Introduction

Hereditary vision impairment can result from a large group of complex eye disorders, including but not limited to retinal dystrophies (RD), optic neuropathy (e.g. glaucoma), lens diseases (e.g. cataract), and disorders related to eye development (e.g. anterior segment dysgenesis). These inherited diseases are generally featured by progressive and chronic visual impairment, genetic heterogeneity, and major clinical overlap among disorders. Many of these disorders also have high clinical and penetrance variability. Further complexity is attributed to their mode of inheritance, as these disorders can be inherited as autosomal recessive, autosomal dominant, X-linked, mitochondrial, and even through digenic and polygenic traits. Genetic studies in endogamous populations have been instrumental in characterizing the molecular basis of diverse vision disorders.

Pakistan has a rich anthropogeneological background and the highest prevalence of consanguineous marriages in the world [1]. The present study ascertained families from the Southern Punjab province in Pakistan with the aim to identify the underlying molecular basis of diverse segregating vision disorders. After clinical phenotyping, whole-exome sequencing was performed on four families, which revealed two novel and two reported variants of known vision impairment genes, AIPL1, FOXE3, MERTK, and GUCY2D. AIPL1 encodes a molecular chaperon for the key phototransduction enzyme called Phosphodiesterase6 (PDE6), and its pathogenic variants are associated with Leber congenital amaurosis, dominant cone-rod dystrophy and recessive retinitis pigmentosa [2]. Transcription factor encoded by FOXE3 has a forked head DNA binding domain, and variants of FOXE3 are responsible for the dysgenesis of anterior segment tissues like lens and cornea resulting in microphthalmia, glaucoma, and aphakia [3]. While MERTK is one of the tyrosine kinase receptors of cell surface membrane expressed in retinal pigment epithelium. Recessive pathogenic variants of MERTK are reported in autosomal recessive retinitis pigmentosa [4]. Finally, GUCY2D encodes for the synthesis of enzyme photoreceptor guanylate cyclase GC-E, needed for the synthesis of second messenger of cGMP. Regulation of GUCY2D is by guanylate cyclase-activating proteins (GCAPs) through intra cellular Ca++ sensation. Pathogenic variants of GUCY2D are reported in association with LCA and dominantly inherited cone rod dystrophies [5]. The results of this study further support the utility of exome sequencing and genetic screening for families with diverse and clinically heterogenous segregating vision disorders in order to catalog the novel disease-causing variants of known genes. This will improve the clinical genetic diagnostic rate as well as help to establish the frequency of previously reported alleles in Pakistani population.

Materials and methods

Results

Clinical phenotyping

After obtaining IRB approval and informed consent from the four large consanguineous families, GCUF01, GCUF02, GCUF04, and GCUF06 (Fig 1A), we enrolled the families from Punjab, Pakistan. The neonatal clinical records of the affected participants were not available at the time of enrollment. Family history considered along with ophthalmological examinations reveal that the affected members of family GCUF01 have severe congenital nystagmus, an equal level of complete blindness irrespective of their ages (10–43 years) since their first year of life. Slit-lamp ophthalmoscopic and visual acuity studies reveal bony spicules in the retina. However, intraocular pressures (IOP) of family members were within the normal range (10–20 mm Hg, Table 1) at the time of testing. A tentative diagnosis of Leber congenital amaurosis (LCA) was assigned to the vision impairment phenotype segregating in family GCUF01. Similarly, phenotypic features were recorded in the affected individuals of family GCUF02, however with a key difference. In contrast to GCUF01, affected individuals of GCUF02 do not have congenital vision impairment. The youngest affected individuals (IV:9; 27 yrs) had sufficient perception of light during examination (Table 1). The pigmentary retinopathy of the retina (Fig 1A) was consistent with retinitis pigmentosa (RP).

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Fig 1. Family pedigrees of vision disorder and causative mutation.

(a) Segregation of probable pathogenic alleles in four families from Pakistan having diverse visual disorders. Empty and filled symbols represent unaffected and affected individuals, respectively. Consanguineous marriages are indicated by double lines. The genotypes (homozygous, heterozygous, and wild type) of the identified alleles are also shown for each of the participating family members. Clinical phenotype/eye picture and fundus of the affected individual with (age) is shown against each pedigree. Asterisks (*) in pedigree drawings of families GCUF02 and GCUF04 mark the individuals drawn twice to show their relations with other family members. Images of the eyes of the affected individuals are also shown, along with the fundoscopic image of an affected individual of family GCUF02 to document the salt and pepper appearance of the retina due to retinitis pigmentosa. (b) Clustal-W sequence alignments of multiple amino acids of orthologous proteins shows evolutionarily conserved variant residues across different species for missense variants of AIPL1 and FOXE3. Protein truncation position of GUCY2D and MERTK is shown as stick diagram.

https://doi.org/10.1371/journal.pone.0239748.g001

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Table 1. Family IDs, gender, age, clinical phenotypes, and ophthalmological remarks of participating individuals.

https://doi.org/10.1371/journal.pone.0239748.t001

The affected members of GCUF04 have congenital nystagmus and photophobia with visual acuity of no finger count in daylight, except V:1 who was able to count fingers in daylight (Table 1). However, in low-light examination, they were able to recognize general objects. V:1 was even able to count fingers in daylight. Macular examination showed neovascularization and typical mottling of para-macular areas. An initial diagnosis of vernal keratoconjunctivitis in both eyes was made by the ophthalmologist who examined the family in Pakistan. In contrast to the above three families, the affected members of GCUF06 reported having congenital anterior segment dysgenesis (ASD), keratoconus, congenital bilateral corneal haze, and apparent microphthalmia (Fig 1A). The youngest affected female (V:2, 6 yrs) had an intraocular pressure of 30/35 (OD/OS) at the time of examination (Table 1).

Genetic screening

Exome sequencing was performed for the proband of each family to determine the probable genetic cause(s) for the diversity among vision disorders segregating in the four families. Autosomal recessive inheritance (both homozygous and compound heterozygous) was assumed from the family trees. Four novel variants were prioritized through online prediction tools: c.465G>T, c.1843A>T, c.289A>G, and c.139delC in AIPL1, MERTK, FOXE3, and GUCY2D, respectively, (Fig 1A, Table 2). They were then validated using Sanger Sequencing in the available family members. These variants included two missense [p.(Gln155His), p.(Ile97Val)], one nonsense [p.(Lys615*)] and one frameshift allele p.(Pro47Thrfs*38)]. Variants identified in this study were present in evolutionarily conserved regions (Fig 1A) of the encoded proteins and were absent or had very low frequencies in gnomAD (Table 2).

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Table 2. Genes, identified novel variants and their ACMG classification.

https://doi.org/10.1371/journal.pone.0239748.t002

Next, to assess the predicted impact of identified variants on the encoded proteins secondary structures, we performed 3D molecular modeling with I-TASSER online programs. We concluded that the p.(Lys615*) nonsense variant of MERTK (Fig 2A) and the p.(Pro47TrhfsTer38) frameshift variant of GUCY2D are likely to yield complete loss of function of both proteins, as the mRNAs harboring these alleles will likely degrade through the nonsense-mediated decay (NMD) machinery [9]. In the unlikely event, MERTK mRNA escapes the NMD, the insertion of nonsense codon at amino acid position 615 is predicted to remove the carboxy tail. In contrast, the p.(Gln155His) variant, found in the GCUF01 family, is predicted to cause the loss of ionic interaction with aspartate residue at position 157 (Fig 2A), and therefore might impact the folding of the protein secondary structure of AIPL1. Neoconservative p.(Ile97Val) variant, located in the forkhead DNA binding domain of FOXE3 transcription factor, is also predicted to cause the loss of hydrogen bond with p.Ile97 (Fig 2B) and might impact DNA binding due to the change in its the domain folding.

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Fig 2. Protein 3D structural modeling of variants identified in this study.

Structures of both wild type and mutated residues (red) are shown. Solid black lines represent the interactions with neighboring amino acids. Ball and stick representation in red shows wild type residue, while the blue and purple represent mutated amino acids. (a) The p.(Gln155His) variant of AIPL1 is predicted to cause loss of hydrogen bond with p.(Asp 157) impacting protein 3D folding and introduces torsion in the mutated protein. (b) The p.(Ile97Val) variant in FOXE3, is predicted to cause loss of ionic interaction and impact proper protein folding. The Isoleucine (Ile) at position 97 is predicted to form a hydrogen bond with p.Ile76, which is lost due to replacement with valine at position 97.

https://doi.org/10.1371/journal.pone.0239748.g002

Discussion

High throughput sequencing (e.g. exome sequencing) has revolutionized genetics screening approaches for Mendelian disorders, including monogenic forms of vision disorders. Combinatorial approaches to identify individuals with actionable variants in highly penetrant monogenic forms of common vision disorders is absolutely essential if genomic medicine is to fulfill its promised purpose. Here, we report the identification of two novel and two reported disease-associated variants of AIPL1, MERTK, FOXE3, and GUCY2D in multiplex Pakistan families (Fig 1A). Identification of these variants is expected to aid in the accurate diagnosis of the vision disorder forms that segregate in the affected individuals, especially in the presence of limited clinical resources available in these families’ catchment areas in Pakistan.

The affected participants of the GCUF01 family are homozygous for c.465G>T (p.(Gln155His)) variant in AIPL1 (Encoded protein: Aryl hydrocarbon receptor (AhR)-interacting protein-like 1 (AIPL1) with three tetratricopeptide motifs). AIPL1 acts as a molecular chaperon for the Phosphodiesterase6 (PDE6) key phototransduction enzyme [10]. Pathogenic variants in AIPL1 are a common (5–10%) cause of LCA in diverse populations [11]. Severe congenital nystagmus and non-progressive vision loss together with a predicted pathogenic variant in AIPL1 is consistent with the ophthalmologist diagnosis of LCA in the affected individuals of the GCUF01family.

Similarly, the RP phenotype in family GCUF02 is co-segregating with a truncating allele (p.(Lys615*)) of Mer tyrosine kinase (MERTK; OMIM#604705). MERTK belongs to a family (Tyro3/Axl/Mer) of tyrosine kinases [12] and is essential for the effective phagocytosis of photoreceptor outer segments by the retinal pigment epithelium [13–15]. Accumulation of cellular debris, due to impaired phagocytosis, hampers the supply of oxygen and nutrient to photoreceptor cells [16]. Pathogenic variants of MERTK are known to cause severe, faster, and early onset RP with worse macular degeneration [17]. The truncating (p.(Lys615*)) variant segregating in the GCUF02 family is predicted to truncate the carboxy tail of the encoded proteins, which would impact the kinase activity, which in turn is needed for the proper phagocytosis of photoreceptor outer segments engulfed by RPE [14, 16].

GUCY2D encodes a cyclic guanosine monophosphate (cGMP)-producing guanylate cyclase enzyme. Calcium and cGMP act as second messengers in phototransduction. The levels of cGMP in light-sensing photoreceptor cells are controlled by the cGMP-hydrolyzing phosphodiesterase and guanylate cyclase enzymes. Variants impacting the function of the GUCY2D enzyme lead to severe retinal dystrophies, including LCA type 1 and autosomal dominant cone-rod dystrophy [5]. The c.139delC truncating variant found in the GCUF04 family is expected to completely abolish the enzymatic activity of GUCY2D, which is consistent with the LCA phenotype observed.

Forkhead box protein E3 (FOXE3) belongs to family of forkhead transcription factors. A-helix of the forkhead motif of FOXE3 binds with a major groove of DNA during transcription [18]. Homozygous variants were found in inherited congenital primary aphakia, sclerocornea, with incomplete penetrance of glaucoma [19–21], while some dominant variants are associated with ocular dysgenesis, cataracts, and Peters’ anomaly [22, 23] in human. The “likely pathogenic” missense variant found in the GCUF06 family is associated with microphthalmia, sclerocornea, and anterior segment aplasia in most of the affected individuals, while one individual (V:2) also had high IOP (Table 1). Unavailability of FA, OCT, and ERG are the main limitations explaining genetic variants in relation to phenotypic diversity in this study. In summary, for families living in remote areas of Pakistan with limited economical resources and sparse health facilities, genetic screenings will likely help in complete diagnosis, family counseling, and disease management.

Acknowledgments

We are thankful to all the participants, their families, and health care professionals for their cooperation in clinical characterization.