Bi-allelic Variants in TONSL Cause SPONASTRIME Dysplasia and a Spectrum of Skeletal Dysplasia Phenotypes

Abstract

SPONASTRIME dysplasia is an autosomal-recessive spondyloepimetaphyseal dysplasia characterized by spine (spondylar) abnormalities, midface hypoplasia with a depressed nasal bridge, metaphyseal striations, and disproportionate short stature. Scoliosis, coxa vara, childhood cataracts, short dental roots, and hypogammaglobulinemia have also been reported in this disorder. Although an autosomalrecessive inheritance pattern has been hypothesized, pathogenic variants in a specific gene have not been discovered in individuals with SPONASTRIME dysplasia. Here, we identified bi-allelic variants in TONSL, which encodes the Tonsoku-like DNA repair protein, in nine subjects (from eight families) with SPONASTRIME dysplasia, and four subjects (from three families) with short stature of varied severity and spondylometaphyseal dysplasia with or without immunologic and hematologic abnormalities, but no definitive metaphyseal striations at diagnosis. The finding of early embryonic lethality in a Tonsl À/À murine model and the discovery of reduced length, spinal abnormalities, reduced numbers of neutrophils, and early lethality in a tonsl À/À zebrafish model both support the hypomorphic nature of the identified TONSL variants. Moreover, functional studies revealed increased amounts of spontaneous replication fork stalling and chromosomal aberrations, as well as fewer camptothecin (CPT)-induced RAD51 foci in subject-derived cell lines. Importantly, these cellular defects were rescued upon re-expression of wild-type (WT) TONSL; this rescue is consistent with the hypothesis that hypomorphic TONSL variants are pathogenic. Overall, our studies in humans, mice, zebrafish, and subject-derived cell lines confirm that pathogenic variants in TONSL impair DNA replication and homologous recombination-dependent repair processes, and they lead to a spectrum of skeletal dysplasia phenotypes with numerous extra-skeletal manifestations.

Model organisms contribute to diagnosis and discovery in the undiagnosed diseases network: current state and a future vision

“…Because organs, cell types, and gene functions are well conserved across vertebrates, analysis of zebrafish mutants provides insights into gene functions in other vertebrates, including humans [ 58 , 59 ]. Zebrafish are used widely to validate candidate human disease genes and elucidate the molecular mechanisms and pathophysiology of disease [ 27 , 28 , 33 , 60 – 62 ] as well as for drug discovery [ 63 ].…”

Zebrafish Models for Human Skeletal Disorders

“…The phenotypes shown by genetically affected zebrafish resemble the symptoms found in human patients. Whereas gene expression studies are insufficient for a sensible model ( dymeclin ; Denais et al, 2011 ), the study of the hypomorph extl3 mutant boxer ( Volpi et al, 2017 ), the morpholino downregulation of ddrgk1 ( Egunsola et al, 2017 ) or nasna ( van Karnebeek et al, 2016 ), and the CRISPR/Cas9 editing of tonsl ( Burrage et al, 2019 ) could be considered appropriate zebrafish models for these four SE(M)D dysplasias ( Supplementary Table 1 ). Although these models do not exactly reproduce the symptoms found in humans, several molecular and cellular results support their great potential.…”

“…Moreover, the facial dysmorphism found in patients with SEMD with intellectual disorders NANS type could be associated with the small head and craniofacial cartilage phenotypes obtained in zebrafish morphants of nasna gene ( van Karnebeek et al, 2016 ). Finally, several skeletal phenotypes have been described in zebrafish with CRISPR/Cas9-edited tonsl gene that resembles those of SPONASTRIME dysplasia ( Burrage et al, 2019 ). Lethality before 20 days post-fertilization (dpf), accelerated vertebra ossification, short body size, and spinal abnormalities in the mutant resemble the spine abnormalities, disproportionately short stature, and exaggerated lumbar lordosis or scoliosis found in humans with SPONASTRIME dysplasia ( Burrage et al, 2019 ).…”

“…Finally, several skeletal phenotypes have been described in zebrafish with CRISPR/Cas9-edited tonsl gene that resembles those of SPONASTRIME dysplasia ( Burrage et al, 2019 ). Lethality before 20 days post-fertilization (dpf), accelerated vertebra ossification, short body size, and spinal abnormalities in the mutant resemble the spine abnormalities, disproportionately short stature, and exaggerated lumbar lordosis or scoliosis found in humans with SPONASTRIME dysplasia ( Burrage et al, 2019 ). These results strongly suggest the potential pre-clinical use of these models in the genetic analysis of human mutant alleles or the search for treatments.…”

Identification of potential pathogenic genes causing occipitalization of the atlas

“…In AGAP1, LRP5, and TONSL, two other rare heterozygous variants were identi ed in different patients. It has been reported that these genes associate with vertebral or Chiari malformations [34][35][36][37][38][39]. The related information is displayed in Table 3.…”