Background
Alport syndrome (AS) is a rare hereditary disease with an incidence of 1/5000–1/50000, involving the basement membranes of the kidneys, ears and eyes. It is characterized by chronic kidney disease, sensorineural hearing loss, and ocular abnormalities [
1], affecting at least 1 in 50,000 individuals [
2]. There are three inheritance patterns: X-linked dominant inheritance of
COL4A5 mutations, autosomal recessive of
COL4A3 or
COL4A4 mutations, and autosomal dominant of
COL4A3 or
COL4A4 mutations, with a percentage of 80, 15, and 5%, respectively [
3,
4]. The
COL4A5 and
COL4A3/COL4A4 genes code for different collagen type IV chains, including α3, α4 and α5 chains, that form heterotrimers in the basement membranes [
5].
Type IV collagen is the main collagen component of the basement membrane, playing an important role in the functioning of basement membrane barrier, and contains six kinds of alpha chains (α1–6) [
6‐
8]. Defects in α3, α4 and α5(IV) chains can cause abnormal formation of mature alpha chains, further disrupting the formation of type IV collagen molecules and changing the structure of the basement membrane. These physiological changes finally results in the dysfunction of organs such as the glomerulus, the retina and the cochlea.
Each alpha chain of type IV collagen contains both collagen and non-collagen regions [
9]. The collagen region is rich in the triple structure of glycine (Gly)-X-Y (X, Y for any amino acid) [
5], and glycine is necessary in the Gly-X-Y triple structure of the collagen domain. It is the only amino acid that is small enough to enter the helix center of collagen.
Earlier studies [
10‐
13] have revealed that hearing loss is a result of alterations of basement membranes in AS patients. The α5(IV) chain is present in the basement membrane and spiral ligament of the whole Corti organ, and the results of immunostaining of the cochlea for antibodies against α5(IV) chain of type IV collagen has been reported [
7]. Mutations in
COL4A5 gene resulted in the abnormality or absence of the corresponding coding product. Therefore, the normal IV collagen network structure cannot be constructed, resulting in basement membrane injury and hearing loss [
8,
14]. Different variant types such as deletions, insertions, splicing variants, direct or indirect nonsense mutations, and missense variants (including Gly or non-Gly substitutions) in
COL4A5 gene have been identified [
3].
Genotype–phenotype correlations in the kidney have been studied for X-linked AS disease [
15,
16]; however, there are fewer studies regarding the human inner ear in AS. Several studies have reported that hearing loss is one of the early signs of AS [
17‐
19]. It has become evident that hearing loss appears at the end of childhood or at the beginning of adolescence in boys with X-linked disease. A continuous progression in hearing loss indicates a poor prognosis [
13]. Most of the reports have shown that hearing loss in AS is sensorineural, progressive and symmetrical [
20], often affecting the middle but especially high frequencies in a moderate or severe extent. The groove-type audiometric curve is present in only about 47.1% of AS patients [
20]. Hearing loss related to X-linked AS mainly occurs in male patients, and the level of hearing loss is always more severe in men than in women [
21]. It has been reported that the risk of developing hearing loss before the age of 30 was about 60% in patients with a missense mutation, whereas it reached 90% for patients with other types of mutations including splice site mutations [
16].
Till date, there is no trend in hearing changes or possible influencing factors on the hearing level have been reported. Therefore, this study aimed to explore the clinical audiological characteristics in male patients with AS and the auditory genotype–phenotype correlations. The results of this study might be useful in predicting the trends in hearing changes and in providing timely interventions for such patients.
Discussion
There are three types of inheritance in AS: X-linked dominant inheritance, autosomal recessive and autosomal dominant inheritance. Among them, X-linked inheritance is the most important type of inheritance. An earlier study [
27] showed that the XLAS patients have dominant genetic characteristics in both males and females. However, the hearing loss was not manifested in all XLAS patients [
15].
This study showed that the hearing impairments of X-linked male patients were characterized as bilateral symmetrical sensorineural deafness, with an incidence of 63.2% (55/87), and was consistent with that of the literature [
15]. The majority of patients presented with mild-to-moderate hearing loss, and 4 presented with moderate-to-severe hearing loss. In our study, hearing loss was usually started in the middle frequencies and gradually affected high frequencies, which was different from that in the paper [
20]. Our results reported that hearing loss usually started at school age and gradually increased with increasing age. However, it maintained a relatively steady platform level of 50–60 dB HL during the teenage years. The shape of the audiometric curve was mainly groove-type (92.73%), which was quite different from the level reported in the literature [
20]. The characteristics of hearing loss and hearing curve in this study are different from those reported in previous literatures, and this may be related to sample size or age distribution of the subjects. The characteristics of groove-type audiometric curve is clinically rare, contributing to the diagnosis of AS. This meant that, if the hearing loss plots as a groove-type audiometric curve, a preliminary diagnosis of AS can be made in children with hematuria or proteinuria before kidney puncture, skin biopsy pathology, or genetic diagnosis. Studies [
28] have shown that the proportion of patients with AS misdiagnosed as other kidney diseases was up to 86%. Combined with the typical groove-type curve of hearing loss, a preliminary early diagnosis can be made. The audiological features and the relationship between hearing and genotype were analyzed as follows.
Hearing loss in male XLAS patients initially involved the middle frequency range and was gradually increased with increasing age. Middle frequency hearing was relatively stable when it had dropped to a certain level, and then high frequency hearing began to decline. It is speculated that this change in audiology is related to cochlear middle basement and basal membrane disease. During the early stages of the disease, damage to middle basement membrane was more obvious than to the basal membrane, and the lesion on the basal membrane was not sufficient to cause high-frequency hearing loss. With increasing time and age, the middle basement and basal membrane lesions gradually became more severe, with the development of hearing loss at higher frequencies. We hypothesized that there was a different expression and metabolism of type IV collagen regulatory genes in the middle and basal membrane of the cochlea, resulting in different manifestations of early middle frequency hearing loss.
COL4A5 gene mutation is the cause of XLAS, and so far, more than 300
COL4A5 mutation sites have been found [
29]. All 87 patients in this study were tested for
COL4A5/6 genotype, and 60 different
COL4A5 pathogenic variants were identified in 63 patients. There are various forms of
COL4A5 gene mutations, including rearrangement, deletion, insertion and point mutations, leading to splicing mutations, frameshift mutations, missense mutations or truncation mutations. Mutations such as rearrangement, nonsense and frameshift mutations led to the abnormal structure or function of type IV collagen, and are also known to be severe mutations in AS [
15,
16]. Our research concluded that missense mutations are less likely to lead to hearing loss than other mutations. In addition, this study also showed that the type of mutation affects the degree of hearing loss and the age of onset. The more severe the mutation type was, the earlier and the more serious was the hearing loss. The audiological phenotype and genotype are closely related. It has been reported that the risk of developing hearing loss before the age of 30 was about 60% in patients with missense mutations, whereas it reached 90% for the other types of mutations together including splice site mutations [
16]. However, this phenomenon has not been observed in our study. The reasons for this might be due to that most of our outpatients are children, and the population included was basically children-only, where one was over 30-years-old. An earlier study showed that the location of the mutation site is also related to the time of occurrence of end-stage renal disease, i.e., the closer the mutation site is to the 5 ‘end of the gene, the earlier the occurrence of the disease [
30]. However, in this study, there was no significant correlation between hearing level or the age of onset of hearing loss and mutation sites.
It has been reported that in patients with mild gene mutations, hearing loss remains mild [
16]. However, in this study, 9 patients with missense mutations had moderate hearing loss, wherein 7 cases had glycine replaced by other amino acids, 1 case of proline replaced by serine, and 1 case of serine replaced by aspartic acid. Even if the amino acid changes are all from glycine to other amino acids, there was a significant difference in the secondary structure of the α5(IV) chain and the clinical phenotype of the kidney. This might be due to the fact that the glycine is located in different positions in the same domain of α5(IV). Moreover, the degree of change of secondary structure is consistent with the severity of the corresponding renal clinical phenotype [
5]. The gene mutation in one patient in this study was a missense mutation in which the glycine was replaced by aspartic acid and the site was located in the exon 49. Glycine is polar and uncharged, whereas aspartic acid is a negatively polar charged molecule. So, it was speculated that the change in the charge properties of amino acids may affect the normal folding of whole α5(IV) chain. This affects the structure of the basement membrane, leading to a change in hearing. Therefore, it is thought that some missense mutations that caused glycine to be replaced by non-identical amino acids may not be mild mutations, but a more serious clinical phenotype [
31‐
33].
The missense mutations in
COL4A5 collagen region, such as the replacement of glycine or arginine, usually result in a more severe disease in the kidney [
34]. In this study, 5 patients with severe mutations had normal hearing, and their ages were 6, 6, 7, 12 and 15 years old, respectively. This cannot be explained by factors such as the severity of mutations and changes in age. The analysis found that the gene mutation sites were located in exons 8, 19, 22, 25, and 33, respectively, with the mutation site closer to the 5′ end. However, in this study, the relationship between hearing and the location of the mutation was statistically analyzed, and there was no obvious correlation between the hearing level and the location of the mutation, which was inconsistent with the previous reports [
30]. Therefore, further studies are warranted.
At the same time, a total of 49 patients were followed up for more than 2 years, where 28 cases showed a decreasing trend in the hearing level of about 5 dB per year. Through observation, it was found that there was no significant difference in the degree of progression of hearing loss between patients with mild mutations and those with severe mutations after hearing loss had occurred. The European Union’s AS Collaborative Group conducted a study on this phenomenon to evaluate the genetic heterogeneity of diseases among different families of the disease and to study the relationship between genotype differences and end-stage renal disease progression. Therefore, it is necessary for us to further expand the sample size and follow-up time to study the relationship between the auditory genotypes and the phenotypes.
Through family studies, we identified the phenomenon of co-segregation of clinical phenotypes and genotypes. The pathogenicity of 17 kinds of gene mutations found in this study has already been reported in the previous studies. We conducted nucleic acid conservation and pathogenicity analysis and amino acid conservation analysis for unreported gene mutations in this study. The PhastCons and PhyloP software showed that the amino acid sequences were almost highly conserved. Polyphen-2 and SIFT gene prediction softwares were used to predict the possible pathogenicities of 20 unreported missense mutations. Polyphen-2 prediction software could not be able to predict the outcome in 2 patients. In one of these 2 cases, SIFT predicted the pathogenicity, while the other patient was non-pathogenic. The remaining 18 unreported missense mutations were likely to be pathogenic. The severe mutations were predicted to be pathogenic. Further cytological and animal experimental studies should be conducted for those novel mutations that discovered gene loci in this study.