreased in 3 donors (4.2%); the protein S level was decreased in 7 donors (9.7%). An elevated factor VIII dosage was shown in 10 donors (13.9%) and hyperhomocysteinemia in 9 donors (12.5%). Concentration of antithrombin III was in the normal range for all study group donors. The factor V Leiden mutation was combined with the heterozygous prothrombin G20210A in 2 cases and with protein S deficiency in one case; 2 healthy donors presented an associated deficiency of protein C and protein S. Although none of these healthy subjects had a previous history of thrombosis, low-molecular-weight heparin was administered to all donors during granulocyte colony-stimulating factor administration to prevent thrombotic events. No donor experienced short or long-term thrombotic diseases after a median follow-up of 29.2 months. Our data do not support this clinical practice because there is no evidence that the combination of granulocyte colony-stimulating factor to previous hypercoagulable conditions results in thrombotic events.

Malpuech syndrome: broadening the clinical spectrum and molecular analysis by array-CGH.

We report on a patient with mental and growth retardation, bilateral cleft lip and palate, hypertelorism, ptosis, hearing loss and mild epispadias, suggestive of Malpuech syndrome. High-resolution karyotype and microarray-CGH using an oligonucleotide array with 75Kb oligo's were normal, excluding Wolf-Hirschhorn syndrome. Long-term follow-up revealed psychiatric manifestations starting at young age.

Reciprocal translocations: a trap for cytogenetists?

We report four cases of subjects with phenotypic abnormalities and mental retardation associated with apparently balanced translocations, two inherited and two de novo, which showed, by molecular analysis, a hidden complexity. All the cases have been analyzed with different molecular techniques, including array-CGH, and in two of them the translocation breakpoints have been defined at the level of base pairs via studies in somatic hybrids containing single derivative chromosomes. We demonstrated that all the translocations were in fact complex rearrangements and that an imbalance was present in three of them, thus accounting for the phenotypic abnormalities. In one case, a Prader-Willi subject, we were not able to determine the molecular cause of his phenotype. This study, while confirming previous data showing unexpected complexity in translocations, further underscores the need for molecular investigations before taking for granted an apparently simple cytogenetic interpretation.

Pulmonary agenesis/hypoplasia, microphthalmia, and diaphragmatic defects: report of an additional case.

Diaphragmatic defects with associated pulmonary hypoplasia/agenesis and anophthalmia/microphthalmia have been reported as part of a complex syndromic phenotype. We report a case with the combination of these malformations.

Identification of 14 rare marker chromosomes and derivatives by spectral karyotyping in prenatal and postnatal diagnosis.

Extra structurally abnormal chromosomes (ESACs) and cryptic rearrangements are often associated with mental retardation and phenotypic abnormalities. In some cases their characterisation, using standard cytogenetic techniques and fluorescence in situ hybridization (FISH), is difficult and time consuming, where a fast and accurate identification is essential, especially when such chromosomal aberrations are found in prenatal diagnosis. A recent molecular technique, spectral karyotyping (SKY), based on the spectral signature of 24 chromosome-specific painting probes labelled with different combinations of five fluorochromes, allows the simultaneous visualisation of all human chromosomes in different colours. We used SKY analysis on 14 cases with rare ESACs or cryptic unbalanced rearrangements found at pre- or postnatal diagnosis. SKY analysis permitted the classification of chromosome rearrangements in all 14 cases analysed in combination with FISH analysis.