Loss-of-function variant in SPIN4 causes an X-linked overgrowth syndrome
Julian C. Lui, Jacob Wagner, Elaine Zhou, Lijin Dong, Kevin M. Barnes, Youn Hee Jee, Jeffrey Baron
Julian C. Lui, Jacob Wagner, Elaine Zhou, Lijin Dong, Kevin M. Barnes, Youn Hee Jee, Jeffrey Baron
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Research Article Development Genetics

Loss-of-function variant in SPIN4 causes an X-linked overgrowth syndrome

Abstract

Overgrowth syndromes can be caused by pathogenic genetic variants in epigenetic writers, such as DNA and histone methyltransferases. However, no overgrowth disorder has previously been ascribed to variants in a gene that acts primarily as an epigenetic reader. Here, we studied a male individual with generalized overgrowth of prenatal onset. Exome sequencing identified a hemizygous frameshift variant in Spindlin 4 (SPIN4), with X-linked inheritance. We found evidence that SPIN4 binds specific histone modifications, promotes canonical WNT signaling, and inhibits cell proliferation in vitro and that the identified frameshift variant had lost all of these functions. Ablation of Spin4 in mice recapitulated the human phenotype with generalized overgrowth, including increased longitudinal bone growth. Growth plate analysis revealed increased cell proliferation in the proliferative zone and an increased number of progenitor chondrocytes in the resting zone. We also found evidence of decreased canonical Wnt signaling in growth plate chondrocytes, providing a potential explanation for the increased number of resting zone chondrocytes. Taken together, our findings provide strong evidence that SPIN4 is an epigenetic reader that negatively regulates mammalian body growth and that loss of SPIN4 causes an overgrowth syndrome in humans, expanding our knowledge of the epigenetic regulation of human growth.

Authors

Julian C. Lui, Jacob Wagner, Elaine Zhou, Lijin Dong, Kevin M. Barnes, Youn Hee Jee, Jeffrey Baron

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Figure 2

Loss of function in Spin4 frameshift variant.

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Loss of function in Spin4 frameshift variant. (A) Schematic diagram show...
(A) Schematic diagram showing the location of the frameshift relative to the 3 Tudor domains. (B) Western blot with anti-FLAG antibody confirmed a change in molecular size of the frameshift SPIN4 variant. The experiment was repeated 3 times with similar results. (C) Tritiated thymidine uptake in HEK293 cells showed that both SPIN1 and SPIN4 WT suppress proliferation. SPIN4 mutant (Mut) did not show any suppression of proliferation compared with empty vector (EV). Consistently, siRNA against SPIN1 or SPIN4 increased proliferation compared with scrambled siRNA. *P < 0.05, 1-way ANOVA, n = 8–14. (D and E) GFP-tagged proteins were transfected in HEK293 cells, and subcellular localization was examined by fractionation followed by anti-GFP Western blot (D) or fluorescence microscopy (E). H2B-GFP was used as a control for chromatin-bound protein. Anti–histone H3 was used to assess purity of chromatin-bound protein fraction. C, cytoplasmic; Nu, nuclear; chr, chromatin-bound. The experiment was repeated 3 times with similar results. (F and G) Histone peptide arrays were used to assess histone binding properties of human SPIN1, SPIN4 WT, and Mut. Each spot on the array contains a peptide portion of a histone that has undergone 1 or more specific posttranslational modifications. Darker color indicates greater binding of the indicated Spin protein to that modified peptide. The upper and lower halves of each array are used as technical replicates. Both SPIN1 and SPIN4 WT bind to modified histone peptides, while SPIN4 Mut showed substantially diminished binding. Peptides were ranked based on average binding to SPIN4 WT across 8 arrays and a box-and-whisker plot was generated. The line inside the box represents the median; upper and lower boundary of the boxes represents the 25th and 75th percentile, respectively; whiskers represent the 5th and 95th percentile (n = 8).