The genome variation results of different patients and their families were classified and summarized by pathogenic genes.
Mutation distribution in suspected LHON patients
In two and one half years, 116 suspected patients with optic neuropathy or LHON were examined to help diagnose 25 cases of LHON (seen in Figure 1) among them. The ratio of male to female in patients with LHON is 4/1 in our investigation. The average age of the patients diagnosed as LHON was 19 years old, their age ranged from 6 to 36 years old. The three common mutant sites of mt.3460, mt.11778 and mt.14484 are main (96%) causes of LHON, and MTND4 m.11778G>A is the most common pathogenic mutation among them, then MTND6 m.14484T>C and MTND1 m.3460G>A. Just one rare mutant that MTND6 m.14502T>C was found in these Chinese patients from central China. Several LHON patients are with incomplete mitochondrial mutation or two mutations.
Pathogenic mutations of the hereditary retinopathy
In the same period, 95 families were examined by using the targeted sequencing technology, which are suspected with retinitis pigmentosa or congenital retinopathy. Partial genealogical trees are shown in Figure S25. We totally identified 68 distinct mutations from 31 different known disease genes in the patients of these families, 37 mutations are novel among them. The results are grouped by related genes found in the retinopathy patients. In this investigation, 57.9% of the families under test have been detected significant mutants (Table 1 and Table 2). The mutations listed in Table 1 are predicted as damaging or disease causing by function analysis software, and a part of the mutations have been studied and reported. The phenotype and mutation of each of these families are co-segregation, respectively. Targeted sequencing of retinopathy related genes provides 45.3% diagnostic rate, and another 12.6% families receive candidate gene mutations with undefined pathogenicity in this study.
Four families (Family 14, 15, 48 and 54) suffering from retinitis pigmentosa caused by RHO mutations, the patients of these families presented with night blindness from childhood, visual field defect or tubular visual field and retinitis pigmentosa. The Sanger sequencing results of mutant sites of Family 14 and Family 15 are presented in Figure S13 and S14 respectively. NDP mutations can lead to familial exudative vitreoretinopathy or Norrie disease. Two families (18 and 46) with FEVR2 have been detected two novel NDP mutations, c.124C>A (p.H42N) (Sanger result in Figure 3) and c.401_402delGA (p.*134Wfs*13). The probands were found that their eyes do not chase things when they a few months old, and no blood vessel area of binocular fundus detected by ophthalmoscopic examination. Male patients of the two families have no other serious visual problems. Two families (32 and 55) diagnosed as Norrie disease have two known NDP mutations, c.343C>T (p.R115X) and c.268C>T (p.R90C). The two months old male patient in Family 32 has vitreous hyperplasia, right microphthalmos and microcorneas, while the male patient in Family 55 is total blindness and atrophy of eyeballs. Mutation of USH2A can cause retinitis pigmentosa with or without syndromic hearing loss. The patients from Family 7 and Family 47 with Usher syndrome, type 2A, they presented with retinitis pigmentosa and hearing impairment. The two patients have different mutations in USH2A gene (Figure S11, S17 and S18). The patient of Family 9 with USH2A mutation just has nonsyndromic retinal diseases. Three families (Family 27, 38 and 51) were detected different RS1 hemizygous mutations in the retinoschisis patients. The results of fundus examination and optical coherence tomography of the congenital retinoschisis patient in Family 38 are shown in Figure S5 and S6. Patients from the two families (Family 1 and 2) can be diagnosed as RP, 38 caused by MERTK gene mutations. These patients are characterized by retinitis pigmentosa, night blindness and visual field loss.
Small deletion and nonsense mutation of CYP4V2 resulted in Bietti crystalline corneoretinal dystrophy of the patients from Family 3 and 4. The Patient’s result of visual electrophysiology in Family 3 is shown in Figure S1. The CYP4V2 c.(802-8)_810delTCATACAGGTCATCGCTinsGC and c.958C>T mutations in Family 4 are shown in Figure S8 and S9. FSCN2 c.72delG causes retinitis pigmentosa, type 30 in two unrelated families (Family 5 and 6). FSCN2 c.72delG Sanger sequencing result is shown in Figure S10. Small deletion and frameshift mutation of PRPF31 resulted in retinitis pigmentosa, type 11 of the patients from Family 12 (PRPF31 c.1074-8_1079delGTACCGGTCCCCAG novel mutation seen in Figure S12) and Family 50. There are four RP patients from three generations of the Family 12. In addition to the symptoms of retinitis pigmentosa, night blindness and tubular visual field, the proband and his father (Figure S25) were also treated with bilateral cataract surgery. There are two families (Family 33 and 52) with family history of RP and night blindness caused by the same mutation, RPGR c.2236_2237delGA. There are two families (Family 43 and 44) with family history of RP and night blindness caused by the different mutations of RP2, and the splicing mutation c.769-2A>G has been reported to pathogenic for RP, the frameshift mutation c.572_582dup11 is novel.
There seventeen families affected by different retinal diseases have been detected pathogenic or likely pathogenic mutations of 17 different related genes. The patient of Family 36 with macular degeneration has poor eyesight. The patient of Family 16 has retinochoroidal coloboma, his results of visual field examination and mutation sequencing is shown in Figure S4. Sanger sequencing result of the mutant site in Family 17 is shown in Figure S15, and the RP proband also combined with cataract when he twenty-six years old. The patient of Family 20 was two years old (Sanger result seen in Figure S20), her full field ERG showed that rod cells had no waves, while scotopic ERG showed that the amplitudes ofαandβwaves decreased. The ophthalmoscopic image and sequencing result of RCS patient from Family 22 are presented in Figure S19. The CNGA1 mutations in Family 45 were validated by Sanger sequencing (Figure S16). The thirty-four years old mother and her daughter have macular degeneration of fundus in Family 34. The forty-one years old patient of Family 35 suffered from retinal detachment, primary vitreous hyperplasia and familial exudative vitreoretinopathy, and his mother with the same FZD4 c.612T>A heterozygous mutation also had the same eye symptoms. Both of the thirty-three years old man and his mother with neurodeatrophia and familial exudative vitreoretinopathy carry the LRP5 c.485_488delACGG heterozygous mutation in Family 37. The three years old girl with congenital horizontal nystagmus has compound heterozygous variation of SLC38A8 in Family 39, and her parents are carriers of the heterozygous variation. The two years old boy is a Leber congenital amaurosis patient, and his parents are the carriers of heterozygous variation of AIPL1 in Family 40. The hemizygous FRMD7 c.910C>T (p.R304X) mutation leaded to Nystagmus of the boy in Family 41, and his mother carries the heterozygous mutation. The five years old boy diagnosed as Leber congenital amaurosis caused by homozygous mutation of GUCY2D c.3177_3178delAC inherited from parents.
Variants of undetermined significance in retinopathy families
The mutations listed in Table 2 are predicted as damaging or associated with the clinical phenotypes of the families, which can be considered as candidate mutants but not fully determined. The families included in Table 2 usually have no family history of hereditary diseases. There are four families (8, 10, 23 and 53) been detected different compound heterozygous mutations of USH2A, the mutations associate with the nonsyndromic phenotype of retinitis pigmentosa of these patients without obvious hearing impairment. The mutations found in the four families are likely pathogenic. The four years old boy in Family 19 was detected compound heterozygous mutation of USH1C, which gene variation can cause Usher syndrome -type 1C characterized as severe hearing impairment and retinitis pigmentosa. The patient with RP and night blindness has suffered from bilateral secretory otitis media, but his bilateral hearing is basically normal. His both ears passed TEOAE (transient evoked otoacoustic Emissions) examination and DPOAE (distortion product otoacoustic emissions) test at the acoustic frequency (1k, 2k, 4k and 8k Hz), but left ear did not pass DPOAE at 0.5k Hz acoustic frequency. I wave latency was slightly longer after 80 dBnHL short-tone stimulation in ABR (auditory brainstem response) test of the boy, and other waves were normal. So the USH1C mutation is associated with the phenotypes, but undetermined significance. The other patients from different families (13, 25, 56, 24, 29 and 31) (Table 2) have candidate gene mutations and corresponding phenotypes, respectively. The Sanger sequencing results of Family 56 is shown in Figure S21 and S22. It should be noted that the RS1 c.240G>C (p.Q80H) mutation not co-separated from the phenotype and genotype in Family 26.
Prenatal diagnosis of families with confirmed patients
There are 11 families with patients confirmed on clinical and genetic level subjected to prenatal molecular diagnosis. The fetuses from Family 15 and 43 were diagnosed as RP on gene level, they then terminated the pregnancy. The fetus from Family 18 was diagnosed as FEVR2 on gene level, but the mother decided to continue pregnancy due to the clinical heterogeneity of the disease. The baby of Family 18 was born as FEVR2 with mild phenotype. The Family 2 obtains healthy offspring through prenatal diagnosis and the third generation of IVF technology. There five families (Famliy 17, 33, 44, 49 and 55) have healthy offsprings through natural pregnancy and prenatal diagnosis. The heterozygous carriers born from two families (Family 42 and 46) are not presented clinical phenotypes of the diseases, respectively.
Family 2: There are two RP patients in Family 2. The thirteen years old sister with patchy defects of visual field and abnormal ERG, she has homozygous mutation of MERTK c.754delC (Figure 4). The ten years old brother’s symptoms was milder than his sister, he also had defects of visual field (Figure S7) and carried the same homozygous mutation. They all suffered from night blindness and visual impairment. Their parents are the carriers of the heterozygous variation of MERTK c.754delC. According to ACMG guidelines, the novel frameshift mutation of MERTK c.754delC should be considered as pathogenic, and its grade (PVS1) is high. This family acquired a healthy boy through three generations of IVF technology (pre-implantation genetic diagnosis).
Family 3: The patient with homogeneous mutation of CYP4V2 c.(802-8)_810delTCATACAGGTCATCGCTinsGC has retinitis pigmentosa and visually impaired. This mutation is known pathogenic for Bietti crystalline corneoretinal dystrophy (Bietti CCD), it involves small deletion and insertion in splicing regions. She has the typical fundus and visual electrophysiological symptoms (Figure S24 and Figure S1). So the patient can be diagnosed as Bietti CCD by combining ocular manifestations and gene mutation.
Family 5: The RP patient in Family 5 all carried known pathogenic mutation, FSCN2 c.72delG. The proband has the typical fundus and visual electrophysiological symptoms (Figure S23 and Figure S2). This mutation is the same genetic cause for Family 6, and it is a common pathogenic mutation for RP, 30.
Family 11: The three years old boy, one of fraternal twins, sought medical advice for night blindness. The patient’s clinical manifestations also included retinal abnormalities, lateral nystagmus and finger stimulating eyeball phenomenon. He has the TULP1 compound heterozygous mutation of c.1318C>T (p.R440X) and c.1142T>G (p.V381G), his parents are heterozygous variants carrier of each of the two mutations. The nonsense mutation c.1318C>T (p.R440X) is known pathogenic for Leber congenital amaurosis, type 15, and the missense mutation c.1142T>G (p.V381G) is novel. c.1142T>G can lead to amino acid substitution of the protein product, it affects the function. The eye examination and mutations are presented in Figure 2. So the boy was diagnosed as LCA 15 by combining clinical manifestations and gene mutation.
Family 18: The one years old boy’s fundus photographs and mutation sequencing results are shown in Figure 3. The cornea was clear in both eyes, the anterior chamber was preserved and the lens was transparent. The fundus examination showed that there was no blood vessel area in both eyes. The temporal epiretinal membrane of the right fundus vascular arch pulled the macula. The mutation of NDP can lead to familial exudative vitreoretinopathy, type 2 (FEVR2), and c.124C>A (p.H42N) is novel for FEVR2. There is one known pathogenic mutation of c.125A>G (p.H42R) at the same location of polypeptide chain of this novel variant. According to ACMG guidelines and related prediction software, the c.124C>A (p.H42N) should be pathogenic. FEVR2 is characterized by no blood vessel area of fundus, but severity of the disease varies. There three persons with c.124C>A (p.H42N) mutation have no blood vessel area in both fundus from this family.
Family 21: This is a consanguinuous marriage family of Chinese Hui (A Chinese minority). The patient presented with retinoschisis, macular edema and night blindness, and he carried the homozygous mutation of NR2E3 c.925C>T (p.R309W). The ophthalmological examination and mutation sequencing results of the patient are shown in Figure 5 and Figure S3. The missense mutation c.925C>T of NR2E3 is novel for Goldmann-Favre syndrome, but the c.925C>G (p.R309G) at same location of mRNA and polypeptide chain is known pathogenic for Goldmann-Favre syndrome and Enhanced S-cone syndrome. Some scholars believe that the severe type of Enhanced S-cone syndrome is Goldmann-Favre syndrome. The patient's condition has worsened over the past 10 years, and he can be diagnosed as Goldmann-Favre syndrome by combining phenotype and genotype.