Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Jan;41(1):277-290.
doi: 10.1002/humu.23927. Epub 2019 Oct 15.

Next-generation Sequencing for the Diagnosis of MYH9-RD: Predicting Pathogenic Variants

Affiliations
Free PMC article

Next-generation Sequencing for the Diagnosis of MYH9-RD: Predicting Pathogenic Variants

Loredana Bury et al. Hum Mutat. .
Free PMC article

Abstract

The heterogeneous manifestations of MYH9-related disorder (MYH9-RD), characterized by macrothrombocytopenia, Döhle-like inclusion bodies in leukocytes, bleeding of variable severity with, in some cases, ear, eye, kidney, and liver involvement, make the diagnosis for these patients still challenging in clinical practice. We collected phenotypic data and analyzed the genetic variants in more than 3,000 patients with a bleeding or platelet disorder. Patients were enrolled in the BRIDGE-BPD and ThromboGenomics Projects and their samples processed by high throughput sequencing (HTS). We identified 50 patients with a rare variant in MYH9. All patients had macrothrombocytes and all except two had thrombocytopenia. Some degree of bleeding diathesis was reported in 41 of the 50 patients. Eleven patients presented hearing impairment, three renal failure and two elevated liver enzymes. Among the 28 rare variants identified in MYH9, 12 were novel. HTS was instrumental in diagnosing 23 patients (46%). Our results confirm the clinical heterogeneity of MYH9-RD and show that, in the presence of an unclassified platelet disorder with macrothrombocytes, MYH9-RD should always be considered. A HTS-based strategy is a reliable method to reach a conclusive diagnosis of MYH9-RD in clinical practice.

Keywords: ACMG guidelines; MYH9-related disorders; clinical diagnosis; genomics; high throughput sequencing; variant classification.

Figures

Figure 1
Figure 1
Schematic representation of the heavy chain A of nonmuscle myosin class IIA (NMMHC‐IIA) and variants position. (a) Schematic representation of NMMHC‐IIA protein. Nonmuscle myosin II A shows a hexameric structure consisting of two heavy chains, namely NMMHC‐IIA, and two pairs of light chains. Each heavy chain includes a N‐terminal globular head domain (HD), a neck region which binds the light chains, and a C‐terminal α‐helical coiled‐coil tail domain (TD), which ends with a nonhelical tail domain (NHTD) involved in the subcellular localization of the protein. The HD includes four subdomains: the N‐terminal SRC‐Homology 3 like motif (SH3), the upper and lower 50 kDa subdomains, that together form the motor domain (MD), and the converter subdomain. In green the globular HD, in violet the neck domain and in blue the coiled coil TD with the NHTD at the 3′‐UTR in orange. (b) Affected exons and variants identified. The most affected exons are highlighted with red stars. The novel variants are shown in bold and the number in brackets is the patient ID number. Colors reflect protein domains. All variants described were confirmed by Sanger sequencing. The * indicates the two mutations identified in the same patient (39). #*,^,°,// represent members of the same family. 3′‐UTR, 3′‐untranslated region
Figure 2
Figure 2
Evolutionary conservation variant analysis. From the outer to the inner circle. MYH9 protein domains: in green the N‐terminal globular head domain (HD), in purple the neck domain, in blue the C‐terminal α‐helical coiled‐coil tail domain (TD) and in orange the 3′‐UTR. Evolutionary conserved regions in the MYH9 protein in gray. All the pathogenic, likely pathogenic and VUS variants affect highly conserved amino acid residues. Variant minor allele frequency (MAF) in gnomAD database is represented by green bars. Smaller is the green bar lower is the allele frequency. Variants present in ClinVar and LOVD are represented by blue bars. The height of each blue bar represents the number of patients previously described with the same variant. Variants in this cohort previously seen in the literature include ‘pathogenic’ and ‘likely pathogenic’ variants, in red and orange, respectively. Novel variants in this cohort include ‘likely pathogenic” variants and VUS in orange and black, respectively. 3′‐UTR, 3′‐untranslated region
Figure 3
Figure 3
Döhle‐like inclusion bodies localization by NMMHC‐IIA immunofluorescence or MGG staining. Light microscopy and immunofluorescence analyses of granulocytes in a healthy control (control), in patients (32 for immunofluorescence and 43 for light microscopy) with a pathogenic variant (pathogenic) and in three patients (28, 29, and 40) with a variant of uncertain significance (VUS). The analysis was performed by two independent centres: Panels I–V show results obtained by centre 1; Panels VI–X show results obtained by centre 2. Both centres used rabbit antihuman NMMHCIIA Ab followed by Alexa‐Fluor 488‐conjugated secondary antibody. Results between the two centers were highly comparable. The patient's sample in which a pathogenic variant was identified shows circular to oval shaped cytoplasmic punctuate spots, classified as type II inclusions (panels II and VII). Patients’ samples in which VUSs were identified show a speckled staining (panels III and VIII and panels V and X, respectively), and many small dots scattered throughout the cytoplasm (panels IV and IX) classified as type III inclusions. Panels XI–XV show May–Grünwald–Giemsa staining. Panels XII and XV show the presence of Döhle‐like bodies (arrowhead) in patients’ samples with a pathogenic variant (XII) and a VUS (XV). NMMHCIIA, nonmuscle myosin of class IIA; VUS, variant of uncertain significance
Figure 4
Figure 4
Cohort Phenotype. (a) HPO terms coded for hematological and (b) nonhematological symptoms. Y axis: HPO terms; X axis: patient ID number (from 1 to 50). Red box: the presence of the phenotypic feature; green box: the presence of NMMHC‐IIA aggregates identified only after centralized immunofluorescence analysis; and blue box: data not available. Pale yellow box: the absence of phenotypic feature. NMMHCIIA, nonmuscle myosin of class IIA

Similar articles

See all similar articles

References

    1. Althaus K., & Greinacher A. (2009). MYH9‐related platelet disorders. Seminars in Thrombosis and Hemostasis, 35, 189–203. - PubMed
    1. Arrondel C., Vodovar N., Knebelmann B., Grünfeld J. P., Gubler M. C., Antignac C., … Heidet L. (2002). Expression of the nonmuscle myosin heavy chain IIA in the human kidney and screening for MYH9 mutations in Epstein and Fechtner syndromes. Journal of the American Society of Nephrology, 13, 65–74. - PubMed
    1. Balduini C. L., Cattaneo M., Fabris F., Gresele P., Iolascon A., Pulcinelli F. M., … Italian Gruppo di Studio delle P. (2003). Inherited thrombocytopenias: A proposed diagnostic algorithm from the Italian Gruppo di Studio delle Piastrine. Haematologica, 88, 582–592. - PubMed
    1. Balduini C. L., Pecci A., & Noris P. (2012). Inherited thrombocytopenias: The evolving spectrum. Hämostaseologie, 32, 259–270. - PubMed
    1. Balduini C. L., Pecci A., & Savoia A. (2011). Recent advances in the understanding and management of MYH9‐related inherited thrombocytopenias. British Journal of Haematology, 154, 161–174. - PubMed
Feedback