Spectrum of variants associated with inherited retinal dystrophies in Northeast Mexico

Inherited retinal dystrophies (IRDs) are characterized by progressive degeneration of photoreceptors, resulting in vision loss that may develop from birth to late middle age [1]. IRDs comprise a variety of overlapping conditions, including retinitis pigmentosa (RP), Stargardt disease/macular dystrophy (STGD/MD), cone-rod dystrophies (CRD), Leber congenital amaurosis (LCA) and syndromic forms such as Usher syndrome. Collectively, they have a prevalence of ~ 1 in 2,000–3,000 people [2, 3] and are estimated to affect up to 5.5 million individuals worldwide [4]. Rod dominant dystrophies, such as RP, present with peripheral vision loss and night blindness [5]. By contrast, cone dominant dystrophies, such as STGD/MD and CRD, present with central vision loss and impaired color perception, photophobia, and nystagmus [5, 6]. As both types of dystrophies progress, rod and cones may undergo degeneration compromising both central and peripheral vision at end stages. LCA is the most severe type of IRD, affecting both photoreceptors and the retinal pigment epithelium, with symptoms appearing during the first year of life [6].

IRDs exhibit both genetic and clinical heterogeneity. All inheritance patterns have been reported among IRDs, including autosomal, X-linked, mitochondrial, or digenic patterns [7, 8]. Currently, more than 200 causative genes have been identified, with the majority being autosomal recessive conditions [7, 8]. IRDs show considerable genetic and allelic heterogeneity [2, 3]. For example, ABCA4 mutations have been associated with the development of STGD, RP, CRD, and age-related macular degeneration [9]. Furthermore, intrafamilial variability is common among RDs [10, 11] and it is partly explained by environmental or genetic modifiers, specifically, mutations in other IRDs genes or single nucleotide variants [3].

Identification of the causative genetic variants is essential to ensure an accurate diagnosis and to provide a reference for genetic counseling [8]. In addition, understanding the molecular mechanism of IRDs is leading to the development of therapeutic interventions that seek to halt the loss of photoreceptors and vision preservation [3, 12]. Several studies have employed next-generation sequencing (NGS) techniques in multiple cohorts of RDs patients, with detection rates of molecular defects in ~ 60% of cases [6, 7]. The overall detection rate is not as high as expected for several reasons, including, but not limited to, variants in intronic sequences, uncharacterized genes, variants affecting mRNA splicing, and structural variants, such as copy number variations, duplications, or inversions [5, 7, 8]. There is a large variability of genes and mutations causing IRDs among different populations, and molecular analysis of understudied groups will allow for the reclassification of variants of unknown significance into pathogenic variants [4, 7]. Currently, there is limited data on the underlying genetic variants in families of Mexican descent. Furthermore, the available research has focused on IRDs patients from central and south Mexico [13, 14]. Therefore, the present study was undertaken to contribute to this growing area of research by analyzing the mutation spectrum of IRDs-associated genes in Northeastern Mexican patients, i.e., the states of Coahuila, Nuevo Leon, and Tamaulipas.

The study protocol was approved by the Institutional Review Board of the School of Medicine at Tecnologico de Monterrey (code P000625-DIMDRET-CEIC-CR001), and all procedures were conducted in compliance with the Declaration of Helsinki. Written informed consent was obtained from all the patients or their legal guardians.

The study population comprised 126 unrelated patients who were selected based on: (1) IRD diagnosis, (2) origin/residence in Northeastern Mexico (Coahuila, Nuevo Leon, and Tamaulipas), and (3) grandparents born in Mexico. Participants were recruited in the following outpatient clinics: Fundación Santos y de la Garza Evia, Fundación Destellos de Luz, Instituto de la Visión of Hospital La Carlota, and from the private practice. IRD diagnosis was based on clinical examination, including uncorrected and corrected visual acuity, fundus examination, visual field testing, fundus autofluorescence, and spectral-domain optical coherence tomography scan. Full-field electroretinography was performed when available. A clinical geneticist collected demographic and familiar data, including family pedigree, age of onset of symptoms, and presence of systemic findings.

DNA sample was extracted from saliva or buccal swab and analyzed with a targeted NGS panel for inherited retinal dystrophies comprising at least 293 genes at Invitae Corp. (San Francisco, CA). Targeted regions were enriched using a hybridization-based protocol and sequenced using Illumina technology. Exon deletions and duplications were assessed using an internal algorithm that compared read-depth for each target sequence in the proband to internal control samples. Classification of variants was based on the American College of Medical Genetics and Genomics (ACMG) guidelines.

Genetic testing was performed on a total of 126 probands with 74 females and 52 males. Probands were natives/residents of Nuevo Leon (94), Tamaulipas (20) and Coahuila (12). The average age of the probands at the time of testing was 39.06 ± 18.64 years (range 4–82 years). The median age at symptoms onset was 13 years (IQ range 17.5) (range 2 months to 70 years). The full demographic and clinical data of the patients is shown in Supplementary material Table 1.

The initial diagnoses in this cohort, according to clinical presentation and examination (Table 2), were: Non-syndromic IRD: RP (53 cases), STGD/MD (21 cases), CRD (15 cases), LCA (3 cases), X-linked retinoschisis (4 cases). Syndromic IRD: Usher 2A syndrome (25 cases), Bardet Biedl syndrome (2 cases), Alstrom syndrome (1 case), 1 case with intellectual disability, short stature, deafness, optic atrophy, and RP, and 1 case with intellectual disability, deafness, coarse facies, and late onset RP.

Cases were classified as solved, partially solved, and unsolved according to a previous work [13] (Table 1). The causative variant detection rate (solved cases) in this cohort was 74.6% (94/126) (Table 2). Partially solved cases were detected in 10/126. A total of 22/126 cases remained unsolved.

Genetic variants for IRDs are present in up to 36% of the world population, when accounting for asymptomatic carriers of recessive mutations [4]. As many of these mutations could be novel in nature and geographically prevalent due to founder effects, the genetic study of IRDs in diverse groups of populations is highly relevant [13]. The enormous genetic and phenotypical heterogeneity of IRDs is reflected in this work. The cohort contains 126 cases, pathogenic or probably pathogenic variants were identified in 94 cases, 10 cases were partially solved cases and 22 persisted as unsolved cases. To the authors’ best knowledge, two previous large cohorts have reported genetic findings in IRDs in patients originating from central and south Mexico [13, 14], so it is important to complete the information for these retinal pathologies in other regions of the country. In addition, it is also important to consider the genetic differences in the northeastern population which could possess a greater proportion of European alleles [86, 87] compared with the central/south Mexican populations.

A similar number of patients were examined across the previous cohorts and the present study (144, 143 and 126 patients respectively) [13, 14]. Regarding gender distribution, while a slight female predilection was found in the present study (56.3 vs 44%), the opposite was reported by Villanueva, et al. [13] (58.3 vs 41.7% of males and females respectively) and no gender distribution was reported by Zenteno, et al. [14]. A comparison of the characteristics of the patients from the three cohorts is shown in Table 2. The most common, pre-sequencing diagnosis was RP across all three cohorts. In addition, the mutation detection rate was similar in all 3 studies, ranging from 70–80% and the most detected mutation type were missense variants across all three studies. On the other hand, the most frequently encountered affected gene in the present study was USH2A (29.78%). This number differs from previous studies on Mexicans, whose reports were 3.5% [13] and 7% [14]. In the other cohorts ABCA4 was more frequently altered [13, 14]. Finally, the proportion of unsolved cases was similar between the present study and Villanueva-Mendoza, et al. [13] (15.3 vs 17.46%) and higher in the cohort from Zenteno, et al. [14] (33.5%). The considerable proportion of unsolved cases could be related to gene panel limitations, including its capability to detect RPGR variants, CNVs, and intronic variants.

The most frequent pathogenic variant of the whole cohort was c.2299del (p.Glu767Serfs*21) in USH2A. This variant is in exon 13 is the subject of a phase 3 therapy clinical trial involving the investigational new drug Ultevursen, an antisense RNA oligonucleotide (NCT05158296). The high prevalence of the c.2299del variant in USH2A found in the present study could be relevant for this therapy if it is approved. There is sufficient clinical evidence that the c.2299del (p.Glu767SerfsTer21) variant is pathogenic and highly prevalent. A recent report on the frequency of this variant in the cases from central and southern Mexico accounts for 7 and 23% of the alleles causing non-syndromic RP and Usher syndrome, respectively [88]. Furthermore, the Genome Aggregation Database v.4.0.0 shows that the frequency of this allele in the admixed Latino population is 0.0014, the highest globally, followed by the 0.001176 frequency in non-Finnish Europeans [89]. Dreyer, et al. reported the c.2299del variant in patients from Europe, North and South America, South Africa, and China and noted that it is associated to a core haplotype suggesting that this mutation is an ancestral mutation spread in Europe and introduced in the Americas after the conquest [90].

Other genetic therapies in development are relevant for this report. The vMCO-010 in phase 2 clinical trial (NCT05417126) and rAAV2tYF-GRK1-RPGR in phase 1/2 clinical trial (NCT03316560) are two promising therapies for patients with STGD/MD and X-Linked RPGR, respectively. On the other hand, no patients with RPE65 variants were found in this cohort, therefore no candidates for the only approved gene therapy for IRDs are reported.

Of all causative variants in this cohort, 14 were novel. Eight of these were missense variants (one pathogenic, five VUS, and two probably pathogenic). Three were CNVs, all classified as pathogenic, two frameshift variants classified as VUS and one splicing classified as probably pathogenic. All VUS are suggested to be disrupting variants but there was not enough evidence to classify them as pathogenic. The three novel CNVs may be explained by the recent developments in NGS detection by NGS suggesting that CNV detection will improve the diagnosis rate.

There were some interesting cases. The first was a case with isodisomy of chromosome 1, which has been already reported [91]. We also detected a patient with type IIIC mucopolysaccharidosis (Sanfilippo C), a 55 years patient, with severe intellectual disability, speech impairment, deafness, coarse facies, motor deterioration and late onset RP. He had an affected sister who suddenly died at 21. This patient has two novel HGSNAT variants, one CNV classified as pathogenic, and one missense classified as VUS. This could be the first case reported in a Mexican patient. Another interesting case was a woman patient with Arts syndrome, an X-linked disorder, who suffers from retinal dystrophy, optical atrophy, deafness, short stature, and intellectual disability. Among the unsolved cases, there is one case with isolated RP and two biallelic variants in CNBG1, c.1676C > A (p.Thr559Lys) and c.1720C > T (p.Leu574Phe), both classified as VUS. Looking into the clinical presentation of this patient and the changes at the protein level, we can conclude that there is a clinical correlation and that those VUS are probably disruptive. Another remarkable case was a 15 years male with juvenile retinoschisis, carrying a VUS in hemizygous state at RS1 identified as c.341C > T (p.Ser114Phe). This variant is highly likely to be the cause of the clinical presentation. Another notable case, was a 58 years male with CRD, with the VUS c.2795 T > G (p.Met932Arg) in heterozygous genotype in GUCY2D. The clinical presentation, the suggestive mother visual symptoms, and the amino acid changes suggest that this variant is highly suspicious of being disruptive as suggested by algorithmic predictions [92].

This study has some limitations, such as the sample size, as there were only 126 patients. Another limitation could be that this panel only encompasses genes in nuclear DNA and very few intronic variants. Sequence changes in the promoter, non-coding exons, and other non-coding regions were not covered. Additionally, no ancestry and founder effect studies were performed.