Introduction
The kidney and ear share developmental origins, with signaling intermediates that play crucial roles in morphogenesis, function, and the specification of cell types. Additionally, both organs possess the same collagen type IV basement membrane structure [1]. Therefore, the robust pathophysiological links exist between hearing loss and kidney diseases [1–3]. Approximately 30% of all genetic hearing loss cases are congenital and syndromic [4], with a subset involved in both the ear and kidney system, including Alport syndrome characterized by glomerulopathy.
Alport syndrome, a collagen type IV basement membrane disorder, is characterized by proteinuria and hematuria, often leading to kidney failure, along with sensorineural hearing loss (SNHL) and ocular abnormalities [5,6]. Alport syndrome is a genetic disease resulting from pathogenic variants in the COL4A3, COL4A4, and COL4A5 genes. Variants in COL4A3 and COL4A4 can lead to Alport syndrome with either an autosomal dominant (autosomal dominant Alport syndrome, ADAS) or an autosomal recessive inheritance pattern (autosomal recessive Alport syndrome, ARAS). Additionally, COL4A5 variants are associated with X-linked Alport syndrome (XLAS) [5,7]. The collagen IV family consists of six alpha chains that form trimers of α1α1α2, α3α4α5, and α5α5α6 [6,8]. These are localized in the auditory and kidney systems, such as basement membranes of the Bowman’s capsule, glomeruli, tubules, cochlea, vestibular system, and ocular structures [6,8]. Therefore, variants in the collagen IV family can lead to disruption or deterioration of these basement membranes, contingent on how the collagen IV variants affect the structural integrity of trimer assembly. Recent in vivo studies have shown that endothelin-1 activation increases extracellular matrix gene expression and thickens the strial capillary basement membranes (SCBMs) in Alport mice, with type IV collagen composition contributing to cellular damage through pericyte detachment and altered cytoskeletal dynamics [9,10]. This process, in turn, disrupts endocochlear potentials essential for hearing function and maintenance, thereby serving as a pathogenic mechanism for auditory phenotype in Alport syndrome.
In human genetics, audiological manifestations of Alport syndrome typically precede SNHL, characterized by symmetric and progressive nature in the middle- and high-frequency ranges and a good ability to discriminate speech, and a hearing level not exceeding 60–70 dB [5,7,8,11]. However, the auditory phenotypes in Alport syndrome cannot be uniformly characterized due to its heterogeneous presentation, which varies according to inheritance pattern, genotypes (e.g., type of mutations), and sex [12], which complicates the firm genotype-phenotype correlations. Furthermore, the phenotypes may be influenced by an allelic hierarchy where mutant alleles or their combinations can exert distinct effects on the clinical manifestations of the syndrome [13].
Despite several reports of auditory phenotypes in Alport syndrome in the literature, there have been no studies specifically addressing clinical phenotypes from audiological perspectives in South Korean patients. Differences in genotypes across ethnic groups may influence how mutant alleles affect auditory phenotypes. In this regard, we comprehensively elaborated on the characteristics of hearing loss associated with Alport syndrome, offering valuable references that could inform strategies for auditory testing and hearing rehabilitation.
Methods
Subjects
We reviewed data from Alport syndrome patients and focused on participants attending the Center for Rare Diseases of Seoul National University Children’s Hospital in Seoul, Korea, between March 2014 and February 2023. Patients lacking audiological documentation or without genetic diagnoses were excluded. We then retrieved demographic data and clinical phenotypes, including past medical history, results from pure-tone audiometry (PTA), and genotypes, from electronic medical records. Ultimately, 49 patients from 47 families were included.
This study was conducted after receiving approval from Institutional Review Board (IRB) of Seoul National University Hospital (No. 2403-086-1520). Approval for the waiver of informed consent has been granted by the IRB.
Genetic analysis
As previously described in the literature [14,15], genetic testing was conducted using Sanger sequencing, targeted panel sequencing, and whole-exome sequencing. Specifically, one family with a COL4A5 deletion predicted by NGS-based copy number variation screening tools underwent whole-genome sequencing (WGS) to further delineate the breakpoints and structural variations. DNA libraries for WGS were prepared from 1 µg of genomic DNA using the TruSeq DNA PCR-Free kit (Illumina). The sequencing was performed on the NovaSeq 6000 platform (Illumina) to produce 151 bp paired-end reads. These sequenced reads were aligned to the GRCh38 reference genome using the BWA-MEM algorithm, and duplicate reads were removed with SAMBLASTER [16,17]. Structural variations were identified with DELLY [18], and the breakpoints of these structural variations were visually inspected and verified using the Integrative Genomics Viewer (IGV; https://igv.org).
In our study, variants such as frameshift, nonsense, canonical splicing, and structural variations were classified as truncated variants. Conversely, missense variants and in-frame deletions/duplications were categorized as non-truncated variants. Hereafter, patients with at least one truncated allele will be referred to as the “truncated group,” while those with biallelic non-truncated alleles will be designated as the “non-truncated group.” The interpretation of variant pathogenicity adhered to the guidelines of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) [19]. The novelty of the variants was determined by the ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) and LitVar2 databases (https://www.ncbi.nlm.nih.gov/research/litvar2/).
Audiological analysis
We analyze PTA results at 0.25 kHz, 0.5 kHz, 1 kHz, 2 kHz, 4 kHz, and 8 kHz to evaluate the hearing thresholds of subjects. Subsequently, mean hearing thresholds at 0.25 kHz and 0.5 kHz were designated as low-frequency hearing levels, those at 1 kHz and 2 kHz as middle-frequency hearing levels, and those at 4 kHz and 8 kHz as high-frequency hearing levels. Hearing loss was defined as a mean hearing level equal to or less than 25 dB, calculated by averaging the thresholds at 0.5 kHz, 1 kHz, 2 kHz, and 4 kHz in the worse ear. We organized age groups into bins using 10-year intervals and conducted analyses on the hearing levels across frequencies. Additionally, we developed linear regression models to estimate the annual progression of hearing loss, depending on sex, genotypes, and inheritance patterns.
Renal function analysis
Estimated glomerular filtration rate (eGFR), coupled with PTA profiles, were retrieved from electric medical records, whenever available. The eGFR was calculated using creatinine with the Cockcroft-Gault (CG), Modification of Diet in Renal Disease (MDRD), Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI), and Schwartz formulas. For patients aged 19 years or younger, the Schwartz formula was primarily used. If the Schwartz result was not available, values calculated using CKD-EPI, MDRD, and CG formulas were used in that order. For adult patients, aged over 19 years, the CKD-EPI formula was applied first, and if it was not available, values calculated using the MDRD and CG formulas were used in that order. The GFR category was defined based on the guidelines provided by the 2024 KDIGO (Kidney Disease: Improving Global Outcomes) clinical practice guideline for the evaluation and management of chronic kidney disease [20]. The history of dialysis and kidney transplantation was also gathered as a possible factor associated with hearing. Afterward, we analyzed the relationship between renal function or dialysis and hearing level in patients without a history of kidney transplantation or before undergoing kidney transplantation. The eGFR and dialysis were not included in the same multivariable analysis due to high multicollinearity.
Statistical analysis
The t test and Wilcoxon signed-rank test were used to evaluate the continuous variables. Additionally, the chi-square test and Fisher exact test were employed for the comparison of categorical variables. Linear regression analysis was conducted to evaluate the changes in hearing level according to the aging process or eGFR decrease in individuals with each inheritance pattern. Analysis of covariance was used to perform adjusted analysis. All statistical analyses were conducted using IBM SPSS version 25.0 (IBM Corp.) or R version 4.3.2 (R Project for Statistical Computing).
Results
Cohort description
The demographics and clinical characteristics of the Alport syndrome cohort are described in Table 1, including 49 patients from 47 families. Among these patients, 29 (59.2%) were male. All were clinically diagnosed with Alport syndrome at an average age of 15.2 ± 1.8 years (range, 4–60 years). The patients were categorized based on their inheritance patterns into ARAS (n = 10 from 10 families), ADAS (n = 8 from seven families), and XLAS (n = 31 from 30 families) (Tables 2, 3).
Genotypes
COL4A5 was the most commonly affected gene (n = 31, 63.3%), followed by COL4A3 (n = 8, 18.4%), COL4A4 (n = 7, 16.3%), and COL4A4/COL4A3 (n = 1, 2.0%) (Fig. 1A). In terms of inheritance patterns, XLAS was the most prevalent (n = 31, 63.3%), followed by ARAS (n = 10, 20.4%), and ADAS (n = 8, 12.8%). In addition, one proband displayed dual genetic etiologies inherited from their parents, exhibiting two pathogenic variants in a trans configuration (n = 1, 2.1%) (Fig. 1B). The genotypes of Alport syndrome were diverse across all mutated genes without evidence of mutational hotspot or founder mutant alleles. Single nucleotide variant was the most frequent variant type of variants in COL4A3 (93.8%), COL4A4 (76.9%), and COL4A5 genes (71.0%), followed by short indels in those genes (COL4A3, 6.2%; COL4A4, 15.4%; and COL4A5, 22.6%) (Fig. 1C). Missense variants occupied largest proportion of variant types in COL4A3 (62.5%), COL4A4 (46.2%), and COL4A5 (48.4%). Frameshift variants were the second most frequent variant type in COL4A3 (31.3%) and COL4A5 (19.4%), while splicing variants were the second largest percentage in COL4A4 (23.1%) (Fig. 1D). Interestingly, two structural variation alleles were identified in our cohort, one from COL4A5 and the other from COL4A4. The position of the entire COL4A5 hemizygous deletion (ChrX:108,420,073–108,422,099) identified through WGS is illustrated using IGV (Supplementary Fig. 1, available online). In this study, the proportion of patients with at least one truncated allele (mono- or bi-allelic), defined by frameshift, nonsense, canonical splicing, and structural variations, was 18 (36.7%). In contrast, the proportion of patients with biallelic non-truncated alleles, defined by missense variants and in-frame deletions or duplications, was 31 (63.3%).
Within this cohort, 60 disease-causing variants were detected (52 when collapsing identical variants once), of which 45 variants were novel. All variants identified herein were classified as either pathogenic or likely pathogenic according to the ACMG-AMP guideline.
Audiological characteristics of autosomal recessive Alport syndrome
Ten affected patients with ARAS (from 10 families) were included. Specifically, COL4A3-related and COL4A4-related ARAS were six and four, respectively. Their average age at ascertainment was 14.5 ± 2.2 years (range, 8–27 years) (Table 4), and the proportion of male was 30.0% (Table 2). A total of 44 audiograms were used to analyze the audiological features of ARAS.
The average age at which hearing loss was documented in ARAS cases was 23.8 ± 1.3 years (range, 8–34 years) (Table 4). The hearing levels of ARAS patients demonstrated a progressive nature, particularly at high frequencies with a down-sloping configuration (Fig. 2A). In their 20s, the mean hearing level was classified as mild hearing loss (33.9 ± 2.8 dB), and by their 30s, the mean hearing level escalated to moderate-to-severe hearing loss (53.9 ± 3.6 dB), beyond serviceable hearing thresholds.
We further examined the impact of presence of truncated alleles on auditory phenotypes in patients with ARAS. Hearing loss occurred at a significantly younger average age in the truncated group (mean age, 17.0 ± 1.7 years; range, 8–24 years) compared to the non-truncated group (mean age, 28.7 ± 2.7 years; range, 24–34 years; p < 0.001) (Table 4). The truncated group exhibited higher hearing levels but showed slow progression at low frequency. Conversely, similar progression at middle and high frequency were noted compared to the non-truncated group (Fig. 2B–D). In detail, the annual progression of hearing loss in the ARAS truncated group occurred in a stepwise manner, showing an increase of 0.9 dB at low frequency, 2.0 dB at middle frequency, and 2.4 dB at high frequency. Conversely, the ARAS non-truncated group experienced an annual progression of hearing loss of 1.8 dB at low frequency, 1.9 dB at middle frequency, and 2.2 dB at high frequency in a stepwise manner. Moreover, the incidence of hearing loss progressively increased with age among ARAS cases, irrespective of the genotype, as detailed in Table 5.
Renal characteristics of autosomal recessive Alport syndrome and their association with hearing level
Among the 44 audiograms of ARAS patients, 33 audiograms had concurrent eGFR records. Of these, 16 audiograms belonged to the truncated group, while 17 audiograms belonged to the non-truncated group. For the audiograms in the truncated group, eight corresponded to patients with GFR category 5, two belonged to patients with chronic kidney disease stage 2, and three belonged to patients with GFR category 1 at the time of measurement. And their mean hearing level was significantly associated with eGFR (B = –0.283, p < 0.001) (Supplementary Fig. 2, available online) and dialysis (p < 0.001). This association was consistent after adjusting for age (eGFR: β = –1.136, p = 0.001; dialysis: β = 1.340, p = 0.005). On the other hand, all in the non-truncated group were classified as GFR category 1 or GFR category 2 at the time of taking audiogram, and their mean hearing level was not significantly associated with eGFR (B = –0.298, p = 0.21), and there were no patients receiving dialysis.
Audiological characteristics of X-linked Alport syndrome
Thirty-one patients with XLAS (from 30 families) were included. A total of 117 audiograms were used for audiological analysis. Their average age at ascertainment was 12.7 ± 1.9 years (range, 4–60 years), and the proportion of male was 88.9% (Table 3).
The average at which hearing loss was documented in XLAS cases was 14.2 ± 0.6 years (range, 7–60 years) (Table 4). The hearing levels of XLAS patients demonstrated a progressive pattern. In their first decade, the mean hearing level was classified as mild hearing loss (31.7 ± 3.3 dB), and by their 20s, the mean hearing level escalated to moderate-to-severe hearing loss (48.8 ± 4.2 dB).
We first conducted further analysis to compare audiological characteristics of XLAS cases, according to sex. Consistent with our hypothesis, male XLAS patients exhibited an earlier onset and age of hearing loss compared to female XLAS patients (Tables 3, 4). Furthermore, a higher incidence of hearing loss was observed in male XLAS cases than in female ones, with a significant difference (p = 0.04) (Table 5). Male XLAS patients also experienced more severe hearing loss over the course of follow-up assessments (Table 2). Notably, in their 10s, male XLAS patients presented with bilateral mild-to-moderate SNHL (35.7 ± 3.3 dB), characterized by a cookie-bite pattern (35.7 ± 3.3 dB). The progression of their hearing loss was especially pronounced at higher frequencies, exhibiting a down-sloping audiogram in their 30s (Fig. 3A–D). When comparing annual hearing loss progression, male XLAS patients demonstrated steeper declines at low (0.5 dB/year), middle (0.7 dB/year), and high frequencies (1.4 dB/year) relative to females (low frequency, 0.4 dB/year; middle frequency, 0.6 dB/year; and high frequency, 0.8 dB/year).
In male XLAS patients, truncated group experienced a markedly rapid deterioration of hearing loss annually at low (0.6 dB/year), middle (1.2 dB/year), and high frequencies (2.3 dB/year) compared to non-truncated group, who showed slower rates of decline at low (0.4 dB/year), middle (0.5 dB/year), and high frequencies (0.9 dB/year), as depicted in Fig. 3E–H.
Renal characteristics of male X-linked Alport syndrome and their association with hearing level
Among the 35 audiograms of male XLAS patients, 24 belonged to patients with eGFR data. Of these, 20 belonged to patients in GFR category 1 or 2, and four belonged to patients in GFR category 5 at the time of measurement. The eGFR (B = –0.156, p = 0.04) and dialysis (B = –0.156, p = 0.002) of the male XLAS truncated group were significantly associated with mean hearing level (Supplementary Fig. 2). However, this relationship is no longer significant after adjusting age (eGFR: β = 0.243, p = 0.40; dialysis: β = –0.076, p = 0.87).
Among the 69 audiograms of the non-truncated group, 37 belonged to patients with GFR category 1 or 2, and six belonged to patients with GFR category 3. Two audiograms corresponded to patients with GFR category 4, and one audiogram corresponded to a patient with GFR category 5. Fifteen audiograms of two non-truncated XLAS patients were excluded due to a history of kidney transplantation. Their eGFR level was not associated with the mean hearing level (B = –0.034, p = 0.61), and there were no patients receiving dialysis.
Autosomal dominant Alport syndrome
During the follow-up assessments, the majority of ADAS cases (n = 7) exhibited hearing levels within the normal range (Supplementary Fig. 3, available online). However, there was an exception with Case 2, who suffered from a definite noise trauma (Supplementary Fig. 3).
Discussion
For the first time, we herein detailed the audiological phenotypes of Alport syndrome in South Korean patients. Audio-gene profile analyses revealed that ARAS cases typically experienced hearing loss during their 10s to 20s, displaying a progressive pattern. The ARAS truncated group had hearing loss at an earlier age, although the progression pattern remained similar regardless of genotypes (truncated vs. non-truncated). Notably, XLAS cases demonstrated varying audiological phenotypes based on sex and genotypes (truncated vs. non-truncated), with the male XLAS truncated group being associated with more severe audiological phenotypes in overall. Additionally, the eGFR of ARAS and male XLAS truncated group showed a linear relationship with hearing level. In contrast, female XLAS cases generally maintained normal hearing until their 40s, and the majority of ADAS cases preserved a normal hearing level throughout the study period. Currently, no treatment exists to prevent or even cure the deterioration of hearing loss in Alport syndrome [21]. Once SNHL is developed, real-world management is limited to auditory rehabilitation, including hearing aids or cochlear implantation [22,23]. Given this, integrating audiological phenotypes and genotypes, as evidenced herein, has elucidated the natural course of hearing loss and appropriate auditory rehabilitation in Alport syndrome.
Patients with Alport syndrome typically pass newborn hearing screenings and most commonly present with SNHL in adolescence or even in early childhood [5,7]. Recent studies have also reported that approximately 70% of Alport syndrome patients receive their diagnosis in their late teens or even as adults due to their progressive nature [8]. Previous studies revealed that in about 64% of ARAS cases, hearing loss occurs before the age of 20 years [24,25]. Additionally, by the age of 27 years, approximately 74% exhibit signs of hearing loss [25]. Our data uncovered that ARAS patients displayed hearing loss at a rate of 33.3% by the age of 20 years and 70.5% by the age of 40 years, which was lower than the prevalences reported in a previous study conducted in Italy or a systematic review with heterogeneous ethnicity, but showed a similar pattern of progressive hearing loss. When focusing on the ARAS truncated group, hearing loss was documented in 62.5% of cases by the age of 20 years and 81.3% by the age of 30 years. The majority of ARAS cases experienced hearing loss during their 10s to 20s, even including cases identified as early as the age of 8 years. Additionally, ARAS cases demonstrated an average hearing loss progression rate of 2 to 3 dB per year across all frequencies. Therefore, despite a post-lingual onset of hearing loss, early evaluation of hearing loss is imperative once ARAS has been genetically confirmed. Given the substantial progression of hearing loss regardless of the genotypes (truncated vs. non-truncated), it’s advised to conduct periodic hearing evaluations every 6 to 12 months from the point of diagnosis into the teenage years. PTA, a noninvasive, standard procedure for assessing hearing thresholds across the 250 to 8,000 Hz frequency range, is feasible from around the age of 5 years to 6 years [26]. Upon confirming hearing loss, it’s critical to evaluate language development, which can be supported by hearing aid rehabilitation [27,28]. Furthermore, in cases of delayed language development, active interventions, such as audio-verbal and music therapy, are recommended to enhance language acquisition [29,30].
Consistent with previous reports, we also observed that audiological manifestations of XLAS are distinct based on sex. Typically, more severe forms of hearing loss tend to occur in males than females [6,12]. Clinically, XLAS in male patients presents symptoms similar to ARAS, with the median onset age for hearing loss occurring in the mid-20s [6,25]. By the age of 15 years, 50% of male patients are diagnosed with hearing loss [12]. Around the age of 30 years, hearing loss occurs in approximately 60% of cases with missense mutations, and up to 90% in cases with other mutations [31]. At the age of 40 years, 90% of male patients experience hearing loss [6,12]. In contrast, female XLAS patients suffered less hearing loss than males. About 12% of female patients experience hearing loss at the age of 40 years [6,12]. Approximately 20% of female XLAS patients undergo hearing impairment in their 60s, while approximately 15% of the general population typically experiences age-related hearing loss in their 60s [12,32]. Our data demonstrated parallel findings regarding the average age at which hearing loss was documented and the proportion of hearing loss across different ages, with a slightly higher proportion of hearing loss at each age. Moreover, male XLAS patients carrying truncation variants experienced a more accelerated progression of hearing loss. In our study, the youngest case of XLAS was a 7-year-old boy with truncated variants. Consequently, beyond the vigilance for ARAS cases, an earlier assessment of hearing loss, coupled with regular hearing evaluations every 6 months and prompt rehabilitation, becomes crucial once a diagnosis of male XLAS is made, especially with truncated variants.
In contrast, ADAS typically exhibits less severe clinical characteristics of kidney impairment and extrarenal manifestations including hearing loss. Hearing loss is less common in ADAS compared to Alport syndrome caused by other collagen IV family [6]. For example, Solanki et al. [33] demonstrated that certain genetic variants, such as p.Gly695Arg (e.g., collagen-related glycine variants) and truncated variants, do not significantly increase the risk of hearing loss. Meanwhile, missense variants or in-frame deletions have been associated with an increased risk of hearing loss, albeit by around 13% [33]. Furthermore, Kamiyoshi et al. [34] reported a low incidence of hearing loss in ADAS, with only one case among the cohort of 25 individuals (age range, 5–82 years; average age, 33.4 years). Aligned with previous studies, only one patient among our ADAS cases showed severe hearing loss due to a history of noise-induced hearing loss caused by gunshots. This case reflects the increased susceptibility of noise-induced trauma in Alport mice, as evidenced by thickened SCBMs and associated loss of endocochlear potentials required to maintain hearing function [10]. In contrast to ARAS and XLAS, ADAS patients had less demand for hearing screening and rehabilitation at an early age. Nonetheless, given a higher prevalence of hearing loss in individuals with missense or in-frame deletion variants [33], early hearing screening for these patients at a younger age might be helpful. Moreover, education regarding the increased risk of noise-induced hearing loss, even in ADAS cases, is crucial for prevention.
This study revealed that renal function was significantly associated with the ARAS truncated group and the male XLAS truncated group, with a more prominent relationship in the ARAS group. Since the kidney and cochlea share a similar structure [1], the severe phenotype of the cochlea can be related to the phenotype of kidney [35,36]. Therefore, physicians should check hearing levels when eGFR decreases in the ARAS and male XLAS truncated groups and monitor eGFR when these patients experience hearing loss progression.
This study had limitations that warrant consideration in future research. The sample size for certain genetic and audiogram profiles was too small to fully assess audiologic characteristics. Upon the results from ARAS cases, it is challenging to definitively conclude whether there are no differences in the progression pattern of hearing loss according to genotypes (truncated vs. non-truncated variants). This lack of significance may be due to the insufficient data for the ARAS truncated group beyond their 30s. Although the number of Alport syndrome patients was limited, we observed a similar trend in hearing loss phenotypes as reported previously, and further delineated the phenotypes in detail. Given the rarity of Alport syndrome, a more comprehensive, multicenter study with a larger cohort is essential to accurately determine the hearing loss phenotypes overall.
We elucidated the characteristics of hearing loss associated with Alport syndrome, focusing on genetic attributes. Our study included 49 Alport syndrome patients from 47 families, identifying 60 disease-causing variants, 45 of which were novel. Audio-gene profiles revealed distinct auditory phenotypes corresponding to genotype and sex. Specifically, the hearing levels of ARAS patients and males with XLAS showed a progressive decline, particularly in the truncated group. Conversely, the majority of females with XLAS and those with ADAS maintained normal hearing levels. This study detailed the auditory phenotypes of Alport syndrome and their relationship with renal phenotype at a tertiary referral center in South Korea, providing valuable references that guide auditory testing and rehabilitation strategies. Nonetheless, a more comprehensive, multicenter study with a larger cohort is essential to accurately determine the hearing loss phenotypes overall.
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