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Lysine-specific methyltransferase 2A (KMT2A) is responsible for methylation of histone H3 (K4H3me) and contributes to chromatin remodeling, acting as “writer” of the epigenetic machinery. Mutations in KMT2A were first reported in Wiedemann–Steiner syndrome (WDSTS). More recently, KMT2A variants have been described in probands with a specific clinical diagnosis comprised in the so-called chromatinopathies. Such conditions, including WDSTS, are a group of overlapping disorders caused by mutations in genes coding for the epigenetic machinery. Among them, Rubinstein–Taybi syndrome (RSTS) is mainly caused by heterozygous pathogenic variants in CREBBP or EP300. In this work, we used next generation sequencing (either by custom-made panel or by whole exome) to identify alternative causative genes in individuals with a RSTS-like phenotype negative to CREBBP and EP300 mutational screening. In six patients we identified different novel unreported variants in KMT2A gene. The identified variants are de novo in at least four out of six tested individuals and all of them display some typical RSTS phenotypic features but also WDSTS specific signs. This study reinforces the concept that germline variants affecting the epigenetic machinery lead to a shared molecular effect (alteration of the chromatin state) determining superimposable clinical conditions.

Rubinstein-Taybi syndrome (RSTS) is a rare multisystem developmental disorder with moderate to severe intellectual disability caused by heterozygous mutations of eitherCREBBPorEP300genes encoding CBP/p300 chromatin regulators. We explored the gene programs and processes underlying the morphological and functional alterations shown by iPSC-derived neurons modeling RSTS to bridge the molecular changes resulting from defective CBP/p300 to cognitive impairment. By global transcriptome analysis, we compared the differentially expressed genes (DEGs) marking the transition from iPSC-derived neural progenitors to cortical neurons (iNeurons) of five RSTS patients carrying privateCREBBP/EP300mutations and manifesting differently graded neurocognitive signs with those of four healthy controls. Our data shows a defective and altered neuroprogenitor to neuron transcriptional program in the cells from RSTS patients. First, transcriptional regulation is weaker in RSTS as less genes than in controls are modulated, including genes of key processes of mature functional neurons, such as those for voltage-gated channels and neurotransmitters and their receptors. Second, regulation is subverted as genes acting at pre-terminal stages of neural differentiation in cell polarity and adhesive functions (members of the cadherin family) and axon extension/guidance (members of the semaphorins and SLIT receptors families) are improperly upregulated. Impairment or delay of RSTS neuronal differentiation program is also evidenced by decreased modulation of the overall number of neural differentiation markers, significantly impacting the initial and final stages of the differentiation cascade. Last, extensive downregulation of genes for RNA/DNA metabolic processes confirms that RSTS is a global transcription disorder, consistent with a syndrome driven by chromatin dysregulation. Interestingly, the morphological and functional alterations we have previously appointed as biomarkers of RSTS iNeurons provide functional support to the herein designed transcriptome profile pointing to key dysregulated neuronal genes as main contributors to patients' cognitive deficit. The impact of RSTS transcriptome may go beyond RSTS as comparison of dysregulated genes across modeled neurodevelopmental disorders could unveil convergent genes of cognitive impairment.

Biallelic mutations in RECQL4 gene, a caretaker of the genome, cause Rothmund-Thomson type-II syndrome (RTS-II) and confer increased cancer risk if they damage the helicase domain. We describe five families exemplifying clinical and allelic heterogeneity of RTS-II, and report the effect of pathogenic RECQL4 variants by in silico predictions and transcripts analyses. Complete phenotype of patients #39 and #42 whose affected siblings developed osteosarcoma correlates with their c.[1048_1049del], c.[1878+32_1878+55del] and c.[1568G>C; 1573delT], c.[3021_3022del] variants which damage the helicase domain. Literature survey highlights enrichment of these variants affecting the helicase domain in patients with cancer outcome raising the issue of strict oncological surveillance. Conversely, patients #29 and #19 have a mild phenotype and carry, respectively, the unreported homozygous c. 3265G>T and c. 3054A>G variants, both sparing the helicase domain. Finally, despite matching several criteria for RTS clinical diagnosis, patient #38 is heterozygous for c. 2412_2414del; no pathogenic CNVs out of those evidenced by high-resolution CGH-array, emerged as contributors to her phenotype.

Item does not contain fulltext Glioblastomas, the most frequent and malignant glial tumors, are known to be phenotypically heterogeneous. A low fraction of glioblastomas is associated with specific chromosomal losses at 1p and 19q, which are commonly found in oligodendrogliomas and are generally considered to be a primary event in the development of these tumors. Subsequent progression of oligodendroglial tumors appears to be triggered by additional molecular features underlying the transition to anaplastic oligodendroglioma and glioblastoma multiforme (GBM) such as deletions of 9p and 10q, and alterations of CDKN2A (p16), which is located at 9p21. These findings strengthen the view that GBM on rare occasions may develop from oligodendroglial differentiated cells. In the present study, we evaluated the newly established MI-4 glioblastoma cell line, which displays 1p and 19q specific alterations targeting preferential regions of allelic loss in glial neoplasms, by array-CGH and fluorescence in situ hybridization (FISH) analyses that were combined to obtain a high resolution map of targeted chromosome rearrangements and copy number changes throughout the genome. Genome-wide and chromosome 19 full coverage array-CGH analysis of the MI-4 cell line revealed that in this particular cell line, 1p-specific loss, including the CDKN2 (p18) gene, is not accompanied by loss of the previously described 19q13.3 tumor suppressor candidate region. Interestingly, the array-CGH (CGHa) profile showed an increase in copy number along most of 19q including the AKT2 oncogene and the KLKs gene family, which have previously been shown to be amplified in pancreatic carcinomas and upregulated in several tumors, respectively. The concomitant 1p partial loss and chromosome 19 alterations, with the +7 and -10-specific GBM markers associated with homozygous deletion of 9p21.3 including CDKN2A (p16), are distinct features of the glioblastoma MI-4 cell line, illustrating its origin from an olidodendroglial tumor. Based on these results, we conclude that the MI-4 glioblastoma cell line might function as a model system for investigations into the behavior of a defined oligodendroglioma subtype.

La sindrome di Rothmund-Thomson (RTS; MIM#268400) è una rara genodermatosi a trasmissione autosomico recessiva, caratterizzata da ampia eterogeneità clinica e genetica (Larizza et al., 2010). Mutazioni bialleliche nel gene RECQL4 (MIM*603780) codificante per una elicasi della famiglia RecQ, sono presenti in 2/3 dei pazienti, e definiscono il sottotipo RTS-II in cui il segno cardine cutaneo del poichiloderma (teleangectasia, ipo- ed iper-pigmentazione, atrofia) ad insorgenza precoce su volto e arti è associato a bassa statura, alterazioni scheletriche, e aumentato rischio di sviluppare tumori, quali l’osteosarcoma nell’infanzia (età media di insorgenza 14 anni) e tumori cutanei in età adulta. E’ stata indicata una correlazione tra mutazioni troncanti che affliggono il dominio elicasico di RECQL4 (aminoacidi 489-662 e 683-850) e la predisposizione al cancro. Riportiamo l’analisi clinica e molecolare di 3 nuovi pazienti: RTS-6f, RTS-6m e RTS-29.

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%).

The parent-of-origin-dependent expression of IGF2 and H19 is controlled by the imprinting center 1 (IC1) consisting of a methylation-sensitive chromatin insulator. IC1 is normally methylated on the paternal chromosome and nonmethylated on the maternal chromosome. We found that 22 cases in a large cohort of patients affected by Beckwith-Wiedemann syndrome (BWS) had IC1 methylated on both parental chromosomes, resulting in biallelic activation of IGF2 and biallelic silencing of H19. These individuals had marked macrosomia and high incidence of Wilms' tumor. A subset of these patients had 1.4- to 1.8-kb deletions with hypermethylation of the remaining IC1 region and fully penetrant BWS phenotype when transmitted maternally. Another subset of individuals with IC1 hypermethylation had a similar clinical phenotype but no mutation in the local vicinity. All these cases were sporadic and in at least two families affected and unaffected members shared the same maternal IC1 allele but not the abnormal maternal epigenotype. Similarly, no IC1 deletion was detected in 10 nonsyndromic Wilms' tumors with IC1 hypermethylation. In conclusion, methylation defects at the IGF2-H19 locus can result from inherited mutations of the imprinting center and have high recurrence risk or arise independently from the sequence context and not transmitted to the progeny. Copyright © 2009 S. Karger AG, Basel.

INTRODUCTION: Defects of prokineticin pathway affect the neuroendocrine control of reproduction, but their role in the pathogenesis of central hypogonadism remains undefined, and the functional impact of the missense PROKR2 variants has been incompletely characterized. MATERIAL AND METHODS: In a series of 246 idiopathic central hypogonadism patients, we found three novel (p.V158I, p.V334M, and p.N15TfsX30) and six already known (p.L173R, p.T260M, p.R268C, p.V274D, p.V331M, and p.H20MfsX23) germline variants in the PROKR2 gene. We evaluated the effects of seven missense alterations on two different prokineticin receptor 2 (PROKR2)-dependent pathways: inositol phosphate-Ca(2+) (Gq coupling) and cAMP (Gs coupling). RESULTS: PROKR2 variants were found in 16 patients (6.5%). Expression levels of variants p.V158I and p.V331M were moderately reduced, whereas they were markedly impaired in the remaining cases, except p.V334M, which was significantly overexpressed. The variants p.T260M, p.R268C, and p.V331M showed no remarkable changes in cAMP response (EC50) whereas the IP signaling appeared more profoundly affected. In contrast, cAMP accumulation cannot be stimulated through the p.L173R and p.V274D, but IP EC50 was similar to wt inp.L173R and increased by 10-fold in p.V274D. The variant p.V334M led to a 3-fold increase of EC50 for both cAMP and IP. CONCLUSION: Our study shows that single PROKR2 missense allelic variants can either affect both signaling pathways differently or selectively. Thus, the integrity of both PROKR2-dependent cAMP and IP signals should be evaluated for a complete functional testing of novel identified allelic variants.

Aim of this study is to investigate the capability of Imatinib to induce an anti-leukemic effect in Core Binding Factor (CBF)-leukemia patients presenting either with extracellular juxtamembrane or kinase KIT mutations. On the basis of a screening analysis for KIT mutations, two patients with a kinase mutation and one with extracellular juxtamembrane mutation, in first or subsequent leukemic relapse, received 400 mg Imatinib twice daily for 30 days. After Imatinib discontinuation, bone marrow cells were re-tested to assess the KIT mutational status and the chromosomal set. In our experience, none of the treated patients had a response by standard criteria; in particular, we did not observe any activity against acute myeloid leukemia (AML) associated with KIT kinase mutations. However, in the patient with extracellular juxtamembrane mutation, Imatinib seems to have some clinical beneficial effect and, most important, is able to abrogate the leukemic subclone carrying the mutation. Whether Imatinib, in combination with other agents, may play a role in the treatment of AML with more sensitive extracellular juxtamembrane KIT mutation remains to be determined.

Background and Objectives. Mutations of KIT receptor tyrosine kinase are involved in the constitutive activation and development of human hematologic malignancies. Gain-of-function mutations in the second intracellular kinase domain (TK2) and in the juxtamembrane domain are described in patients with core binding factor acute myeloid leukemia (CBFL) and are associated with leukocytosis. We evaluated the incidence of KIT mutation in 52 adult patients with de novo CBFL and in 49 FLT3/ITD-negative childhood patients with de novo acute myeloid leukemia (AML), excluding cases of acute promyelocytic leukemia. Design and Methods. In order to analyze the role of KIT in CBFL we examined the KIT mutations in 52 adult CBFL, including 15 previously reported patients, and in 49 non-APL childhood AML patients using sensitive detection methods. We correlated our findings with the presence of trisomy 4 and investigated the relationship of the extra chromosome 4 with KIT mutations. Results. Several kinds of gain-of-function KIT mutations were found in 24 of the 52 (46.1%) adult CBFL cases and 6 of the 49 (12.2%) non-APL childhood AML patients. KIT mutations were detected in 4 of the 8 adult patients and one childhood AML case bearing trisomy of chromosome 4 as either the sole cytogenetic aberration or a karyotypic aberration additional to t(8;21). In three of the trisomy 4 cases we demonstrated that trisomy 4 leads to duplication of the KIT mutated allele. Interpretation and Conclusions. These results underline that the KIT gene is activated in AML characterized by distinct cytogenetic and molecular genetic patterns and represents the most frequently mutated target in adult CBFL.

Rett syndrome is an X-linked neurodevelopmental dominant disorder that affects almost exclusively girls. The vast majority of cases are sporadic and are caused by de novo mutations in the MECP2 gene, located in Xq28. Only few familial cases have been reported: in four cases, the mother was an asymptomatic carrier and in other four cases, the germline mosaicism in the mother was postulated. Owing to the above reported cases of germline mosaicism, we decided to offer prenatal diagnosis to all expectant mothers with a Rett daughter despite the absence of the causative mutation in parents' blood. We describe here the outcome of the first nine cases of prenatal diagnosis followed by our center. In eight cases, the fetus did not carry the mutation. In one case, the female fetus did carry the same mutation of the affected sister. The couple decided to interrupt the pregnancy and to devolve fetal tissues for research purposes. Our results indicate that prenatal diagnosis should be proposed to all couples with a Rett daughter, even when the mutation is apparently de novo. Moreover, one positive prenatal test among the first nine cases indicates that germline mosaicism may be seriously considered for the assessment of recurrence risk during genetic counseling.

First described in 1971, partial trisomy 6p is uncommon and generally secondary to a familial reciprocal translocation. The proximal breakpoint of the reported cases varies from p11 to p25. We here report on a patient with moderate mental retardation, craniofacial and pigmentary anomalies, proteinuria, and hyperglycemia who was found to have a mosaic karyotype 46,X,add(Y)(q12)/45,X. Fluorescence in situ hybridization (FISH) enabled us to identify that the additional material on Yqh derived from 6p and to define the rearrangement as der(Y)t(Y;6)(q12;p22). To the best of our knowledge, this is the first case of trisomy 6p22-pter without an associated deleted segment; the second breakpoint of the rearrangement is in Yqh. Precise mapping of the centromeric breakpoint of the trisomic 6p segment allowed a more convincing correlation between partial 6p trisomy and clinical phenotype to be addressed. In particular, the proteinuria often observed in 6p trisomic patients could be assigned to the 6p22-6pter region.

The tyrosine kinase receptor, KIT, and its ligand, KL are important regulators of germ cell development. The aim of this study was to examine in detail the expression of the genes encoding these proteins (White and Steel, respectively) during the fetal period (14.5-18.5 days post coitum, dpc) and the two weeks after birth in mouse ovaries using the highly sensitive in situ reverse-transcriptase polymerase chain reaction (in situ RT-PCR). KL and KIT mRNAs were not detected in 14.5-15.5 dpc ovaries but, between 16.5 and 17.5 dpc, most of the oocytes in the outer regions of the ovaries positively stained for both mRNAs. The majority of the co-expressing oocytes were identified at the zygotene/pachytene stage of meiotic prophase I. At 18.5 dpc, positive staining for KL mRNA was present only in the somatic cells in the outer regions of the ovaries. At birth, faint KL mRNA-labelled somatic cells were mainly found in the central region of the ovaries and, by P7-14, a higher level of expression was detected in the follicle cells of one- and two-layered growing follicles. Between 17.5 dpc and birth, most of the oocytes expressed KIT mRNA and, from P7 onward, there was a considerable accumulation of transcripts in the growing oocytes. The results of in situ RT-PCR were confirmed by RT-PCR on purified populations of oocytes, and at protein level by means of immunohistochemistry. The co-expression of KL and KIT in a fraction of fetal oocytes suggests that the KL/KIT system, besides the well known paracrine functions on germ cells, may exert a novel autocrine role during the mid-stage of the oocyte meiotic prophase. The possibility that this autocrine loop plays a role in sustaining the survival of fetal oocytes in this stage is supported by the finding that the addition to the culture medium of anti-KL or anti-KIT antibodies led to a significant increase in oocyte apoptosis in the absence of exogenous KL.

Recent evidence has indicated a role for the two early response genes c-fos and c-jun in transcriptional regulation of genes acting in osmoregulation. On this basis we investigated their expression in response to hypertonic stress in the human embryonal EUE epithelial cell line. EUE cells have proven to be a useful tool for studying long-term in vitro adaptation to hypertonic stress. After culturing EUE cells in hypertonic medium a marked c-fos induction was observed, both at the mRNA and the protein level. Northern analysis of fos-mRNA showed a peak expression at 4 h, followed by a progressive decline till complete extinction at 8 h. Immunofluorescence analysis of FOS protein evidenced a similar, although slightly delayed kinetics of expression. Conversely, neither c-jun nor c-myc up-regulation could be detected. The treatment of EUE cells with cycloheximide led to superinduction of c-fos expression, (with high levels up to 12 h), and to a c-jun expression that was just detectable. Hypertonic stimulation of the transformed cell lines A549, MCF7 and JR induced both c-fos and c-jun only in JR cells. Hypertonic shock was also effective in inducing c-fos expression in fetal human diploid fibroblasts, although the response was earlier and more transient than in EUE cells. These findings indicate that c-fos is a primary response gene in hypertonic stress-activated cells, although the pattern and kinetics of its induction may differ according to the type of cell.

Trisomy of chromosome 12 is a nonrandom chromosomal change in pituitary adenomas, particularly prolactinomas. This and the finding that prolactin-secreting pituitary adenomas develop in transgenic mice overexpressing the wild-type HMGA2 gene (which maps to 12q14-15) prompted us to investigate HMGA2 rearrangements and expression in human prolactinomas. By dual-color interphase fluorescence in situ hybridization analysis using HMGA2-specific PACs and BACs, we found that the HMGA2 locus was amplified in seven of the eight prolactinoma samples examined. The cytogenetic manifestations of elevated HMGA2 concentrations ranged from simple trisomy to tetrasomy 12 and der(12) chromosomes to marker chromosomes bearing 12q14-15-derived regions. Reverse transcription-PCR, Western blot and immunohistochemical analysis showed HMGA2 overexpression in a number of prolactinomas bearing rearrangement of regions 12q14-15. These data suggest a critical role of the HMGA2 overexpression in the generation of prolactin-secreting pituitary adenomas in humans.

Two distinct marker chromosomes, presenting with intercalated C- and distamycin A-Dapi-positive regions, were observed in a metastatic and a primary melanoma. To establish the origin of these heterochromatic sequences, we performed in situ hybridization analysis using specific probes for human repetitive DNA. The marker of the primary melanoma, m2, a der 16 chromosome resulting from the translocation of the 1q12-qter segment to band q23 of chromosome 16, showed specific hybridization with Sau3A but not with EcoRI sequences at the interstitial C-band. Thus the origin of this region from the normal chromosome 1 pericentromeric heterochromatin, containing both EcoRI and Sau3A sequences, could be established. On the other hand, the marker of the metastatic melanoma, m1, a der 1 chromosome showing an abnormally banded region inserted between 1q11 and 1q21-qter, failed to give any hybridization signals at the C- and distamycin A-Dapi-positive band when the same EcoRI and Sau3A probes were used. Furthermore, no hybridization was observed using either a probe for SatIII-specific sequences (QP23), mapping to chromosome 9 heterochromatic block, or LS6BB, a ribosomal DNA probe. From these data we speculate that more complex molecular rearrangements may have occurred during the transposition of heterochromatin from its original site to m1. The heterochromatin change found in m1 may be related to advanced stages of malignancy.

Microcell production by means of Colcemid-induced micronucleation and subsequent enucleation with the density gradient technique was adjusted for use with the murine T-lymphoma line ESb-M. Modification of the standard protocol for a cell type on which no experiments had previously been performed required careful monitoring of the multiple steps in the procedure in order to optimize the final microcell yield. Traditional microscopic verification may sometimes be ambiguous, due to the lack of a clear cutoff point between small whole cells and cell fragments; in these conditions, the level of variability increases, thus impairing quantitative estimations. Flow cytometric (FCM) analysis of DNA content and size of donor cells and microcells was therefore applied in parallel to provide additional quantitative information. The FCM results supplemented the microscopic data in assessing which fraction recovered from the gradient has the lowest percentage of contaminant whole cells; however, FCM analysis may provide more statistically significant data due to the large size of the sample examined. Moreover, FCM is of prospective use in providing the basis for subsequent sorting of either pure microcells or specific subpopulations of defined DNA content and size.

The role of collagen type III and IV (Coll III, IV) in Duchenne muscular dystrophy (DMD) was investigated by the indirect immunofluorescence technique (IIF) both in muscle biopsies and derived cell cultures. Ten dystrophic cases were studied and compared with twelve suitable control cases, extending the IIF analysis to two other representative non-collagenous proteins of the extracellular matrix (ECM), namely fibronectin (FN) and laminin (LM). DMD biopsies generally displayed a thickening of endomysial and/or perimysial connective, as compared to control specimens. All the markers analyzed were found to contribute to this connective proliferation, although from a quantitative point of view the relative involvement decreases progressively from FN through Coll III and IV to LM. However, due to Coll IV selective endomysial localization, Coll IV alteration can be considered a specific indicator of a muscle cell defect. Results from DMD muscle cultures revealed no significant changes in comparison to control cultures, except for Coll IV. A well-organized, Coll IV-containing pericellular matrix was noted in a fraction of DMD cultures as a unique feature seen in no control culture. This alteration was again considered significant since Coll IV is the only marker clearly associated with the myotube component still expressed in vitro. The negative results obtained on the other marker proteins should not however be considered definitive, due to the lack in the simplified in vitro system used of humoral and/or neuronal factors which may be needed to express gene defect(s) in differentiated cells.

Background: Chromosome 13q deletion is associated with varying phenotypes, which seem to depend on the location of the deleted segment. Although various attempts have been made to link the 13q deletion intervals to distinct phenotypes, there is still no acknowledged consensus correlation between the monosomy of distinct 13q regions and specific clinical features. Methods: 14 Italian patients carrying partial de novo 13q deletions were studied. Molecular–cytogenetic characterisation was carried out by means of array-comparative genomic hybridisation (array-CGH) or fluorescent in situ hybridisation (FISH). Results: Our 14 patients showed mental retardation ranging from profound–severe to moderate–mild: eight had central nervous system (CNS) anomalies, including neural tube defects (NTDs), six had eye abnormalities, nine had facial dysmorphisms and 10 had hand or feet anomalies. The size of the deleted regions varied from 4.2 to 75.7 Mb. Conclusion: This study is the first systematic molecular characterisation of de novo 13q deletions, and offers a karyotype–phenotype correlation based on detailed clinical studies and molecular determinations of the deleted regions. Analyses confirm that patients lacking the 13q32 band are the most seriously affected, and critical intervals have been preliminarily assigned for CNS malformations. Dose-sensitive genes proximal to q33.2 may be involved in NTDs. The minimal deletion interval associated with the Dandy–Walker malformation (DWM) was narrowed to the 13q32.2–33.2 region, in which the ZIC2 and ZIC5 genes proposed as underlying various CNS malformations are mapped.