Clinical, genetic and structural delineation of RPL13-related spondyloepimetaphyseal dysplasia suggest extra-ribosomal functions of eL13

Abstract

Spondyloepimetaphyseal dysplasia with severe short stature, RPL13-related (SEMD-RPL13), MIM#618728), is a rare autosomal dominant disorder characterized by short stature and skeletal changes such as mild spondylar and epimetaphyseal dysplasia affecting primarily the lower limbs. The genetic cause was first reported in 2019 by Le Caignec et al., and six disease-causing variants in the gene coding for a ribosomal protein, RPL13 (NM_000977.3) have been identified to date. This study presents clinical and radiographic data from 12 affected individuals aged 2-64 years from seven unrelated families, showing highly variable manifestations. The affected individuals showed a range from mild to severe short stature, retaining the same radiographic pattern of spondylar- and epi-metaphyseal dysplasia, but with varying severity of the hip and knee deformities. Two new missense variants, c.548 G>A, p.(Arg183His) and c.569 G>T, p.(Arg190Leu), and a previously known splice variant c.477+1G>A were identified, confirming mutational clustering in a highly specific RNA binding motif. Structural analysis and interpretation of the variants' impact on the protein suggests that disruption of extra-ribosomal functions of the protein through binding of mRNA may play a role in the skeletal phenotype of SEMD-RPL13. In addition, we present gonadal and somatic mosaicism for the condition.

Fig. 1. eL13 variants mapped onto a three-dimensional (3D) protein structure of eL13.

a CryoEM structure of the Homo sapiens ribosome (PDB 6olg). b Enlarged view of eL13 (in red) shown in detail, interacting with 28S rRNA (orange) and ribosomal proteins eL36 (blue) and eL27a (green). c The location of the missense variants clustered on the alpha helix 7 (H7), which is a conserved region of the protein. The missense variants are either on the hydrophobic side of the helix, in contact with eL36 (A178E, A185P), or on the opposite positively charged surface, that binds the negatively charged 28S rRNA (R183H/P, R190L). The eL13 insertion point is located near the variable linker connecting helices 5 and 6. Substitutions of hydrophobic residues to proline or glutamic acid, would break the helix or introduce repulsive negative charges, while those of positively charged arginines would directly disrupt RNA binding. d Schematic view of eL13 secondary structure topology, indicating the location of the insertion and missense variants. The RNA binding regions across eL13 are shown in pink and eL13 28S rRNA binding is mediated by H1, beta-hairpin B1 and H7. Pathogenic eL13 variants cluster on helix 7 (H7), while the insertion targets the variable linker region connecting helix 5 and 6 (H5-H6). e Close-up view of the RNA-binding motif mutated in SEMD-RPL13. 28S rRNA adopts a dsRNA hairpin structure, stabilized by a wobble G.U base pair (U975.G980), recognized by eL13 R183-R185 arginine-fork, that strongly binds to the nucleic phosphate backbone. The hairpin is further recognized by R190 binding to unpaired G979. Note the sharp bend in the helix at the interaction loci mediated by G·U wobble pair. f Schematic representation of the previously reported RPL13 (NM_000977.3) variants (in black) along with the three disease-causing variants identified in this study (lower part). Previously unreported variants identified in this study are depicted in red.

Fig. 2. Pedigrees and clinical pictures of affected individuals.

a Pedigrees of the families. In families 1, 2, and 5, the affected individuals (filled symbols and indicated with +) were heterozygous for c.548G>A in RPL13. Individuals marked with WT were tested and showed the wild-type allele. In family 4, the affected individuals were heterozygous for c.569G>A. Note that family 4 has two affected siblings, but parents were not carriers of the variant in blood DNA, suggesting parental gonadal mosaicism. In family 6 and 7, the affected individuals marked with + were heterozygous for the c.477+1G>A variant. Note that in family 6, gray color indicates mosaicism in blood DNA for the variant. In family 7, the older half-brother, mother, and maternal grandmother had suggestive clinical features but variant testing is pending. The pedigree of individual 3 is not included in this article as her family members did not agree for its publication. b Clinical pictures of proband 2-II:2, nine years. c Proband 4-II:4, 15 years. d Proband 1-III:4, three years, and his father 1-II:6 (e) and uncle 1-II:5 (f). g Half-siblings from family 7: aged 6 years and nine months and 2 years and 7 months, respectively. Note the variable severity of limb deformities. Written consent was obtained for publication of the photographs.

Fig. 3. Lateral radiographs and sagittal MRI of the lumbar spine.

a Radiograph of Patient 1-III:4 at age 3 years showing mild platyspondyly. b MRI of Patient 3 at age 7 years showing mild platyspondyly with irregular end plates and lumbosacral lordosis. c Radiograph of Patient 4-II:1 at 18 years of age showing mild platyspondyly with endplate modification. d Radiograph of Patient 4-II:4 at 15 years of age showing mild modification of vertebral end plates. Platyspondyly is not seen. e Radiograph of Patient 5-III:1 at age 7 years showing mild platyspondyly with severe lumbosacral lordosis and irregular end plates, and short neural arches of the lower lumbar spine and spinal canal stenosis. f Radiograph of Patient 6-II:1 at 7 years old, showing subtle irregularities of the vertebral end plates. g Radiograph of Patient 7-III:2 at 8 years showing vertebral bodies with central notches and anterior ossification defects. h Radiograph of Patient 7-III:3 at 4 years showing increased lumbosacral lordosis and vertebral bodies with anterior ossification defects and irregular end plates.

Fig. 4. Radiographs of the pelvis.

a Patient 1-III:4, radiograph at 3 years; showing unossified capital femoral epiphyses, and metaphyseal irregularities of the proximal femora with short femoral necks and coxa vara, and irregular acetabula. b Patient 2-II:2, radiograph at 9 years; showing flat, irregular capital femoral epiphyses, metaphyseal dysplasia of the proximal femora with short necks and coxa vara, and normal acetabula. c Patient 3, radiograph at 11 years; showing short femoral neck, severely flat capital femoral epiphyses, severe coxa vara and shallow acetabula, together with high-rising greater trochanters. d Patient 4-II:1, radiograph at 25 years; showing short femoral necks with mild coxa vara. The capital femoral epiphyses are not deformed, while the joint spaces are narrow. e Patient 4-II:4, radiograph at 15 years; showing severely flat capital femoral epiphyses and short femoral necks with mild coxa vara and high-rising greater trochanters. f Patient 5-III:1, radiograph at 10 years; showing short femoral neck, severely flat capital femoral epiphyses, severe coxa vara, and shallow acetabula. g Patient 5-II:2 the radiograph at 42 years showing flat capital femoral epiphyses, short femoral necks with mild coxa vara and high-rising greater trochanters, and narrow joint spaces. h Patient 6-II:1, radiograph at 7 years; showing short femoral necks, very small and flat capital femoral epiphyses, metaphyseal irregularities of the proximal femora, and severe coxa vara. i Patient 7-III:2, radiograph at 6 years 10 months; showing short femoral necks, flat capital femoral epiphysis of the left and delayed capital femoral epiphyseal ossification of the right, and severe metaphyseal irregularities of the proximal femora. j Patient 7-III:3, radiograph at 2 years and 3 months; showing short femoral necks, delayed ossification of both capital femoral epiphyses and severe metaphyseal irregularities of the proximal femora.

Fig. 5. Knee radiographs.

a Patient 1-III:4, radiograph at age 3 years; showing irregular metaphyses and small epiphyses of the distal femora and proximal tibiae with mild genu varum. b Patient 3, radiograph at age 11 years; showing genu varum and defective ossification of the medial aspect of the proximal tibial epiphysis. 8-plate guided growth was inserted to restore the knee deformity. c Patient 4-II:1 radiograph at age 18 years; showing mild epiphyseal dysplasia. d Patient 4-II:4, radiograph at age 15 years; showing mild epiphyseal dysplasia. e Patient 5-III:1, radiograph at age 3 years; showing small epiphyses and metaphyseal irregularities of the distal femora and proximal tibiae with bilateral genu varum. f Patient 5-II:2, radiograph at age 42 years; showing premature degenerative joint disease. g Patient 6-II:1, radiograph at 7 years; showing metaphyseal changes of the knees and mild genu varum. h Patient 7-III:2, radiograph at 7 years; showing mild metaphyseal changes and mildly flat epiphyses of the knees, genu valgum. 8-plate guided growth surgery partially restored severe genu valgum. i Patient 7-III:3, radiograph at 2 years; showing mild metaphyseal changes, mildly flat epiphyses of the knees and genu varum.