Disruption of PIKFYVE causes congenital cataract in human and zebrafish

  1. Shaoyi Mei
  2. Yi Wu
  3. Yan Wang
  4. Yubo Cui
  5. Miao Zhang
  6. Tong Zhang
  7. Xiaosheng Huang
  8. Sejie Yu
  9. Tao Yu  Is a corresponding author
  10. Jun Zhao  Is a corresponding author
  1. Shenzhen Eye Institute, Shenzhen Eye Hospital Affiliated to Jinan University, China
  2. Shenzhen Key Laboratory for Neuronal Structural Biology, Biomedical Research Institute, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center, China
  3. State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, China
  4. Department of Ophthalmology, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University; The first Affiliated Hospital, Southern University of Science and Technology), China
6 figures, 3 tables and 1 additional file

Figures

Pedigree structure and ocular manifestations of the cataract family.

(A) Pedigree of the family with congenital cataract. Squares denote males and circles denote females; Symbols crossed by a line indicate deceased individuals. Filled symbols indicate affected individuals, while open symbols indicate unaffected individuals. All affected family members had bilateral congenital cataract. The arrow denotes the proband. The individuals marked with an asterisk (∗) are analyzed by whole-exome sequencing. Genotypes of the PIKFYVE variant (p.G1943E) are indicated below each symbol (+, wild-type allele; −, p.G1943E variant allele). (B) Slit-lamp photographs showing the transparent lens of an unaffected individual (IV-6) and the nuclear pulverulent cataract in the left eye of the proband (III-9).

Figure 2 with 2 supplements
The PIKFYVE variant identified from the congenital cataract family.

(A) Sanger sequencing chromatogram showing the cDNA sequences from a healthy control and a cataract patient. The heterozygous c.5828G>A missense variant in the patient is indicated by the red arrow. (B) A schematic diagram showing the human PIKFYVE domains. The p.G1943E variant in the PIPK domain is indicated by the red arrow. (C) Protein sequence alignment of PIKFYVE orthologs in vertebrates. The black triangle denotes the conserved glycine at position 1943. (D) Western blot analysis of PIKFYVEWT and PIKFYVEG1943E expression in HEK293T cells that were transiently transfected with either pCS2(+)-CMV-PIKFYVEWT or pCS2(+)-CMV-PIKFYVEG1943E. The protein levels were normalized by GAPDH expression. Experiments were repeated three times. (E) Predicted structure model of the p.G1943E variant form of PIKFYVE PIPK domain generated by the PHYPRE2 server (http://www.sbg.bio.ic.ac.uk/~phyre2/html/). N-lobe, C-lobe, and the hinge linker are shown in gold, cyan, and red, respectively. The variant residue E1943 is shown in sticks and labeled with green. The negatively charged residue D1872 close to E1943 side chain is also shown in sticks. (F) A schematic demonstrating the organization of PIKFYVE PIPK domain. N-lobe, C-lobe, and the hinge linker are shown in gold, cyan, and red, respectively. The position of the p.G1943E variant is labeled with a yellow star. (G) Surface electrostatic potential comparison of the PIPK domain of PIKFYVE between wild-type (WT) and p.G1943E variant. The electrostatic potentials are presented as heatmaps from red to blue, and the electrostatic potential scales are shown in the lower panel. See Figure 2—source data 1 for details.

Figure 2—figure supplement 1
Expression of PIKFYVE in human lens capsule.

(A) RT-PCR results of lens capsules from three individuals. NC represents negative control without cDNA. (B) Quantitative RT-PCR shows the relative expression of PIKFYVE compared with GAPDH.

Figure 2—figure supplement 2
A schematic diagram showing the distribution of PIKFYVE variants.

(A) The genomic loci of identified variants of PIKFYVE from sporadic cataract patients. (B) The amino acid changes of identified variants and their positions in PIKFYVE protein.

Figure 2—figure supplement 2—source data 1

Raw data for the clinical manifestation of seven patients with PIKFYVE variants in Figure 2—figure supplement 2.

https://cdn.elifesciences.org/articles/71256/elife-71256-fig2-figsupp2-data1-v1.xlsx
Figure 3 with 3 supplements
Disruption of the PIPK domain of Pikfyve in zebrafish caused early-onset cataract.

(A) A schematic diagram showing the generated pikfyveΔ8 mutant allele. The underlined base pairs are the sgRNA target. The deleted base pairs are shown in dark blue while inserted ones are shown in red. The stop codon introduced in the mutant form is shown in the grey box. (B) Representative images showing the lens of sibling and pikfyveΔ8 mutants at 5 dpf. (C) Representative differential interference contrast (DIC) images showing the lens of pikfyve+/+, pikfyve+/Δ8, and pikfyveΔ8/Δ8 embryos at 3 dpf and 5 dpf. The scale bars represent 10 μm in (B) and (C). (D) Quantification of vacuole number in the lens of pikfyve+/+, pikfyve+/Δ8, and pikfyveΔ8/Δ8 embryos at 3 dpf (n=7 for pikfyve+/+; n=10 for pikfyve+/Δ8; n=7 for pikfyveΔ8/Δ8) and 5 dpf (n=7 for pikfyve+/+; n=11 for pikfyve+/Δ8; n=6 for pikfyveΔ8/Δ8). (E) Representative images of HLEB3 cells treated with DMSO or PIKFYVE inhibitor YM201636 for 4 hr. The scale bars represent 25 μm. (F) Quantification of the vacuole numbers in (E). ****, p<0.0001, Student’s t-test. All experiments were repeated three times. See Figure 3—source data 1 for details.

Figure 3—figure supplement 1
Characterization of pikfyve-deficient zebrafish mutants.

(A) Gross morphology of 5-dpf and 7-dpf siblings and pikfyveΔ8 mutants. The scale bars represent 500 μm. (B) Survival rate of pikfyveΔ8 mutants (n=48) and siblings (n=50).

Figure 3—figure supplement 2
Ectopic overexpression of PIKFYVEG1943E failed to induce cataract defect in zebrafish.

(A) Schematic view of constructs in which the WT and G1943E variant form of PIKFYVE were expressed under the control of the ubiquitously expressed ubiquitin promoter. (B) Representative images showing the lens of 5-dpf WT, Tg(ubi:PIKFYVEEWT) and Tg(ubi:PIKFYVEG1943E) zebrafish embryos. The scale bars represent 20 μm. All experiments were repeated three times.

Figure 3—figure supplement 3
The G1943E variant form of PIKFYVE is less efficient to rescue the vacuole defect in pikfyve-deficient zebrafish mutants.

(A) Representative images showing the lens of 5-dpf siblings, pikfyveΔ8, pikfyveΔ8;Tg(ubi:PIKFYVEWT), and pikfyveΔ8;Tg(ubi:PIKFYVEG1943E) zebrafish. The scale bars represent 20 μm. (B) Whole-mount in situ hybridization (WISH) analysis of human PIKFYVE transcripts in 2-dpf Tg(ubi:PIKFYVEWT) and Tg(ubi:PIKFYVEG1943E) zebrafish. The scale bars represent 200 μm. See Figure 3—figure supplement 3—source data 1 for details.

Detailed characterization of cataract phenotypes in pikfyveΔ8 mutants.

(A) Confocal imaging of the lens of 5-dpf siblings and pikfyveΔ8 mutants in Tg(cryaa:DsRed) transgenic background. (B) Hematoxylin-eosin (HE) staining of 5-dpf siblings and pikfyveΔ8 mutant zebrafish lens after cryostat section. (C) ZL-1 antibody and DAPI staining of 5-dpf siblings and pikfyveΔ8 mutant zebrafish lens. (D) Transmission electron microscope (TEM) images of the lens of siblings and pikfyveΔ8 mutants at 3 dpf and 5 dpf. ASS, autophagy lysosome; LD, lipid droplet; N, nucleus. All results were confirmed in three different individuals. All the scale bars represent 10 μm.

Figure 5 with 1 supplement
Characterization of vacuoles in pikfyveΔ8 mutants.

(A) Time-lapse imaging indicating the dynamic changes of vacuole formation in the lens of 4-dpf pikfyveΔ8 mutants. White arrows indicate the fusion process of two small vacuoles. (B) Representative images showing the lens of 3.5-dpf siblings and pikfyveΔ8 mutants injected with gfp-rab5c mRNA. (C) Representative images showing lens of 3.5-dpf siblings and pikfyveΔ8 mutants injected with gfp-rab7 mRNA. (D) Representative images showing lens of 3.5-dpf siblings and pikfyveΔ8 mutants injected with gfp-rab11a mRNA. (E) Representative images showing lens of 3.5-dpf siblings and pikfyveΔ8 mutants injected with mcherry-lc3b mRNA. All experiments were repeated three times. All the scale bars represent 10 μm.

Figure 5—figure supplement 1
Characterization of lysosomes in microglia and lens of pikfyveΔ8 mutant zebrafish.

Confocal images of the lens and microglia of 4-dpf pikfyveΔ8 mutants in Tg(mpeg1:dsredx) background after LysoSensor staining. The results were confirmed in more than three different individuals. The scale bars represent 20 μm.

Figure 6 with 1 supplement
Baf-A1 partially rescued the vacuole defect in the lens of pikfyveΔ8 mutant zebrafish.

(A) Representative confocal images of the lens of 4-dpf pikfyveΔ8 mutants treated with DMSO or Baf-A1 for 4.5 hr. (B) Quantification of the vacuole numbers in the lens of 4-dpf siblings and pikfyveΔ8 mutant embryos treated with DMSO or Baf-A1 (n=10 for sibling groups; n=7 for mutant groups). (C) Confocal images of the lens of 4-dpf pikfyveΔ8 mutants with no treatment or after 4.5 hr treatment with DMSO or Baf-A1. (D) Quantification of the vacuole numbers in (C) (n=6 for each group). All experiments were repeated three times. All the scale bars represent 20 μm. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001, Student’s t-test. See Figure 6—source data 1 for details.

Figure 6—figure supplement 1
Overexpression of trpml1 failed to rescue the lens defects in pikfyve Δ8 mutants.

(A) Confocal images of the lens of 4-dpf pikfyveΔ8 mutants and pikfyveΔ8 mutants injected with trpml1 mRNA. The scale bars represent 20 μm. (B) Quantification of the vacuole number in the lens of 4-dpf pikfyveΔ8 mutants and pikfyveΔ8 mutants injected with trpml1 mRNA. n.s., not significant, p>0.05, Student’s t-test. See Figure 6—figure supplement 1—source data 1 for details.

Tables

Table 1
Clinical characteristics of living patients in the cataract family.
PatientGenderAge at diagnosis (years)BCVA before surgery (OD, OS)Cataract type (OU)Surgery (Y/N)BCVA after surgery (OD, OS)
Ⅱ-1F9Y20/160, 20/125
Ⅱ-4F1220/40, 20/32Nuclear pulverulentNNA
Ⅱ-8M420/100, LPNuclear pulverulentY20/40, FC
Ⅲ-1M4720/25, 20/20Peripheral cortical punctateNNA
Ⅲ-5M620/40, 20/40Nuclear pulverulentY20/20, 20/20
Ⅲ-6M720/40, 20/50Nuclear pulverulentY20/16, 20/16
Ⅲ-9M620/40, 20/66Nuclear pulverulentY20/28, 20/25
Ⅳ-1M2220/20, 20/20Peripheral cortical punctateNNA
Ⅳ-3M320/30, 20/25Nuclear pulverulentNNA
Ⅳ-5M220/125, 20/125Nuclear and Y-suturalNNA
  1. BCVA: best corrected visual acuity; OD: eye, oculus dexter; OS: eye, oculus sinister; OU: eye, oculus uterque; Y: yes; N: no; F: female; M: male; LP: light perception; FC: finger counting; —: unknown; NA: not applicable.

Table 2
Candidate variants identified from whole-exome sequencing in the cataract family.
GeneSNP IDChromosome position (bp; hg19)cDNA changeAmino acid changeMAF(gnomAD)PolyPhenGERPCADD
PIKFYVErs771244880chr2:209217490c.5828G>Ap.G1943E0.000020.9995.0926.00
NPHS1rs114849139chr19:36330456c.2869C>Gp.V957L0.0010.9874.2714.34
FPR1rs78488639chr19:52249959c.289G>Tp.L97M0.0080.7952.5510.70
  1. MAF: minor allele frequency; gnomAD: genome aggregation database; PolyPhen: polymorphism phenotyping; GERP: evidence of evolutionary conservation; CADD: combined annotation dependent depletion.

Key resources table
Reagent type (species) or resourceDesignationSource or
reference
IdentifiersAdditional information
Cell line (Homo sapiens)HEK293TLaboratory cell bank
of Shenzhen PKU-HKUST Medical
Center
Initially ordered
from ATCC by
colleagues in
HKUST
Cell line (H. sapiens)HLEB3ATCCATCC CRL-11421, RRID:CVCL_6367
Strain, strain background (Danio rerio)pikfyveΔ8 mutantThis paperMaintained in Shenzhen PKU-HKUST Medical Center
Strain, strain background (D. rerio)Tg(ubi:PIKFYVEWT)This paperMaintained in Shenzhen PKU-HKUST Medical Center
Strain, strain background (D. rerio)Tg(ubi:PIKFYVEG1943E)This paperMaintained in Shenzhen PKU-HKUST Medical Center
Strain, strain background (D. rerio)Tg(cryaa:DsRed;il-1b:GFP-F)doi.10.1242/dmm.014498
Chemical compound, drugYM201636Selleck, ChinaS1219800 nM for 4 hr
Chemical compound, drugbafilomycin A1MedChemExpress, NJHY-1005581 μM for 4.5 hr
AntibodyAnti-PIKFYVE antibody (rabbit polyclonal)Abcam, UKCat# ab137907WB (1:1000)
AntibodyAnti-GAPDH antibody (rabbit polyclonal)Abcam, UKCat# ab9485, RRID:AB_307275WB (1:1000)
AntibodyAnti-Rabbit IgG H&L (goat polyclonal)Abcam, UKCat# ab205718, RRID:AB_2819160WB (1:5000)
AntibodyAnti-Lens Fiber Cell Marker antibody [ZL-1] (mouse monoclonal)Abcam, UKCat# ab185979IF (1:100)
AntibodyAnti-Mouse-555 secondary antibody (donkey polyclonal)Thermo Fisher Scientific, USACat# A-31570, RRID:AB_2536180IF (1:400)
Sequence-based reagent (H. sapiens)Human PIKFYVE genotyping fwdThis paperPCR primerTTTTGACCTTCTCTTGATTAGAGG
Sequence-based reagent (H. sapiens)Human PIKFYVE genotyping revThis paperPCR primerAAATATGGCCTAGTAACCAAAGTTAAA
Sequence-based reagent (H. sapiens)Human PIKFYVE cDNA cloning fwdThis paperPCR primerATGGCCACAGATGATAAGAC
Sequence-based reagent (H. sapiens)Human PIKFYVE cDNA cloning revThis paperPCR primerTCAGCAATTCAGACCCAAGCCTG
Sequence-based reagent (H. sapiens)Human PIKFYVE qPCR fwdThis paperPCR primerCGTCCCCAACACTGGACTCTGC
Sequence-based reagent (H. sapiens)Human PIKFYVE qPCR revThis paperPCR primerCCCTGGCCTCCTTCTGCTCTCTC
Sequence-based reagent (H. sapiens)Human GAPDH qPCR fwdThis paperPCR primerCGAGATCCCTCCAAAATCAA
Sequence-based reagent (H. sapiens)Human GAPDH qPCR revThis paperPCR primerGTCTTCTGGGTGGCAGTGAT
Sequence-based reagent (D. rerio)Zebrafish pikfyve genotyping fwdThis paperPCR primerGAGAACCTGCTCAAACTGGTGC
Sequence-based reagent (D. rerio)Zebrafish pikfyve genotyping revThis paperPCR primerAGATTTGACCACCATCTCCAGC

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  1. Shaoyi Mei
  2. Yi Wu
  3. Yan Wang
  4. Yubo Cui
  5. Miao Zhang
  6. Tong Zhang
  7. Xiaosheng Huang
  8. Sejie Yu
  9. Tao Yu
  10. Jun Zhao
(2022)
Disruption of PIKFYVE causes congenital cataract in human and zebrafish
eLife 11:e71256.
https://doi.org/10.7554/eLife.71256