Background: Although the p.C759F (c.2276G>T, p.Cys759Phe) variant in the USH2A gene has been identified in association with retinal degeneration by several authors, its pathogenicity has been questioned once by the publication of two unaffected homozygotes from a single family. Objectives: The objective of the study was to ascertain the role of p.C759F in hereditary retinal disease. Methods: We examined 87 research articles reporting on patients carrying this variant and then used this information as primary data for a series of meta-analytical tests. Results: Independent statistical analyses showed that p.C759F (i) is highly enriched in patients with respect to healthy individuals, (ii) represents a clear-cut recessive allele causing disease when it is in trans with other mutations, (iii) is pathogenic in homozygotes. Conclusions: Our results confirm that p.C759F is a bona fide mutation, leading to retinal blindness according to a recessive pattern of inheritance.

Retinitis pigmentosa (RP) is a genetic condition that affects exclusively the retina. It is characterized by progressive loss of vision, in turn due to the degeneration of the rod and cone photoreceptors [1]. Usher syndrome (USH) is a rare monogenic and recessive disorder, characterized by RP and sensorineural hearing loss [2]. Depending on the severity of the auditory deficit, the presence or absence of vestibular dysfunction, and the age of onset of RP, USH is further classified into four subtypes [3, 4]. Patients affected by the most frequent form, type 2 (USH2), generally display congenital moderate-to-severe hearing impairment that is non-progressive. With an overall count of over 1,000 pathogenic variants [5], USH2A is the most commonly mutated gene [6] in both USH and non-syndromic RP (MIM *608400) [7‒11]. Generally speaking, it has been observed that truncating mutations, in a homozygous state or in compound heterozygosity with other truncating mutations, are associated with the syndromic form of the disease, whereas missense mutations, regardless of the nature of the mutation detected in trans, can be linked to non-syndromic RP or with RP and mild hearing impairment [8, 11]. In particular, the c.2276G>T (NM_206933.4) variant, corresponding to the p.C759F missense (p.Cys759Phe; NC_000001.10:g.216420460C>A, hg19; NC_000001.11:g.216247118C>A, hg38), was originally described to be associated with non-syndromic RP [12] and later found to be one of the most frequent pathogenic mutations in USH2A overall, according to cohort studies including hundreds of patients (e.g., [10]). However, p.C759F was also reported to be present homozygously in two asymptomatic individuals from a single family [13, 14], in apparent disagreement with such extensive genetic screens.

Recognizing p.C759F as a clear-cut pathogenic variant has very important consequences for correct genetic counseling, medical genetics research, and potential clinical interventions, including enrollment of patients in gene-based clinical trials [15, 16]. In this study, we reviewed all existing literature about p.C759F and performed an unsupervised genotype versus phenotype meta-analysis to evaluate the pathogenic potential of this variant.

Literature Search

PubMed, LitVar [17], ClinVar [5], and the LOVD [18] database were mined to identify journal articles containing data that were relevant to our study. For PubMed, we used keywords and MESH headings such as USH2A, usherin, usher syndrome type 2, c.2276G>T, Cys759Phe and C759F, etc. (the exact search terms are listed in the suppl. Methods; for all online suppl. material, see https://doi.org/10.1159/000535545). Papers from LitVar, ClinVar, and LOVD were retrieved by searching directly for our variant of interest. Articles were included in our collection if they (i) were published between January 1, 2000, and January 1, 2022, (ii) were written in English, and (iii) were peer-reviewed. Genotypes were retrieved from primary data that were present in any part of these papers (main text, figures, tables, suppl. material, etc.), obtained by gene panel sequencing, whole-exome sequencing, whole-genome sequencing (WGS), or targeted Sanger sequencing.

Population Analyses

To compute the theoretical number of patients with recessive RP and USH, we took advantage from the data reported by Hanany et al. [6] on the frequency of recessive inherited retinal dystrophies (IRDs) mutations (at the heterozygous state) in the general world population. Specifically, this study compiled the genetic landscape of all variants associated with these diseases for six main ethnic groups, based on data extracted from the gnomAD database [19]. Using a matrix of allele frequencies, the authors calculated the genetic prevalence for each gene in association with IRDs, in every population, by simulating the chances of heterozygous carriers to meet and generate affected offspring carrying biallelic mutations.

In this work, we repeated the same calculations by considering, however, variants in recessive RP and Usher genes only (excluding p.C759F). We then assessed the theoretical number of affected individuals by multiplying the obtained genetic prevalence by the number of individuals of a given population and, finally, of the whole world.

To calculate the theoretical number of compound heterozygotes for p.C759F and another mutation (p.C759F/mut) in the general population, we first identified all known USH2A mutations by considering all variants categorized as pathogenic or likely pathogenic mutations in ClinVar (data collection of Jan 9, 2022), with the exception of p.C759F. We then retrieved their frequencies from the gnomAD database and summed such frequencies (online suppl. Table 1). The product of this sum and p.C759F’s frequency, multiplied by 2, would result in the frequency of people with the p.C759F/mut genotype, according to the Hardy-Weinberg law. Similarly, the frequency of individuals who are homozygous for p.C759F in the general population was obtained by computing the square of this allele’s frequency in gnomAD, which corresponds to the q2 value in the Hardy-Weinberg equation.

Statistical Analysis

Individuals were stratified according to their different genotypes: p.C759F homozygotes, compound heterozygotes for p.C759F and another mutation, or monoallelic for p.C759F. To determine the pathogenicity of the p.C759F variant, 2 × 2 contingency tables were assessed by Pearson’s χ2 tests, and specifically by the χ2 test function in R, with default options. All tests are summarized in Tables 1–4. All allele frequencies from the general population were derived from the gnomAD database v2.1.1 [19].

Literature Search

In total, 434 articles matched the search criteria described in the Methods (online suppl. Tables 2–5; online suppl. Fig. 1). Of those, 87 reported original primary data on p.C759F, identified mainly by targeted sequencing, whole-exome sequencing, and, in fewer instances, whole-genome sequencing. A total of 667 individuals carried the variant (online suppl. Table 6), of whom 564 individuals were ophthalmic patients (mostly with non-syndromic RP and USH2) and 103 individuals were unaffected. More specifically, 58 out of the 564 patients were homozygous for p.C759F, 389 were biallelic (or presumed biallelic) for p.C759F and another mutation (Fig. 1), and 117 were monoallelic for p.C759F (heterozygous for p.C759F with no other recognized USH2A mutation). Notably, 24 ophthalmic patients were reported to carry p.C759F in trans with deep intronic variants or CNVs in USH2A. Concerning unaffected individuals, 101 were heterozygous for p.C759F and corresponded in all instances but one to relatives of patients carrying p.C759F; none was a compound heterozygote for p.C759F and another mutation, whereas 2 were the discordant homozygous cases mentioned above [13, 14].

Fig. 1.

Schematic representation of the USH2A protein, usherin. All pathogenic or likely pathogenic variants reported so far in patients with p.C759F are shown. Nomenclature for all variants is the same as the one reported in the original publication that describes them.

Fig. 1.

Schematic representation of the USH2A protein, usherin. All pathogenic or likely pathogenic variants reported so far in patients with p.C759F are shown. Nomenclature for all variants is the same as the one reported in the original publication that describes them.

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Empirical Assessment of Pathogenicity

As an empirical, non-statistical test for pathogenicity of p.C759F, we compared the number of homozygous patients from our literature search versus gnomAD, a database of healthy individuals. As mentioned, our collection of articles revealed that 58 patients (out of 564 affected individuals) were homozygous for p.C759F, whereas none of the ∼140,000 healthy controls from gnomAD displayed this genotype. This simple assessment indicates that a person who is homozygous for p.C759F (e.g., a newborn) is very likely to develop retinal disease at some point in their life, based on unrefined epidemiological data alone.

Allelic Enrichment in Patients versus Controls

A similar, more rigorous test could be performed by comparing p.C759F’s allele frequency in the general population [19] versus the one detected in the largest unsupervised genetic screening of patients with IRDs performed so far, reported by Perea-Romero et al. [10]. According to the gnomAD database, in 64,301 healthy non-Finnish European (NFE) individuals (128,602 alleles) the p.C759F allele was present 182 times. Notably, no homozygotes were reported. Conversely, in the study by Perea-Romero et al. [10], this allele was detected 138 times in 4,386 Spanish patients with RP or syndromic retinal degenerations (8,772 alleles) (Table 1). This represents an ∼11-fold enrichment (odds ratio, OR = 11.3) of p.C759F in patients versus controls, which is also extremely significant from a statistical standpoint (χ2 = 718; p value = 3.5 × 10−158), indicating a very clear association of the p.C759F allele with disease. Similar calculations performed by using data from individuals of Southern European ancestry (SEU) from gnomAD as controls (instead of non-Finnish Europeans) gave comparable results (∼7-fold enrichment, χ2 = 112; p value = 4.0 × 10−26, Table 1).

Table 1.

Allelic enrichment of p.C759F in patients versus controls

Number of p.C759F alleles at position c.2276Number of wt alleles at position c.2276Total
Patients (Perea-Romero et al.) 138 8,634 8,772 
Controls (gnomAD-NFE) 182 128,420 128,602 
Controls (gnomAD-SEU) 26 11,544 11,570 
Number of p.C759F alleles at position c.2276Number of wt alleles at position c.2276Total
Patients (Perea-Romero et al.) 138 8,634 8,772 
Controls (gnomAD-NFE) 182 128,420 128,602 
Controls (gnomAD-SEU) 26 11,544 11,570 

NFE test: χ2 = 718, p value = 3.5 × 10−158, OR = 11.3.

SEU test: χ2 = 112, p value = 4.0 × 10−26, OR = 7.10.

Is p.C759F a Recessive Mutation?

Next, we wanted to assess whether p.C759F had all the characteristics of a bona fide Mendelian, recessive mutation. In other words, we set out to investigate whether this variant is pathogenic whenever it is in trans with another known recessive mutation in USH2A (the analysis of homozygous genotypes is described below). If p.C759F was a benign DNA variant, then the frequency of individuals with recognized mutations in USH2A in trans with respect to p.C759F would roughly be the same in patients versus controls, as it would be the case for any other non-pathogenic variants. To avoid any ascertainment bias, we refrained from directly scoring the data on compound heterozygotes we retrieved from our literature search versus controls since this search was, by design, specifically selecting for p.C759F genotypes. Instead, we assessed the number of individuals in a projected world population who are expected to carry a clear-cut mutation in USH2A in trans with respect to p.C759F (p.C759F/mut). By merging the information on pathogenicity of USH2A variants, as reported in ClinVar [5], and their frequency, retrieved from the full gnomAD database (ALL) [19], we estimated 39,082 individuals to have such genotypes, according to the Hardy-Weinberg equation and assuming the world population to be 7.6 billion people (see Methods). That is, roughly, one individual out of 200,000 from the general population.

We then repeated the same calculation by considering patients only. According to the data reported by Hanany et al. [6], also based on frequencies from gnomAD, worldwide (i.e., out of 7.6 billion people) there should be 1,561,234 individuals suffering from USH or recessive RP. Again, if p.C759F was a benign allele, then the proportion of compound heterozygotes (p.C759F/mut) among patients would be roughly the same as the one detected in controls. In our literature search, which certainly does not include the assessment of the world global population, we detected 365 patients who had this genotype (we excluded compound heterozygotes with deep intronic variants or CNVs in USH2A since these mutations were not considered by Hanany et al. [6]). In other words, in the world there are at least 365 patients with USH2 or recessive RP carrying p.C759F in trans with a mutation (mut). This would correspond to a minimal value of 365/1,561,234 = 1 person out of 4,277 individuals. Even after considering this substantial underestimation in the number of patients with this genotype over the globe, the enrichment between patients carrying p.C759F/mut (1:∼4,000) with respect to controls with the same genotype (1:∼200,000) is ∼45-fold higher. The p value associated with such enrichment was lower than the smallest number that can be represented by a computer with a typicalfloating-point number notation (i.e., < 5.0 × 10−324), and its corresponding χ2 value was 15,681 (Table 2). Hence, p.C759F results in a disease phenotype whenever it is in trans with another mutation (i.e., in compound heterozygosity), therefore acting as a classical recessive allele, with an alternative hypothesis being in the range of one event over several billions of billions.

Table 2.

Test for p.C759F being a recessive mutation

Individuals with p.C759F/mut in USH2AIndividuals with other genotypesTotal
Patients 365 1,560,869 1,561,234 
Controls (gnomAD-ALL) 39,082 7,599,960,918 7,600,000,000 
Individuals with p.C759F/mut in USH2AIndividuals with other genotypesTotal
Patients 365 1,560,869 1,561,234 
Controls (gnomAD-ALL) 39,082 7,599,960,918 7,600,000,000 

χ2 = 15,681, p value <5.0 × 10−324, OR = 45.5.

Is p.C759F Pathogenic in the Homozygous State?

Finally, we assessed whether p.C759F is pathogenic in homozygotes. To test this hypothesis, we repeated the same calculations described above with respect to the p.C759F/p.C759F genotype in patients versus controls. Again, if this genotype was benign, then the percentage of homozygotes should not be different in individuals from the general population versus patients. As mentioned, we refrained from using data on p.C759F frequency from our literature search since in this dataset affected individuals with p.C759F were specifically selected, and therefore statistics on these numbers may be subject to bias. Instead, we estimated the number of p.C759F homozygous individuals in the general population by using the information from the full gnomAD database (ALL). Since the reported frequency of p.C759F is 9.68 × 10−4, the frequency of homozygotes would be the square of this number (by the Hardy-Weinberg equation), i.e., 9.36 × 10−7, or 7,117 people in a world population of 7.6 billion individuals. This value corresponds approximately to one out of 1,000,000 unaffected individuals. As before, the frequency of individuals suffering from USH and recessive RP should be 2.11 × 10−4 (or 1,561,234 people in the world), according to the data published by Hanany et al. [6] and to gnomAD. Our literature findings, again, based on just a very small fraction of the world population, revealed that on our planet there are at least 58 patients who are also homozygotes for p.C759F. This corresponds to a minimal value of 58/1,561,234 patients, i.e., 1 person out of 26,918 affected individuals. Hence, the enrichment between patients having this genotype (1:∼27,000) with respect to controls with the same genotype (1:∼1,000,000) is approximately 40-fold higher, with an associated p value of less than 5.0 × 10−324 and a χ2 value of 2,131 (Table 3).

Table 3.

Test for p.C759F being pathogenic in homozygotes (meta-analysis test)

Individuals with p.C759F/p.C759FIndividuals with other genotypesTotal
Patients 58 1,561,176 1,561,234 
Controls (gnomAD-ALL) 7,117 7,599,992,883 7,600,000,000 
Individuals with p.C759F/p.C759FIndividuals with other genotypesTotal
Patients 58 1,561,176 1,561,234 
Controls (gnomAD-ALL) 7,117 7,599,992,883 7,600,000,000 

χ2 = 2,131, p value <5.0 × 10−324, OR = 39.7.

We could obtain the same results by considering hard experimental data from unsupervised screenings of cohorts of patients versus data from the general, unaffected population. For instance, the study by Perea-Romero et al. [10] identified 21 p.C759F homozygotes among a cohort of 4,386 unrelated patients with RP or syndromic retinal degeneration, corresponding approximately to one individual out of 209. Based on gnomAD data, in healthy non-Finnish Europeans (NFE) p.C759F’s frequency is ∼1.41 × 10−3 (precisely: 0.001415), corresponding to 1,473 homozygotes in Europe (∼735,800,000 people) (Table 4). This is in turn equivalent to a frequency of ∼2 homozygous individuals per million people from this continent, which is far less than one person (precisely: 0.0088 persons) in a cohort of 4,386 subjects. Finding a ∼2,400-fold enrichment of p.C759F/p.C759F in patients versus controls (i.e., 21 people vs. 0.0088) is obviously very significant (χ2 = 47,149; p < 5.0 × 10−324), indicating that homozygosity for p.C759F is clearly pathogenic. The same analysis, performed with genotype data retrieved from Southern Europeans (SEU) and applied to the number of inhabitants of Spain (∼46,800,000), produced similar data (χ2 = 17,411; p < 5.0 × 10−324, Table 4).

Table 4.

Test for p.C759F being pathogenic in homozygotes (cohort tests)

Individuals with p.C759F/p.C759FIndividuals with other genotypesTotal
Patients (Perea-Romero et al.21 4,365 4,386 
Controls (gnomAD-NFE) 1,473 735,798,527 735,800,000 
Controls (gnomAD-SEU) 236 46,799,764 46,800,000 
Individuals with p.C759F/p.C759FIndividuals with other genotypesTotal
Patients (Perea-Romero et al.21 4,365 4,386 
Controls (gnomAD-NFE) 1,473 735,798,527 735,800,000 
Controls (gnomAD-SEU) 236 46,799,764 46,800,000 

NFE test: χ2 = 47,149, p value <5.0 × 10−324, OR = 2,403.

SEU test: χ2 = 17,411, p value <5.0 × 10−324, OR = 954.

The p.C759F mutation in the USH2A gene has been identified for the first time in the year 2000, in association with non-syndromic RP in 18 patients from 10 families [12]. Since then, this allele has been reported in more than 500 additional patients with IRD in several studies, including a few investigations involving large and very large cohorts [8, 10, 20]. These revealed p.C759F to be a major cause of RP without hearing loss [7, 8, 10, 21] and, based on the number of submissions to ClinVar, the most frequent missense mutation in USH2A overall.

However, the literature also reports a single consanguineous family with two asymptomatic siblings, found to be homozygous for p.C759F by targeted mutational screening, and two affected siblings, who were heterozygotes for the same missense variant [13]. Twelve years later, these two affected individuals were found to be homozygous for the p.R560C (NM_000283.4) variant in PDE6B, by the use of an NGS panel of 26 retinal disease genes and Sanger sequencing [14].

Over the years, pathogenicity of p.C759F has been investigated specifically at least four times, on individual cohorts. DuPont et al. [21] analyzed 982 non-Asian American patients with RP and showed a significant enrichment of this allele in such patients. In separate studies, Lenassi et al. [8], Blanco-Kelly et al. [7], and Perez-Carro et al. [11] also examined patients carrying variants in USH2A in individuals from the UK, Canada, and Spain, concluding that p.C759F is indeed a pathogenic mutation.

In this paper, we confirm the pathogenic nature of the p.C759F variant by performing a few meta-analytical tests on primary data from 87 articles, reporting on 667 individuals with this mutation, over different cohorts of patients. First, we showed that in our collection of papers homozygosity for p.C759F is reported to be associated with diseases in 58 cases out of 60 (∼97%), resulting in a high probability for individuals with this genotype, including pre-asymptomatic infants or children, to develop retinal degeneration at some point in their life. Although, strictly speaking, this is just an empirical observation that is also not free from ascertainment bias, it provides a practical and evidence-based estimate for p.C759F being pathogenic in the homozygous state.

Second, we tested whether p.C759F, independently of its zygosity or assortment with other alleles in USH2A, was enriched in patients versus controls. To this end, we compared the frequency of this variant in the largest cohort of naive patients with IRDs that is presently available [10] versus controls from the gnomAD database. Our analysis shows that p.C759F is significantly enriched in patients’ genotypes, with odds ratios of 11.3 and 7.10, in non-Finnish Europeans and in Southern Europeans, respectively, with extremely significant p values, indicating that this allele is clearly detrimental for retinal health.

Third, we assessed whether p.C759F would act as a classical, recessive Mendelian mutation, i.e., whether it would be pathogenic whenever in trans with another clear-cut USH2A mutation. This analysis, based on the null hypothesis that p.C759F is not a mutation and hence should not behave differently in patients versus controls, showed in fact that p.C759F/mut genotypes are enriched in patients by at least 45 times, again with a highly significant p value. This result clearly shows that p.C759F is not a benign rare variant but a true recessive mutation leading to pathological consequences in compound heterozygosity with other USH2A mutations.

Lastly, we tackled the very central question of whether homozygosity for p.C759F leads to retinal degeneration. We performed this analysis by two independent tests. In the first one, as for the previous assessment of compound heterozygosity, we estimated the number of individuals from the world population who should carry p.C759F homozygously, based on the experimentally determined frequency of this allele in various cohorts. According to these data, in the general population there should be 1 individual in a million with this genotype. Conversely, our literature search, which certainly does not include all patients in the world, shows that at least ∼1 in 27,000 patients with retinal degenerations is a homozygote for p.C759F. Once more, this enrichment is highly significant and confirms that homozygosity for this missense variant is pathogenic. In the second test, we performed a similar analysis, by using however experimental data only [10]. In a way, although it is limited to the Spanish population, this analysis is perhaps more accurate than the previous one, since it does not test a computed lower limit for genotypes in patients (e.g., the one detected by our literature search), but assesses the actual assortment of genetic variants in a real cohort of patients. Again, homozygosity for p.C759F was enriched as much as 2,400 times (or 954 times) in patients versus non-Finnish European controls (or in Southern European controls), with extremely significant p values, clearly rejecting the hypothesis that the p.C759F/p.C759F genotype is benign.

The results of our study are therefore univocal and in agreement with other recent analyses supporting pathogenicity for p.C759F. For example, the latest article from the RUSH2A project, assessing genotype-phenotype association for patients with variants exclusively in USH2A, identifies p.C759F as the most common missense mutation, specifically in relation to non-syndromic RP [22]. In addition, the ClinVar database labels p.C759F as “pathogenic” (three stars), following the manual curation of experimental data by an FDA-recognized team of scientists (ACMG codes applied: BS1, PS4, PM3_Strong, PP1_Strong, PP4, and PP3). As a comparison, the most frequent USH2A mutation, the c.2299del inactivating variant, has two stars. Finally, Reurink et al. [23] recently generated the first in vivo model for p.C759F and demonstrated that homozygous knock-in zebrafish animals exhibit reduced electroretinogram b-wave amplitudes and mislocalized rhodopsin, which are both typical signs of IRD.

Identifying p.C759F as a true pathogenic mutation is an extremely important matter since patients carrying this allele may be eligible for enrollment in clinical trials (and possible future therapy), involving, for instance, the skipping of exon 13 in USH2A transcripts [15, 16]. This meta-analysis on data from original research articles unequivocally confirms pathogenicity for p.C759F and supports the conclusions reached independently by multiple authors on their individual cohorts, over more than 20 years of research. In addition, our results clearly show that p.C759F acts as a classical, bona fide Mendelian mutation that causes disease with complete penetrance and should therefore be recognized as such in both medical research and genetic counseling.

The authors would like to acknowledge Virginie G. Peter and Francesco Paolo Ruberto for help with data collection.

An ethics statement is not applicable because this study is based exclusively on published literature.

The authors have no conflicts of interest to declare.

This work was supported by the Swiss National Science Foundation (Grants # 176097 and 204285).

Carlo Rivolta conceived the project, performed data analysis, and wrote the manuscript; Ji Hoon Han and Francesca Cancellieri performed data collection, data extraction, data analysis, and synthesis and wrote the manuscript; Irene Perea-Romero and Carmen Ayuso provided experimental data; and Mathieu Quinodoz curated statistical analyses. All authors read, edited, and approved the manuscript.

Additional Information

Ji Hoon Han and Francesca Cancellieri contributed equally to this work.

All data have been extracted from published literature, which has been appropriately referenced. All primary data are therefore publicly available via their original sources. Further inquiries can be directed to the corresponding author.

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