Lucia Cavelier

Total RNA sequencing reveals nascent transcription and widespread co-transcriptional splicing in the human brain

Transcriptome sequencing allows for analysis of mature RNAs at base pair resolution. Here we show that RNA-seq can also be used for studying nascent RNAs undergoing transcription. We sequenced total RNA from human brain and liver and found a large fraction of reads (up to 40%) within introns. Intronic RNAs were abundant in brain tissue, particularly for genes involved in axonal growth and synaptic transmission. Moreover, we detected significant differences in intronic RNA levels between fetal and adult brains. We show that the pattern of intronic sequence read coverage is explained by nascent transcription in combination with co-transcriptional splicing. Further analysis of co-transcriptional splicing indicates a correlation between slowly removed introns and alternative splicing. Our data show that sequencing of total RNA provides unique insight into the transcriptional processes in the cell, with particular importance for normal brain development.

Comparative genomic analysis of human and chimpanzee indicates a key role for indels in primate evolution.

Sequence comparison of humans and chimpanzees is of interest to understand the mechanisms behind primate evolution. Here we present an independent analysis of human chromosome 21 and the high-quality BAC clone sequences of the homologous chimpanzee chromosome 22. In contrast to previous studies, we have used global alignment methods and Ensembl predictions of protein coding genes (n = 224) for the analysis. Divergence due to insertions and deletions (indels) along with substitutions was examined separately for different genomic features (coding, noncoding genic, and intergenic sequence). The major part of the genomic divergence could be attributed to indels (5.07%), while the nucleotide divergence was estimated as 1.52%. Thus the total divergence was estimated as 6.58%. When excluding repeats and low-complexity DNA the total divergence decreased to 2.37%. The chromosomal distribution of nucleotide substitutions and indel events was significantly correlated. To further examine the role of indels in primate evolution we focused on coding sequences. Indels were found within the coding sequence of 13% of the genes and approximately half of the indels have not been reported previously. In 5% of the chimpanzee genes, indels or substitutions caused premature stop codons that rendered the affected transcripts nonfunctional. Taken together, our findings demonstrate that indels comprise the majority of the genomic divergence. Furthermore, indels occur frequently in coding sequences. Our results thereby support the hypothesis that indels may have a key role in primate evolution.

High modal number and triple trisomies are highly correlated favorable factors in childhood B-cell precursor high hyperdiploid acute lymphoblastic leukemia treated according to the NOPHO ALL 1992/2000 protocols

Between 1992 and 2008, 713 high hyperdiploid acute lymphoblastic leukemias in children aged 1–15 years were diagnosed and treated according to the Nordic Society for Pediatric Hematology and Oncology acute lymphoblastic leukemia 1992/2000 protocols. Twenty (2.8%) harbored t(1;19), t(9;22), der(11q23), or t(12;21). The median age of patients with “classic” high hyperdiploidy was lower than that of patients with translocation-positive high hyperdiploidy (P<0.001). Cases with triple trisomies (+4, +10, +17), comprising 50%, had higher modal numbers than the triple trisomy-negative cases (P<0.0001). The probabilities of event-free survival and overall survival were lower for those with white blood cell counts ≥50×109/L (P=0.017/P=0.009), ≥5% bone marrow blasts at day 29 (P=0.001/0.002), and for high-risk patients (P<0.001/P=0.003), whereas event-free, but not overall, survival, was higher for cases with gains of chromosomes 4 (P<0.0001), 6 (P<0.003), 17 (P=0.010), 18 (P=0.049), and 22 (P=0.040), triple trisomies (P=0.002), and modal numbers >53/55 (P=0.020/0.024). In multivariate analyses, modal number and triple trisomies were significantly associated with superior event-free survival in separate analyses with age and white blood cell counts. When including both modal numbers and triple trisomies, only low white blood cell counts were significantly associated with superior event-free survival (P=0.009). We conclude that high modal chromosome numbers and triple trisomies are highly correlated prognostic factors and that these two parameters identify the same subgroup of patients characterized by a particularly favorable outcome.

DNA methylation-based subtype prediction for pediatric acute lymphoblastic leukemia

BackgroundWe present a method that utilizes DNA methylation profiling for prediction of the cytogenetic subtypes of acute lymphoblastic leukemia (ALL) cells from pediatric ALL patients. The primary aim of our study was to improve risk stratification of ALL patients into treatment groups using DNA methylation as a complement to current diagnostic methods. A secondary aim was to gain insight into the functional role of DNA methylation in ALL.ResultsWe used the methylation status of ~450,000 CpG sites in 546 well-characterized patients with T-ALL or seven recurrent B-cell precursor ALL subtypes to design and validate sensitive and accurate DNA methylation classifiers. After repeated cross-validation, a final classifier was derived that consisted of only 246 CpG sites. The mean sensitivity and specificity of the classifier across the known subtypes was 0.90 and 0.99, respectively. We then used DNA methylation classification to screen for subtype membership of 210 patients with undefined karyotype (normal or no result) or non-recurrent cytogenetic aberrations (‘other’ subtype). Nearly half (n = 106) of the patients lacking cytogenetic subgrouping displayed highly similar methylation profiles as the patients in the known recurrent groups. We verified the subtype of 20% of the newly classified patients by examination of diagnostic karyotypes, array-based copy number analysis, and detection of fusion genes by quantitative polymerase chain reaction (PCR) and RNA-sequencing (RNA-seq). Using RNA-seq data from ALL patients where cytogenetic subtype and DNA methylation classification did not agree, we discovered several novel fusion genes involving ETV6, RUNX1, and PAX5.ConclusionsOur findings indicate that DNA methylation profiling contributes to the clarification of the heterogeneity in cytogenetically undefined ALL patient groups and could be implemented as a complementary method for diagnosis of ALL. The results of our study provide clues to the origin and development of leukemic transformation. The methylation status of the CpG sites constituting the classifiers also highlight relevant biological characteristics in otherwise unclassified ALL patients.

Paediatric B-cell precursor acute lymphoblastic leukaemia with t(1;19)(q23;p13) : clinical and cytogenetic characteristics of 47 cases from the Nordic countries treated according to NOPHO protocols

The translocation t(1;19)(q23;p13)/der(19)t(1;19) is a risk stratifying aberration in childhood B‐cell precursor acute lymphoblastic leukaemia (BCP ALL) in the Nordic countries. We have identified 47 children/adolescents with t(1;19)/der(19)t(1;19)‐positive BCP ALL treated on two successive Nordic Society of Paediatric Haematology and Oncology (NOPHO) protocols between 1992 and 2007 and have reviewed the clinical and cytogenetic characteristics of these cases, comprising 1·8% of all cases. The translocation was balanced in 15 cases (32%) and unbalanced in 29 cases (62%). The most common additional chromosome abnormalities were del(9p), i(9q), del(6q), and del(13q). The median age was 7 years, the median white blood cell (WBC) count was 16 × 109/l, and the female/male ratio was 1·2. The predicted event‐free survival (EFS) at 5 and 10 years was 0·79, whereas the predicted overall survival (OS) at 5 and 10 years was 0·85 and 0·82, respectively. Nine patients had a bone marrow relapse after a median of 23 months; no patient had a central nervous system relapse. Additional cytogenetic abnormalities, age, gender, WBC count or whether the t(1;19) was balanced or unbalanced did not influence EFS or OS. Compared to cases with t(12,21) and high hyperdiploidy, EFS was similar, but overall survival was worse in patients with t(1;19)/der(19)t(1;19) (P = 0·004).

Genome-wide sequencing for the identification of rearrangements associated with Tourette syndrome and obsessive-compulsive disorder

BackgroundTourette Syndrome (TS) is a neuropsychiatric disorder in children characterized by motor and verbal tics. Although several genes have been suggested in the etiology of TS, the genetic mechanisms remain poorly understood.MethodsUsing cytogenetics and FISH analysis, we identified an apparently balanced t(6,22)(q16.2;p13) in a male patient with TS and obsessive-compulsive disorder (OCD). In order to map the breakpoints and to identify additional submicroscopic rearrangements, we performed whole genome mate-pair sequencing and CGH-array analysis on DNA from the proband.ResultsSequence and CGH array analysis revealed a 400 kb deletion located 1.3 Mb telomeric of the chromosome 6q breakpoint, which has not been reported in controls. The deletion affects three genes (GPR63, NDUFA4 and KLHL32) and overlaps a region previously found deleted in a girl with autistic features and speech delay. The proband’s mother, also a carrier of the translocation, was diagnosed with OCD and shares the deletion. We also describe a further potentially related rearrangement which, while unmapped in Homo sapiens, was consistent with the chimpanzee genome.ConclusionsWe conclude that genome-wide sequencing at relatively low resolution can be used for the identification of submicroscopic rearrangements. We also show that large rearrangements may escape detection using standard analysis of whole genome sequencing data. Our findings further provide a candidate region for TS and OCD on chromosome 6q16.

The frequency and prognostic impact of dic(9;20)(p13.2;q11.2) in childhood B-cell precursor acute lymphoblastic leukemia: results from the NOPHO ALL-2000 trial.

The dic(9;20)(p13.2;q11.2) is reported to be present in ∼2% of childhood B-cell precursor acute lymphoblastic leukemia (BCP ALL). However, it easily escapes detection by G-banding analysis and its true prevalence is hence unknown. We performed interphase fluorescence in situ hybridization analyses—in a three-step manner—using probes for: (i) CDKN2A at 9p21, (ii) 20p and 20q subtelomeres and (iii) cen9 and cen20. Out of 1033 BCP ALLs diagnosed from 2001 to 2006, 533 were analyzed; 16% (84/533) displayed 9p21 deletions, of which 30% (25/84) had dic(9;20). Thus, dic(9;20)-positivity was found in 4.7% (25/533), making it the third most common genetic subgroup after high hyperdiploidy and t(12;21)(p13;q22). The dic(9;20) was associated with a female predominance and an age peak at 3 years; 18/25 (72%) were allocated to non-standard risk treatment at diagnosis. Including cases detected by G-banding alone, 29 dic(9;20)-positive cases were treated according to the NOPHO ALL 2000 protocol. Relapses occurred in 24% (7/29) resulting in a 5-year event-free survival of 0.69, which was significantly worse than for t(12;21) (0.87; P=0.002) and high hyperdiploidy (0.82; P=0.04). We conclude that dic(9;20) is twice as common as previously surmised, with many cases going undetected by G-banding analysis, and that dic(9;20) should be considered a non-standard risk abnormality.

Efficient cellular fractionation improves RNA sequencing analysis of mature and nascent transcripts from human tissues

BackgroundThe starting material for RNA sequencing (RNA-seq) studies is usually total RNA or polyA+ RNA. Both forms of RNA represent heterogeneous pools of RNA molecules at different levels of maturation and processing. Such heterogeneity, in addition to the biases associated with polyA+ purification steps, may influence the analysis, sensitivity and the interpretation of RNA-seq data. We hypothesize that subcellular fractions of RNA may provide a more accurate picture of gene expression.ResultsWe present results for sequencing of cytoplasmic and nuclear RNA after cellular fractionation of tissue samples. In comparison with conventional polyA+ RNA, the cytoplasmic RNA contains a significantly higher fraction of exonic sequence, providing increased sensitivity in expression analysis and splice junction detection, and in improved de novo assembly of RNA-seq data. Conversely, the nuclear fraction shows an enrichment of unprocessed RNA compared with total RNA-seq, making it suitable for analysis of nascent transcripts and RNA processing dynamics.ConclusionOur results show that cellular fractionation is a more rapid and cost effective approach than conventional polyA+ enrichment when studying mature RNAs. Thus, RNA-seq of separated cytosolic and nuclear RNA can significantly improve the analysis of complex transcriptomes from mammalian tissues.

Donor or Recipient Origin of Posttransplant Lymphoproliferative Disorders Following Solid Organ Transplantation

Previous studies of donor or recipient origin of posttransplant lymphoproliferative disorders (PTLDs) following solid organ transplantation (SOT) have either been small or with selected patient groups. We studied tumor origin in a population‐based cohort of 93 patients with PTLD following SOT. Tumor origin of PTLD tissue was analyzed by fluorescence in situ hybridization of the sex chromosomes in cases of sex mismatch between donor and recipient (n = 41), or HLA genotyping in cases of identical sex but different HLA type (n = 52). Tumor origin of PTLD could be determined in 67 of the 93 cases. All 67 PTLDs were of recipient origin. They were found in recipients of kidney (n = 38), liver (n = 12), heart (n = 10) and lung (n = 7). The most common recipient‐derived lymphomas were monomorphic B‐cell PTLDs (n = 45), monomorphic T cell PTLDs (n = 9), indolent lymphomas (n = 6), and polymorphic PTLD (n = 4). Half of the recipient‐derived PTLDs were Epstein–Barr virus‐positive. Twelve of the recipient‐derived PTLDs were located in the grafts: in four cases exclusively and in eight cases in combination with disseminated disease outside the graft. Tumor origin was indeterminable in 26 cases, probably due to low DNA quality. We conclude that the vast majority of PTLDs after SOT was of recipient origin.

Splicing in the Human Brain

It has become increasingly clear over the past decade that RNA has important functions in human cells beyond its role as an intermediate translator of DNA to protein. It is now known that RNA plays highly specific roles in pathways involved in regulatory, structural, and catalytic functions. The complexity of RNA production and regulation has become evident with the advent of high-throughput methods to study the transcriptome. Deep sequencing has revealed an enormous diversity of RNA types and transcript isoforms in human cells. The transcriptome of the human brain is particularly interesting as it contains more expressed genes than other tissues and also displays an extreme diversity of transcript isoforms, indicating that highly complex regulatory pathways are present in the brain. Several of these regulatory proteins are now identified, including RNA-binding proteins that are neuron specific. RNA-binding proteins also play important roles in regulating the splicing process and the temporal and spatial isoform production. While significant progress has been made in understanding the human transcriptome, many questions still remain regarding the basic mechanisms of splicing and subcellular localization of RNA. A long-standing question is to what extent the splicing of pre-mRNA is cotranscriptional and posttranscriptional, respectively. Recent data, including studies of the human brain, indicate that splicing is primarily cotranscriptional in human cells. This chapter describes the current understanding of splicing and splicing regulation in the human brain and discusses the recent global sequence-based analyses of transcription and splicing.