إبراهيم عبدالله فائع المازني

البحوث المنشورة

Inherited Thrombocytopenia: Update on Genes and Genetic Variants Which may be Associated with Bleeding

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6593073/

19/06/2019

Inherited thrombocytopenia (IT) is comprised of a group of hereditary disorders characterized by a reduced platelet count as the main feature, and often with abnormal platelet function, which can subsequently lead to impaired haemostasis. Inherited thrombocytopenia results from genetic mutations in genes implicated in megakaryocyte differentiation and/or platelet formation and clearance. The identification of the underlying causative gene of IT is challenging given the high degree of heterogeneity, but important due to the presence of various clinical presentations and prognosis, where some defects can lead to hematological malignancies. Traditional platelet function tests, clinical manifestations, and hematological parameters allow for an initial diagnosis. However, employing Next-Generation Sequencing (NGS), such as Whole Genome and Whole Exome Sequencing (WES) can be an efficient method for discovering causal genetic variants in both known and novel genes not previously implicated in IT. To date, 40 genes and their mutations have been implicated to cause many different forms of inherited thrombocytopenia. Nevertheless, despite this advancement in the diagnosis of IT, the molecular mechanism underlying IT in some patients remains unexplained. In this review, we will discuss the genetics of thrombocytopenia summarizing the recent advancement in investigation and diagnosis of IT using phenotypic approaches, high-throughput sequencing, targeted gene panels, and bioinformatics tools.

A comprehensive bioinformatic analysis of 126 patients with an inherited platelet disorder to identify both sequence and copy number genetic variants

https://onlinelibrary.wiley.com/doi/full/10.1002/humu.24114

15/09/2020

Inherited bleeding disorders (IBDs) comprise an extremely heterogeneous group of diseases that reflect abnormalities of blood vessels, coagulation proteins, and platelets. Previously the UK‐GAPP study has used whole‐exome sequencing in combination with deep platelet phenotyping to identify pathogenic genetic variants in both known and novel genes in approximately 40% of the patients. To interrogate the remaining “unknown” cohort and improve this detection rate, we employed an IBD‐specific gene panel of 119 genes using the Congenica Clinical Interpretation Platform to detect both single‐nucleotide variants and copy number variants in 126 patients. In total, 135 different heterozygous variants in genes implicated in bleeding disorders were identified. Of which, 22 were classified pathogenic, 26 likely pathogenic, and the remaining were of uncertain significance. There were marked differences in the number of reported variants in individuals between the four patient groups: platelet count (35), platelet function (43), combined platelet count and function (59), and normal count (17). Additionally, we report three novel copy number variations (CNVs) not previously detected. We show that a combined single‐nucleotide variation (SNV)/CNV analysis using the Congenica platform not only improves detection rates for IBDs, suggesting that such an approach can be applied to other genetic disorders where there is a high degree of heterogeneity.

Novel gene variants in patients with platelet‐based bleeding using combined exome sequencing and RNAseq murine expression data

https://onlinelibrary.wiley.com/doi/full/10.1111/jth.15119

06/10/2020

Background: The UK Genotyping and Phenotyping of Platelets study has recruited and analyzed 129 patients with suspected heritable bleeding. Previously, 55 individuals had a definitive genetic diagnosis based on whole exome sequencing (WES) and platelet morphological and functional testing. A significant challenge in this field is defining filtering criteria to identify the most likely candidate mutations for diagnosis and further study.

Objective: Identify candidate gene mutations for the remaining 74 patients with platelet‐based bleeding with unknown genetic cause, forming the basis of future re‐recruitment and further functional testing and assessment.

Methods: Using python‐based data frame indexing, we first identify and filter all novel and rare variants using a panel of 116 genes known to cause bleeding across the full cohort of WES data. This identified new variants not previously reported in this cohort. We then index the remaining patients, with rare or novel variants in known bleeding genes against a murine RNA sequencing dataset that models proplatelet‐forming megakaryocytes.

Results: Filtering against known genes identified candidate variants in 59 individuals, including novel variants in several known genes. In the remaining cohort of “unknown” patients, indexing against differentially expressed genes revealed candidate gene variants in several novel unreported genes, focusing on 14 patients with a severe clinical presentation.

Conclusions: We identified candidate mutations in a cohort of patients with no previous genetic diagnosis. This work involves innovative coupling of RNA sequencing and WES to identify candidate variants forming the basis of future study in a significant number of undiagnosed patients.

A novel RUNX1 exon 3 – 7 deletion causing a familial platelet disorder

https://www.tandfonline.com/doi/full/10.1080/09537104.2021.1887470

22/02/2021

Familial Platelet Disorder with associated Myeloid Malignancy (FPDMM) is a rare inherited disorder confirmed with the presence of a pathogenic germline RUNX1 variant and is thought to be heavily underdiagnosed. RUNX1 has also been found to be mutated in up to 10% of adult AML cases and other cell malignancies. We performed targeted next-generation sequencing and subsequent MLPA analysis in a kindred with multiple affected individuals with low platelet counts and a bleeding history. We detected a novel heterozygous exon 3–7 large deletion in the RUNX1 gene in all affected family members which is predicted to remove all of the Runt-homology DNA-binding domain and a portion of the Activation domain. Our results show that the combination of targeted NGS and MLPA analysis is an effective way to detect copy number variants (CNVs) which would be missed by conventional sequencing methods. This precise diagnosis offers the possibility of accurate counseling and clinical management in such patients who could go onto develop other cell malignancies.

المؤتمرات العلمية

3rd European Congress on Thrombosis and Haemostasis (ECTH 2019)

02-04/10/2019

Glasgow, United Kingdom

Oral presentation

Functional investigation of a CD36 variant in patients with an inherited bleeding disorder

Background: Inherited thrombocytopenia (IT) is comprised of a group of hereditary disorders characterised by   reduced platelet counts as the main feature, and often with abnormal variable bleeding diathesis. CD36, also known as platelet glycoprotein 4 (GPIV), is an integral membrane protein and a major receptor which binds thrombospondin, collagen and oxidized low-density lipoprotein among others. CD36 has several functions of clinical significance such as roles in malaria, angiogenesis, thrombocytopenia, obesity and cancer, but precise mechanisms are still unclear.

Aim: This study aimed to investigate the functional role of a nonsense sequence variant in CD36 in two siblings with an inherited bleeding disorder.

Methods: Exome sequencing data was analysed from two siblings with a history of bleeding and low platelet counts. Patient platelet lysates were used to perform Western blotting. To gain insight into the functional consequences of this variant, site direct mutagenesis was carried out on WT CD36 to generate mutant versions (with the stop gain truncated version of CD36) of the CD36 expression construct and transfected into Jurkat T cells. To elucidate the effect of the CD36 variant on downstream signalling pathways, a nuclear factor of activated T-cells (NFAT) transcriptional reporter assay was performed on transfected Jurkat T cells with CD36 WT and mutant constructs. Subsequently, cell lysates from the transfected cells were used to perform Western blotting and determine the expression on the cell surface by flow cytometry.

Results: Exome sequencing analysis identified a heterozygous stop gain variant at amino acid 325, leading to a truncated protein in CD36. The truncated CD36 protein was demonstrated by Western blotting in both of patient platelet lysates and transfected cell lysates. NFAT-luciferase assay was activated in the CD36 WT, but not in the mutated constructs. WT CD36, but not the mutant constructs, was detected by flow cytometry.

Summary/Conclusion: Investigation of the effect of downstream signalling pathways has given novel insights into how a genetic variant of CD36 in patients with an inherited thrombocytopenia affects CD36 function.

https://uksacb.org/wp-content/uploads/wp_dndcf7_uploads/ECTH2019-ABSTRACT-BOOK-1.pdf

BSHT Annual Scientific Meeting

24/01/2020

Birmingham, United Kingdom

Poster presentation

Functional investigation of a cd36 variant in patients with an inherited bleeding disorder

Background: Inherited thrombocytopenia (IT) is comprised of a group of hereditary disorders characterised by   reduced platelet counts as the main feature, and often with abnormal variable bleeding diathesis. CD36, also known as platelet glycoprotein 4 (GPIV), is an integral membrane protein and a major receptor which binds thrombospondin, collagen and oxidized low-density lipoprotein among others. CD36 has several functions of clinical significance such as roles in malaria, angiogenesis, thrombocytopenia, obesity and cancer, but precise mechanisms are still unclear.

Aim: This study aimed to investigate the functional role of a nonsense sequence variant in CD36 in two siblings with an inherited bleeding disorder.

Methods: Exome sequencing data was analysed from two siblings with a history of bleeding and low platelet counts. Patient platelet lysates were used to perform Western blotting. To gain insight into the functional consequences of this variant, site direct mutagenesis was carried out on WT CD36 to generate mutant versions (with the stop gain truncated version of CD36) of the CD36 expression construct and transfected into Jurkat T cells. To elucidate the effect of the CD36 variant on downstream signalling pathways, a nuclear factor of activated T-cells (NFAT) transcriptional reporter assay was performed on transfected Jurkat T cells with CD36 WT and mutant constructs. Subsequently, cell lysates from the transfected cells were used to perform Western blotting and determine the expression on the cell surface by flow cytometry.

Results: Exome sequencing analysis identified a heterozygous stop gain variant at amino acid 325, leading to a truncated protein in CD36. The truncated CD36 protein was demonstrated by Western blotting in both of patient platelet lysates and transfected cell lysates. NFAT-luciferase assay was activated in the CD36 WT, but not in the mutated constructs. WT CD36, but not the mutant constructs, was detected by flow cytometry.

Summary/Conclusion: Investigation of the effect of downstream signalling pathways has given novel insights into how a genetic variant of CD36 in patients with an inherited thrombocytopenia affects CD36 function.

https://bsht.org.uk/wp-content/uploads/2020/02/BSHT-2020-programme-book-for-website.pdf