3.1 Clinical characteristics of patients with LOXHD1 variants
In this study, three affected children from three non-consanguineous Chinese families were identified. All of these families have negative family histories of congenital or early-onset HL. The detailed clinical features of these patients are listed in Table 1.
All patients presented with a prelingual onset hearing loss, and the diagnosed age varied from 2 to 8 years old. They all had bilateral, moderate to severe sensorineural HL (Fig. 1A). DPOAEs of all patients were absent bilaterally. PTA revealed that patient #1 and #2 had severe HL. ABR and ASSR revealed that patients #3 had bilateral moderate HL, and no increases in interpeak latencies were observed. The hearing loss was found to worsen with age in patient #1, with three years’ follow-up. Speech audiometry showed that the speech recognition thresholds (SRT) were 80 dB and 85 dB in the left and right ear of patient #1; 65 dB and 100 dB in the left and right ear of patient #2 (Fig. 1B). Moreover, a maximum of 95% and 100% speech recognition was attained at 110 dB sound level in the better ear of patient #1 and patient #2. Speech in the noise test showed that 50% speech recognition was attained at the signal-to-noise of 5 dB with 100 dB for patient #1, while 60% speech recognition was attained at signal-to-noise 5 dB with 100 dB for patient #2.
None of the affected individuals complained of tinnitus and vertigo. Besides, none of the family members complained of eye pain, glare, or blurred vision. Otoscopic examination and external ear inspection were normal in all patients. The MRI and CT of the brain and temporal bones of the left and right ears for patient #1 and patient #2 demonstrated to be normal (Fig. 1C). No known causes of acquired HL were found in our patients, such as prenatal infection, TORCH (toxoplasmosis, rubella, CMV, HSV) infection, aminoglycoside exposure, otoacoustic trauma, meningitis.
None had delayed motor development or dysmorphology. Besides, the Wechsler Intelligence Scale test for children (WISC-III) was performed on patients #2, and the score was 44 of Verbal IQ, 98 of Performance IQ, 63 of full-scale IQ.
3.2 Variation detection and functional analysis
We identified six novel LOXHD1 variants. All the variants were detected in a compound heterozygous state (Fig. 1D). The variants consisted of one nonsense, two splicing, and three missense variants. The six variants were all located in a well-conserved site (Fig. 2A). Besides, all variants are located in PLAT domains of LOXHD1 protein, with one except (c.4814T>C, p.Met1605Thr) (Fig. 3A). Based on the ACMG guidelines, the variants were categorized into two pathogenic, three likely pathogenic, and one uncertain significance (Table 2).
Molecular models for the homeodomain of wild-type and variant-containing LOXHD1 protein were developed and adopted a β sandwich fold containing PLAT domains. The two missense variants (c.6038G>A, p.Gly2013Glu; c.1891C>T, p.Arg631Cys) were predicted to change the polarity with surrounding active site residues and consequently destabilize the β sandwich structures (Fig. 2B).
The two splicing variants were evaluated in silico using the web tool HSF. The variant (c.3351-1C>A) was predicted to alter the wide-type acceptor sites and may affect splicing by HSF; variants c.4212+5G>A were predicted to alternate the original splicing donor site and then affected the splicing (Supplementary Fig. 1).
The missense variant (c.4814T>C, p.Met1605Thr) was absent in the ExAC, 1000 Genomes, and gnomAD databases and highly conserved across different species. Unfortunately, the Met1605Thr protein template cannot be obtained from Swiss-model servers, and more evidence for its pathogenicity was not obtained. Thus, the pathogenicity of this variant is uncertain.
3.3 Literature review of published cases
To date, a total of 111 cases with DFNB77 were reported, including the three cases in this study (Supplementary Table 2). We found that most patients’ onset age was less than ten years old (68.5% for < 5 years and 14.4% for 5-10 years). There have 16.2% of patients shown severe HL, and 50.5% shown profound HL. 28.8% of patients were described with progressive HL, and 56.2 % of them occurred in adulthood (Table 3). The complete audiometric data were divided into four groups by age (<10, 10-19, 20-55, and >55 years old), and we found that the main audiogram of DFNB77 among different age groups was a high-frequency down-sloping configuration (Fig. 4). The degree of HL was observed increased in all frequencies in adulthood and more severe in elderhood. We further compared the genotype and phenotype among different populations, and we found that the genotype of LOXHD1, the severity of HL, the type of HL, and the audiogram had significant differences (P <0.05) (Table 4). The profound HL, serious form HL, and gently sloping audiogram HL in the Middle East are obviously common than others.
A total of 85 variants of LOXHD1 were reported with the 111 reported cases, including 19 nonsense, 6 frameshifts, 2 deletions, 1 indel, 13 splice-site variants, 43 missense, and 1 synonymous variant (Fig. 3A). Among them, 67 (79%) variants were located in the PLAT domains, 13 (19%) of which were in the PLAT14 domain (Fig. 3B). The most common mutations were c.4212+1G>A; c.4714C>T, Arg1572Ter; and c.4480C>T, Arg1494Ter with a frequency of 14.8%, 8.1%, and 6.3%, respectively. The LOXHD1 variants in 65 (59%) patients were in a compound heterozygous state (Table 5). However, in the Middle East, all LOXHD1 variants were in the homozygous state (Table 4)
3.3.3 Genotype-phenotype correlations analysis
About 60% of variants were associated with a milder auditory phenotype, with residual hearing at low frequencies (250 Hz, 500 Hz). Moreover, 19% of the variants that cause serious phenotype were located in the PLAT14 domain, indicating that it is the most frequent domain with a serious phenotype (Fig. 3B). 55% of the variants with milder phenotype were missense variants (Fig. 3C). We further compared the different combinations of LOXHD1 mutations. The results showed that compared with homozygous variants of LOXHD1, individuals with heterozygous compound variants had a significantly milder phenotype, especially individuals carrying one missense and one splicing or bi-allelic missense variants (P <0.05) (Table 5).