Non-enzymatic digoxigenin labeling of allele-specific oligonucleotides for the detection of the sickle cell mutation

Abstract Abstract 1653 Background: Sickle cell disease (SCD) is a monogenic disorder with phenotypic heterogeneity, possibly determined by polymorphisms (SNPs) in genes whose products modify the pathophysiology of the disease. Priapism is one of the most common vaso-occlusive complications of SCD, and it occurred in more than 30% of males. The Klotho (KL) gene appears to be associated with vascular function and nitric oxide biology and the presence of SNPs could affect its function. Association between KL and priapism in SCD patients was suggested by Nolan et al. in 2004. However, other authors could not confirm this finding (Elliot et al., 2007). Objective: We decided to evaluate the relevance of SNPs rs2249358, rs211234 and rs9536314 to the occurrence of priapism in patients with SCD followed at Outpatient clinic at Escola Paulista de Medicina/UNIFESP. Methods: Forty male patients with SCD were enrolled, 39 (97.5%) with sickle cell anemia (SS) and one (2.5%) SC hemoglobinopathy. The manifestation of priapism was identified through analyses of medical records. The SNP rs2249358 was identified by PCR followed by restriction with XbaI. The other SNPs, rs211234 and rs9536314, were analyzed by allele specific PCR. Statistical analysis: t test, Chi2 or Fisher. This study was approved by Ethical Committee, and all patients agreed in participate. Results: The median age of the patient was 28.5 years-old (20-68 y.o.). Fourteen out of 40 patients had priapism (35%), each one with SS disease. The group of patients with priapism were older (32.5 y.o., 25–68 y.o.) than the group without this manifestation (27.5 y.o., 20–56 y.o.) (p=0.03). There was no statistical difference in the distribution of the SNPs rs211234 and rs9536314 between the two groups of patients (p=0.51 and p= 0.09, respectively). Regarding the distribution of SNP rs2249358, the group with priapism presented 8 individuals (57.1%) with GG genotype, 5 (35.7%) with AA and 1 (7.17%) with AG, whereas in the group without priapism, the distribution was different: 5 (19.2%) with GG, 7 (26.9%) with AA and 14 (53.8%) with AG genotype (p=0.0212). When we compare the presence of at least one A allele (AA or A-) with the G allele in homozygosis (GG), we observed that the A allele has a protector effect (OR: 0.1786; CI: 0.04232–0.7535) (p=0.031). Conclusions: In a relatively small group of patients with SCD, it was observed a significant proportion of individuals with priapism, which reinforces the importance of this manifestation. We also observed correlation between SNP rs2249358 of KL gene and priapism, as suggested previously. Disclosures: No relevant conflicts of interest to declare.

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Silent β-Thalassemia Mutations at −101 (C>T) and −71 (C>T) and Their Coinheritance with the Sickle Cell Mutation in Bahrain

Hemoglobin ◽

10.3109/03630269.2013.785434 ◽

2013 ◽

Vol 37 (4) ◽

pp. 369-377 ◽

Cited By ~ 2

Author(s):

Nabeel J. Al Moamen ◽

Fawzia Mahdi ◽

Ebtihal Salman ◽

Thabet Ahmed ◽

Ruqaya Abbas ◽

...

Keyword(s):

Sickle Cell ◽

Cell Mutation

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Detection of sickle-cell mutation by electrophoresis of partial RNA:DNA hybrids following solution hybridization

Gene ◽

10.1016/0378-1119(85)90110-6 ◽

1985 ◽

Vol 39 (1) ◽

pp. 77-83 ◽

Cited By ~ 2

Author(s):

Frederick S. Jones ◽

Jacob I. Grimberg ◽

Stuart G. Fischer ◽

John P. Ford

Keyword(s):

Sickle Cell ◽

Cell Mutation ◽

Rna:Dna Hybrids

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Assessment of Erythroid Chimerism in Sickle Cell Patients Undergoing HSCT By Digital Droplet PCR

Blood ◽

10.1182/blood.v124.21.563.563 ◽

2014 ◽

Vol 124 (21) ◽

pp. 563-563

Author(s):

Lihui Yin ◽

Rupa A Udani ◽

Mary Parlow ◽

Daniel B Bellissimo ◽

Joel A Brochstein

Keyword(s):

Sickle Cell ◽

Conflicts Of Interest ◽

Hematopoietic Stem ◽

Post Transplant ◽

Digital Droplet Pcr ◽

Blood Disorder ◽

The Status ◽

Droplet Pcr ◽

Allele Specific ◽

Donor Genotype

Abstract Sickle cell disease (SCD) is an inherited autosomal recessive blood disorder associated with significant morbidity. Nonmyeloablative allogeneic hematopoietic stem cell transplantation (HSCT) is increasingly used in severely affected patients with SCD. These transplants result in mixed hematopoietic chimerism so accurate chimerism testing is important for monitoring the status of the transplant. Reconstitution of the erythroid compartment is essential. Since red cells lack a nucleus, DNA-based chimerism assays do not directly assess the chimerism in the erythroid compartment. Several studies have shown that the chimerism in the white cells and erythroid cells can be very different (W, C., etal. Exp. Hematol. 2003, 31:924; Andreanni, M., etal. Haematologica, 2011, 96:128). We developed a procedure for quantification of chimerism in the erythroid compartment of blood using RNA-based digital droplet PCR (ddPCR). The sensitivity and specificity of the assay was evaluated then tested post-transplant samples from sickle cell patients. Total RNA is reverse transcribed and amplified in a two-step RT-PCR approach. The PCR reaction containing allele-specific hydrolysis probes is partitioned into ~15,000 droplets then amplified. The copy number of HbA and HbS transcripts from cells of the erythroid lineage is determined with ddPCR (BioRad, QX-200). The %HbA and the %Donor can be calculated using the donor genotype (A/A or A/S). The assay is designed to have a sensitivity of 1% with donor genotype of A/A and 5% with donor genotype of A/S. Each post-transplant sample is tested in duplicate along with an AA control, SS control, AS control and 1% sensitivity controls (5 controls total). Thirty AA or SS samples were tested to assess assay specificity. The average %HbA detected in a SS sample was 0.03%; the average %HbS in a AA sample was as 0.02%. The background signal is significantly below the cutoff for 1% sensitivity. Using contrived samples with low, medium, and high %HbA, we demonstrate the assay is accurate and linear to 0-2%HbA across the reportable range of 0%HbA to 100%HbA. The % CV of the minor allele for samples in the range of 10%-90%HbA, were equal or below 15%. A total of 11 post-transplant samples from 7 transplanted sickle cell patients were tested, and the results were compared to DNA-based/FISH chimerism, if available. Our results were comparable with DNA-based chimerism (Table 1). Five of the samples had slightly higher %donor in the erythroid compartment compared to the white cell compartment. The erythroid chimerism reflected changes in chimerism status: the decrease then increase in chimerism (P2), stable chimerism (P5) and graft failure (P7). Abstract 563. Table 1: Erythroid chimerism status of post-transplant SCD patients Patient # Donor Genotype Sample # %HbA %Donor %Recipient %Donor by FISH/STR P1 AS 1 52 100 0 95 P2 AA 1 66 66 34 72 2 44 44 56 50 3 100 100 0 95 P3 AS 1 54 100 0 85 P4 AA 1 100 100 0 100 P5 AS 1 14 28 72 15 2 12 24 76 15 P6 AS 1 57 100 0 95 P7 AA 1 0 0 100 Rejecting graft 2 0 0 100 Rejecting graft Assessment of the chimerism in the erythroid lineage may be a better indicator of donor erythropoiesis. We describe an accurate and sensitive assay for monitoring erythroid chimerism and the effects of post-transplant therapies in sickle cell patients undergoing HSCT. This assay also demonstrates the feasibility measuring erythroid chimerism detection in other hematologic disorders, such as thalassemia. Disclosures No relevant conflicts of interest to declare.

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