scholarly journals Inherited human Apollo deficiency causes severe bone marrow failure and developmental defects

Blood ◽  
2022 ◽  
Author(s):  
Laëtitia Kermasson ◽  
Dmitri Churikov ◽  
Aya Awad ◽  
Riham Smoom ◽  
Elodie Lainey ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Telomere Length ◽  
Catalytic Domain ◽  
Bone Marrow Failure ◽  
Premature Aging ◽  
Compound Heterozygous ◽  
Gene Correction ◽  
Developmental Defects ◽  
A Genome ◽  
Telomere Biology

Inherited bone marrow failure syndromes (IBMFS) represent a group of disorders typified by impaired production of one or several blood cell types. The telomere biology disorders dyskeratosis congenita (DC) and its severe variant Høyeraal-Hreidarsson (HH) syndrome are rare IBMFS characterized by bone marrow failure, developmental defects, and various premature aging complications associated with critically short telomeres. Here we identified biallelic variants in the gene encoding the 5'-to-3' DNA exonuclease Apollo/SNM1B in three unrelated patients presenting with a DC/HH phenotype consisting of early onset hypocellular bone marrow failure, B and NK lymphopenia, developmental anomalies, microcephaly and/or intrauterine growth retardation. All three patients carry a homozygous or compound heterozygous (in combination with a null-allele) missense variant affecting the same residue L142 (L142F or L142S) located in the catalytic domain of Apollo. Apollo-deficient cells from patients exhibited spontaneous chromosome instability and impaired DNA repair that was complemented by CRISPR/Cas9-mediated gene correction. Furthermore, patients' cells showed signs of telomere fragility that were however not associated with global reduction of telomere length. Unlike patients' cells, human Apollo KO HT1080-cell lines showed strong telomere dysfunction accompanied by excessive telomere shortening, suggesting that the L142S and L142F Apollo variants are hypomorphic. Collectively, these findings define human Apollo as a genome caretaker and identify biallelic Apollo variants as a genetic cause of a hitherto unrecognized severe IBMFS combining clinical hallmarks of DC/HH with normal telomere length.

Longitudinal Changes In Telomere Length In Patients with Dyskeratosis Congenita

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2230-2230
Author(s):  
Blanche P Alter ◽  
Neelam Giri ◽  
Peter M. Lansdorp ◽  
Gabriela M. Baerlocher ◽  
Philip S Rosenberg ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Telomere Length ◽  
Bone Marrow Failure ◽  
Dyskeratosis Congenita ◽  
Premature Aging ◽  
Bone Marrow Failure Syndrome ◽  
Cross Sectional ◽  
Telomere Shortening ◽  
Early Presentation ◽  
Telomere Attrition

Abstract Abstract 2230 Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome with a complex clinical phenotype, including dysplastic nails, lacy reticular pigmentation, and oral leukoplakia (the diagnostic triad). Numerous other physical abnormalities may be present, in addition to cytopenias due to bone marrow failure, and a high risk of leukemia or solid tumors. However, many patients have no physical findings at diagnosis. Patients with DC have very short telomeres, and approximately one-half have a mutation in one of six genes important in telomere biology. Telomere length in leukocyte subsets, measured by automated flow fluorescence in situ hybridization (flow-FISH), is both sensitive and specific for identifying individuals with DC. Telomeres consist of nucleotide repeats and a protein complex at chromosome ends that are critical in chromosomal stability which shorten during normal cell division. Cross-sectional studies of normal individuals suggest that telomere length decreases with age in a sigmoid pattern from birth to old age. In a cross-sectional analysis of 26 patients with DC, we previously observed that telomere length appeared to be stable or even to slightly increase with age (BP Alter et al, Blood 110:149, 2007). Similar results were shown in 23 different DC patients by others (M Bessler et al, FEBS Lett 2010 in press). We speculated that these data were influenced by early presentation (or recognition) of clinically more severe patients, while patients with similar telomere length who were clinically milder were identified at older ages. In this pilot study, we examined, for the first time, the longitudinal age-association of telomere attrition in nine patients with DC who were followed for five to seven years (currently 8 – 50 years of age). These include three patients with mutations in TERC, and two each with TINF2, TERT, and DKC1 mutations. When first studied, four had normal hematopoiesis, three moderate cytopenias, one was receiving androgens, and one was on transfusions. Subsequently, one with normal hematopoiesis developed mild thrombocytopenia, one who was on transfusions responded to androgens, and one with moderate aplastic anemia became severe. In all cases, telomere length decreased with age. In a linear regression model, the average annual decrease in telomere length in lymphocytes was 167 base pairs/year (bp/yr) + 104, similar to the rate in granulocytes, 159 + 92 bp/yr. According to the literature, the rate of telomere attrition in longitudinal studies in normal blood is ∼45-50 bp/yr, albeit by methods other than flow-FISH; the rate of telomere shortening appears to decrease with increasing age. The average patient Z-scores at the beginning of the study were -3.9 standard deviations below the median for age in healthy normal controls, and were -4.3 at the end, consistent with the impression that DC patient telomeres shorten somewhat more than expected from normal aging. These data support the hypothesis that the earlier cross-sectional results for patients with DC indeed were influenced by the cross-sectional rather than longitudinal nature of the data. The current longitudinal data suggest that telomere shortening could possibly be accelerated in patients with DC, but larger studies are required. Our results indicate that patients with DC have telomeres that are much shorter than normal for their age, and that over time they continue to shorten, consistent with DC being classified as a disorder of premature aging. Disclosures: Lansdorp: Repeat Diagnostics: Equity Ownership.

Telomere Length Measurement by Flow-FISH Distinguishes Dyskeratosis Congenita from Other Bone Marrow Failure Syndromes.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 183-183
Author(s):  
Blanche P. Alter ◽  
Gabriela Baerlocher ◽  
Sharon A. Savage ◽  
Stephen Jacob Chanock ◽  
Babette B. Weksler ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Telomere Length ◽  
Sensitivity And Specificity ◽  
Bone Marrow Failure ◽  
Correct Diagnosis ◽  
White Blood Cells ◽  
Dyskeratosis Congenita ◽  
Length Measurement ◽  
Specific Method ◽  
Telomere Biology

Abstract This study was designed to evaluate the utility of flow-FISH telomere length measurement in white blood cells (WBC) as a screening test for Dyskeratosis congenita (DC). We studied 26 patients: 17 with DC, 1 silent carrier (clinically normal; mutation in TERC), 4 with the Hoyeraal-Hreidarsson variant (HH), and 4 with Revesz Syndrome. Five had mutations in DKC1, 5 in TERC, and 2 in TERT. 23 had hematologic abnormalities, 19 had 2 or 3 of the DC diagnostic triad (lacey pigmentation, dyskeratotic nails, and leukoplakia), and 4 had soft signs of DC. We evaluated 54 first-degree relatives of DC patients, 16 Fanconi Anemia patients (FA), 14 with Diamond-Blackfan Anemia (DBA), 5 with Shwachman Diamond Syndrome (SDS), and 10 with other possibly inherited cytopenias (Other). Telomere length was measured in granulocytes and lymphocyte subsets by automated multicolor flow-FISH; results were compared with age-matched values from 400 normal controls. “Very low (VL)” telomere length was defined as a mean telomere length below the normal first percentile for age and specific WBC type. We observed VL telomeres in all subsets in the silent carrier, all HH and Revesz patients, and 15/17 with DC. Eight of 51 DC relatives had VL telomeres in granulocytes versus 2/54 with VL telomeres in lymphocytes. The sensitivities for distinguishing a DC patient from an unaffected relative were 92% in lymphocytes and 96% in granulocytes; the specificities were 96% and 98%, respectively; the sensitivity and specificity for VL telomeres in both cell types were 96% and 96%. The silent carrier with a TERC mutation developed thrombocytopenia, hypocellular marrow, and a cytogenetic clone during follow-up. The 2 DC relatives with VL telomeres in lymphocytes were from a family without a known mutant gene; they may also be silent carriers. The latter possibility disqualified an HLA-matched sibling as a donor for DC-related aplastic anemia, because of engraftment concerns; another sibling donor with normal telomere length was selected. VL granulocyte telomeres were observed in 5/16 FA, 3/14 DBA, 1/5 SDS, and 1/10 Other patients, versus 2/16 FA, 1/14 DBA, 1/5 SDS, and 0/10 Other in lymphocytes, and in both lineages in only 1 each of FA, DBA, and SDS. The sensitivity and specificity for distinguishing DC from non-DC patients using VL telomeres in both lineages were 96% and 93%, respectively. Only DC patients had consistently VL telomeres in all cell subsets. Flow-FISH telomere length measurement provides a sensitive and specific method for identifying patients with DC among families, regardless of mutation status, and distinguishes patients with DC from those with other inherited or acquired marrow failure syndromes. It may also help to detect silent carriers, and facilitate identification of mutations in other telomere biology genes. Our data suggest that the diagnostic triad, soft physical findings and/or bone marrow failure may not be required for the diagnosis of DC. Correct diagnosis of DC will enhance genetic counseling and hematologic management.

A Novel hTERC Deletion Manifesting with Features of Dyskeratosis Congenita and Genetic Anticipation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4157-4157
Author(s):  
Stan Benke ◽  
D. S. Houston ◽  
Inderjeet Dokal ◽  
Tom Vulliamy
Keyword(s):  
Bone Marrow ◽  
Telomere Length ◽  
Peripheral Blood ◽  
Autosomal Dominant ◽  
Bone Marrow Failure ◽  
Macrocytic Anemia ◽  
Phenotypic Expression ◽  
Dyskeratosis Congenita ◽  
Sequencing Analysis ◽  
Genetic Anticipation

Abstract The gene encoding the RNA component of human telomerase (hTERC) is mutated in families with the autosomal dominant form of dyskeratosis congenita (DC). The phenomenon of genetic anticipation has recently been reported to accompany this form of DC, with disease severity increasing in offspring of affected individuals. It has been postulated that anticipation in these families relates to the adverse impact of hTERC mutations on inherited telomere length, with progressive telomere shortening seen in succeeding generations (Nat Gen2004; 36:447). We describe here a novel hTERC mutation, with affected individuals presenting in adulthood with mild mucocutaneous abnormalities, bone marrow failure and a pattern of penetrance supporting the presence of disease anticipation. The proband in the family studied presented at age 49 with squamous cell carcinoma of the tongue and a history of oral leukoplakia which he had developed at age 30. Peripheral blood on presentation was remarkable only for a mild macrocytic anemia. During treatment of his malignancy, severe and irreversible bone marrow hypoplasia was precipitated by a single cycle of cisplatinum chemotherapy. The patient’s brother at age 25 had been previously diagnosed with severe aplastic anemia; this was refractory to standard immunosuppression with cyclosporine and antithymocyte globulin. No somatic abnormailites were identified in this patient. Testing for Fanconi anemia in both siblings was negative. Direct sequencing analysis of hTERC in these patients revealed both to be heterozygous for a novel hTERC mutation (79 deletion C). Further studies among family members documented heterozygosity for the mutation in the mother of these two siblings. At age 77, she displayed none of the mucocutaneous signs associated with DC, while the only abnormality seen in her peripheral blood was an elevated mean corpuscular volume. The hTERC mutation seen in this family most likely exerts its effects through disruption of the pseudoknot domain. The findings of an individual with normal longevity, minimal phenotypic expression and affected offspring are further evidence of genetic anticipation being an important feature of autosomal dominant DC. Correlation with determination of telomere length has been initiated.

The Role of Telomerase Activity and Telomere Length in Cellular Sensitivity to DNA Damaging Agents.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4192-4192
Author(s):  
Greg T. Rice ◽  
Michael A. Beasley ◽  
Ike I. Akabogu ◽  
Erik R. Westin ◽  
Dale A. Winnike ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Telomere Length ◽  
Bone Marrow Failure ◽  
Skin Pigmentation ◽  
Cytotoxic Agents ◽  
Cell Surface Expression ◽  
Cell Transplant ◽  
Telomere Shortening ◽  
Hematopoietic Stem ◽  
Increased Sensitivity

Abstract Dyskeratosis congenita (DC) is a premature aging syndrome characterized by progressive bone marrow failure, abnormal skin pigmentation and nail dystrophy. We have described an autosomal dominant form of DC (AD DC) in a large three-generation kindred that is due to a mutation in the gene encoding human telomerase RNA (hTR). While telomere shortening is a normal consequence of the aging process, DC patients display extremely short telomeres in many somatic cell types, including hematopoietic cells, and they often suffer from bone marrow failure. Allogeneic hematopoietic stem cell transplant (HSCT) remains the only curative therapy for marrow failure in DC. However, HSCT in DC is generally poorly tolerated and associated with significant morbidity, perhaps as a consequence of increased sensitivity of dividing cells to cytotoxic agents during myeloablative therapy. To test this hypothesis, we characterized lymphocytes from various AD DC patients and age matched controls that had been placed in long term culture following in vitro exposure to irradiation (137Cs) and varying doses of Taxol, Adriamycin, and Etoposide. Cell proliferation and viability were quantified by direct visual counting on a hemocytometer, and flow cytometry was employed to assess apoptosis and cell surface expression of senescent markers. In addition to DC lymphocytes having a decreased proliferative capacity and higher basal apoptotic levels, an increased sensitivity to irradiation, Taxol, Adriamycin, and Etoposide was noted. These results suggest that telomere shortening may be an important factor in determining cellular tolerance to cytotoxic therapy and support the concept of reduced intensity HSCT regimens in both aged individuals and DC patients. Further studies have been initiated to determine whether reconstitution of telomere length in DC cells alters response to cytotoxic agents.

Homozygous and Compound Heterozygous Mutations in the Telomerase Reverse Transcriptase Gene in Two Families with Spontaneous Dyskeratosis Congenita and Idiopathic Aplastic Anemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1675-1675
Author(s):  
Hong-Yan Du ◽  
Elena Pumbo ◽  
Peter Manley ◽  
David B. Wilson ◽  
Philip Mason ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Autosomal Recessive ◽  
Amino Acid Change ◽  
Autosomal Dominant ◽  
Bone Marrow Failure ◽  
Gene Mutations ◽  
Dyskeratosis Congenita ◽  
Compound Heterozygous ◽  
The Family ◽  
Tert Gene

Abstract Dyskeratosis congenita (DC) is a rare inherited bone marrow failure syndrome. Classically, DC presents with progressive bone marrow failure, abnormal skin pigmentation, nail dystrophy, and mucosal leukoplakia. The pattern of inheritance in families with DC suggests an X-linked recessive, an autosomal dominant, and an autosomal recessive form of DC. However, in the majority of patients the occurrence of the disease is sporadic or the family history is unknown. Mutations in four different genes have been associated with DC so far. Mutations in DKC1 have been shown to account for the X-linked form of DC and DKC1 de novo mutations account for about one third of male patients with sporadic disease. Mutations in the telomerase RNA TERC and in the catalytic subunit of telomerase, TERT, have been shown to be responsible for the autosomal dominant form of DC. Interestingly, patients with heterozygous mutations in TERC and TERT often show a milder form of disease and a later age of onset and often lack the classic mucocutaeous features, thus are classified as atypical DC. Very recently homozygosity for a mutation in NOP10 has been identified in one family with autosomal recessive disease. The products of the genes mutated in DC are all components of the telomerase complex, suggesting that disease in patients with DC is caused by a defect in telomere maintenance. Here we investigated two patients, one UPN # 199.001 presenting with the classic manifestations of DC and the other UPN# 284.001 presenting with progressive bone marrow failure but no other clinical features suggestive of DC. In both patients the telomeres measured in peripheral blood mononuclear cells were very short, being defined as being below the 1st percentile. Mutation analysis in the genes associated with DC revealed that patient 199.001 was homozygous for a novel TERT (C2110T) gene mutation, causing an amino acid change (P704S) within the RT domain of TERT. Both parents were heterozygous for the C to T transition. Interestingly however, the father was in addition heterozygote for a second mutation in TERT (C1234T; H412Y) a mutation which has previously been described and has been shown to reduce telomerase activity by 50%. Investigations of the family revealed that the parent’s were distantly related, explaining the same TERT sequence alteration in both parents. Both arms of the family contained members with pulmonary fibrosis. In the second patient 284.001 we identified two different novel TERT gene mutations. One A2537G causes the amino acid change Y846C in the RT domain of TERT whereas the other C2628G causes H876Q also in the RT domain. One of the mutations was inherited from each parent and the parent with the A2537G mutation also had very short telomeres. These two families illustrate that the pattern of inheritance in patients with DC may be complex and show for the first time that homozygous or compound heterozygous TERT gene mutation may be associated with DC. Co-dominance of the three different TERT gene mutations and the inheritance of short telomeres have possibly contributed to development of disease in these patients who were thought to have sporadic DC and idiopathic aplastic anemia.

Study of the Correlation between Phenotype - Including Acute Leukemia/MDS- and Genotype in Shwachman Diamond Syndrome in 60 Patients from the French SCN Registry.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3301-3301
Author(s):  
Jean Donadieu ◽  
B. Beaupain ◽  
S. Beaufils ◽  
V. Gandemer ◽  
J.P. Fermant ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Leukemia ◽  
Survival Data ◽  
Bone Marrow Failure ◽  
Great Majority ◽  
Rank Test ◽  
Compound Heterozygous ◽  
Missense Mutations ◽  
Specialized School ◽  
Shwachman Diamond Syndrome

Abstract Shwachman Diamond syndrome (SDS) is a rare multi organ genetic disease bearing a very high risk of haematological complications i.e. MDS/leukaemia and Bone Marrow Failure. The aim of this study is to explore genotype predisposition of the major complications observed in SDS’s patients and to explore prognosis factors of MDS/leukaemia. Methods: Among 90 SDS patients included in the French Severe Chronic Neutropenia Registry, SBDS gene was screened in 63 patients and mutations have been found in 60 patients. Cut-off date was july 30th, 2007. Differences between groups of patients were analysed as survival data, by log rank test. The medical events analysed were: death (n=6), myelodysplasia or acute leukemia (n=6), bone marrow failure (n=6), all hematological events combined (n=12), the use of G-CSF as infectious prophylaxis (n= 11), the necessity of an orthopedic surgery (n=4) and the necessity of nutritional medical support (parenteral or enteral feeding, by mean of gastrostomy (n= 5), intrauterine growth retardation (n=19) and finally, major development retardation if it leads to a specialized school (n=10). Results: Mutations were found in 60 patients (35 males, 25 females) belonging to 54 distinct families (in 6 families, two siblings were genotyped). The median age at last analysis was 10.3 years (0.5yr-38.6 yr). The great majority of patients present the recurrent genotype K62X/C84fs (n=38, 68%) while 19 other mutations were founded, which could be classified in truncating mutations leading to premature stop codons (nonsense, frameshift or splicing defect; n=8) or missense mutations (n=11). We compared patients with truncating mutations on both alleles to compound heterozygous patients carrying at least one missense mutation. Even if differences were observed for the distribution of events between genotype subgroups of patients, none of them raised statistically significance. However, to date, all leukemia has been observed in the group of patients with “truncating” mutations. The genotype of patients with leukemia was [K62X]+[C84fs] in 5 and [C84fs]+[V93fs] in one; while the genotype of patients with BM failure was [K62X]+[C84fs] (n=2), [C84fs]+[624+1G>C], [C84fs]+[C119R], K62X/undetermined, and [C84fs]+[E99fs], [C84fs]+[E44fs]. Among the 6 pairs of siblings tested, four had a similar outcome and two pairs were discordant for the haematological events (leukaemia in one family, Bone marrow failure in the second family). Further, we have analysed genotype, gender, G-CSF therapy and initial Neutrophils and monocytes count, Hemoglobin level, Platelet level as risk factors of Leukemia/MDS. In a multivariate model, none of these features predicts Leukemia/MDS in SDS patients. Conclusion: The genotype of SDS did not appear to be correlated with clinical presentation or outcome. It remains possible than patients without truncating mutations (about 18%) may have a low rate of leukaemia but our survey lack of statistical powerful to demonstrate this hypothesis. We also failed to determine prognostic factors of Leukemia/MDS in SDS patients.

Age-Adjusted Telomere Length Is Not Associated with Variation in Hematological Parameters in Ageing Women

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3102-3102
Author(s):  
Isabelle Fleury ◽  
Sylvie Provost ◽  
Claude Belisle ◽  
Lambert Busque
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Transplantation ◽  
Telomere Length ◽  
Marrow Transplantation ◽  
Copy Number ◽  
Bone Marrow Failure ◽  
Telomere Maintenance ◽  
Hematological Parameter ◽  
Allogeneic Bone ◽  
Potential Biomarker

Abstract Background. Telomeres play a crucial role in maintaining physical integrity of chromosomes. In the absence of telomerase, telomere length (TL) shortens with each cell division up to a critical threshold where cellular senescence occurs. TL is inversely correlated with age, is longer in women than in men, and demonstrates a strong heritability. Normal blood counts are maintained through out life by an extraordinary number of cell divisions rendering telomere maintenance primordial to prevent stem cell exhaustion. In fact, some cases of bone marrow failure syndromes, such as aplastic anemia and dyskeratosis congenital, have been linked to mutation in the telomerase gene; and stressed hematopoiesis, such at it occurs during the first year following allogeneic bone marrow transplantation induces TL shortening. We hypothesized that individuals with shorter TL may have lower blood counts and a decreased bone marrow reserve. The evaluation of TL as a potential biomarker of ageing hematopoiesis is important in the context of bone marrow transplantation performed with increasingly old donors. Methods. We measured TL in 1583 women, predominantly aged over 60, all originating from 288 French-Canadian families using a real-time quantitative PCR method that measures the number of telomere repeats relatively to the copy number of a single copy number gene. Telomeres were adjusted for age. Pearson or Spearman correlations were used to determine association between age-adjusted TL (aTL) and hematological parameter according to, respectively, whether or not a normal distribution was observed for these data. A Bonferroni correction was further applied to set the statistical significance threshold. Results. aTL varied significantly between individuals of the cohort, but no correlation was detected with hemoglobin levels (−0,001; p=0,978), mean corpuscular volume (−0,031; p=0,403); leucocytes (0,055; p=0,139); neutrophils (0,078; p=0,036), monocytes, (0,059; p=0,113), eosinophils (−0,032; p=0,394) and platelets (0,030; p=0,428) counts. Conclusion. Based on our analysis, TL do not predict blood cells counts in ageing women and may not be a useful biomarker for donor selection. This could also suggest that there is a threshold beyond which TL has an effect on hematopoiesis and that point was not reached in our cohort.

Common Recurrent DKC1 Mutation A353V Does Not Support Telomere Maintenance in Induced Pluripotent Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 50-50
Author(s):  
Baiwei Gu ◽  
Jason A. Mills ◽  
Jian-meng Fan ◽  
Deborah L. French ◽  
Monica Bessler ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Telomere Length ◽  
Pluripotent Stem Cells ◽  
Skin Fibroblast ◽  
Bone Marrow Failure ◽  
Ips Cells ◽  
Telomere Maintenance ◽  
Fibroblast Cells ◽  
Induced Pluripotent

Abstract Abstract 50 Dyskeratosis Congenita (DC) is a rare bone marrow failure syndrome showing considerable genetic and clinical heterogeneity. The most common form is the X-linked form due to mutations in the DKC1 gene encoding dyskerin, a protein important in telomere maintenance and ribosomal RNA biogenesis. Six other genes, all of whose products are involved in telomere maintenance, have been shown to be mutated in DC, together the seven genes accounting for about half of the known cases. The X-linked form can cause severe disease for which therapeutic options are limited. It is known that mutant dyskerin destabilizes telomerase RNA leading to rapidly shortening telomeres, accelerated stem cell aging and bone marrow failure. However the precise mechanism by which this occurs is not known. So far studies of the cell biology of DC stem and progenitor cells have been hampered by their scarcity in patients and their short life span and attempts to create mouse models have suffered from differences in telomere biology between mouse and human. An alternative approach that has recently become feasible is the production of induced pluripotent stem cells (iPSC) from patient fibroblasts that can then be used to investigate disease pathogenesis. Accordingly we generated iPSC from skin fibroblast from X-linked DC patients carrying DKC1 mutations Q31E, δ37A and 353V, and by using the classical OCT4, KLF4, SOX2 and cMYC 4-transcription factor system. Of particular interest is the A353V mutation since this is a recurrent mutation and accounts for about 40% of DKC1 mutations. In total, we obtained two Q31E clones, three δ37 clones and eight A353V clones. We found that all these DKC1 mutant iPS cells express decreased levels of dyskerin, in agreement with our mouse studies that show mutant proteins are relatively unstable. Mutant iPSC have very low levels of TERC (only 20–30% of the levels in WT iPSC) while TERT expression is the same as in WT cells. By using the TRAP assay, we found that both A353V and δ37 iPSC showed dramatically decreased telomerase activity; only 10–20 % compared to WT iPSC. After measuring the telomere length of both patient skin fibroblast cells and DKC1 mutant iPSC, we found A353V and δ37 iPSC lost the ability to elongate the telomere end during iPSC reprogramming while WT iPSC showed significantly increased telomere length compared to WT skin fibroblast cells. These results indicated that DKC1 iPSC are defective in telomere maintenance. In terms of ribosome biogenesis, we found that some snoRNA expression was slightly decreased including H/ACA snoRNAs E2, E3, U69, ACA10 and scaRNAs U90 and U93 while all C/D snoRNA we investigated were unchanged compared with WT iPS cells. We also found that DKC1 mutant iPS cells did not show significantly changes in ribosomal profiles or in the kinetics of rRNA processing. Together these results suggest that the iPSC faithfully reproduce the molecular features of the human disease and will prove to be a useful tool in investigations of the pathogenesis and treatment of DC. Disclosures: Bessler: Alexion Phamaceutical: Membership on an entity's Board of Directors or advisory committees; National Organization for Rare Dieases: Speakers Bureau.

Mesodermal Development From Reprogrammed Fanconi Anemia Cells Is Affected by ALDH2 Enzymatic Activity

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 648-648
Author(s):  
Naoya Suzuki ◽  
Asuka Hira ◽  
Akira Niwa ◽  
Megumu Saito ◽  
Keitaro Matsuo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Dna Damage ◽  
Developmental Stage ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
A Genome ◽  
The Impact ◽  
Mesodermal Development

Abstract Abstract 648 Introduction Fanconi anemia (FA) is a genome instability disorder with clinical characteristics including progressive bone marrow failure (BMF), developmental abnormalities, and increased occurrence of leukemia and cancer. To date 15 genes have been implicated in FA, and their products form a common DNA repair network often referred to as “FA pathway”. Following DNA damage or replication stress, the FA pathway is activated, leading to the monoubiquitination of FANCD2 and FANCI proteins (the ID complex). The monoubiquitinated ID complex is loaded on damaged chromatin with subnuclear foci formation, and mediates homologous recombination. Since cells derived from FA patients are hypersensitive to treatments that induce DNA interstrand cross-links (ICLs), the FA pathway has been considered to function in ICL repair. However, it still remains unclear what type of endogenous DNA damage is repaired through the FA pathway and is the cause of phenotypes in FA patients. Recent studies have suggested that cells deficient in the FA pathway are also sensitive to formaldehyde and acetaldehyde. Aldehydes may create DNA adducts including ICLs or protein DNA crosslinking. These results raise a possibility that the FA pathway prevents BMF by mitigating genotoxicity due to endogenous aldehydes. It has been known that ALDH2 deficiency resulting from Glu487Lys substitution (A allele) is prevalent in East Asian populations. While the Glu487 form (G allele) is proficient in aldehyde catabolism, even the GA heterozygote displayed strongly reduced catalysis because ALDH2 is a tetrameric enzyme and the variant form can suppress the activity in a dominant negative manner. Therefore some Japanese FA patients are expected to be deficient in ALDH2, providing an opportunity to test role of ALDH2 and aldehyde metabolism in human FA patients. Results and discussion In FA fetus, p53/p21 axis has already activated in fetal liver (Ceccaldi, Cell stem cell, 2012), indicating the possibility that hematopoietic defects in FA patients originates from an earlier developmental stage. Since human hematopoietic system originates from embryonic mesoderm, we set out to estimate the role of ALDH2 and FANCA pathway during early embryogenesis. For this, we reprogrammed somatic cells from a patient with ALDH2 GA genotype and observed their in vitro mesodermal differentiation. We first introduced reprogramming factors into fibroblasts by episomal vectors, and obtained colonies which are morphologically compatible with human induced pluripotent stem cells (iPSCs). These iPSC-like cells (designated as FA-iPLCs) showed close similarity to conventional ES/iPSCs regarding marker gene expressions and differentiation ability into three germ layers. We obtained gene-complemented FA-iPLCs (designated as cFA-iPLCs) for control study. To evaluate the impact of ALDH2 activity on iPSC- or iPLC-derived mesodermal differentiation, we next adapted the previously reported serum-free monolayer culture system. Both FA- and cFA-iPLCs showed similar differentiation manners with conventional embryonic stem cells and iPSCs, and percentages of KDR+ mesodermal progenitors including KDR+CD34+ common hemoangiogenic progenitors were comparable. Notably, ALDH2 agonist Alda1 did increase only FA-iPLC-derived mesodermal progenitors but not cFA-iPLCs. These data supported the hypothesis that mesodermal development towards hematopoietic cells in human can be affected by ALDH2 activity in the absence of FA pathway. To confirm the hypothesis, next we set out to assess whether the variation in ALDH2 affects symptoms in Japanese FA patients. Strikingly, we found that progression of BMF was strongly accelerated in heterozygous carrier of the variant A allele compared to homozygous GG patients. Furthermore we looked at occurrence of leukemia and/or myelodysplasia and the somatic developments. Interestingly, these were not significantly difference between patients with each variation of ALDH2, indicating the possibility that aldehydes affect only in early hematopoietic development, not other mesodermal tissues. Overall, our results from FA-iPLCs and clinical study indicate that the variation in ALDH2 affects the occurrence of bone marrow failure in FA patients, and that hematopoietic defect in FA patients is caused by aldehydes in early mesodermal developmental stage. Disclosures: No relevant conflicts of interest to declare.

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