Background

Iron overload is the primary cause of morbidity in transfusion-dependent thalassemia. Increase in iron causes mitochondrial dysfunction under experimental conditions, but the occurrence and significance of mitochondrial damage is not understood in patients with thalassemia.

Methods

Mitochondrial DNA (mtDNA) to nuclear DNA copy number (Mt/N) and frequency of the common 4977-bp mitochondrial deletion (ΔmtDNA⁴⁹⁷⁷) were quantified using a quantitative PCR assay on whole blood samples from 38 subjects with thalassemia who were receiving regular transfusions.

Results

Compared with healthy controls, Mt/N and ΔmtDNA⁴⁹⁷⁷ frequency were elevated in thalassemia (P = 0.038 and P < 0.001, respectively). ΔmtDNA⁴⁹⁷⁷ was increased in the presence of either liver iron concentration > 15 mg/g dry-weight or splenectomy, with the highest levels observed in subjects who had both risk factors (P = 0.003). Myocardial iron (MRI T2* < 20 ms) was present in 0%, 22%, and 46% of subjects with ΔmtDNA⁴⁹⁷⁷ frequency < 20, 20-40, and > 40/1 × 10⁷ mtDNA, respectively (P = 0.025). Subjects with Mt/N values below the group median had significantly lower Matsuda insulin sensitivity index (5.76 ± 0.53) compared with the high Mt/N group (9.11 ± 0.95, P = 0.008).

Conclusion

Individuals with transfusion-dependent thalassemia demonstrate age-related increase in mtDNA damage in leukocytes. These changes are markedly amplified by splenectomy and are associated with extrahepatic iron deposition. Elevated mtDNA damage in blood cells may predict the risk of iron-associated organ damage in thalassemia.

Carbon monoxide (CO) production from heme catabolism is increased with hemolysis. A portable end-tidal CO (ETCO) monitor was used to analyze breath samples in 16 children with sickle cell anemia (SCA, 5-14 years). Median (range) ETCO for SCA was 4.35 ppm (1.8-9.7) versus 0.80 ppm (0.2-2.3) for controls (P < 0.001). ETCOc >2.1 ppm provided sensitivity and specificity of 93.8% (69.8-99.8%) for detecting SCA. ETCO correlated with reticulocytosis (P = 0.015) and bilirubin (P = 0.009), and was 32% lower in children receiving hydroxyurea (P = 0.09). Point-of-care ETCO analysis may prove useful for non-invasive monitoring of hemolysis and as a screening test for SCA.

Iron overload is the primary cause of mortality and morbidity in thalassemia major despite advances in chelation therapy. We performed a pilot clinical trial to evaluate the safety and efficacy of combined therapy with deferasirox (DFX, 20-30 mg/kg daily) and deferoxamine (DFO, 35-50mg/kg on 3-7 days/week) in 22 patients with persistent iron overload or organ damage. In the 18 subjects completing 12 months of therapy, median liver iron concentration decreased by 31% from 17.4 mg/g (range 3.9-38.2mg/g) to 12.0mg/g (range 0.96-26.7 mg/g, p<0.001). Median ferritin decreased by 24% from 2465 ng/mL (range 1110-10,700 ng/mL) to 1875 ng/mL (range 421-5800 ng/mL, p=0.002). All 6 subjects with elevated myocardial iron showed improvement in MRI T2* (p=0.031). The mean±S.E. plasma non-transferrin-bound iron (NTBI) declined from 3.10±0.25μM to 2.15±0.29μM (p=0.028). The administration of DFX during infusion of DFO further lowered NTBI (-0.28±0.08 μM, p=0.004) and labile plasma iron (LPI, -0.03±0.01 μM, p=0.006). The simultaneous administration of DFO and DFX rapidly reduced systemic and myocardial iron, and provided an excellent control of the toxic labile plasma iron species without an increase in toxicity.

Aim

To develop a non-invasive prenatal test for beta-hemoglobinopathies based on analyzing maternal plasma by using next generation sequencing.

Methods

We applied next generation sequencing (NGS) of maternal plasma to the non-invasive prenatal testing (NIPT) of autosomal recessive diseases, sickle cell disease and beta-thalassemia. Using the Illumina MiSeq, we sequenced plasma libraries obtained via a Twist Bioscience probe capture panel covering 4 Kb of chromosome 11, including the beta-globin (HBB) gene and >450 genomic single-nucleotide polymorphisms (SNPs) used to estimate the fetal fraction (FF). The FF is estimated by counting paternally transmitted allelic sequence reads present in the plasma but absent in the mother. We inferred fetal beta-globin genotypes by comparing the observed mutation (Mut) and reference (Ref) read ratios to those expected for the three possible fetal genotypes (Mut/Mut; Mut/Ref; Ref/Ref), based on the FF.

Results

We bioinformatically enriched the FF by excluding reads over a specified length via in-silico size selection (ISS), favoring the shorter fetal reads, which increased fetal genotype prediction accuracy. Finally, we determined the parental HBB haplotypes, which allowed us to use the read ratios observed at linked SNPs to help predict the fetal genotype at the mutation site(s). We determined HBB haplotypes via Oxford Nanopore MinION sequencing of a 2.2 kb amplicon and aligned these sequences using Soft Genetics' NextGENe LR software.

Conclusion

The combined use of ISS and HBB haplotypes enabled us to correctly predict fetal genotypes in cases where the prediction based on variant read ratios alone was incorrect.