Danai Bem

Loss-of-Function Mutations in RAB18 Cause Warburg Micro Syndrome

Warburg Micro syndrome and Martsolf syndrome are heterogenous autosomal-recessive developmental disorders characterized by brain, eye, and endocrine abnormalities. Previously, identification of mutations in RAB3GAP1 and RAB3GAP2 in both these syndromes implicated dysregulation of the RAB3 cycle (which controls calcium-mediated exocytosis of neurotransmitters and hormones) in disease pathogenesis. RAB3GAP1 and RAB3GAP2 encode the catalytic and noncatalytic subunits of the hetrodimeric enzyme RAB3GAP (RAB3GTPase-activating protein), a key regulator of the RAB3 cycle. We performed autozygosity mapping in five consanguineous families without RAB3GAP1/2 mutations and identified loss-of-function mutations in RAB18. A c.71T > A (p.Leu24Gln) founder mutation was identified in four Pakistani families, and a homozygous exon 2 deletion (predicted to result in a frameshift) was found in the fifth family. A single family whose members were compound heterozygotes for an anti-termination mutation of the stop codon c.619T > C (p.X207QextX20) and an inframe arginine deletion c.277_279 del (p.Arg93 del) were identified after direct gene sequencing and multiplex ligation-dependent probe amplification (MLPA) of a further 58 families. Nucleotide binding assays for RAB18(Leu24Gln) and RAB18(Arg93del) showed that these mutant proteins were functionally null in that they were unable to bind guanine. The clinical features of Warburg Micro syndrome patients with RAB3GAP1 or RAB3GAP2 mutations and RAB18 mutations are indistinguishable, although the role of RAB18 in trafficking is still emerging, and it has not been linked previously to the RAB3 pathway. Knockdown of rab18 in zebrafish suggests that it might have a conserved developmental role. Our findings imply that RAB18 has a critical role in human brain and eye development and neurodegeneration.

Evaluation of participants with suspected heritable platelet function disorders including recommendation and validation of a streamlined agonist panel

Light transmission aggregometry (LTA) is used worldwide for the investigation of heritable platelet function disorders (PFDs), but interpretation of results is complicated by the feedback effects of ADP and thromboxane A(2) (TxA(2)) and by the overlap with the response of healthy volunteers. Over 5 years, we have performed lumi-aggregometry on 9 platelet agonists in 111 unrelated research participants with suspected PFDs and in 70 healthy volunteers. Abnormal LTA or ATP secretion test results were identified in 58% of participants. In 84% of these, the patterns of response were consistent with defects in Gi receptor signaling, the TxA(2) pathway, and dense granule secretion. Participants with defects in signaling to Gq-coupled receptor agonists and to collagen were also identified. Targeted genotyping identified 3 participants with function-disrupting mutations in the P2Y(12) ADP and TxA(2) receptors. The results of the present study illustrate that detailed phenotypic analysis using LTA and ATP secretion is a powerful tool for the diagnosis of PFDs. Our data also enable subdivision at the level of platelet-signaling pathways and in some cases to individual receptors. We further demonstrate that most PFDs can be reliably diagnosed using a streamlined panel of key platelet agonists and specified concentrations suitable for testing in most clinical diagnostic laboratories.

Enrichment of FLI1 and RUNX1 mutations in families with excessive bleeding and platelet dense granule secretion defects

We analyzed candidate platelet function disorder genes in 13 index cases with a history of excessive bleeding in association with a significant reduction in dense granule secretion and impaired aggregation to a panel of platelet agonists. Five of the index cases also had mild thrombocytopenia. Heterozygous alterations in FLI1 and RUNX1, encoding Friend leukemia integration 1 and RUNT-related transcription factor 1, respectively, which have a fundamental role in megakaryocytopoeisis, were identified in 6 patients, 4 of whom had mild thrombocytopenia. Two FLI1 alterations predicting p.Arg337Trp and p.Tyr343Cys substitutions in the FLI1 DNA-binding domain abolished transcriptional activity of FLI1. A 4-bp deletion in FLI1, and 2 splicing alterations and a nonsense variation in RUNX1, which were predicted to cause haploinsufficiency of either FLI1 or RUNX1, were also identified. Our findings suggest that alterations in FLI1 and RUNX1 may be common in patients with platelet dense granule secretion defects and mild thrombocytopenia.

Mutation Spectrum in RAB3GAP1, RAB3GAP2, and RAB18 and Genotype–Phenotype Correlations in Warburg Micro Syndrome and Martsolf Syndrome

Warburg Micro syndrome and Martsolf syndrome (MS) are heterogeneous autosomal‐recessive developmental disorders characterized by brain, eye, and endocrine abnormalities. Causative biallelic germline mutations have been identified in RAB3GAP1, RAB3GAP2, or RAB18, each of which encode proteins involved in membrane trafficking. This report provides an up to date overview of all known disease variants identified in 29 previously published families and 52 new families. One‐hundred and forty‐four Micro and nine Martsolf families were investigated, identifying mutations in RAB3GAP1 in 41% of cases, mutations in RAB3GAP2 in 7% of cases, and mutations in RAB18 in 5% of cases. These are listed in Leiden Open source Variation Databases, which was created by us for all three genes. Genotype–phenotype correlations for these genes have now established that the clinical phenotypes in Micro syndrome and MS represent a phenotypic continuum related to the nature and severity of the mutations present in the disease genes, with more deleterious mutations causing Micro syndrome and milder mutations causing MS. RAB18 has not yet been linked to the RAB3 pathways, but mutations in all three genes cause an indistinguishable phenotype, making it likely that there is some overlap. There is considerable genetic heterogeneity for these disorders and further gene identification will help delineate these pathways.

Use of next‐generation sequencing and candidate gene analysis to identify underlying defects in patients with inherited platelet function disorders

Inherited platelet function disorders (PFDs) are heterogeneous, and identification of the underlying genetic defects is difficult when based solely on phenotypic and clinical features of the patient.

Mice Lacking the ITIM-Containing Receptor G6b-B Exhibit Macrothrombocytopenia and Aberrant Platelet Function

An inhibitory receptor ensures that megakaryocytes produce proper numbers of functional platelets. Controlling Platelet Production Megakaryocytes reside in the bone marrow, where they produce platelets, cell fragments that form clots to prevent blood loss at sites of damage to the vasculature. Platelets and megakaryocytes share many activating receptors on their surface, but unlike platelets, megakaryocytes fail to become activated when exposed to components of the extracellular matrix. Mazharian et al. found that mice deficient in the immunoreceptor tyrosine–based inhibition motif–containing receptor G6b-B had fewer and larger platelets than did their wild-type counterparts. In addition, G6b-B–deficient mice exhibited increased bleeding in response to damage and had activated megakaryocytes, which resulted in the production of defective platelets. Together, these data suggest that G6b-B dampens activating signals in megakaryocytes to enable the generation of the appropriate number of functional platelets. Platelets are highly reactive cell fragments that adhere to exposed extracellular matrix (ECM) and prevent excessive blood loss by forming clots. Paradoxically, megakaryocytes, which produce platelets in the bone marrow, remain relatively refractory to the ECM-rich environment of the bone marrow despite having the same repertoire of receptors as platelets. These include the ITAM (immunoreceptor tyrosine–based activation motif)–containing collagen receptor complex, which consists of glycoprotein VI (GPVI) and the Fc receptor γ-chain, and the ITIM (immunoreceptor tyrosine–based inhibition motif)–containing receptor G6b-B. We showed that mice lacking G6b-B exhibited macrothrombocytopenia (reduced platelet numbers and the presence of enlarged platelets) and a susceptibility to bleeding as a result of aberrant platelet production and function. Platelet numbers were markedly reduced in G6b-B–deficient mice compared to those in wild-type mice because of increased platelet turnover. Furthermore, megakaryocytes in G6b-B–deficient mice showed enhanced metalloproteinase production, which led to increased shedding of cell-surface receptors, including GPVI and GPIbα. In addition, G6b-B–deficient megakaryocytes exhibited reduced integrin-mediated functions and defective formation of proplatelets, the long filamentous projections from which platelets bud off. Together, these findings establish G6b-B as a major inhibitory receptor regulating megakaryocyte activation, function, and platelet production.

Whole exome sequencing identifies genetic variants in inherited thrombocytopenia with secondary qualitative function defects

Inherited thrombocytopenias are a heterogeneous group of disorders characterized by abnormally low platelet counts which can be associated with abnormal bleeding. Next-generation sequencing has previously been employed in these disorders for the confirmation of suspected genetic abnormalities, and more recently in the discovery of novel disease-causing genes. However its full potential has not yet been exploited. Over the past 6 years we have sequenced the exomes from 55 patients, including 37 index cases and 18 additional family members, all of whom were recruited to the UK Genotyping and Phenotyping of Platelets study. All patients had inherited or sustained thrombocytopenia of unknown etiology with platelet counts varying from 11×109/L to 186×109/L. Of the 51 patients phenotypically tested, 37 (73%), had an additional secondary qualitative platelet defect. Using whole exome sequencing analysis we have identified “pathogenic” or “likely pathogenic” variants in 46% (17/37) of our index patients with thrombocytopenia. In addition, we report variants of uncertain significance in 12 index cases, including novel candidate genetic variants in previously unreported genes in four index cases. These results demonstrate that whole exome sequencing is an efficient method for elucidating potential pathogenic genetic variants in inherited thrombocytopenia. Whole exome sequencing also has the added benefit of discovering potentially pathogenic genetic variants for further study in novel genes not previously implicated in inherited thrombocytopenia.

Characterization of multiple platelet activation pathways in patients with bleeding as a high-throughput screening option: use of 96-well Optimul assay

Up to 1% of the population have mild bleeding disorders, but these remain poorly characterized, particularly with regard to the roles of platelets. We have compared the usefulness of Optimul, a 96-well plate-based assay of 7 distinct pathways of platelet activation to characterize inherited platelet defects in comparison with light transmission aggregometry (LTA). Using Optimul and LTA, concentration-response curves were generated for arachidonic acid, ADP, collagen, epinephrine, Thrombin receptor activating-peptide, U46619, and ristocetin in samples from (1) healthy volunteers (n = 50), (2) healthy volunteers treated with antiplatelet agents in vitro (n = 10), and (3) patients with bleeding of unknown origin (n = 65). The assays gave concordant results in 82% of cases (κ = 0.62, P < .0001). Normal platelet function results were particularly predictive (sensitivity, 94%; negative predictive value, 91%), whereas a positive result was not always substantiated by LTA (specificity, 67%; positive predictive value, 77%). The Optimul assay was significantly more sensitive at characterizing defects in the thromboxane pathway, which presented with normal responses with LTA. The Optimul assay is sensitive to mild platelet defects, could be used as a rapid screening assay in patients presenting with bleeding symptoms, and detects changes in platelet function more readily than LTA. This trial was registered at www.isrctn.org as #ISRCTN 77951167.

VPS33B regulates protein sorting into and maturation of α-granule progenitor organelles in mouse megakaryocytes.

Arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome is caused by deficiencies in the trafficking proteins VPS33B or VIPAR, and is associated with a bleeding diathesis and a marked reduction in platelet α-granules. We generated a tamoxifen-inducible mouse model of VPS33B deficiency, Vps33b(fl/fl)-ER(T2), and studied the platelet phenotype and α-granule biogenesis. Ultrastructural analysis of Vps33b(fl/fl)-ER(T2) platelets identified a marked reduction in α-granule count and the presence of small granule-like structures in agreement with the platelet phenotype observed in ARC patients. A reduction of ∼65% to 75% was observed in the α-granule proteins von Willebrand factor and P-selectin. Although platelet aggregation responses were not affected, a defect in δ-granule secretion was observed. Under arteriolar shear conditions, Vps33b(fl/fl)-ER(T2) platelets were unable to form stable aggregates, and tail-bleeding measurement revealed a bleeding diathesis. Analysis of bone marrow-derived megakaryocytes (MKs) by conventional and immuno-electron microscopy from Vps33b(fl/fl)-ER(T2) mice revealed a reduction in mature type-II multivesicular bodies (MVB II) and an accumulation of large vacuoles. Proteins that are normally stored in α-granules were underrepresented in MVB II and proplatelet extensions. These results demonstrate that abnormal protein trafficking and impairment in MVB maturation in MKs underlie the α-granule deficiency in Vps33b(fl/fl)-ER(T2) mouse and ARC patients.

SLFN14 mutations underlie thrombocytopenia with excessive bleeding and platelet secretion defects

Inherited thrombocytopenias are a group of disorders that are characterized by a low platelet count and are sometimes associated with excessive bleeding that ranges from mild to severe. We evaluated 36 unrelated patients and 17 family members displaying thrombocytopenia that were recruited to the UK Genotyping and Phenotyping of Platelets (GAPP) study. All patients had a history of excessive bleeding of unknown etiology. We performed platelet phenotyping and whole-exome sequencing (WES) on all patients and identified mutations in schlafen 14 (SLFN14) in 12 patients from 3 unrelated families. Patients harboring SLFN14 mutations displayed an analogous phenotype that consisted of moderate thrombocytopenia, enlarged platelets, decreased ATP secretion, and a dominant inheritance pattern. Three heterozygous missense mutations were identified in affected family members and predicted to encode substitutions (K218E, K219N, and V220D) within an ATPase-AAA-4, GTP/ATP-binding region of SLFN14. Endogenous SLFN14 expression was reduced in platelets from all patients, and mutant SLFN14 expression was markedly decreased compared with that of WT SLFN14 when overexpressed in transfected cells. Electron microscopy revealed a reduced number of dense granules in affected patients platelets, correlating with a decreased ATP secretion observed in lumiaggregometry studies. These results identify SLFN14 mutations as cause for an inherited thrombocytopenia with excessive bleeding, outlining a fundamental role for SLFN14 in platelet formation and function.