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CASE REPORTS
Identification of biallelic germline variants of SRP68 in a sporadic case with severe congenital neutropenia
Congenital neutropenia (CN) are a heterogeneous group of very rare diseases characterized by profound and chronic neutropenia possibly associated with co- morbidities.1 Variants in about 25 genes implicating dis- tinct pathophysiological pathways have been identified in CN.1 Recently, co-translational targeting of nascent secretory and membrane proteins to the endoplasmic reticulum (ER) mediated by the signal recognition particle (SRP) complex has been implicated in the pathogenesis of both CN and Shwachman-Diamond-like syndrome.2,3 In humans, the SRP complex is composed of six proteins (SRP9, SRP14, SRP19, SRP54, SRP68, and SRP72) assem- bled on a 7S RNA molecule.4 The SRP54 protein with its GTPase activity constitutes the key element of this com- plex and regulates the entire physiological process.5 We recently reported germline SRP54 variants in a large number of sporadic and familial cases with autosomal dominant CN.3 Apart from SRP54, germline heterozy- gous variants of SRP72 have been described in a few cases with aplastic anemia and myelodysplasia.6,7
Here, we report a sporadic case of severe CN associat-
ed with biallelic pathogenic variants of SRP68, implicat- ing for the first time the SRP68 protein in the pathogen- esis of CN. Specifically, we investigated the functional consequences of SRP68 defect on granulopoiesis and on ER homeostasis (see the Online Supplementary Appendix). The patient is a Caucasian boy referred for an anal abscess at 6 weeks of age. Blood counts showed severe neutropenia (white blood cells 6.1x109/L, neutrophils 0.2x109/L), elevated monocyte count (1.7x109/L), anemia (hemoglobin 7.5 g/dL) associated with iron deficiency and moderate thrombocytopenia (platelets 149x109/L). Serial blood counts (n=41) confirmed the persistent and profound neutropenia (0.200x109/L [range: 0-1.800]). The bone marrow examination showed a maturation arrest at the promyelocytic stage and major features of dysgranu- lopoiesis. Promyelocytes displayed numerous condensed granulations, abnormal nuclei, clumped chromatin and the absence of cytoplasmic vacuoles; the remaining neu- trophils were highly dystrophic (Figure 1A). In the first year of life, neutropenia was associated with profound anemia and thrombocytopenia which recovered later. The level of hemoglobin and the platelet count were respectively 8.7 g/dL [range, 5.5-11.3] and 132x109/L [range, 43-318]). From diagnosis to the last follow-up at the age of 5 years, he had a prophylactic antibiotherapy and was treated with granulocyte colony-stimulating fac-
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Figure 1. A sporadic case with severe congenital neutropenia and loss-of-function variants of SRP68. (A) Cytology analysis after May Grunwald-Giemsa staining of patient’s bone marrow. Pictures represent granulocytic precursors (left) and neutrophils (right) (original magnification x100). (B) Sanger sequencing confir- mation of the splice site variant c.184+2T>C located in intron 1 of SRP68. F: forward strand; R: reverse strand. (C) Confirmation of the deletion of exon 1 of SRP68 by quantitative polymerase chain reaction (PCR) using SYBR-Green. (D) Predicted alternate cryptic splice site located 37 bp upstream of the splice donor site shown on SRP68 sequence and confirmed by RT-PCR using RNA extracted from fibroblasts and Sanger sequencing of the shorter product (176 bp). (E) Expression level of SRP68 protein in fibroblasts evaluated by western blot analysis
haematologica | 2021; 106(4)