Markus F et al. (2021), A novel missense variant in the EML1 gene assoc...

ECB-ART-49090

J Hum Genet 2021 Dec 01;6612:1159-1167. doi: 10.1038/s10038-021-00947-5.

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A novel missense variant in the EML1 gene associated with bilateral ribbon-like subcortical heterotopia leads to ciliary defects.

Markus F , Kannengießer A , Näder P , Atigbire P , Scholten A , Vössing C , Bültmann E , Korenke GC , Owczarek-Lipska M , Neidhardt J .

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Heterotopia is a brain malformation caused by a failed migration of cortical neurons during development. Clinical symptoms of heterotopia vary in severity of intellectual disability and may be associated with epileptic disorders. Abnormal neuronal migration is known to be associated with mutations in the doublecortin gene (DCX), the platelet-activating factor acetylhydrolase gene (PAFAH1B1), or tubulin alpha-1A gene (TUBA1A). Recently, a new gene encoding echinoderm microtubule-associated protein-like 1 (EML1) was reported to cause a particular form of subcortical heterotopia, the ribbon-like subcortical heterotopia (RSH). EML1 mutations are inherited in an autosomal recessive manner. Only six unrelated EML1-associated heterotopia-affected families were reported so far. The EML1 protein is a member of the microtubule-associated proteins family, playing an important role in microtubule assembly and stabilization as well as in mitotic spindle formation in interphase. Herein, we present a novel homozygous missense variant in EML1 (NM_004434.2: c.692G>A, NP_004425.2: p.Gly231Asp) identified in a male RSH-affected patient. Our clinical and molecular findings confirm the genotype-phenotype associations of EML1 mutations and RSH. Analyses of patient-derived fibroblasts showed the significantly reduced length of primary cilia. In addition, our results presented, that the mutated EML1 protein did not change binding capacities with tubulin. The data described herein will expand the mutation spectrum of the EML1 gene and provide further insight into molecular and cellular bases of the pathogenic mechanisms underlying RSH.

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	Fig. 1. Clinical manifestations of RSH in the patient and familiar co-segregation analyses the novel missense variant in the EML1 gene.A The pedigree of the family showing the RSH-affected family member (P, II.6). The parents are third-grade cousins. Co-segregation analyses of the novel missense variant (NM_001008707.1: c.746G>A) in the EML1 gene revelated that the healthy parents (I.1 and I.2) and the brother (UB, II.2) were carriers, while the RSH-affected patient (P, II.6) showed both mutated alleles. B Sagittal and axial T2 weighted images at age 3 weeks demonstrate complete callosal agenesis, bilateral enlargement of the occipital horns (colpocephaly), hypoplastic basal ganglia, and a complex cortical malformation. The polymicrogyric cortex is thin and irregular in all lobes. Bilaterally an undulating ribbon-like heterotopic band of gray matter is visible in subcortical location partially reaching the periventricular margin. C Multiple protein sequence alignment of EML1 proteins showed that the amino acid residue 231 is highly conserved across species (NP_004425.2, gray). Squares—males, circles—females, black solid square—patient affected with RSH, solid white icons—healthy individuals, double line—consanguinity, P—RSH-affected patient, UB—unaffected brother, #—family members available for further analyzes including Sanger sequencing, AA—amino acid, asterisk indicates highly conserved AA positions
	Fig. 2. Comparison of known and novel mutations in EML1.A Tolerance landscape analyses applying MetaDome showed tolerant (blue), neutral (yellow), and intolerant (red) regions for missense variants. B Schematic representation of domains of the EML1 protein located to newly identified mutation (M3: p.Gly231Asp) to the HELP motive (green) of EML1. Previously reported EML1 mutations are shown (NC1: p.Arg138, MC1: p.Thr243Ala, M2: p.Trp225Arg, N2: p.Arg523, M4: p.Val254Met, MC2: p.Gly439Asp/p.Gly478Val) [11, 27, 28]. C The EML1 protein contains two β-propeller structures building the TAPE domain (yellow) intimately associated with HELP motif (green). The presented EML1 structure includes the protein structure from amino acid 175 to 815 (NP_004425.2). Previously reported mutation sites located in the HELP motif are highlighted as ball-and-stick model in the 3D model and in the zoomed-in area. LC low complexity region, CC coiled coil region, HELP hydrophobic EMAP-like protein motif (green), WD WD40 repeats, N nonsense, M missense, C compound heterozygous mutations, 1–4—patient’s number
	Fig. 3. Immunocytochemical detection of EML1 in patient-derived fibroblasts and measurements of primary cilia length.A Immunocytochemical detection of EML1 in skin fibroblasts derived from the RSH-affected patient (P) and controls (C1; C2 similar results, data not shown) was performed with antibodies against EML1 (green, PA5-21294,), α-tubulin (magenta, T9026), and DAPI (blue) in four independent replicates. The scale bar corresponds to 20 µm. B Cilia axonemes and basal bodies were stained (in patient-derived and control fibroblasts) using acetylated D-tubulin (magenta) and y-tubulin (green), respectively. A scale bar corresponds to 3 µm. C Cilia lengths were measured in skin fibroblast from the RSH-affected patient (P) and control cell lines (C1, C2) by measuring the length of D-tubulin signals. At least 240 cilia were measured per cell line. We found no significant difference (p > 0.05) in the cilia length between the controls, while a highly significant reduction of cilia length (p ≤ 0.001) was observed in the patient when compared to the control cell lines. Symbols: α-Tub α tubulin, γ-Tub γ tubulin, D-Tub D-tubulin, n.s. not significant, Asterisk indicates highly significant
	Fig. 4. Western blot and co-immune precipitation analyses of the EML1 protein.A WB analyses of EML1 protein isolated from fibroblasts derived from the RSH-affected patient (P) and unrelated controls (C1, C2; independent replicates: 3). GAPDH served as a loading control. B Overexpression of wild-type EML1231Gly and mutated EML1231Asp in HEK-293T. A mock control (empty vector), a control for transfection reagent (PEI), and wild-type protein form HEK-293T cells were used as controls. Using whole-cell lysate EML1 protein was precipitated by EML1 antibody (PA5-21294) and WB analyses were performed with EML1 (sc-390841) and α-tubulin (T9026) antibodies. P—RSH-affected patient, C1-2—control 1-2, HEK WT—HEK wild type, Mock—empty vector, PEI—polyethylenimine (transfection reagent), EML1231Gly—overexpressed EML1 reference protein, EML1231Asp—overexpressed EML1 mutated protein, kDa—kilo Dalton

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