PMC8667665_01
Clinical findings of family 1. (A) Family tree. (B-F) Proband and onychodystrophy. In fingers (C,D), absence of first- and fifth-digits fingernails, index fingers aplasia, finger-like thumbs, and shorter fifth fingers. In toes (E,F), absent toenails on the first and second digits, and hypoplasia toenails on the third to fifth digits of right foot; Left foot, absent toenails on the digits 1-3, rudimentary digits 4-5. Hand X-ray: distal phalanges absence of the index finger; hypoplasia of the distal phalanx of feet's fifth digits. (G-K) Phenotypes of onychodystrophy of the mother are similar to the proband.
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F1
In family 1, the 6-year-old boy (Figures 1A,B) was first referred to the clinic when he was 20-month-old due to congenital hearing-loss. X-ray analysis revealed bilateral absence of the distal phalanges of the index finger (Figures 1C,D). His feet examination and roentgenograms showed bilateral hypoplasia of the distal phalanx of the fifth digits (Figures 1E,F). Audiological evaluations by behavioral audiometry, auditory brainstem response (ABR), multifrequency steady-state evoked potential (ASSR), and distorted product otoacoustic emission (DPOAE) revealed bilateral profound sensorineural hearing loss (SNHL) (Supplementary Figure S1). The 20-year-old mother (Figure 1G) with normal cognitive function also showed similar phenotypes, such as bilateral profound congenital sensorineural hearing loss and onychodystrophy (Figures 1H,I). Her toenail development was severely affected, showing no toenails at all (Figures 1J,K).
PMC8667665_02
Clinical findings of family 2. (A) Family tree. (B-F) Proband and onychodystrophy. Absent fingernails on the first- and fifth-digits hypoplastic fingernails. Toenails are totally absent. Hand and foot X-rays show normal phalanx development. (G-K) Phenotypes of onychodystrophy of the father are similar to the proband.
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F2
I). In addition, his foot X-ray showed hypoplasia of the distal phalanx in his second right toe (Figures 2J,K). WES analysis of the affected spouse in family 2 (individual II-2, Figure 2A) identified a missense variant (c.733A>G, p.M245V in the KIF11 gene) (GenBank acc. no., NM_004523).
PMC8667665_03
Clinical findings of family 2. (A) Family tree. (B-F) Proband and onychodystrophy. Absent fingernails on the first- and fifth-digits hypoplastic fingernails. Toenails are totally absent. Hand and foot X-rays show normal phalanx development. (G-K) Phenotypes of onychodystrophy of the father are similar to the proband.
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F2
I). In addition, his foot X-ray showed hypoplasia of the distal phalanx in his second right toe (Figures 2J,K). WES analysis of the affected spouse in family 2 (individual II-2, Figure 2A) identified a missense variant (c.733A>G, p.M245V in the KIF11 gene) (GenBank acc. no., NM_004523).
Mutation analysis of TJP2. (A) Sanger sequencing chromatograms show heterozygous variants in the affected son and mother. (B) All TJP2 mutations curated in HGMD are mapped to the encoded TJP2 protein; Blue arrows: Variants detected in cases with hearing-loss; Red arrow: p.D530E in the PDZ3 motif detected in the study. Another variant (p.A112T) that caused hearing loss is mapped to PDZ1 motif. (C) Schematic of the TJP2 three-dimensional protein structure, the p.D530E position is indicated by an arrow. How the variants in PDZ motifs affect the protein structure are currently unknown. (D) The p.D530 residue is evolutionarily conserved from zebrafish to human. (E) Agarose gel electrophoresis shows PCR products of TJP2 mRNA between c.1307 and 1712. M, marker; C, normal control sample; P, patient's sample. (F) Sanger sequencing chromatograms show the TJP2 cDNA sequencing from normal control (i) and patient sample (ii).
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F3
To confirm the predicted splicing site, RT-PCR analysis was conducted to examine the c.1590T>G-containing region using the proband's peripheral blood lymphocyte cells and normal control sample. The 406-bp RT-PCR product as a normal control was clearly detected by electrophoresis and further confirmed by Sanger sequencing (Figures 3E,F, arrowhead in lane C), while PCR product in patient was shown as multiple bands (lane P) and no single peak in Sanger sequencing, suggesting a mixed PCR product due to a splicing event. Quantification analysis using ImageJ showed at least 2-fold higher expression of 406-bp RT-PCR product (arrowhead) in control group (lane C) than that in patient (lane P), suggesting a potential effect of mutation on the accurate splicing of pre-mRNA into mRNA (Figure 3E).
Mutation analysis of KIF11. (A) Three hearing-loss associated variants are located in the KISc domain (SmartMotif). (B) Based on the human KIF11 kinesin domain 3D-structure (PDB, 3K5E), all the three hearing-loss associated variants are located in the same beta-sheet in the Kinesin motor domain.
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F4
Unexpectedly, we found that these two hearing-loss-associated variants and the current variant in KIF11 (Figure 4A) are all mapped to a small region within a single beta-sheet (amino acids 235-245, Figure 4B) in the catalytic domain of Kinesin motor (Smart Motif: mart.embl-heidelberg.de; PDB database: 3K5E), suggesting a specific portion of the protein implicated in hearing function.