Christopher J. Penkett

A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders

Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached, which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants, short insertions/deletions, and large copy number variants (though not inversions) which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples, respectively, from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology, whereas the remainder had an a priori highly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only 8 of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD.

A gain-of-function variant in DIAPH1 causes dominant macrothrombocytopenia and hearing loss

Macrothrombocytopenia (MTP) is a heterogeneous group of disorders characterized by enlarged and reduced numbers of circulating platelets, sometimes resulting in abnormal bleeding. In most MTP, this phenotype arises because of altered regulation of platelet formation from megakaryocytes (MKs). We report the identification of DIAPH1, which encodes the Rho-effector diaphanous-related formin 1 (DIAPH1), as a candidate gene for MTP using exome sequencing, ontological phenotyping, and similarity regression. We describe 2 unrelated pedigrees with MTP and sensorineural hearing loss that segregate with a DIAPH1 R1213* variant predicting partial truncation of the DIAPH1 diaphanous autoregulatory domain. The R1213* variant was linked to reduced proplatelet formation from cultured MKs, cell clustering, and abnormal cortical filamentous actin. Similarly, in platelets, there was increased filamentous actin and stable microtubules, indicating constitutive activation of DIAPH1. Overexpression of DIAPH1 R1213* in cells reproduced the cytoskeletal alterations found in platelets. Our description of a novel disorder of platelet formation and hearing loss extends the repertoire of DIAPH1-related disease and provides new insight into the autoregulation of DIAPH1 activity.

A dominant gain-of-function mutation in universal tyrosine kinase SRC causes thrombocytopenia, myelofibrosis, bleeding, and bone pathologies

E527K hyperactive SRC results in megakaryocytes with increased podosome formation, thrombocytopenia, myelofibrosis, bleeding, and bone pathologies. SRC shows its stripes The nonreceptor tyrosine kinase SRC is a proto-oncogene that has been associated with cancer progression. Now, Turro et al. find a gain-of-function mutation in SRC in nine patients with myelofibrosis, bleeding, and bone disorders. This mutation prevented SRC from inhibiting itself, and the overactive SRC resulted in enhanced tyrosine phosphorylation in a zebrafish model as well as in patient-derived cells. In patients with myelofibrosis, this SRC mutation was associated with increased outgrowth of myeloid and megakaryocyte colonies, with abnormal platelet production, which could be rescued by SRC kinase inhibition. These findings may be important for understanding the severe bleeding in cancer patients treated with Src family kinase inhibitors. The Src family kinase (SFK) member SRC is a major target in drug development because it is activated in many human cancers, yet deleterious SRC germline mutations have not been reported. We used genome sequencing and Human Phenotype Ontology patient coding to identify a gain-of-function mutation in SRC causing thrombocytopenia, myelofibrosis, bleeding, and bone pathologies in nine cases. Modeling of the E527K substitution predicts loss of SRC’s self-inhibitory capacity, which we confirmed with in vitro studies showing increased SRC kinase activity and enhanced Tyr419 phosphorylation in COS-7 cells overexpressing E527K SRC. The active form of SRC predominates in patients’ platelets, resulting in enhanced overall tyrosine phosphorylation. Patients with myelofibrosis have hypercellular bone marrow with trilineage dysplasia, and their stem cells grown in vitro form more myeloid and megakaryocyte (MK) colonies than control cells. These MKs generate platelets that are dysmorphic, low in number, highly variable in size, and have a paucity of α-granules. Overactive SRC in patient-derived MKs causes a reduction in proplatelet formation, which can be rescued by SRC kinase inhibition. Stem cells transduced with lentiviral E527K SRC form MKs with a similar defect and enhanced tyrosine phosphorylation levels. Patient-derived and E527K-transduced MKs show Y419 SRC–positive stained podosomes that induce altered actin organization. Expression of mutated src in zebrafish recapitulates patients’ blood and bone phenotypes. Similar studies of platelets and MKs may reveal the mechanism underlying the severe bleeding frequently observed in cancer patients treated with next-generation SFK inhibitors.

Mutations in CACNA2D4 Cause Distinctive Retinal Dysfunction in Humans

movement, movement toward the optic disc, and movement beyond the optic disc. No movement was observed in 11 of 21 eyes (52.4%) with RD; movement toward the optic disc in 7 eyes (33.3%) with RD and 1 eye (12.5%) with RFs; and movement beyond the optic disc in 3 eyes (14.3%) with RD and 7 eyes (87.5%) with RFs. Nerve fiber layer forms were classified into 3 groups: uniform NFL thickening, formation of 2 distinct NFL bundles, and formation of 1 large NFL bundle. Uniform NFL thickening occurred in 17 of 21 eyes (81.0%) with RD; 2 distinct NFL bundles in 3 eyes (14.3%) with RD and 2 eyes (25.0%) with RFs; and 1 large NFL bundle in 1 eye (4.8%) with RD and 6 eyes (75.0%) with RFs. In this study, SS-OCT showed the detailed structures of the retinal deformities progressing from RD to RFs in patients with FEVR. The 3 characteristic findings associated with severity ranging from RD to RFs (Table 1) were a long, tapering sensory retina and slippage of the sensory retina, movement of the sensory retina, and formation of NFL bundles (Fig 2). A long, tapering sensory retina has been reported in cases with ßzone parapapillary atrophy with axial length elongation, whereas slippage of the sensory retina has not. A possible explanation may be that the difference between the axial length elongation results in retinal, choroidal, and scleral traction and the fibrovascular changes in eyes with FEVR result only in retinal traction. Retinal movement from the contralateral side of the lesion to the optic disc has been reported in fundus photographs of RFs in eyes with FEVR. In addition, SS-OCT clarified that the material covering the optic disc was structurally preserved sensory retina. The NFL bundles progressed from only thickening to 2 distinct bundles to 1 large bundle and resulted in unique RFs that differed from other folds, which are composed mostly of all retinal layers owing to excessive retinal tissue, such as papillomacular folds with microphthalmos. Because the epiretinal membrane, which affects the NFL and other retinal layers by local traction on the retina in pediatric and adult patients, does not induce RFs, only the retinal traction during the pediatric time frame is insufficient to cause RFs. The unique NFL bundles may develop in a limited manner under severe traction caused by fibrovascular tissues in the peripheral retina during that period. We observed only structural changes in the area around the optic disc in patients with FEVR. Further studies are warranted, including functional analysis of the NFL and the other retinal layers, structural analysis of the peripheral retina, and comparative analysis of subjects with other diseases such as retinopathy of prematurity. The current results showed that NFL thickening occurred in the early stage of RD and that the bundled NFL was the major component of the RFs. In the peripheral retina, FEVR has a characteristic circumferential vascular maldevelopment, which leads to traction in the anteroposterior direction and circumferentially. Anteroposterior traction may cause changes in the sensory retina including a long, tapering form, slippage, and movement, whereas the circumferential traction may develop NFL bundles.

A comprehensive high-throughput sequencing test for the diagnosis of inherited bleeding, thrombotic and platelet disorders

Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached, which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants, short insertions/deletions, and large copy number variants (though not inversions) which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples, respectively, from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology, whereas the remainder had an a priori highly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only 8 of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD.

Epigenetic analysis of regulatory T cells using multiplex bisulfite sequencing

This work was supported by Wellcome Trust Grant 096388, JDRF Grant 9-2011-253, the National Institute for Health Research Cambridge Biomedical Research Centre (BRC) and Award P01AI039671 (to LSW. and JAT.) from the National Institute of Allergy and Infectious Diseases (NIAID). CW is supported by the Wellcome Trust (089989). The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of NIAID or the National Institutes of Health. The Cambridge Institute for Medical Research is in receipt of Wellcome Trust Strategic Award 100140. We gratefully acknowledge the participation of all NIHR Cambridge BioResource volunteers. We thank the Cambridge BioResource staff for their help with volunteer recruitment. We thank members of the Cambridge BioResource SAB and Management Committee for their support of our study and the National Institute for Health Research Cambridge Biomedical Research Centre for funding. We thank Fay Rodger and Ruth Littleboy for running the Illumina MiSeq in the Molecular Genetics Laboratories, Addenbrookes Hospital, Cambridge. This research was supported by the Cambridge NIHR BRC Cell Phenotyping Hub. In particular, we wish to thank Anna Petrunkina Harrison, Simon McCallum, Christopher Bowman, Natalia Savinykh, Esther Perez and Jelena Markovic Djuric for their advice and support in cell sorting. We also thank Helen Stevens, Pamela Clarke, Gillian Coleman, Sarah Dawson, Jennifer Denesha, Simon Duley, Meeta Maisuria-Armer and Trupti Mistry for acquisition and preparation of samples.

Rare variants in GP1BB are responsible for autosomal dominant macrothrombocytopenia

The von Willebrand receptor complex, which is composed of the glycoproteins Ibα, Ibβ, GPV, and GPIX, plays an essential role in the earliest steps in hemostasis. During the last 4 decades, it has become apparent that loss of function of any 1 of 3 of the genes encoding these glycoproteins (namely, GP1BA, GP1BB, and GP9) leads to autosomal recessive macrothrombocytopenia complicated by bleeding. A small number of variants in GP1BA have been reported to cause a milder and dominant form of macrothrombocytopenia, but only 2 tentative reports exist of such a variant in GP1BB By analyzing data from a collection of more than 1000 genome-sequenced patients with a rare bleeding and/or platelet disorder, we have identified a significant association between rare monoallelic variants in GP1BB and macrothrombocytopenia. To strengthen our findings, we sought further cases in 2 additional collections in the United Kingdom and Japan. Across 18 families exhibiting phenotypes consistent with autosomal dominant inheritance of macrothrombocytopenia, we report on 27 affected cases carrying 1 of 9 rare variants in GP1BB.

Platelet function is modified by common sequence variation in megakaryocyte super enhancers

Linking non-coding genetic variants associated with the risk of diseases or disease-relevant traits to target genes is a crucial step to realize GWAS potential in the introduction of precision medicine. Here we set out to determine the mechanisms underpinning variant association with platelet quantitative traits using cell type-matched epigenomic data and promoter long-range interactions. We identify potential regulatory functions for 423 of 565 (75%) non-coding variants associated with platelet traits and we demonstrate, through ex vivo and proof of principle genome editing validation, that variants in super enhancers play an important role in controlling archetypical platelet functions.

Complex Structural Variants Resolved by Short-Read and Long-Read Whole Genome Sequencing in Mendelian Disorders

Complex structural variants (cxSVs) are genomic rearrangements comprising multiple structural variants, typically involving three or more breakpoint junctions. They contribute to human genomic variation and can cause Mendelian disease, however they are not typically considered during genetic testing. Here, we investigate the role of cxSVs in Mendelian disease using short-read whole genome sequencing (WGS) data from 1,324 individuals with neurodevelopmental or retinal disorders from the NIHR BioResource project. We present four cases of individuals with a cxSV affecting Mendelian disease-associated genes. Three of the cxSVs are pathogenic: a de novo duplication-inversion-inversion-deletion affecting ARID1B in an individual with Coffin-Siris syndrome, a deletion-inversion-duplication affecting HNRNPU in an individual with intellectual disability and seizures, and a homozygous deletion-inversion-deletion affecting CEP78 in an individual with cone-rod dystrophy. Additionally, we identified a de novo duplication-inversion-duplication overlapping CDKL5 in an individual with neonatal hypoxic-ischaemic encephalopathy. Long-read sequencing technology used to resolve the breakpoints demonstrated the presence of both a disrupted and an intact copy of CDKL5 on the same allele; therefore, it was classified as a variant of uncertain significance. Analysis of sequence flanking all breakpoint junctions in all the cxSVs revealed both microhomology and longer repetitive sequences, suggesting both replication and homology based processes. Accurate resolution of cxSVs is essential for clinical interpretation, and here we demonstrate that long-read WGS is a powerful technology by which to achieve this. Our results show cxSVs are an important although rare cause of Mendelian disease, and we therefore recommend their consideration during research and clinical investigations.

Abnormal differentiation of B cells and megakaryocytes in patients with Roifman syndrome

Background: Roifman syndrome is a rare inherited disorder characterized by spondyloepiphyseal dysplasia, growth retardation, cognitive delay, hypogammaglobulinemia, and, in some patients, thrombocytopenia. Compound heterozygous variants in the small nuclear RNA gene RNU4ATAC, which is necessary for U12‐type intron splicing, were identified recently as driving Roifman syndrome. Objective: We studied 3 patients from 2 unrelated kindreds harboring compound heterozygous or homozygous stem II variants in RNU4ATAC to gain insight into the mechanisms behind this disorder. Methods: We systematically profiled the immunologic and hematologic compartments of the 3 patients with Roifman syndrome and performed RNA sequencing to unravel important splicing defects in both cell lineages. Results: The patients exhibited a dramatic reduction in B‐cell numbers, with differentiation halted at the transitional B‐cell stage. Despite abundant B‐cell activating factor availability, development past this B‐cell activating factor–dependent stage was crippled, with disturbed minor splicing of the critical mitogen‐activated protein kinase 1 signaling component. In the hematologic compartment patients with Roifman syndrome demonstrated defects in megakaryocyte differentiation, with inadequate generation of proplatelets. Platelets from patients with Roifman syndrome were rounder, with increased tubulin and actin levels, and contained increased &agr;‐granule and dense granule markers. Significant minor intron retention in 354 megakaryocyte genes was observed, including DIAPH1 and HPS1, genes known to regulate platelet and dense granule formation, respectively. Conclusion: Together, our results provide novel molecular and cellular data toward understanding the immunologic and hematologic features of Roifman syndrome. Graphical abstract: Figure. No caption available.