Biallelic splicing variants in the nucleolar 60S assembly factor RBM28 cause the ribosomopathy ANE syndrome

Alopecia, neurologic defects, and endocrinopathy (ANE) syndrome is a rare ribosomopathy known to be caused by a p.(Leu351Pro) variant in the essential, conserved, nucleolar large ribosomal subunit (60S) assembly factor RBM28. We report the second family of ANE syndrome to date and a female pediatric ANE syndrome patient. The patient presented with alopecia, craniofacial malformations, hypoplastic pituitary, and hair and skin abnormalities. Unlike the previously reported patients with the p.(Leu351Pro) RBM28 variant, this ANE syndrome patient possesses biallelic precursor messenger RNA (pre-mRNA) splicing variants at the 5′ splice sites of exon 5 (ΔE5) and exon 8 (ΔE8) of RBM28 ({"type":"entrez-nucleotide","attrs":{"text":"NM_018077.2","term_id":"187960108","term_text":"NM_018077.2"}}NM_018077.2:c.541+1_541+2delinsA]; 946G > T]). In silico analyses and minigene splicing experiments in cells indicate that each splice variant specifically causes skipping of its respective mutant exon. Because the ΔE5 variant results in an in-frame 31 amino acid deletion (p.(Asp150_Lys180del)) in RBM28 while the ΔE8 variant leads to a premature stop codon in exon 9, we predicted that the ΔE5 variant would produce partially functional RBM28 but the ΔE8 variant would not produce functional protein. Using a yeast model, we demonstrate that the ΔE5 variant does indeed lead to reduced overall growth and large subunit ribosomal RNA (rRNA) production and pre-rRNA processing. In contrast, the ΔE8 variant is comparably null, implying that the partially functional ΔE5 RBM28 protein enables survival but precludes correct development. This discovery further defines the underlying molecular pathology of ANE syndrome to include genetic variants that cause aberrant splicing in RBM28 pre-mRNA and highlights the centrality of nucleolar processes in human genetic disease.

Ribosome biogenesis (RB) is the essential cellular process in which the complex macromolecular ribosomal machinery is manufactured and assembled, enabling protein translation (1–4). Both ribosomal RNA (rRNA) and ribosomal protein (RP) components must be correctly synthesized, processed, modified, folded, translocated, and ultimately joined in the cytoplasm to engage in global protein synthesis (1–3). For eukaryotes, four rRNA molecules (1, 5) and about 80 RPs (1, 6, 7) form the core of the mature small (40S) and large (60S) ribosomal subunits. The demand for ribosomes during the cell cycle is immense: in a growing yeast cell, more than 30 ribosomes are synthesized per second (8), while in a growing HeLa cell, this figure balloons to 125 ribosomes per second (9). Over 200 trans-acting assembly factors are necessary to achieve the fast and accurate ribosome assembly required to meet this tremendous cellular translational demand (1).Given that up to 80% of cellular metabolism is devoted to RB (10), it is unsurprising that defects in RB factors are causative of a class of human diseases known as ribosomopathies (1, 11–16). Though not fully understood, tissue-specific defects are the hallmark of ribosomopathies (11, 17). Tissues formed from hematopoietic or neural crest cell lineages are disproportionately affected, resulting in anemia, neutropenia, and leukemia, bone marrow failure diseases including Diamond–Blackfan Anemia (DBA) (18–23) and Shwachman–Diamond syndrome (24–26), craniofacial, dermatological, and neurological diseases including Treacher Collins syndrome (27–29) and postaxial acrofacial dysostosis (30), and alopecia, neurologic defects, and endocrinopathy (ANE) syndrome (31–34).ANE syndrome (OMIM: 612079) (31, 35) is a rare ribosomopathy defined by heterogeneous clinical features of variable severity including alopecia, neurological deformities, intellectual disability, and hormonal deficiencies with pubertal delay. In the only ANE syndrome case report published to date, Nousbeck and coworkers studied five brothers of consanguineous parentage with variable ANE syndrome features, finding that ANE syndrome patient tissue samples had quantifiably fewer ribosomes and qualitatively dysmorphological rough endoplasmic reticula versus healthy control samples (31). All five patients were found to carry a homozygous missense variant (p.(Leu351Pro); L > P) in RBM28 (31), a known essential 60S assembly factor orthologous to yeast Nop4 (36–39). Follow-up studies further defined the clinical extent of endocrinopathy (32) and the biochemical mechanisms of hair and skin defects (33) and of inhibited ribosome biogenesis (34, 40) due to impaired function of RBM28 or its yeast homolog, Nop4. However, due to the rarity of the disease and lack of sufficient animal model studies, further investigation of ANE syndrome has been limited.We report a female pediatric patient in the second family of ANE syndrome to date, unrelated to the family in the original case report (31). The ANE syndrome patient has a clinical presentation consistent with the definition of ANE syndrome but possesses differing genetic variants and molecular pathology. Using in vivo techniques, we demonstrate that the patient’s compound heterozygous splicing variants in RBM28 create one hypomorphic (ΔE5) and one null (ΔE8) allele with respect to overall growth and 60S pre-rRNA processing functions. By elucidating the pathology of an ANE syndrome patient, our results bolster and extend our understanding of this rare ribosomopathy and reinforce the importance of proper nucleolar function in human health and disease.