USH followed an autosomal recessive inheritance pattern in all patients (Fig. 1). Bone-spicule hyperpigmentation, attenuated arteries and sensorineural deafness signs were consistently observed in all patients. Based on the classification of visual acuity according to the World Health Organization (International Classification of Disease 11, 2018), three patients were legally blind, and one patient had severe hearing impairment. Optical coherence tomography (OCT) imaging suggested significantly diminished macular ganglion cell complex (GCC) thickness, reduced macular thickness and disorganized outer segment structure (Fig. 2e). Optical coherence tomography angiography (OCTA) revealed a decreasing macular capillary flow density in the superficial and deep capillary plexus, while the foveal avascular zone (FAZ) was also enlarged (Fig. 2f). Fundus autofluorescence (FAF) images collected from patients are shown in Figs. 2&3. ERG showed no response from the rod cells (Fig. 3f). One patient (F5-III-1) had a hearing aid when she was five years old (Fig. 3e). The clinical findings of each patient are summarized in Table 1.
Targeted exome sequencing data analysis
Targeted exome sequencing (TES) of 381 genes implicated in inherited retinal degeneration (IRDs) was performed. On average, 2.7 GB of raw data were generated per targeted exome, of which 90.30% to 94.58% had quality scores above Q30. Each targeted exome contained approximately 5,554,000 reads, while an average of 4,502,000 reads remained after adapter trimming. Overall, the mapped reads achieved more than 99.89% coverage of the targeted regions. The average sequencing depths of the targeted regions ranged from 274.42X to 754.24X (Supplemental table 1). Overall, 99.45% to 99.77% of targeted exons exhibited coverage > 10X and 97.63% to 99.49% had coverage > 20X. Approximately 3,000 variants were identified for each sample by using the SOAPsnp program. After excluding the SNPs released of the 1000 Genomes Project with a MAF>0.05 and variants reported in HapMap 28, the variants were further narrowed down to 26–223, respectively. USH is inherited in an autosomal recessive pattern, so homozygous and compound heterozygous variants were considered for analysis. Among the 370 substitutions, fewer than 20 candidate mutations were further selected by using functional prediction and conservation analysis and consistency with the genetic transmission mode. By following the described sample filtering strategy, we successfully identified candidate mutations in all patients.
Confirmation of pathogenic mutations by familial validation and Sanger sequencing
We identified disease-causing mutations in five families with sporadic USH2 (Table 2). In the five pedigrees, the probands were all diagnosed with typical USH2 based on their clinical features. In family F1, the proband (F1-II-3) carried a homozygous USH2A mutation (c.8559-2A>G, splice site), which was subsequently transmitted to the healthy (heterozygous) daughter (F1-III-1), and we reconfirmed that no consanguineous marriage had occurred. In family F2, the TES results from the proband showed two compound heterozygous USH2A mutations (R626X and c.8559-2A>G), and the unaffected son harboured a heterozygous USH2A mutation (R626X). In pedigree F3, the proband (F3-II-2) was found to carry two mutations (P350R and exon 61dup). This condition was further confirmed by intrafamilial validation with Sanger sequencing and MLPA. One mutation (P3505R) was transmitted from the unaffected mother (F3-I-2), and it is conceivable that the mutation (USH2A exon 61dup) was inherited from her (now-deceased) father (F3-I-1), as her unaffected brother also harboured this mutation. In family F4, a compound heterozygous mutation (c.8559-2A>G and c.11389+3A>T) was identified in the proband (F4-II-3). Sanger sequencing of the coding region further confirmed that the mutation c.8559-2A>G was derived from an unaffected mother (F4-I-2), while two unaffected siblings (F4-II-1 and F4-II-2) had no mutation. In the last pedigree, F5, the proband (F5-III-1) was found to carry three USH2A gene mutations. Two novel heterozygous mutations, c.4397-2A>T and c.4555G>A (G1519R), were confirmed in her unaffected father (F5-II-3), and the same two mutations (c.4397-2A>T and c.4555G>A ) were present in the maternal allele. Another splicing mutation, c.11389+3A>T, was identified in the unaffected mother (F5-II-4). Thus, all identified mutations were successfully validated in five USH families via expanded TES, Sanger sequencing and cosegregation analysis.