MIR204 n.37C>T variant as a cause of chorioretinal dystrophy variably associated with iris coloboma, early‐onset cataracts and congenital glaucoma

Four members of a three‐generation Czech family with early‐onset chorioretinal dystrophy were shown to be heterozygous carriers of the n.37C>T in MIR204. The identification of this previously reported pathogenic variant confirms the existence of a distinct clinical entity caused by a sequence change in MIR204. Chorioretinal dystrophy was variably associated with iris coloboma, congenital glaucoma, and premature cataracts extending the phenotypic range of the condition. In silico analysis of the n.37C>T variant revealed 713 novel targets. Additionally, four family members were shown to be affected by albinism resulting from biallelic pathogenic OCA2 variants. Haplotype analysis excluded relatedness with the original family reported to harbour the n.37C>T variant in MIR204. Identification of a second independent family confirms the existence of a distinct MIR204‐associated clinical entity and suggests that the phenotype may also involve congenital glaucoma.

Interestingly, the ocular co-expression of TRPM3 and miR-204 is upregulated by PAX6, a master controller of eye development. 4,5 Oculocutaneous albinism type 2 (OCA2, MIM #203200) is an autosomal recessive disorder in which the biosynthesis of melanin pigment is reduced in skin, hair and eyes. In cases when only the eyes are affected, the term ocular albinism is used. Typical features of OCA2 comprise foveal hypoplasia, iris translucency, and hypopigmented fundus. 6 The disease may be caused by homozygous or compound heterozygous pathogenic variants in the OCA2 gene (MIM *611409).
OCA2 has 24 exons (23 coding), spanning almost 345 kb of genomic DNA in the region of 15q11. 2-q12 7 In this study, we report a three-generation family with the occurrence of an early-onset chorioretinal dystrophy variably associated with iris coloboma, premature cataracts, and congenital glaucoma in four members. Heterozygous variant n.37C>T in MIR204 was detected. In addition, molecular investigation resolved the presence of albinism in the spouse of the proband and his three children by identifying OCA2 pathogenic variants, including one daughter with dual a diagnosis.

| miRNA target analysis
To investigate the putative functional consequences of the n.37C>T variant on its target genes, we applied miRNA target prediction tool miRDB (version 6.0). 14,15 Both sequences WT hsa-miR-204-5p (uucccuuugucauccuaugccu) as well as n.37C>T hsa-miR-204-5p (uuccuuuugucauccuaugccu) were submitted to the miRDB. The generated list of genes including target scores for each variant was used to create a heatmap (R version 4.1.2 and pheatmap package). All the predicted targets have scores between 50 and 100, which are assigned by the computational algorithm. A predicted target with score >80 is considered likely to be real. If the score is <60, it is recommended to provide additional supporting evidence.
The functional annotation was generated by the Database for Annotation, Visualisation and Integrated Discovery (DAVID tool, version 2021) 16

| Additional molecular genetic findings
The spouse of the proband was found to be a compound heterozy- It has been predicted to generate an mRNA transcript with novel splicing between exons 2 and 20, thus resulting in premature truncation and a functionally null OCA2 allele p.(Ser77Hisfs*7). 18 This SV has been repeatedly observed in compound heterozygosity with pathogenic variants in patients diagnosed with albinism. 18  The c.1327G>A in OCA2 is classified as pathogenic using following ACMG criteria: PM2_Supporting (low frequency in population databases), PM3_Strong (detected in trans with a pathogenic/likely pathogenic variant in ≥40 individuals with albinism), [19][20][21][22][23] PM1_Supporting (located in a mutational hotspot), PS4_Moderate (prevalence of the variant in affected individuals is significantly increased compared to controls; ≥10 homozygous probands, including the current study, with consistent phenotypes have been reported), 19,24,25 PS3_Supporting (well-established functional studies show damaging effect on the gene or gene product), 3 PP4_Supporting (patient's phenotype or family history is highly specific for a disease with a single genetic aetiology).

| Clinical features in the extended family
The 74-year-old mother of the proband (individual I:2, Figure 1 Table 1).
The proband had four children. The youngest one, a 6-year-old girl (individual III:4, Figure 1), was noted to suffer from congenital glaucoma for which she had been treated elsewhere. She underwent several antiglaucoma procedures in both eyes (Table 1). Our examination revealed bilateral macular atrophic lesions with pigmented edges.
In addition, her fundus appeared pale bilaterally. There were no signs of premature cataracts. She also developed band keratopathy in both eyes ( Figure 2G,H). BCVA at the age of 6 was 0.06 and light projection in the right and left eye, respectively.
The oldest child, 15-year-old female (individual III:1, Figure 1    ( Figure S5A), presumably having no or little impact on the preferential processing of the -3p or -5p miRNA strands in the miRNA molecular machinery. The n.37C>T variant gives a rise to the miRNA molecule (n.37C>T hsa-miR-204-5p) that possesses a change in its seed sequence on the 4th position when compared to wild type (WT hsa-miR-204-5p; Figure S5A). n.37C>T, led us to screen genes associated with congenital glaucoma.
As none pathogenic/likely pathogenic variants were identified we propose that congenital glaucoma is likely a further phenotypic sign of MIR204-associated disease.
Changes in seed sequence, which is essential for the miRNA binding to the target mRNA, may lead to binding disruption and/or alterations to pre-miRNA processing. 27,28 Notably, the expression of both pre-miRNA, as well as processed -3p/-5p forms of the MIR204 n.37C>T variant was not affected when compared to the wild type,