Page 7 - L. Genetics and genomics

Identification of CHPF2 as a novel candidate gene
for progressive nonsyndromic hearing loss.

Kyumin Kim¹, Byunghwa Noh¹, Heon Yung Gee², Jae Young Choi¹, Jinsei Jung¹*
,
¹Department of Otorhinolaryngology, Yonsei University College of Medicine, Seoul 03722, Republic of Korea,
²Department of Pharmacology, Yonsei University College of Medicine, Seoul 03722, Republic of Korea

BACKGROUND AIM

Hearing loss is the most common sensory disorder in humans. Hearing loss caused by autosomal dominant diseases ＊To investigate novel causes of progressive hearing loss with autosomal dominant
accounts for 22-25% of hereditary hearing loss, and about 30 causative genes have been identified. However, the inheritance.
exact association and mechanism for this are not yet known. In addition, there are many related genes that have not
been discovered yet, and research is underway to discover them. In this study, we uncovered CHPF2 as a newly ＊To elucidate the molecular pathomechanism of hearing loss related with the mutation in
discovered deafness gene through whole exome sequencing in a family where affected individuals showed CHPF2
progressive hearing loss with the onset from 20s to 40s. (YUHL cohort)
Therefore, we studied how it affects Othith and hair cells when injected into zebrafish embryos through Mo injection
targeting chpf2.
METHODS

- Variant filtering process for the identification of causative mutations. - MO injection
1. calculating the concentration based on the Mo
sequence information {chpf2 e2i2 (ata aat gtt tcc tca
cct gat gct t; 25bp DNA)}, prepare a mixture.
2. obtaining a fertilized egg from an adult zebrafish,
about 4~6nL microinjection is performed into the one-
celled fertilized egg. (Through a pre-test, the
appropriate concentration of MO was set to 4-6 ng.)
3. incubation for 5 days at 28 degrees, stain with Yo-
pro1 staining solution (2uM) and wash.
4. Anesthetize with MS-222 (Tricaine), fix in 3%
methyl cellulose, and observe hair cells in neuromast
under a fluorescence microscope or confocal.
RESULTS
Control MO injected
(B)
(A)

Figure 1. Family group of patients who develop autosomal dominant progressive hearing loss
(A) Family tree of patients with autosomal dominant inheritance. (B) Pure tone hearing test results for sensorineural Figure 3. Analysis of otholith of zebrafish larvae by morpholino (chpf2 e2i2).
hearing loss in the 30s and 40s. YUHL1-24 was normal, and YUHL-12 showed bilateral sensorineural hearing loss, After injecting 4 ug of MO into a fertilized egg in one cell state, the change in otholith was observed.
which gradually progressed from the 30s. Morphologically, there was no difference between the control group and the exposed group for otholith.
(A) Control
CHPF2 MO injected
c.1966_1967insC;p.Arg656Profs*10 #1 #2 #3 #1 #2 #3 #4

11 9 10 8 10 7 8

11 8 11 7 8 8 4
(B)
11 16 6 9 8 6 8
26.6% reduce
(B)
Figure 4. Analysis of hair cell damaged by morpholino
(chpf2 e2i2) using confocal microscope.
(A) As a result of observing the hair cells of zebrafish larva
using a confocal microscope, (B) it was confirmed that the hair
cells decreased by 26.6% in the exposure group injected with
MO compared to the control group.
Figure 2. Discovery of new causative gene mutations in patients with progressive hearing loss through
next-generation sequencing methods.
The autosomal dominant CHPF2 C.1966_1967insC;p.Arg656Profs*10 gene mutation was discovered through
next-generation sequencing.
CONCLUSION REFERENCES ACKNOWLEDGEMENTS
In this study, we found CHPF2, a newly discovered deaf gene, through 1. Jinsei Jung, Han Sang Kim, Min Goo Lee, Eun Jin Yang, Jae Young This research was supported by the Bio & Medical Technology
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onset of infected individuals in their 20s to 40s. Sensorineural Hearing Loss and Vestibular Disorder, Shows Impaired funded by the Korean government (MSIP&MOHW)
Cochlin Post-Translational Cleavage and Secretion. Human Mutation (No. 2019M3E5D5066690)
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As a result, we found that the number of hair cells in the neural mast was potential target of BMP signaling; Am J Hum Genet. 2010
significantly reduced in the MO injection group compared to the control, Dec10;87(6):757-67. Contact information
while the morphology of the otolith did not differ between the two groups.
3. Hiroyasu Ogawa,et al.(2012) Chondroitin sulfate synthase-2 is
-> Taken together, we consider that the mutation in CHPF2 causes necessary for chain extension of chondroitin sulfate but not critical for Department of Otorhinolaryngology, Yonsei University College of
autosomal dominant nonsyndromic sensorineural hearing loss, which skeletal development. PLoS ONE 2012 vol. 7 (8) pp. e43806 Medicine ABMRC Building, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, Korea
may be caused by the hair cell degeneration in the inner ear. (03722) e-mail : h20394@yuhs.ac Tel : +82-10-6281-2327
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