Shwachman-Diamond syndrome with clonal interstitial deletion of the long arm of chromosome 20 in bone marrow: haematological features, prognosis and genomic instability.

In Shwachman-Diamond syndrome (SDS), deletion of the long arm of chromosome 20, del(20)(q), often acquired in bone marrow (BM), may imply a lower risk of developing myelodysplastic syndrome/acute myeloid leukaemia (MDS/AML), due to the loss of the EIF6 gene. The genes L3MBTL1 and SGK2, also on chromosome 20, are in a cluster of imprinted genes, and their loss implies dysregulation of BM function. We report here the results of array comparative genomic hybridization (a-CGH) performed on BM DNA of six patients which confirmed the consistent loss of EIF6 gene. Interestingly, array single nucleotide polymorphisms (SNPs) showed copy neutral loss of heterozygosity for EIF6 region in cases without del(20)(q). No preferential parental origin of the deleted chromosome 20 was detected by microsatellite analysis in six SDS patients. Our patients showed a very mild haematological condition, and none evolved into BM aplasia or MDS/AML. We extend the benign prognostic significance of del(20)(q) and loss of EIF6 to the haematological features of these patients, consistently characterized by mild hypoplastic BM, no or mild neutropenia, anaemia and thrombocytopenia. Some odd results obtained in microsatellite and SNP-array analysis demonstrate a peculiar genomic instability, in an attempt to improve BM function through the acquisition of the del(20)(q).

Parental origin of the deletion del(20q) in Shwachman-Diamond patients and loss of the paternally derived allele of the imprinted L3MBTL1 gene.

Shwachman-Diamond syndrome (SDS) (OMIM 260400) is a rare autosomal recessive disease characterized by exocrine pancreatic insufficiency, skeletal, and hematological abnormalities and bone marrow (BM) dysfunction. Mutations in the SBDS gene cause SDS. Clonal chromosome anomalies are often present in BM, i(7)(q10) and del(20q) being the most frequent ones. We collected 6 SDS cases with del(20q): a cluster of imprinted genes, including L3MBTL1 and SGK2 is present in the deleted region. Only the paternal allele is expressed for these genes. Based on these data, we made the hypothesis that the loss of this region, in relation to parental origin of deletion, may be of relevance for the hematological phenotype. By comparing hematological data of our 6 cases with a group of 20 SDS patients without evidence of del(20q) in BM, we observed a significant difference for Hb levels (P < 0.012), and a difference slightly above the significance level for RBC counts (P < 0.053): in both cases the values were higher in patients with del(20q). We also report preliminary evidence for an increased number of BFU-E colonies in cases with paternal deletion, data on the presence of the deletion in colonies and in mature circulating lymphocytes. © 2016 Wiley Periodicals, Inc.

Bioarchaeological and paleogenomic profiling of the unusual Neolithic burial from Grotta di Pietra Sant'Angelo (Calabria, Italy).

The Neolithic burial of Grotta di Pietra Sant'Angelo (CS) represents a unique archaeological finding for the prehistory of Southern Italy. The unusual placement of the inhumation at a rather high altitude and far from inhabited areas, the lack of funerary equipment and the prone deposition of the body find limited similarities in coeval Italian sites. These elements have prompted wider questions on mortuary customs during the prehistory of Southern Italy. This atypical case requires an interdisciplinary approach aimed to build an integrated bioarchaeological profile of the individual. The paleopathological investigation of the skeletal remains revealed the presence of numerous markers that could be associated with craft activities, suggesting possible interpretations of the individual's lifestyle. CT analyses, carried out on the maxillary bones, showed the presence of a peculiar type of dental wear, but also a good density of the bone matrix. Biomolecular and micromorphological analyses of dental calculus highlight the presence of a rich Neolithic-like oral microbiome, the composition of which is consistent with the presence pathologies. Finally, paleogenomic data obtained from the individual were compared with ancient and modern Mediterranean populations, including unpublished high-resolution genome-wide data for 20 modern inhabitants of the nearby village of San Lorenzo Bellizzi, which provided interesting insights into the biodemographic landscape of the Neolithic in Southern Italy.

Donor Cell Acute Myeloid Leukemia after Hematopoietic Stem Cell Transplantation for Chronic Granulomatous Disease: A Case Report and Literature Review.

The patient reported here underwent hematopoietic stem cell transplantation (HSCT) due to chronic granulomatous disease (CGD) caused by biallelic mutations of the NCF1 gene. Two years later, he developed AML, which was unexpected and was recognized via sex-mismatched chromosomes as deriving from the donor cells; the patient was male, and the donor was his sister. Donor cell leukemia (DCL) is very rare, and it had never been reported in patients with CGD after HSCT. In the subsequent ten years, the AML relapsed three times and the patient underwent chemotherapy and three further HSCTs; donors were the same sister from the first HSCT, an unrelated donor, and his mother. The patient died during the third relapse. The DCL was characterized since onset by an acquired translocation between chromosomes 9 and 11, with a molecular rearrangement between the MLL and MLLT3 genes-a quite frequent cause of AML. In all of the relapses, the malignant clone had XX sex chromosomes and this rearrangement, thus indicating that it was always the original clone derived from the transplanted sister's cells. It exhibited the ability to remain quiescent in the BM during repeated chemotherapy courses, remission periods and HSCT. The leukemic clone then acquired different additional anomalies during the ten years of follow-up, with cytogenetic results characterized both by anomalies frequent in AML and by different, non-recurrent changes. This type of cytogenetic course is uncommon in AML.

Ubiquitin-proteasome-rich cytoplasmic structures in neutrophils of patients with Shwachman-Diamond syndrome.

BACKGROUND: Shwachman-Diamond syndrome is an autosomal recessive disorder in which severe bone marrow dysfunction causes neutropenia and an increased risk of leukemia. Recently, novel particulate cytoplasmic structures, rich in ubiquitinated and proteasomal proteins, have been detected in epithelial cells and neutrophils from patients with Helicobacter pylori gastritis and several epithelial neoplasms. DESIGN AND METHODS: Blood neutrophils from 13 cases of Shwachman-Diamond syndrome - ten with and three without SBDS gene mutation - and ten controls were investigated by confocal microscopy and ultrastructural immunocytochemistry using antibodies against ubiquitinated proteins, proteasomes, p62 protein, and Helicobacter pylori VacA, urease and outer membrane proteins. RESULTS: Many extensively disseminated particulate cytoplasmic structures, accounting for 22.78 ± 5.57% (mean ± standard deviation) of the total cytoplasm, were found in blood neutrophils from mutated Shwachman-Diamond syndrome patients. The particulate cytoplasmic structures showed immunoreactivity for polyubiquitinated proteins and proteasomes, but no reactivity for Helicobacter pylori products, which are present in particulate cytoplasmic structures of Helicobacter pylori-positive gastritis. Neutrophils from patients with Shwachman-Diamond syndrome frequently showed p62-positive autophagic vacuoles and apoptotic changes in 5% of cells. No particulate cytoplasmic structures were observed in most control neutrophils; however, in a few cells from two cases we noted focal development of minute particulate cytoplasmic structures, accounting for 0.74 ± 0.56% of the total cytoplasm (P<0.001 versus particulate cytoplasmic structures from mutated Shwachman-Diamond syndrome patients). Neutrophils from non-mutated Shwachman-Diamond-syndrome-like patients resembled controls in two cases, and a third case showed particulate cytoplasmic structure patterns intermediate between those in controls and those in mutated Shwachman-Diamond syndrome patients. CONCLUSIONS: Particulate cytoplasmic structures are a prominent feature of neutrophils from patients with Shwachman-Diamond syndrome. They may help us to understand the mechanism of granulocyte dysfunction and the neoplastic risk of the disease.

Autologous bone marrow-derived mesenchymal stromal cells in the treatment of fistulising Crohn's disease.

OBJECTIVE: External fistulas represent a disabling manifestation of Crohn's disease with a difficult curability and a high relapse rate despite a large therapeutic armamentarium. Stem cell therapy is a novel and promising approach for treatment of chronic inflammatory conditions. We therefore investigated the feasibility, safety and efficacy of serial intrafistular injections of autologous bone marrow-derived mesenchymal stromal cells (MSCs) in the treatment of fistulising Crohn's disease. PATIENTS AND METHODS: We enrolled 12 consecutive outpatients (eight males, median age 32 years) refractory to or unsuitable for current available therapies. MSCs were isolated from bone marrow and expanded ex vivo to be used for both therapeutic and experimental purposes. Ten patients (two refused) received intrafistular MSC injections (median 4) scheduled every 4 weeks, and were monitored by surgical, MRI and endoscopic evaluation for 12 months afterwards. The feasibility of obtaining at least 50×106 MSCs from each patient, the appearance of adverse events, and the efficacy in terms of fistula healing and reduction of both Crohn's disease and perianal disease activity indexes were evaluated. In addition, the percentage of both mucosal and circulating regulatory T cells expressing FoxP3, and the ability of MSCs to influence mucosal T cell apoptosis were investigated. RESULTS: MSC expansion was successful in all cases; sustained complete closure (seven cases) or incomplete closure (three cases) of fistula tracks with a parallel reduction of Crohn's disease and perianal disease activity indexes (p < 0.01 for both), and rectal mucosal healing were induced by treatment without any adverse effects. The percentage of mucosal and circulating regulatory T cells significantly increased during the treatment and remained stable until the end of follow up (p < 0.0001 and p < 0.01, respectively). Furthermore, MSCs have been proven to affect mucosal T cell apoptotic rate. CONCLUSIONS: Locally injected MSCs represent a feasible, safe and beneficial therapy in refractory fistulising Crohn's disease.

Phenotypic Variation in Two Siblings Affected with Shwachman-Diamond Syndrome: The Use of Expert Variant Interpreter (eVai) Suggests Clinical Relevance of a Variant in the KMT2A Gene.

Introduction. Shwachman-Diamond Syndrome (SDS) is an autosomal-recessive disorder characterized by neutropenia, pancreatic exocrine insufficiency, skeletal dysplasia, and an increased risk for leukemic transformation. Biallelic mutations in the SBDS gene have been found in about 90% of patients. The clinical spectrum of SDS in patients is wide, and variability has been noticed between different patients, siblings, and even within the same patient over time. Herein, we present two SDS siblings (UPN42 and UPN43) carrying the same SBDS mutations and showing relevant differences in their phenotypic presentation. Study aim. We attempted to understand whether other germline variants, in addition to SBDS, could explain some of the clinical variability noticed between the siblings. Methods. Whole-exome sequencing (WES) was performed. Human Phenotype Ontology (HPO) terms were defined for each patient, and the WES data were analyzed using the eVai and DIVAs platforms. Results. In UPN43, we found and confirmed, using Sanger sequencing, a novel de novo variant (c.10663G > A, p.Gly3555Ser) in the KMT2A gene that is associated with autosomal-dominant Wiedemann−Steiner Syndrome. The variant is classified as pathogenic according to different in silico prediction tools. Interestingly, it was found to be related to some of the HPO terms that describe UPN43. Conclusions. We postulate that the KMT2A variant found in UPN43 has a concomitant and co-occurring clinical effect, in addition to SBDS mutation. This dual molecular effect, supported by in silico prediction, could help to understand some of the clinical variations found among the siblings. In the future, these new data are likely to be useful for personalized medicine and therapy for selected cases.

Case Report: Heterozygous Germline Variant in EIF6 Additional to Biallelic SBDS Pathogenic Variants in a Patient With Ribosomopathy Shwachman-Diamond Syndrome.

Background: Shwachman-Diamond syndrome (SDS) is a rare autosomal recessive ribosomopathy mainly characterized by exocrine pancreatic insufficiency, skeletal alterations, neutropenia, and a relevant risk of hematological transformation. At least 90% of SDS patients have pathogenic variants in SBDS, the first gene associated with the disease with very low allelic heterogeneity; three variants, derived from events of genetic conversion between SBDS and its pseudogene, SBDSP1, provided the alleles observed in about 62% of SDS patients. Methods: We performed a reanalysis of the available WES files of a group of SDS patients with biallelic SBDS pathogenic variants, studying the results by next bioinformatic and protein structural analysis. Parallelly, careful clinical attention was given to the patient focused in this study. Results: We found and confirmed in one SDS patient a germline heterozygous missense variant (c.100T>C; p.Phe34Leu) in the EIF6 gene. This variant, inherited from his mother, has a very low frequency, and it is predicted as pathogenic, according to several in silico prediction tools. The protein structural analysis also envisages the variant could reduce the binding to the nascent 60S ribosomal. Conclusion: This study focused on the hypothesis that the EIF6 germline variant mimics the effect of somatic deletions of chromosome 20, always including the locus of this gene, and similarly may rescue the ribosomal stress and ribosomal dysfunction due to SBDS mutations. It is likely that this rescue may contribute to the stable and not severe hematological status of the proband, but a definite answer on the role of this EIF6 variant can be obtained only by adding a functional layer of evidence. In the future, these results are likely to be useful for selected cases in personalized medicine and therapy.

The frequent and clinically benign anomalies of chromosomes 7 and 20 in Shwachman-diamond syndrome may be subject to further clonal variations.

BACKGROUND: An isochromosome of the long arm of chromosome 7, i(7)(q10), and an interstitial deletion of the long arm of chromosome 20, del(20)(q), are the most frequent anomalies in the bone marrow of patients with Shwachman-Diamond syndrome, which is caused in most cases by mutations of the SBDS gene. These clonal changes imply milder haematological symptoms and lower risk of myelodysplastic syndromes and acute myeloid leukaemia, thanks to already postulated rescue mechanisms. RESULTS: Bone marrow from fourteen patients exhibiting either the i(7)(q10) or the del(20)(q) and coming from two large cohorts of patients, were subjected to chromosome analyses, Fluorescent In Situ Hybridization with informative probes and array-Comparative Genomic Hybridization. One patient with the i(7)(q10) showed a subsequent clonal rearrangement of the normal chromosome 7 across years. Four patients carrying the del(20)(q) evolved further different del(20)(q) independent clones, within a single bone marrow sample, or across sequential samples. One patient with the del(20)(q), developed a parallel different clone with a duplication of chromosome 3 long arm. Eight patients bore the del(20)(q) as the sole chromosomal abnormality. An overall overview of patients with the del(20)(q), also including cases already reported, confirmed that all the deletions were interstitial. The loss of material varied from 1.7 to 26.9 Mb and resulted in the loss of the EIF6 gene in all patients. CONCLUSIONS: Although the i(7)(q) and the del(20)(q) clones are frequent and clinically benign in Shwachman Diamond-syndrome, in the present work we show that they may rearrange, may be lost and then reconstructed de novo, or may evolve with independent clones across years. These findings unravel a striking selective pressure exerted by SBDS deficiency driving to karyotype instability and to specific clonal abnormalities.

Microarray expression studies on bone marrow of patients with Shwachman-Diamond syndrome in relation to deletion of the long arm of chromosome 20, other chromosome anomalies or normal karyotype.

BACKGROUND: Clonal chromosome changes are often found in the bone marrow (BM) of patients with Shwachman-Diamond syndrome (SDS). The most frequent ones include an isochromosome of the long arm of chromosome 7, i (7)(q10), and an interstitial deletion of the long arm of chromosome 20, del (20)(q). These two imbalances are mechanisms of somatic genetic rescue. The literature offers few expression studies on SDS. RESULTS: We report the expression analysis of bone marrow (BM) cells of patients with SDS in relation to normal karyotype or to the presence of clonal chromosome anomalies: del (20)(q) (five cases), i (7)(q10) (one case), and other anomalies (two cases). The study was performed using the microarray technique considering the whole transcriptome (WT) and three gene subsets selected as relevant in BM functions. The expression patterns of nine healthy controls and SDS patients with or without chromosome anomalies in the bone marrow showed clear differences. CONCLUSIONS: There is a significant difference between gene expression in the BM of SDS patients and healthy subjects, both at the WT level and in the selected gene sets. The deletion del (20)(q), with the EIF6 gene consistently lost, even in patients with the smallest losses of material, changes the transcription pattern: a low proportion of abnormal cells led to a pattern similar to SDS patients without acquired anomalies, whereas a high proportion yields a pattern similar to healthy subjects. Hence, the benign prognostic value of del (20)(q). The case of i (7)(q10) showed a transcription pattern similar to healthy subjects, paralleling the positive prognostic role of this anomaly as well.

The route to development of myelodysplastic syndrome/acute myeloid leukaemia in Shwachman-Diamond syndrome: the role of ageing, karyotype instability, and acquired chromosome anomalies.

An investigation of 22 new patients with Shwachman-Diamond syndrome (SDS) and the follow-up of 14 previously reported cases showed that (i) clonal chromosome changes of chromosomes 7 and 20 were present in the bone marrow (BM) of 16 out of 36 cases, but if non-clonal changes were taken into account, the frequency of anomalies affecting these chromosomes was 20/36: a specific SDS karyotype instability was thus confirmed; (ii) the recurrent isochromosome i(7)(q10) did not include short arm material, whereas it retained two arrays of D7Z1 alphoid sequences; (iii) the deletion del(20)(q11) involved the minimal region of deletion typical of myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML); (iv) only one patient developed MDS, during the rapid expansion of a BM clone with a chromosome 7 carrying additional material on the short arms; (v) the acquisition of BM clonal chromosome anomalies was age-related. We conclude that karyotype instability is part of the natural history of SDS through a specific mutator effect, linked to lacking SBDS protein, with consequent clonal anomalies of chromosomes 7 and 20 in BM, which may eventually promote MDS/AML with the patients' ageing.

Dietary treatment in adult-onset type II glycogenosis.

Eight patients with adult-onset type II glycogenosis (GSD II), all carrying the IVS1-13T

Familial myelodysplastic syndromes, monosomy 7/trisomy 8, and mutator effects.

A family is reported, in which two sisters presented with myelodysplastic syndrome (MDS), namely refractory anemia with excess of blasts in transformation (RAEB-t), and refractory anemia (RA). Bone marrow chromosome changes were present in both: trisomy and tetrasomy 8 (with a pericentric inversion of one chromosome 8) in the older sister, and monosomy 7 (with clones with additional trisomies 19 and 21) in the younger one. Molecular data were obtained on the parental chromosome involved in these numerical anomalies, which proved to be of paternal origin in these cases. The observations of this family, and a review of familial cases of MDS/acute myeloid leukemia (AML), led us to consider that they may be divided into two groups: those which arise on the basis of a Mendelian predisposing disorder exerting a mutator effect, often with the acquisition of monosomy 7, and those in which no specific Mendelian predisposing disease is recognized, as the familial monosomy 7 cases and the one reported here. We postulate that in these families an inherited mutator effect is present and that it causes a karyotype instability, which leads to MDS/AML, often through the acquisition of monosomy 7 and trisomy 8.

Living-floors and structures from the Lower Paleolithic to the Bronze Age in Italy.

New researches have been performed on the analysis of some Italian dwelling structures dating from the Lower Paleolithic to Bronze Age. Different methods have been applied to each study according to the extensions of the areas explored. The following sites have been analyzed: Isernia La Pineta (Molise), Visogliano (Trieste) - Lower Paleolithic; Grotta del Cavallo (Lecce), Grotta Grande and Riparo del Molare (Salerno) - Middle Paleolithic; Grotta di Fumane (Verona), Riparo Tagliente (Verona), Grotta Continenza (Fucino L'Aquila), San Bartolomeo (Maiella Mountain, Abruzzo) - Upper Paleolithic; Mondeval de Sora (Belluno), Alpe Veglia (Verbania) and Grotta Edera (Aurisina, Trieste) -Mesolithic; Cala Giovanna Piano (Pianosa Island, Livorno), Contraguda (Perfugas, Sassari), Colle Santo Stefano (Fucino, L'Aquila), Catignano (Pescara), Settefonti (L'Aquila) - Neolithic; Castellaro Lagusello (Monzambano, Mantua) - Bronze Age.

Hereditary Hemorrhagic Telangiectasia: Breakpoint Characterization of a Novel Large Deletion in ACVRL1 Suggests the Causing Mechanism.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular dysplasia. Mutations in either ENG or ACVRL1 account for around 85% of cases, and 10% are large deletions and duplications. Here we present a large novel deletion in ACVRL1 gene and its molecular characterization in a 3 generation Italian family. We employed short tandem repeats (STRs) analysis, direct sequencing, multiplex ligation-dependant probe amplification (MLPA) analysis, and 'deletion-specific' PCR methods. STRs Analysis at ENG and ACVRL1 loci suggested a positive linkage for ACVRL1. Direct sequencing of this gene did not identify any mutations, while MLPA identified a large deletion. These results were confirmed and exactly characterized with a 'deletion-specific' PCR: the deletion size is 4,594 bp and breakpoints in exon 3 and intron 8 show the presence of short direct repeats of 7 bp [GCCCCAC]. We hypothesize, as causative molecular mechanism, the replication slippage model. Understanding the fine mechanisms associated with genomic rearrangements may indicate the nonrandomness of these events, highlighting hot spots regions. The complete concordance among MLPA, STRs analysis and 'deletion-specific PCR' supports the usefulness of MLPA in HHT molecular analysis.