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TPO gene mutations. (A) p.Cys296Alafs*21 (c.886delT) in exon 8 (reverse sequence) and (B) p.Gln660Glu (C.1978C>G) in exon 11 and p.Arg665Trp (c.1993C>T) in exon 11 (reverse sequence); (C) Pedigrees of the family and THS values at diagnosis (* cordocentesis). The p.Cys296Alafs*21 (c.886delT) and the p.Gln660Glu (C.1978C>G) mutation was detected in both patients (II.1 and II.2) and the father (I.1). The p.Arg665Trp (c.1993C>T) mutation was detected in both patients and the mother (I.2).
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Thep.Arg665Trp mutation was present in the mother's DNA, and the p.Gln660Glu and p.Cys296Alafs*21 mutations were found in the father's DNA ( Figure 1 ).
PMC8251258_02
TPO gene mutations. (A) p.Cys296Alafs*21 (c.886delT) in exon 8 (reverse sequence) and (B) p.Gln660Glu (C.1978C>G) in exon 11 and p.Arg665Trp (c.1993C>T) in exon 11 (reverse sequence); (C) Pedigrees of the family and THS values at diagnosis (* cordocentesis). The p.Cys296Alafs*21 (c.886delT) and the p.Gln660Glu (C.1978C>G) mutation was detected in both patients (II.1 and II.2) and the father (I.1). The p.Arg665Trp (c.1993C>T) mutation was detected in both patients and the mother (I.2).
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Thep.Arg665Trp mutation was present in the mother's DNA, and the p.Gln660Glu and p.Cys296Alafs*21 mutations were found in the father's DNA ( Figure 1 ).
(A) Western blot of HEK293 extracts from cells transfected with wild-type or mutant TPOs. Lane: 1) pCDNA 3.1; Lane 2) Arg665Trp-TPO, 103 kDa; Lane 3) delT668-TPO, 35 kDa; Lane 4) TPO-WT; 103 kDa; Lane 5) molecular weight marker Thermo Scientific Page Ruler (Thermo Scientific, Carlsbad, CA). All strains expressed the endogenous control alpha-tubulin (53 kDa). (B) Extracellular enzymatic activity of HEK293 cells expressing wild-type or mutant TPOs. Enzymatic activity was assessed using the Amplex UltraRed reagent. lambda
EXCITATION = 530 nm and lambda
EMISSION = 560 nm. The values are expressed as a percentage of the TPO-WT transfected cells' activity (*p < 0.05).
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Considering the height and body mass indices, no growth retardation was observed according to World Health Organization (5-19 years old) ( Supplemental Figure 2 ), and there were no motor problems detected in either child during the medical follow-up. In comparison, the Arg665Trp-TPO missense mutant and TPO-WT migrate at a molecular weight of 103 kDa ( Figure 2A ). Arg665Trp-TPO, and delT886-TPO. As shown in  Figure 2B , the TPO activities of Arg665Trp-TPO and delT886-TPO are significantly lower than TPO-WT levels.
Cellular localization of wild-type and mutant TPOs. Immunofluorescence, using anti-TPO RPE5379- (Abcam) and AlexaFluor 594 (Invitrogen), under permeabilized conditions, Panel (A) TPO-WT, Panel (B) delT668-TPO, Panel (C) Arg665Trp-TPO, and (D) pcDNA and impermeabilized conditions Panel (E) TPO-WT, Panel (F) delT668-TPO, Panel (G) Arg665Trp-TPO, and Panel (H) pcDNA transfected HEK293 cells. Images were acquired with a Leica TCS SP8 confocal microscope at 63x magnification.
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Under non-permeabilizing conditions ( Figures 3E-H ), TPO-WT was localized along the cell membrane. Notably, we observed reduced membrane staining in cells transfected with delT886-TPO and Arg665Trp-TPO, indicating that these mutant proteins are not correctly inserted into the membrane ( Figures 3F, G ).
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