Marco Cipolli

Evaluation of energy metabolism and calcium homeostasis in cells affected by Shwachman-Diamond syndrome.

Isomorphic mutation of the SBDS gene causes Shwachman-Diamond syndrome (SDS). SDS is a rare genetic bone marrow failure and cancer predisposition syndrome. SDS cells have ribosome biogenesis and their protein synthesis altered, which are two high-energy consuming cellular processes. The reported changes in reactive oxygen species production, endoplasmic reticulum stress response and reduced mitochondrial functionality suggest an energy production defect in SDS cells. In our work, we have demonstrated that SDS cells display a Complex IV activity impairment, which causes an oxidative phosphorylation metabolism defect, with a consequent decrease in ATP production. These data were confirmed by an increased glycolytic rate, which compensated for the energetic stress. Moreover, the signalling pathways involved in glycolysis activation also appeared more activated; i.e. we reported AMP-activated protein kinase hyper-phosphorylation. Notably, we also observed an increase in a mammalian target of rapamycin phosphorylation and high intracellular calcium concentration levels ([Ca2+]i), which probably represent new biochemical equilibrium modulation in SDS cells. Finally, the SDS cell response to leucine (Leu) was investigated, suggesting its possible use as a therapeutic adjuvant to be tested in clinical trials.

Incidence of Shwachman–Diamond syndrome

To the Editor: Shwachman–Diamond syndrome (SDS) (OMIM 260400) is a rare autosomal recessive disease first reported in 1964 [1,2]. Diagnostic criteria have been updated [3] to cope with a highly variable phenotype including abnormalities in exocrine pancreas, bones and bone marrow [4–6], an increased risk for myelodisplastic syndrome and leukemia and almost specific bone marrow chromosome abnormalities [7]. Data concerning the incidence of the disorder, all obtained before the identification of the SBDS gene [8], are scanty and outdated. Kent et al. [9] reported an incidence of 1/200,000; in 1995 a slightly higher estimate, 1/150,000, was suggested by LozadaMunoz and Del Pilar Aliaga, as cited by Cipolli [10]. Goobie et al. [11] estimated the incidence of SDS as 1/76,563. The collaboration of the laboratories performing molecular diagnoses of SDS in Italy (A.M., E.N.) allowed us to collect the birth dates for all Italian patients in whom the presence of two mutations in the SBDS gene was confirmed. We are well aware that some cases with a clinical diagnosis of SDS may have not been studied by molecular analyses, so that our ascer-

Italian Children Go to School with a Hydration Deficit

Background and aims: Fluid requirements of children vary as a function of gender and age. To our knowledge, there is very little literature on the hydration status of Italian children. We assessed morning hydration status in a large sample of 515 Italian school children aged 9 to 11 years. Methods: Recruited children completed a questionnaire on fluid and food intake at breakfast and collected a urine sample the very same day after breakfast. Breakfast food and fluid nutritional composition was analysed and urine osmolality was measured using a cryoscopic osmometer. Results: More than two thirds of the children had urine osmolality above 800 mOsmol/kg while 35.0% had urine osmolality over 1000 mOsmol/kg. This was more frequent in boys than in girls (71.9% versus 62.5%; p=0.02). Total water intake (water coming from both food and fluid) as well as total fluid intake at breakfast were significantly and inversely correlated with urine osmolality. Conclusions: Almost two thirds of the children in this large cohort had evidence of a hydration deficit when they went to school in the morning, despite breakfast intake. Children’s fluid intake at breakfast does not suffice to maintain an adequate hydration status for the whole morning.

Diffuse alterations in grey and white matter associated with cognitive impairment in Shwachman-Diamond syndrome: Evidence from a multimodal approach

Shwachman–Diamond syndrome is a rare recessive genetic disease caused by mutations in SBDS gene, at chromosome 7q11. Phenotypically, the syndrome is characterized by exocrine pancreatic insufficiency, bone marrow dysfunction, skeletal dysplasia and variable cognitive impairments. Structural brain abnormalities (smaller head circumference and decreased brain volume) have also been reported. No correlation studies between brain abnormalities and neuropsychological features have yet been performed. In this study we investigate neuroanatomical findings, neurofunctional pathways and cognitive functioning of Shwachman–Diamond syndrome subjects compared with healthy controls. To be eligible for inclusion, participants were required to have known SBDS mutations on both alleles, no history of cranial trauma or any standard contraindication to magnetic resonance imaging. Appropriate tests were used to assess cognitive functions. The static images were acquired on a 3 × 0 T magnetic resonance scanner and blood oxygen level-dependent functional magnetic resonance imaging data were collected both during the execution of the Stroop task and at rest. Diffusion tensor imaging was used to assess brain white matter. The Tract-based Spatial Statistics package and probabilistic tractography were used to characterize white matter pathways. Nine participants (5 males), half of all the subjects aged 9–19 years included in the Italian Shwachman–Diamond Syndrome Registry, were evaluated and compared with nine healthy subjects, matched for sex and age. The patients performed less well than norms and controls on cognitive tasks (p = 0.0002). Overall, cortical thickness was greater in the patients, both in the left (+10%) and in the right (+15%) hemisphere, significantly differently increased in the temporal (left and right, p = 0.04), and right parietal (p = 0.03) lobes and in Brodmann area 44 (p = 0.04) of the right frontal lobe. The greatest increases were observed in the left limbic-anterior cingulate cortex (≥43%, p < 0.0004). Only in Brocas area in the left hemisphere did the patients show a thinner cortical thickness than that of controls (p = 0.01). Diffusion tensor imaging showed large, significant difference increases in both fractional anisotropy (+37%, p < 0.0001) and mean diffusivity (+35%, p < 0.005); the Tract-based Spatial Statistics analysis identified six abnormal clusters of white matter fibres in the fronto-callosal, right fronto-external capsulae, left fronto-parietal, right pontine, temporo-mesial and left anterior–medial–temporal regions. Brain areas activated during the Stroop task and those active during the resting state, are different, fewer and smaller in patients and correlate with worse performance (p = 0.002). Cognitive impairment in Shwachman–Diamond syndrome subjects is associated with diffuse brain anomalies in the grey matter (verbal skills with BA44 and BA20 in the right hemisphere; perceptual skills with BA5, 37, 20, 21, 42 in the left hemisphere) and white matter connectivity (verbal skills with alterations in the fronto-occipital fasciculus and with the inferior-longitudinal fasciculus; perceptual skills with the arcuate fasciculus, limbic and ponto-cerebellar fasciculus; memory skills with the arcuate fasciculus; executive functions with the anterior cingulated and arcuate fasciculus).

Further characterization of Shwachman–Diamond syndrome: Psychological functioning and quality of life in adult and young patients

To assess psychosocial functioning and quality of life in a representative group of adult and young patients with Shwachman–Diamond syndrome (SDS), all patients 3 years old and over included in the Italian SDS Registry were investigated using an ad‐hoc questionnaire for information about demography, education, socialization, rehabilitation therapy, and standardized questionnaires [SF‐36, Child Behavior Check‐List (CBCL)] for quality of life and behavior. Results were compared with those of a Cystic Fibrosis (CF) patient group, matched for age and sex. Eighty‐one percent of patients answered. All but one adult patient lived with their parents, 24% had independent income, and 57% had a drivers license. Different levels (from mild to severe) of cognitive impairment were reported by 76% of the adults and by 65% of the young patients. These data are significantly lower than those of the CF group. Both groups present low scores in the emotional and mental health evaluations at SF‐36, but SDS patients reported significantly more limitations in physical functioning (PF) and more body pain (BP) experiences. As reported by parents at CBCL, young SDS patients show more “social problems” (in the clinical area 31% SDS vs. 6% CF), “attention deficits disorder” (29% SDS vs. 0%CF), and “somatic complaints” (24% SDS vs. 12% CF). Psychosocial functioning is impaired in the majority of SDS patients, significantly more than in patients affected by CF.

New insights into the Shwachman-Diamond Syndrome-related haematological disorder: hyper-activation of mTOR and STAT3 in leukocytes

Shwachman-Diamond syndrome (SDS) is an inherited disease caused by mutations of a gene encoding for SBDS protein. So far little is known about SBDS exact function. SDS patients present several hematological disorders, including neutropenia and myelodysplastic syndrome (MDS), with increased risk of leukemic evolution. So far, the molecular mechanisms that underlie neutropenia, MDS and AML in SDS patients have been poorly investigated. STAT3 is a key regulator of several cellular processes including survival, differentiation and malignant transformation. Moreover, STAT3 has been reported to regulate neutrophil granulogenesis and to induce several kinds of leukemia and lymphoma. STAT3 activation is known to be regulated by mTOR, which in turn plays an important role in cellular growth and tumorigenesis. Here we show for the first time, to the best of our knowledge, that both EBV-immortalized B cells and primary leukocytes obtained from SDS patients present a constitutive hyper-activation of mTOR and STAT3 pathways. Interestingly, loss of SBDS expression is associated with this process. Importantly, rapamycin, a well-known mTOR inhibitor, is able to reduce STAT3 phosphorylation to basal levels in our experimental model. A novel therapeutic hypothesis targeting mTOR/STAT3 should represent a significant step forward into the SDS clinical practice.

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.

Immunophenotypic analysis of hematopoiesis in patients suffering from Shwachman-Bodian-Diamond Syndrome.

Shwachman–Diamond syndrome is a rare disorder characterized by exocrine pancreatic insufficiency, skeletal abnormalities, and bone marrow failure, with high risk of leukemic evolution. The aim of the study was the immunophenotypic characterization of bone marrow cells from patients with Shwachman–Diamond syndrome to assess the maturation pathway of blood progenitor cells and to identify the presence of recurrent abnormalities.

Structural variation in SBDS gene, with loss of exon 3, in two Shwachman-Diamond patients.

Shwachman-Diamond Syndrome (SDS) is an autosomal recessive disease (#260400) characterized by pancreatic exocrine insufficiency, hematological abnormalities, bone marrow failure with increased risk for acute myelodysplasia and acute myeloid leukaemia and skeletal alterations. As suggested by international guidelines [1], the SDS clinical diagnosis is confirmed by detection of bi-allelic pathogenic variants in SBDS gene, localized at 7q11.21 [2,3]. Targeted mutation analysis of exon 2 by PCR-RFLP discloses in at least 90% of SDS patients one of the three common mutations, (c.185_184TANCT, c.258+2TNC and the combination of [c.183_184TANCT:258+2TNC] on one allele). Two of the commonmutations are observed concomitantly in approximately 62% of SDS patients [3]. In our laboratories, since 2003, we performed SBDSmolecular analysis confirming the clinical diagnosis in 115 patients, and all of them harboured at least one of the common mutations in exon 2. We recently found two families, each of themwith two affected children, identified with their Unique Patient Number (UPN) as UPN 42, UPN 43 and UPN 61, UPN 76, carrying only the [c.258+2TNC]mutation; no other DNA changes was found after complete sequencing analysis of all five exons of SBDS and their flanking intronic regions. In both families, the clinical picture of the patients completely fits the diagnostic criteria: in all of them, at clinical diagnosis, both hematological manifestations (neutropenia, thrombocytopenia and hypocellular bone marrow) and exocrine pancreas dysfunction (with reduced levels of fecal elastase and serum lipase) were present. No patient presented skeletal defects but a growth delay was a common symptom. UPN 61 underwent bone marrow transplantation at age 2 following an EBV infection requiring blood transfusions. Western-blot analysis using SBDS antibody did not demonstrate any SBDS protein in the three buffy coats available, suggesting the presence of a more complex change on the second allele (Fig. 1). On genomicDNAof the four patientswe performed a deletion/duplication analysis by long-range PCRwith the specific primers designed by Boocock et al. [2], but matching them differently to obtain longer amplicons containing more exons. When using the primers [forward for exon 2 and reverse for exon 4], that amplify the region from exon 2 to exon 4, we observed in all patients the expected band of 3756bp and the presence of a second smaller band of approximately 3000bp (Fig. 2). We then extracted the DNA from the smaller band and used it as template in a new long-range PCR experiment, with the same primers.

Absence of acquired copy number neutral loss of heterozygosity (CN-LOH) of chromosome 7 in a series of 10 patients with Shwachman-Diamond syndrome

We report that acquired copy number neutral loss of heterozygosity (CN-LOH) of chromosome 7 was not identified in a series of 10 patients with Shwachman–Diamond syndrome (SDS). Shwachman–Diamond syndrome (Online Mendelian Inheritance in Man reference 260400) is a rare autosomal recessive disease first reported in 1964 (Bodian et al,1964; Shwachman et al,1964) and characterized by exocrine pancreatic insufficiency, skeletal abnormalities and bone marrow (BM) dysfunction with an increased risk to develop myelodysplastic syndrome and/or acute myeloid leukaemia (MDS/ AML). Almost 90% of SDS cases are caused by two common mutations, c. [183_184TA>CT] and c.[258 + 2T>C], in exon 2 of the SBDS gene, localized on chromosome 7. Clonal chromosome anomalies are often found in the BM of SDS patients; the most frequent are an isochromosome for long arms of chromosome 7, i(7)(q10) and an interstitial deletion of long arms of chromosome 20, del(20)(q11) (Maserati et al, 2009). An earlier study analysed BM DNA of eight cases with the i(7)(q10) and demonstrated that all of them carried a double dose of the c.[258 + 2T>C] (Minelli et al, 2009). This result suggested that, as the c.[258 + 2T>C] mutation still allows the production of some amount of normal protein (Austin et al, 2005), this could contribute to the low incidence of MDS/AML observed in this subset of SDS patients. Recently, Parikh et al (2012) described acquired CN-LOH for most of 7q in a patient with SDS, also showing that the clone of BM cells with CN-LOH contained two copies of the gene with the c.[258 + 2T>C] mutation. The presence of the CN-LOH in BM cells mimics the presence of i(7)(q10): both mechanisms produce a duplication of the c.[258 + 2T>C] mutation, together with the probable associated positive effects. Parikh et al (2012) also outlined that the genetic variation of the CN-LOH could provide an explanation for clonal expansion of the affected haematopoietic progenitor cell. Consequently, we investigated if, to provide the cell with an extra copy of the c.[258 + 2T>C] mutation, this could be a common mechanism in addition to the typical i(7)(q10) frequently observed in BM from SDS patients. We collected BM samples from 10 SDS patients in whom the cytogenetic analysis demonstrated a normal karyotype (eight cases) or the presence of the 20q deletion (two cases). The genotypes were c.[258 + 2T>C]/[183_184TA>CT] in seven cases and c.[258 + 2T>C]/[others] in three cases. Morphological analysis of the BM aspirate, available for six patients, showed normal or hypoplastic cellularity in three, and normal or hyperplastic cellularity with minimal decrease of erythropoiesis, megakaryopoiesis and myelopoiesis in the remaining three. These data are in keeping with the karyotype-phenotype correlation discussed by Pressato et al (2012). Microsatellite analysis, performed as reported in Minelli et al (2009), demonstrated a normal dosage between paternal and maternal alleles, excluding the presence of uniparental disomy related to chromosome 7, in all BM samples examined (Table I and Fig 1A). The method is able to identify the presence of the chromosomal anomaly if it is present in at least 20% of cells (Minelli et al, 2009). To verify the reliability of the method, we sequenced exon 2 of the SBDS gene from the BM of two SDS patients with 50–70% cells with i(7)(q10) and, as expected, found that the mutant peak (C) was higher than the wild-type peak (T) (Fig 1B). Exon 2 of the SBDS gene was then sequenced using BM and peripheral blood samples and, in all cases analysed, the mutant peak (C) and the wild-type peak (T) for the c.[258 + 2T>C] mutation were exactly the same height in both samples (Fig 1C) whereas Parikh et al (2012) observed an higher peak for the mutated allele when BM and fibroblasts were compared. Thus, no evidence for CN-LOH was found in any of the patients studied. If the hypothesis that an increased level of the c.[258 + 2T>C] mutation on a SDS background (c.[183_184TA>CT]/c.[258 + 2T>C]) provides a selective advantage, we would expect to identify cases in whom a clone showing a (partial) loss of the c.[183_184TA>CT] or an homozygous c.[258 + 2T>C] is present.