Josef Davidsson

The DNA methylome of pediatric acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy, with high hyperdiploidy [51-67 chromosomes] and the t(12;21)(p13;q22) [ETV6/RUNX1 fusion] representing the most frequent abnormalities. Although these arise in utero, there is long latency before overt ALL, showing that additional changes are needed. Gene dysregulation through hypermethylation may be such an event; however, this has not previously been investigated in a detailed fashion. We performed genome-wide methylation profiling using bacterial artificial chromosome arrays and promoter-specific analyses of high hyperdiploid and ETV6/RUNX1-positive ALLs. In addition, global gene expression analyses were performed to identify associated expression patterns. Unsupervised cluster and principal component analyses of the chromosome-wide methylome profiles could successfully subgroup the two genetic ALL types. Analysis of all currently known promoter-specific CpG islands demonstrated that several B-cell- and neoplasia-associated genes were hypermethylated and underexpressed, indicating that aberrant methylation plays a significant leukemogenic role. Interestingly, methylation hotspots were associated with chromosome bands predicted to harbor imprinted genes and the tri-/tetrasomic chromosomes in the high hyperdiploid ALLs were less methylated than their disomic counterparts. Decreased methylation of gained chromosomes is a previously unknown phenomenon that may have ramifications not only for the pathogenesis of high hyperdiploid ALL but also for other disorders with acquired or constitutional numerical chromosome anomalies.

Relapsed childhood high hyperdiploid acute lymphoblastic leukemia: presence of preleukemic ancestral clones and the secondary nature of microdeletions and RTK-RAS mutations.

Although childhood high hyperdiploid acute lymphoblastic leukemia is associated with a favorable outcome, 20% of patients still relapse. It is important to identify these patients already at diagnosis to ensure proper risk stratification. We have investigated 11 paired diagnostic and relapse samples with single nucleotide polymorphism array and mutation analyses of FLT3, KRAS, NRAS and PTPN11 in order to identify changes associated with relapse and to ascertain the genetic evolution patterns. Structural changes, mainly cryptic hemizygous deletions, were significantly more common at relapse (P<0.05). No single aberration was linked to relapse, but four deletions, involving IKZF1, PAX5, CDKN2A/B or AK3, were recurrent. On the basis of the genetic relationship between the paired samples, three groups were delineated: (1) identical genetic changes at diagnosis and relapse (2 of 11 cases), (2) clonal evolution with all changes at diagnosis being present at relapse (2 of 11) and (3) clonal evolution with some changes conserved, lost or gained (7 of 11), suggesting the presence of a preleukemic clone. This ancestral clone was characterized by numerical changes only, with structural changes and RTK-RAS mutations being secondary to the high hyperdiploid pattern.

Array based characterization of a terminal deletion involving chromosome subband 15q26.2: an emerging syndrome associated with growth retardation, cardiac defects and developmental delay

BackgroundSubtelomeric regions are gene rich and deletions in these chromosomal segments have been demonstrated to account for approximately 2.5% of patients displaying mental retardation with or without association of dysmorphic features. However, cases that report de novo terminal deletions on chromosome arm 15q are rare.MethodsIn this study we present the first example of a detailed molecular genetic mapping of a de novo deletion in involving 15q26.2-qter, caused by the formation of a dicentric chromosome 15, using metaphase FISH and tiling resolution (32 k) genome-wide array-based comparative genomic hybridization (CGH).ResultsAfter an initial characterization of the dicentric chromosome by metaphase FISH, array CGH analysis mapped the terminal deletion to encompass a 6.48 megabase (Mb) region, ranging from 93.86–100.34 Mb on chromosome 15.ConclusionIn conclusion, we present an additional case to the growing family of reported cases with 15q26-deletion, thoroughly characterized at the molecular cytogenetic level. In the deleted regions, four candidate genes responsible for the phenotype of the patient could be delineated: IGFR1, MEF2A, CHSY1, and TM2D3. Further characterization of additional patients harboring similar 15q-aberrations might hopefully in the future lead to the description of a clear cut clinically recognizable syndrome.

Deletion of the SCN gene cluster on 2q24.4 is associated with severe epilepsy: An array-based genotype-phenotype correlation and a comprehensive review of previously published cases.

PURPOSE To characterize a deletion of chromosome 2q at the molecular level in a patient suffering from severe epilepsy resembling severe myoclonic epilepsy of infancy/Dravets syndrome (SMEI/DS) and to correlate other cases harboring deletions in the same region to morphological and clinical data. METHODS Array-based comparative genomic hybridization (array CGH) was performed on DNA from the patient. Forty-three previously published cases reporting deletions within region 2q21-q31 were collected and analyzed regarding their cytogenetic and clinical data. RESULTS A del(2)(q24.3q31.1) was detected in the patient, spanning a 10.4-megabase (Mb) region between 165.18 and 175.58Mb, harboring 47 genes. FISH analysis was performed, confirming this deletion. Twenty-two of the 43 previously published cases were seizure-positive. The most common dysmorphic features were ear abnormalities, microcephaly, micrognathia and brachysyndactyly for all patients as well as for solely the seizure-positive and -negative ones. For the 22 seizure-positive cases chromosome subband 2q24.3 constituted the smallest commonly deleted region among the majority of the cases, where subbands 2q22.1 and 2q33.3 represented the most proximal and distal breakpoint, respectively. CONCLUSIONS Based on the early age of presentation and the severity of the epilepsy reported for the majority of the seizure-positive cases it was concluded that SMEI/DS could be the epileptic encephalopathy associated with deletions within the 2q22.1-q33.3 region, due to haploinsuffiency of SCN1A and/or complete or partial deletion of other voltage-gated sodium channel genes caused by the aberration. Furthermore, our study supports that array CGH is a competent technique for screening SCN1A mutation-negative patients diagnosed with SMEI/DS-like epilepsies and dysmorphic features, generating rapid and high-resolution data of genomic imbalances present in the patients.

Gain-of-function SAMD9L mutations cause a syndrome of cytopenia, immunodeficiency, MDS and neurological symptoms

Several monogenic causes of familial myelodysplastic syndrome (MDS) have recently been identified. We studied 2 families with cytopenia, predisposition to MDS with chromosome 7 aberrations, immunodeficiency, and progressive cerebellar dysfunction. Genetic studies uncovered heterozygous missense mutations in SAMD9L, a tumor suppressor gene located on chromosome arm 7q. Consistent with a gain-of-function effect, ectopic expression of the 2 identified SAMD9L mutants decreased cell proliferation relative to wild-type protein. Of the 10 individuals identified who were heterozygous for either SAMD9L mutation, 3 developed MDS upon loss of the mutated SAMD9L allele following intracellular infections associated with myeloid, B-, and natural killer (NK)-cell deficiency. Five other individuals, 3 with spontaneously resolved cytopenic episodes in infancy, harbored hematopoietic revertant mosaicism by uniparental disomy of 7q, with loss of the mutated allele or additional in cisSAMD9L truncating mutations. Examination of 1 individual indicated that somatic reversions were postnatally selected. Somatic mutations were tracked to CD34+ hematopoietic progenitor cell populations, being further enriched in B and NK cells. Stimulation of these cell types with interferon (IFN)-α or IFN-γ induced SAMD9L expression. Clinically, revertant mosaicism was associated with milder disease, yet neurological manifestations persisted in 3 individuals. Two carriers also harbored a rare, in trans germ line SAMD9L missense loss-of-function variant, potentially counteracting the SAMD9L mutation. Our results demonstrate that gain-of-function mutations in the tumor suppressor SAMD9L cause cytopenia, immunodeficiency, variable neurological presentation, and predisposition to MDS with -7/del(7q), whereas hematopoietic revertant mosaicism commonly ameliorated clinical manifestations. The findings suggest a role for SAMD9L in regulating IFN-driven, demand-adapted hematopoiesis.

The t(X;7)(q22;q34) in paediatric T-cell acute lymphoblastic leukaemia results in overexpression of the insulin receptor substrate 4 gene through illegitimate recombination with the T-cell receptor beta locus

The t(X;7)(q22;q34), a translocation not previously reported in a neoplastic disorder, was identified and molecularly characterised in a paediatric T‐cell acute lymphoblastic leukaemia (T‐ALL), subsequently shown also to harbour a deletion of 6q, a STIL/TAL1 fusion and an activating NOTCH1 mutation. The t(X;7) was further investigated using fluorescence in situ hybridisation (FISH), real‐time quantitative polymerase chain reaction (RQ‐PCR) and Western blot analyses. FISH revealed a breakpoint at the T‐cell receptor beta locus at 7q34 and mapped the corresponding breakpoint to Xq22.3. The latter region contains only two known genes, namely insulin receptor substrate 4 (IRS4) and collagen, type IV, alpha 5 (COL4A5), the expressions of which were analysed by the use of RQ‐PCR. COL4A5 was not differentially expressed in the t(X;7)‐positive sample compared to five T‐ALL controls. However, a marked, 1000‐fold overexpression of IRS4 was identified. Western blot analysis with a monoclonal antibody against IRS4 showed overexpression also at the protein level. Considering that forced expression of several members of the IRS family has been shown to result in increased cell proliferation, for example in haematopoietic cells, we hypothesise that the IRS4 up‐regulation in T‐ALL is pathogenetically important as a mitogenic stimulus.

BRAF mutations are very rare in B- and T-cell pediatric acute lymphoblastic leukemias

We read with interest the study by Gustafsson et al.1 regarding BRAF mutations in childhood acute lymphoblastic leukemia (ALL). They investigated exons 11 and 15 of BRAF and exons 1 and 2 of NRAS in 29 cases (25 pre-B ALL, 3 T-cell ALL and 1 undifferentiated ALL), and identified six (21%) BRAF mutations and seven (24%) NRAS mutations. The frequencies of BRAF mutations seemed to be particularly common in T-cell ALL (two out of three; 67%); only four (16%) of the pre-B ALL harbored a BRAF mutation. All mutations were located in exon 15—three L597Q and one V590I among the pre-B ALL and one V600E and one G596S in the T-cell ALL. Hou et al.2 recently demonstrated that the L597Q mutation is a functional oncogene, at least in an in vitro experimental system. In the study by Gustafsson et al.,1 and as expected based on previous studies,3, 4 the BRAF and NRAS mutations were generally mutually exclusive. The high frequency of BRAF mutations was unexpected since such mutations otherwise have been demonstrated to be very rare in hematologic malignancies. In fact, previous analyses revealed no BRAF mutations in 53 cell lines from leukemias and lymphomas,3 65 multiple myelomas/plasma cell leukemias,5 21 multiple myelomas6 or in 149 acute myeloid leukemias (AML).7, 8 We know of only three additional studies reporting acquired BRAF mutations in malignant hematologic disorders. In a series of 164 B- and T-cell lymphomas, Lee et al.9 detected BRAF mutations in 2.5%. The same group10 also reported such mutations in 20% of B-lineage ALL, 9% of acute biphenotypic leukemia and 4% of AML; all the investigated patients were adults. Finally, Christiansen et al.,11 who analyzed 140 treatment-related myelodysplastic syndromes and AML, found three t-AML with a BRAF mutation; all these were adult acute monoblastic leukemias with t(9;11)(p21;q23). Interestingly, germ line mutations in the closely related RAF1 (previously CRAF) were recently reported in two patients who developed t-AML.8 Hence, there may be an association between mutations in RAF genes and t-AML. It may also be noteworthy that among the 17 BRAF-positive hematologic malignancies reported to date,1, 9, 10, 11 only four (24%)—one T-cell ALL1 and three t-AML11—have carried the highly activating V600E amino-acid substitution, which results in a 500-fold activation of the BRAF protein, inducing a pronounced RAS-RAF-MEK-ERK signaling.3

Constitutional trisomy 8 mosaicism as a model for epigenetic studies of aneuploidy

BackgroundTo investigate epigenetic patterns associated with aneuploidy we used constitutional trisomy 8 mosaicism (CT8M) as a model, enabling analyses of single cell clones, harboring either trisomy or disomy 8, from the same patient; this circumvents any bias introduced by using cells from unrelated, healthy individuals as controls. We profiled gene and miRNA expression as well as genome-wide and promoter specific DNA methylation and hydroxymethylation patterns in trisomic and disomic fibroblasts, using microarrays and methylated DNA immunoprecipitation.ResultsTrisomy 8-positive fibroblasts displayed a characteristic expression and methylation phenotype distinct from disomic fibroblasts, with the majority (65%) of chromosome 8 genes in the trisomic cells being overexpressed. However, 69% of all deregulated genes and non-coding RNAs were not located on this chromosome. Pathway analysis of the deregulated genes revealed that cancer, genetic disorder, and hematopoiesis were top ranked. The trisomy 8-positive cells displayed depletion of 5-hydroxymethylcytosine and global hypomethylation of gene-poor regions on chromosome 8, thus partly mimicking the inactivated X chromosome in females.ConclusionsTrisomy 8 affects genes situated also on other chromosomes which, in cooperation with the observed chromosome 8 gene dosage effect, has an impact on the clinical features of CT8M, as demonstrated by the pathway analysis revealing key features that might explain the increased incidence of hematologic malignancies in CT8M patients. Furthermore, we hypothesize that the general depletion of hydroxymethylation and global hypomethylation of chromosome 8 may be unrelated to gene expression regulation, instead being associated with a general mechanism of chromatin processing and compartmentalization of additional chromosomes.

dup(19)(q12q13.2): array-based genotype-phenotype correlation of a new possibly obesity-related syndrome.

Small supernumerary marker chromosomes (sSMCs) derived from the near‐centromeric area of chromosome 2 are very rare. In addition, duplications of the 2p11.2→q11.2 region have displayed considerable variability between patients harboring and lacking clinical findings. Moreover, constitutional duplication of the 19q12→q13.2 region has previously only been described in two cases and was associated with delay of developmental milestones, corpus callosum anomalies, and obesity. Herein, we present a genotype–phenotype correlation in a patient harboring two sSMCs derived from chromosomes 2 and 14 or 22, respectively. The DNA was studied using G‐banding, fluorescence in situ hybridization techniques, and array‐based comparative genomic hybridization. A 48,XX,+der(2)del(2)(p11)del(2)(q11.2),+der(14)t(14;19)(q11;q12)del(19)(q13.31) or 48,XX,+der(2)del(2)(p11)del(2)(q11.2),+der(22)t(22;19)(q11;q12)del(19)(q13.31) was detected in the patient. The sSMC 14;19 or 22;19, with its centromere originating from either chromosome 14 or 22, encompassed a 13.56 megabase (Mb) 19q derived region, harboring 263 genes, and the sSMC 2 a 2.71 Mb region including 29 genes. The patient had symptoms including a ventral septal defect, bilateral grade IV urinary reflux, corpus callosum agenesis, microphthalmia, and obesity. The 19q segment contained the genes AKT2, CEACAM1, CEBPA, LIPE, and TGFB1 which are involved in adipose tissue homeostasis and insulin resistance, and could potentially contribute to the obese phenotype observed. Array‐based genetic characterization and long‐term clinical evaluation with attention toward weight gain in patients with chromosome 19q duplications might in the future lead to the description of a obesity‐associated genetic syndrome, something that could have implications in management and treatment of patients carrying a dup(19)(q12q13.2). Whether the der(2)(p11q11.2) contributes to the phenotype remains inconclusive.

Tiling resolution array-based comparative genomic hybridisation analyses of acute lymphoblastic leukaemias in children with Down syndrome reveal recurrent gain of 8q and deletions of 7p and 9p.

Tiling resolution array-based comparative genomic hybridisation analyses of acute lymphoblastic leukaemias in children with Down syndrome reveal recurrent gain of 8q and deletions of 7p and 9p