Molecular defects in the motor adaptor BICD2 cause proximal spinal muscular atrophy with autosomal-dominant inheritance

Abstract

The most common form of spinal muscular atrophy (SMA) is a recessive disorder caused by deleterious SMN1 mutations in 5q13, whereas the genetic etiologies of non-5q SMA are very heterogeneous and largely remain to be elucidated. In a Bulgarian family affected by autosomal-dominant proximal SMA, we performed genome-wide linkage analysis and whole-exome sequencing and found a heterozygous de novo c.320C>T (p.Ser107Leu) mutation in bicaudal D homolog 2 (Drosophila) (BICD2). Further analysis of BICD2 in a cohort of 119 individuals with non-5q SMA identified a second de novo BICD2 mutation, c.2321A>G (p.Glu774Gly), in a simplex case. Detailed clinical and electrophysiological investigations revealed that both families are affected by a very similar disease course, characterized by early childhood onset, predominant involvement of lower extremities, and very slow disease progression. The amino acid substitutions are located in two interaction domains of BICD2, an adaptor protein linking the dynein molecular motor with its cargo. Our immunoprecipitation and localization experiments in HeLa and SH-SY5Y cells and affected individuals' lymphoblasts demonstrated that p.Ser107Leu causes increased dynein binding and thus leads to accumulation of BICD2 at the microtubule-organizing complex and Golgi fragmentation. In addition, the altered protein had a reduced colocalization with RAB6A, a regulator of vesicle trafficking between the Golgi and the endoplasmic reticulum. The interaction between p.Glu744Gly altered BICD2 and RAB6A was impaired, which also led to their reduced colocalization. Our study identifies BICD2 mutations as a cause of non-5q linked SMA and highlights the importance of dynein-mediated motility in motor neuron function in humans.

Figure 1

Pedigrees, Clinical Features, and Electropherograms of Individuals with BICD2 Mutations (A and D) Pedigree structure and haplotype analysis of families 177 (A) and 584 (D). The probands are denoted by an arrow. Squares and circles symbolize males and females, respectively, and symbols with diagonal black lines represent deceased individuals. Filled symbols denote family members affected by SMA, and open symbols indicate unaffected individuals. Individuals with an asterisk were selected for exome sequencing. The disease-segregating haplotype on chromosome 9q is indicated in black. Short-tandem-repeat (STR) markers are shown in order from p-ter (top) to q-ter (bottom). Markers of the genome-wide linkage panel are represented in bold. (B and C) The clinical features of person 177.III.1 show hypotrophy of proximal and distal muscles of the lower limbs (B) and scapular winging (C). (E) The electropherograms of heterozygous BICD2 mutations c.320C>T (p.Ser107Leu) and c.2321A>G (p.Glu774Gly).

Figure 2

Coimmunoprecipitation of BICD2 and Its Interactors Dynein and RAB6A (A) Schematic representation of the structure of BICD2. The Ser107 residue is positioned in the N-terminal region that interacts with dynein, whereas the Glu774 amino acid lies within the C-terminal domain that interacts with RAB6A. (B) Lysates of SH-SY5Y cells stably expressing BICD2-V5 wild-type and altered proteins were used for immunoprecipitation (IP) with antibodies against dynein intermediate chain 1 (DIC) and V5. The p.Ser107Leu altered BICD2-V5 coprecipitated more with DIC than did the wild-type protein in reciprocal experiments. TUBB was used for showing equal loading of the lysates. (C) Lysates of lymphoblasts from members of family 177 were immunoprecipitated with DIC antibody and show an increased BICD2 load in the samples of the three affected individuals compared to the healthy relative (177.III.2). GAPDH was used for showing equal loading of the lysates. (D) Immunoprecipitation (IP) of stable SH-SY5Y cell extracts with RAB6A antibody demonstrates that there is less interaction between RAB6A and p.Glu774Gly altered BICD2-V5 than between RAB6A and the wild-type. The amount of coprecipitated p.Ser107Leu BICD2-V5 was comparable to that of the wild-type. GAPDH was used for showing equal loading of the lysates.

Figure 3

Colocalization of BICD2 and RAB6A Is Reduced by the SMA-Causing BICD2 Alterations HeLa cells expressing BICD2-EGFP (green) were stained for RAB6A (red). p.Glu774Gly altered BICD2-EGFP is decreased in RAB6A-positive regions. Also, the p.Ser107Leu altered protein shows reduced colocalization with RAB6A, but not to the same extent as p.Glu774Gly. In addition, the p.Ser107Leu altered BICD2 shows an intense punctual staining in the perinuclear region (arrow). Graphs and error bars represent the mean and SEM. The Pearson’s correlation coefficient (calculated with the ImageJ Coloc2 plugin) is determined in 62, 62, and 61 cells expressing wild-type, p.Ser107Leu, and p.Glu774Gly proteins, respectively, from three independent transfection and immunostaining experiments. ^∗p < 0.05, ^∗∗∗p < 0.001. The scale bar represents 5 μm.

Figure 4

The p.Ser107Leu Substitution in the Dynein Binding Domain Induces BICD2 Accumulation at the MTOC Compared to wild-type and p.Glu774Gly altered BICD2-EGFP, p.Ser107Leu altered BICD2-EGFP (green) shows increased localization at the MTOC (visualized with γ-tubulin, red). The graph and error bars represent the mean and SEM of mean EGFP intensity at the MTOC (white bars) and in the rest of the cell (black bars) measured in 34, 37, and 17 HeLa cells expressing wild-type, p.Ser107Leu, and p.Glu774Gly proteins, respectively. ^∗p < 0.05. The scale bar represents 10 μm. The following abbreviations is used: NS, not significant.

Figure 5

p.Ser107Leu Altered BICD2 Leads to Golgi Fragmentation SH-SY5Y cells stably expressing BICD2-EGFP (green) were immunostained with the Golgi marker Giantin (red). Compared to wild-type and p.Glu774Gly mutant cells, cells with p.Ser107Leu altered BICD2 show a dispersed Golgi apparatus. In addition, in p.Ser107Leu mutant cells, a perinuclear accumulation of BICD2 is apparent (asterisk). Golgi dispersal is represented as a ratio between the minimal rectangle area around the Giantin-positive structures (segmented in ImageJ by automatic intensity thresholding) and the total area of the cell. The graph and error bars represent the mean and SEM of Golgi dispersal measured in 131, 103, and 104 SH-SY5Y cells expressing wild-type, p.Ser107Leu, and p.Glu774Gly proteins, respectively. ^∗∗∗p < 0.001. The scale bar represents 5 μm. The following abbreviation is used: NS, not significant.