A Scalable Computational Approach for Simulating Complexes of Multiple Chromosomes.

Significant efforts have been recently made to obtain the three-dimensional (3D) structure of the genome with the goal of understanding how structures may affect gene regulation and expression. Chromosome conformational capture techniques such as Hi-C, have been key in uncovering the quantitative information needed to determine chromatin organization. Complementing these experimental tools, co-polymers theoretical methods are necessary to determine the ensemble of three-dimensional structures associated to the experimental data provided by Hi-C maps. Going beyond just structural information, these theoretical advances also start to provide an understanding of the underlying mechanisms governing genome assembly and function. Recent theoretical work, however, has been focused on single chromosome structures, missing the fact that, in the full nucleus, interactions between chromosomes play a central role in their organization. To overcome this limitation, MiChroM (Minimal Chromatin Model) has been modified to become capable of performing these multi-chromosome simulations. It has been upgraded into a fast and scalable software version, which is able to perform chromosome simulations using GPUs via OpenMM Python API, called Open-MiChroM. To validate the efficiency of this new version, analyses for GM12878 individual autosomes were performed and compared to earlier studies. This validation was followed by multi-chain simulations including the four largest human chromosomes (C1-C4). These simulations demonstrated the full power of this new approach. Comparison to Hi-C data shows that these multiple chromosome interactions are essential for a more accurate agreement with experimental results. Without any changes to the original MiChroM potential, it is now possible to predict experimentally observed inter-chromosome contacts. This scalability of Open-MiChroM allow for more audacious investigations, looking at interactions of multiple chains as well as moving towards higher resolution chromosomes models.

The functional epigenetic landscape of aberrant gene expression in molecular subgroups of newly diagnosed multiple myeloma.

BACKGROUND: Multiple Myeloma (MM) is a hematological malignancy with genomic heterogeneity and poor survival outcome. Apart from the central role of genetic lesions, epigenetic anomalies have been identified as drivers in the development of the disease. METHODS: Alterations in the DNA methylome were mapped in 52 newly diagnosed MM (NDMM) patients of six molecular subgroups and matched with loci-specific chromatin marks to define their impact on gene expression. Differential DNA methylation analysis was performed using DMAP with a ≥10% increase (hypermethylation) or decrease (hypomethylation) in NDMM subgroups, compared to control samples, considered significant for all the subsequent analyses with p<0.05 after adjusting for a false discovery rate. RESULTS: We identified differentially methylated regions (DMRs) within the etiological cytogenetic subgroups of myeloma, compared to control plasma cells. Using gene expression data we identified genes that are dysregulated and correlate with DNA methylation levels, indicating a role for DNA methylation in their transcriptional control. We demonstrated that 70% of DMRs in the MM epigenome were hypomethylated and overlapped with repressive H3K27me3. In contrast, differentially expressed genes containing hypermethylated DMRs within the gene body or hypomethylated DMRs at the promoters overlapped with H3K4me1, H3K4me3, or H3K36me3 marks. Additionally, enrichment of BRD4 or MED1 at the H3K27ac enriched DMRs functioned as super-enhancers (SE), controlling the overexpression of genes or gene-cassettes. CONCLUSIONS: Therefore, this study presents the underlying epigenetic regulatory networks of gene expression dysregulation in NDMM patients and identifies potential targets for future therapies.

The impact of cytogenetics on duration of response and overall survival in patients with relapsed multiple myeloma (long-term follow-up results from BSBMT/UKMF Myeloma X Relapse [Intensive]): a randomised, open-label, phase 3 trial.

The Myeloma X trial (ISCRTN60123120) registered patients with relapsed multiple myeloma. Participants were randomised between salvage autologous stem cell transplantation (ASCT) or weekly cyclophosphamide following re-induction therapy. Cytogenetic analysis performed at trial registration defined t(4;14), t(14;16) and del(17p) as high-risk. The effect of cytogenetics on time to progression (TTP) and overall survival was investigated. At 76 months median follow-up, ASCT improved TTP compared to cyclophosphamide (19 months (95% confidence interval [95% CI] 16-26) vs. 11 months (9-12), hazard ratio [HR]: 0·40, 95% CI: 0·29-0·56, P < 0·001), on which the presence of any single high-risk lesion had a detrimental impact [likelihood ratio test (LRT): P = 0·011]. ASCT also improved OS [67 months (95% CI 59-not reached) vs. 55 months (44-67), HR: 0·64, 95% CI: 0·42-0·99, P = 0·0435], with evidence of a detrimental impact with MYC rearrangement (LRT: P = 0·021). Twenty-one (24·7%) cyclophosphamide patients received an ASCT post-trial, median OS was not reached (95% CI: 39-not reached) for these participants compared to 31 months (22-39), in those who did not receive a post-trial ASCT. The analysis further supports the benefit of salvage ASCT, which may still be beneficial after second relapse in surviving patients. There is evidence that this benefit reduces in cytogenetic high-risk patients, highlighting the need for targeted study in this patient group.

Integrative Variation Analysis Reveals that a Complex Genotype May Specify Phenotype in Siblings with Syndromic Autism Spectrum Disorder.

It has been proposed that copy number variations (CNVs) are associated with increased risk of autism spectrum disorder (ASD) and, in conjunction with other genetic changes, contribute to the heterogeneity of ASD phenotypes. Array comparative genomic hybridization (aCGH) and exome sequencing, together with systems genetics and network analyses, are being used as tools for the study of complex disorders of unknown etiology, especially those characterized by significant genetic and phenotypic heterogeneity. Therefore, to characterize the complex genotype-phenotype relationship, we performed aCGH and sequenced the exomes of two affected siblings with ASD symptoms, dysmorphic features, and intellectual disability, searching for de novo CNVs, as well as for de novo and rare inherited point variations-single nucleotide variants (SNVs) or small insertions and deletions (indels)-with probable functional impacts. With aCGH, we identified, in both siblings, a duplication in the 4p16.3 region and a deletion at 8p23.3, inherited by a paternal balanced translocation, t(4, 8) (p16; p23). Exome variant analysis found a total of 316 variants, of which 102 were shared by both siblings, 128 were in the male sibling exome data, and 86 were in the female exome data. Our integrative network analysis showed that the siblings' shared translocation could explain their similar syndromic phenotype, including overgrowth, macrocephaly, and intellectual disability. However, exome data aggregate genes to those already connected from their translocation, which are important to the robustness of the network and contribute to the understanding of the broader spectrum of psychiatric symptoms. This study shows the importance of using an integrative approach to explore genotype-phenotype variability.

Clinical features in adult patient with Wolf-Hirschhorn syndrome.

The Wolf-Hirschhorn syndrome (WHS) encompasses deletions at the distal part of the short arm of one chromosome 4 (4p16 region). Clinical signs frequently include a typical facial appearance, mental retardation, intrauterine and postnatal growth retardation, hypotonia with decreased muscle bulk and seizures besides congenital heart malformations, midline defects, urinary tract malformations and brain, hearing and ophthalmologic malformations. Pathogenesis of WHS is multigenic and many factors are involved in prediction of prognosis such as extent of deletion, the occurrence of severe chromosome anomalies, the severe of seizures, the existence of serious internal, mainly cardiac, abnormalities and the degree of mental retardation. The phenotype of adult with WHS is in general similar to that of childhood being facial dysmorphism, growth retardation and mental retardation the rule in both adults and children. Avoid long-term complications and provide rehabilitation programs and genetic counseling may be essential in these patients.

Modified cIg-FISH protocol for multiple myeloma in routine cytogenetic laboratory practice.

The International Myeloma Working Group recommends that fluorescence in situ hybridization (FISH) be performed on specifically identified plasma cells (PC). This is because chromosomal abnormalities are not frequently detected by traditional karyotyping due to the low proliferative rate of PC in multiple myeloma (MM). Conventional FISH enhances the sensitivity but lacks the specificity, as it does not distinguish PC from other hematopoetic cells. To fulfill this recommendation, PC need to be selected either by flow cytometry or immunomagnetic bead-based PC sorting or by concomitant labeling of the cytoplasmic immunoglobulin light chain, which allows for unambiguous identification. These techniques require expertise, time, and funding and are not easily incorporated into the routine workflow of the cytogenetic laboratory. We have modified and refined the technique using fixed cell pellets to achieve nicely separated and easily identifiable PC. With immunostaining and subsequent FISH (i.e., cytoplasmic immunoglobulin FISH, cIg-FISH), this technique can be easily incorporated into every cytogenetic laboratory. Twenty samples from patients with MM were subjected to routine FISH, cIg-FISH, and chromosomal karyotyping and the results were compared. Three FISH probes, which enabled detection of the t(4;14), t(14;16) and deletion of TP53, were used to validate this modified technique successfully.

[Wolf-Hirschhorn syndrome. A series of 27 patients: their epidemiological and clinical characteristics. The current situation of the patients and the opinions of their caregivers regarding the diagnostic process]. / Sindrome de Wolf-Hirschhorn. Serie de 27 pacientes: caracteristicas epidemiologicas y clinicas. Situacion actual de los pacientes y opinion de sus cuidadores respecto al proceso diagnostico.

INTRODUCTION: Wolf-Hirschhorn syndrome (WHS) is a chromosome pathology produced by a deletion in the distal region of the short arm of chromosome 4. It is characterised by the presence of a peculiar phenotype, delayed growth, delayed psychomotor development and epilepsy. AIMS: To describe the characteristics of a series of children with WHS, including the mean amount of time spent on reaching the diagnosis, and to evaluate the opinion of the families about the diagnostic process. PATIENTS AND METHODS: The researchers contacted the National WHS Association and, through them, contact was established with 29 families affected by the condition. Information was collected about the clinical features of the child and the opinion about the diagnostic process, and the families were asked to present medical reports that confirmed the information they had given. Once a database of information about the patients had been created, it was submitted to a statistical analysis. RESULTS: Information was obtained on 27 families. The mean age of the patients is currently 6.94 ± 6.37 years. The mean age of diagnosis was 14.34 months. Delayed intrauterine growth exists in 92.6% of the pregnancies. Epilepsy is present in 92.6% of patients, 44.4% of them in monotherapy. Delayed psychomotor/cognitive development exists in all the patients. Thirty-three per cent of them can walk unaided. The parents rated the treatment offered by physicians with a mean score of 7.25 ± 2.17 and the information they were provided with was given a score of 6.29 ± 2.11. CONCLUSIONS: No references have been found regarding the mean age of diagnosis for WHS. In our sample there are important variations in this respect, possibly influenced by the phenotype of the case and the doctor's own experience. The clinical characteristics are similar to the ones that were expected. The estimated degree of dependence is high and, in contrast, the quality of the information received by the family is low.

TITLE: Sindrome de Wolf-Hirschhorn. Serie de 27 pacientes: caracteristicas epidemiologicas y clinicas. Situacion actual de los pacientes y opinion de sus cuidadores respecto al proceso diagnostico.Introduccion. El sindrome de Wolf-Hirschhorn (SWH) es una cromosomopatia producida por una delecion en la region distal del brazo corto del cromosoma 4. Se caracteriza por la presencia de un fenotipo peculiar, retraso en el crecimiento, retraso del desarrollo psicomotor y epilepsia. Objetivos. Describir las caracteristicas de una serie de niños con SWH, incluido el tiempo medio empleado para el diagnostico, y valorar la opinion de las familias sobre el proceso diagnostico. Pacientes y metodos. Se contacto con la Asociacion Nacional de SWH y, a traves de ella, con 29 familias afectadas. Se recogio informacion sobre la clinica del niño y la opinion sobre el proceso diagnostico, y se solicitaron informes medicos que confirmaran la informacion facilitada. Constituida una base de datos de pacientes, se procedio a su analisis estadistico. Resultados. Se obtuvo informacion de 27 familias. Los pacientes presentan una edad media actual de 6,94 ± 6,37 años. La edad media de diagnostico fue de 14,34 meses. Existe retraso del crecimiento intrauterino en el 92,6% de los embarazos. Un 92,6% de los pacientes presenta epilepsia, el 44,4% de ellos en monoterapia. Existe retraso del desarrollo psicomotor/cognitivo en todos los pacientes. Camina sin ayuda el 33%. Los padres califican con una nota media de 7,25 ± 2,17 el trato ofrecido por los facultativos y de 6,29 ± 2,11 la informacion recibida. Conclusiones. No se han encontrado referencias a la edad media de diagnostico para el SWH. En nuestra muestra, existen variaciones importantes en este aspecto, posiblemente condicionadas por el fenotipo del caso y la experiencia del medico. Las caracteristicas clinicas son similares a las esperadas. El grado de dependencia estimado es alto y la calidad de la informacion recibida por la familia, baja.

Loss of heterozygosity 4q24 and TET2 mutations associated with myelodysplastic/myeloproliferative neoplasms.

Chromosomal abnormalities are frequent in myeloid malignancies, but in most cases of myelodysplasia (MDS) and myeloproliferative neoplasms (MPN), underlying pathogenic molecular lesions are unknown. We identified recurrent areas of somatic copy number-neutral loss of heterozygosity (LOH) and deletions of chromosome 4q24 in a large cohort of patients with myeloid malignancies including MDS and related mixed MDS/MPN syndromes using single nucleotide polymorphism arrays. We then investigated genes in the commonly affected area for mutations. When we sequenced TET2, we found homozygous and hemizygous mutations. Heterozygous and compound heterozygous mutations were found in patients with similar clinical phenotypes without LOH4q24. Clinical analysis showed most TET2 mutations were present in patients with MDS/MPN (58%), including CMML (6/17) or sAML (32%) evolved from MDS/MPN and typical MDS (10%), suggesting they may play a ubiquitous role in malignant evolution. TET2 mutations affected conserved domains and the N terminus. TET2 is widely expressed in hematopoietic cells but its function is unknown, and it lacks homology to other known genes. The frequency of mutations in this candidate myeloid regulatory gene suggests an important role in the pathogenesis of poor prognosis MDS/MPN and sAML and may act as a disease gene marker for these often cytogenetically normal disorders.

A large patient study confirming that facioscapulohumeral muscular dystrophy (FSHD) disease expression is almost exclusively associated with an FSHD locus located on a 4qA-defined 4qter subtelomere.

Facioscapulohumeral muscular dystrophy (FSHD), an autosomal dominant disorder, represents the third most common human muscular dystrophy. The FSHD disease locus, at chromosome 4q35, is associated with large contractions of the polymorphic repeat sequence array D4Z4. In addition to FSHD disease association with large D4Z4 deletions, a biased interaction with a specific 4qter subtelomeric sequence has been described in patients. Two distinct 4qter subtelomeres, defined as types 4qA and 4qB, have been identified and shown to be equally prevalent in the Caucasian population. In almost all 4q35-linked patients with FSHD, however, disease expression only occurs when large D4Z4 deletions are located on 4qA-defined 4qter subtelomeres. Conversely, large D4Z4 repeat contractions situated on 4qB-defined subtelomeres either are not disease-causing or exhibit a greatly reduced disease penetrance. This study was initiated to confirm this direct FSHD disease association data by measuring the frequency of type 4qA-defined and 4qB-defined subtelomeric sequences in a large cohort of 164 unrelated patients with FSHD from Turkey and the UK, all known to have large D4Z4 deletions. An almost complete association was found between large D4Z4 repeat array deletions located on 4qA-defined 4qter subtelomeres and disease expression in our large FSHD patient cohort. The observed failure of probes 4qA and 4qB to hybridise to two patient-derived DNA samples confirms the presence of an additional rare type of 4qter subtelomeric sequence in humans.

Two consecutive immunophenotypic switches in a child with MLL-rearranged acute lymphoblastic leukemia.

An 18-month-old girl was diagnosed with pre-pre-B ALL/t(4;11) leukemia, which during the treatment and after matched bone marrow transplantation (BMT), underwent two consecutive switches from lymphoid to myeloid lineage and vice versa. The high expression of HOXA9 and FLT3 genes remaining genotypically stable in a leukemia throughout phenotypic switches, suggests that this leukemia may have originated as a common B/myeloid progenitors.

Simultaneous localization of MLL, AF4 and ENL genes in interphase nuclei by 3D-FISH: MLL translocation revisited.

BACKGROUND: Haematological cancer is characterised by chromosomal translocation (e.g. MLL translocation in acute leukaemia) and two models have been proposed to explain the origins of recurrent reciprocal translocation. The first, established from pairs of translocated genes (such as BCR and ABL), considers the spatial proximity of loci in interphase nuclei (static "contact first" model). The second model is based on the dynamics of double strand break ends during repair processes (dynamic "breakage first" model). Since the MLL gene involved in 11q23 translocation has more than 40 partners, the study of the relative positions of the MLL gene with both the most frequent partner gene (AF4) and a less frequent partner gene (ENL), should elucidate the MLL translocation mechanism. METHODS: Using triple labeling 3D FISH experiments, we have determined the relative positions of MLL, AF4 and ENL genes, in two lymphoblastic and two myeloid human cell lines. RESULTS: In all cell lines, the ENL gene is significantly closer to the MLL gene than the AF4 gene (with P value < 0.0001). According to the static "contact first" model of the translocation mechanism, a minimal distance between loci would indicate a greater probability of the occurrence of t(11;19)(q23;p13.3) compared to t(4;11)(q21;q23). However this is in contradiction to the epidemiology of 11q23 translocation. CONCLUSION: The simultaneous multi-probe hybridization in 3D-FISH is a new approach in addressing the correlation between spatial proximity and occurrence of translocation. Our observations are not consistent with the static "contact first" model of translocation. The recently proposed dynamic "breakage first" model offers an attractive alternative explanation.

Genome-wide scan for white matter hyperintensity: the Framingham Heart Study.

BACKGROUND AND PURPOSE: White matter hyperintensity (WMH) volume is associated with aging and cerebrovascular disease and has been demonstrated to have a high heritability in the Framingham Heart Study as well as in other studies. We performed a genome-wide linkage analysis to identify chromosomal regions that may harbor genes influencing WMH in a family-based sample of the Framingham Heart Study. METHODS: Brain magnetic resonance scans were performed, and WMH and total cranial volume (TCV) were quantified as previously described on 2259 cohort and offspring participants. The outcome used for linkage analysis was an age specific (within 10-year age groups) z-score for the natural logarithm of the ratio of WMH to TCV. This z-score was based on 2230 individuals after excluding 26 participants with neurological conditions other than stroke and 3 individuals whose ages were out of range. Variance component linkage analysis included 747 individuals (mean age=62.16+/-12.43 years) with both magnetic resonance measure and genotype information in 237 families. Mean percent WMH to TCV was 0.098+/-0.175 with a range of 0.00025% to 1.37% in the linkage analysis subjects. RESULTS: A maximum multipoint logarithm of the odds (LOD) score=3.69, which indicates significant evidence of linkage, was observed at 4 cM on chromosome 4. A suggestive peak with LOD=1.78 was observed at 95 cM on chromosome 17. CONCLUSIONS: We have significant evidence that a gene influencing WMH volume is located on chromosome 4 of the human genome.

The hypereosinophilic syndrome: fluorescence in situ hybridization detects the del(4)(q12)-FIP1L1/PDGFRA but not genomic rearrangements of other tyrosine kinases.

BACKGROUND AND OBJECTIVES: According to WHO criteria, the idiopathic hypereosinophilic syndrome (HES) is defined as persistent eosinophilia (>1.5x10(9)/L) without underlying causes, which is associated with signs or symptoms of organ involvement. Increased bone marrow blasts (>5%) or cytogenetic/genetic markers indicate chronic eosinophilic leukemia (CEL). A cryptic deletion of 4q12, i.e. del(4)(q12), producing the FIP1L1/PDGFRA fusion gene, identifies a distinct CEL subgroup (4q-/CEL). Our aims were: a) to use interphase-fluorescent in situ hybridization (FISH) to detect the cryptic 4q12 deletion; b) to compare the clinico-hematologic features of 4q-/CEL with other HES; c) to investigate whether PDGFRB, FGFR1, ABL1, and ETV6-activated tyrosine kinases are rearranged in CEL/HES. DESIGN AND METHODS: This multicenter study included 20 patients fulfilling the WHO criteria for HES and 6 patients without signs/symptoms of end-organ involvement. Double-color FISH was applied in all cases to investigate del(4)(q12). Further interphase-FISH assessed whether PDGFRB/5q33, FGFR1/8p11, ABL1/9q34, and ETV6/12p13, undergo rearrangements in HES. RESULTS: Ten of the 26 patients (9 males and 1 female) had a cryptic del(4)(q12)-FIP1L1/PDGFRA which was confirmed by reverse transcription polymerase chain reaction (RT-PCR) analysis in four. Hepatomegaly and splenomegaly were significantly more frequent in these 10 than in the other 16 patients. Seven of these 10 patients received imatinib mesylate therapy and all achieved hematologic remission. In 3 of the patients interphase-FISH and RT-PCR demonstrated cytogenetic and molecular remission. Improvements were observed in signs and symptoms of cardiac and central nervous system involvement in 2 and 1 patient, respectively. Rearrangements of PDGFRB, FGFR1, ABL1, or ETV6 were not detected in this study. INTERPRETATION AND CONCLUSIONS: FISH is a reliable diagnostic test for differentiating 4q-/CEL from other forms of HES, allowing an early diagnosis of good responders to imatinib mesylate therapy. For the first time we show that PDGFRB, FGFR1, ABL1 and ETV6 are not rearranged in HES and 4q-/CEL cases we studied.

CD221 (IGF-1R) is aberrantly expressed in multiple myeloma, in relation to disease severity.

We investigated the expression of insulin-like growth factor-1 receptor (CD221) in normal, reactive and malignant plasma cells. We show that CD221 is aberrantly expressed on human myeloma cells, that higher levels of CD221 are observed in patients and human myeloma cell lines with the most aggressive 14q32 translocations, and that CD221 expression has a negative prognostic impact in patients with multiple myeloma.

The 4q subtelomere harboring the FSHD locus is specifically anchored with peripheral heterochromatin unlike most human telomeres.

This paper investigates the nuclear localization of human telomeres and, specifically, the 4q35 subtelomere mutated in facioscapulohumeral dystrophy (FSHD). FSHD is a common muscular dystrophy that has been linked to contraction of D4Z4 tandem repeats, widely postulated to affect distant gene expression. Most human telomeres, such as 17q and 17p, avoid the nuclear periphery to reside within the internal, euchromatic compartment. In contrast, 4q35 localizes at the peripheral heterochromatin with 4p more internal, generating a reproducible chromosome orientation that we relate to gene expression profiles. Studies of hybrid and translocation cell lines indicate this localization is inherent to the distal tip of 4q. Investigation of heterozygous FSHD myoblasts demonstrated no significant displacement of the mutant allele from the nuclear periphery. However, consistent association of the pathogenic D4Z4 locus with the heterochromatic compartment supports a potential role in regulating the heterochromatic state and makes a telomere positioning effect more likely. Furthermore, D4Z4 repeats on other chromosomes also frequently organize with the heterochromatic compartment at the nuclear or nucleolar periphery, demonstrating a commonality among chromosomes harboring this subtelomere repeat family.

Prader-Willi syndrome with an unusually large 15q deletion due to an unbalanced translocation t(4;15).

Prader-Willi syndrome (PWS) is a neurobehavioral disorder caused by deletions in the 15q11-q13 region, by maternal uniparental disomy of chromosome 15 or by imprinting defects. Structural rearrangements of chromosome 15 have been described in about 5% of the patients with typical or atypical PWS phenotype. An 8-year-old boy with a clinical diagnosis of PWS, severe neurodevelopmental delay, absence of speech and mental retardation was studied by cytogenetic and molecular techniques, and an unbalanced de novo karyotype 45,XY,der(4)t(4;15)(q35;q14),-15 was detected after GTG-banding. The patient was diagnosed by SNURF-SNRPN exon 1 methylation assay, and the extent of the deletions on chromosomes 4 and 15 was investigated by microsatellite analysis of markers located in 4qter and 15q13-q14 regions. The deletion of chromosome 4q was distal to D4S1652, and that of chromosome 15 was located between D15S1043 and D15S1010. Our patient's severely affected phenotype could be due to the extent of the deletion, larger than usually seen in PWS patients, although the unbalance of the derivative chromosome 4 cannot be ruled out as another possible cause. The breakpoint was located in the subtelomeric region, very close to the telomere, a region that has been described as having the lowest gene concentrations in the human genome.

FSHD in Chinese population: characteristics of translocation and genotype-phenotype correlation.

Current studies of facioscapulohumeral muscular dystrophy (FSHD) are confined to the white population. The authors surveyed 110 healthy individuals and 27 families with FSHD including 55 patients and 74 relatives by pulsed-field gel electrophoresis. The authors report the characteristics of translocation and genotype-phenotype correlation, and their results indicate 4q to 10q translocation contributes to the occurrence of de novo mutation. This leads to a more severe phenotype in the Chinese population comparing to EcoRI allele sizes and the intersexual difference.