Loss of function of FAM177A1, a Golgi complex localized protein, causes a novel neurodevelopmental disorder

Abstract

Purpose: The function of FAM177A1 and its relationship to human disease is largely unknown. Recent studies have demonstrated FAM177A1 to be a critical immune-associated gene. One previous case study has linked FAM177A1 to a neurodevelopmental disorder in 4 siblings.

Methods: We identified 5 individuals from 3 unrelated families with biallelic variants in FAM177A1. The physiological function of FAM177A1 was studied in a zebrafish model organism and human cell lines with loss-of-function variants similar to the affected cohort.

Results: These individuals share a characteristic phenotype defined by macrocephaly, global developmental delay, intellectual disability, seizures, behavioral abnormalities, hypotonia, and gait disturbance. We show that FAM177A1 localizes to the Golgi complex in mammalian and zebrafish cells. Intersection of the RNA sequencing and metabolomic data sets from FAM177A1-deficient human fibroblasts and whole zebrafish larvae demonstrated dysregulation of pathways associated with apoptosis, inflammation, and negative regulation of cell proliferation.

Conclusion: Our data shed light on the emerging function of FAM177A1 and defines FAM177A1-related neurodevelopmental disorder as a new clinical entity.

Keywords: Developmental Delay; FAM177A1; Macrocephaly; Neurodevelopment; Zebrafish.

Figure 1.. Genetic findings of individuals harboring homozygous variants in FAM177A1.

(A) Pedigrees representing affected individuals (shaded black) in the three families reported in this cohort, as well as variants in FAM177A1 and predicted protein changes in individuals tested. (B) Gene schematic of the location of variants and deletions in the mRNA and predicted protein changes.

Figure 2.. Deletions in FAM177A1 gene result in altered mRNA expression and loss of protein expression in individuals A1 and A2.

(A) RNA sequencing reads from Family A showing near complete absence of reads covering exons 4, 5, and the 3’UTR (blue arrows and bars). (B) Sashimi plot of RNA-sequencing data from Family A showing splicing from exon 3 into the downstream gene, PPP2R3C (blue arrows). (C) Exon-level quantifications showing the number of RNA-sequencing reads covering each exon of FAM177A1 in Individuals A1 (blue) and A2 (green); background distributions represent exon-level quantifications across an internal control cohort (D) Western blot analysis of FAM177A1 and GAPDH levels in iPSCs derived from fibroblasts of healthy donor, Individual A1, and Individual A2.

Figure 3.. Subcellular localization of zebrafish fam177a1a-mNeonGreen and human FAM177A1-mNeonGreen in zebrafish embryos and mammalian COS7 cell line.

Developing Notochord and enveloping layer (EVL) in the embryos. (A-F) Embryos co-injected with fam177a1a-mNeonGreen and GM130-tdTomato RNAs at one-cell stage and imaged at 12 hpf. (G-L) Embryos co-injected with FAM177A1-mNeonGreen and GM130-tdTomato RNAs at embryo one-cell stage and imaged at 12 hpf. (A, D) Zebrafish fam177a1a-mNeonGreen. (B, E, H, K) GM130-tdTomato. (C, F, I, L) Merged images. (G, J) Human FAM177A1-mNeongGreen. Scale bars, 20 mm. (M, N, O) Human FAM177A1 is predominantly in the Golgi complex in mammalian cells. COS7 cell lines expressing FAM177A1-GFP. Note the co-localization with a marker of the trans-Golgi complex, GalT (Galactosyltransferase, shown in magenta). Image is z-stacked image showing the max projection of all signals. Dotted white lines indicate cell margins, and nucleus (n) respectively. Scale bar = 10 mm.

Figure 4.. Generation of zebrafish fam177a1a, fam177a1b and fam177a1a/b geneless mutants.

(A) Genetic locus of fam177a1a and the sequences of geneless alleles. (B) Genetic locus of fam177a1b and the sequences of the geneless alleles. (C) Gene expression of fam177a1a and fam177a1a/b compared to WT as measured by quantitative RNA sequencing in the fam177a1a and fam177a1b genetic background (KO- knockout −/−, CPM- copies per million).

Figure 5.. Morphometric analysis of zebrafish fam177a1a, fam177a1b and fam177a1a/b geneless mutants.

(A) Measurement of body length from swim bladder to the end of the tail in WT, fam177a1a, fam177a1b and fam177a1a/b mutants at 25 hpf. (B-E) Lateral views at 25 hpf. (B) WT. (C) fam177a1a single mutant. (D) fam177a1b single mutant. (E) fam177a1a/b double mutant. Scale bar, 200 mm.

Figure 6.. Intracellular distribution of the Golgi complex in developing notochord during late gastrulation.

Dorsal view, anterior to the left. Brackets indicate notochord area. (A-F) Embryos injected with H2B-GFP, memGFP and GM130-tdTomato RNAs at one-cell stage and imaged at 11 hpf. (G-L) Embryos injected with H2B-GFP and memGFP RNAs at one-cell stage, fixed at 11 hpf, and stained with ɑ-GM130 antibody. (A-C, G-I) WT embryos. (D-F, J-L) fam177a1a/b double mutant embryos. (A, D, G, J) Expression of H2B-GFP in the cell nuclei and memGFP in cell membranes. (B, E) Expression of GM130-tdTomato in the Golgi vesicles. (H, K) Detection of the endogenous GM130 protein by immunohistochemistry (C, F, I, L) Merged images. Scale bar, 20 ums.

Figure 7.. Bulk RNA-sequencing analysis of WT, fam177a1a and fam177a1a/b replicates in zebrafish 8dpf.

(A) Heatmap of correlation of three independent RNA samples. The color represents the strength and direction of correlation. Three replicates of each genotype. (B) Principal component analysis (PCA) using RNA-sequencing data. Percent variability within each principal component is listed on each axis.

Figure 8.. RNA-seq analysis FAM177A-deficient and WT fibroblasts.

(A) Heatmap of correlation within the human RNA samples from the affected participants’ fibroblasts and control fibroblasts. (B) Principal component analysis using human RNA-seq data.

Figure 9.. Gene Ontology (GO) enrichment analysis of differentially expressed genes among human and zebrafish RNA-sequencing.

GO terms pertaining to biological processes (BP) and human phenotype ontology (HP) are listed on the y-axis. The color of the bubbles indicates significance of the enrichment (log10(Pvalue). The hit ratio (hit_ratio) refers to the size of the bubble and indicates the relative number of genes associated with the term. (A) Up-regulated genes. (B) Down-regulated genes.