PKHD1L1, A Gene Involved in the Stereociliary Coat, Causes Autosomal Recessive Nonsyndromic Hearing Loss

Identification of genes associated with nonsyndromic hearing loss is a crucial endeavor, given the substantial number of individuals who remain without a diagnosis after even the most advanced genetic testing. PKHD1L1 was established as necessary for the formation of the cochlear hair-cell stereociliary coat and causes hearing loss in mice and zebrafish when mutated. We sought to determine if biallelic variants in PKHD1L1 also cause hearing loss in humans. Exome sequencing was performed on DNA of three families segregating autosomal recessive moderate to severe nonsyndromic sensorineural hearing loss. Compound heterozygous missense p.[(Gly129Ser)];p.[(Gly1314Val)], homozygous missense p.(His2479Gln) and nonsense p.(Arg3381Ter) variants were identified in PKHD1L1 that were predicted to be damaging using in silico pathogenicity prediction methods. In vitro functional analysis of two missense variants was performed using purified recombinant PKHD1L1 protein fragments. We then evaluated protein thermodynamic stability with and without the missense variants found in one of the families. In vitro functional assessment indicated that both engineered PKHD1L1 mutant p.(Gly129Ser) and p.(Gly1314Val) constructs significantly reduced the folding and structural stabilities of the expressed protein fragments, providing further evidence to support pathogenicity of these variants. In silico molecular modelling using AlphaFold2 and protein sequence alignment analysis were carried out to further explore potential variant effects on protein folding and stability and exposed key structural features that might suggest PKHD1L1 protein destabilization. Multiple lines of evidence collectively associate PKHD1L1 with nonsyndromic mild-moderate to severe sensorineural hearing loss. PKHD1L1 testing in individuals with mild-moderate hearing loss may identify further affected families. ### Competing Interest Statement Go Hun Seo is an employee of 3Billion, Inc. ### Funding Statement This work was supported by NIDCD K08 DC019716 to AES, the De Garay Family Fund to MAK with support from the Boston Childrens Rare Disease Cohort Initiative, and the German Research Foundation (DFG) VO 2138/7-1 grant 469177153 to BV, and by NIH R01DC017166 (NIDCD) and R01DC020190 (NIDCD) to AAI. Funding for work in Pakistan was provided by the University of the Punjab (SN). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Institutional review boards of Boston Childrens Hospital (IRB P-00031494), University Medical Center Goettingen (No. 3/2/16), and the School of Biological Sciences, University of Punjab, Lahore, Pakistan (IRB No. 00005281) gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors