Whole Genome Sequencing of 10,000 Rare Disease Patients Reveals Digenic Inheritance of Blood Disorders

Inherited bleeding, thrombotic and platelet disorders (BPDs) affect approximately 3M people worldwide and an appreciable portion have a disorder of megakaryopoiesis and the production and function of platelets, including the formation of granules. While genetic variants in 76 genes have been implicated in BPDs, many patients remain without a molecular diagnosis (Lentaigne et al, Simeoni et al). We hypothesised that some of these disorders may be caused by compound inheritance of variants in two different genes, a mode of inheritance thus far never implicated in BPDs.

For the pilot phase of the 100 000 Genomes Project we have sequenced the whole genomes of 10,000 individuals consisting of probands with molecularly unexplained rare disorders and their close relatives, with 3,000 having inherited disorders of the blood and immune system. We searched for causal variants in known BPD-related genes and employed a new statistical method for Bayesian evaluation of rare variants in Mendelian disease (BeviMed) (Greene et al) to identify novel marginal associations between rare variants and disease status. Where the identified variants could not individually explain the phenotype in full within the pedigrees, we searched for additional variants affecting other BPD-related genes or novel genes identified using BeviMed.

First, we have identified a large pedigree in which certain members with mild thrombocytopenia (lower 10^th percentile of the population distribution) are affected by a single variant encoding a premature stop at residue 69 in the major isoform of the tropomyosin gene TPM4 expressed in megakaryocytes (Pleines et al). Other members of the family have distinctly more severe macrothrombocytopenia (with platelet counts as low as 24x10⁹/l) and this is due to inheritance of a second variant in the actinin gene ACTN1 leading to a Thr340Met substitution, demonstrating a compound additive effect of variants in two genes encoding cytoskeletal proteins important for actin polymerization and thereby causing inadequate platelet formation. It is noteworthy that both of these genes have been identified in a genome wide association study of the count and mean volume of platelets (Gieger et al). Second, we used BeviMed to identify genes containing variants that are marginally associated with a syndrome defined by a platelet granule defect combined with familial autism (Bijl et al). The strongest association is due to splice variants in a granule-related gene on Chr17p13 but in all four unrelated cases at least one additional variant is required to explain the observed segregation patterns. Two of these unrelated cases harbor variants in two other marginally associated genes at Chr9q32 and Chr4q31 and a third harbors a de novo copy number variant. These additional variants likely explain why the children in three of the families are affected by this syndrome while their parents are not.

In conclusion, we have used whole genome sequencing, pedigree building, detailed platelet phenotyping and new association approaches to identify the first cases of digenic inheritance of BPDs. Our results illustrate the cooperative role of different cytoskeletal proteins in platelet formation and cement the role of granule biology in the function of both platelets and neurons.

References:

• Lentaigne et al (2016) Inherited platelet disorders: towards DNA-based diagnosis. Blood127(23) 2814-2823.

• Simeoni et al (2016) A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders. Blood127(23) 2793-2803.

• Greene et al (2016) Bayesian evaluation of variant involvement in Mendelian disease. http://cran.r-project.org/web/packages/BeviMed.

• Pleines et al (2016) Tropomyosins regulate platelet biogenesis. (Under submission).

• Gieger et al (2011) New gene functions in megakaryopoiesis and platelet formation. Nature480(7376) 201-208

• Bijl et al (2015) Platelet studies in autism spectrum disorder patients and first-degree relatives. Molecular Autism6:57.