Heterozygous variants in MYH10 associated with neurodevelopmental disorders and congenital anomalies with evidence for primary cilia-dependent defects in Hedgehog signaling

Abstract

Purpose: Nonmuscle myosin II complexes are master regulators of actin dynamics that play essential roles during embryogenesis with vertebrates possessing 3 nonmuscle myosin II heavy chain genes, MYH9, MYH10, and MYH14. As opposed to MYH9 and MYH14, no recognizable disorder has been associated with MYH10. We sought to define the clinical characteristics and molecular mechanism of a novel autosomal dominant disorder related to MYH10.

Methods: An international collaboration identified the patient cohort. CAS9-mediated knockout cell models were used to explore the mechanism of disease pathogenesis.

Results: We identified a cohort of 16 individuals with heterozygous MYH10 variants presenting with a broad spectrum of neurodevelopmental disorders and variable congenital anomalies that affect most organ systems and were recapitulated in animal models of altered MYH10 activity. Variants were typically de novo missense changes with clustering observed in the motor domain. MYH10 knockout cells showed defects in primary ciliogenesis and reduced ciliary length with impaired Hedgehog signaling. MYH10 variant overexpression produced a dominant-negative effect on ciliary length.

Conclusion: These data presented a novel genetic cause of isolated and syndromic neurodevelopmental disorders related to heterozygous variants in the MYH10 gene with implications for disrupted primary cilia length control and altered Hedgehog signaling in disease pathogenesis.

Keywords: Hedgehog signaling; MYH10; Neurodevelopmental disorder; Nonmuscle myosin; Primary cilia.

Figure 1.. Examples of dysmorphology observed in individuals with MYH10 variants.

(A-E) Images obtained from individual 2 at age 19 years. Note the highly dysplastic thorax and scoliosis in (A,B), significant hypertelorism (C), preauricular pit (D), and 5^th finger clinodactyly (E). (F-I) Images obtained from individual 14 at age 2 years demonstrating hypertelorism (G), broad nasal root (G), upturned nares (G), depressed nasal bridge (H), low set ears (H), broad toes (I), and 5^th toe clinodactyly (I).

Figure 2.. Mapping of MYH10 variants, structural modeling, and pathogenic predictions support deleterious effects of the identified missense variants.

(A) Schematic of MYH10 protein structure demarcating the SH3, motor, and myosin tail domains. Variants are mapped to protein structure. All variants reported using transcript NM_001256012.3. (B) Alignment of vertebrate MYH10 protein sequences across species showing high conservation within the ‘hot spot’ region. Affected residues are delineated in red. Note the overlap between the p.(R740Q) variant identified in the cohort and the p.(R740C) missense variant described in the mouse knock-in model. (C) Alignment of human NMHCII proteins including MYH10, MYH9, and MYH14. Residues affected by missense variants are denoted in red. Note that the p.(W37C) and p.(R740Q) variants affect analogous residues that when affected are defined as pathogenic in the MYH9- and MYH14-related disorders. (D) In the middle is a model of MYH10 (numbering based on NM_001256012.3 / P35580–4) amino acids 1–1363 with a 180-degree rotation of the y-axis. The amino acid color is based on paralog conservation with 13 other human proteins, such that red is functionally conserved 100%, orange conserved functionally, and yellow some signs of conservation. On the outside are the zoom in view of each variant, often labeled in blue. For each variant is shown below the image the predictions of damage from various tools and to the right the conservation in other human paralog members in addition to the 181 vertebrate MYH10 sequences.

Figure 3.. MYH10 knockout cells exhibit defects in primary ciliogenesis and ciliary length with evidence for a dominant-negative effect of patient-specific variants.

(A-L) Immunofluorescent analysis of primary cilia labeled with acetylated tubulin (green, A-D, I-L) and ARL13B (red, E-L) in parental NIH/3T3 (A, E, I), WT-1 (B, F, J), KO-1 (C, G, K), and KO-2 (D, H, L) lines. Nuclei labeled with DAPI (blue, E-L). Scale bar in A (5 um). (M) Analysis of primary ciliogenesis in control and knockout NIH/3T3 cell lines. The percent of cells with primary cilia are expressed between lines. (N) Violin plots of cilia length measurements between control and knockout NIH/3T3 cell lines. (U) Analysis of the percentage of cells with primary cilia in control and knockout RPE-1 cell lines. (O-Z) Immunofluorescent analysis of primary cilia in NIH/3T3 cells with stable overexpression of empty vector (pcDNA3; O, S, W), HA-tagged wildtype MYH10 (WT; P, T, X), HA-tagged p.(R740Q) (Q, U, Y), and HA-tagged p.(E1740G) (R, V, Z) and stained with antibodies against HA (green; O-R, W-Z) and ARL13B (red; S-Z). Nuclei are labeled with DAPI (blue; S, Z). Scale bar in O (5 um). (AA) Quantitation of the percent of ciliated cells between stable cell lines. (BB) Violin plots of primary cilia length measurements between stable NIH/3T3 cell lines. For ciliogenesis quantification, data was collected from 3 random fields of view (>50 cells/field of view) from maximum intensity projections of Z-stack images. Each experiment independently repeated at least N=3. For ciliary length quantitation, 100 cilia were measured from maximal intensity projected Z-stacks for each condition and each experiment was independently repeated at least N=3.