GATA1 Mutations in Clonal Disorders of Children with and without Down Syndrome.

Abstract Down Syndrome (DS) children are 10–20 times folder likely to develop acute leukemia (AL) within the first four years of life compared to general pediatric population. Recently acquired somatic mutations in GATA1 gene on chromosome X have been described in most cases of DS AML and congenital TMD of DS carry the same type of mutations in exon 2 of GATA1. Here we report the preliminary results of GATA1 mutation in AL with and without DS children. The aim of this study is to provide insights in the relationships of GATA1 mutations and trisomy 21 in leukemogenesis process. GATA1 mutations were assayed in genomic DNA in 34 children with DS and AL, 2 with transient myeloproliferative disorder (TMD), 3 with myelodisplastic syndrome. Sequential sample including 2 pre-diagnosis in neonate period and 1 year before diagnosis were available in two children and 40 randomly selected DS children without known hematological disorder. A rare case of a non-DS neonate with TMD and clonal trisomy 21 were also examined. Genomic DNA was extracted and the exon 2 of GATA1 was PCR amplified as described by Wechsler et al. PCR products were sequenced in both directions and analyzed in a MegaBACE 1000 automated sequencer. Presently, GATA 1 mutations were found in 7 cases of AL, in all TMD cases with DS and none MDS case of DS. Interesting, a neonate girl with no phenotypic features of DS, but TMD features whose karyotype revealed 47, XX, +21/46, XX mosaics. A G-to-T transversion was detected which is predicted to result in a premature stop codon (c.119G>T; p.Glu67X) at the time of onset of TMD. However this same mutation was not detcted at 5 years of age.To our knowledge, this is the first reported case of TMD without DS with a detected GATA1 mutation. The presence of both somatically acquired abnormalities probably confers a proliferative advantage to the cell, resulting in TMD. We postulated in this case that both genetic abnormalities were temporary because of the non self-renewing nature of the progenitor that first had a non-disjunction event and this progenitor and the proliferative clone eventually disappeared. Therefore, even the proliferative advantage that the combination of trisomy 21 and GATA1 mutation confer, maintenance of these genetic changes are necessary for full leukemic transformation and persistence.

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GATA1 Mutation In Transient Leukemia (TL) and Myeloid Leukemia of Down Syndrome

Blood ◽

10.1182/blood.v116.21.1725.1725 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1725-1725

Author(s):

Katarina Reinhardt ◽

Alford Kate ◽

Katarina Bohmer ◽

Paresh Vyas ◽

Reinhardt Dirk

Keyword(s):

Down Syndrome ◽

Myeloid Leukemia ◽

Stop Codon ◽

Direct Sequencing ◽

Mutation Screening ◽

Patient Specific ◽

Exon 2 ◽

Transient Leukemia ◽

Almost All ◽

Gata1 Mutation

Abstract Abstract 1725 Children up to the age of 5 years with trisomy 21 (T21, Down Syndrome) are at a 400-fold excess risk of developing myeloid leukemia (ML-DS). ∼5% of newborns with T21 develop a transient leukemia (TL). The megakaryoblastic phenotype by morphology and immunophenotyping is similar in both leukemias. Mutations in hematopoietic transcription factor GATA1 gene leading to expression of N-terminal truncated protein (GATA1s) have been detected in almost all TL and ML-DS patients and is the diagnostic genetic hallmark of these diseases. Aims: Fast and accurate identification is required to:confirm the diagnosis of TL or ML-DSconfirm the diagnosis of a GATA1s positive leukemia in children with no or little stigmata of Down Syndrome (T21 mosaic)monitor minimal residual disease (MRD)determine the pattern of GATA1 mutation in TL and ML-DS. Patients: Here we report the largest cohort of children (n=229) with TL (n=129) and ML-DS (n=100). The blast percentage of blasts were significant different (TL 43±3% vs. ML-DS 29 ±2%, p<0.03). Methods: The GATA1 mutation screening have been performed in two laboratories, the central reference of the AML-BFM Study Group (Hannover, Germany; TL n=90, ML-DS n=63) and at the Weatherall Institute of Molecular Medicine (Oxford, UK; TL n=39, ML-DS n=37). The AML-BFM Lab conducted direct sequencing. If this failed, sequencing was repeated with sorted blasts. If the result remained negative, subcloning of the blasts was performed (21 out of 137 patients). The Oxford lab screened all samples by WAVE and direct sequencing. The lower limit of blasts which allowed for successful detection of a GATA1 mutation was 2%. Results: GATA1 mutations were identified in 125 out of 129 (96%) newborns with TL and in 97/100 (97%) children with ML-DS. In 99% of cases GATA1 mutations were detected in exon 2; only in 2 cases were exon 3 mutations identified. GATA1 mutation were identified in 13 children with Down mosaic and acute leukemia (TL n=8; ML-DS n=5). The detection of GATA1 prevents intensive chemotherapy in newborns with TL and allowed reduced intensity chemotherapy to be administered in infants with ML-DS. The mutations are diverse: deletions (37%), point mutations (24%), duplications (23%) and insertions (16%). With exception of substitutions, which were uniquely detected in TL (n=2; 1.6%), no differences between TL and ML-DS have been observed. Mutations were predicted to result in a stop codon(66%), affect splicing (16%), produce a frameshift that produced a subsequent stop codon (7%), or alter the start codon (3%). No differences in these predicted outcomes was present between TL and DS-ML. Summary: Rapid detection of GATA1 mutations is possible in almost all children with T1 and mosaic T21 who develop TL or ML-DS with these approaches, even in samples where the blast count is as low as 2%. Mutation detection and conformation of the correct diagnosis is critical to ensure appropriate therapy is administered and to allow patient specific MRD monitoring. Disclosures: No relevant conflicts of interest to declare.

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Exploring the Pathogenesis of Down Syndrome-Related Myeloproliferative Disorders Using iPSCs

Blood ◽

10.1182/blood.v124.21.868.868 ◽

2014 ◽

Vol 124 (21) ◽

pp. 868-868

Author(s):

Nishinaka Yoko ◽

Akira Niwa ◽

Mitsujiro Osawa ◽

Akira Watanabe ◽

Tatsutoshi Nakahata ◽

...

Keyword(s):

Down Syndrome ◽

B Lymphocytes ◽

Trisomy 21 ◽

Morphological Difference ◽

Chromosome 21 ◽

Fish Analysis ◽

Transcription Activator ◽

Differentiation Potential ◽

Exon 2 ◽

Gata1 Mutation

Abstract Down syndrome (DS) is a congenital syndrome due to the trisomy of chromosome 21. Transient myeloproliferative disorder (TMD) is its hematopoietic complication, affecting approximately 10% of DS-neonates. TMD is characterized by transient abnormal proliferation of blastic cells, and importantly, all TMD blasts bear mutations in GATA1 gene. Although TMD usually resolves spontaneously within 3 months after birth, twenty to thirty percent of TMD patients develop acute megakaryoblastic leukemia (AMKL) within several years afterward. This leukemogenic transition is considered as a good model for multi-step tumorigenesis. According to this putative multi-step model, the first hit should be additional chromosome 21, the second one is mutations on GATA1 gene which is requisite to the onset of TMD, and the “unknown” third hits are required for the progression into AMKL. However, it is still unclear that 1) how GATA1 mutation promotes TMD development, 2) what kinds of third hit are required for the onset of AMKL, and 3) why GATA1-mutated progenitors prevails during embryonic hematopoiesis only in trisomy 21 patients? In order to address these issues, a strictly controlled isogenic cell panels that can reproduce human emboryonic hematopoietic development is needed. Human induced pluripotent stem cells (iPSCs) derived from DS patients are a promising platform for this, but so far there is no report regarding GATA1-mutated TMD-associated iPSCs. Therefore, we set out to establish an iPSC panel that covers each genomic status of chromosome 21 and GATA1 gene. For this, we established both GATA1 mutant and wildtype clones from both trisomy 21 and disomy 21 clones. And we also established TMD patient derived iPSCs. First, we established isogenic iPSCs derived from EB virus immortalized B-lymphocytes of 2 mosaic DS patients. Frequency of trisomy 21 cells evaluated by FISH analysis and G-banding were 93% and 94% for each patient. We reprogrammed these cells by introducing 5 episomal vectors, pCE-hOCT3/4, pCE-hSK, pCE-hUL, pCE-mp53DD and pCXB-EBNA1, under feeder free condition. We genotyped each iPSC clones by digital-PCR analysis and found that the frequency of trisomy iPSC clones were comparable to that of trisomy cells in original EBV immortalized B-lymphocytes. There is no morphological difference between disomy and trisomy iPSC clones in both patients. We next introduced disease-associated GATA1 mutation into established isogenic trisomy and disomy iPS clones using transcription activator-like effector nuclease (TALEN) technology. We introduced a frameshift mutation in exon 2, which causes premature termination of the full-length transcript originated from 1st ATG and exclusively produces the shorter isoform of GATA1 (GATA1s) transcribed from 2nd ATG. Next, we obtained peripheral blood mononuclear cells (PBMCs) from a TMD patient in order to establish TMD-blast-derived iPSCs. Eighty-nine percent of nuclear cells in the PBMC fraction was CD117+CD45+ blastic cells, whereas only 5.6% were non-blast cells including CD3 positive T-lymphocytes, CD11b positive myeloid lineage cells and CD19 positive B-lymphocytes. The CD117+ cells showed TMD/AMKL blast-like appearance such as coarse choromatin pattern with nucleolus and bleb-like structures. We sorted out CD45+CD117+ blastic cells and CD45+CD117- non-blastic cells and successfully established iPSC clones from both populations. In conclusion, we successfully established a comprehensive panel of iPSC clones for evaluating the hematopoietic consequence associated with the GATA1 genotype and the ploidy of chromosome 21. We are currently evaluating hematopoietic differentiation potential of each clone and exploring the underlying pathophysiology of TMD/AMKL by using this platform. We believe that comprehensive understanding of TMD and AMKL pathogenesis provides a fruitful insight into our understanding of human leukemogenesis. Disclosures No relevant conflicts of interest to declare.

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GATA1 Mutations in Newborns with Down Syndrome.

Blood ◽

10.1182/blood.v108.11.641.641 ◽

2006 ◽

Vol 108 (11) ◽

pp. 641-641

Author(s):

Claudio Sandoval ◽

Sharon R. Pine ◽

Charlotte Druschel ◽

Somasundaram Jayabose ◽

Qianxu Guo ◽

...

Keyword(s):

New York ◽

Down Syndrome ◽

Single Strand Conformation Polymorphism ◽

Protein Product ◽

Polymorphism Analysis ◽

Exon 2 ◽

Blood Spots ◽

Increased Risk ◽

Significant Difference ◽

Gata1 Mutation

Abstract Somatic mutations of the GATA1 gene have been detected in almost all cases of Down syndrome (DS)-associated acute megakaryoblastic leukemia (AMKL) and transient leukemia (TL). There is emerging evidence that the protein product of GATA1 mutations, GATA1s, directly contributes to leukemogenesis. Although an in utero origin of GATA1 mutations is established, a comprehensive study using a large number of cases is required to determine the overall incidence and clinical relevance of GATA1 mutations in DS newborns. We screened 575 DS infants born between January 1997 and December 1999 for GATA1 exon 2 mutations by single-strand conformation polymorphism analysis of PCR products. We used Gunthrie cards obtained from the New York Congenital Malformation Registry. Registry data was blinded until after the GATA1 mutation analyses were completed. Twenty-eight (4.9%) infants were identified as having a GATA1 mutation. There was no significant difference in the frequency of GATA1 mutations based on gender or maternal average age (p = 0.93 and 0.31, respectively). There was no significant difference in the GATA1 mutation frequency between those classified as black, white, or Asian, but Hispanics had a borderline non-significant increase in frequency of GATA1 mutations compared to non-Hispanics (8.5% compared to 4.0%, p=0.06). Based on data from the New York Cancer Registry reviewed in 2005, two of the patients with a GATA1 mutation subsequently developed leukemia; one patient developed AMKL and the other had a leukemia of unspecified phenotype. Out of the 547 GATA1-negative patients, there was only one case of leukemia, but it was AML excluding AMKL. These results confirm a pre-natal origin of GATA1 mutations in DS patients. The frequency in this study was lower than the 10 percent previously reported (2 of 21 DS blood spots). Obtaining Gunthrie card blood spots for GATA1 mutation analysis serves as a relatively non-invasive screening approach. The presence of a GATA1 mutation at the time of birth might serve as a biomarker for an increased risk of developing DS-related AMKL.

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High Frequency of GATA1 Mutations in Childhood Non-Down Syndrome Acute Megakaryoblastic Leukemia

Blood ◽

10.1182/blood.v120.21.888.888 ◽

2012 ◽

Vol 120 (21) ◽

pp. 888-888 ◽

Cited By ~ 1

Author(s):

Katarina Reinhardt ◽

C. Michel Zwaan ◽

Michael Dworzak ◽

Jasmijn D.E. de Rooij ◽

Gertjan Kaspers ◽

...

Keyword(s):

Transcription Factor ◽

Down Syndrome ◽

Trisomy 21 ◽

Mutation Screening ◽

Study Group ◽

Acute Megakaryoblastic Leukemia ◽

Monosomy 7 ◽

Megakaryoblastic Leukemia ◽

Exon 1 ◽

Gata1 Mutation

Abstract Abstract 888 Introduction: Pediatric acute megakaryoblastic leukemia (AMKL) occurred in 6.6% (84/1271) of the children enrolled to the AML-BFM98 and 2004 studies. Despite a similar phenotype in morphology and immunophenotype, AMKL shows a heterogenous cytogenetic distribution (normal karyotype 23%, complex karyotype 21%, t(1;22) 9%; MLL-rearrangement 8%; monosomy 7 5%, trisomy 8 5%; other aberrations 29%). Mutations of the hematopoietic transcription factor GATA1 have been identified in almost all children suffering myeloid leukemia of Down syndrome (ML-DS). In addition, GATA1 mutations (GATA1mut) could be identified in children with trisomy 21 mosaic. Here, AMKL without evidence of Down syndrome or Down syndrome mosaic were analyzed for mutations in exon 1, 2 or 3 of the transcription factor GATA1. Patients: Seventy-one children from the AML-BFM Study group (n=51; 2000–2011), the Netherlands (n=10), France (n=3) and Scandinavia (n=7) were included. Within the AML-BFM Group the 51 analyzed patients showed similar characteristics compared to the total cohort of 84 children with AMKL of the AML-BFM 98 and 2004 studies. AMKL was confirmed according to the WHO classification by genetics (t(1;22)); morphology and immunophenotyping. Table 1a) summarizes the patientxs characteristics and b) the cytogenetic results. Methods: For GATA1 mutation screening genomic DNA was amplified by PCR reaction for exon 1, 2, and 3. PCR amplicons were analyzed by direct sequencing or following denaturing high-performance liquid chromatography (WAVE). Results: Seven different GATA1 mutations were detected in 8 children (11.1%; table 2). In all GATA1mut leukemia, a trisomy 21 within the leukemic blasts could be detected. Seven out of these 8 children and all other 64 AMKL patients have been treated with intensive chemotherapy regimens according the study group protocols. The results are given in table 2. All achieved continuous complete remission (CCR; 0.4 to 4.2 years). In one newborn with typical morphology and immunophenotype a GATA1mut associated transient leukemia was supposed. The child achieved CCR (follow-up 6 years). In total, allogeneic stem cell transplantation in 1st CR was performed in 6 children with AMKL (GATA1mut leukemia n=1). Conclusions: GATA1 mutations occurred in 11% of children with AMKL without any symptoms or evidence of trisomy 21 or trisomy 21 mosaic. GATA1 mutations are associated with a trisomy 21 within the leukemic blasts. Although non-response occurred, prognosis was significant better compared to other AMKL. Therefore, analysis of GATA1 mutation in infant AMKL is strongly recommended. Whether treatment reduction similar to ML-DS Down syndrome is feasible needs to be confirmed. Disclosures: No relevant conflicts of interest to declare.

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A new mutation at exon 2 of hprt1 locus causing Lesch-Nyhan Syndrome.

Innovaciencia Facultad de Ciencias Exactas Físicas y Naturales ◽

10.15649/2346075x.362 ◽

2015 ◽

Vol 3 (1) ◽

pp. 18-21

Author(s):

Adriana María Gil Zapata ◽

Adriana Castillo Pico ◽

Leonor Gusmão ◽

António Amorim ◽

Fernando Rodríguez Sanabria

Keyword(s):

Genetic Counseling ◽

Inborn Error ◽

Stop Codon ◽

Premature Stop Codon ◽

Dna Fragments ◽

Hprt Gene ◽

New Mutation ◽

Exon 2 ◽

The Family ◽

Hypoxanthine Guanine Phosphoribosyl Transferase

Introduction: Lesch-Nyhan síndrome (LNS) is an X-linked recessive inborn error of metabolism, due to deficiency of the enzyme Hypoxanthine-guanine-phosphoribosyl transferase (HGPRT; EC.2.4.2.8) resulting in hyperuricemia, neurological and behavioural disturbances. In the present work, we report the results of the study of a Colombian family, where LNS was previously clinically and biochemically diagnosed. Material and Methods: The full HPRT gene, including 9 exons and 8 introns, was amplified on eight separate DNA fragments. Both strands, forward and reverse, of the amplified DNA fragments were analyzed and the obtained sequences were compared with those deposited at National Center for Biotechnology Information. Results and conclusions: Sequence analysis allowed the detection of new LNS causing mutation, an adenine deletion in exon 2 of HPRT1 gene resulting in a frameshift which determines a premature stop codon. This study, besides adding a new mutation to the already large spectrum of disease causing variation at HPRT, allows therefore providing genetic counseling for the family as well as prenatal diagnosis.

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Analysis of GATA1 Mutations in Down Syndrome Infants with Transient Abnormal Myelopoiesis and Clinical Impacts of GATA1 Mutation Types: A Report from the JPLSG TAM-10 Study

Blood ◽

10.1182/blood.v128.22.2865.2865 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2865-2865

Author(s):

Kiminori Terui ◽

Tsutomu Toki ◽

Asahito Hama ◽

Hideki Muramatsu ◽

Daisuke Hasegawa ◽

...

Keyword(s):

Down Syndrome ◽

Genomic Dna ◽

Sanger Sequencing ◽

Rt Pcr ◽

Mrna Isoforms ◽

Blood Samples ◽

Transient Abnormal Myelopoiesis ◽

Splicing Mutations ◽

Targeted Ngs ◽

Gata1 Mutation

Abstract Introduction: Approximately 5-10% of neonates with Down syndrome (DS) develop transient abnormal myelopoiesis (TAM). Almost all patients with TAM have GATA1 mutations resulting in the exclusive expression of a truncated protein (GATA1s). Although TAM patients exhibit various hematological abnormalities including circulating blasts, leukocytosis and thrombocytopenia, these abnormalities have been also reported in DS neonates without TAM. Therefore, analysis of GATA1 mutations is very important in the diagnosis of TAM. However, standard procedures to detect GATA1 mutations have not been established. Most of GATA1 mutations occur within the exon 2 and the surrounding sequences, but types of the mutations are varied, including insertions, deletions, duplications and point mutations. We previously reported that the expression levels of GATA1s were varied depending on types of mutations and might be associated with phenotypes of TAM including white blood cell (WBC) counts at diagnosis and a risk of progression to myeloid leukemia of DS (Kanezaki et al., Blood 2010). However, these findings have not been confirmed by other groups and effects of GATA1 mutation types on other clinical features of TAM have not been investigated. Patients and Methods: One hundred sixty-seven patients were enrolled in TAM-10 study and blood samples were available in 166 patients. GATA1 mutations were analyzed by Sanger sequencing using genomic DNA and complementary DNA (cDNA) prepared from peripheral blood. Expression patterns of GATA1 mRNA isoforms were examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Targeted next-generation sequencing (NGS) were performed for patients in whom GATA1 mutations were not detected by Sanger sequencing. GATA1 mutations were classified into 3 groups according to the predicted consequences, splicing error (SE), loss of the first methionine (LOM) and premature termination codon (PTC). Blood smears were centrally reviewed. Patients whose smears were prepared more than 14 days after the onsets of TAM were excluded from the morphological analyses. Differences in clinical parameters among the 3 mutation groups were analyzed using the Fisher's exact test, Kruskal-Wallis test or Steel-Dwass test. Results: Mean age at sample collection, WBC count and blast percentage of blood samples were 8 days (range, 0-70 days), 22,100 µ/l (range, 4,400-422,000 µ/l), and 28.5% (range, 0-95%), respectively. GATA1 mutations were identified in 153 of 166 patients (92%) by Sanger sequencing. Although GATA1 mutations were not detected in 13 patients, splicing mutations were suspected in 7 patients because of the lack of the full-length GATA1 mRNA isoforms. In 12 of these 13 patients, blast percentages of the samples were less than 5%. GATA1 mutations were identified after targeted NGS in 10 of 13 patients negative for GATA1 mutations by Sanger sequencing. Of note, splicing mutations were confirmed after targeted NGS in all 7 patients suspected of having splicing mutations by RT-PCR. Collectively, GATA1 mutations were identified in 163 of 166 patients (98%). After exclusion of patients with multiple mutations (n=14) and internal deletion mutations (n=5), 39, 13 and 92 patients were classified into the SE, LOM and PTC groups, respectively. WBC counts at diagnosis were not significantly different among the 3 groups. However, the incidences of eosinophilia (>1,500 µ/l) were significantly different among the 3 groups (P<0.0001) and eosinophilia was more frequent in the SE (14/31, 45%) and LOM (4/11, 36%) groups than in the PTC (6/76, 8%) group (P<0.0001 and P=0.041, respectively). The levels of alanine aminotransferase (ALT) at diagnosis were also different among the 3 groups (P<0.00001) and the difference was statistically significant between the SE (median, 69; range, 11-468) and PTC group (median, 16; range, 3-380; P<0.00001). Conclusion: These results suggest that Sanger sequencing using cDNA as well as genomic DNA is rapid and sensitive method to detect GATA1 mutations and that targeted NGS is useful for detection of GATA1 mutations in patients with low blast percentages. GATA1 mutation types may affect some clinical features of TAM including the numbers of eosinophils and the levels of ALT. Because estimated expression levels of GATA1s are higher in SE and LOM groups than PTC group, high GATA1s expression might be associated with eosinophilia and increased levels of ALT in TAM. Disclosures No relevant conflicts of interest to declare.

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Heterogenous Expression of Glycoprotein lb, IX and V in Platelets from Two Patients with Bernard-Soulier Syndrome Caused by Different Genetic Abnormalities

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649956 ◽

1995 ◽

Vol 74 (06) ◽

pp. 1411-1415 ◽

Cited By ~ 49

Author(s):

Masaaki Noda ◽

Kingo Fujimura ◽

Toshiro Takafuta ◽

Takeshi Shimomura ◽

Tetsuro Fujlmoto ◽

...

Keyword(s):

Complex Analysis ◽

Transmembrane Domain ◽

Stop Codon ◽

Expression Patterns ◽

Premature Stop Codon ◽

Surface Expression ◽

Coding Region ◽

Genetic Changes ◽

Pcr Products ◽

Bernard Soulier Syndrome

SummaryBernard-Soulier syndrome (BSS) is a rare inherited bleeding disorder, which is caused by deficiency or decrease of the platelet GPIb/IX/V complex. Analysis of two patients with BSS by How cytometry of the blood revealed different expression patterns of the components of the GPIb/IX/V complex. In case 1, GPIX was completely absent but residual amounts of GPIbα and GPV were detectable; in case 2, GPIbα was completely absent. We amplified the coding regions of GPIbα, GPIbß, GPV, and GPIX from the patients’ genomic DNA with the polymerase chain reaction (PCR) and sequenced the PCR products. In case 1, we identified a point mutation in the GPIX coding region that changes the codon for tryptophan-126 (TGG) to a nonsense codon (TGA). In case 2, we found a deletion of nucleotide within seven adenine repeats at the position of 1932 to 1938 in the coding region of GPIbα, which causes a frame shift that results in 58 altered amino acids and a premature stop codon. These genetic changes alter the transmembrane domain of GPIX or GPIbα and, therefore, would prevent proper insertion of the proteins in the plasma membrane. Thus, abnormality of a single component protein (GPIX or GPIbα) alters the assembly of the GPIb/IX/V complex and causes heterogenous surface expression of GPIbα, GPV and GPIX.

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GATA1 Mutations in Transient Leukemia and Myeloid Leukemia in “Down” Syndrome.

Blood ◽

10.1182/blood.v112.11.923.923 ◽

2008 ◽

Vol 112 (11) ◽

pp. 923-923 ◽

Cited By ~ 1

Author(s):

Katarina Reinhardt ◽

Katarina Boehmer ◽

Jan-Henning Klusmann ◽

Manuela Germeshausen ◽

Annette Sander ◽

...

Keyword(s):

Down Syndrome ◽

Trisomy 21 ◽

Myeloid Leukemia ◽

Patient Specific ◽

Exon 2 ◽

Megakaryoblastic Leukemia ◽

Risk Of Death ◽

Splicing Mutations ◽

Transient Leukemia

Abstract Introduction: Newborns with trisomy 21 have a 5 to 10% risk to develop transient leukemia (TL). More than 20% of these infants progress to myeloid leukemia of Down syndrome (ML-DS) within the first 4 years of life. Mutations of the hematopoietic transcription factor GATA1 have been identified in almost all patients with TL and ML-DS. Here we report the biological and follow up data of a large cohort of children with proven GATA1 mutation reported to date either as TL (n=43) or ML-DS (n=28). Results: GATA1 mutations (point mutations, insertion, deletion, duplication; including 45 mutations not yet published) were identified in 42/43 TL (98%) and in 23/28 ML-DS (83%) patients. n age madian gastation week WBC/μl blasts % outcome 1PB: peripheral blood; BM: bone marrow transient leukemia 43 3 days (0 to 57) 37 (31 to 40) 33450 (1000 to 321000) 45 (7 to 91) death n = 3, 7%  ML-DS n = 9, 22% ML-DS 28 1-3 yrs (0.8 to 3) 38 (37 to 38) 4900 (1000 to 160000) PB1 7(1-87)  BM1 24 (4-78) death n = 2, 7%  relapse n=0 In 9 patients multiple mutations were noted in the same clone as confirmed by subcloning. In one patient with TL two different GATA1 mutations were detected in two independent clones. When this patient progressed to ML-DS only the minor clone was present. The majority of the mutations was localized in exon 2 (n=59). Only a few mutations could be found in intron 1 and 2 (n=5) or in exon 3 (n=1). As a result, these mutations led to the introduction of a premature stop codon within exon 2 (n=40), frameshift (n=14), altered splicing (n=7), or lack of an initiation codon (n=4). Interestingly, children with TL and splicing mutations were significantly older at diagnosis than patients with other mutations (day 38 vs. day 3 p <0.05). No differences between mutational types were evident regarding gestational age, white blood cell count, platelet count, hemoglobin levels, or risk of death or ML-DS. In children with a myeloproliferative disease (MPD; n=7) or acute megakaryoblastic leukemia (AMKL; n=1) without stigmata of Down syndrome, GATA1 mutations could be detected. All of them were diagnosed as trisomy 21 mosaic. In this group the frequency of frameshift and altered splice mutations (5/7 vs. 9/36) was significantly higher compared to those with premature stop codons (2/7 vs. 27/36); pFishers exact =0.03). In 20 children (TL n=13, ML-DS n=7) the GATA1 mutant clone has been prospectively monitored by quantitative PCR using patient specific TaqMan probes. Seventeen TL patients showed decreasing minimal residual disease (MRD) levels and became negative (<10−4) during follow-up, whereas three children, who later developed ML-DS, remained positive at all time points. After two treatment elements all ML-DS patients had undetectable levels of GATA1s. After a median follow up of 1.5 years (0.9 to 2 years), no child suffered relapse however, the follow-up is much too short to draw definitive conclusions. Conclusion: In conclusion, we confirmed the high frequency of GATA1 mutations in children with TL or ML-DS. The occurrence of splicing mutations correlated with the age at diagnosis underlining the biologic relevance of the kind of mutation. We demonstrated the feasibility of a leukemia specific monitoring of MRD. As those children with sustaining detectable levels of GATA1s progressed to leukemia, these results might have therapeutic consequences for TL and later for ML-DS. In addition it may serve as a proof of principle for the feasibility of MRD monitoring in other AML-associated mutations. The identification of GATA1s positive MPD and AMKL in children without obvious stigmata of Down syndrome, all confirmed as trisomy 21 mosaic, implicate the necessity of GATA1s diagnostics in all newborn and infants with megakaryoblastic leukemia.

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Transient and Myeloid Leukemia in Children with Down Syndrome Mosaic

Blood ◽

10.1182/blood.v118.21.2539.2539 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2539-2539

Author(s):

Alexandra Kolenova ◽

Katarina Reinhardt ◽

Michaela Nathrath ◽

Claudia Rossig ◽

Arend von Stackelberg ◽

...

Keyword(s):

Down Syndrome ◽

Stem Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Trisomy 21 ◽

Myeloid Leukemia ◽

Patient Specific ◽

Allogenic Stem Cell Transplantation ◽

Gata1 Mutation

Abstract Abstract 2539 Introduction: Transient leukemia (TL) occurs in 5 to 10% of newborns with Down syndrome (DS). In almost all cases it resolves spontaneously within 3 months, but 20–25% of the children develop myeloid leukemia (ML-DS) until the age of 4 years. TL and ML-DS can occur also in children without any clinical signs of Down syndrome, but with constitutional trisomy 21 due to mosaicism. It can be difficult to diagnose TL or ML-DS in these children and the treatment strategies have not been defined. Patients/Material: Between 1/2002 and 7/2011, 15 newborns and infants were diagnosed with DS mosaic. Nine of them presented with TL and 8 children suffered from ML-DS; 2 of them with a history of TL (table 1). In children without any stigmata the special morphology and immunophenotype of blasts triggered the screening for GATA1 mutation and trisomy 21 mosaic. Diagnostic work-up was performed according to standard guidelines: morphology, immunophenotyping (IP), cytogenetics and FISH (trisomy 21), molecular genetics (GATA 1 mutation screening). Screening of GATA1 mutations was done with direct sequencing of PCR product (Exon1, Exon2, and Exon3). For monitoring of GATA1 mutant clone qPCR have been used with patient specific TaqMan probes and primers. Mosaic was detected by cytogenetics or FISH in bone marrow, blood and/or fibroblasts. Results: All newborns with TL achieved complete remission (CR). Due to clinical symptoms caused by the leukemic blasts, in 3 children low-dose cytarabine was applied. One patient died due to cardiovascular failure. In all patients GATA 1 mutation was confirmed. Minimal residual disease by qPCR (mutation-specific probes) or immunophenotyping (IP) revealed negativity in 3 out of 3 children monitored (follow-up 2 to 10.1 yrs). Two children with (unknown) trisomy 21 mosaic were diagnosed as acute megakaryoblastic leukemia (AMKL) and treated according the high risk arm of the AML-BFM 2004 including allogeneic stem cell transplantation (one child), GATA1 mutation was identified retrospectively. Both children are alive in CR. Six children with ML-DS were initially treated according the AML-BFM protocol. After ML-DS was confirmed, therapy was continued with the intensity reduced schedule according to the ML-DS 2006 protocol. All children are still in CR (follow-up 1.5 to 6.7 years, median 2.4 yrs). This was confirmed by MRD-monitoring, which achieved negativity after two treatment elements (qPCR <10−4 n=3; IP <10−3 n=6). In one child a distinct refractory myeloid leukemia population (GATA1mut negative/trisomy 21 negative) arose after the 1st induction. Due to treatment refractory, allogenic stem cell transplantation was applied. Conclusions: GATA1 mutated leukemia has to be excluded in all young children with AMKL (<5years old) to prevent overtreatment. Treatment with reduced intensity protocol like ML-DS 2006 seems to be effective and sufficient in children with trisomy 21 mosaic and GATA1 mutated ML-DS. Disclosures: No relevant conflicts of interest to declare.

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