Nieves Perdigones

Telomere biology and translational research.

Origins. The appreciation that there might be something special about chromosome ends began in the 1930s through 2 independent observations. Herman Muller, working with fruit flies, noticed that x rays caused chromosome breakages and that the broken ends subsequently fused with each other. He noticed that the real ends of chromosomes never took part in these fusion events, and he concluded chromosome ends were sealed in some way. At about the same time, Barbara McClintock observed in maize that dicentric chromosomes (chromosomes with 2 centromeres) broke at mitosis and that the broken ends fusedwith each other. Again, the natural chromosome ends were not involved. She also noticed that the fusion events did not happen in embryonic cells; the broken ends here were ‘‘healed’’ somehow. In 1961, Hayflick found he could grow normal human diploid cells, but they would divide only a limited num-

Single nucleotide polymorphism array analysis of bone marrow failure patients reveals characteristic patterns of genetic changes.

The bone marrow failure syndromes (BMFS) are a heterogeneous group of rare blood disorders characterized by inadequate haematopoiesis, clonal evolution, and increased risk of leukaemia. Single nucleotide polymorphism arrays (SNP‐A) have been proposed as a tool for surveillance of clonal evolution in BMFS. To better understand the natural history of BMFS and to assess the clinical utility of SNP‐A in these disorders, we analysed 124 SNP‐A from a comprehensively characterized cohort of 91 patients at our BMFS centre. SNP‐A were correlated with medical histories, haematopathology, cytogenetic and molecular data. To assess clonal evolution, longitudinal analysis of SNP‐A was performed in 25 patients. We found that acquired copy number‐neutral loss of heterozygosity (CN‐LOH) was significantly more frequent in acquired aplastic anaemia (aAA) than in other BMFS (odds ratio 12·2, P < 0·01). Homozygosity by descent was most common in congenital BMFS, frequently unmasking autosomal recessive mutations. Copy number variants (CNVs) were frequently polymorphic, and we identified CNVs enriched in neutropenia and aAA. Our results suggest that acquired CN‐LOH is a general phenomenon in aAA that is probably mechanistically and prognostically distinct from typical CN‐LOH of myeloid malignancies. Our analysis of clinical utility of SNP‐A shows the highest yield of detecting new clonal haematopoiesis at diagnosis and at relapse.

Acquired copy number neutral loss of heterozygosity of chromosome 7 associated with clonal haematopoiesis in a patient with Shwachman‐Diamond syndrome

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by bone marrow (BM) failure, pancreatic insufficiency, and skeletal abnormalities. Mutations in the SBDS gene on chromosome arm 7q, explain 90% of SDS cases (Boocock et al, 2003). SBDS is essential for the assembly of mature ribosomes. Frequently SDS patients are compound heterozygotes for two common SBDS mutations (the 183_184 TA→CT and 258+2 T→C mutations). The null 183_184 TA→CT mutation results in a stop codon while the 258+2 T→C mutation causes a splicing error with only small amounts of full length protein produced (Austin et al, 2005). Homozygosity for the 183_184 TA→CT mutation has never been found. Here we describe acquired copy number neutral loss of heterozygosity (CN-LOH) for most of 7q in an SDS patient. The clone of BM cells with CN-LOH contained 2 copies of the gene with the 258+2 T→C mutation, which increases the level of SBDS protein providing the likely explanation for clonal expansion of the affected haematopoietic progenitor cell. Neutropenia was found in a neonatal female when a complete blood count was performed due to concerns of neonatal sepsis. In addition to haematological and infectious issues, this patient also developed exocrine pancreatic insufficiency. At four months of age she was diagnosed with SDS, which was confirmed by gene sequencing. A BM aspiration and biopsy were performed on the patient at 13 months of age and a follow-up examination was performed a year later. Karyotypes were prepared, and single nucleotide polymorphism (SNP) analysis of DNA isolated from the BM aspirates was accomplished with the HumanHap610 (first BM) or Omni1-Quad (second BM aspirate) genotyping beadchip (Illumina, San Diego, CA). The patient and her unaffected parents were recruited for a research study at the Childrens Hospital of Philadelphia, Pennsylvania, USA. Samples from skin, BM and peripheral blood (PB) from the patient as well as PB from the parents were obtained after written informed consent. The Ethics Committee of the Hospital approved the study in accordance with the declaration of Helsinki. SNP analysis of DNA from the parents PB was performed with the Illumina Human Omni1-Quad gene chip. Data analysis was performed with GenomeStudio Software (Illumina). Plots of two parameters, the log2R ratio and the B allele frequency, provided information regarding copy number and genotype. The comparison between the genotype data of the parents and the CN-LOH region in the patient was performed with GenomeStudio Software. Polymerase chain reaction and Sanger sequencing of BM DNA and DNA from cultured skin fibroblasts and PB was carried out by standard procedures (Boocock et al, 2003, Sanger and Coulson 1975). The BM aspirate and biopsy performed on the patient at 13 months of age, showed normal cellularity, with normal erythropoiesis and megakaryopoiesis, with a decrease in myeloid progenitors. Standard cytogenetic analysis revealed a normal karyotype 46,XX. Genome wide (GW) SNP array analysis revealed CN-LOH in 20% of the cells from a region just below the centromere of chromosome 7 to the end of 7q (Figure 1). A similar percentage of cells with 7q CN-LOH and no other cytogenetic abnormalities were identified on a second BM one year later. Figure 1 B allele frequency results and LOH analysis from BM of CHOP256.01 at chromosome 7 Genotype analysis of the parents showed that the mother was heterozygous for the 183_184 TA→CT mutation and the father was heterozygous for the 258+2 T→C mutation. The patient was heterozygous for both mutations. Analysis of 683 informative markers from 7q showed that the LOH was due to loss of the maternal alleles. The comparison of the 258+2 T→C sequencing peaks between fibroblast and BM samples from the patient showed that the 258+2 C peak was slightly higher than the 258+2 T peak in the BM sample but not in the skin sample (Figure 2). These results indicate that the cells with LOH of 7q detected in the BM of the patient have two copies of the SBDS 258+2 T→C mutation. Figure 2 Chromatograph of the SBSD 258+2 T→C mutation from BM(A) and skin (B) Our results are consistent with clonal expansion of cells, in the BM of an SDS patient, that have undergone a genetic event producing LOH for 7q, including the SBDS gene. Genetic analysis shows that the expanded clone contains 2 copies of the hypomorphic SBDS mutation while the constitutional genotype is compound heterozygous for 258+2 T→C and 183_184 TA→CT. This is the third example of somatic genomic changes leading to clonal expansion in the BM of SDS patients. Clonal cytogenetic abnormalities affecting 7q have been repeatedly observed in the BM of SDS patients (Dror et al, 2002, Shimamura 2006, Smith et al, 2002). The most frequent is an isochromosome 7q (i(7)(q10)), leading to 3 copies of 7q. Interestingly, all of the compound heterozygous SDS patients with i(7)(q10) clones analysed so far have an extra copy of the 258+2 T→C mutated gene (Minelli et al, 2009). In those cases, as in the case described here, there is an increase in the copy number of the gene producing some active protein. Another common cytogenetic event in SDS is an interstitial deletion in 20q removing a region that includes the EIF6 gene (Pressato et al, 2012). It was recently shown that SBDS couples with the GTPase EFL1 to cause the release of EIF6 from the pre-60S ribosome subunit, an essential step in the formation of the mature ribosome (Finch et al, 2011). It was hypothesized that the decreased amount of EIF6 in these cells, in the context of SBDS deficiency, improved ribosome biogenesis. Again the deletion is associated with clonal expansion. Interestingly SDS has an increased likelihood of progression to myelodysplastic syndrome/acute myeloid leukaemia that is frequently associated with numerical and structural chromosome 7 anomalies. To the contrary, both i(7)(q10) and the 20q deletion in patients with SDS are associated with low risk of transformation of the affected cell clone (Minelli et al, 2009, Pressato, et al, 2012), though this relationship has been questioned (Dror et al, 2002). Interestingly this patient is another example of an emerging paradigm whereby, in the context of the aplastic BM, haematopoietic progenitor cells that partially overcome the disease causing genetic lesion due to acquired genomic changes have a survival or growth advantage, leading to clonal expansion. Somatic genomic changes and clonal expansion have been observed in other BM failure syndromes (May 2011). These changes often improve blood cell production and in some cases decrease the risk of leukaemic transformation. Such “natural gene therapy” may be instructive concerning possible drug targets.

Clonal Hematopoiesis in Patients with Dyskeratosis Congenita

Dyskeratosis congenita (DC) is a rare inherited telomeropathy most frequently caused by mutations in a number of genes all thought to be involved in telomere maintenance. The main causes of mortality in DC are bone marrow failure as well as malignancies including leukemias and solid tumors. The clinical picture including the degree of bone marrow failure is highly variable and factors that contribute to this variability are poorly understood. Based on the recent finding of frequent clonal hematopoiesis in related bone marrow failure syndromes, we hypothesized that somatic mutations may also occur in DC and may contribute at least in part to the variability in blood production. To evaluate for the presence of clonal hematopoiesis in DC, we used a combination of X‐inactivation, comparative whole exome sequencing (WES) and single nucleotide polymorphism array (SNP‐A) analyses. We found that clonal hematopoiesis in DC is common, as suggested by skewed X‐inactivation in 8 out of 9 female patients compared to 3 out of 10 controls, and by the finding of acquired copy neutral loss‐of‐heterozygosity on SNP‐A analysis. In addition, 3 out of 6 independent DC patients were found to have acquired somatic changes in their bone marrow by WES, including a somatic reversion in DKC1, as well as missense mutations in other protein coding genes. Our results indicate that clonal hematopoiesis is a common feature of DC, and suggest that such somatic changes, though commonly expected to indicate malignancy, may lead to improved blood cell production or stem cell survival. Am. J. Hematol. 91:1227–1233, 2016.

A Family with Hoyeraal-Hreidarsson Syndrome and Four Variants in Two Genes of the Telomerase Core Complex

We describe an African American family with Hoyeraal–Hreidarrson syndrome (HHS) in which 2 TERT mutations (causing P530L and A880T amino acid changes) and two in the DKC1 variants (G486R and A487A) were segregating. Both genes are associated with dyskeratosis congenita and HHS. It was important to determine the importance of these mutations in disease pathogenesis to counsel family members. From genetic analysis of family members, telomere length and X‐inactivation studies we concluded that compound heterozygosity for the TERT mutations was the major cause of HHS and the DKC1 G486R variant is a rare African variant unlikely to cause disease. Pediatr Blood Cancer 2013; 60: E4–E6.

Common polymorphic deletion of glutathione S-transferase theta predisposes to acquired aplastic anemia: Independent cohort and meta-analysis of 609 patients

Acquired aplastic anemia (AA) is a rare life‐threatening bone marrow failure syndrome, caused by autoimmune destruction of hematopoietic stem and progenitor cells. Epidemiologic studies suggest that environmental exposures and metabolic gene polymorphisms contribute to disease pathogenesis. Several case–control studies linked homozygous deletion of the glutathione S‐transferase theta (GSTT1) gene to AA; however, the role of GSTT1 deletion remains controversial as other studies failed to confirm the association. We asked whether a more precise relationship between the GSTT1 null polymorphism and aplastic anemia could be defined using a meta‐analysis of 609 aplastic anemia patients, including an independent cohort of 67 patients from our institution. We searched PubMed, Embase, and the Cochrane Database for studies evaluating the association between GSTT1 null genotype and development of AA. Seven studies, involving a total of 609 patients and 3,914 controls, fulfilled the eligibility criteria. Meta‐analysis revealed a significant association of GSTT1 null genotype and AA, with an OR = 1.74 (95% CI 1.31–2.31, P < 0.0001). The effect was not driven by any one individual result, nor was there evidence of significant publication bias. The association between AA and GSTT1 deletion suggests a role of glutathione‐conjugation in AA, possibly through protecting the hematopoietic compartment from endogenous metabolites or environmental exposures. We propose a model whereby protein adducts generated by reactive metabolites serve as neo‐epitopes to trigger autoimmunity in aplastic anemia. Am. J. Hematol. 88:862–867, 2013.

Abstract 2977: Most patients with acquired aplastic anemia develop clonal hematopoiesis early in disease

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Clonal hematopoiesis is an expansion of hematopoietic stem cells, caused by somatic mutations or epigenetic changes that confer a growth advantage to the host cell. Although recently recognized as a phenomenon of aging, clonal hematopoiesis has been traditionally associated with pre-cancerous states and malignant transformation. Acquired aplastic anemia (AA), a non-neoplastic autoimmune blood disorder occurring in children and adults, has been associated with clonal hematopoietic disorders; transformation to myelodysplastic syndrome (MDS) or acute leukemia is a late complication in 10-15% of AA patients. Based on the association of AA with clonal disorders, we hypothesized that clonal hematopoiesis is a general phenomenon in AA, and can be seen in the majority of AA patients, including children. To evaluate somatic genetic changes in AA, we used a combination of single nucleotide polymorphism array (SNP-A) genotyping and comparative whole exome sequencing of paired bone marrow aspirates and skin in twenty nine patients with AA. All somatic mutations were validated by bi-directional Sanger sequencing. The median age of diagnosis was 14 years (range 1.5-65). Patients were analyzed at a median of 1.1 years from diagnosis. None of the patients had histopathological evidence of MDS at the time of analysis. Somatic mutations were identified in the majority of patients, including patients with pediatric-onset AA. Three patients (10%) had somatic loss-of-function mutations in HLA class I alleles. Although MDS-associated mutations were identified in 2 of 29 patients, the majority of mutations were not in genes associated with MDS and hematologic malignancies. Pathway analysis of mutated genes revealed an enrichment of genes in pathways of immunity and transcriptional regulation. Comparison of somatic mutations in AA to a patient with a 30-year history of AA who progressed to MDS revealed that, unlike in AA, which was characterized by diverse and frequently oligoclonal hematopoiesis, progression to MDS was associated with an expansion of a dominant clone carrying multiple classical mutations linked to malignancy: pathogenic mutations in SUZ12 (homozygous for the mutated region due to copy number-neutral loss of heterozygosity (CN-LOH) at the chromosomal region 17q11.2qter), ASXL1, RUNX1, and PHF6. In conclusion, our data show that clonal hematopoiesis emerges in the majority of patients with AA, including children and young adults, can be detected early in disease, and has a mutational spectrum largely distinct from MDS. Our results highlight that in the absence of morphologic features of myelodysplasia, the presence of clonal hematopoiesis with somatic mutations cannot be used to distinguish MDS from AA. Future longitudinal studies of clonal hematopoiesis in AA will help to explain differences in patients’ disease course, and will enable personalized treatment approaches in AA. Citation Format: Daria V. Babushok, Nieves Perdigones, Juan C. Perin, Timothy S. Olson, Wenda Ye, Jacquelyn J. Roth, Curt Lind, Carine Cattier, Yimei Li, Helge Hartung, Michele E. Paessler, Dale M. Frank, Hongbo M. Xie, Tracy M. Busse, Shanna Cross, Gregory M. Podsakoff, Dimitrios Monos, Jaclyn A. Biegel, Philip J. Mason, Monica Bessler. Most patients with acquired aplastic anemia develop clonal hematopoiesis early in disease. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2977. doi:10.1158/1538-7445.AM2015-2977

Case Report: Paroxysmal nocturnal hemoglobinuria in a woman heterozygous for G6PD A-

We describe a case of paroxysmal nocturnal hemoglobinuria (PNH) in a woman who is heterozygous for the glucose-6-phosphate dehydrogenase A- ( G6PDA-) allele. PNH is associated with one or more clones of cells that lack complement inhibition due to loss of function somatic mutations in the PIGA gene. PIGA encodes the enzyme phosphatidylinositol glycan anchor biosynthesis, class A, which catalyses the first step of glycosylphosphatidylinisotol ( GPI) anchor synthesis. Two GPI anchored red cell surface antigens regulate complement lysis. G6PD catalyses the first step of the pentose phosphate pathway and enzyme variants, frequent in some populations have been selected because they confer resistance to malaria, are associated with hemolysis in the presence of oxidizing agents including several drugs. The patient had suffered a hemolytic attack after taking co-trimoxazole, a drug that precipitates hemolysis in G6PD deficient individuals. Since both G6PD and PIGA are X-linked we hypothesized that the PIGA mutation was on the X-chromosome carrying the G6PDA- allele. Investigations showed that in fact the PIGA mutation was on the X-chromosome carrying the normal G6PD B allele. We speculate that complement activation on G6PD A- red cells exposed to Bactrim might have triggered complement activation inducing the lysis of G6PD B PNH Type II red blood cells or that the patient may have had a PNH clone expressing G6PDA- at the time of the hemolytic episode.

Using induced human pluripotent stem cells to study Diamond-Blackfan anemia: an outlook on the clinical possibilities.

The recent development of induced human pluripotent stem cells (iPS cells) from differentiated adult cells has been heralded as potentially a major advance in medicine. iPS cells can be genetically manipulated and differentiated into a variety of adult tissues. The technology can be used to model disease pathogenesis, to screen for drugs that may be used to treat diseases and, perhaps one day, to produce healthy tissues to replace those affected by disease. Here we discuss these possibilities in the bone marrow failure syndrome Diamond Blackfan Anemia.