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Case Reports
impair ALAS2 mRNA regulation in response to iron lev- els. We hypothesized that under iron limiting condi- tions, IRP would not repress ALAS2 mRNA translation, with consequent overproduction of ALAS2 protein. This predicted increase in ALAS2 protein levels was indeed observed by immunoblotting for ALAS2 from cell lysates obtained from cultured and differentiated CD34+ erythroid precursors from the proband and the mother (II.5) who only bears the ALAS2 IRE mutation (Figure 1C). Therefore, our data supports the hypothesis that the 38U>C mutation in the ALAS2 IRE increases ALAS2 protein expression and modifies the severity of EPP caused by a confirmed CLPX mutation. In contrast, the ALAS2 gain-of-function in XLPP is caused by increased enzymatic activity in the protein.14
Sequencing of the ALAS2 gene in other family mem- bers showed that this mutation does not co-segregate with porphyrin accumulation (Figure 1A and data not shown). The proband inherited the -38U>C change from the maternal branch (II:5; II:6; I3, Figure 1A). Those relatives have higher than normal ZnPP levels and are asymptomatic (Table 1). Methylation analysis confirmed the absence of skewed X inactivation in rela- tives (II.5 and II.6) and the proband (III.2) who carried the IRE mutation.
Previously we and others described the important role of CLPX in heme biosynthesis through its regulation of ALAS2 turnover and enzyme activity.11,15 The G298D mutation in the CLPX gene, present in heterozygosity in this family (proband, father and paternal uncle), decreases the proteolytic activity of the CLPXP pro- tease, causing accumulation of ALAS2.11 However, in comparison to the mild photosensitivity presented by the paternal family, the proband presents with a full and severe EPP phenotype due to the combination of the paternal CLPX G298D mutation and the maternal ALAS2 -38T>C mutation present in the 5’ IRE of ALAS2 gene.
Collectively, our data suggests that the ALAS2 IRE mutation is the modifier necessary for the clinical pres- entation of EPP in the proband. The IRE/IRP-dependent down regulation of ALAS2 during iron deficiency is impaired and contributes to ALA overproduction and increases PPIX level in erythrocytes especially during iron deficiency. This postulate was supported clinically by the beneficial effect of the oral iron therapy when the patient was anemic (Table 1).
In summary, we have identified the first described mutation in the IRE of ALAS2 gene that contributes to porphyrin accumulation in erythrocytes above a thresh- old leading to overt EPP. These data strongly support the role of ALAS2 as an important modifier gene trigger- ing the clinical manifestations in erythropoietic disor- ders and extend the involvement of its IRE/IRP system to human heme metabolism and erythropoietic por- phyria.
Sarah Ducamp,1,2* Sara Luscieti,3* Xènia Ferrer-Cortès,4,5 Gaël Nicolas,1,2 Hana Manceau,2,6 Katell Peoc’h,2,6
Yvette Y. Yien,7 Caroline Kannengiesser,1,3
Laurent Gouya,1,2,6 Herve Puy,1,2,6# and Mayka Sanchez4,5#
1Centre de Recherche sur l’inflammation, INSERM U1149 CNRS ERL Université Paris Diderot, site Bichat, Sorbonne Paris Cité, Paris, France; 2Laboratory of Excellence, GR-EX, Paris, France; 3Institute of Predictive and Personalized Medicine of Cancer (IMPPC), Badalona, Barcelona, Spain; 4Universitat Internacional de Catalunya (UIC), Department of Basic Sciences, Iron metabolism: Regulation and Diseases, Sant Cugat del Vallès, Barcelona, Spain; 5BloodGenetics S.L. Diagnostics in Inherited
Blood Diseases, Esplugues de Llobregat, Barcelona; Spain; 6Centre Français des Porphyries, AP-HP, Hôpital Louis Mourier, Colombes, France and 7Department of Biological Sciences, University of Delaware, Newark, DE, USA
*SD and SL contributed equally as co-first authors. #HP and MS contributed euqally as co-senior authors. Correspondence: HERVE’ PUY - herve.puy@aphp.fr MAYKA SANCHEZ - msanchez@uic.es doi:10.3324/haematol.2020.272450
Received: September 23, 2020.
Accepted: January 22, 2021.
Pre-published: Fabruary 18, 2021.
Disclosures: no conflicts of interest to disclose.
Contributions: MS, LG and HP developed the study concept and designed the research; KP, LG and HP recruited family mem- bers and collected patients’ data; SD and HM performed patients DNA sequencing; Ckdid the X inactivation and CGH array experiments;
SL performed the IRE/IRP functional EMSA; HM did the ery- throid progenitor cell culture; GN perfomed immunoblot experi- ments;
XF-C, YYY, HP and MS wrote the manuscript and all authors participated in data discussion, analyzed data, read and approved the manuscript.
Acknowledgments: we are very grateful to all parent family who kindly contributed to this study. We thank Drs. Carole Beaumont, Zoubida Karim and Saïd Lyoumi for helpful feedback, discussions, and editorial assistance.
Funding: this study was partially supported by grant SAF2015-70412-R, and grant RTI-2018-101735-B-I100, MCI/AEI/FEDER, EU from the Spanish Secretary of Research, Development and Innovation (MINECO); grant DJCLS-R-14/04 from the Deutsche Josep Carreras Leukämie-Stiftung, 2014 SGR225 (GRE) from Generalitat de Catalunya to MS; SD and HM were supported by the Laboratory of excellence, BR-Ex, Paris, France;
the labex GR-Ex (reference ANR-11- LABX-0061) is funded by the program “Investissements d’avenir” of the French National Research Agency (reference ANR-11-IDEX-0005-02); YYY is supported by the National Institutes of Health (R03DK118307).
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