Biomedical subjects

Mariano Rocchi

Publications and source records attributed to Mariano Rocchi.

At least 37 records · Page 2Linked to original sources

PubMed2005

Progressive proximal expansion of the primate X chromosome centromere.

Previous studies of the pericentromeric region of the human X chromosome short arm (Xp) revealed an age gradient from ancient DNA that contains expressed genes to recent human-specific DNA at the functional centromere. We analyzed the finished sequence of this human genomic region to investigate its evolutionary history. Phylogenetic analysis of >1,500 alpha-satellite monomers from the region revealed the presence of five physical domains, each containing monomers from a distinct phylogenetic clade. The most distal domain contains long interspersed nucleotide element repeats that were active >35 million years ago, whereas the four proximal domains contain more recently active long interspersed nucleotide element repeats. An out-of-register, unequal recombination (i.e., crossover) detected at the edge of the X chromosome-specific alpha-satellite array (DXZ1) may reflect the most recent of a series of punctuating events during evolution that resulted in a proximal physical expansion of the X centromere. The first 18 kb of this array has 97-99% pairwise identity among all 2-kb repeat units. To perform more detailed evolutionary comparisons, we sequenced the junction between the ancient DNA of Xp and the primate-specific alpha satellite in chimpanzee, gorilla, orangutan, vervet, macaque, and baboon. The striking conservation found in all cases supports the ancestral nature of the alpha satellite at this location. These studies demonstrate that the primate X centromere appears to have evolved through repeated expansion events occurring within the central, active region of centromeric DNA, with the newly added sequences then conferring centromere function.

Animals↗

PubMed2005

Punctuated duplication seeding events during the evolution of human chromosome 2p11.

Primate genomic sequence comparisons are becoming increasingly useful for elucidating the evolutionary history and organization of our own genome. Such studies are particularly informative within human pericentromeric regions--areas of particularly rapid change in genomic structure. Here, we present a systematic analysis of the evolutionary history of one approximately 700-kb region of 2p11, including the first autosomal transition from pericentromeric sequence to higher-order alpha-satellite DNA. We show that this region is composed of segmental duplications corresponding to 14 ancestral segments ranging in size from 4 kb to approximately 115 kb. These duplicons show 94%-98.5% sequence identity to their ancestral loci. Comparative FISH and phylogenetic analysis indicate that these duplicons are differentially distributed in human, chimpanzee, and gorilla genomes, whereas baboon has a single putative ancestral locus for all but one of the duplications. Our analysis supports a model where duplicative transposition events occurred during a narrow window of evolution after the separation of the human/ape lineage from the Old World monkeys (10-20 million years ago). Although dramatic secondary dispersal events occurred during the radiation of the human, chimpanzee, and gorilla lineages, duplicative transposition seeding events of new material to this particular pericentromeric region abruptly ceased after this time period. The multiplicity of initial duplicative transpositions prior to the separation of humans and great-apes suggests a punctuated model for the formation of highly duplicated pericentromeric regions within the human genome. The data further indicate that factors other than sequence are important determinants for such bursts of duplicative transposition from the euchromatin to pericentromeric regions.

Animals↗

PubMed2005

Submicroscopic deletions in an acute myeloid leukemia case with a four-way t(8;11;16;21).

The t(8;21)(q22;q22) rearrangement is observed in about 15% of acute myelocitic leukemia (AML) cases, while variant t(8;21) translocations are detected in 6-10% of AML patients positive for the 5'RUNX1/3'CBFA2T1 fusion gene. We report a detailed molecular cytogenetic analysis of a four-way variant t(8;11;16;21)(q22;q14;q12;q22) performed by fluorescence in situ hybridization with specific BAC and PAC clones. The study demonstrated the loss of several megabases belonging to chromosomes 11 and 16 whereas no deletion was detected on der(21). These findings suggest that a precise breakpoint characterization could identify submicroscopic genomic deletions whose meaning remains to be defined.

Acute Disease↗

PubMed2005

Molecular cytogenetic study of instability at 1q21 approximately q32 in adult acute lymphoblastic leukemia.

In the present paper, we report a molecular cytogenetic study of 1q abnormalities associated with t(8;14)(q24;q32) in an adult common B acute lymphoblastic leukemia case with FAB-L2 morphology. The use of appropriate molecular cytogenetic probes allowed us to detect 13 different subclones showing heterogeneous chromosome 1 abnormalities. A complex pattern of rearrangements consisting of translocations, duplications, and inversions was observed. Breakage-fusion-bridge cycle and jumping translocation are hypothesized to have been involved in generating the large number of aberrations we detected.

Chromosome Aberrations↗

PubMed2004

Evolution of human IgH3'EC duplicated structures: both enhancers HS1,2 are polymorphic with variation of transcription factor's consensus sites.

The enhancer complex regulatory region at the 3' of the immunoglobulin heavy cluster (IgH3'EC) is duplicated in apes along with four constant genes and the region is highly conserved throughout humans. Both human IgH3'ECs consist of three loci high sensitive (HS) to DNAse I with enhancer activity. It is thus possible that the presence of structural divergences between the two IgH3'ECs and of relative polymorphisms correspond to functional regulatory changes. To analyse the polymorphisms of these almost identical regions, it resulted mandatory to identify the presence of divergent sequences, in order to select distinctive primers for specific PCR genomic amplifications. To this aim, we first compared the two entire IgH3'ECs in silicio, utilising the updated GenBank (GB) contigs, then we analysed the two IgH3'ECs by cloning and sequencing amplicons from independent genomes. In silicio analysis showed that several inversions, deletions and short insertions had occurred after the duplication. We analysed in detail, by sequencing specific regions, the polymorphisms occurring in enhancer HS1,2-A (which lies in IgH3'EC-1, 3' to the Calpha-1 gene) and in enhancer HS1,2-B (which lies in IgH3'EC-2, 3' to Calpha-2). Polymorphisms are due to the repetition (occurring one to four times) of a 38-bp sequence present at the 3' of the core of enhancers HS1,2. The structure of both human HS1,2 enhancers has revealed not yet described polymorphic features due to the presence of variable spacer elements separating the 38-bp repetitions and to variable external elements bordering the repetition cluster. We found that one of the external elements gave rise to a divergent allele 3 in the two clusters. The frequency of the different alleles of the two loci varies in the Italian population and allele 3 of both loci are very rare. The analysis of the Callicebus moloch, Gorilla gorilla and Pan troglodytes HS1,2 enhancers showed the transformation from the ancestral structure with the 31- to the 17-bp external element in hominids. The relevance of the polymorphisms in the HS1,2 enhancers is due to the variable number of binding sites for the transcription factors: NF-kappaB, CMYB, BSAP1/2, AP1/4, E47, MyoD and muE5 and thus to the possible influence of these variations on switch, production of Ig and on maturation of B cells.

Animals↗

PubMed2004

Evolutionary history of chromosome 20.

The evolutionary history of human chromosome 20 in primates was investigated using a panel of human BAC/PAC probes spaced along the chromosome. Oligonucleotide primers derived from the sequence of each human clone were used to screen horse, cat, pig, and black lemur BAC libraries to assemble, for each species, a panel of probes mapping to chromosomal loci orthologous to the loci encompassed by the human BACs. This approach facilitated marker-order comparison aimed at defining marker arrangement in primate ancestor. To this goal, we also took advantage of the mouse and rat draft sequences. The almost perfect colinearity of chromosome 20 sequence in humans and mouse could be interpreted as evidence that their form was ancestral to primates. Contrary to this view, we found that horse, macaque, and two New World monkeys share the same marker-order arrangement from which the human and mouse forms can be derived, assuming similar but distinct inversions that fully account for the small difference in marker arrangement between humans and mouse. The evolutionary history of this chromosome unveiled also two centromere repositioning events in New World monkey species.

Animals↗

PubMed2004

The structure and evolution of centromeric transition regions within the human genome.

An understanding of how centromeric transition regions are organized is a critical aspect of chromosome structure and function; however, the sequence context of these regions has been difficult to resolve on the basis of the draft genome sequence. We present a detailed analysis of the structure and assembly of all human pericentromeric regions (5 megabases). Most chromosome arms (35 out of 43) show a gradient of dwindling transcriptional diversity accompanied by an increasing number of interchromosomal duplications in proximity to the centromere. At least 30% of the centromeric transition region structure originates from euchromatic gene-containing segments of DNA that were duplicatively transposed towards pericentromeric regions at a rate of six-seven events per million years during primate evolution. This process has led to the formation of a minimum of 28 new transcripts by exon exaptation and exon shuffling, many of which are primarily expressed in the testis. The distribution of these duplicated segments is nonrandom among pericentromeric regions, suggesting that some regions have served as preferential acceptors of euchromatic DNA.

Animals↗

PubMed2004

Insertion of telomeric repeats at intrachromosomal break sites during primate evolution.

Short blocks of telomeric-like DNA (Interstitial Telomeric Sequences, ITSs) are found far from chromosome ends. We addressed the question as to how such sequences arise by comparing the loci of 10 human ITSs with their genomic orthologs in 12 primate species. The ITSs did not derive from expansion of pre-existing TTAGGG units, as described for other microsatellites, but appeared suddenly during evolution. Nine insertion events were dated along the primate evolutionary tree, the dates ranging between 40 and 6 million years ago. Sequence comparisons suggest that in each case the block of (TTAGGG)n DNA arose as a result of double-strand break repair. In fact, ancestral sequences were either interrupted precisely by the tract of telomeric-like repeats or showed the typical modifications observed at double-strand break repair sites such as short deletions, addition of random sequences, or duplications. Similar conclusions were drawn from the analysis of a chimpanzee-specific ITS. We propose that telomeric sequences were inserted by the capture of a telomeric DNA fragment at the break site or by the telomerase enzyme. Our conclusions indicate that human ITSs are relics of ancient breakage rather than fragile sites themselves, as previously suggested.

Animals↗

PubMed2004

Identification of a commonly amplified 4.3 Mb region with overexpression of C8FW, but not MYC in MYC-containing double minutes in myeloid malignancies.

Double minutes (dmin), the cytogenetic hallmark of genomic amplification, are found in approximately 1% of karyotypically abnormal acute myeloid leukemias (AML) and myelodysplastic syndromes (MDS). The MYC gene at 8q24 has been reported to be amplified in the majority of the cases, and generally it has been assumed that MYC is the target gene. However, only a few studies have focused on the extent of the amplicon or on the expression patterns of the amplified genes. We have studied six cases (five AML and one MDS) with MYC-containing dmin. Detailed fluorescence in situ hybridization analyses identified a common 4.3 Mb amplicon, with clustered proximal and distal breakpoints, harboring eight known genes (C8FW, NSE2, POU5FLC20, MYC, PVT1, AK093424, MGC27434 and MLZE). The corresponding region was deleted in one of the chromosome 8 homologues in five of the six cases, suggesting that the dmin originated through extra replication (or loop-formation)--excision--amplification. Northern blot analysis revealed that MYC was not overexpressed. Instead, the C8FW gene, encoding a phosphoprotein regulated by mitogenic pathways, displayed increased expression. These results exclude MYC as the target gene and indicate that overexpression of C8FW may be the functionally important consequence of 8q24 amplicons in AML and MDS.

Acute Disease↗

PubMed2004

A fluorescence in situ hybridization study of complex t(9;22) in two chronic myelocytic leukemia cases with a masked Philadelphia chromosome.

The t(9;22)(q34;q11) is evident in more than 90% of patients with chronic myelocytic leukemia (CML) and gives rise to the Philadelphia chromosome (Ph). Approximately 5%-10% of CML patients show variant translocations involving other chromosomes in addition to chromosomes 9 and 22. In some variant translocations, additional material is transferred on der(22), resulting in a masked Ph chromosome. In this paper, we report two apparently Ph-negative (Ph-) CML cases showing a t(7;9;22)(q22;q34;q11) and a t(8;9;22)(q12;q34;q11), respectively. A detailed molecular cytogenetic characterization was performed by fluorescence in situ hybridization (FISH), which disclosed the presence of the 5'BCR/3'ABL fusion gene on the der(7) and der(8) chromosomes, respectively. Derivative (22) appeared as a masked Ph chromosome in both cases. FISH analysis with appropriate BAC/PAC clones allowed us to precisely characterize the complex chromosomal rearrangements that were not detected by conventional cytogenetic analysis.

Chromosomes, Human, Pair 22↗

PubMed2004

A chronic myelocytic leukemia case bearing deletions on the three chromosomes involved in a variant t(9;22;11).

Genomic deletions on the derivative chromosomes bearing the reciprocal fusion gene have recently been reported in chronic myelocytic leukemia (CML). We here describe a CML case with a variant rearrangement t(9;22;11)(q34;q11;q13) showing the loss of chromosome 11 sequences in addition to der(9) deletions. Known tumor suppressor genes involved in apoptosis and in the control of cell proliferation were found to be mapped to the lost sequences. Our findings indicate that genomic deletions may occur also on the third derivative chromosome in variant t(9;22).

Chromosomes, Human, Pair 11↗

PubMed2004

Insertions generating the 5'RUNX1/3'CBFA2T1 gene in acute myeloid leukemia cases show variable breakpoints.

Translocation t(8;21)(q22;q22) is a common karyotypic abnormality detected in about 15% of acute myeloid leukemia (AML) cases. The rearrangement results in fusion of the RUNX1 (also known as AML1) and CBFA2T1 (also known as ETO) genes, generating a 5'RUNX1/3'CBFA2T1 transcriptionally active fusion gene on derivative chromosome 8, but some cases with ins(21;8) and ins(8;21) have been observed. However, a detailed breakpoint characterization of the insertion events has never been reported. In the present article, we describe six insertion events among 82 (7.3%) AML cases characterized by the RUNX1/CBFA2T1 fusion. Using FISH experiments with appropriate bacterial artificial chromosome (BAC) and P1 artificial chromosome (PAC) probes, we were able to perform a detailed molecular cytogenetic characterization of one case with ins(8;21) and five with ins(21;8). Our analysis revealed that insertions generating the 5'RUNX1/3'CBFA2T1 gene showed variable breakpoints; the size of the inserted elements ranged from 2.4 to 44 Mb.

Core Binding Factor Alpha 2 Subunit↗

PubMed2004

A novel FISH assay for SS18-SSX fusion type in synovial sarcoma.

Synovial sarcoma is a morphologically, clinically and genetically distinct entity that accounts for 5-10% of all soft tissue sarcomas. The t(X;18)(p11.2;q11.2) is the cytogenetic hallmark of synovial sarcoma and is present in more than 90% of the cases. It produces three types of fusion gene formed in part by SS18 from chromosome 18 and by SSX1, SSX2 or, rarely, SSX4 from the X chromosome. The SS18-SSX fusions do not seem to occur in other tumor types, and it has been shown that in synovial sarcoma a clear correlation exists between the type of fusion gene and histologic subtype and, more importantly, clinical outcome. Previous analyses regarding the type of fusion genes have been based on PCR amplification of the fusion transcript, requiring access to good-quality RNA. In order to obtain an alternative tool to diagnose and follow this malignancy, we developed a fluorescence in situ hybridization (FISH) assay that could distinguish between the two most common fusion genes, that is, SS18-SSX1 and SS18-SSX2. The specificity of the selected bacterial artificial chromosome clones used in the detection of these fusion genes, as well as the sensitivity of the analysis in metaphase and interphase cells, was examined in a series of 28 synovial sarcoma samples with known fusion gene status. In all samples, the type of fusion was correctly identified by FISH. Thus, the assay described here should be useful for clarifying unresolved chromosome markers and for identifying fusion gene status in samples from which RNA of sufficient quality for PCR could not be extracted.

Antigens, Neoplasm↗

PubMed2004

A double cryptic chromosome imbalance is an important factor to explain phenotypic variability in Wolf-Hirschhorn syndrome.

A total of five Wolf-Hirschhorn syndrome (WHS) patient with a 4p16.3 de novo microdeletion was referred because of genotype-phenotype inconsistencies, first explained as phenotypic variability of the WHS. The actual deletion size was found to be about 12 Mb in three patients, 5 Mb in another one and 20 Mb in the last one, leading us to hypothesize the presence of an extrachromosome segment on the deleted 4p. A der(4)(4qter --> p16.1::8p23 --> pter) chromosome, resulting from an unbalanced de novo translocation was, in fact, detected in four patients and a der(4)(4qter --> q32::4p15.3 --> qter) in the last. Unbalanced t(4;8) translocations were maternal in origin, the rec(4p;4q) was paternal. With the purpose of verifying frequency and specificity of this phenomenon, we investigated yet another group of 20 WHS patients with de novo large deletions (n = 13) or microdeletions (n = 7) and with apparently straightforward genotype-phenotype correlations. The rearrangement was paternal in origin, and occurred as a single anomaly in 19 out of 20 patients. In the remaining patient, the deleted chromosome 4 was maternally derived and consisted of a der(4)(4qter --> 4p16.3::8p23 --> 8pter). In conclusions, we observed that 20% (5/25) of de novo WHS-associated rearrangements were maternal in origin and 80% (20/25) were paternal. All the maternally derived rearrangements were de novo unbalanced t(4;8) translocations and showed specific clinical phenotypes. Paternally derived rearrangements were usually isolated deletions. It can be inferred that a double, cryptic chromosome imbalance is an important factor for phenotypic variability in WHS. It acts either by masking the actual deletion size or by doubling a quantitative change of the genome.

Abnormalities, Multiple↗

PubMed2004

Inverted duplications: how many of them are mosaic?

The best-known situation indissolubly linked to mosaicism is the uniparental disomy where a trisomic or monosomic zygote develops at least one cell line with 46 chromosomes. The mosaicism normal/abnormal cell lines may remain confined to placenta or persist in the embryo. Here, we describe a second situation that might also be indissolubly linked to a mosaic condition or at least to a confined placental mosaicism. We analysed the case of a mosaicism del(8p)/inv dup(8p) found in prenatal diagnosis. We had already demonstrated that the first product of the abnormal meiotic recombination at the basis of the inv dup rearrangements is a dicentric chromosome. Its breakage leads to the formation of a deleted and an inv dup chromosome. Although we had previously assumed that the dicentric underwent a breakage at meiosis II so that the zygote inherited the inv dup chromosome, our findings and those of others indeed indicate that the dicentric may be inherited in the zygote and that it might persist as such in early postzygotic stages, then undergoing different breakages in different cells leading to different abnormal chromosomes, either deleted or duplicated. Selection versus the most viable cell line(s) results either in a confined placental mosaicism with the inv dup cell line as the only one present in the embryo or in children with both the deleted and the inv dup cell lines. Phenotype/karyotype relationships in inv dup rearrangements must also take into account the influence of the other abnormal cell line during embryogenesis.

Aborted Fetus↗

PubMed2004

Derivative chromosome 9 deletions in chronic myeloid leukemia are associated with loss of tumor suppressor genes.

It has recently been postulated that the absence of a single tumor suppressor gene (TSG) allele can provide a selective advantage for an emerging tumor cell. We have characterized the precise extension of the deletion on der(9) in 20 chronic myeloid leukemia (CML) cases using FISH analysis with an appropriate set of BAC/PAC probes to attempt a better definition of TSGs encompassed by these genomic deletions. Chromosome 9 deletions on the der(9) were detected in 15 (75%) cases; the TSG PTGES gene was lost in 11 (73%) cases. Chromosome 22 deletions on der(9) were found in 18 (90%) of the analysed cases; two TSGs were found located inside the deleted sequences of chromosome 22: SMARCB1 and GSTT1. These TSGs were found deleted in 16 (89%) cases bearing deletions of chromosome 22. Fourteen (70%) patients were treated with IFN-alpha therapy: 12 did not obtain complete haematologic remission (CHR) and 2 were not evaluable for response. Therefore, the patients did not respond to the IFN-alpha treatment started Glivec obtaining CHR and major cytogenetic response (MCR). The observation that deletions on der(9) are associated with the loss of TSGs suggests their possible involvement in the CML outcome, mediated by a haplo-insufficiency mechanism.

Adult↗

PubMed2004

Analysis of segmental duplications and genome assembly in the mouse.

Limited comparative studies suggest that the human genome is particularly enriched for recent segmental duplications. The extent of segmental duplications in other mammalian genomes is unknown and confounded by methodological differences in genome assembly. Here, we present a detailed analysis of recent duplication content within the mouse genome using a whole-genome assembly comparison method and a novel assembly independent method, designed to take advantage of the reduced allelic variation of the C57BL/6J strain. We conservatively estimate that approximately 57% of all highly identical segmental duplications (>or=90%) were misassembled or collapsed within the working draft WGS assembly. The WGS approach often leaves duplications fragmented and unassigned to a chromosome when compared with the clone-ordered-based approach. Our preliminary analysis suggests that 1.7%-2.0% of the mouse genome is part of recent large segmental duplications (about half of what is observed for the human genome). We have constructed a mouse segmental duplication database to aid in the characterization of these regions and their integration into the final mouse genome assembly. This work suggests significant biological differences in the architecture of recent segmental duplications between human and mouse. In addition, our unique method provides the means for improving whole-genome shotgun sequence assembly of mouse and future mammalian genomes.

Animals↗

PubMed2004

Recurrent sites for new centromere seeding.

Using comparative FISH and genomics, we have studied and compared the evolution of chromosome 3 in primates and two human neocentromere cases on the long arm of this chromosome. Our results show that one of the human neocentromere cases maps to the same 3q26 chromosomal region where a new centromere emerged in a common ancestor of the Old World monkeys approximately 25-40 million years ago. Similarly, the locus in which a new centromere was seeded in the great apes' ancestor was orthologous to the site in which a new centromere emerged in the New World monkeys' ancestor. These data suggest the recurrent use of longstanding latent centromeres and that there is an inherent potential of these regions to form centromeres. The second human neocentromere case (3q24) revealed unprecedented features. The neocentromere emergence was not accompanied by any chromosomal rearrangement that usually triggers these events. Instead, it involved the functional inactivation of the normal centromere, and was present in an otherwise phenotypically normal individual who transmitted this unusual chromosome to the next generation. We propose that the formation of neocentromeres in humans and the emergence of new centromeres during the course of evolution share a common mechanism.

Animals↗