Uroporphyrinogen III synthase erythroid promoter mutations in adjacent GATA1 and CP2 elements cause congenital erythropoietic porphyria

Solis, Constanza; Aizencang, G.; Astrin, Kenneth H.; Bishop, David F.; Desnick, Robert J.

doi:10.1172/jci10642

Cited by 85 publications

(89 citation statements)

References 41 publications

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“…We selected single nucleotide mutations implicated in X-linked sideroblastic anemia (XLSA) (21,22), congenital erythropoietic porphyria (CEP) (20), and pyruvate kinase deficiency (PKD) (23,43) that were predicted to disrupt a GATA1 binding site (BS) (Fig. S1).…”

Section: Resultsmentioning

confidence: 99%

“…In several Mendelian erythroid disorders (MEDs), NC genetic variants have been identified in the DNA-binding motif (WGATAR) of the hematopoietic master regulator, GATA1 (20)(21)(22)(23)(24)(25). GATA1 is both necessary for proper erythropoiesis (3,26) and sufficient to reprogram alternative lineages toward an erythroid fate (27,28).…”

Section: Mendelian Erythroid Disordersmentioning

confidence: 99%

“…Given these deficiencies, we interrogated three regions harboring rare variants implicated in distinct MEDs that are predicted to disrupt GATA1 motifs, using a combination of experimental and computational approaches to gain further insight into GATA1 transcriptional activity (20)(21)(22)(23)43).…”

Section: Significancementioning

confidence: 99%

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Insight into GATA1 transcriptional activity through interrogation ofciselements disrupted in human erythroid disorders

Wakabayashi

Ulirsch

Ludwig

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Whole-exome sequencing has been incredibly successful in identifying causal genetic variants and has revealed a number of novel genes associated with blood and other diseases. One limitation of this approach is that it overlooks mutations in noncoding regulatory elements. Furthermore, the mechanisms by which mutations in transcriptional cis-regulatory elements result in disease remain poorly understood. Here we used CRISPR/Cas9 genome editing to interrogate three such elements harboring mutations in human erythroid disorders, which in all cases are predicted to disrupt a canonical binding motif for the hematopoietic transcription factor GATA1. Deletions of as few as two to four nucleotides resulted in a substantial decrease (>80%) in target gene expression. Isolated deletions of the canonical GATA1 binding motif completely abrogated binding of the cofactor TAL1, which binds to a separate motif. Having verified the functionality of these three GATA1 motifs, we demonstrate strong evolutionary conservation of GATA1 motifs in regulatory elements proximal to other genes implicated in erythroid disorders, and show that targeted disruption of such elements results in altered gene expression. By modeling transcription factor binding patterns, we show that multiple transcription factors are associated with erythroid gene expression, and have created predictive maps modeling putative disruptions of their binding sites at key regulatory elements. Our study provides insight into GATA1 transcriptional activity and may prove a useful resource for investigating the pathogenicity of noncoding variants in human erythroid disorders.GATA1 | cis-regulatory elements | noncoding mutations | Mendelian erythroid disorders W hole-exome sequencing (WES) and targeted sequencing approaches have greatly accelerated our ability to identify causal genetic lesions in both previously implicated and novel genes underlying monogenic disorders (1, 2). In hematology, WES has been extremely useful for identifying unknown genetic etiologies for various disorders, such as those affecting red blood cell (RBC) production, including Diamond-Blackfan anemia and congenital dyserythropoietic anemia (3-5), disorders of RBC structure and function (6, 7), and disorders affecting other aspects of hematologic function (2, 8). Despite this considerable success, however, more than 50% of cases of presumed monogenic diseases are refractory to current WES approaches (9). Although resolving these remaining cases will benefit from improvements in exome capture (10), read alignment (11), and variant annotation methodologies (11), the importance of genetic variation occurring within regulatory elements (REs) outside of the traditionally investigated coding sequences in hematologic and other diseases is being increasingly appreciated (12).Whole-genome sequencing (WGS) approaches are becoming progressively more available and affordable, but separating pathogenic genetic variation from benign or unrelated mutations remains especially difficult outside of protein-coding gen...

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Section: Resultsmentioning

confidence: 99%

Section: Mendelian Erythroid Disordersmentioning

confidence: 99%

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Insight into GATA1 transcriptional activity through interrogation ofciselements disrupted in human erythroid disorders

Wakabayashi

Ulirsch

Ludwig

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Molecular study of the UROS gene in CEP patients has highlighted a variety of mutations spreading all along the 10 coding exons, including missense or nonsense mutations, splicing defects, and large deletions/insertions, as well as mutations in the erythroid-specific promoter region (6,7). In disease related to protein dysfunction, it is diagnostically and therapeutically essential to understand the multiple mechanisms that explain the pathogenicity of specific mutants.…”

mentioning

confidence: 99%

Therapeutic potential of proteasome inhibitors in congenital erythropoietic porphyria

Blouin

Duchartre

Costet

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Congenital erythropoietic porphyria (CEP) is a rare autosomal recessive disorder characterized by uroporphyrinogen III synthase (UROS) deficiency resulting in massive porphyrin accumulation in blood cells, which is responsible for hemolytic anemia and skin photosensitivity. Among the missense mutations actually described up to now in CEP patients, the C73R and the P248Q mutations lead to a profound UROS deficiency and are usually associated with a severe clinical phenotype. We previously demonstrated that the UROS C73R mutant protein conserves intrinsic enzymatic activity but triggers premature degradation in cellular systems that could be prevented by proteasome inhibitors. We show evidence that the reduced kinetic stability of the UROS P248Q mutant is also responsible for increased protein turnover in human erythroid cells. Through the analysis of EGFP-tagged versions of UROS enzyme, we demonstrate that both UROS C73R and UROS P248Q are equally destabilized in mammalian cells and targeted to the proteasomal pathway for degradation. We show that a treatment with proteasomal inhibitors, but not with lysosomal inhibitors, could rescue the expression of both EGFP-UROS mutants. Finally, in CEP mice (Uros P248Q/P248Q ) treated with bortezomib (Velcade), a clinically approved proteasome inhibitor, we observed reduced porphyrin accumulation in circulating RBCs and urine, as well as reversion of skin photosensitivity on bortezomib treatment. These results of medical importance pave the way for pharmacologic treatment of CEP disease by preventing certain enzymatically active UROS mutants from early degradation by using proteasome inhibitors or chemical chaperones.protein misfolding | Günther's disease | enzyme instability | heme biosynthetic pathway | pharmacological therapy

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“…In the case of erythroid disorders, previous studies have identified GATA1 binding site mutations located in noncoding regions of a number of genes implicated in human erythroid disorders: ALAS2 in X-linked sideroblastic anemia (25,26), PKLR in pyruvate kinase deficiency (27), and UROS in congenital erythropoietic porphyria (28). These noncoding variants provide a unique opportunity to examine the necessity of these elements using modern genome editing tools, such as CRISPR/Cas9 (29).…”

Section: Insight From Rare Disorders Of Erythropoiesismentioning

confidence: 99%

Society for Pediatric Research 2015 Young Investigator Award: genetics of human hematopoiesis—what patients can teach us about blood cell production

Wakabayashi

Sankaran

2015

Pediatr Res

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Blood cell production or hematopoiesis is one of the most well-understood paradigms of cell differentiation in the body. The majority of work on hematopoiesis comes from studies that have primarily been conducted in mice, zebrafish, or other valuable model systems. However, it is clear that such model organisms may not consistently and faithfully mimic what is observed in humans with blood disorders. Moreover, there is significant divergence between species that is increasingly being appreciated at the genomic level. As a result, there is an opportunity to use observations in humans to provide a refined view of hematopoiesis. Here, we discuss vignettes from our work that illustrate how insight from human genetics can improve our understanding of blood cell production and identify promising therapeutic approaches for blood disorders.

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Uroporphyrinogen III synthase erythroid promoter mutations in adjacent GATA1 and CP2 elements cause congenital erythropoietic porphyria

Cited by 85 publications

References 41 publications

Insight into GATA1 transcriptional activity through interrogation ofciselements disrupted in human erythroid disorders

Insight into GATA1 transcriptional activity through interrogation ofciselements disrupted in human erythroid disorders

Therapeutic potential of proteasome inhibitors in congenital erythropoietic porphyria

Society for Pediatric Research 2015 Young Investigator Award: genetics of human hematopoiesis—what patients can teach us about blood cell production

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