Retinitis pigmentosa with iris coloboma due to miR-204 gene variant in a Chinese family

Abstract

Purpose

To characterize the phenotype and genotype of a Chinese family with autosomal-dominant retinitis pigmentosa (RP) accompanied by iris coloboma.

Methods

The proband, a 34-year-old male, was examined with his family by using fundus photography, optical coherence tomography (OCT), autofluorescence, and full-field electroretinography (ffERG). Genetic analyses were conducted through whole-exome sequencing (WES) to screen for variations.

Results

Three members of this Chinese family were shown to be bilateral iris coloboma. The male proband and his mother exhibited typical RP feature. The proband's late grandfather had been documented manifestation of iris coloboma. The mode of inheritance was confirmed to be autosomal dominance. Through linkage analysis and WES, a heterozygous variation in the miR-204 gene (n.37C>T), a noncoding RNA gene, was identified in these three members.

Conclusions

In this third independent and the first Asian family, the existence of a miR-204 variant associated with RP accompanied by iris coloboma was confirmed. Our findings reinforce the significance of miR-204 as an important factor influencing visual function in the retina. When phenotypes like RP accompanied by iris coloboma in an autosomal-dominant pattern, including in Chinese patients, miR-204 aberrations should be considered.

INTRODUCTION

Retinitis pigmentosa (RP) is an inherited retinal degenerative condition marked by the progressive loss of retinal photoreceptor cells and the retinal pigment epithelium (RPE) (Pagon, 1988).Typically, RP is transmitted via autosomal-dominant (AD, 15%–25%), autosomal-recessive (AR, 5–20%), and X-linked (XL, 5%–15%) modes (Tsang & Sharma, 2018a). Patients with AD-RP usually manifest the disease late in life, progress slowly, and spare the macula for an extended period (Tsang & Sharma, 2018a). Microphthalmia, anophthalmia, and coloboma (MAC) encompass a spectrum of developmental eye defects (Williamson & FitzPatrick, 2014). Both RP and MAC frequently result from genetic variations in coding regions, leading to impaired or lost protein function. MicroRNAs (miRNAs) are a class of noncoding RNAs that regulate gene expression, primarily through posttranscriptional silencing of complementary mRNA targets (Zuzic et al., 2019). Clinical instances of RP caused by miRNA gene variations are relatively rare (Zuzic et al., 2019), and the co-occurrence of RP and MAC is even less common. This report describes a Chinese family presenting with RP and iris coloboma linked to a miR-204 gene variation (n.37C>T) inherited in an AD pattern.

METHODS Subjects

A four-generation family with RP accompanied by iris coloboma presented at the Shaanxi Eye Hospital of Xi'an People's Hospital (Xi'an Fourth Hospital) in China. The proband was examined with his mother and son by a full medical, ophthalmic, and family history combined with clinical examination. Medical records for other family members were reviewed with the patients' written consent. Ophthalmic examination included fundus photography, optical coherence tomography (OCT), autofluorescence, and full-field electroretinography (ffERG). Ethical approval and informed consent were obtained from all study participants. The research adhered to the tenets of the Declaration of Helsinki.

Exome sequencing and validation

To elucidate the molecular basis of this phenotype, we enrolled five family members for genetic analysis. After obtaining informed consent, whole-exome sequencing (WES) was conducted. Whole-exome capture was carried out using SureSelectXT Human All Exon V6 Kit (Agilent Technologies, CA, USA) according to the manufacturer's protocol. Whole-exome DNA libraries were then sequenced on the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA). Local realignments, quality control text, and variant calling were assembled using the Genome Analysis Toolkit (GATK). All sequencing reads were mapped against a human reference genome (UCSC hg19/GRCH37) with Burroughs–Wheeler Aligner (BWA). The single-nucleotide polymorphism (SNP), small insertions or deletions (InDels), and copy number variations (CNV) were also achieved with GATK. Variants were annotated with Annovar (http://annovar.openbioinformatics.org/) and evaluated by referring to Online Mendelian Inheritance in Man (OMIM), Human Gene Mutation Database (HGMD), ClinVar databases, and according to the criteria and guidelines of the American College of Medical Genetics and Genomics (ACMG, 2015) (Richards et al., 2015) genetic variation, and the clinical phenotype of the patients. Sorting Intolerant from Tolerant (SIFT), Polymorphism Phenotyping v2 (Polyphen2), and Mutation Taster were used to predict the potential effect on the protein function. Strong candidates were subsequently confirmed by Sanger sequencing. The variant's nomenclature followed the Human Genome Variation Society (HGVS) policy (https://varnomen.hgvs.org/).

RESULTS Clinical features of the family

A 34-year-old Chinese male presented with long-standing poor vision and photophobia since childhood. Diagnosed in 2016 with congenital iris colobomas and RP, he experienced a noticeable decline in vision in both eyes over the past 2 years. His social history was noncontributory. Family history revealed his mother suffering from congenital iris colobomas and RP; one of his sons exhibited strabismus, photophobia, and iris coloboma; and his late grandfather also had iris coloboma and poor vision. No other family members reported ocular symptoms. The patient's best corrected visual acuity (BCVA) was 20/100 in the right eye and 20/125 in the left eye. Examination revealed bilateral inferior iris coloboma and posterior subcapsular lens opacity (Figure 1a). Fundus examination showed classic RP signs, including bone spicule-like pigmentation in the peripheral retina and attenuated arterioles (Figure 1b). OCT indicated thinning of the outer retinal layers with a blurred photoreceptor ellipsoid zone (EZ) in the foveal regions and disappearance of EZ beyond these areas in both eyes (Figure 1c). Fundus autofluorescence (FAF) images showed scattered hypoautofluorescent spots in the peripheral retina (Figure 1d). ffERG revealed extinguished waveforms (Figure 1e). The patient's mother, diagnosed with RP and iris coloboma, had undergone cataract surgery 10 years prior and had an intraocular lens implant (Figure 2). His 2-year-old son exhibited iris coloboma (Figure S1) but no noticeable retinal abnormalities (Figure 3). Eye examinations of the patient's younger sister and another 8-year-old son showed no significant findings.

FIGURE 1. Clinical findings of the proband: (a) slit lamp biomicroscope photographs depicting bilateral inferior iris coloboma with posterior subcapsular lens opacity. (b) Fundus photos showing bone spicule-like pigmentation in the peripheral retina and attenuated arterioles. (c) OCT examination revealing thinning and loss of the outer retinal layers. (d) FAF indicating scattered hypofluorescent lesions in the peripheral retina. (e) ffERG presenting extinguished waves under both scotopic and photopic conditions.

FIGURE 2. Clinical findings of the proband's mother: (a) slit lamp biomicroscope photographs displaying bilateral iris coloboma with intraocular lenses. (b) Fundus photos revealing extensive bone spicule-like pigmentation in the peripheral retina, a pale optic disc, and attenuated arterioles. (c) OCT examination demonstrating thinning of the outer retinal layers and an epiretinal membrane in the right eye. (d) FAF indicating scattered hypofluorescent lesions in the peripheral retina. (e) ffERG showing extinguished waveforms.

FIGURE 3. Clinical findings of the proband's young son: (a) fundus photos depicting no significant retinal abnormalities. (b) Dark-adapted 3.0 ERG presenting normal waveforms.

Molecular genetic findings

A heterozygous variation n.37C>T, Chr9: 73424964G>A in the miR-204 gene (NR_029621.1) was identified. The variation was reported as a pathogenic variant in the ClinVar database and as a damaging mutation (DM) in the HGMD database. The variant was inherited from his mother and passed to the proband's son (Figure 4), confirming the diagnosis of AD-RP in the proband.

FIGURE 4. Family pedigree and genetic analysis: (a) pedigree chart—The proband is marked with an arrow. The proband, his mother, and his young son have congenital iris colobomas. The proband was diagnosed with RP in 2016 and began experiencing significant vision deterioration at 32 years of age. His mother, who started losing vision in her 30s, was also diagnosed with RP. The proband's late grandfather had been documented cases of iris coloboma and poor vision. Filled circles and squares represent affected individuals, while open circles and squares denote unaffected individuals. Asterisks (*) indicate individuals who have undergone genetic testing. Squares represent males, and circles represent females. (b) Sanger Sequencing: The proband (II:2) inherited the heterozygous variation n.37C>T from his mother (I:2) and passed it to his young son (III:2).

DISCUSSION

RP represents the most prevalent form of inherited retinal degeneration (IBD), affecting approximately 1.5 million individuals globally. Its worldwide prevalence ranges from about 1 in 9000 to 1 in 750, with around 1 in 3500 reported in China (Chen et al., 2022; Koyanagi et al., 2019). This condition, resulting from various inherited patterns, leads to the progressive death of photoreceptor cells and consequent vision loss. Patients with AD-RP typically show symptoms later in life (Tsang & Sharma, 2018a). X-linked RP (XLRP) is often regarded as one of the most severe forms, predominantly affecting males (Tsang & Sharma, 2018b). AR-RP tends to present with a severity that lies somewhere between AD-RP and XLRP (Tsang & Sharma, 2018a, 2018b).In the case of this Chinese family, the male proband exhibited classic RP fundus changes. He began experiencing significant vision deterioration at 32 years of age. His mother, who started losing vision in her 30s, was also diagnosed with RP. The proband's young son, aged 2, presented with strabismus, photophobia, and iris coloboma, yet exhibited a normal fundus, possibly due to his early age. The proband's late grandfather also had been documented cases of iris coloboma and poor vision. Thus, the inheritance pattern in this family was suggested to be dominant.

The pathogenesis of RP remains incompletely understood but is thought to involve genetic variants that impair the renewal and shedding of the outer segmental disc membrane of rod photoreceptors. These variants can also affect the phototransduction pathway, rhodopsin metabolism, mRNA formation and splicing, and transcription factor functionality (Hartong et al., 2006). To date, over 100 pathogenic genes have been identified, each linked to specific functions within photoreceptors, such as cell motility and signaling, transduction pathways, structural support, and metabolism (Tsang & Sharma, 2018c). The advent of next-generation sequencing technologies, particularly WES, has enabled molecular diagnosis in 60%–80% of RP cases, uncovering numerous genes involved in retinal development and signaling pathways (Salmaninejad et al., 2019). Pathogenic sequence variants are primarily found in protein-coding genes, with rare occurrences in noncoding RNA genes (such as promoters, lincRNA, and microRNA) (Karali & Banfi, 2019; Zuzic et al., 2019).MicroRNAs (miRNAs) play a crucial role in modulating gene expression, regulating intracellular signaling pathways, and other biological processes (Bereimipour et al., 2021). Diseases caused by miRNA variations have recently gained attention. In a healthy human retina, miR-204 is expressed in the ganglion cell layer (GCL), inner nuclear layer (INL), outer nuclear layer (ONL), retinal pigment epithelium (RPE), and ARPE-19 cell lines (Shiels, 2020). The pathogenic variant in miR-204 was first reported in a case of retinal degeneration associated with ocular coloboma, characterized by iris keyhole-shaped defects, sometimes accompanied by congenital cataract (Conte et al., 2015). In 2023, this pathogenic variant was observed also in a Czech family and associated with congenital glaucoma, besides RP and iris coloboma (Jedlickova et al., 2023). In our patient, WES revealed a miR-204 n.37C>T variant. Both the proband and his mother exhibited iris coloboma and cataracts, with the miR-204 variant inherited from his mother, who had congenital iris coloboma in both eyes. This variant was also present in the proband's son, who displayed iris coloboma. Consequently, pedigree analysis with the miR204(n.37C>T) variant aligns with a dominant inheritance pattern.

The development of ocular structures and retinal cells necessitates precise spatial and temporal organization. Disruptions in these dynamics can result in congenital ocular and retinal defects, often leading to visual impairment (Krueger & Morris, 2022). A known cause of congenital ocular coloboma is the failure to close the choroid fissure (Ohuchi et al., 2019). Approximately two-thirds of iris coloboma cases are inherited as an AD trait (Ohuchi et al., 2019). A probable homozygous mutation was reported to result in a stillborn fetus (Ohuchi et al., 2019). Currently, mutations in 29 genes are recognized as causing AD retinitis pigmentosa (ADRP) (Daiger et al., 2018), and over 80 genes have been implicated in microphthalmia, anophthalmia, and coloboma (MAC) (Ohuchi et al., 2019). These genes are exclusively protein-coding, and no overlap has been found between these two gene datasets. Retinal degeneration co-occurring with iris coloboma is exceptionally rare and tends to be linked with abnormalities in visual system development. It is also observed in few syndromic inherited eye diseases. CHARGE syndrome, an AD genetic disorder, is caused by variants in the chromodomain helicase DNA binding protein 7 (CHD7), a critical gene for normal development (Krueger & Morris, 2022). Ocular features of CHARGE syndrome are MAC, including IBD (Hsu et al., 2014). Additionally, systemic manifestations, such as choanal atresia, heart defects, genital abnormalities, growth retardation, and ear malformation are variably present in CHARGE syndrome patients (Hsu et al., 2014). Bardet–Biedl syndrome, another condition rarely displaying iris coloboma associated with RP (Héon et al., 2005), is a rare AR ciliopathy. It is characterized by retinal dystrophy, obesity, postaxial polydactyly, renal dysfunction, learning difficulties, and hypogonadism (Melluso et al., 2023).Notably, only miR-204 has been reported in the literature to be associated with these ocular manifestations rather than entire syndromes (Conte et al., 2015; Jedlickova et al., 2023). MiR-204 is a miRNA highly expressed in the eye, starting from early stages of development (Bereimipour et al., 2021; Conte et al., 2010). Given miR-204's critical role in the eye, it is unsurprising that pathogenic variants in the miR-204 gene can lead to severe eye defects.

While numerous genetic variants are known to cause visual impairment and blindness, there is a growing understanding of the complex interplay between gene regulation and retinal pathologies. MiR-204 is not alone in this respect; researchers believe that other miRNAs may also give rise to ocular diseases. However, genetic diseases caused by miRNA variants that follow Mendelian inheritance patterns are still scarcely reported (Conte et al., 2015). Mencía et al. (2009) were the first to describe a patient with AD progressive hearing loss caused by a miRNA variant. Hughes et al. (2011) later identified a variant of miR-184 linked to keratoconus and early-onset cataracts. MiR-204 is the first reported miRNA variant with a causal role in inherited retinal dystrophies in two Caucasian families (Conte et al., 2015; Jedlickova et al., 2023). Other miRNAs have been linked to IBD only in mouse models. Alterations in miRNA expression, such as in the miR-183/96/182 clusters, are observable in animal models of IBD, suggesting a foundational role rather than a direct pathogenic agent in photoreceptor degeneration (Zuzic et al., 2019).MiR-204 is located within intron 8 of the transient receptor potential melastatin channel 3 gene (TRPM3) on chromosome 9q21.12 (Shiels, 2020). Deletions within 9q21, encompassing the TRPM3 gene, have been rarely reported to cause ocular phenotypes. More commonly, they are associated with intellectual disability, epilepsy, delayed speech, autistic behaviors, and facial dysmorphology (Boudry-Labis et al., 2013; Pua et al., 2014). TRPM3 was first identified in a light-sensitive retinal mutant of Drosophila in 1969 (Cosens & Manning, 1969), and later studies revealed that the TRPM3 gene hosts miR-204, acting as both a tumor suppressor and a negative regulator of posttranscriptional gene expression (Shiels, 2020). As early as 2010, miR-204 was found to be highly expressed in human retinal pigment epithelium, playing a role in its differentiation and eye development (Conte et al., 2010). The first case of a British family with n.37C>T dominant variants in miR-204 leading to inherited retinal dystrophies and MAC features was reported in 2015 (Conte et al., 2015). This discovery underscored miR-204's importance in photoreceptor development. Wild-type (wt) miR-204 is necessary for proper lens and RPE development, interacting with Pax6, a key regulator of eye development (Conte et al., 2010). The n.37C>T variant in miR-204 is thought to gain function by creating novel, aberrant recognition sites in genes not typically targeted by miR-204, including Lef1, which plays a role in retina (Cho & Cepko, 2006; Fotaki et al., 2013) and RPE differentiation (Conte et al., 2015; Westenskow et al., 2009). However, the precise molecular mechanism underlying miR-204-associated diseases remains to be fully elucidated.

In summary, miR-204 emerges as a pivotal factor influencing visual function within the retina. The occurrence of retinal dystrophy combined with ocular tissue defects due to miR-204 variants is a rarity in clinical settings. This report marks the first documented instance of a Chinese family exhibiting AD-RP and MAC associated with a miR-204 variant. Our findings significantly contribute to identifying miR-204 as a critical gene responsible for RP. In clinical cases where RP and iris coloboma coexist, consideration should be given to this disease and potential miRNA aberrations.

AUTHOR CONTRIBUTIONS

L.Z.: concept and study design. Data interpretation. Drafing, revision and fnal approval of manuscript. H-L.Z.:data collection. Drafing, revision and fnal approval of manuscript. H-Y.W.: study supervision. Concept and study design. Data interpretation. Revision and final approval of manuscript. J.W.:data analysis and interpretation. R.W.:data analysis. Z-L.C.: data collection. Q-F.W..: data collection.

ACKNOWLEDGMENTS

This study was supported by the Key Research and Development Program of Shaanxi Province, China (2023-YBSF-537); the Natural Science Basis Research Plan in Shaanxi Province of China (2022JM-514); the General Cultivation Project of Municipal Health Commission in Xi'an, China (2024 ms18); Bethune·Lumitin Research Funding for the young and middle-aged Ophthalmologists (BJ-LM2021011J); Research Incubation Fund of Xi'an People's Hospital (Xi'an Fourth Hospital) (ZD-7 and ZD-8).

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

ETHICS STATEMENT

Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.