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. 2023 Jul 21;13(1):11776.
doi: 10.1038/s41598-023-38909-w.

Phenotypic and molecular basis of SIX1 variants linked to non-syndromic deafness and atypical branchio-otic syndrome in South Korea

Somin Lee #  1 , Yejin Yun #  1 , Ju Hyuen Cha  1 , Jin Hee Han  2 , Dae Hee Lee  3 , Jae-Jin Song  2 , Moo Kyun Park  1 , Jun Ho Lee  1 , Seung Ha Oh  1 , Byung Yoon Choi  2 , Sang-Yeon Lee  4   5   6
Affiliations

Phenotypic and molecular basis of SIX1 variants linked to non-syndromic deafness and atypical branchio-otic syndrome in South Korea

Somin Lee et al. Sci Rep. .

Abstract

Branchio-oto-renal (BOR)/branchio-otic (BO) syndrome is a rare disorder and exhibits clinically heterogenous phenotypes, marked by abnormalities in the ear, branchial arch, and renal system. Sporadic cases of atypical BOR/BO syndrome have been recently reported; however, evidence on genotype-phenotype correlations and molecular mechanisms of those cases is lacking. We herein identified five SIX1 heterozygous variants (c.307dupC:p.Leu103Profs*51, c.373G>A:p.Glu125Lys, c.386_391del:p.Tyr129_Cys130del, c.397_399del:p.Glu133del, and c.501G>C:p.Gln167His), including three novel variants, through whole-exome sequencing in five unrelated Korean families. All eight affected individuals with SIX1 variants displayed non-syndromic hearing loss (DFNA23) or atypical BO syndrome. The prevalence of major and minor criteria for BOR/BO syndrome was significantly reduced among individuals with SIX1 variants, compared to 15 BOR/BO syndrome families with EYA1 variants. All SIX1 variants interacted with the EYA1 wild-type; their complexes were localized in the nucleus except for the p.Leu103Profs*51 variant. All mutants also showed obvious but varying degrees of reduction in DNA binding affinity, leading to a significant decrease in transcriptional activity. This study presents the first report of SIX1 variants in South Korea, expanding the genotypic and phenotypic spectrum of SIX1 variants, characterized by DFNA23 or atypical BO syndrome, and refines the diverse molecular aspects of SIX1 variants according to the EYA1-SIX1-DNA complex theory.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Clinical phenotypes of the five unrelated Korean families segregated with SIX1 variants and functional characterization of all the SIX1 variants. (a) The pedigrees of the five unrelated Korean families with SIX1 variants. The clinical phenotypes were summarized based on major and minor diagnostic criteria for BOR/BO syndrome. (b) Protein domain and conservation maps. The residues of the five SIX1 variants were in the SIX1 SD or HD domains. One truncating variant (p.Leu103Profs*51) was in the SIX1 SD domain. All the variants’ residues were well-conserved among the SIX1 orthologs in various species. (c) SIX1 variants reported thus far in the literature. The upper color (purple) represents the SIX1 variants that have been studied functionally, while the lower color (blue) denotes SIX1 variants that have not yet been characterized functionally. This study presents five variants. Among them, three variants (p.Leu103Profs*51, p.Tyr129_Cys130del, and p.Gln167His) are novel, and two variants (p.Glu125Lys and p.Glu133del) have previously been reported. The Glu133del variant has undergone functional studies.
Figure 2
Figure 2
Comparison of clinical profiles between the SIX1 and EYA1 variants using an in-house database (a–d) A comparative analysis of the prevalence of major diagnostic criteria for BOR syndrome among individuals with SIX1 and EYA1 variants. (e, f) A comparison evaluating the prevalence of minor diagnostic criteria for BOR syndrome between individuals with SIX1 and EYA1 variants. (h) Phenotypic features associated with SIX1 variants in relation to DFNA23 or atypical BO syndrome.
Figure 3
Figure 3
Three-dimensional modeling and structural analysis. All four SIX1 mutants in the homeodomain produce structural changes that sterically inhibit DNA binding, possibly compromising structure stability. (a) SIX1 is important for the overall DNA interaction processes of the HD domain. (b) Modeling by superimposing the SIX1-DNA complex over the AbdB/Exd-DNA complex revealed close alignment with the SIX1 HD domain. (c) The hydrogen bonds between the DNA phospho-backbone and p.Gln167 stabilize the DNA duplex. (d) The bulky side chain of the p.Gln167His variant expels itself from the DNA binding cleft, leading to a loss of hydrogen bonds.
Figure 4
Figure 4
Western blot analysis for the SIX1 wild-type, frameshift, and missense variants by transient transfection of HEK293 cells. (a) Expression of SIX1 wild-type and mutants was detected by western blotting of HEK293 cells. The SIX1 wild-type and missense variants have a molecular weight of 42 kDa. The molecular weight of the frameshift variant is 21 kDa. (b) Expression of SIX1 wild-type and mutants co-transfected with EYA1 wild-type was detected by western blotting in HEK293 cells. The SIX1 wild-type and missense variants have a molecular weight of 42 kDa, and that of the frameshift variant is 21 kDa. The original immunoblots (uncropped, full length membranes with membrane edges visible, and standard protein size markers and expected molecular weight labeled) were all provided in Fig. S5.
Figure 5
Figure 5
Subcellular localization of SIX1 wild-type and mutants. Immunofluorescence of HEK293 cells co-transfected with C-terminally Myc-DDK-tagged SIX1 wild-type, p.Leu103Pfs*51, p.Glu125Lys, p.Tyr129_Cys130del, p.Gln167His, p.Glu133del, and C-terminally 6xHis-tagged EYA1 wild-type. Cells were immunostained with anti-Myc and anti-His (green) antibodies. In co-transfections with EYA1 wild-type, all SIX1 mutants and wild-type were localized in the nucleus, except for p.Leu103Profs*51.
Figure 6
Figure 6
Protein–protein interactions between EYA1 and SIX1 variants. HEK293 cells were transfected with His-EYA1 and Myc-DDK-SIX1 plasmids for 24 h. Whole-cell lysates were collected and subjected to protein–protein interaction assays with TALON resin to His-tag protein pull-down. The original immunoblots (uncropped, full length membranes with membrane edges visible, and standard protein size markers and expected molecular weight labeled) were all provided in Fig. S6.
Figure 7
Figure 7
DNA binding assay for SIX1 wild-type variants to measure the binding affinity of SIX1 protein to DNA. (a) HEK293 cells were transfected with the SIX1 variant plasmids, pCMV6-SIX1 wild-type-Myc-DDK, pCMV6-SIX1 p.Leu103Profs*51-Myc-DDK, pCMV6-SIX1 p.Glu125Lys-Myc-DDK, pCMV6-SIX1 p.Tyr129_Cys130del Myc-DDK, pCMV6-SIX1 p.Glu133del-Myc-DDK, and pCMV6-SIX1 p.Gln167His-Myc-DDK, and nuclear extracts were obtained. The original immunoblots (uncropped, full length membranes with membrane edges visible, and standard protein size markers and expected molecular weight labeled) were all provided in Fig. S7. (b) Anti-Myc antibody (2276S, CST) was incubated with nuclear extracts to quantify the binding affinity of SIX1 protein to DNA colorimetrically. Nuclear extracts of non-transfected cells were used as a control. The experiment was conducted in triplicate, and data were analyzed by one-way ANOVA.
Figure 8
Figure 8
Transcriptional activity based on the luciferase reporter assay. The transcriptional efficiency of SIX1 wild-type in transfected HEK293T cells was highest using the luciferase vector system (pGL4.12[luc2CP], 2 µg). All SIX1 mutants significantly reduce the transcriptional activity required to regulate downstream target gene expression, even the EYA1 co-transfected mutants. The significance is higher in the MYOG Luc-vector than in the Single Luc-vector. SIX1 wild-type enhanced luciferase activity by approximately four-fold. In contrast, SIX1 mutants enhanced luciferase activity by less than two-fold, demonstrating a significantly poorer ability to induce transcription than the SIX1 wild-type.
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