Lucia Nacci

Novel recurrent chromosome anomalies in Shwachman-Diamond syndrome.

Two chromosome anomalies are frequent in the bone marrow (BM) of patients with Shwachman–Diamond syndrome (SDS): 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 anomalies are associated with a lower risk of developing myelodysplasia (MDS) and/or acute myeloid leukemia. The chromosome anomalies may be due to an SDS‐specific karyotype instability, reflected also by anomalies that are not clonal, but found in single cells in the BM or in peripheral blood (PB).

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.

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.

Whole exome sequencing discloses heterozygous variants in the DNAJC21 and EFL1 genes but not in SRP54 in 6 out of 16 patients with Shwachman-Diamond Syndrome carrying biallelic SBDS mutations

leukemia. Leukemia, 26, 2360–2366. National Cancer Institute. (2018) SEER Cancer Statistics Review 1975-2014. Section 28. Childhood cancer by site: incidence, survival, and mortality. Available at: https://seer.cancer.gov/ csr/1975_2015/results_merged/sect_28_child hood_cancer.pdf. [Accessed Jun 26, 2018] Stam, R.W., Schneider, P., de Lorenzo, P., Valsecchi, M.G., den Boer, M.L. & Pieters, R. (2007) Prognostic significance of high-level FLT3 expression in MLL-rearranged infant acute lymphoblastic leukemia. Blood, 110, 2774–2775. Stone, R.M., Mandrekar, S.J., Sanford, B.L., Laumann, K., Geyer, S., Bloomfield, C.D., Thiede, C., Prior, T.W., D€ ohner, K., Marcucci, G., LoCoco, F., Klisovic, R.B., Wei, A., Sierra, J., Sanz, M.A., Brandwein, J.M., de Witte, T., Niederwieser, D., Appelbaum, F.R., Medeiros, B.C., Tallman, M.S., Krauter, J., Schlenk, R.F., Ganser, A., Serve, H., Ehninger, G., Amadori, S., Larson, R.A. & D€ ohner, H. (2017) Midostaurin plus chemotherapy for acute myeloid leukemia with a FLT3 mutation. The New England Journal of Medicine, 377, 454–464. Weisberg, E., Boulton, C., Kelly, L.M., Manley, P., Fabbro, D., Meyer, T., Gilliland, D.G. & Griffin, J.D. (2002) Inhibition of mutant FLT3 receptors in leukemia cells by the small molecule tyrosine kinase inhibitor PKC412. Cancer Cell, 1, 433– 443. Zwaan, C.M., Kolb, E.A., Reinhardt, D., Abrahamsson, J., Adachi, S., Aplenc, R., De Bont, E.S., De Moerloose, B., Dworzak, M., Gibson, B.E., Hasle, H., Leverger, G., Locatelli, F., Ragu, C., Ribeiro, R.C., Rizzari, C., Rubnitz, J.E., Smith, O.P., Sung, L., Tomizawa, D., van den Heuvel-Eibrink, M.M., Creutzig, U. & Kaspers, G.J. (2015) Collaborative efforts driving progress in pediatric acute myeloid leukemia. Journal of Clinical Oncology, 33, 2949–2962.