PKHD1L1, a gene involved in the stereocilia coat, causes autosomal recessive nonsyndromic hearing loss

Abstract

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 four families segregating autosomal recessive nonsyndromic sensorineural hearing loss. Compound heterozygous p.[(Gly129Ser)];p.[(Gly1314Val)] and p.[(Gly605Arg)];p[(Leu2818TyrfsTer5)], 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 and performed a minigene splicing assay for another variant. In silico molecular modeling using AlphaFold2 and protein sequence alignment analysis were carried out to further explore potential variant effects on structure. In vitro functional assessment indicated that both engineered PKHD1L1 p.(Gly129Ser) and p.(Gly1314Val) mutant constructs significantly reduced the folding and structural stabilities of the expressed protein fragments, providing further evidence to support pathogenicity of these variants. Minigene assay of the c.1813G>A p.(Gly605Arg) variant, located at the boundary of exon 17, revealed exon skipping leading to an in-frame deletion of 48 amino acids. In silico molecular modeling 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.

PKHD1L1 is required for stereocilia bundle maintenance, durable hearing function and resilience to noise exposure

“…As we have determined that PKHD1L1-defficient bundles are more susceptible to noise-induced damage, the variability in the acoustic environment between the animal facilities experienced by PKHD1L1-defficient mice used in these studies may account for the differences seen in hearing thresholds. Interestingly, audiogram profiles of human patients with non-syndromic hearing loss and biallelic PKHD1L1 variants were also diverse (Redfield et al, 2024). As PKHD1L1 variants may render hair cells more vulnerable to damage due to environmental factors, it is likely that in addition to the potential differences in the damaging effect of distinct PKHD1L1 variants at the protein level, hearing loss profiles in affected probands may also reflect differing levels of acoustic exposure throughout their lives.…”

“…With increasing evidence to support PKHD1L1 as a human deafness gene (Lewis et al, 2023; Redfield et al, 2024), the function of PKHD1L1 requires further study. The role of the developmental stereocilia coat remains unclear.…”

“…As PKHD1L1 variants may render hair cells more vulnerable to damage due to environmental factors, it is likely that in addition to the potential differences in the damaging effect of distinct PKHD1L1 variants at the protein level, hearing loss profiles in affected probands may also reflect differing levels of acoustic exposure throughout their lives. With increasing evidence to support PKHD1L1 as a human deafness gene (Lewis et al, 2023;Redfield et al, 2024), the function of PKHD1L1 requires further study. The role of the developmental stereocilia coat remains unclear.…”

“…(Makrogkikas et al, 2023). In humans, we recently reported biallelic variants of PKHD1L1 in four unrelated families with congenital non-syndromic sensorineural hearing loss (Redfield et al, 2024). These PKHD1L1 variants were predicted to be damaging in silico, and some of the putative disease-causing mutations were shown to reduce the thermodynamic stabilities of PKHD1L1 protein fragments in vitro (Redfield et al, 2024).…”

“…In humans, we recently reported biallelic variants of PKHD1L1 in four unrelated families with congenital non-syndromic sensorineural hearing loss (Redfield et al, 2024). These PKHD1L1 variants were predicted to be damaging in silico, and some of the putative disease-causing mutations were shown to reduce the thermodynamic stabilities of PKHD1L1 protein fragments in vitro (Redfield et al, 2024). Furthermore, a large-scale exome study of 532 individuals recently identified the PKHD1L1 gene as a possible contributor to hearing outcomes, with a higher variant load in older adults with hearing loss compared to the non-hearing loss group (Lewis et al, 2023).…”