Enhanced Gene-Disease Association and Carrier Screening by Homozygous Mutation Carriers: A Study of Genotypes and Clinical Phenotypes in 17 IRD Families with Homozygous Mutations in Northern China

Enhanced Gene-Disease Association and Carrier Screening by Homozygous Mutation Carriers: A Study of Genotypes and Clinical Phenotypes in 17 IRD Families with Homozygous Mutations in Northern China | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Article Enhanced Gene-Disease Association and Carrier Screening by Homozygous Mutation Carriers: A Study of Genotypes and Clinical Phenotypes in 17 IRD Families with Homozygous Mutations in Northern China Xue Fan, Zhen Li, Lingzhi Sha, Xunlun Sheng, Weining Rong This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4776885/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Jan, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract In this study, patients with inherited retinal dystrophies (IRDs) who visited Ningxia Eye Hospital from January 2015 to September 2023 were analyzed. Through whole genome exon sequencing (WES) and Sanger verification, 17 probands carrying homozygous mutations were detected. The association between the genotype and clinical phenotype of patients with homozygous variants was analyzed. Among all the patients, 3 patients (17.6%) had a family history of consanguineous marriage, and the onset age of 5 patients(29.41%) was less than 10 years. According to 12 patients (70.59% ), they had the best corrected visual acuity (monocular) < 0.3. 3 were blind, 9 with moderate to severe visual impairment, and 2 with mild visual impairment. 16 homozygous variants were detected on 9 different genes, of which 7 were de novo homozygous variants, including frameshift variants, missense variants, and a copy number variant. These mutations are related to clinical phenotypes such as Usher syndrome type II (USHⅡ), Stargardt disease (STGD), Retinitis Pigmentosa (RP), Leber congenital amaurosis (LCA), and Bardet-Biedl syndrome (BBS) respectively. The results of the study indicate that more than 80% of homozygous variant carriers originated from non-consanguineous families, emphasizing the significance of genetic screening for individuals who lack a family history of consanguineous marriage and no obvious clinical phenotypes, but who may carry genetic pathogenic variants for genetic diseases. Furthermore, analysis of patient genotypes and clinical phenotypes further expanded the spectrum of mutations in pathogenic genes for IRDs and the spectrum of clinical phenotypes for variants in known genes, and identifying couples at high fertility risk and individuals with moderate or greater severity IRDs can provide a basis for genetic counseling, reproductive decision-making, disease prevention, and management. Biological sciences/Genetics Health sciences/Diseases/Eye diseases Inherited retinal dystrophies Homozygous variation Genotype Clinical phenotype Consanguineous marriage Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Inherited retinal dystrophies (IRDs) are the most common and serious clinical blinding eye disease, and the leading cause of blindness in children and young adults worldwide [ 1 ] , for which there is no effective treatment yet. The incidence of IRDs is about 1:4000 in developed countries [ 2 ] , and in China, epidemiological surveys show that the incidence of retinitis pigmentosa (RP) is as high as 1:1000 in people over 40 years old [ 3 ] . In recent years, new ophthalmic drugs for gene therapy have stepped into the "fast track of transformation", bringing bright hope to IRD patients. The prerequisite for achieving IRD gene therapy is to identify the pathogenic genes and make an accurate diagnosis. Currently, there are still the following problems in the clinical diagnosis of IRDs: numerous pathogenic genes, complex and diverse clinical phenotypes, intricate relationships between genotypes and clinical phenotypes, and the lack of professional genetic knowledge among ophthalmologists lead to misdiagnosis, underdiagnosis, and mistreatment of a large number patients with IRDs. The ClinGen guidelines state that two evidences are supporting the genetic link to disease: genetics (e.g. case studies) and experiments (e.g. animal models) [ 4 ] [ 5 ] . To demonstrate the superiority of genetic evidence, the maximum score of experimental evidence (6 points) was only half that of gene evidence (12 points). This highlights the importance of reporting more cases with the same clinical phenotype to increase the credibility of the gene-disease association. As compared to compound heterozygous variants, homozygous variants are less common. The homozygous variant refers to the same variant present in both alleles at the same locus on a pair of homologous chromosomes. Since two alleles on the same chromosome have the same function, the homozygous variant can more fully express the loss of function and the phenotypic effects of the mutation. Meanwhile, patients with a homozygous variant will inherit the variant stably from their offspring, all of whom will carry the same variant, resulting in an increased incidence of genetic disease in their offspring. Therefore, screening tests for individuals who have no obvious genetic disease phenotype but may carry pathogenic variants of genetic disease genes is an effective way to reduce the incidence of hereditary diseases. In this study, the genotypes and clinical phenotypes of 17 IRD patients who carry homozygous variants were studied, and we performed screening tests on IRD probands’ parents who carried homozygous variants. The aim is to expand the spectrum of IRD pathogenic gene variants, and investigate the relationship between homozygous variants and clinical phenotypes of IRDs, to provide a basis for genetic counseling, reproductive decision-making, disease prevention, and management. Results In this study, 17 patients with homozygous variants were collected, and 3 had a family history of consanguineous marriage. The average age was 21.23 years old (ranging from infancy to 51 years). 29.41% (5/17) of patients developed symptoms before 10 years. The best-corrected visual acuity (in one eye) of 70.59%(12/17) patients was less than 0.3 and 35.29% (6/17) with best-corrected visual acuity (in one eye) better than 0.5 (Table 1 ). According to the visual impairment standards established by WHO in 2019, 3 were blind, 9 had moderate to severe visual impairment, 3 had mild visual impairment, and 2 were close but did not meet the criteria for visual impairment. A total of 16 variants were detected in 17 patients, including 9 genes: USH2A, CYP4V2, PROM1, RP1, CNGA1, PRPH2, ABCA4, CRB1 , and BBS9 , which were involved in 6 inherited retinal diseases: Retinitis pigmentosa (RP), Usher syndrome (USH), Bietti crystalline chorioretinal dystrophy (BCD), Stargardt disease (STGD), Leber's congenital amaurosis (LCA), and Bardet-Biedl Syndrome (BBS). This study identified 7 novel variants: including 4 frameshift mutations - c.10179delG (p.Met3393fs) of USH2A gene, c.544dupC (p.Gln182fs) of PROM1 gene, c.265del (p.Leu89Phefs*4) and c.253del (p. Leu85Phefs*4) of CNGA1 gene; 2 missense variants - c.1363G > C (p.Gly455Arg) of PROM1 gene, c.1363G > C (p.Gly455Arg) of PRPH2 gene; and 1 copy number variant - copy number loss of BBS9 gene located in the region of chr7. 33404663–33409821(Table 2 ), all of which were pathogenic/likely pathogenic(Table 3 ). The clinical diagnosis was Usher syndrome type II (USHⅡ), Stargardt disease (STGD), Retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), and Bardet-Biedl syndrome (BBS) respectively. The parents of all the probands had normal clinical phenotypes and carried the same heterozygous variants of the above-mentioned related genes. Analysis of the pathogenicity and clinical phenotypes of newly identified homozygous variants Family 4 The proband was a 39-year-old male, who complained of progressive binocular vision loss accompanied by night blindness for 19 years, and he denied family history and consanguineous marriage history. The BCVA was 0.3 in the right eye and 0.4 in the left eye (Table 1 ). No abnormalities were observed in the anterior segment of both eyes. The fundus examination revealed the boundaries of the optic discs were clear and the color was waxy yellow, with no reflection in the macular fovea, the retinal vessels were attenuated, and bone spicule-like pigment deposits were visible on the peripheral retina. OCT showed the disappearance of the light reflection signals in the local ellipsoid zone and interdigitation zone of the fovea, along with the atrophy of the retinal pigment epithelium. ERG showed significant impairment of cone and rod cell function in both eyes. As validated by the whole exome sequencing and Sanger sequencing, the proband carried a homozygous frameshift variant c.10179delG (p.Met3393fs) in USH2A gene, which resulted in a premature translational-termination codon (PTC) due to a frameshift at the 3393 codon (Fig. 1 ). According to the ACMG guidelines, the frameshift variant was considered as Pathogenic Very Strong (PVS1), while the absence of this variant in the normal population database was considered as Pathogenic Moderate (PM2), the proband's clinical symptoms were consistent with Usher Syndrome, a monogenic genetic disease, which was considered as Pathogenic Supporting (PP4). Therefore, the variant c.10179delG (p.Met3393fs) was classified as Pathogenic (PVS1 + PM2 + PP4). The proband had poor hearing since childhood and was eventually diagnosed as Usher syndrome type II. Family 7 The proband of family 7, age of 7, complained of progressive bilateral vision reduced, and his parents denied family history and consanguineous marriage history. BCVA was 0.25 in the right eye and 0.15 in the left eye (Table 1 ). The anterior segment was normal and fundus examination showed pale optic discs with clear borders in both eyes. No reflection in the macular fovea, oval atrophy, and yellowish-white patchy exudation were observed in the macular area, showing a bull's-eye change. OCT indicated a significant thinning of the macular fovea, disappearance of the outer nuclear layer and ellipsoid zone, and atrophy of the retinal pigment epithelium. Fundus angiography reveals 'worm-eaten-like' fluorescent spots around the macular region. As validated by the whole exome sequencing and Sanger sequencing, a homozygous frameshift variant c.544dupC (p.Gln182fs) was detected in PROM1 gene of the proband (Fig. 2 ). According to the ACMG guidelines, the frameshift variant was considered as Pathogenic Very Strong (PVS1), the variant was not detected in the normal population database or the known variant databases was considered as Pathogenic Moderate (PM2), the proband's clinical symptoms were consistent with Stargardt disease, a monogenic genetic disease, which was considered as Pathogenic Supporting (PP4). Therefore, such a variant was classified as pathogenic (PVS1 + PM2 + PP4) based on the standards and guidelines for the interpretation of sequence variants. The proband was finally diagnosed with Stargardt disease. Family 8 The proband of family 8 was a 32-year-old female who presented with bilateral vision loss accompanied by night blindness for 17 years, and she denied any family history or consanguineous marriage history. BCVA was CF in the right eye and HM in the left eye (Table 1 ). The anterior segment was normal and fundus examination showed the waxy yellow optic discs with clear borders in both eyes. No reflection in the macular fovea, the retinal vessels were attenuated, and bone spicule-like pigment deposits were visible on the peripheral retina. OCT showed mild thinning of the macular region. ERG indicated significant impairment of cone and rod cell function in both eyes. As validated by the whole exome sequencing and Sanger sequencing, a homozygous missense variant c.1363G > C (p.Gly455Arg) was detected PROM1 gene of the proband and his parents carried the same heterozygous variant respectively. This variant resulted in a change of the 455th codon from encoding glycine to encoding arginine (Fig. 3 ). According to the ACMG guidelines, this variant was not detected in the normal population database or in the known variant databases, which was considered as Pathogenic Moderate(PM2). Moreover, proteomic conservation analysis suggested that the p.G455R mutation results in the substitution of a nonpolar, uncharged glycine at site 455 with a nonpolar, positively charged arginine. The N atom of the main chain forms a hydrogen bond with the O atom of the large, nonpolar, uncharged phenylalanine at site 451, with a hydrogen bond distance of 3.0 Å (the distances of normal protein structures is 2.9 Å). The O atom of the main chain forms a hydrogen bond with the N atom of the nonpolar, uncharged glycine at site 459, with a hydrogen bond distance of 2.7 Å (the distances of normal protein structures is 3.0 Å). These changes in amino acid interactions lead to alterations to the structure and function of the protein after the mutation (Fig. 3 ). A variety of bioinformatics computing software indicated the deleterious impact of this variant (PP3_Supporting) (Table 3 ). The genotype and clinical phenotypic co-segregation in family members were considered as supportive evidence (PP1), the phenotype of the variant carriers was highly consistent with the monogenic genetic disease (RP), which was considered supportive evidence (PP4), gene testing report from a reliable and authoritative source considered this variant as Pathogenic, which serves as additional supportive evidence (PP5). Therefore, such a variant was classified as Pathogenic (PM2 + PP3 + PP1 + PP4 + PP5) based on the standards and guidelines for the interpretation of sequence variants. The problem was ultimately diagnosed with binocular RP. Family 12 The proband of family 12 was a 51-year-old female, who complained of progressive night blindness and vision decline for 20 years in both eyes, and she denied family history and consanguineous marriage history. BCVA was 0.5 in the right eye and 1.0 in the left eye (Table 1 ). No abnormalities were observed in the anterior segment of both eyes. Fundus examination showed the waxy yellow optic discs with clear borders in both eyes. No reflection in the macular fovea, the retinal vessels were attenuated, and bone spicule-like pigment deposits were visible on the peripheral retina. OCT showed the disappearance of the light reflection signals in the local ellipsoid zone and interdigitation zone at the macular center in the right eye, along with atrophy of the retinal pigment epithelium. ERG showed significant impairment of cone and rod cell function in both eyes. As validated by the whole exome sequencing and Sanger sequencing, a homozygous frameshift deletion variant c.265del (p.Leu89Phefs*4) was detected in CNGA1 gene of the proband, which led to a premature stop codon at position 4 of the new reading frame, and caused a frameshift beginning with codon Leucine 89, changing this amino acid to a Phenylalanine. The daughter of the proband carried the same heterozygous variant with normal clinical phenotype (Fig. 4 ). According to the ACMG guidelines, this variant was considered as Pathogenic Very Strong (PVS1), the variant was not detected in the normal population database or in the known variant databases was considered as Pathogenic Moderate (PM2), The genotype and clinical phenotypic co-segregation in family members was considered as supportive evidence (PP1). The proband's clinical symptoms were consistent with retinitis pigmentosa, a monogenic genetic disease, which was considered Pathogenic Supporting (PP4). Therefore, such a variant was classified as Pathogenic (PVS1 + PM2 + PP1 + PP4) based on the standards and guidelines for the interpretation of sequence variants. The proband was diagnosed with binocular RP. Family 13 The proband of family 13 was a 20-year-old male, who complained of the left eye blurred vision accompanied by night blindness, and he denied family history and consanguineous marriage history. BCVA was 0.8 in the right eye and 0.1 in the left eye (Table 1 ). Exotropia in both eyes, with IOL in the left eye. Fundus examination showed the waxy yellow optic discs with clear borders in both eyes. No reflection in the macular fovea, the retinal vessels were attenuated, and bone spicule-like pigment deposits were visible on the peripheral retina. OCT examination showed the disappearance of the light reflection signals in the local ellipsoid zone and interdigitation zone at the macular center, along with atrophy of the retinal pigment epithelium in the left eye. As validated by the whole exome sequencing and Sanger sequencing, a homozygous frameshift deletion variant c.253del (p.Leu85Phefs*4) was detected in the CNGA1 gene of the proband, which led to a premature stop codon at position 4 of the new reading frame, and caused a frameshift beginning with codon Leucine 85, changing this amino acid to a Phenylalanine. The mother of the proband carried the same heterozygous variant with normal clinical phenotype (Fig. 5 ). According to the ACMG guidelines, this variant was Pathogenic Very Strong (PVS1), the variant was not detected in the normal population database or in the known variant databases was considered as Pathogenic Moderate (PM2). The proband's clinical symptoms were consistent with retinitis pigmentosa, a monogenic genetic disease, which was considered Pathogenic Supporting (PP4). Therefore, such a variant was classified as Pathogenic (PVS1 + PM2 + PP4) based on the standards and guidelines for the interpretation of sequence variants. The proband was ultimately diagnosed with binocular RP. Family 14 The proband of family 14 was a 6-year-old male, whose parents complained that the proband had poor vision in both eyes since childhood, they denied family history and consanguineous marriage history. BCVA was 0.02 in both eyes (Table 1 ). The proband exhibited horizontal nystagmus in both eyes, difficulty fixation, and eye poking, with no obvious abnormalities observed in the anterior segment. Fundus examination showed the boundaries of the optic discs were clear and the color was pale, the light reflection of the foveal centralis was not visible. OCT examination showed a mild elevation of the ellipsoid zone and interdigitation zone in the macular region of the right eye. As validated by the whole exome sequencing and Sanger sequencing, the homozygous variant c.640T > A (p.Cys214Ser) was detected in PRPH2 gene of the proband, which resulted in a change of 214th codon from encoding Cysteine to Serine (Fig. 6 ). According to the ACMG guidelines, this variant was not detected in the normal population database or in the known variant databases, which was considered as Pathogenic Moderate(PM2). A variety of bioinformatics computing software indicated the deleterious impact of this variant (PP3_Supporting)., Moreover, proteomic conservation analysis suggested that the p.C241S mutation results in the substitution of a polar, uncharged cysteine at site 241 with a polar, uncharged serine. The N atom of the main chain and the hydroxyl group of the side chain form hydrogen bonds with the O atom of the nonpolar, uncharged phenylalanine at site 211, with hydrogen bond distances of 3.2 and 2.7 Å (the distances of normal protein structures are 3.1 Å). The O atom of the main chain forms a hydrogen bond with the N atom of the side chain of the nonpolar, uncharged tryptophan at site 246, with a hydrogen bond distance of 3.1 Å (the distances of normal protein structures is 3.2 Å). These changes in amino acid interactions lead to alterations in the structure and function of the mutated protein (Fig. 6 ). The genotype and clinical phenotypic co-segregation in family members was considered as supportive evidence (PP1), the proband's clinical symptoms were consistent with LCA, a monogenic genetic disease, which was considered as Pathogenic Supporting (PP4), gene testing report from a reliable and authoritative source considered this variant as Pathogenic, which serves as additional supportive evidence (PP5). Therefore, such a variant was classified as Pathogenic (PM2 + PP1 + PP3 + PP4 + PP5) based on the standards and guidelines for the interpretation of sequence variants. The proband was ultimately diagnosed with binocular LCA. Family 17 The proband of family 17 was a 7-year-old normal color, fundus tessellation with no light reflection of foveal centralis. OCT examination showed no obvious structure of the fovea centralis (Fig. 7 ). ERG showed significant impairment of cone and rod cell function in both eyes. The proband was born with hexadactyly in both hands and surgical treatment had been performed. At the age of 7, the proband was 133 cm in height, 44 kg in weight, and body mass index (BMI) was 24.87 kg/m2. The proband's parents complained that she usually had poor memory and slow response, and the intelligence test suggested subnormal intelligence. As validated by the whole exome sequencing and Sanger sequencing, a homozygous copy number deletion located in the region of chr7:33404663–33409821 was found in the BBS9 gene of the proband. The size of the homozygous copy number deletion was 5159bp, which covered exon 17, part of intron 16, and part of intron 17 in the BBS9 gene, resulting in an alteration in protein length. It was confirmed from Sanger sequencing analysis that the BBS9 gene variant was homozygous in affected individuals and heterozygous carriers in normal parents, supporting autosomal recessive inheritance (Fig. 7 ). The proband was eventually diagnosed with Bardet-Biedl syndrome (BBS). This variant does not disrupt the reading frame but likely results in a possible change in protein length. Such copy number deletion variant, being nonpolymorphic, was not found in the DGV Normal Population Copy Number Variant Database. No copy number variants with a similar segment size were found in the Decipher database. According to the ACMG guidelines, such deletion is of uncertain significance. Discussion Due to the genetic characteristics of autosomal recessive inherited diseases, we generally believe that homozygous variant patients are more likely to appear in families with consanguineous marriages. Before this, research on homozygous variants often focused primarily on families with consanguineous marriages [ 15 ] [ 16 ] [ 17 ] , however, in this study, 82.35% of the proband’s parents were non-consanguineous marriages. We speculate that some variants may have a higher carrier rate in the general population, and non-consanguineous couples may come from different genetic backgrounds and carry more diverse recessive genes, which increases the risk of homozygous variant offsprings from non-consanguineous families. When each member of a non-consanguineous couple carries recessive genes, these recessive genes become homozygous during the formation of gametes, resulting in the development of mutant traits in the offspring, such mutations may lead to genetic disorders such as intellectual disabilities, delayed development, and congenital malformations. We can reduce the incidence of homozygous mutations in consanguineous families by avoiding consanguineous marriages [ 18 ] . However, it is impossible to avoid the occurrence of homozygous patients in non-consanguineous families. Therefore, it is necessary to further analyze the variants with high carrier frequencies in the normal population, which shall help us better prevent and intervene in the occurrence of hereditary diseases. Through genetic counseling and testing, it is possible to understand the genetic status and family medical history of both couples and predict the risk of offspring. On this basis, targeted prevention and intervention measures can be taken, such as prenatal consultation, prenatal diagnosis, etc., to predict the incidence probability of the next generation and produce healthy offspring [ 19 ] . At present, there is a growing emphasis on carrier screening for monogenic genetic diseases both at home and abroad. Screening of individuals who have no obvious genetic disease phenotypes but may carry pathogenic variants of genetic disease genes, or common pathogenic genes, can further reduce the prevalence of offspring. Therefore, it is very necessary to continuously explore the relationship between genes and diseases, which can provide a basis for preconception carrier screening. It has been found from previous studies that patients with homozygous variants appear to exhibit a more severe clinical phenotype and earlier age of onset. For instance, homozygous variants in the MERTK gene can lead to a more severe form of retinitis pigmentosa [ 20 ] ; homozygous variants in PTPN11gene resulted in Noonan syndrome (NS) patients with more pronounced short stature and slower growth rates compared to previously reported patients with heterozygous variants [ 21 ] ; homozygous variations in the PRPH2gene often result in a more severe LCA phenotype, and the patients usually have onset of disease before the age of 10 [ 22 ] [ 23 ] . The clinical phenotypes of the proband in this study were also consistent with this finding, as compared with the patients with heterozygous variants in the PRPH2 gene showing earlier onset and a more severe form of LCA compared to those with heterozygous variants. Such finding is also borne out by animal experiments. For instance, zebrafish with homozygous variants in the ZNF408 gene exhibit more severe retinal vascular abnormalities compared to those with heterozygous variants [ 24 ] . However, not all patients with the homozygous variants are consistent with this conclusion. We have observed that more than 70% of the patients in our study had an age of onset after 10 years old, but nearly 50% had a BCVA ≤ 0.3 in at least one eye before the age of 40. Therefore, the results of our study suggest that the majority of patients with homozygous variants do not show an early age of onset, but the proportion of moderate-to-severe visual impairment and blindness in working age at a high level. 7 new variants were identified in this study. Previous studies have shown that the short isoform a of the USH2A gene is expressed in the retina and the cochlea, whereas the long isoform b is expressed only in the retina [ 25 ] . The truncated mutations lead to premature termination of translation and result in nonsense-mediated mRNA decay causing truncation or even deletion of proteins, which potentially leads to more severe phenotypes compared to protein changes caused by nontruncated missense mutations [ 26 ] [ 27 ] . In this study, a new homozygous frameshift variant c.10179delG (p.Met3393fs) was detected in the USH2A gene in the Family 4 Proband, and the patient was diagnosed with Usher syndrome because of the clinical presentation of RP combined with deafness. In the retina, PROM1 is mainly located in the outer segment of the retinal photoreceptor and plays a key role in the morphogenesis of the photoreceptor outer segment membrane discs [ 28 ] . Throughout literature, PROM1 gene variants have been associated with multiple diseases with overlapping phenotypes, such as retinitis pigmentosa, cone-rod dystrophy, Stargardt-like macular dystrophy, bull’s-eye macular dystrophy, and Leber congenital amaurosis [ 29 ] [ 30 ] . Depending on the type of variants, the age of onset, initial symptoms, and severity of the disease can vary among patients. In this study, two probands carrying the PROM1 gene variant had chief complaints of reduced vision and night blindness, respectively, and were diagnosed with Stargardt's disease and RP, respectively, in combination with fundus and ERG examinations. The PROM1 gene variant can be inherited in both recessive and dominant patterns [ 31 ] . As compared with the retinal dystrophies caused by dominant PROM1 gene variants, the patients with retinal dystrophies associated with recessive PROM1 gene variants, such as CORD and Leber congenital amaurosis (LCA), have an earlier age of onset, more severe clinical phenotype, and can present with vision decline at an early stage of the disease [ 30 ] , which is highly consistent with the clinical phenotypes of the patients in this study. Homozygous frameshift deletion variant c.265del (p.Leu89Phefs*4) and c.253del (p.Leu85Phefs*4) of CNGA1 gene were detected in the probands of Family 12 and Family 13, and they were clinically diagnosed as RP. As shown by previous studies, variants in this gene are strongly associated with RP. This study further expands the spectrum of variants of CNGA1 gene. Diseases caused by variants of PRPH2 gene are dominantly inherited predominantly and only a few are autosomal recessive inheritance [ 32 ] . Currently, only one case of autosomal recessive RP due to the PRPH2 gene has been reported, but the detailed clinical manifestations of this patient have not been reported [ 33 ] . In this study, the homozygous missense variant c.640T > A (p.Cys214Ser) was detected in the PRPH2 gene of Family 14 Proband. The affected child had poor binocular vision since childhood, with a BCVA of 0.02 in both eyes at the age of 6, but did not show any symptoms of night blindness. Due to the overlap of phenotypes, it is easy to ignore the differentiation between RP and LCA. However, patients with LCA usually have severe visual impairment at birth, and the lesion often involves the outer retina, combined with the symptoms of binocular horizontal nystagmus, difficulty fixation, and eye poking, the patient in this case was diagnosed with LCA. Patients carrying a heterozygous variant of the PRPH2 gene generally have an age of onset after 30 years old [ 34 ] [ 35 ] . The patient in this case was born with visual impairment, which may be preliminary inferred that patients with homozygous variants of the PRPH2 gene have an earlier age of onset and a more severe clinical phenotype. This study further confirmed that variants in the PRPH2 gene can lead to recessive IRDs. The BBSome complex is a key regulator of the ciliary membrane proteome, which is mainly involved in the process of ciliogenesis and intraflagellar transport [ 36 ] . The protein encoded by the BBS9 gene is an important component of ciliary structure. In previously reported cases, BBS9-related diseases only led to Bardet-Biedl syndrome [ 37 ] . The proband of family 17 in this study presented typical BBS symptoms at first visit, and a homozygous copy number deletion in the chr7:33404663–33409821 region of the BBS9 gene was detected in the WES. To further clarify such a variant, we verified it by real-time fluorescent quantitative PCR (qPCR). So far, there are almost no reports of BBS9 copy number variant, and most reports are mainly homozygous variants. The patients exhibit polydactyly, obesity, intellectual disability, and associated ocular symptoms such as vision loss, night blindness, and nystagmus from early childhood [ 37 ] [ 38 ] [ 39 ] , which are reported to be similar to the phenotype of the proband of family 17. There are still 30–40% of IRDs that cannot be explained by routine genetic testing, of which copy number variants account for 9% [ 40 ] [ 41 ] . Therefore, in genetic diagnosis, when whole exome sequencing shows negative results, and the patient has a suspicious clinical phenotype, other different forms of variants, such as copy number variant, should be further considered to identify the pathogenic variant. In this study, homozygous variants were found to have a higher incidence in non-consanguineous families, suggesting that heterozygous variants of some genes have a higher carrier rate in the normal population. We also identified 7 new pathogenic variants and analyzed their possible pathogenic mechanisms, emphasizing the non-negligible impact of copy number variants on the disease. Cases with homozygous variants are relatively rare, but patients with homozygous variants can exclude the influence of other heterozygous variants to clarify whether the relevant variants are the direct cause of the patient's disease, which reinforces the supporting evidence for the association of Mendelian disease variants and the clinical phenotypes. It also emphasizes the importance of screening for carriers with no family history of consanguineous marriage and no obvious phenotype of genetic disease. Timely detection of couples at high risk of having children and individuals with moderate or greater severity IRDs can provide a basis for genetic counseling, reproductive decision-making, disease prevention, and management. Materials and methods Subjects The study complied with the Declaration of Helsinki, and it was approved and reviewed by the Human Research Ethics Committee of the People's Hospital of Ningxia Hui Autonomous Region(No. 2022-KJCG-006). In this study, we need to publish the facial information of participants to support the presentation of research results, and all participants included in the study obtained informed consent from the patients or their legal guardians and signed the relevant informed consent forms. We have taken measures to protect the privacy of participants, including but not limited to anonymization processes and restricting access to the information.Participants have the right to withdraw their consent at any time, and we will immediately cease the use of their facial information and remove it from published materials where possible.The publication of facial information in this study has been reviewed and approved by the Ethics Committee on Human Research at People Hospital in the Ningxia Hui Autonomous Region. This study included a total of 17 patients diagnosed with IRDs from January 2015 to September 2023 in Ningxia Eye Hospital and they were confirmed to carry homozygous variants by WES. The current medical history, past medical history, personal history, family history, and marital history of the probands were asked and recorded in detail, and the family tree was drawn. Clinical evaluation The complete ophthalmic examinations were performed on IRD patients, including slit-lamp microscopy, indirect ophthalmoscopy, uncorrected visual acuity (UCVA), best corrected visual acuity (BCVA), chromoscopy (Fifth Edition Color Blindness Examination Chart, Zi-Ping Yu), scanning laser ophthalmoscope (Optos DaytonaP200T), fundus photography (TOPCON, Japan, TRC-NW300), optical coherence tomography (OCT) (HD-OCT4000, Carl Zeiss Meditec, USA), electroretinogram (Roland Consult Stasche, Finger GumbHD-14770, Germany), perimetry (Humphrey Field Analyzer 750i, Germany). According to the patient's clinical phenotype and the characteristics of pathogenic genes, the necessary general examinations were performed, such as hearing tests, etc. Diagnostic criteria for visual impairment: According to the visual impairment standards established by WHO in 2019 [ 42 ] , the best corrected visual acuity (BCVA) of the better-seeing eye is less than 0.5 but equal to or greater than 0.3is classified as mild visual impairment, if which being less than 0.3 but equal to or greater than 0.1, it is classified as moderate visual impairment, if which being less than 0.1 but equal to or greater than 0.05, it is classified as severe visual impairment, and if which is less than 0.05 is classified as blindness. Methods Genomic DNA extraction 5 ml of peripheral venous blood was collected from all participants, and genomic DNA was extracted by using the Qiamp Blood Mini Kit DNA extraction kit (Qiagen, Germany) with standard protocol after the concentration and purity were detected by UV spectrophotometer and 1.5% agarose gel electrophoresis, stored the DNA in a -20°C refrigerator. Whole exome sequencing Whole exome sequencing (WES) capture was performed by using Agilent SureSelect Exon Capture Kit, and sequencing was performed with a high-throughput sequencer (Illumina) at a depth of 100×. The original sequencing data were processed by Illumina base-calling Software 1.7 and then compared the data with the human genome DNA reference sequence (NCBI build 37.1) of the National Center for Biotechnology Information (NCBI). Single nucleotide variants (SNV), insertion and deletion variants (Indel) were analyzed by SOAP software ( http://soap.genomics.org.cn ) and BWA software ( http://bio-bwa . sourceforge.net/), to obtain all the variants occurring in the DNA sequences in the samples. Then filtered out the high-frequency variant sites with Minor Allele Frequency (MAF) > 1% in the database (db135), and filtered out the variants that do not affect the structure and function of proteins. After step-by-step filtering, the homozygous variants shared by all patients in the family were screened to identify candidate pathogenic variations. Sanger sequencing was used for candidate pathogenic variants to exclude false positives, and further co-segregation of genotypes and phenotypes was validated among normal family members. Fluorescence quantitative PCR Fluorescence quantitative PCR was performed to verify the detected copy number variant. Genomic DNA (gDNA) from the peripheral blood of family members was extracted using a blood genomic DNA extraction kit (Beijing Tiangen Biochemical Technology Co., Ltd.). Primers were designed for the gene under investigation. The qPCR reaction system was prepared according to the NovoStart® SYBR qPCR SuperMix Plus kit. The 20-µL system contained 10 µL of 2×NovoStart® SYBR qPCR SuperMix Plus, 0.5 µL each of forward and reverse primers, an equal volume of 1 µL of gDNA (10 ng), and 8 µL of water. An initial denaturation (95°C for 1 min) followed by 40-cycle amplification (95°C for 20 s, 60°C for 20 s) program was performed on a Roche Light Cycler II 480 real-time fluorescence quantitative PCR instrument, with 3 replicates per reaction. The copy numbers of genes were calculated using the 2-ΔΔCt method, normalized by the Ct values of the internal reference genes, and healthy individuals served as reference. For autosomes, a relative copy number around 2 indicates a normal sample, while a relative copy number value around 1 indicates a sample with a 1 copy number deletion. Pathogenicity analysis of genetic variants The American College of Medical Genetics and Genomics (ACMG) established Standards and Guidelines for Interpretation of Sequence Variants in 2015, which were used to evaluate the pathogenicity of novel variations for genetic variation. MAF<0.005 was used as the criteria to exclude benign variants by reference to the databases for East Asian populations Allele frequencies available with 1000 Genomes Project (1000G, http://browser.1000genomes.org ) and Exome Aggregation Consortium ( http://exac.broadinstitute.org/ ). Polyphen2( http://genetics.bwh.harvard.edu/pph2 ), SIFT( http://sift.jcvi.org),REVEL(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5065685/ ), CADD ( https://cadd.gs.washington.edu/score ) and Mutation Taster ( http://mutationtaster.org/ ) were used for pathogenicity prediction. Measurements of the conservation of gene sequences across species in evolution have been made using websites like GERP++ ( https://bio.tools/gerp ). Variants were classified as uncertain clinical significance when at least 1 of 4 predictions had a benign outcome or when there was insufficient evidence of pathogenicity. When all predictions turned out to be accurate, variations were categorized as potentially pathogenic when used in conjunction with further data. Pathogenic variants were defined as frameshift, nonsense, and variants with experimental proof of causing loss of protein function. For the conservativeness study of variant loci, the online analysis tool Multalin ( http://sacs.ucsf.edu/cgi-bin/multalin.py ) was employed. Interpretation rules of copy number variants (CNV) referred to the 2019 Edition of the ACMG Guidelines for Interpretation and Reporting of Copy Number Variants [ 43 ] . Alphafoldwas used to construct the normal protein structure, and Pymol 2.3 software was used to make a visualized analysis of the mutant protein. Declarations Acknowledgements : The authors thank all patients and their family members for their participation. Ethics approval and consent to participate : The Ethics Committee on Human Research at People Hospital in the Ningxia Hui Autonomous Region accepted and examined our work (reference number: 2022-KJCG-006), which adhered to the Declaration of Helsinki. Each participant or their legal guardians provided their written informed permission before to taking part. Authors' contributions : Xue Fan and Weining Rong wrote the main manuscript, Zhen Li and Lingzhi Sha collected cases data and followed up patients. Weining Rong and Xunlun Sheng polished the article. All authors reviewed the manuscript. Availability of data and materials ：The datasets generated and analyzed during the current study are available in the [Banklt] repository (BankIt (https://www.ncbi.nlm.nih.gov/nuccore/) ID: PQ038085、PQ038086、PQ038087、PQ045748、PQ045749、PQ045750). Competing interests : I declare that the authors have no competing interests as defined by Clinical Genetics or other interests that might be perceived to influence the results and discussion reported in this paper. Funding : This work was supported by the National Natural Science Foundation of China(82260206), the training project of the scientific innovation commanding talented person in Ningxia Hui Autonomous Region(2020GKLRLX13), Major achievement transformation project of Ningxia Hui Autonomous Region(2022CJE09011), the key research development project of Ningxia Hui Autonomous Region(2024BEG02017). References Scholl, H. P. N. et al. Emerging therapies for inherited retinal degeneration. Sci Transl Med 8, 368rv6 (2016). Jespersgaard, C. et al. Molecular genetic analysis using targeted NGS analysis of 677 individuals with retinal dystrophy. Sci Rep 9, 1219 (2019). Sen, P. et al. Prevalence of retinitis pigmentosa in South Indian population aged above 40 years. Ophthalmic Epidemiol 15, 279–281 (2008). Smith, E. D. et al. Classification of Genes: Standardized Clinical Validity Assessment of Gene-Disease Associations Aids Diagnostic Exome Analysis and Reclassifications. 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Tissue-specific genotype-phenotype correlations among USH2A-related disorders in the RUSH2A study. Hum Mutat 43, 613–624 (2022). Yanardag, S., Rhodes, S., Saravanan, T., Guan, T. & Ramamurthy, V. Prominin 1 is crucial for the early development of photoreceptor outer segments. Sci Rep 14, 10498 (2024). Michaelides, M. et al. The PROM1 mutation p.R373C causes an autosomal dominant bull’s eye maculopathy associated with rod, rod-cone, and macular dystrophy. Invest Ophthalmol Vis Sci 51, 4771–4780 (2010). Ragi, S. D. et al. Compound heterozygous novel frameshift variants in the PROM1 gene result in Leber congenital amaurosis. Cold Spring Harb Mol Case Stud 5, a004481 (2019). Del Pozo-Valero, M. et al. Expanded Phenotypic Spectrum of Retinopathies Associated with Autosomal Recessive and Dominant Mutations in PROM1. Am J Ophthalmol 207, 204–214 (2019). Conley, S. M. & Naash, M. I. Gene therapy for PRPH2-associated ocular disease: challenges and prospects. Cold Spring Harb Perspect Med 4, a017376 (2014). Perea-Romero, I. et al. Genetic landscape of 6089 inherited retinal dystrophies affected cases in Spain and their therapeutic and extended epidemiological implications. Sci Rep 11, 1526 (2021). Wawrocka, A. et al. Novel variants identified with next-generation sequencing in Polish patients with cone-rod dystrophy. Mol Vis 24, 326–339 (2018). Cheng, J. et al. A novel splicing mutation in the PRPH2 gene causes autosomal dominant retinitis pigmentosa in a Chinese pedigree. J Cell Mol Med 23, 3776–3780 (2019). Pigino, G. Intraflagellar transport. Curr Biol 31, R530–R536 (2021). Zhang, Y., Xu, M., Zhang, M., Yang, G. & Li, X. A Novel BBS9 Mutation Identified via Whole-Exome Sequencing in a Chinese Family with Bardet-Biedl Syndrome. Biomed Res Int 2021, 4514967 (2021). Tang, H.-Y., Xie, F., Dai, R.-C. & Shi, X.-L. Novel homozygous protein-truncating mutation of BBS9 identified in a Chinese consanguineous family with Bardet-Biedl syndrome. Mol Genet Genomic Med 9, e1731 (2021). Suárez-González, J., Seidel, V., Andrés-Zayas, C., Izquierdo, E. & Buño, I. Novel biallelic variant in BBS9 causative of Bardet-Biedl syndrome: expanding the spectrum of disease-causing genetic alterations. BMC Med Genomics 14, 91 (2021). Schneider, N. et al. Inherited retinal diseases: Linking genes, disease-causing variants, and relevant therapeutic modalities. Prog Retin Eye Res 89, 101029 (2022). Zampaglione, E. et al. Copy-number variation contributes 9% of pathogenicity in the inherited retinal degenerations. Genet Med 22, 1079–1087 (2020). World Health Organization. World Report on Vision. Geneva, 180 p. (2019). Riggs, E. R. et al. Technical standards for the interpretation and reporting of constitutional copy-number variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics (ACMG) and the Clinical Genome Resource (ClinGen). Genet Med 22, 245–257 (2020). Tables Tables 1 to 3 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files MainTables.docx Cite Share Download PDF Status: Published Journal Publication published 24 Jan, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 15 Oct, 2024 Reviews received at journal 12 Oct, 2024 Reviewers agreed at journal 02 Oct, 2024 Reviews received at journal 03 Sep, 2024 Reviewers agreed at journal 19 Aug, 2024 Reviewers invited by journal 18 Aug, 2024 Editor assigned by journal 12 Aug, 2024 Editor invited by journal 11 Aug, 2024 Submission checks completed at journal 11 Aug, 2024 First submitted to journal 21 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4776885","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":350240407,"identity":"dff1cbac-dcd7-4a9d-961d-4f461b21577f","order_by":0,"name":"Xue Fan","email":"","orcid":"","institution":"Ningxia Hui Autonomous Region Peoples Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xue","middleName":"","lastName":"Fan","suffix":""},{"id":350240408,"identity":"5cf28ecb-6ff6-41dd-b4c7-8fe21e3f3a84","order_by":1,"name":"Zhen Li","email":"","orcid":"","institution":"Ningxia Hui Autonomous Region Peoples Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhen","middleName":"","lastName":"Li","suffix":""},{"id":350240409,"identity":"b11bd64f-84bc-4043-beea-7c2c46c86f97","order_by":2,"name":"Lingzhi Sha","email":"","orcid":"","institution":"Ningxia Hui Autonomous Region Peoples Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lingzhi","middleName":"","lastName":"Sha","suffix":""},{"id":350240411,"identity":"f3583413-add4-44c2-9b98-fe159a5c77db","order_by":3,"name":"Xunlun Sheng","email":"","orcid":"","institution":"Gansu Aier Optometry Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xunlun","middleName":"","lastName":"Sheng","suffix":""},{"id":350240413,"identity":"ec2db762-7b20-4564-8606-735a710f82a0","order_by":4,"name":"Weining Rong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIiWNgGAWjYPACCQY2IGnwocKGh5+9gQQthTPOpMlI9hwgwa7PvC2HbQxuOOBXZd7ee/g1T4VFHp90+8UNvA3neRhuMDB++JiDW4vMmXNp1jxnJIrZZM4UG0juuM3DOLuBWXLmNjyekMgxM85tk0hsk8hJMzA8c5uHWeYAGzMvQS3/wFrSfyS2neNhk0ggqMX4cW4DSEv6AYODbQd4eAhq4TljxvznGNgWBsOGM8k8EjwHm/H7hb3H+OOMmrrE+TPSHxj/qbCztz/efPDDRzxagIBNAkLzGEAFGBvwqgcC5g8Qmv0BIZWjYBSMglEwQgEA4chQN5h3TJ0AAAAASUVORK5CYII=","orcid":"","institution":"Ningxia Hui Autonomous Region Peoples Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Weining","middleName":"","lastName":"Rong","suffix":""}],"badges":[],"createdAt":"2024-07-21 13:14:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4776885/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4776885/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-87844-5","type":"published","date":"2025-01-24T15:57:06+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":65790253,"identity":"d617b13e-3ca4-4e92-b167-40e24ae5f34f","added_by":"auto","created_at":"2024-10-02 17:06:53","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2378944,"visible":true,"origin":"","legend":"\u003cp\u003eMutation sequence analysis and clinical examination of the family 4: (\u003cstrong\u003eA\u003c/strong\u003e)Pedigree of the family 4: The filled black symbol represents the affected member, and the arrow denotes the proband. (\u003cstrong\u003eB\u003c/strong\u003e) Sequence chromatograms of identified mutations. (\u003cstrong\u003eC\u003c/strong\u003e)The fundus of both eyes: The color of the optic was waxy yellow, the retinal vessels were attenuated, and bone spicule-like pigment deposits were visible on the peripheral retina, OCT showed the disappearance of the light reflection signals in the local ellipsoid zone and interdigitation zone of fovea, along with atrophy of the retinal pigment epithelium. (\u003cstrong\u003eD\u003c/strong\u003e)ERG: Significant impairment of cone and rod cell function in both eyes. (\u003cstrong\u003eE\u003c/strong\u003e)The homology of amino acid sequences between human USH2A and other species, the amino acid at positions 3393 is highly conserved among species, and the mutated residues 3393 is boxed and indicated.\u003c/p\u003e","description":"","filename":"figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4776885/v1/74c3c801b945503469cac6a1.jpg"},{"id":65789934,"identity":"0b15c1cd-2056-4353-9aef-61de01a9356e","added_by":"auto","created_at":"2024-10-02 16:58:48","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2030071,"visible":true,"origin":"","legend":"\u003cp\u003eMutation sequence analysis and clinical examination of the family 7: (\u003cstrong\u003eA\u003c/strong\u003e)Pedigree of the family 7: The filled black symbol represents the affected member, and the arrow denotes the proband. (\u003cstrong\u003eB\u003c/strong\u003e)The fundus of both eyes: The color of the optic was pale in both eyes, oval atrophy and yellowish-white patchy exudation were observed in the macular area, showing a bull's-eye change, OCT indicated a significant thinning of the macular fovea, disappearance of the outer nuclear layer and ellipsoid zone, along with atrophy of the retinal pigment epithelium, fundus angiography reveals 'worm-eaten-like' fluorescent spots around the macular region. (\u003cstrong\u003eC\u003c/strong\u003e)Sequence chromatograms of identified mutations. (\u003cstrong\u003eD\u003c/strong\u003e)The homology of amino acid sequences between human PROM1 and other species, the amino acid at positions 182 is highly conserved among species, and the mutated residues 182 is boxed and indicated.\u003c/p\u003e","description":"","filename":"figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4776885/v1/36290e151d68ba41f3b7395e.jpg"},{"id":65790202,"identity":"7a6546d6-e663-4314-88f4-b6413dd99c44","added_by":"auto","created_at":"2024-10-02 17:06:48","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2458639,"visible":true,"origin":"","legend":"\u003cp\u003eMutation sequence analysis and clinical examination of the family 8: (\u003cstrong\u003eA\u003c/strong\u003e)Pedigree of the family 8:The filled black symbol represents the affected member, and the arrow denotes the proband. (\u003cstrong\u003eB\u003c/strong\u003e)The fundus of both eyes: The color of the optic was waxy yellow, the retinal vessels were attenuated, and bone spicule-like pigment deposits were visible on the peripheral retina, OCT showed mild thinning of the macular region. (\u003cstrong\u003eC\u003c/strong\u003e)ERG: Significant impairment of cone and rod cell function in both eyes. (\u003cstrong\u003eD\u003c/strong\u003e)Sequence chromatograms of identified mutations. (\u003cstrong\u003eE\u003c/strong\u003e)Proteomic conservation analysis suggested that the p.G455R mutation results in the substitution of a nonpolar, uncharged glycine at site 455 with a nonpolar, positively charged arginine, which leads to alterations to the structure and function of the protein after the mutation. (\u003cstrong\u003eF\u003c/strong\u003e)The homology of amino acid sequences between human PROM1 and other species, the amino acid at positions 455 is highly conserved among species, and the mutated residues 455 is boxed and indicated.\u003c/p\u003e","description":"","filename":"figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4776885/v1/8e40584cbaa0afa204dbb2ae.jpg"},{"id":65790696,"identity":"f91ed2b4-959e-49a7-9106-5606c0378bdb","added_by":"auto","created_at":"2024-10-02 17:14:48","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2058962,"visible":true,"origin":"","legend":"\u003cp\u003eMutation sequence analysis and clinical examination of the family 12: (\u003cstrong\u003eA\u003c/strong\u003e)Pedigree of the family 12:The filled black symbol represents the affected member, and the arrow denotes the proband. (\u003cstrong\u003eB\u003c/strong\u003e)The fundus of both eyes: The color of the optic was waxy yellow in both eyes, the retinal vessels were attenuated, and bone spicule-like pigment deposits were visible on the peripheral retina, OCT showed the disappearance of the light reflection signals in the local ellipsoid zone and interdigitation zone at the macular center in the right eye, along with atrophy of the retinal pigment epithelium. (\u003cstrong\u003eC\u003c/strong\u003e)ERG: Significant impairment of cone and rod cell function in both eyes. (\u003cstrong\u003eD\u003c/strong\u003e)Sequence chromatograms of identified mutations. (\u003cstrong\u003eE\u003c/strong\u003e)The homology of amino acid sequences between human CNGA1 and other species, the amino acid at positions 89 is highly conserved among species, and the mutated residues 89 is boxed and indicated.\u003c/p\u003e","description":"","filename":"figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4776885/v1/0ad0a8e56d4fdf6d7f3481af.jpg"},{"id":65789938,"identity":"c8575508-a2f9-475c-b194-f44619e06563","added_by":"auto","created_at":"2024-10-02 16:58:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":220932,"visible":true,"origin":"","legend":"\u003cp\u003eMutation sequence analysis and clinical examination of the family 13: (\u003cstrong\u003eA\u003c/strong\u003e)Pedigree of the family 13:The filled black symbol represents the affected member, and the arrow denotes the proband. (\u003cstrong\u003eB\u003c/strong\u003e)The fundus of both eyes: The color of the optic was waxy yellow in both eyes, the retinal vessels were attenuated, and bone spicule-like pigment deposits were visible on the peripheral retina, OCT examination showed the disappearance of the light reflection signals in the local ellipsoid zone and interdigitation zone at the macular center, along with atrophy of the retinal pigment epithelium in the left eye. (\u003cstrong\u003eC\u003c/strong\u003e)Sequence chromatograms of identified mutations. (\u003cstrong\u003eD\u003c/strong\u003e)The homology of amino acid sequences between human CNGA1 and other species, the amino acid at positions 85 is highly conserved among species, and the mutated residues 85 is boxed and indicated.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4776885/v1/71587c705878e0697c0d8158.png"},{"id":65789935,"identity":"21d2871d-03da-448f-89e0-b30867105aa5","added_by":"auto","created_at":"2024-10-02 16:58:48","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2303943,"visible":true,"origin":"","legend":"\u003cp\u003eMutation sequence analysis and clinical examination of the family 14: (\u003cstrong\u003eA\u003c/strong\u003e)Pedigree of the family 14: The filled black symbol represents the affected member, and the arrow denotes the proband. (\u003cstrong\u003eB\u003c/strong\u003e)The fundus of both eyes: The color of the optic was pale, OCT showed a mild elevation of the ellipsoid zone and interdigitation zone in the macular region of the the right eye. (\u003cstrong\u003eC\u003c/strong\u003e)Sequence chromatograms of identified mutations. (\u003cstrong\u003eD\u003c/strong\u003e)The homology of amino acid sequences between human PRPH2 and other species. The amino acid at positions 214 is highly conserved among species, and the mutated residues 214 is boxed and indicated. (\u003cstrong\u003eE\u003c/strong\u003e)Proteomic conservation analysis suggested that the p.C241S mutation results in the substitution of a polar, uncharged cysteine at site 241 with a polar, uncharged serine, which leads to alterations in the structure and function of the mutated protein.\u003c/p\u003e","description":"","filename":"figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4776885/v1/43f8db1aa229fb37224c0d6f.jpg"},{"id":65789940,"identity":"ee2bc404-622e-400e-994b-03f49f8c76ef","added_by":"auto","created_at":"2024-10-02 16:58:48","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3387573,"visible":true,"origin":"","legend":"\u003cp\u003eMutation sequence analysis and clinical examination of the family 17: (\u003cstrong\u003eA\u003c/strong\u003e)Pedigree of the family 17: The filled black symbol represents the affected member, double horizontal lines indicate consanguineous marriage, and the arrow denotes the proband. (\u003cstrong\u003eB\u003c/strong\u003e)The fundus of both eyes: OCT examination showed no obvious structure of the fovea centralis. (\u003cstrong\u003eC\u003c/strong\u003e)ERG: Significant impairment of cone and rod cell function in both eyes. (\u003cstrong\u003eD\u003c/strong\u003e)Atient appearance: Epicanthus, wide eye distance, overweight, exotropia, hands have been polydactyly surgery. (\u003cstrong\u003eE\u003c/strong\u003e)Breakpoint analysis report and QPCR analysis chart.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4776885/v1/9a2b40789ee6068f8742de5e.jpg"},{"id":74858305,"identity":"0776af9f-1b96-4147-b98f-2a8a4c23a73c","added_by":"auto","created_at":"2025-01-27 16:07:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15582724,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4776885/v1/87582c06-bbdd-46ed-a148-0b848efdc04c.pdf"},{"id":65790201,"identity":"c4073223-1411-41c3-b415-7defa8ec837d","added_by":"auto","created_at":"2024-10-02 17:06:48","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":36282,"visible":true,"origin":"","legend":"","description":"","filename":"MainTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-4776885/v1/bb28cd749868c3e07da4047f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhanced Gene-Disease Association and Carrier Screening by Homozygous Mutation Carriers: A Study of Genotypes and Clinical Phenotypes in 17 IRD Families with Homozygous Mutations in Northern China","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInherited retinal dystrophies (IRDs) are the most common and serious clinical blinding eye disease, and the leading cause of blindness in children and young adults worldwide\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e, for which there is no effective treatment yet. The incidence of IRDs is about 1:4000 in developed countries\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e, and in China, epidemiological surveys show that the incidence of retinitis pigmentosa (RP) is as high as 1:1000 in people over 40 years old\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. In recent years, new ophthalmic drugs for gene therapy have stepped into the \"fast track of transformation\", bringing bright hope to IRD patients. The prerequisite for achieving IRD gene therapy is to identify the pathogenic genes and make an accurate diagnosis. Currently, there are still the following problems in the clinical diagnosis of IRDs: numerous pathogenic genes, complex and diverse clinical phenotypes, intricate relationships between genotypes and clinical phenotypes, and the lack of professional genetic knowledge among ophthalmologists lead to misdiagnosis, underdiagnosis, and mistreatment of a large number patients with IRDs. The ClinGen guidelines state that two evidences are supporting the genetic link to disease: genetics (e.g. case studies) and experiments (e.g. animal models)\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. To demonstrate the superiority of genetic evidence, the maximum score of experimental evidence (6 points) was only half that of gene evidence (12 points). This highlights the importance of reporting more cases with the same clinical phenotype to increase the credibility of the gene-disease association. As compared to compound heterozygous variants, homozygous variants are less common. The homozygous variant refers to the same variant present in both alleles at the same locus on a pair of homologous chromosomes. Since two alleles on the same chromosome have the same function, the homozygous variant can more fully express the loss of function and the phenotypic effects of the mutation. Meanwhile, patients with a homozygous variant will inherit the variant stably from their offspring, all of whom will carry the same variant, resulting in an increased incidence of genetic disease in their offspring. Therefore, screening tests for individuals who have no obvious genetic disease phenotype but may carry pathogenic variants of genetic disease genes is an effective way to reduce the incidence of hereditary diseases. In this study, the genotypes and clinical phenotypes of 17 IRD patients who carry homozygous variants were studied, and we performed screening tests on IRD probands\u0026rsquo; parents who carried homozygous variants. The aim is to expand the spectrum of IRD pathogenic gene variants, and investigate the relationship between homozygous variants and clinical phenotypes of IRDs, to provide a basis for genetic counseling, reproductive decision-making, disease prevention, and management.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIn this study, 17 patients with homozygous variants were collected, and 3 had a family history of consanguineous marriage. The average age was 21.23 years old (ranging from infancy to 51 years). 29.41% (5/17) of patients developed symptoms before 10 years. The best-corrected visual acuity (in one eye) of 70.59%(12/17) patients was less than 0.3 and 35.29% (6/17) with best-corrected visual acuity (in one eye) better than 0.5 (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). According to the visual impairment standards established by WHO in 2019, 3 were blind, 9 had moderate to severe visual impairment, 3 had mild visual impairment, and 2 were close but did not meet the criteria for visual impairment. A total of 16 variants were detected in 17 patients, including 9 genes: \u003cem\u003eUSH2A, CYP4V2, PROM1, RP1, CNGA1, PRPH2, ABCA4, CRB1\u003c/em\u003e, and\u003cem\u003eBBS9\u003c/em\u003e, which were involved in 6 inherited retinal diseases: Retinitis pigmentosa (RP), Usher syndrome (USH), Bietti crystalline chorioretinal dystrophy (BCD), Stargardt disease (STGD), Leber\u0026apos;s congenital amaurosis (LCA), and Bardet-Biedl Syndrome (BBS). This study identified 7 novel variants: including 4 frameshift mutations - c.10179delG (p.Met3393fs) of \u003cem\u003eUSH2A\u003c/em\u003egene, c.544dupC (p.Gln182fs) of \u003cem\u003ePROM1\u003c/em\u003e gene, c.265del (p.Leu89Phefs*4) and c.253del (p. Leu85Phefs*4) of \u003cem\u003eCNGA1\u003c/em\u003e gene; 2 missense variants - c.1363G\u0026thinsp;\u0026gt;\u0026thinsp;C (p.Gly455Arg) of \u003cem\u003ePROM1\u003c/em\u003e gene, c.1363G\u0026thinsp;\u0026gt;\u0026thinsp;C (p.Gly455Arg) of \u003cem\u003ePRPH2\u003c/em\u003egene; and 1 copy number variant - copy number loss of \u003cem\u003eBBS9\u003c/em\u003e gene located in the region of chr7. 33404663\u0026ndash;33409821(Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), all of which were pathogenic/likely pathogenic(Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The clinical diagnosis was Usher syndrome type II (USHⅡ), Stargardt disease (STGD), Retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), and Bardet-Biedl syndrome (BBS) respectively. The parents of all the probands had normal clinical phenotypes and carried the same heterozygous variants of the above-mentioned related genes.\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eAnalysis of the pathogenicity and clinical phenotypes of newly identified homozygous variants\u003c/h2\u003e\n \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e\n \u003ch2\u003eFamily 4\u003c/h2\u003e\n \u003cp\u003eThe proband was a 39-year-old male, who complained of progressive binocular vision loss accompanied by night blindness for 19 years, and he denied family history and consanguineous marriage history. The BCVA was 0.3 in the right eye and 0.4 in the left eye (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). No abnormalities were observed in the anterior segment of both eyes. The fundus examination revealed the boundaries of the optic discs were clear and the color was waxy yellow, with no reflection in the macular fovea, the retinal vessels were attenuated, and bone spicule-like pigment deposits were visible on the peripheral retina. OCT showed the disappearance of the light reflection signals in the local ellipsoid zone and interdigitation zone of the fovea, along with the atrophy of the retinal pigment epithelium. ERG showed significant impairment of cone and rod cell function in both eyes. As validated by the whole exome sequencing and Sanger sequencing, the proband carried a homozygous frameshift variant c.10179delG (p.Met3393fs) in \u003cem\u003eUSH2A\u003c/em\u003e gene, which resulted in a premature translational-termination codon (PTC) due to a frameshift at the 3393 codon (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). According to the ACMG guidelines, the frameshift variant was considered as Pathogenic Very Strong (PVS1), while the absence of this variant in the normal population database was considered as Pathogenic Moderate (PM2), the proband\u0026apos;s clinical symptoms were consistent with Usher Syndrome, a monogenic genetic disease, which was considered as Pathogenic Supporting (PP4). Therefore, the variant c.10179delG (p.Met3393fs) was classified as Pathogenic (PVS1\u0026thinsp;+\u0026thinsp;PM2\u0026thinsp;+\u0026thinsp;PP4). The proband had poor hearing since childhood and was eventually diagnosed as Usher syndrome type II.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003ch3\u003eFamily 7\u003c/h3\u003e\n\u003cp\u003eThe proband of family 7, age of 7, complained of progressive bilateral vision reduced, and his parents denied family history and consanguineous marriage history. BCVA was 0.25 in the right eye and 0.15 in the left eye (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The anterior segment was normal and fundus examination showed pale optic discs with clear borders in both eyes. No reflection in the macular fovea, oval atrophy, and yellowish-white patchy exudation were observed in the macular area, showing a bull\u0026apos;s-eye change. OCT indicated a significant thinning of the macular fovea, disappearance of the outer nuclear layer and ellipsoid zone, and atrophy of the retinal pigment epithelium. Fundus angiography reveals \u0026apos;worm-eaten-like\u0026apos; fluorescent spots around the macular region. As validated by the whole exome sequencing and Sanger sequencing, a homozygous frameshift variant c.544dupC (p.Gln182fs) was detected in \u003cem\u003ePROM1\u003c/em\u003egene of the proband (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). According to the ACMG guidelines, the frameshift variant was considered as Pathogenic Very Strong (PVS1), the variant was not detected in the normal population database or the known variant databases was considered as Pathogenic Moderate (PM2), the proband\u0026apos;s clinical symptoms were consistent with Stargardt disease, a monogenic genetic disease, which was considered as Pathogenic Supporting (PP4). Therefore, such a variant was classified as pathogenic (PVS1\u0026thinsp;+\u0026thinsp;PM2\u0026thinsp;+\u0026thinsp;PP4) based on the standards and guidelines for the interpretation of sequence variants. The proband was finally diagnosed with Stargardt disease.\u003c/p\u003e\n\u003ch3\u003eFamily 8\u003c/h3\u003e\n\u003cp\u003eThe proband of family 8 was a 32-year-old female who presented with bilateral vision loss accompanied by night blindness for 17 years, and she denied any family history or consanguineous marriage history. BCVA was CF in the right eye and HM in the left eye (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The anterior segment was normal and fundus examination showed the waxy yellow optic discs with clear borders in both eyes. No reflection in the macular fovea, the retinal vessels were attenuated, and bone spicule-like pigment deposits were visible on the peripheral retina. OCT showed mild thinning of the macular region. ERG indicated significant impairment of cone and rod cell function in both eyes. As validated by the whole exome sequencing and Sanger sequencing, a homozygous missense variant c.1363G\u0026thinsp;\u0026gt;\u0026thinsp;C (p.Gly455Arg) was detected \u003cem\u003ePROM1\u003c/em\u003egene of the proband and his parents carried the same heterozygous variant respectively. This variant resulted in a change of the 455th codon from encoding glycine to encoding arginine (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). According to the ACMG guidelines, this variant was not detected in the normal population database or in the known variant databases, which was considered as Pathogenic Moderate(PM2). Moreover, proteomic conservation analysis suggested that the p.G455R mutation results in the substitution of a nonpolar, uncharged glycine at site 455 with a nonpolar, positively charged arginine. The N atom of the main chain forms a hydrogen bond with the O atom of the large, nonpolar, uncharged phenylalanine at site 451, with a hydrogen bond distance of 3.0 \u0026Aring; (the distances of normal protein structures is 2.9 \u0026Aring;). The O atom of the main chain forms a hydrogen bond with the N atom of the nonpolar, uncharged glycine at site 459, with a hydrogen bond distance of 2.7 \u0026Aring; (the distances of normal protein structures is 3.0 \u0026Aring;). These changes in amino acid interactions lead to alterations to the structure and function of the protein after the mutation (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). A variety of bioinformatics computing software indicated the deleterious impact of this variant (PP3_Supporting) (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The genotype and clinical phenotypic co-segregation in family members were considered as supportive evidence (PP1), the phenotype of the variant carriers was highly consistent with the monogenic genetic disease (RP), which was considered supportive evidence (PP4), gene testing report from a reliable and authoritative source considered this variant as Pathogenic, which serves as additional supportive evidence (PP5). Therefore, such a variant was classified as Pathogenic (PM2\u0026thinsp;+\u0026thinsp;PP3\u0026thinsp;+\u0026thinsp;PP1\u0026thinsp;+\u0026thinsp;PP4\u0026thinsp;+\u0026thinsp;PP5) based on the standards and guidelines for the interpretation of sequence variants. The problem was ultimately diagnosed with binocular RP.\u003c/p\u003e\n\u003ch3\u003eFamily 12\u003c/h3\u003e\n\u003cp\u003eThe proband of family 12 was a 51-year-old female, who complained of progressive night blindness and vision decline for 20 years in both eyes, and she denied family history and consanguineous marriage history. BCVA was 0.5 in the right eye and 1.0 in the left eye (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). No abnormalities were observed in the anterior segment of both eyes. Fundus examination showed the waxy yellow optic discs with clear borders in both eyes. No reflection in the macular fovea, the retinal vessels were attenuated, and bone spicule-like pigment deposits were visible on the peripheral retina. OCT showed the disappearance of the light reflection signals in the local ellipsoid zone and interdigitation zone at the macular center in the right eye, along with atrophy of the retinal pigment epithelium. ERG showed significant impairment of cone and rod cell function in both eyes. As validated by the whole exome sequencing and Sanger sequencing, a homozygous frameshift deletion variant c.265del (p.Leu89Phefs*4) was detected in \u003cem\u003eCNGA1\u003c/em\u003egene of the proband, which led to a premature stop codon at position 4 of the new reading frame, and caused a frameshift beginning with codon Leucine 89, changing this amino acid to a Phenylalanine. The daughter of the proband carried the same heterozygous variant with normal clinical phenotype (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). According to the ACMG guidelines, this variant was considered as Pathogenic Very Strong (PVS1), the variant was not detected in the normal population database or in the known variant databases was considered as Pathogenic Moderate (PM2), The genotype and clinical phenotypic co-segregation in family members was considered as supportive evidence (PP1). The proband\u0026apos;s clinical symptoms were consistent with retinitis pigmentosa, a monogenic genetic disease, which was considered Pathogenic Supporting (PP4). Therefore, such a variant was classified as Pathogenic (PVS1\u0026thinsp;+\u0026thinsp;PM2\u0026thinsp;+\u0026thinsp;PP1\u0026thinsp;+\u0026thinsp;PP4) based on the standards and guidelines for the interpretation of sequence variants. The proband was diagnosed with binocular RP.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eFamily 13\u003c/h2\u003e\n \u003cp\u003eThe proband of family 13 was a 20-year-old male, who complained of the left eye blurred vision accompanied by night blindness, and he denied family history and consanguineous marriage history. BCVA was 0.8 in the right eye and 0.1 in the left eye (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Exotropia in both eyes, with IOL in the left eye. Fundus examination showed the waxy yellow optic discs with clear borders in both eyes. No reflection in the macular fovea, the retinal vessels were attenuated, and bone spicule-like pigment deposits were visible on the peripheral retina. OCT examination showed the disappearance of the light reflection signals in the local ellipsoid zone and interdigitation zone at the macular center, along with atrophy of the retinal pigment epithelium in the left eye. As validated by the whole exome sequencing and Sanger sequencing, a homozygous frameshift deletion variant c.253del (p.Leu85Phefs*4) was detected in \u003cem\u003ethe CNGA1\u003c/em\u003e gene of the proband, which led to a premature stop codon at position 4 of the new reading frame, and caused a frameshift beginning with codon Leucine 85, changing this amino acid to a Phenylalanine. The mother of the proband carried the same heterozygous variant with normal clinical phenotype (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). According to the ACMG guidelines, this variant was Pathogenic Very Strong (PVS1), the variant was not detected in the normal population database or in the known variant databases was considered as Pathogenic Moderate (PM2). The proband\u0026apos;s clinical symptoms were consistent with retinitis pigmentosa, a monogenic genetic disease, which was considered Pathogenic Supporting (PP4). Therefore, such a variant was classified as Pathogenic (PVS1\u0026thinsp;+\u0026thinsp;PM2\u0026thinsp;+\u0026thinsp;PP4) based on the standards and guidelines for the interpretation of sequence variants. The proband was ultimately diagnosed with binocular RP.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eFamily 14\u003c/h3\u003e\n\u003cp\u003eThe proband of family 14 was a 6-year-old male, whose parents complained that the proband had poor vision in both eyes since childhood, they denied family history and consanguineous marriage history. BCVA was 0.02 in both eyes (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The proband exhibited horizontal nystagmus in both eyes, difficulty fixation, and eye poking, with no obvious abnormalities observed in the anterior segment. Fundus examination showed the boundaries of the optic discs were clear and the color was pale, the light reflection of the foveal centralis was not visible. OCT examination showed a mild elevation of the ellipsoid zone and interdigitation zone in the macular region of the right eye. As validated by the whole exome sequencing and Sanger sequencing, the homozygous variant c.640T\u0026thinsp;\u0026gt;\u0026thinsp;A (p.Cys214Ser) was detected in \u003cem\u003ePRPH2\u003c/em\u003egene of the proband, which resulted in a change of 214th codon from encoding Cysteine to Serine (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). According to the ACMG guidelines, this variant was not detected in the normal population database or in the known variant databases, which was considered as Pathogenic Moderate(PM2). A variety of bioinformatics computing software indicated the deleterious impact of this variant (PP3_Supporting)., Moreover, proteomic conservation analysis suggested that the p.C241S mutation results in the substitution of a polar, uncharged cysteine at site 241 with a polar, uncharged serine. The N atom of the main chain and the hydroxyl group of the side chain form hydrogen bonds with the O atom of the nonpolar, uncharged phenylalanine at site 211, with hydrogen bond distances of 3.2 and 2.7 \u0026Aring; (the distances of normal protein structures are 3.1 \u0026Aring;). The O atom of the main chain forms a hydrogen bond with the N atom of the side chain of the nonpolar, uncharged tryptophan at site 246, with a hydrogen bond distance of 3.1 \u0026Aring; (the distances of normal protein structures is 3.2 \u0026Aring;). These changes in amino acid interactions lead to alterations in the structure and function of the mutated protein (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). The genotype and clinical phenotypic co-segregation in family members was considered as supportive evidence (PP1), the proband\u0026apos;s clinical symptoms were consistent with LCA, a monogenic genetic disease, which was considered as Pathogenic Supporting (PP4), gene testing report from a reliable and authoritative source considered this variant as Pathogenic, which serves as additional supportive evidence (PP5). Therefore, such a variant was classified as Pathogenic (PM2\u0026thinsp;+\u0026thinsp;PP1\u0026thinsp;+\u0026thinsp;PP3\u0026thinsp;+\u0026thinsp;PP4\u0026thinsp;+\u0026thinsp;PP5) based on the standards and guidelines for the interpretation of sequence variants. The proband was ultimately diagnosed with binocular LCA.\u003c/p\u003e\n\u003ch3\u003eFamily 17\u003c/h3\u003e\n\u003cp\u003eThe proband of family 17 was a 7-year-old normal color, fundus tessellation with no light reflection of foveal centralis. OCT examination showed no obvious structure of the fovea centralis (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). ERG showed significant impairment of cone and rod cell function in both eyes. The proband was born with hexadactyly in both hands and surgical treatment had been performed. At the age of 7, the proband was 133 cm in height, 44 kg in weight, and body mass index (BMI) was 24.87 kg/m2. The proband\u0026apos;s parents complained that she usually had poor memory and slow response, and the intelligence test suggested subnormal intelligence. As validated by the whole exome sequencing and Sanger sequencing, a homozygous copy number deletion located in the region of chr7:33404663\u0026ndash;33409821 was found in \u003cem\u003ethe BBS9\u003c/em\u003e gene of the proband. The size of the homozygous copy number deletion was 5159bp, which covered exon 17, part of intron 16, and part of intron 17 in \u003cem\u003ethe BBS9\u003c/em\u003e gene, resulting in an alteration in protein length. It was confirmed from Sanger sequencing analysis that \u003cem\u003ethe BBS9\u003c/em\u003e gene variant was homozygous in affected individuals and heterozygous carriers in normal parents, supporting autosomal recessive inheritance (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). The proband was eventually diagnosed with Bardet-Biedl syndrome (BBS). This variant does not disrupt the reading frame but likely results in a possible change in protein length. Such copy number deletion variant, being nonpolymorphic, was not found in the DGV Normal Population Copy Number Variant Database. No copy number variants with a similar segment size were found in the Decipher database. According to the ACMG guidelines, such deletion is of uncertain significance.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDue to the genetic characteristics of autosomal recessive inherited diseases, we generally believe that homozygous variant patients are more likely to appear in families with consanguineous marriages. Before this, research on homozygous variants often focused primarily on families with consanguineous marriages\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e, however, in this study, 82.35% of the proband\u0026rsquo;s parents were non-consanguineous marriages. We speculate that some variants may have a higher carrier rate in the general population, and non-consanguineous couples may come from different genetic backgrounds and carry more diverse recessive genes, which increases the risk of homozygous variant offsprings from non-consanguineous families. When each member of a non-consanguineous couple carries recessive genes, these recessive genes become homozygous during the formation of gametes, resulting in the development of mutant traits in the offspring, such mutations may lead to genetic disorders such as intellectual disabilities, delayed development, and congenital malformations. We can reduce the incidence of homozygous mutations in consanguineous families by avoiding consanguineous marriages\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. However, it is impossible to avoid the occurrence of homozygous patients in non-consanguineous families. Therefore, it is necessary to further analyze the variants with high carrier frequencies in the normal population, which shall help us better prevent and intervene in the occurrence of hereditary diseases. Through genetic counseling and testing, it is possible to understand the genetic status and family medical history of both couples and predict the risk of offspring. On this basis, targeted prevention and intervention measures can be taken, such as prenatal consultation, prenatal diagnosis, etc., to predict the incidence probability of the next generation and produce healthy offspring\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. At present, there is a growing emphasis on carrier screening for monogenic genetic diseases both at home and abroad. Screening of individuals who have no obvious genetic disease phenotypes but may carry pathogenic variants of genetic disease genes, or common pathogenic genes, can further reduce the prevalence of offspring. Therefore, it is very necessary to continuously explore the relationship between genes and diseases, which can provide a basis for preconception carrier screening.\u003c/p\u003e \u003cp\u003eIt has been found from previous studies that patients with homozygous variants appear to exhibit a more severe clinical phenotype and earlier age of onset. For instance, homozygous variants in the MERTK gene can lead to a more severe form of retinitis pigmentosa\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e; homozygous variants in PTPN11gene resulted in Noonan syndrome (NS) patients with more pronounced short stature and slower growth rates compared to previously reported patients with heterozygous variants\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e; homozygous variations in the PRPH2gene often result in a more severe LCA phenotype, and the patients usually have onset of disease before the age of 10\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. The clinical phenotypes of the proband in this study were also consistent with this finding, as compared with the patients with heterozygous variants in the PRPH2 gene showing earlier onset and a more severe form of LCA compared to those with heterozygous variants. Such finding is also borne out by animal experiments. For instance, zebrafish with homozygous variants in the ZNF408 gene exhibit more severe retinal vascular abnormalities compared to those with heterozygous variants\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. However, not all patients with the homozygous variants are consistent with this conclusion. We have observed that more than 70% of the patients in our study had an age of onset after 10 years old, but nearly 50% had a BCVA\u0026thinsp;\u0026le;\u0026thinsp;0.3 in at least one eye before the age of 40. Therefore, the results of our study suggest that the majority of patients with homozygous variants do not show an early age of onset, but the proportion of moderate-to-severe visual impairment and blindness in working age at a high level.\u003c/p\u003e \u003cp\u003e7 new variants were identified in this study. Previous studies have shown that the short isoform a of the \u003cem\u003eUSH2A\u003c/em\u003e gene is expressed in the retina and the cochlea, whereas the long isoform b is expressed only in the retina\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. The truncated mutations lead to premature termination of translation and result in nonsense-mediated mRNA decay causing truncation or even deletion of proteins, which potentially leads to more severe phenotypes compared to protein changes caused by nontruncated missense mutations\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. In this study, a new homozygous frameshift variant c.10179delG (p.Met3393fs) was detected in the \u003cem\u003eUSH2A\u003c/em\u003egene in the Family 4 Proband, and the patient was diagnosed with Usher syndrome because of the clinical presentation of RP combined with deafness.\u003c/p\u003e \u003cp\u003eIn the retina, \u003cem\u003ePROM1\u003c/em\u003e is mainly located in the outer segment of the retinal photoreceptor and plays a key role in the morphogenesis of the photoreceptor outer segment membrane discs\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Throughout literature, \u003cem\u003ePROM1\u003c/em\u003e gene variants have been associated with multiple diseases with overlapping phenotypes, such as retinitis pigmentosa, cone-rod dystrophy, Stargardt-like macular dystrophy, bull\u0026rsquo;s-eye macular dystrophy, and Leber congenital amaurosis\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Depending on the type of variants, the age of onset, initial symptoms, and severity of the disease can vary among patients. In this study, two probands carrying the \u003cem\u003ePROM1\u003c/em\u003e gene variant had chief complaints of reduced vision and night blindness, respectively, and were diagnosed with Stargardt's disease and RP, respectively, in combination with fundus and ERG examinations. The \u003cem\u003ePROM1\u003c/em\u003e gene variant can be inherited in both recessive and dominant patterns\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. As compared with the retinal dystrophies caused by dominant \u003cem\u003ePROM1\u003c/em\u003e gene variants, the patients with retinal dystrophies associated with recessive \u003cem\u003ePROM1\u003c/em\u003e gene variants, such as CORD and Leber congenital amaurosis (LCA), have an earlier age of onset, more severe clinical phenotype, and can present with vision decline at an early stage of the disease\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e, which is highly consistent with the clinical phenotypes of the patients in this study. Homozygous frameshift deletion variant c.265del (p.Leu89Phefs*4) and c.253del (p.Leu85Phefs*4) of \u003cem\u003eCNGA1\u003c/em\u003e gene were detected in the probands of Family 12 and Family 13, and they were clinically diagnosed as RP. As shown by previous studies, variants in this gene are strongly associated with RP. This study further expands the spectrum of variants of \u003cem\u003eCNGA1\u003c/em\u003egene. Diseases caused by variants of \u003cem\u003ePRPH2\u003c/em\u003egene are dominantly inherited predominantly and only a few are autosomal recessive inheritance\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Currently, only one case of autosomal recessive RP due to \u003cem\u003ethe PRPH2\u003c/em\u003e gene has been reported, but the detailed clinical manifestations of this patient have not been reported\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. In this study, the homozygous missense variant c.640T\u0026thinsp;\u0026gt;\u0026thinsp;A (p.Cys214Ser) was detected in \u003cem\u003ethe PRPH2\u003c/em\u003e gene of Family 14 Proband. The affected child had poor binocular vision since childhood, with a BCVA of 0.02 in both eyes at the age of 6, but did not show any symptoms of night blindness. Due to the overlap of phenotypes, it is easy to ignore the differentiation between RP and LCA. However, patients with LCA usually have severe visual impairment at birth, and the lesion often involves the outer retina, combined with the symptoms of binocular horizontal nystagmus, difficulty fixation, and eye poking, the patient in this case was diagnosed with LCA. Patients carrying a heterozygous variant of \u003cem\u003ethe PRPH2\u003c/em\u003e gene generally have an age of onset after 30 years old\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. The patient in this case was born with visual impairment, which may be preliminary inferred that patients with homozygous variants of \u003cem\u003ethe PRPH2\u003c/em\u003e gene have an earlier age of onset and a more severe clinical phenotype. This study further confirmed that variants in the \u003cem\u003ePRPH2\u003c/em\u003e gene can lead to recessive IRDs.\u003c/p\u003e \u003cp\u003eThe BBSome complex is a key regulator of the ciliary membrane proteome, which is mainly involved in the process of ciliogenesis and intraflagellar transport\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. The protein encoded by the BBS9 gene is an important component of ciliary structure. In previously reported cases, BBS9-related diseases only led to Bardet-Biedl syndrome\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. The proband of family 17 in this study presented typical BBS symptoms at first visit, and a homozygous copy number deletion in the chr7:33404663\u0026ndash;33409821 region of the BBS9 gene was detected in the WES. To further clarify such a variant, we verified it by real-time fluorescent quantitative PCR (qPCR). So far, there are almost no reports of BBS9 copy number variant, and most reports are mainly homozygous variants. The patients exhibit polydactyly, obesity, intellectual disability, and associated ocular symptoms such as vision loss, night blindness, and nystagmus from early childhood\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e, which are reported to be similar to the phenotype of the proband of family 17. There are still 30\u0026ndash;40% of IRDs that cannot be explained by routine genetic testing, of which copy number variants account for 9%\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. Therefore, in genetic diagnosis, when whole exome sequencing shows negative results, and the patient has a suspicious clinical phenotype, other different forms of variants, such as copy number variant, should be further considered to identify the pathogenic variant.\u003c/p\u003e \u003cp\u003eIn this study, homozygous variants were found to have a higher incidence in non-consanguineous families, suggesting that heterozygous variants of some genes have a higher carrier rate in the normal population. We also identified 7 new pathogenic variants and analyzed their possible pathogenic mechanisms, emphasizing the non-negligible impact of copy number variants on the disease. Cases with homozygous variants are relatively rare, but patients with homozygous variants can exclude the influence of other heterozygous variants to clarify whether the relevant variants are the direct cause of the patient's disease, which reinforces the supporting evidence for the association of Mendelian disease variants and the clinical phenotypes. It also emphasizes the importance of screening for carriers with no family history of consanguineous marriage and no obvious phenotype of genetic disease. Timely detection of couples at high risk of having children and individuals with moderate or greater severity IRDs can provide a basis for genetic counseling, reproductive decision-making, disease prevention, and management.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSubjects\u003c/h2\u003e \u003cp\u003eThe study complied with the Declaration of Helsinki, and it was approved and reviewed by the Human Research Ethics Committee of the People's Hospital of Ningxia Hui Autonomous Region(No. 2022-KJCG-006). In this study, we need to publish the facial information of participants to support the presentation of research results, and all participants included in the study obtained informed consent from the patients or their legal guardians and signed the relevant informed consent forms. We have taken measures to protect the privacy of participants, including but not limited to anonymization processes and restricting access to the information.Participants have the right to withdraw their consent at any time, and we will immediately cease the use of their facial information and remove it from published materials where possible.The publication of facial information in this study has been reviewed and approved by the Ethics Committee on Human Research at People Hospital in the Ningxia Hui Autonomous Region. This study included a total of 17 patients diagnosed with IRDs from January 2015 to September 2023 in Ningxia Eye Hospital and they were confirmed to carry homozygous variants by WES. The current medical history, past medical history, personal history, family history, and marital history of the probands were asked and recorded in detail, and the family tree was drawn.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eClinical evaluation\u003c/h2\u003e \u003cp\u003eThe complete ophthalmic examinations were performed on IRD patients, including slit-lamp microscopy, indirect ophthalmoscopy, uncorrected visual acuity (UCVA), best corrected visual acuity (BCVA), chromoscopy (Fifth Edition Color Blindness Examination Chart, Zi-Ping Yu), scanning laser ophthalmoscope (Optos DaytonaP200T), fundus photography (TOPCON, Japan, TRC-NW300), optical coherence tomography (OCT) (HD-OCT4000, Carl Zeiss Meditec, USA), electroretinogram (Roland Consult Stasche, Finger GumbHD-14770, Germany), perimetry (Humphrey Field Analyzer 750i, Germany). According to the patient's clinical phenotype and the characteristics of pathogenic genes, the necessary general examinations were performed, such as hearing tests, etc. Diagnostic criteria for visual impairment: According to the visual impairment standards established by WHO in 2019\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e, the best corrected visual acuity (BCVA) of the better-seeing eye is less than 0.5 but equal to or greater than 0.3is classified as mild visual impairment, if which being less than 0.3 but equal to or greater than 0.1, it is classified as moderate visual impairment, if which being less than 0.1 but equal to or greater than 0.05, it is classified as severe visual impairment, and if which is less than 0.05 is classified as blindness.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMethods\u003c/h2\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003eGenomic DNA extraction\u003c/h2\u003e \u003cp\u003e5 ml of peripheral venous blood was collected from all participants, and genomic DNA was extracted by using the Qiamp Blood Mini Kit DNA extraction kit (Qiagen, Germany) with standard protocol after the concentration and purity were detected by UV spectrophotometer and 1.5% agarose gel electrophoresis, stored the DNA in a -20\u0026deg;C refrigerator.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eWhole exome sequencing\u003c/h2\u003e \u003cp\u003eWhole exome sequencing (WES) capture was performed by using Agilent SureSelect Exon Capture Kit, and sequencing was performed with a high-throughput sequencer (Illumina) at a depth of 100\u0026times;. The original sequencing data were processed by Illumina base-calling Software 1.7 and then compared the data with the human genome DNA reference sequence (NCBI build 37.1) of the National Center for Biotechnology Information (NCBI). Single nucleotide variants (SNV), insertion and deletion variants (Indel) were analyzed by SOAP software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://soap.genomics.org.cn\u003c/span\u003e\u003cspan address=\"http://soap.genomics.org.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and BWA software ( \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bio-bwa\u003c/span\u003e\u003cspan address=\"http://bio-bwa\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. sourceforge.net/), to obtain all the variants occurring in the DNA sequences in the samples. Then filtered out the high-frequency variant sites with Minor Allele Frequency (MAF)\u0026thinsp;\u0026gt;\u0026thinsp;1% in the database (db135), and filtered out the variants that do not affect the structure and function of proteins. After step-by-step filtering, the homozygous variants shared by all patients in the family were screened to identify candidate pathogenic variations. Sanger sequencing was used for candidate pathogenic variants to exclude false positives, and further co-segregation of genotypes and phenotypes was validated among normal family members.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eFluorescence quantitative PCR\u003c/h2\u003e \u003cp\u003eFluorescence quantitative PCR was performed to verify the detected copy number variant. Genomic DNA (gDNA) from the peripheral blood of family members was extracted using a blood genomic DNA extraction kit (Beijing Tiangen Biochemical Technology Co., Ltd.). Primers were designed for the gene under investigation. The qPCR reaction system was prepared according to the NovoStart\u0026reg; SYBR qPCR SuperMix Plus kit. The 20-\u0026micro;L system contained 10 \u0026micro;L of 2\u0026times;NovoStart\u0026reg; SYBR qPCR SuperMix Plus, 0.5 \u0026micro;L each of forward and reverse primers, an equal volume of 1 \u0026micro;L of gDNA (10 ng), and 8 \u0026micro;L of water. An initial denaturation (95\u0026deg;C for 1 min) followed by 40-cycle amplification (95\u0026deg;C for 20 s, 60\u0026deg;C for 20 s) program was performed on a Roche Light Cycler II 480 real-time fluorescence quantitative PCR instrument, with 3 replicates per reaction. The copy numbers of genes were calculated using the 2-ΔΔCt method, normalized by the Ct values of the internal reference genes, and healthy individuals served as reference. For autosomes, a relative copy number around 2 indicates a normal sample, while a relative copy number value around 1 indicates a sample with a 1 copy number deletion.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003ePathogenicity analysis of genetic variants\u003c/h2\u003e \u003cp\u003eThe American College of Medical Genetics and Genomics (ACMG) established Standards and Guidelines for Interpretation of Sequence Variants in 2015, which were used to evaluate the pathogenicity of novel variations for genetic variation. MAF\u0026lt;0.005 was used as the criteria to exclude benign variants by reference to the databases for East Asian populations Allele frequencies available with 1000 Genomes Project (1000G, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://browser.1000genomes.org\u003c/span\u003e\u003cspan address=\"http://browser.1000genomes.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and Exome Aggregation Consortium (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://exac.broadinstitute.org/\u003c/span\u003e\u003cspan address=\"http://exac.broadinstitute.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Polyphen2(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://genetics.bwh.harvard.edu/pph2\u003c/span\u003e\u003cspan address=\"http://genetics.bwh.harvard.edu/pph2\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), SIFT(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://sift.jcvi.org),REVEL(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5065685/\u003c/span\u003e\u003cspan address=\"http://sift.jcvi.org),REVEL(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5065685/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), CADD (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cadd.gs.washington.edu/score\u003c/span\u003e\u003cspan address=\"https://cadd.gs.washington.edu/score\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and Mutation Taster (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://mutationtaster.org/\u003c/span\u003e\u003cspan address=\"http://mutationtaster.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were used for pathogenicity prediction. Measurements of the conservation of gene sequences across species in evolution have been made using websites like GERP++ (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bio.tools/gerp\u003c/span\u003e\u003cspan address=\"https://bio.tools/gerp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Variants were classified as uncertain clinical significance when at least 1 of 4 predictions had a benign outcome or when there was insufficient evidence of pathogenicity. When all predictions turned out to be accurate, variations were categorized as potentially pathogenic when used in conjunction with further data. Pathogenic variants were defined as frameshift, nonsense, and variants with experimental proof of causing loss of protein function. For the conservativeness study of variant loci, the online analysis tool Multalin (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://sacs.ucsf.edu/cgi-bin/multalin.py\u003c/span\u003e\u003cspan address=\"http://sacs.ucsf.edu/cgi-bin/multalin.py\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was employed. Interpretation rules of copy number variants (CNV) referred to the 2019 Edition of the \u003cem\u003eACMG Guidelines for Interpretation and Reporting of Copy Number Variants\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. Alphafoldwas used to construct the normal protein structure, and Pymol 2.3 software was used to make a visualized analysis of the mutant protein.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e: The authors thank all patients and their family members for their participation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e: The Ethics Committee on Human Research at People Hospital in the Ningxia Hui Autonomous Region accepted and examined our work (reference number: 2022-KJCG-006), which adhered to the Declaration of Helsinki. Each participant or their legal guardians provided their written informed permission before to taking part.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e: Xue Fan and Weining Rong wrote the main manuscript, Zhen Li and Lingzhi Sha collected cases data and followed up patients. Weining Rong and Xunlun Sheng polished the article. All authors reviewed the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e：The datasets generated and analyzed during the current study are available in the [Banklt] repository (BankIt (https://www.ncbi.nlm.nih.gov/nuccore/) ID: PQ038085、PQ038086、PQ038087、PQ045748、PQ045749、PQ045750).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e: I declare that the authors have no competing interests as defined by Clinical Genetics or other interests that might be perceived to influence the results and discussion reported in this paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: This work was supported by the National Natural Science Foundation of China(82260206), the training project of the scientific innovation commanding talented person in Ningxia Hui Autonomous Region(2020GKLRLX13), Major achievement transformation project of Ningxia Hui Autonomous Region(2022CJE09011), the key research development project of Ningxia Hui Autonomous Region(2024BEG02017).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eScholl, H. 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R. \u003cem\u003eet al.\u003c/em\u003e Technical standards for the interpretation and reporting of constitutional copy-number variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics (ACMG) and the Clinical Genome Resource (ClinGen). Genet Med 22, 245\u0026ndash;257 (2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables ","content":"\u003cp\u003eTables 1 to 3 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Inherited retinal dystrophies, Homozygous variation, Genotype, Clinical phenotype, Consanguineous marriage","lastPublishedDoi":"10.21203/rs.3.rs-4776885/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4776885/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this study, patients with inherited retinal dystrophies (IRDs) who visited Ningxia Eye Hospital from January 2015 to September 2023 were analyzed. Through whole genome exon sequencing (WES) and Sanger verification, 17 probands carrying homozygous mutations were detected. The association between the genotype and clinical phenotype of patients with homozygous variants was analyzed. Among all the patients, 3 patients (17.6%) had a family history of consanguineous marriage, and the onset age of 5 patients(29.41%) was less than 10 years. According to 12 patients (70.59% ), they had the best corrected visual acuity (monocular)\u0026thinsp;\u0026lt;\u0026thinsp;0.3. 3 were blind, 9 with moderate to severe visual impairment, and 2 with mild visual impairment. 16 homozygous variants were detected on 9 different genes, of which 7 were de novo homozygous variants, including frameshift variants, missense variants, and a copy number variant. These mutations are related to clinical phenotypes such as Usher syndrome type II (USHⅡ), Stargardt disease (STGD), Retinitis Pigmentosa (RP), Leber congenital amaurosis (LCA), and Bardet-Biedl syndrome (BBS) respectively. The results of the study indicate that more than 80% of homozygous variant carriers originated from non-consanguineous families, emphasizing the significance of genetic screening for individuals who lack a family history of consanguineous marriage and no obvious clinical phenotypes, but who may carry genetic pathogenic variants for genetic diseases. Furthermore, analysis of patient genotypes and clinical phenotypes further expanded the spectrum of mutations in pathogenic genes for IRDs and the spectrum of clinical phenotypes for variants in known genes, and identifying couples at high fertility risk and individuals with moderate or greater severity IRDs can provide a basis for genetic counseling, reproductive decision-making, disease prevention, and management.\u003c/p\u003e","manuscriptTitle":"Enhanced Gene-Disease Association and Carrier Screening by Homozygous Mutation Carriers: A Study of Genotypes and Clinical Phenotypes in 17 IRD Families with Homozygous Mutations in Northern China","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-02 16:58:43","doi":"10.21203/rs.3.rs-4776885/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-15T09:48:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-12T20:34:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"59797430158155134344443125868448409443","date":"2024-10-02T07:02:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-03T10:12:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"283134121483733037866154556967088228917","date":"2024-08-19T06:48:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-19T01:01:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-12T06:10:55+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-08-11T09:43:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-11T09:13:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-07-21T13:13:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0a00afe8-1204-4e24-b24e-a1a86b6232ca","owner":[],"postedDate":"October 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":37184387,"name":"Biological sciences/Genetics"},{"id":37184388,"name":"Health sciences/Diseases/Eye diseases"}],"tags":[],"updatedAt":"2025-01-27T15:59:39+00:00","versionOfRecord":{"articleIdentity":"rs-4776885","link":"https://doi.org/10.1038/s41598-025-87844-5","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-01-24 15:57:06","publishedOnDateReadable":"January 24th, 2025"},"versionCreatedAt":"2024-10-02 16:58:43","video":"","vorDoi":"10.1038/s41598-025-87844-5","vorDoiUrl":"https://doi.org/10.1038/s41598-025-87844-5","workflowStages":[]},"version":"v1","identity":"rs-4776885","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4776885","identity":"rs-4776885","version":["v1"]},"buildId":"cTy_lsJlmDsVRNrSptgXS","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}