Maria Francesca Piras

Aims: Recently a subset of mantle cell lymphoma (MCL) with predominant extranodal disease and frequent involvement of mucosa‐associated lymphoid tissues (MALT‐MCL) has been described, showing a more indolent course and favourable prognosis than classical nodal MCL (CN‐MCL). Herein we detail the clinicopathological features of a single centre series of 12 MCL patients with primary MALT presentation, including immunogenetic profile and mutational landscape. Methods and results: The most frequently affected extranodal sites were nasopharynx and Waldeyer's ring (five patients, 41.7%), gastrointestinal tract (three patients), ocular adnexa and salivary glands (two patients each), and a single case with maxillary sinus involvement. All patients were treated, achieving a complete (seven patients) or partial response (five patients). Isolated extranodal relapse occurred in three patients; three patients died of disease. At median 12‐years follow‐up, OS was 11.3 years; 10‐years OS and PFS were 65.5% and 54.7%, respectively. Waldeyer's ring involvement was associated with increased deaths for disease and shorter 10‐years OS (33.3% vs 85.7% in patients lacking Waldeyer's ring involvement). All cases carried t (11;14)(q13;q32)‐ CCND1‐IGH translocation whereas IGH clonal rearrangements were detected in eight patients; six cases, including all patients with Waldeyer's ring involvement, carried unmutated or minimally mutated rearrangements, whereas two cases involving stomach and ocular adnexa showed highly mutated rearrangements. Targeted NGS mutational analysis identified ATM mutations in two cases but did not reveal recurrent alterations involving TP53 , CDKN2A or CCND1 . Conclusions: Our data seem to confirm that MALT‐MCL shares histological, phenotypic and IGHV rearrangements with CN‐MCL, but pursues a more indolent clinical course than CN‐MCL, thus suggesting a putative different genetic pathway.

Centromeres, the chromosomal loci responsible for segregation during cell division, play a key role in genome evolution and speciation. While centromere function is highly conserved and epigenetically defined by CENP-A, the underlying DNA sequences are among the most rapidly evolving. Although mammalian centromeres are typically associated with satellite DNA, we previously showed that equids carry numerous satellite-free centromeres. Here, we investigate centromere and karyotype evolution in Tapirus indicus, a non-equid perissodactyl with exceptional karyotypic plasticity. Through CENP-A ChIP-seq analysis on the same individual for which a near-gapless diploid genome assembly generated by the Vertebrate Genome Project was available, we identify 23 canonical satellite-based centromeres, two completely satellite-free centromeres, and one centromere with a very low content of satellite tracts. The unconventional centromeres arose through centromere repositioning, thereby redefining the evolutionary prevalence of satellite-free centromeres across mammals. Comparative genomic analysis uncovers evolutionary hotspots for satellite-free centromere formation across Perissodactyla. Finally, analysis of CENP-B binding shows that T. indicus displays uncoupling between CENP-A and CENP-B, a feature previously observed only in equids. These findings reveal that high centromere plasticity is not unique to equids and support a broader model in which centromere plasticity and CENP-B uncoupling contribute to karyotype evolution in mammals.

We present telomere-to-telomere genome assemblies of a Thoroughbred horse and a donkey derived from their mule offspring. Now adopted and annotated by NCBI as reference genomes, these assemblies resolve previously inaccessible regions, including satellite arrays, duplications, and telomeres. Equids are known to exhibit an uncoupling between satellite DNA and centromeric function. The completeness of these assemblies enabled annotation of both satellite-based and satellite-free centromeres, as well as non-centromeric satellite loci, revealing notable centromeric plasticity. They also allowed detailed characterization of the variable binding domains of CENP-A—the epigenetic determinant of centromere identity—and CENP-B, whose association with CENP-A, previously considered typical based on a few model organisms, is absent in equids. Comparative analyses of satellite repeats and centromere positions provide new insights into the accelerated karyotypic reshuffling in equid evolution. These assemblies represent foundational resources for equid genomics and support ongoing initiatives such as the Equine Pangenome Project.

The transcription of human telomeres gives rise to a family of long noncoding RNAs, named TERRA. We previously showed that TERRA transcription is driven by CpG island promoters that are composed of stretches of three types of repeats. Using the human genome assembly that was available at that time, putative promoter sequences were localized at several subtelomeres. In this work, using the T2T-CHM13v2.0 human reference genome, we found that 39 out of 46 subtelomeres contain TERRA promoters and grouped them in classes depending on their organization. We then discovered 106 intrachromosomal TERRA-like promoters, adjacent to interstitial telomeric sequences (ITSs) or far away from them. Forty-seven of these promoters are flanked and may regulate the transcription of coding genes, ncRNAs or pseudogenes. Comparative sequence analysis showed that interstitial and subtelomeric promoters belong to a previously undescribed family of segmental duplications deriving from common ancestral sequences. RT-PCR experiments in seven cell lines demonstrated that TERRA transcripts can be synthesized from ITSs. TERRA expression was always low in primary fibroblasts and HeLa cells, while highly variable in the other two telomerase-positive (HT1080 and HEK293) and in the three telomerase-negative ALT cell lines (GM847, U2OS, and VA13). The analysis of RNA-seq data from U2OS, HeLa, and HEK293 cells showed that 205 ITSs were transcribed in at least one cell line. The fraction of transcribed ITSs and the level of their transcription increased with the length of the telomeric repeat stretch. Given the large number of transcribed ITSs, we propose that these loci contribute significantly to the production of the TERRA pool.

Centromeres, the chromosomal loci responsible for proper segregation during cell division, play a key role in genome evolution and speciation. While centromere function is highly conserved and epigenetically defined by CENP-A, the underlying DNA sequences are among the most rapidly evolving. Although mammalian centromeres are typically associated with satellite DNA, we previously showed that equids carry numerous satellite-free centromeres. In this study, we investigated centromere and karyotype evolution in the endangered Tapirus indicus , a non-equid Perissodactyl with exceptional karyotypic plasticity. Through CENP-A ChIP-seq analysis on the same individual for which a near-gapless diploid genome assembly was available, we identified both canonical satellite-based centromeres and three satellite-free centromeres, emerging from centromere repositioning and representing the first such centromeres described in a non-equid Perissodactyl species. Comparative genomic analysis uncovered evolutionary hotspots for satellite-free centromere formation across Perissodactyla. Finally, analysis of CENP-B binding showed that T. indicus displays uncoupling between CENP-A and CENP-B, a feature previously observed only in equids. These findings reveal that high centromere plasticity is not unique to equids and support a broader model in which centromere plasticity and CENP-B uncoupling contribute to karyotype evolution in mammals.

While the centromeric function is conserved and epigenetically specified by CENP-A, centromeric DNA, typically composed of satellite repeats, is highly divergent and rapidly evolving. In the species of the genus Equus (horses, asses and zebras), also known as equids, the numerous centromeres devoid of satellite repeats enabled us to carry out molecular analysis of centromeric chromatin establishing a unique model system for mammalian centromere biology. In this review, after a brief description of the rapid evolution of equids, we outline one of our most relevant initial discoveries: the position of CENP-A binding domains is variable among individuals giving rise to epialleles which are inherited as Mendelian traits. This positional variability was recently confirmed in human centromeres whose repetitive DNA organization could be analyzed thanks to telomere-to-telomere (T2T) genome assemblies. Another unexpected observation was that, in equids, CENP-B does not bind the centromeric core and is uncoupled from CENP-A and CENP-C. CENP-B is absent from the majority of chromosomes while the CENP-B binding DNA sequence (CENP-B box) is comprised within a satellite that is localized at pericentromeric or terminal positions. Finally, comparative molecular and cytogenetic analyses of satellite-free centromeres revealed that the birth of neocentromeres during the evolution of this genus occurred through two alternative mechanisms: centromere repositioning and Robertsonian fusion. These events played a key role in karyotype reshuffling and speciation. Investigating centromere organization in equids provided new insights into the complexity of centromere organization across the vast biodiversity of the mammalian world, where the majority of species remain understudied.

The transcription of human telomeres gives rise to a family of long noncoding RNAs, named TERRA. We previously showed that TERRA transcription is driven by CpG island promoters that are composed by stretches of three types of repeats. Using the human genome assembly that was available at that time, putative promoter sequences were localized at several subtelomeres. In this work, using the T2T-CHM13v2.0 human reference genome, we found that 39 out of 46 subtelomeres contain TERRA promoters and grouped them in classes depending on their organization. We then discovered 106 intrachromosomal TERRA-like promoters, adjacent to interstitial telomeric sequences (ITSs) or far away from them. Fortyseven of these promoters are flanked and may regulate the transcription of coding genes, ncRNAs or pseudogenes. Comparative sequence analysis showed that interstitial and subtelomeric promoters belong to a previously undescribed family of segmental duplications deriving from common ancestral sequences. RT-PCR experiments in seven cell lines demonstrated that TERRA transcripts can be synthesized from ITSs. TERRA expression was always low in primary fibroblasts and HeLa cells while highly variable in the other two telomerase positive (HT1080 and HEK293) and in the three telomerase negative ALT cell lines (GM847, U2OS and VA13). The analysis of RNA-seq data from U2OS, HeLa and HEK293 cells showed that 205 ITSs were transcribed in at least one cell lines. The fraction of transcribed ITSs and the level of their transcription increased with the length of the telomeric repeat stretch. Given the large number of transcribed ITSs, we propose that these loci contribute significantly to the production of the TERRA pool.

While CENP-A is the epigenetic determinant of the centromeric function, the role of CENP-B, a centromeric protein binding a specific DNA sequence, the CENP-B-box, remains elusive. In the few mammalian species analyzed so far, the CENP-B box is contained in the major satellite repeat that is present at all centromeres, with the exception of the Y chromosome. We previously demonstrated that, in the genus Equus, numerous centromeres lack any satellite repeat. In four Equus species, CENP-B is expressed but does not bind the majority of satellite-based centromeres, or the satellite-free ones, while it is localized at several ancestral, now-inactive, centromeres. Centromeres lacking CENP-B are functional and recruit normal amounts of CENP-A and CENP-C. The absence of CENP-B is related to the lack of CENP-B boxes rather than to peculiar features of the protein itself. CENP-B boxes are present in a previously undescribed repeat which is not the major satellite bound by CENP-A. Comparative sequence analysis suggests that this satellite was centromeric in the equid ancestor, lost centromeric function during evolution, and gave rise to a shorter CENP-A bound repeat not containing the CENP-B box but enriched in dyad symmetries. We propose that the uncoupling between CENP-B and CENP-A may have played a role in the extensive evolutionary reshuffling of equid centromeres. This study provides new insights into the complexity of centromere organization in a largely biodiverse world where the majority of mammalian species still have to be studied.

Background: While CENP-A is the epigenetic determinant of the centromeric function, the role of CENP-B, the sole centromeric protein binding a specific DNA sequence (CENP-B-box), remains elusive. In the few mammalian species analyzed so far, the CENP-B box is contained in the major satellite repeat that is present at all centromeres. We previously demonstrated that, in the genus Equus , some centromeres lack any satellite repeat. Results: Here, we show that, in four Equus species, CENP-B is expressed but does not bind the numerous satellite-free and the majority of satellite-based centromeres while it is localized at several ancestral now inactive centromeres. The absence of CENP-B is related to the lack of CENP-B boxes rather than to peculiar features of the protein itself. CENP-B boxes are comprised in a previously undescribed repeat which is not the major satellite bound by CENP-A. Comparative sequence analysis suggests that this satellite was centromeric in the equid ancestor, lost centromeric function during evolution and gave rise to a short CENP-A bound repeat not containing the CENP-B box but being enriched in dyad symmetries. Centromeres lacking CENP-B are functional and recruit normal amounts of the centromeric proteins CENP-A and CENP-C. Conclusions: We propose that the uncoupling between CENP-B and CENP-A may have played a role in the evolutionary reshuffling of equid centromeres. This study provides new insights into the complexity of centromere organization in a largely biodiverse world where the majority of mammalian species still have to be studied.