Poichiloderma con Neutropenia (PN) : una rara sindrome di predisposizione alle mielodisplasie (MDS ): definizione dello spettro clinico e di quello mutazionale del gene C16orf57 e scanning preliminare di pazienti MDS

Poikiloderma with Neutropenia (PN; OMIM#604173) is a rare autosomal recessive genodermatosis characterized by early onset poi-kiloderma and severe non cyclic neutropenia which leads to recurrent infections and bone marrow alterations predisposing to myelodisplastic syndrome (MDS) and, eventually, to acute myeloid leukaemia (AML). C16orf57 has recently been identified as causative gene for PN in our laboratory by means of next generation sequencing (NGS) techniques. 37 PN patients are reported in literature with loss of function muta-tions in C16orf57, including 10 cases clinically and molecularly char-acterized in our laboratory, six of whom are described in this work. All our patients carry biallelic mutations in C16orf57 including six splicing mutations, four non in frame deletions and one early truncat-ing mutation, most of them in homozygous state. The geographical distribution of the six most frequent recurrent muta-tions points out mutational clusters restricted to defined geographical areas suggesting a founder effect the likely hypothesis to explain mu-tation clusters restricted to specific ethnic groups. Transcripts analysis highlights that mutant transcripts are detectable even in the presence of early truncating mutations, suggesting they are relatively stable and potentially translatable in aberrant proteins. C16orf57 protein structure and function are unknown but the presence of two similarly spaced histidine containing tetrapeptides H-X-T/S-X, which identified the active site, and the bioinformatic prediction, which recognized an internally 2-fold symmetric structure organized around histidines H120 and H208, suggest that C16orf57 protein belongs to the phosphoesterase superfamily which encompasses enzymes in-teracting with RNA substrates and involved in rRNA processing. Depending on this model, the effects of the 19 described C16orf57 mutations are the loss of the correct protein folding, and/or the de-struction of the symmetric structure leading to the catalytic activity depletion. The possible involvement of C16orf57 in ribosomes synthesis and as-sembly interconnects PN to other rare inherited syndromes such as Shwachman-Diamond syndrome, Diamond-Blackfan Anemia, Disk-eratosis Congenita and Cartilage-Hair Hypolasia, grouped in the cate-gory of “Ribosomopathies” because of the role of their causative genes in ribogenesis. Indeed, all these diseases are rare MDS predisposing syndromes, as PN. Given that 6 C16orf57-positive PN patients has developed bone mar-row alterations, 10 a myelodysplastic syndrome since the second dec-ade of life and 2 AML, and that defects in ribogenesis have been re-ported for all acquired MDS subtypes and in the context or rare inher-ited syndromes, C16orf57 is a candidate gene in acquired MDS aeti-ology: actually, alterations of C16orf57 activity in controlling myeloid cells homeostasis may contribute to neoplastic transformation. To investigate whether disruption of the gene may also concur to the more common acquired myelodisplastic syndrome, a collection of 111 bone marrow DNA samples from a clinical and cytogenetically well characterized consecutive cohort including MDS, myelodysplas-tic/myeloproliferative neoplasms (MPN/MDS), AML, acquired neu-tropenia and MDS evolving from neutropenia cases has been investi-gated by direct sequencing of C16orf57. Three not reported sequence variations, all in heterozygous state, have been identified: i) c.-56G>A in the 5’UTR region, in a patient with RCMD and 8+, ii) c.450-67dupT in IVS3, in two different patients with RAEB2 and del5q and iii) c.587+21A>G in IVS4 in two patients with normal karyotype and one with MDS/MPN and the other with RAEB1. No malignant tissue analysis confirms the germline status of all variations. To assess if c.-56G>A and c.587+21A>G are mutations or susceptibility variants, a panel of 111 matched controls has been analysed. All controls are WT for c.-56G>A variant but a sample results heterozygous for c.587+21A>G suggesting it is a rare polymorphism. Even if it is a preparatory study, this work represents the first explora-tion of the possible involvement of C16orf57 in MDS aetiology. The cohort will be extend: actually, even if we have not identified causa-tive C16orf57 mutations, we can’t exclude the role of this gene in a very small subset of cases or in a particular subtype of MDS. A paedi-atric cohort of acquired MDS/AML, more similar to PN patients as age of onset, will be analysed too.

Questo lavoro è finalizzato ad espandere le conoscenze cliniche e molecolari della sindrome rara Poichilodema con Neutropenia (PN) causata da mutazioni del gene C16orf57, identificato nel 2010 nel nostro laboratorio grazie a tecniche di sequenziamento di nuova generazione. Orfano fino a tale data, il gene C16orf57, molto conservato ed espresso ubiquitariamente con consistenti livelli nelle cellule mieloidi, è risultato mutato in 37 pazienti PN comprensivi di dieci da noi caratterizzati sotto il profilo clinico e molecolare, sei dei quali sono oggetto di questa tesi. In base al coinvolgimento del compartimento ematopoietico in individui con mutazioni germinali di C16orf57, documentato dalla predisposizione allo sviluppo in giovane età di sindromi mielodisplastiche (MDS) e leucemia mieloide acuta (AML), C16orf57 è un potenziale candidato all'eziologia delle MDS acquisite attraverso difetti della sua funzione nelle cellule mieloidi. Questo razionale ha motivato l' analisi di C16orf57 in una casistica di oltre cento casi di MDS/AML che ha portato all'identificazione di 3 varianti non riportate con possibile significato di fattori di rischio. Seppur preliminare questo studio rappresenta la prima esplorazione circa il coinvolgimento di C16orf57 nell'eziologia delle MDS.