Molecular Diagnosis of Inherited Coagulation and Bleeding Disorders

Abstract

Diagnosis of inherited bleeding disorders (IBDs) remains challenging, especially in the case of inherited platelet disorders, due to the heterogeneity of the clinical and laboratory phenotype, the limited specificity of platelet function tests, and the large number of potential culprit genes. Unraveling the underlying molecular defect provides the definitive diagnosis of IBDs, facilitating prognosis and clinical care, which are especially important for severe clinical syndromes and those that may be associated with an increased risk of malignancy. Until recently, Sanger sequencing of candidate genes has been the only method of molecular diagnosis, but this approach is time-consuming and costly and requires phenotype-based identification of any obvious candidate gene(s). Nowadays, high-throughput sequencing (HTS) allows the simultaneous and rapid investigation of multiple genes at a manageable cost. This HTS technology that includes targeted sequencing of prespecified genes, whole-exome sequencing, or whole-genome sequencing, is revolutionizing the genetic diagnosis of human diseases. Through its extensive implementation in research and clinical practice, HTS is rapidly improving the molecular characterization of IBDs. However, despite the availability of this powerful approach, many patients still do not receive a diagnosis. As IBDs are complex and rare diseases, development of more advanced laboratory assays, improvements in bioinformatic pipelines, and the formation of multidisciplinary teams are encouraged to advance our understanding of IBDs.

Characterization of the Platelet Phenotype Caused by a Germline RUNX1 Variant in a CRISPR/Cas9-Generated Murine Model

“…32 The implementation of HTS platforms in routine clinical practice has enabled the identification of RUNX1 variants in some patients with suspected IPD who lack a specific phenotype. 7,10 However, the definitive diagnosis of FPD/AML in these patients requires an accurate assessment of the pathogenicity of their RUNX1 variants. 16,17 In this context, the p.Leu56Ser alteration in RUNX1 has been found in some patients with clinical and laboratory suspicion of FPD/AML, but is also a relatively frequent variant in the general population (up to 0.017 in the European non-Finnish population, according to GnomAD).…”

“…6 As in most inherited platelet disorders (IPDs), RUNX1related disorder (RUNX1-RD) lacks a specific clinical and laboratory phenotype, which makes genetic diagnosis essential. 7 The introduction of high-throughput sequencing (HTS) techniques into mainstream genetic diagnostic practice for IPDs has shown that molecular variations in TFs are far more frequent than previously believed. [8][9][10] To date, more than 130 families with RUNX1 variants have been reported.…”

Identification of novel variants in ten patients with Hermansky-Pudlak syndrome by high-throughput sequencing

“…The most commonly reported subtypes are HPS-1, HPS-3, and HPS-4. The HPS1 gene was first screened by SS in patients with severe phenotypes and in Puerto Rican patients [4,18], while founder mutations in HPS3 were analyzed in patients with mild phenotypes and in Ashkenazi Jewish patients [2,19]. In addition, the prevalence of HPS-4, one of the severe forms of the disease, is relatively high in different populations, such as Japanese, or inhabitants of Mediterranean regions, among others [1,4,19,20].…”

“…Public databases and in silico platforms provides extensive information. Finally, variants were classified according to the American College of Medical Genetics and Genomics recommendations [9,[15][16][17][18]. The causative genetic variants were also confirmed in the patients and screened in the available family members by Sanger sequencing (SS).…”

Diagnosis of Inherited Platelet Disorders on a Blood Smear

“…The spread of next-generation sequencing (NGS) greatly contributed to the unraveling of the genetic basis of several forms of IPD, and thus immediately became a significant diagnostic tool in the field. Using a small amount of blood with minor risk of pre-analytical artifacts, it can provide patients with a highly accurate diagnosis [4][5][6][7][66][67][68][69][70][71][72]. As it becomes cheaper to perform, it is not inconceivable that NGS could become the standard above other diagnostic techniques.…”