MACF1 Mutations Encoding Highly Conserved Zinc-Binding Residues of the GAR Domain Cause Defects in Neuronal Migration and Axon Guidance

Abstract

To date, mutations in 15 actin-or microtubule-associated genes have been associated with the cortical malformation lissencephaly and variable brainstem hypoplasia. During a multicenter review, we recognized a rare lissencephaly variant with a complex brainstem malformation in three unrelated children. We searched our large brain-malformation databases and found another five children with this malformation (as well as one with a less severe variant), analyzed available whole-exome or -genome sequencing data, and tested ciliogenesis in two affected individuals. The brain malformation comprised posterior predominant lissencephaly and midline crossing defects consisting of absent anterior commissure and a striking W-shaped brainstem malformation caused by small or absent pontine crossing fibers. We discovered heterozygous de novo missense variants or an in-frame deletion involving highly conserved zinc-binding residues within the GAR domain of MACF1 in the first eight subjects. We studied cilium formation and found a higher proportion of mutant cells with short cilia than of control cells with short cilia. A ninth child had similar lissencephaly but only subtle brainstem dysplasia associated with a heterozygous de novo missense variant in the spectrin repeat domain of MACF1. Thus, we report variants of the microtubule-binding GAR domain of MACF1 as the cause of a distinctive and most likely pathognomonic brain malformation. A gain-of-function or dominant-negative mechanism appears likely given that many heterozygous mutations leading to protein truncation are included in the ExAC Browser. However, three de novo variants in MACF1 have been observed in large schizophrenia cohorts.Microtubules (MTs) and filamentous actin form key structural components of the cytoskeleton, a dynamic intracellular structure that is essential for several basic cell functions, including migration, the formation of cellular processes (including axons and dendrites), axonal guidance, and vesicular trafficking. Mammalian genomes contain two spectraplakins-MACF1 (also known as ACF7) and DST (also known as BPAG1)-that function as actin-MT cross-linkers and essential integrators and modulators of cytoskeletal processes. [1][2][3] MACF1 is a large gene that expresses many isoforms, several of which are brain specific, through alternative splicing. The N terminus of the major isoforms contains two calponin-homology (CH) domains that bind actin, a plakin domain, and a long spectrin-repeat rod domain that confers flexibility. The C terminus of all isoforms functions as a MT binding domain and contains two calcium-binding EF-hand domains, a zinc-binding GAR (growth-arrest specific 2 or Gas2-related) domain, a positively charged Gly-Ser-Arg (GSR) region, and an EB1-binding SxIP domain. 1-3

Much More Than a Scaffold: Cytoskeletal Proteins in Neurological Disorders

“…Different LIS-1 mutations give rise to a spectrum of phenotypes with graded severity [287], being the duplications that included LIS-1 and adjacent genes the strongest. MACF1 gene mutations also cause a rare lissencephaly with complex brainstem malformation [245]. The severity of the phenotype depends on the spectrum of mutations: patients with mutations located in the spectrin repeat domain present subtle brainstem dysplasia as compared to patients with variants located in the GAR domain or nearby, which affect the microtubule binding site.…”

In trans variant calling reveals enrichment for compound heterozygous variants in genes involved in neuronal development and growth.

“…Recently, highly penetrant de novo MACF1 mutations were identified in several patients with a newly characterized lissencephaly with a complex brain malformation (Dobyns et al, 2018). This new phenotype highlights MACF1 mutations' variable impact on disease pathogenesis.…”

Centrosome and ciliary abnormalities in fetal akinesia deformation sequence human fibroblasts

“…MACF1 is a MT plus end-binding protein shown to promote persistent MT growth and to connect the actin and microtubule cytoskeletal network 77 . Moreover, MACF1 has been shown to be critical for cilia length and ciliogenesis in several cell types and in fibroblasts from patients with pathogenic variants in MACF1 78,79 . This novel interaction of rapsyn with MACF1 can be the link to its role in ciliogenesis.…”