Novel KLHL26 variant associated with a familial case of Ebstein’s anomaly and left ventricular noncompaction

Samudrala SSK, North LM, Stamm KD, Earing MG, Frommelt MA, Willes R, Tripathi S, Dsouza NR, Zimmermann MT, Mahnke DK, Liang HL, Lund M, Lin CW, Geddes GC, Mitchell ME, Tomita-Mitchell A.

Mol Genet Genomic Med. 2020 Apr;8(4):e1152. doi: 10.1002/mgg3.1152. Epub 2020 Jan 27.

PMID: 31985165 Free PMC Article

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Background: Ebstein’s anomaly (EA) is a rare congenital heart disease of the tricuspid valve and right ventricle. Patients with EA often manifest with left ventricular noncompaction (LVNC), a cardiomyopathy. Despite implication of cardiac sarcomere genes in some cases, very little is understood regarding the genetic etiology of EA/LVNC. Our study describes a multigenerational family with at least 10 of 17 members affected by EA/LVNC.

Methods: We performed echocardiography on all family members and conducted exome sequencing of six individuals. After identifying candidate variants using two different bioinformatic strategies, we confirmed segregation with phenotype using Sanger sequencing. We investigated structural implications of candidate variants using protein prediction models.

Results: Exome sequencing analysis of four affected and two unaffected members identified a novel, rare, and damaging coding variant in the Kelch-like family member 26 (KLHL26) gene located on chromosome 19 at position 237 of the protein (GRCh37). This variant region was confirmed by Sanger sequencing in the remaining family members. KLHL26 (c.709C > T p.R237C) segregates only with EA/LVNC-affected individuals (FBAT p < .05). Investigating structural implications of the candidate variant using protein prediction models suggested that the KLHL26 variant disrupts electrostatic interactions when binding to part of the ubiquitin proteasome, specifically Cullin3 (CUL3), a component of E3 ubiquitin ligase.

Conclusion: In this familial case of EA/LVNC, we have identified a candidate gene variant, KLHL26 (p.R237C), which may have an important role in ubiquitin-mediated protein degradation during cardiac development.


Figure 1 Pedigree of family with members affected by EA/LVNC. EA/LVNC‐affected members are denoted black. VII:5 was a stillborn male, VIII:2 was a premature birth, and VIII:7 was an IUFD; all were affected with cardiac malformations. VIII:8 was an IUFD at 8 weeks, also believed to be of cardiac origin. VI:9 does not fit the complete criteria for EA but is within the spectrum of the phenotype. The proband (VIII:5) is denoted by a dotted circle. Exome sequencing was performed on all encircled family members (VI:9, VII:7, VII:8, VIII:1, VIII:5, and VIII:7). EA/LVNC, Ebstein’s anomaly with left ventricular noncompaction; IUFD, intrauterine fetal death


Figure 2 Structural Model of KLHL26‐CUL3 complex. (a) Our structural model is colored according to domain, depicting the predicted interaction with CUL3 mediated by the BACK and BTB domains. Residues outside of established domains by sequence comparison are colored white. Residues in the BTB domain that flank a site of additional sequence due to alternative splicing are circled. (b) Magnification of the BACK domain defines the location of R237 and its proximity to the CUL3 binding surface. (c). The electrostatic surface of KLHL26‐CUL3 interaction, wherein R237 is circled. (d) The analogous image of 2C for C237, wherein the electrostatic surface is more neutral and partially negative. (e) A 180° rotated view of the electrostatic surface of KLHL26‐CUL3 interaction; the gold circle marks the location of R237. (f) The positive patch on CUL3 is seen across from R237 (parts of KLHL26 that occlude the CUL3 surface are hidden). This patch is created by an HKH sequence whose sidechains fan out radially, contributing to a positive crescent‐shaped region, leaving the backbone oxygen atoms to make a negative surface patch


Figure 3 Proposed mechanism of KLHL26 interaction with CUL3. CUL3 is a core subunit of E3 ubiquitin ligase. Its N‐terminal domain binds to a receptor protein that confers substrate specificity – in this case KLHL26. The C‐ terminal domain binds a RING protein that recruits E2. NEDD8 brings substrate and E2 to the correct conformation for ubiquitination. In KLHL26, most likely the BTB domain interacts with CUL3 and the Kelch domain acts as the substrate‐recognition module