Functional variability among human clones of induced pluripotent stem cells (hiPSCs) remains a limitation in assembling high-quality biorepositories. Beyond inter-person variability, the root cause of intra-person variability remains unknown. Mitochondria guide the required transition from oxidative to glycolytic metabolism in nuclear reprogramming. Moreover, mitochondria have their own genome (mitochondrial DNA [mtDNA]). Herein, we performed mtDNA next-generation sequencing (NGS) on 84 hiPSC clones derived from a cohort of 19 individuals, including mitochondrial and non-mitochondrial patients. The analysis of mtDNA variants showed that low levels of potentially pathogenic mutations in the original fibroblasts are revealed through nuclear reprogramming, generating mutant hiPSCs with a detrimental effect in their differentiated progeny. Specifically, hiPSC-derived cardiomyocytes with expanded mtDNA mutations non-related with any described human disease, showed impaired mitochondrial respiration, being a potential cause of intra-person hiPSC variability. We propose mtDNA NGS as a new selection criterion to ensure hiPSC quality for drug discovery and regenerative medicine.

The ␣-thalassemias are a group of hereditary disorders caused by reduced synthesis of the ␣-chain of hemoglobin. We have developed and tested an ␣-thalassemia assay that uses both multiplex ligationdependent probe amplification (MLPA) with Luminex-based detection and deletion PCR technologies. The MLPA assay consisted of 20 probes, 15 of which hybridized to the ␣-globin gene cluster and 5 that served as control probes. A PCR assay was developed to confirm the presence of heterozygous/homozygous 3.7-kb and 4.2-kb deletions. MLPA and PCR results were compared to Southern blot (SB) results from 758 and 133 specimens, respectively. Lastly, MLPA and PCR results were reviewed and summarized from 5386 clinically tested specimens. SB and MLPA results were concordant in 678/687 (99%) specimens. PCR detected all deletions detected by SB with no false positives. No deletions or duplications were identified in 2630 (49%) clinically tested specimens. Extra ␣-globin copies were identified in 76 patients. A deletion of one or two ␣-globin genes was identified in 1251 (23%) and 1349 (25%) specimens, respectively, including 15 different genotypes. A deletion of three (hemoglobin H) and four ␣-globin genes (Hb Bart's) was observed in 65 or 3 specimens, respectively. Six patients had a deletion within the ␣-globin regulatory region MCS-R2. Thus, MLPA plus deletion PCR identify multiple ␣-globin gene deletions/duplications in patients being tested for ␣-thalassemia. The ␣-thalassemias are a group of recessively inherited disorders caused by reduced or absent synthesis of the ␣-chain of hemoglobin (Hb). Approximately 5% of the world's population is thought to be affected by ␣-thalassemia, making it one of the most common genetic disorders.1 Because of its selective advantage, the carrier rate of ␣-thalassemia is extremely high (Ͼ90%) in tropical and subtropical regions with a history of endemic malaria. 2The phenotype of patients with ␣-thalassemia is variable depending on the number of dysfunctional or absent ␣-globin genes. Patients with one dysfunctional ␣-globin gene (silent carrier) are often asymptomatic and unaware they have the genetic disorder unless they have a complete blood count examined. Similarly, patients with a deletion of two of the four ␣-globin genes (␣-thalassemia trait) may be asymptomatic and require no specific treatment. Hemoglobin H (three ␣-globin genes deleted/dysfunctional) is often more serious and is characterized by microcytic hypochromic hemolytic anemia, splenomegaly, and jaundice. All four of the ␣-globin alleles are dysfunctional in patients with Hb Bart's, the most severe and often fatal form of ␣-thalassemia. 2,3The two genes responsible for synthesis of the ␣-chain of Hb, HBA1 and HBA2, are located in the telomeric region on chromosome 16 (16p13.3) and encode ␣ 1 -globin and ␣ 2 -globin, respectively.2 These two genes are embedded within two highly homologous 4-kb duplication units that can be subdivided into separate homology blocks, written X, Y, and Z.2 Misalignment and recombination within ...

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