The structure, function and evolution of a complete human chromosome 8.

The complete assembly of each human chromosome is essential for understanding human biology and evolution1,2. Here we use complementary long-read sequencing technologies to complete the linear assembly of human chromosome 8. Our assembly resolves the sequence of five previously long-standing gaps, including a 2.08-Mb centromeric α-satellite array, a 644-kb copy number polymorphism in the ß-defensin gene cluster that is important for disease risk, and an 863-kb variable number tandem repeat at chromosome 8q21.2 that can function as a neocentromere. We show that the centromeric α-satellite array is generally methylated except for a 73-kb hypomethylated region of diverse higher-order α-satellites enriched with CENP-A nucleosomes, consistent with the location of the kinetochore. In addition, we confirm the overall organization and methylation pattern of the centromere in a diploid human genome. Using a dual long-read sequencing approach, we complete high-quality draft assemblies of the orthologous centromere from chromosome 8 in chimpanzee, orangutan and macaque to reconstruct its evolutionary history. Comparative and phylogenetic analyses show that the higher-order α-satellite structure evolved in the great ape ancestor with a layered symmetry, in which more ancient higher-order repeats locate peripherally to monomeric α-satellites. We estimate that the mutation rate of centromeric satellite DNA is accelerated by more than 2.2-fold compared to the unique portions of the genome, and this acceleration extends into the flanking sequence.

A 590 kb deletion caused by non-allelic homologous recombination between two LINE-1 elements in a patient with mesomelia-synostosis syndrome.

Mesomelia-synostoses syndrome (MSS) is a rare, autosomal-dominant, syndromal osteochondrodysplasia characterized by mesomelic limb shortening, acral synostoses, and multiple congenital malformations due to a non-recurrent deletion at 8q13 that always encompasses two coding-genes, SULF1 and SLCO5A1. To date, five unrelated patients have been reported worldwide, and MMS was previously proposed to not be a genomic disorder associated with deletions recurring from non-allelic homologous recombination (NAHR) in at least two analyzed cases. We conducted targeted gene panel sequencing and subsequent array-based copy number analysis in an 11-year-old undiagnosed Japanese female patient with multiple congenital anomalies that included mesomelic limb shortening and detected a novel 590 Kb deletion at 8q13 encompassing the same gene set as reported previously, resulting in the diagnosis of MSS. Breakpoint sequences of the deleted region in our case demonstrated the first LINE-1s (L1s)-mediated unequal NAHR event utilizing two distant L1 elements as homology substrates in this disease, which may represent a novel causative mechanism of the 8q13 deletion, expanding the range of mechanisms involved in the chromosomal rearrangements responsible for MSS.

HMSN Lom in 12 Czech patients, with one unusual case due to uniparental isodisomy of chromosome 8.

Hereditary motor and sensory neuropathy-type Lom (HMSNL), also known as CMT4D, a demyelinating neuropathy with late-onset deafness is an autosomal recessive disorder threatening Roma population worldwide. The clinical phenotype was reported in several case reports before the gene discovery. HMSNL is caused by a homozygous founder mutation p.Arg148* in the N-Myc downstream-regulated gene 1. Here, we report findings from the Czech Republic, where HMSNL was found in 12 Czech patients from eight families. In these 12 patients, 11 of the causes were due to p.Arg148* mutation inherited from both parents by the autosomal recessive mechanism. But in one case, the recessive mutation was inherited only from one parent (father) and unmasked owing to an uniparental isodisomy of the entire chromosome eight. The inherited peripheral neuropathy owing to an isodisomy of the whole chromosome pointed to an interesting, less frequent possibility of recessive disease and complications with genetic counseling.

ZBTB7A mutations in acute myeloid leukaemia with t(8;21) translocation.

The t(8;21) translocation is one of the most frequent cytogenetic abnormalities in acute myeloid leukaemia (AML) and results in the RUNX1/RUNX1T1 rearrangement. Despite the causative role of the RUNX1/RUNX1T1 fusion gene in leukaemia initiation, additional genetic lesions are required for disease development. Here we identify recurring ZBTB7A mutations in 23% (13/56) of AML t(8;21) patients, including missense and truncating mutations resulting in alteration or loss of the C-terminal zinc-finger domain of ZBTB7A. The transcription factor ZBTB7A is important for haematopoietic lineage fate decisions and for regulation of glycolysis. On a functional level, we show that ZBTB7A mutations disrupt the transcriptional repressor potential and the anti-proliferative effect of ZBTB7A. The specific association of ZBTB7A mutations with t(8;21) rearranged AML points towards leukaemogenic cooperativity between mutant ZBTB7A and the RUNX1/RUNX1T1 fusion.

DOES THE PATTERN OF CLONAL EVOLUTION IN THE KARYOTYPE OF PATIENTS WITH ACUTE MYELOID LEUKEMIA AND MYELODYSPLASTIC SYNDROMES DEPEND ON THE TYPE OF THE PRIMARY CHROMOSOMAL ABERRATIONS?

The aim of our study was to define if the type of primary chromosomal aberrations (CA) of the karyotype of patients with Acute myeloid leukemia (AML) and Myelodysplastic syndromes (MDS) determines the way and the rate of karyotype development. Conventional cytogenetic analysis was carried out on 248 AML and 105 MDS patients at diagnosis. Clonal evolution (CE) was found in 40% (51 of 128) of AML patients and in 47.5% (19 of 40) of MDS patients having CA in their karyotype. The first pattern we established was for the most frequent CA which initiate CE in 28 patients with a complex karyotype. These CA were non-balansed rearrangements in the following regions: 5q, 7q, 11q, 3q, monosomy 5, monosomy 7. The second pattern of CE was regarding the most frequent aneuploidias (+8, +11, +21, -Y, and the third pattern concerned balanced CA. We found significant difference in the distribution of karyotypes in different stages of progression between the first and the other two groups (p < 0.001). No statistical difference was found between the patterns in the second and the third group CA (p > 0.5).

Cluster analysis of multiplex ligation-dependent probe amplification data in choroidal melanoma.

PURPOSE: To determine underlying correlations in multiplex ligation-dependent probe amplification (MLPA) data and their significance regarding survival following treatment of choroidal melanoma (CM). METHODS: MLPA data were available for 31 loci across four chromosomes (1p, 3, 6, and 8) in tumor material obtained from 602 patients with CM treated at the Liverpool Ocular Oncology Center (LOOC) between 1993 and 2012. Data representing chromosomes 3 and 8q were analyzed in depth since their association with CM patient survival is well-known. Unsupervised k-means cluster analysis was performed to detect latent structure in the data set. Principal component analysis (PCA) was also performed to determine the intrinsic dimensionality of the data. Survival analyses of the identified clusters were performed using Kaplan-Meier (KM) and log-rank statistical tests. Correlation with largest basal tumor diameter (LTD) was investigated. RESULTS: Chromosome 3: A two-cluster (bimodal) solution was found in chromosome 3, characterized by centroids at unilaterally normal probe values and unilateral deletion. There was a large, significant difference in the survival characteristics of the two clusters (log-rank, p<0.001; 5-year survival: 80% versus 40%). Both clusters had a broad distribution in LTD, although larger tumors were characteristically in the poorer outcome group (Mann-Whitney, p<0.001). Threshold values of 0.85 for deletion and 1.15 for gain optimized the classification of the clusters. PCA showed that the first principal component (PC1) contained more than 80% of the data set variance and all of the bimodality, with uniform coefficients (0.28±0.03). Chromosome 8q: No clusters were found in chromosome 8q. Using a conventional threshold-based definition of 8q gain, and in conjunction with the chromosome 3 clusters, three prognostic groups were identified: chromosomes 3 and 8q both normal, either chromosome 3 or 8q abnormal, and both chromosomes 3 and 8q abnormal. KM analysis showed 5-year survival figures of approximately 97%, 80%, and 30% for these prognostic groups, respectively (log-rank, p<0.001). All MLPA probes within both chromosomes were significantly correlated with each other (Spearman, p<0.001). CONCLUSIONS: Within chromosome 3, the strong correlation between the MLPA variables and the uniform coefficients from the PCA indicates a lack of evidence for a signature gene that might account for the bimodality we observed. We hypothesize that the two clusters we found correspond to binary underlying states of complete monosomy or disomy 3 and that these states are sampled by the complete ensemble of probes. Consequently, we would expect a similar pattern to emerge in higher-resolution MLPA data sets. LTD may be a significant confounding factor. Considering chromosome 8q, we found that chromosome 3 cluster membership and 8q gain as traditionally defined have an indistinguishable impact on patient outcome.

Genomic analysis of clonal eosinophils by CGH arrays reveals new genetic regions involved in chronic eosinophilia.

To assess the presence of genetic imbalances in patients with myeloproliferative neoplasms (MPNs), 38 patients with chronic eosinophilia were studied by array comparative genomic hybridization (aCGH): seven had chronic myelogenous leukaemia (CML), BCR-ABL1 positive, nine patients had myeloproliferative neoplasia Ph- (MPN-Ph-), three had a myeloid neoplasm associated with a PDGFRA rearrangement, and the remaining two cases were Lymphoproliferative T neoplasms associated with eosinophilia. In addition, 17 patients had a secondary eosinophilia and were used as controls. Eosinophilic enrichment was carried out in all cases. Genomic imbalances were found in 76% of all MPN patients. Losses on 20q were the most frequent genetic abnormality in MPNs (32%), affected the three types of MPN studied. This study also found losses at 11q13.3 in 26% of patients with MPN-Ph- and in 19p13.11 in two of the three patients with an MPN associated with a PDGFRA rearrangement. In addition, 29% of patients with CML had losses on 8q24. In summary, aCGH revealed clonality in eosinophils in most MPNs, suggesting that it could be a useful technique for defining clonality in these diseases. The presence of genetic losses in new regions could provide new insights into the knowledge of these MPN associated with eosinophilia.

High-resolution genomic analysis of the 11q13 amplicon in breast cancers identifies synergy with 8p12 amplification, involving the mTOR targets S6K2 and 4EBP1.

The chromosomal region 11q13 is amplified in 15-20% of breast cancers; an event not only associated with estrogen receptor (ER) expression but also implicated in resistance to endocrine therapy. Coamplifications of the 11q13 and 8p12 regions are common, suggesting synergy between the amplicons. The aim was to identify candidate oncogenes in the 11q13 region based on recurrent amplification patterns and correlations to mRNA expression levels. Furthermore, the 11q13/8p12 coamplification and its prognostic value, was evaluated at the DNA and the mRNA levels. Affymetrix 250K NspI arrays were used for whole-genome screening of DNA copy number changes in 29 breast tumors. To identify amplicon cores at 11q13 and 8p12, genomic identification of significant targets in cancer (GISTIC) was applied. The mRNA expression levels of candidate oncogenes in the amplicons [RAD9A, RPS6KB2 (S6K2), CCND1, FGF19, FGF4, FGF3, PAK1, GAB2 (11q13); EIF4EBP1 (4EBP1), PPAPDC1B, and FGFR1 (8p12)] were evaluated using real-time PCR. Resulting data revealed three main amplification cores at 11q13. ER expression was associated with the central 11q13 amplification core, encompassing CCND1, whereas 8p12 amplification/gene expression correlated to S6K2 in a proximal 11q13 core. Amplification of 8p12 and high expression of 4EBP1 or FGFR1 was associated with a poor outcome in the group. In conclusion, single nucleotide polymorphism arrays have enabled mapping of the 11q13 amplicon in breast tumors with high resolution. A proximal 11q13 core including S6K2 was identified as involved in the coamplification/coexpression with 8p12, suggesting synergy between the mTOR targets S6K2 and 4EBP1 in breast cancer development and progression.

The NRG1 gene is frequently silenced by methylation in breast cancers and is a strong candidate for the 8p tumour suppressor gene.

Neuregulin-1 (NRG1) is both a candidate oncogene and a candidate tumour suppressor gene. It not only encodes the heregulins and other mitogenic ligands for the ERBB family, but also causes apoptosis in NRG1-expressing cells. We found that most breast cancer cell lines had reduced or undetectable expression of NRG1. This included cell lines that had translocation breaks in the gene. Similarly, expression in cancers was generally comparable to or less than that in various normal breast samples. Many non-expressing cell lines had extensive methylation of the CpG island at the principal transcription start site at exon 2 of NRG1. Expression was reactivated by demethylation. Many tumours also showed methylation, whereas normal mammary epithelial fragments had none. Lower NRG1 expression correlated with higher methylation. Small interfering RNA (siRNA)-mediated depletion of NRG1 increased net proliferation in a normal breast cell line and a breast cancer cell line that expressed NRG1. The short arm of chromosome 8 is frequently lost in epithelial cancers, and NRG1 is the most centromeric gene that is always affected. NRG1 may therefore be the major tumour suppressor gene postulated to be on 8p: it is in the correct location, is antiproliferative and is silenced in many breast cancers.

Characterization of recurrent homogeneously staining regions in 72 breast carcinomas.

Cytogenetic analyses were performed on 223 breast carcinomas, of which 60% contained homogeneously staining regions (hsr), an intrachromosomal cytogenetic feature of gene amplification. The precise hsr localization could be determined for 123 hsr from 72 cases. The juxtacentromeric region of chromosome 8, band 11q13, and the whole of chromosome 17 were frequently involved. For 28 cases, the origin of the DNA sequences forming HSR could be investigated by chromosome painting, comparative genomic hybridization, and/or Southern blotting. Sequences from chromosomes 11 and 17 were mostly found within hsr located on chromosomes 11 and 17, respectively. In contrast, sequences from chromosome 8 were rarely found within hsr localized on chromosome 8. These observations suggest that different mechanisms lead to hsr formation in breast cancer. Band 11 q13 and the 17p chromosome arm may correspond to sites of in situ amplification driven by deletions distal to the amplification target genes. hsr in the region 17q2, which is also a frequent site of in situ amplification, takes place without the occurrence of a distal deletion. The short arm of chromosome 8 is often deleted, but frequently becomes the site of hsr formed elsewhere in the genome.

Prostatic intraepithelial neoplasia (PIN) and other prostatic lesions as risk factors and surrogate endpoints for cancer chemoprevention trials.

The most efficient strategy for chemoprevention clinical trials are short-term studies which focus on surrogate endpoint biomarkers (SEBs) in high-risk target populations. High-grade prostatic intraepithelial neoplasia (PIN) is the most likely precursor of prostate cancer, and is found in a significant number of routine contemporary needle biopsies without cancer. The frequency and extent of PIN are decreased with androgen deprivation therapy, suggesting that it is a suitable endpoint biomarker for modulation. Potential SEBs for screening chemopreventive agents for prostate cancer in short-term Phase II trials include (1) histologic premalignant lesions, such as high-grade PIN; (2) biochemical markers, including prostate-specific antigen (PSA) serum concentration; and (3) morphometric markers, including nuclear texture, shape, and roundness; size and number of nucleoli; and number of apoptotic bodies; (4) proliferation markers, including MIB-1 and PCNA; (5) genetic markers, including nuclear DNA content (ploidy), oncogene c-erbB-2 (HER-2/neu) expression, fluorescence in situ hybridization for chromosome 8; and PSA-producing cells in the blood detected by reverse transcriptase polymerase chain reaction; and (6) differentiation markers, such as microvessel density as a determinant of angiogenesis. Each of these endpoint biomarkers is measured easily and accurately in serum or in tissue specimens such as formalin-fixed, paraffin-embedded needle biopsies, and may be modifiable by intervention. The clinical utility of these biomarkers as modulatable endpoints in prostate cancer chemoprevention needs to be demonstrated in future clinical trials.

[Structural characteristics of the human lipoprotein lipase gene]. / Données structurales sur le gène de la lipoprotéine lipase (LPL) humaine.

Lipoprotein lipase plays a major role in the metabolism of circulating triglyceride-rich lipoproteins. In relation with this study, the fundamental results concerning the structure of human LPL gene are first summarized. Sequencing of this gene enabled us to characterize an Alu sequence. Interest of these repetitive sequences is exposed.