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Introduction: Azacitidine is at present the standard of treatment of myelodysplastic syndromes (MDS). We demonstrated that MDS patients responsive to azacitidine have significantly higher expression of the azacitidine-activating enzyme uridine-cytidine kinase-1 (UCK1) in bone marrow mononuclear cells (Valencia et al. Leukemia 2014). Correlation of the expression of nucleoside transporter, activating and disactivating enzymes with clinical and in vitro response to hypomethylating drugs azacitidine and decitabine has been suggested by several authors. Yet, the crucial role of these enzymes has to be ascertained, as well as their possible different importance in determining resistance. Objectives: To confirm that the cellular expression of nucleoside metabolizing enzymes plays a major role in cellular resistance and significantly impacts on clinical response to azacitidine. Methods: Two cell lines, SKM1 sensitive (SKM1-S) and SKM1 resistant (SKM1-R) to azacitidine (Cluzeau et al. Oncogene 2014) were analyzed for expression of UCK1, UCK2, hENT1, hCNT3, RRM1 and RRM2 by quantitative PCR, using an ABI GeneAmp® 5700. The specific oligonucleotides and TaqMan® probes were acquired among the Assay-on-Demand® Gene Expression Products (Applied Biosystems). Corresponding proteins were quantitated by western blotting in both cell lines. The expression of UCK1 and UCK2 was blunted by siRNAs in order to determine their role in in vitro sensitivity to azacitidine. For UCK2 silencing in SKM1-S, specific siRNAs were used (OriGene Technologies, Inc., Rockville, MD, USA). SKM1-S cells were cultured at a density of 1x106 cells/ml in 9 ml of RPMI 1940 medium. After 72 h of transfection, cells were treated for further 48h with azacitidine at the concentrations of 0,1 and 1 μM. After assessment of effective gene silencing, apoptosis and DNA methylation were evaluated by Annexin V test, MSP and pyrosequencing in cells exposed to azacitidine. In addition, the expression of nucleoside metabolizing enzymes was evaluated prospectively in IPSS high risk MDS patients treated with azacitidine 75mg/m2/7 days every 28 days. Results: SKM1-R cells did not express UCK1, UCK2, hENT1, hCNT3, RRM1 and RRM2. Corresponding proteins were also not expressed. In order to clarify the specific role of UCK2 protein in the mechanisms of resistance to azacitidine, we knocked-down UCK2. A reduction of apoptosis was observed in UCK2-silenced SKM-1 S after azacitidine 0.1 μM treatment: 31%±0,85% Annexin V-positive cells versus 21%±0,35% in non-silenced control SKM1-S cultures (P= 0.054). Hypomethylation induced by in vitro azacitidine treatment was also hampered by absence of expression of UCK2. Quite surprisingly, in MDS cases, gene expression of UCK1, UCK2, hENT1, hCNT3, RRM1 and RRM2 in primary cellsdid not predict different clinical response to azacitidine. Conclusions: We demonstrated that UCK1, UCK2, hENT1, hCNT3, RRM1 and RRM2 and the corresponding proteins are absent in an in vitro generated azacitidine-resistant cell line SKM1-R. These abnormalities could be major determinants of the induced resistance to the nucleoside analogue. Supporting the importance of these enzymes in resistance to nucleosides, and the major role played by UCK2, its silencing induced by synthetic siRNAs significantly decreased azacitidine effects. Prospective evaluation of the predictive role of cellular expression of genes involved in azacitidine metabolism is ongoing in a larger cohort of MDS patients. Establishing the mechanisms of resistance to azacitidine could lead us to optimize treatment of MDS patients whose scarce susceptibility could be predictable. Disclosures Santini: Celgene: Consultancy, Honoraria, Research Funding; Amgen: Other: advisory board; Novartis: Consultancy, Honoraria; Onconova: Other: advisory board; Janssen: Consultancy, Honoraria; Astex: Other: advisory board.

To the editor: We read with interest the article by Frederiksen et al about the increased risk of developing hematologic and nonhematologic cancers in a large cohort of Danish patients with myeloproliferative neoplasms (MPNs).[1][1] In a previous study[2][2] we reported that MPN patients have a 3.

Introduction: Accumulating evidence implicates innate immune activation in the pathobiology of myelodysplastic syndromes (MDS) and its inflammatory bone marrow microenvironment. Excess elaboration of S100A9 accompanied by secondary induction of TNFα and IL-1β, characterize the inflammatory milieu found in lower risk MDS. Erythroid stimulating agents (ESA) and lenalidomide (LEN) are erythropoietic promoters with known anti-inflammatory properties. To date, the role of such inflammation parameters in endogenous erythropoietin (Epo) regulation and response to such treatments has not been investigated. Herein, we investigated the role of these inflammatory cytokines on Epo elaboration and response to ESA and LEN in MDS. Materials and Methods: The HepG2 hepatoma cell line was used to investigate Epo elaboration in vitro. Serum collected from 311 MDS patients from 3 centers (AOU Careggi, University of Firenze; Saint Louis hospital in Paris; H. Lee Moffitt Cancer Center in Tampa) were investigated. 125 were obtained prior to ESA treatment and 186 prior to LEN or LEN+EPO treatment. ELISAs for S100A9, S100A8, TNFα, IL1β and EPO were performed. Clinical data were analyzed from all patients. Spearman's correlation, Mann-Withney and Jonckheere-Terpstra tests were used for analysis of continuous variables. Chi-square test was used for analysis of non-continuous variables. Results: Median age of the patients was 76 years [41-94]. IPSS was low, intermediate-1, intermediate-2 and high risk in 47%, 49%, 3% and 1% of patients, respectively; whereas 28%, 42%, 22%, 7% and 1% of patients were very low, low, intermediate, high and very high risk according to IPSS-R. Serum S100A9 concentration was significantly higher in patients with lower risk versus higher risk MDS (17,798 pg/ml vs 15,291 pg/ml, p=0.01). No significant difference was observed for TNFα and IL1 β according to IPSS or IPSS-R. ELISA quantitation following 24h in vitro stimulation of HepG2 cells with S100A9 (10 to 20 µg/ml), TNFα (10 to 100 ng/ml) or IL1β (10 to 100ng/ml) showed that each cytokine significantly suppressed Epo elaboration in a concentration-dependent manner. To validate these findings, we assessed the relationship between serum concentration of these inflammatory cytokines and Epo level in MDS patients. We observed a significant negative correlation between TNFα and Epo concentration (r=-0.158, p=0.01), and a corresponding significant negative correlation between S100A9 and Epo (r=-0.143, p=0.01). We also found a significant positive correlation between S100A9 and S100A8 (r=0.757, p Conclusion: These findings demonstrate that S100A9 and its NFκ-B transcriptional target, TNFα, directly suppress Epo elaboration and endocrine response to anemia in MDS. These inflammatory proteins may serve as rational biomarkers for response to lenalidomide and/or ESA treatment and merit prospective validation. Therapeutic strategies that either neutralize S100A9 or suppress elaboration of this alarmin may improve erythropoiesis in MDS by restoring Epo response and suppressing innate immune effectors. Disclosures Fenaux: Janssen: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Santini:celgene, Janssen, Novartis, Onconova: Honoraria, Research Funding. List:Celgene Corporation: Honoraria, Research Funding.

Introduction: Myelodysplastic Syndromes (MDS) are clonal neoplasms. Whether the transforming event occurs in a myeloid committed cell or in earlier hematopoietic progenitor/stem cell it is still not ascertained. We evaluated the repopulating ability and stem cell potential of hypoxia maintained primary bone marrow (BM) progenitors derived from Myelodysplastic Syndromes (MDS) patients, and their capacity of engraftment in sublethally irradiated NOD-SCID mice. Methods: Thirty eight BM samples were obtained at diagnosis from MDS patients (WHO: 9 RA, 12 RCMD, 7 RAEB, 8 AML/post MDS, 2 Del(5)q). Mononuclear BM cells were isolated and cultured with TPO, FLT3-L, SCF, IL-3 in severe hypoxic conditions (0,3%O2, 5%CO2, 95%N2), for 10-13 days (LC1). The stem cell potential of these cultures was explored by transferring cells to growth-permissive secondary cultures in normoxia (LC2), in the presence of SCF, G-CSF, IL-6, IL-3, and according to the Culture-Repopulating Ability (CRA) assay methodology, cell proliferation was evaluated daily. Expression of CD34, CD38, CD117, CD133 was determined before and after hypoxia cultures, and when present, the persistence of chromosomal aberrations was analyzed by FISH at various stages of cell culture. In parallel, MDS mononuclear BM cells were intravenously injected in sublethally irradiated NOD-SCID mice before hypoxic culture and after LC1. Mice were sacrificed at day 56 and 90 after graft. The presence of CD45+ human cells in the peripheral blood of injected mice was evaluated by flow cytometry analysis every two weeks, and quantitated to evaluate the repopulating ability and the different engraftment capacity of cells before hypoxic culture and after hypoxic incubation. Mice marrow trephines as well as spleens were also evaluated morphologically to test the engraftment of human MDS cells. Results: In all 38 MDS cases studied, cultured cell number decreased of one log or more after 10-13 days of culture in hypoxic conditions. In 12/38 MDS cases we observed a significant repopulating ability at day 17 of LC2, all classified as Low/INT-1 IPSS risk category. In IPSS high and INT-2 risk cases, repopulating ability according to CRA method was absent; CD34+ cells were always decreased after hypoxia and did not coexpress CD38, confirming the selection of earlier hematopoietic progenitor cells. We demonstrated the persistence of chromosomal aberrations in CD34pos/CD38neg cells after hypoxic culture in 5 MDS cases (del5q, +8, del20q, -Y, complex karyotype). Sublethally irradiated NOD-SCID mice transplanted with hypoxia cultured cells showed in peripheral blood an higher percentage of CD45+ human cell than mice transplanted with non hypoxia selected cells. In particular, mice transplanted with hypoxia cultured BM mononuclear cells derived from a 5q- MDS patient showed an higher percentage of CD45+ human cells than mice transplanted with non-hypoxia selected cells at day 28 after transplant (5,5% vs 0,56 %); mice transplanted with hypoxia cultured BM mononuclear cells derived from a CRDM MDS patient showed an higher percentage of CD45+ human cells than mice transplanted with cells before hypoxia at day 56 (13,3% vs 1,4%), and at day 71 (43,05% vs 12,7%). Mice bone marrow cytometry analysis confirmed higher percentage of CD45+ cells in bone marrow derived from mice transplanted with hypoxia cultured cells than non-hypoxia cultured (1,9 % vs 0,1%). Conclusions: Severe hypoxic culture conditions are maintaining a sub-population of MDS cells endowed with increased repopulating capacity in vitro and in vivo. Most importantly, in our cohort of cases studied, repopulating ability was observed exclusively in IPSS lower risk MDS cases. Disclosures Santini: celgene, Janssen, Novartis, Onconova: Honoraria, Research Funding.

Myelodysplastic syndromes (MDS) and the related disorder chronic myelomonocytic leukemia (CMML) are characterized by abnormal DNA hypermethylation and the DNA methyltransferase inhibitors (DMTis) azacytidine and decitabine (DAC) are frequently used as frontline therapy in these patients. However, DMTis are ineffective for ~50% of the patients who must still undergo treatment for at least 6 months before they can be deemed resistant. Therefore, it is of critical importance to identify baseline molecular differences associated with DMTi sensitivity that can help (i) to improve patient risk-stratification at diagnosis and (ii) identify the underlying mechanisms of resistance to these agents. Previous efforts to identify baseline DNA methylation differences at promoter regions between DMTi responders and non-responders have not been successful, so we hypothesized that any potential differences would be located distally from promoter regions. For this purpose we studied 40 CMML patients at diagnosis, all of whom had been uniformly treated with DAC 20 mg/m2/day x 5 days as frontline therapy. After 6 cycles of therapy patients were classified as responders (n=19, hematological improvement or better), or non-responders (n=21, stable or progressive disease). Mutational analysis showed no significant differences in the frequency of mutations in TET2, ASXL1, DNMT3A, RUNX1, TP53, JAK2, KIT, KRAS, EZH2, IDH1/2 and spliceosome genes. Using Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) we analyzed the baseline methylation status at ~3M CpG sites across the genome of 39/40 CMML patients. We identified 158 statistically significant differentially methylated regions (DMRs) (FDR Disclosures No relevant conflicts of interest to declare.

The nucleoside analog (na) 5-aza-2’-deoxycytidine (dac) has good, but heterogeneous, efficacy in the therapy of chronic myelomonocytic leukemia (cmml). Given that no standard therapy has been identified so far for cmml, there is urgent need to identify molecular markers that could support the choice of dac therapy and identify patients more prone to respond. Recently, we demonstrated that in mds patients the expression of uck1, involved in the activation of azacitidine, may influence the clinical response to this treatment (valencia et al, leukemia 2013). In the present study, we assessed the role of dac metabolizing enzymes as well as genetic alterations common to cmml patients in response to dac in a uniformly and prospectively treated cohort of cmml patients. Methods: Patients Forty cmml patients were treated with dac 20mg/m2/day for 5 days every 28 d. DNA and rna were extracted from bm mononuclear cells of 19 pts defined as responders to dac (r), and 21 as non-responders (nr). Hematological response was evaluated according to iwg 2006 criteria. Gene mutation. the fifteen most frequently mutated genes in cmml were sequenced at a mean depth of coverage of 520x (range 169–714x). Functional studies. the role of two main genes involved in dac metabolism: dck (dac activation) and dctd (dac deactivation) was evaluated by silencing both genes with sirnas. The experiments were performed in the mds-skm1 cell line and in primary cells of 6 cmml cases exposed in vitro to dac 10um for 48h. Gene expression. the expression level of genes hent1, hent2, dctd, hcnt3, cn-ii, dck and cda, involved in dac metabolism was evaluated by qrt-pcr in 38/40 cmml patients and by rnaseq in 14/40 patients. CDNA libraries were sequenced using the hiseq 2000. The counts of endogenous genes were normalized by ercc spike-in library controls, and differential expression analysis was performed using edger (v3.4.2) glm model. The differentially expressed genes were identified at the fdr cutoff of 0.05 and absolute fold change greater than 2. Results: in skm-1 cells and in cmml primary mononuclear cells, dctd silencing increased dac- induced apoptosis (annexin v-positive cells 20.2%±0.8% vs control 13.8%±0.5%; p=0.01). dck silencing led to a decrease in dac-induced apoptosis (annexin v-positive cells 8.8%±0.1% vs control 13.8%±0.5%; p=0.05). We could not detect by rnaseq a significant difference in the expression levels of na metabolizing enzymes between responders and non responders to dac. qrt-pcr confirmed these observations. The mutational frequencies (figure) in this cohort of cmml cases were: srsf2 50%, tet2 44.7%, asxl1 39.4%, nras 18.4%, runx1 and dnmt3a 10.5%, u2af1 and tp53 7.8%, kras, jak2 and kit 5.2%, ezh2, idh1, idh2 and sf3b1 2.6%. No single genetic alteration was significantly associated with shorter overall survival or resistance to dac. Conclusions: although we could clearly demonstrate that in vitro expression of dac metabolizing enzymes influenced response to dac, clinical resistance does not appear to be directly correlated with the expression of genes involved in dac metabolism nor to specific gene mutations and is likely determined by other clinical/molecular variables that remain to be identified. Figure 1 Figure 1. Disclosures Finelli: Janssen: Speaker, Speaker Other; Novartis: Speaker, Speaker Other; Celgene: Research Funding, Speaker Other.

Abstract 2810 Azacitidine is a pyrimidine nucleoside analogue of cytidine and it is at present the standard treatment for myelodysplastic syndromes (MDS). A large number of studies performed to evaluate the mechanism of resistance/response to nucleoside chemotherapeutics like cytosine arabinoside or gemcitabine, have demonstrated the importance of the levels of expression of the enzymes involved in their metabolism. Despite the high clinical interest in azacitidine, little is known about its intracellular metabolism and scanty data are available in literature. Recently, differences in the expression of these enzymes has been reported in cell lines differentially sensitive to hypomethylating agents. Clinical responses to azacitidine are heterogeneous, mainly due to clinical characteristics of MDS patients, so there is a need to identify molecular markers that could predict and/or monitor the efficacy of this therapy. A better understanding of the biological mechanisms involved in the activation and DNA uptake of azacitidine is therefore necessary to possibly identify responsive MDS patients. Objectives: To measure the expression levels of the 5 enzymes involved in azacitidine metabolism and to correlate their gene expression with the clinical response to the drug. To test possible causes of differences in gene expression. Material and Methods: DNA and RNA was extracted from mononuclear cells of 39 IPSS high risk MDS patients treated with at least 6 cycles of azacitidine 75mg/m2/7 days every 28 days. Of them, 14 patients were responders and 25 non-responders to azacitidine according to IWG 2006 criteria. Gene expression was assessed with quantitative PCR, using an ABI GeneAmp® 5700. The specific oligonucleotides and TaqMan® probes of every selected gene will be acquired among the Assay-on-Demand® Gene Expression Products (Applied Biosystems). Promoter methylation of UCK1 was evaluated by methylation specific PCR using specific primers. The UCK1 coding region was sequenced by Sanger method. Results: Gene expression of hENT1, UCK1, DCK, RNR1 and RNR2 was compared between the groups of responders and non-responders. Individually, none of the genes was differentially expressed in responders versus non-responders. Nevertheless, a trend for lower expression in non-responders was shown for the UCK1 gene (median responders: 12.4 versus median non responders: 10.7; P = 0.095). In order to verify whether the presence of aberrant methylation could impact in UCK1 gene expression, we measured the methylation status of a putative CpG island in the UCK1 gene promoter region. The CpG island was unmethylated in all the samples analyzed, including responders and non-responder cases, as well as healthy donors. Moreover, to verify whether the difference in gene expression could be due to nucleotide variants in the gene sequence, we sequenced the UCK1 sequence. We found one polymorphic locus (rs2296957) in exon 6 and additional five intronic SNPs (rs3904060 and rs7867616, Intron 1; rs2296956 and rs189473964, Intron 3; rs150900763, 5' UTR) at the intron/exon boundaries. The presence of an individual genotype at these loci was not associated with therapy response and UCK1 gene expression. Conclusions: Our results show that absence of clinical response to azacitidine could be partly correlated with lower expression of UCK1. This is most likely causing a scarce azacitidine monophosphorylation by UCK1 in non-responders patients. The methylation status of the UCK1 gene promoter is not affecting gene expression. In addition, the genetic polymorphisms found in the UCK1 sequence do not associate with therapy response nor with gene expression. Further studies on larger series of MDS cases are needed to confirm these results which could help driving therapeutic decisions. Disclosures: No relevant conflicts of interest to declare.

Abstract 4615 Introduction. Alterations in chromatin organization are a common mechanism of leukemogenesis. Drugs affecting epigenetics have been proposed as therapy of hematopoietic neoplasms. We have previously shown selective activity of histone deacetylase (HDAC) inhibitors on AML1/ETO positive cells (Barbetti, et al- Oncogene, 2007). The AML1/ETO multiprotein complex induces and stabilizes the local DNA methylation pattern by recruiting HDACs and DNA methyl-transferase-1 (DNMT1), resulting in the stable silencing of AML1-controlled genes, like IL3. Our hypothesis is that DNMT inhibitors (DNMTi) 5-Azacytidine (AZA) and Decitabine (DAC) exert a targeted action on AML1/ETO positive leukemic cells. Methods. We used U937-A/E-9/14/18 cell line, carrying a ponasterone-A inducible construct of AML1/ETO (kindly provided by M. Luebbert). We analyzed the effects of AZA and DAC at different doses (0.01, 0.1, 1 and 10μM), evaluating cell viability and apoptosis (by Annexin V assay, and by WB caspase 9, 8, 3 -cleavage). Cells were then treated with AZA 0.1–1μM or DAC 0.1–0.01μM for 24h. We determined by chromatin immunoprecitation (ChIP) assay the association of the IL3 promoter with H4acetylated, H3K4me3, H3K9me2 and H3K27me3 and the quantitative expression of IL3 by Q-RT PCR, calculated by a comparative threshold cycle method, using the formula 2̂(−ΔΔCt). Other promoters controlled by AML1/ETO, like those of GM-CSF, MPO and CEBPalpha were also evaluated. Methylation of IL3 promoter was evaluated by Methylation Specific PCR (MSP). Confirmatory experiments were carried out in the human leukemic cell line Kasumi-1 naturally expressing AML1/ETO. Results. Although both DNMTi were active on leukemic cells, irrespective of the presence of AML1/ETO expression, the dose at which this effect was evident was differing of one logarithm. AZA 0.1μM significantly increased the percentage of apoptotic cells only in U937 AML1/ETO positive cell line. We observed that untreated apoptotic cells percentage was 4.72% (±0.55 Standard Deviation: SD) while after AZA 0.1μM was 5.70% (± 0.24 SD) (P0.05). These data were confirmed by the induction of cleavage of caspase 9, 8 and 3. DAC 0.01μM significantly induced apoptosis cells only in U937 AML1/ETO positive cells (NT= 6.27% ±0.24 SD; DAC 0.01μM = 7.50% ±0.57 SD; P Given these results, we investigated whether this different response was correlated with chromatin rearrangements directly involving the AML1/ETO gene. We evaluated IL3 expression after AZA and DAC treatments, which induced a significant increase, higher in AML1/ETO-expressing than in non-expressing U937 cells. In fact, in AML1/ETO positive cells the amount of IL3 mRNA 2̂(−ΔΔCt) after AZA and DAC respect to NT was 10.5 ± 0.3 DS and 6.1± 0.5 SD, respectively. In AML1/ETO negative cells, after AZA was 5.70 ±2.7 SD (P>0.05) and after DAC 2.28 ± 0.4 SD (P>0.05), both respect to NT. By ChIP assay we showed that in untreated leukemic cells, both in U937 AML1/ETO-positive and negative cells, at baseline H3K4me3 was not associated with the IL3 promoter, while H3K9me2 was associated with it. H4-acetylated became associated to the IL3 promoter after AZA and DAC treatments but exclusively in U937 cells expressing AML1/ETO. On the other hand, H3K27me3 was co-immunoprecipitated with the IL3 promoter in U937 AML1/ETO negative and in U937 AML1/ETO positive untreated cells. AZA and DAC treatments led to the dissociation of this modified histone from the IL-3 promoter indicating the modification of chromatin rearrangement (Fig 1). Conclusions. Our observations demonstrate that AML1/ETO positive cells are more sensitive to DNMTi than cells not carrying such chimeric gene, in terms of biological effects like cytotoxicity and induction of apoptosis. The dissociation of the IL3 promoter from H3K27me3 and its association with H4acetylated upon DNMTi exposure is observed exclusively in U937 AML1/ETO-positive cells and clearly indicates that the epigenetic mechanism of action of these drugs is exerted selectively on cells harbouring the chimeric protein. This result could support a working hypothesis to develop a treatment for AML1/ETO-positive leukaemias including DNMTi in the therapeutic armamentarium. Disclosures: Santini: Janssen: Honoraria; Celgene: Honoraria; Novartis: Honoraria.

DNA amplification combined with hybridization with 32P-labeled synthetic oligonucleotide probes has been used to identify base substitutions in the 5′ promoter region of the A gamma globin gene in members of eleven families from China, Sardinia, Canada, and the United States who had a heterozygosity for the A gamma-beta+-hereditary persistence of fetal hemoglobin (HPFH), and in members of six black families with a possible G gamma-beta+-HPFH heterozygosity. All three known A gamma types were observed, ie, the British type (-198, T----C), the Chinese type (-196, C----T), and the Green type (-117, G----A); the latter has been found in a black family. Of the six families with G gamma-beta+-HPFH, three had C----G at -202 and none T----C -175. Conditions for hybridization of amplified DNA with the specific probes are provided and the usefulness of the technique is discussed. The increase in numbers of A gamma(G gamma)-beta+-HPFH heterozygotes with specific base substitutions greatly enhances the probability of a direct correlation between these substitutions and the increase in the production of a specific gamma chain.

DNA amplification combined with hybridization with 32P-labeled synthetic oligonucleotide probes has been used to identify base substitutions in the 5′ promoter region of the A gamma globin gene in members of eleven families from China, Sardinia, Canada, and the United States who had a heterozygosity for the A gamma-beta+-hereditary persistence of fetal hemoglobin (HPFH), and in members of six black families with a possible G gamma-beta+-HPFH heterozygosity. All three known A gamma types were observed, ie, the British type (-198, T----C), the Chinese type (-196, C----T), and the Green type (-117, G----A); the latter has been found in a black family. Of the six families with G gamma-beta+-HPFH, three had C----G at -202 and none T----C -175. Conditions for hybridization of amplified DNA with the specific probes are provided and the usefulness of the technique is discussed. The increase in numbers of A gamma(G gamma)-beta+-HPFH heterozygotes with specific base substitutions greatly enhances the probability of a direct correlation between these substitutions and the increase in the production of a specific gamma chain.