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. 2022 Mar 1;107(3):750-754.
doi: 10.3324/haematol.2021.279689.

GNE-related thrombocytopenia: evidence for a mutational hotspot in the ADP/substrate domain of the GNE bifunctional enzyme

Roberta Bottega  1 , Antonio Marzollo  2 , Maddalena Marinoni  3 , Emmanouil Athanasakis  1 , Ilaria Persico  4 , Anna Monica Bianco  1 , Michela Faleschini  1 , Erica Valencic  1 , Daniela Simoncini  3 , Linda Rossini  5 , Fabio Corsolini  6 , Martina La Bianca  1 , Giuseppe Robustelli  3 , Maria Gabelli  5 , Massimo Agosti  3 , Alessandra Biffi  5 , Paolo Grotto  7 , Valeria Bozzi  8 , Patrizia Noris  9 , Alberto B Burlina  10 , Adamo Pio D'Adamo  11 , Alberto Tommasini  11 , Flavio Faletra  1 , Annalisa Pastore  12 , Anna Savoia  13
Affiliations

GNE-related thrombocytopenia: evidence for a mutational hotspot in the ADP/substrate domain of the GNE bifunctional enzyme

Roberta Bottega et al. Haematologica. .
No abstract available

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Figures

Figure 1.
Figure 1.
Identification of novel mutations of the GNE gene. (A) Pedigrees and segregation analysis in the two families F1 and F2. (B) Electropherograms of exon 9 showing the c.1546_1547delinsAG and c.1724C>G substitutions in probands P1 and P2, respectively. Sanger sequencing was performed using the following primers: 9F/5’-TTCTAGAAATCTTTAAGGTGCTATGG-3’ and 9R/5’-CCACCTGACCATGTTGAAGA-3’. (C) Protein multiple alignments, showing conservation through different species at residues (in red) affected by the p.Val516Arg and p.Thr575Arg mutations. H. sapiens (NP_001121699.1), P. troglodytes (XP_003312121.1), M. mulatta (XP_001082113.2), C. lupus (XP_003431623.1), B. taurus (NP_001178072.2), M. musculus (NP_056643.3), R. norvegicus (NP_446217.1), G. gallus (NP_001026603.2), D. rerio (NP_957177.1), and X. tropicalis (NP_001072728.1). (D) Western blot and of total lysates from lymphoblast cells of P1 and P2. Total protein lysates were prepared from these cells using M-PERTM Mammalian Protein Extraction Reagent (Thermo Fisher Scientific). Protein quantification shows only a partial expression (39% and 79%, respectively) of GNE expression compared to wild-type (CTRL) (***P<0,002). Actin was used as a loading control for protein quantification. The antibodies were used as follows: anti-GNE (Santa Cruz Biotechnology, sc-376057, 1:500) and anti-β-actin (Santa Cruz Biotechnology, sc-47778, 1:4,000) as primary antibodies, anti-mouse immunoglobulin conjugated with horseradish peroxidase (HRP) (Bethyl, A90-116P, 1:10,000) as a secondary antibody. Statistical analysis was performed using the t-test. Error bars represent the standard deviation of 4 independent experiments.
Figure 2.
Figure 2.
Blood, bone marrow features, and response to transfusion and treatment. (A) Sialotransferrin profile determined by ion-exchange chromatography using a commercial kit (CDT in Serum, Recipe München). The different isoforms were pointed out by UV detection at 460 nm and quantified by the “area percent method” (i.e., the relative abundance of each isoform is expressed as the percentage ratio of the peak area compared to the sum of the areas of all the peaks). (B) Peripheral blood smear of P2 showing enlarged platelets. (C to E) Bone marrow aspirates with an increased number of megakaryocytes at different stages of maturation. May-Grünwald-Giemsa staining; original magnification 100X (B), 10X (C), 20X (D), and 40X (E). (F) Response to platelet transfusion. Median platelet count before platelet transfusion and up to 7 days following transfusion are shown for both patients (P1 and P2’s specific values are indicated in brackets). (G) Time course of platelet count in response to treatments for P1 (left) and P2 (right) in response to romiplostim administration at different dosages. The dark grey bar indicates a period of complete transfusion dependency, with transfusion every 5-7 days. Values of platelets measured within 7 days after platelet transfusion are not shown.
Figure 3.
Figure 3.
Localization of the GNE mutations associated with thrombocytopenia and three-dimensional structure of the GNE enzyme. (A) Among the 15 mutations (Online Supplementary Table S3), the 7 associated with myopathy are indicated below the schematic representation of the protein structure; the other 8 are depicted above the protein (in red, the novel mutations reported in this paper). All the mutations identified in patients are in a homozygous state except those represented in matched colored boxes that are heterozygous biallelic GNE mutations found in a single patient. H188Y and N550S (light grey boxes) are homozygous mutations identified in the same patient. UDP-GlcNAc 2-epimerase: UDP-N-acetylglucosamine 2-epimerase; ManNAc kinase: N-acetylmannosamine; NH2: NH2-terminus; COOH: COOH-terminus; RUF: a region with unknown function; NES: putative nuclear export signal; AS: allosteric site. Nomenclature of mutations was referred to the NM_001128227.3 transcript. (B) The overall structure (left) and the zoom of the enzymatic pocket (right) of GNE. The side chains of the positions affected by the mutations discussed in this article are explicitly indicated. The structure (Protein Data Bank [PDB] entry 2yhy) corresponds to the GNE complex with ManNAc and ADP. The structure was analyzed by PyMOL and MOLMOL graphic support tools. The degree of exposure of the residues affected by mutations was established by DSSP (Define Secondary Structure of Proteins) analysis.
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References

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