Roberto Valli

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.

Shwachman syndrome as mutator phenotype responsible for myeloid dysplasia/neoplasia through karyotype instability and chromosomes 7 and 20 anomalies

An investigation of 14 patients with Shwachman syndrome (SS), using standard and molecular cytogenetic methods and molecular genetic techniques, showed that (1) the i(7)(q10) is not, or not always, an isochromosome but may arise from a more complex mechanism, retaining part of the short arm; (2) the i(7)(q10) has no preferential parental origin; (3) clonal chromosome changes, such as chromosome 7 anomalies and del(20)(q11), may be present in the bone marrow (BM) for a long time without progressing to myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML); (4) the del(20)(q11) involves the minimal region of deletion typical of MDS/AML; (5) the rate of chromosome breaks is not significantly higher than in controls, from which it is concluded that SS should not be considered a breakage syndrome; (6) a specific kind of karyotype instability is present in SS, with chromosome changes possibly found in single cells or small clones, often affecting chromosomes 7 and 20, in the BM. Hence, we have confirmed our previous hypothesis that the SS mutation itself implies a mutator effect that is responsible for MDS/AML through these specific chromosome anomalies. This conclusion supports the practice of including cytogenetic monitoring in the follow‐up of SS patients.

Familial platelet disorder with propensity to acute myelogenous leukemia: Genetic heterogeneity and progression to leukemia via acquisition of clonal chromosome anomalies

Familial platelet disorder with propensity to acute myelogenous leukemia, or FPD/AML (OMIM #601399), is a rare autosomal dominant condition, with only 12 families reported. It is characterized by qualitative and quantitative platelet defects and predisposition to the development of myeloid malignancies. Causal mutations have been identified in the RUNX1 gene (also known as AML1, CBFA2) in the 11 families so far analyzed. RUNX1 is a gene frequently involved in the pathogenesis of sporadic leukemia and myelodysplastic syndromes, through acquired chromosome rearrangements and point mutations. We report an Italian family with three members affected with FPD/AML, two sibs and their father, who developed myelodysplastic syndromes (which in one subsequently evolved into AML). Direct sequencing and polymorphisms haplotype analysis of the region of chromosome 21 where RUNX1 is mapped demonstrated that FPD/AML in this family was not caused by any mutation of the RUNX1 gene, thus providing evidence for the genetic heterogeneity of this disorder. Cytogenetic studies showed monosomy 7 in the marrow of all the three affected subjects, as well as an independent clone with trisomy 8 in the father. The importance of mutator effects in the pathogenesis of familial myeloid malignancies characterized by relevant chromosome changes, in the presence or absence of an underlying Mendelian disorder, has already been suggested. Our results and a review of the cytogenetic literature led us to postulate that mutations also causing FPD/AML may have a mutator effect that could give origin to myelodysplastic syndromes and acute myeloid leukemias through acquired chromosome changes.

Deletion of chromosome 20 in bone marrow of patients with Shwachman‐Diamond syndrome, loss of the EIF6 gene and benign prognosis

Bianchi, V., Robles, R., Alberio, L., Furlan, M. & Lammle, B. (2002) Von Willebrand factor-cleaving protease (ADAMTS13) in thrombocytopenic disorders: a severely deficient activity is specific for thrombotic thrombocytopenic purpura. Blood, 100, 710–713. Coppo, P., Bengoufa, D., Veyradier, A., Wolf, M., Bussel, A., Millot, G.A., Malot, S., Heshmati, F., Mira, J.P., Boulanger, E., Galicier, L., DureyDragon, M.A., Fremeaux-Bacchi, V., Ramakers, M., Pruna, A., Bordessoule, D., Gouilleux, V., Scrobohaci, M.L., Vernant, J.P., Moreau, D., Azoulay, E., Schlemmer, B., Guillevin, L. & Lassoued, K. (2004) Severe ADAMTS13 deficiency in adult idiopathic thrombotic microangiopathies defines a subset of patients characterized by various autoimmune manifestations, lower platelet count, and mild renal involvement. Medicine (Baltimore), 83, 233–244. Froehlich-Zahnd, R., George, J.N., Vesely, S.K., Terrell, D.R., Aboulfatova, K., Dong, J.F., Luken, B.M., Voorberg, J., Budde, U., Sulzer, I., Lammle, B. & Kremer Hovinga, J.A. (2011) Evidence for a role of anti-ADAMTS13 autoantibodies despite normal ADAMTS13 activity in recurrent thrombotic thrombocytopenic purpura. Haematologica. Epub ahead of print. doi: 10.3324/haematol.2011.051433. Hovinga, J.A., Vesely, S.K., Terrell, D.R., Lammle, B. & George, J.N. (2010) Survival and relapse in patients with thrombotic thrombocytopenic purpura. Blood, 115, 1500–1511; quiz 1662. Legendre, C.M., Babu, S., Furman, R.R., Sheerin, N.S., Cohen, D.J., Gaber, O., Eitner, F., Delmas, Y., Loirat, C., Greenbaum, L.A. & Zimmerhackl, L.B. (2010) Safety & efficacy of eculizumab in aHUS patients resistant to plasma therapy: interim analysis from a phase II trial. Journal of the American Society of Nephrology, 21, 93A. Mache, C.J., Acham-Roschitz, B., Fremeaux-Bacchi, V., Kirschfink, M., Zipfel, P.F., Roedl, S., Vester, U. & Ring, E. (2009) Complement inhibitor eculizumab in atypical hemolytic uremic syndrome. Clin J Am Soc Nephrol, 4, 1312– 1316. Remuzzi, G. (2003) Is ADAMTS-13 deficiency specific for thrombotic thrombocytopenic purpura? No. J Thromb Haemost, 1, 632– 634. Remuzzi, G., Galbusera, M., Noris, M., Canciani, M.T., Daina, E., Bresin, E., Contaretti, S., Caprioli, J., Gamba, S., Ruggenenti, P., Perico, N. & Mannucci, P.M. (2002) von Willebrand factor cleaving protease (ADAMTS13) is deficient in recurrent and familial thrombotic thrombocytopenic purpura and hemolytic uremic syndrome. Blood, 100, 778–785. Salmon, J.E., Heuser, C., Triebwasser, M., Liszewski, M.K., Kavanagh, D., Roumenina, L., Branch, D.W., Goodship, T., Fremeaux-Bacchi, V. & Atkinson, J.P. (2011) Mutations in complement regulatory proteins predispose to preeclampsia: a genetic analysis of the PROMISSE cohort. PLoS Med, 8, e1001013. Tsai, H.M. (2003) Is severe deficiency of ADAMTS-13 specific for thrombotic thrombocytopenic purpura? Yes. Journal of Thrombosis and Haemostasis, 1, 625–631.

Evaluating chromosomal mosaicism by array comparative genomic hybridization in hematological malignancies: the proposal of a formula

Array-based comparative genomic hybridization (aCGH) has proven indispensable to the study of unbalanced constitutional and acquired chromosomal anomalies, but its sensitivity for detecting mosaicism is still not well established. On the basis of the ADM2 algorithm used for microarray image analysis with one of the most widely used oligomer-based aCGH platforms [the whole genome 244K system by Agilent Technologies (Santa Clara, CA)] we suggest a formula to infer the percentage of cells bearing a chromosome imbalance in cases with constitutional or acquired mosaicism. Three examples of acquired mosaicism in which this formula was applied are reported together with parallel fluorescence in situ hybridization (FISH) to interphase nuclei with informative probes. Although some approximation affects both the results inferred from aCGH and FISH data, the proposed formula was successful in the three patients studied.

Monosomy 7 in myeloid malignancies: parental origin and monitoring by real-time quantitative PCR

Monosomy 7 in myeloid malignancies: parental origin and monitoring by real-time quantitative PCR

Different loss of material in recurrent chromosome 20 interstitial deletions in Shwachman-Diamond syndrome and in myeloid neoplasms

BackgroundAn interstitial deletion of the long arms of chromosome 20, del(20)(q), is frequent in the bone marrow (BM) of patients with myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), and myeloproliferative neoplasms (MPN), and it is recurrent in the BM of patients with Shwachman-Diamond syndrome (SDS), who have a 30-40% risk of developing MDS and AML.ResultsWe report the results obtained by microarray-based comparative genomic hybridization (a-CGH) in six patients with SDS, and we compare the loss of chromosome 20 material with one patient with MDS, and with data on 92 informative patients with MDS/AML/MPN and del(20)(q) collected from the literature.ConclusionsThe chromosome material lost in MDS/AML/MPN is highly variable with no identifiable common deleted regions, whereas in SDS the loss is more uniform: in 3/6 patients it was almost identical, and the breakpoints that we defined are probably common to most patients from the literature. In some SDS patients less material may be lost, due to different distal breakpoints, but the proximal breakpoint is in the same region, always leading to the loss of the EIF6 gene, an event which was related to a lower risk of MDS/AML in comparison with other patients.

DNA isolation protocol effects on nuclear DNA analysis by microarrays, droplet digital PCR, and whole genome sequencing, and on mitochondrial DNA copy number estimation

Potential bias introduced during DNA isolation is inadequately explored, although it could have significant impact on downstream analysis. To investigate this in human brain, we isolated DNA from cerebellum and frontal cortex using spin columns under different conditions, and salting-out. We first analysed DNA using array CGH, which revealed a striking wave pattern suggesting primarily GC-rich cerebellar losses, even against matched frontal cortex DNA, with a similar pattern on a SNP array. The aCGH changes varied with the isolation protocol. Droplet digital PCR of two genes also showed protocol-dependent losses. Whole genome sequencing showed GC-dependent variation in coverage with spin column isolation from cerebellum. We also extracted and sequenced DNA from substantia nigra using salting-out and phenol / chloroform. The mtDNA copy number, assessed by reads mapping to the mitochondrial genome, was higher in substantia nigra when using phenol / chloroform. We thus provide evidence for significant method-dependent bias in DNA isolation from human brain, as reported in rat tissues. This may contribute to array “waves”, and could affect copy number determination, particularly if mosaicism is being sought, and sequencing coverage. Variations in isolation protocol may also affect apparent mtDNA abundance.

High variability of genomic instability and gene expression profiling in different HeLa clones.

The HeLa cell line is one of the most popular cell lines in biomedical research, despite its well-known chromosomal instability. We compared the genomic and transcriptomic profiles of 4 different HeLa batches and showed that the gain and loss of genomic material varies widely between batches, drastically affecting basal gene expression. Moreover, different pathways were activated in response to a hypoxic stimulus. Our study emphasizes the large genomic and transcriptomic variability among different batches, to the point that the same experiment performed with different batches can lead to distinct conclusions and irreproducible results. The HeLa cell line is thought to be a unique cell line but it is clear that substantial differences between the primary tumour and the human genome exist and that an indeterminate number of HeLa cell lines may exist, each with a unique genomic profile.

Cytogenetic monitoring in Shwachman-Diamond syndrome: a note on clonal progression and a practical warning.

We analyzed the results of periodic chromosome analyses performed on bone marrow of 22 patients with Shwachman-Diamond syndrome (SDS), 8 directly observed and 14 from the literature, selected because of changes in the cytogenetic picture during the course of the disease. This study points out some features of the cytogenetic evolution in SDS relevant for prognostic evaluation but never noted in the literature. In particular, the lack of any clonal progression and the frequent appearance of independent clones with chromosomal changes different from the one initially discovered, with possible severe prognostic implications, are reported.