A systematic review of inherited retinal dystrophies in Pakistan: updates from 1999 to April 2023

Background Inherited retinal degenerations (IRDs) are a group of rare genetic conditions affecting retina of the eye that range in prevalence from 1 in 2000 to 1 in 4000 people globally. This review is based on a retrospective analysis of research articles reporting IRDs associated genetic findings in Pakistani families between 1999 and April 2023. Methods Articles were retrieved through survey of online sources, notably, PubMed, Google Scholar, and Web of Science. Following a stringent selection criterion, a total of 126 research articles and conference abstracts were considered. All reported variants were cross-checked and validated for their correct genomic nomenclature using different online resources/databases, and their pathogenicity scores were explained as per ACMG guidelines. Results A total of 277 unique sequence variants in 87 distinct genes, previously known to cause IRDs, were uncovered. In around 70% cases, parents of the index patient were consanguineously married, and approximately 88.81% of the detected variants were found in a homozygous state. Overall, more than 95% of the IRDs cases were recessively inherited. Missense variants were predominant (41.88%), followed by Indels/frameshift (26.35%), nonsense (19.13%), splice site (12.27%) and synonymous change (0.36%). Non-syndromic IRDs were significantly higher than syndromic IRDs (77.32% vs. 22.68%). Retinitis pigmentosa (RP) was the most frequently observed IRD followed by Leber’s congenital amaurosis (LCA). Altogether, mutations in PDE6A gene was the leading cause of IRDs in Pakistani families followed by mutations in TULP1 gene. Conclusion In summary, Pakistani families are notable in expressing recessively inherited monogenic disorders including IRDs likely due to the highest prevalence of consanguinity in the country that leads to expression of rare pathogenic variants in homozygous state. Supplementary Information The online version contains supplementary material available at 10.1186/s12886-024-03319-7.


Introduction
Inherited retinal dystrophies (IRDs) are a group of clinically and genetically diverse eye disorders ranging in prevalence from 1 in 2000 to 1 in 4000 people globally [1,2].IRDs are broadly divided into two categories (i) non-syndromic, and (ii) syndromic types depending upon absence or presence of extra-ocular manifestations, respectively.Major clinical symptoms of non-syndromic IRDs include, but not limited to, night blindness or nyctalopia, color vision deficiency, photophobia, nystagmus, reduced visual acuity, day vision loss as well as central or peripheral vision loss.Syndromic IRDs, on the other hand, are known for systemic findings such as obesity, polydactyly, renal abnormalities, deafness, speech, and intellectual disability together with ocular symptoms [3][4][5][6].Globally, mutations in over 300 distinct genes have thus far been associated with all forms of IRDs [3].IRDs follow all modes of Mendelian inheritance such as autosomal recessive [7], autosomal dominant [8], X-linked [9], mitochondrial [10] as well as digenic [11] and oligogenic patterns [12].Accordingly, IRDs are classified into various subtypes depending upon disease onset, mode of inheritance, rate of disease progression, clinical presentation, part of retina affected (rods, cones, retinal pigment epithelium, inner retina and choroid), and/or involvement of extra ocular phenotypes [13].Non-syndromic and syndromic IRDs are briefly explained in the following sections.

Non-syndromic IRDs Cone or cone-rod dystrophy (CD/CRD)
Cone and cone-rod dystrophies (CD/CRD) are a rare form of retinal dystrophies with a worldwide prevalence rate of 1:40,000 [14].CD/CRD are progressive disorders of cone and rod photoreceptor cells in retina presenting clinical and genetic heterogeneity [4].Major clinical symptoms of CD include reduced day vision, color vision deficiency, reduced visual acuity, and photophobia [15].Similarly, CRD is characterized by cone dysfunction at first resulting in progressive loss of day vision.This is followed by rods dysfunction eventually leading to night blindness (nyctalopia).However, symptoms such as photophobia, color vision deficiency, and legal blindness overlap between CD and CRD.The electroretinogram (ERG) shows both cone and rod dysfunction and is non-recordable in advanced case [16].CD/CRD may transmit as autosomal recessive, autosomal dominant or X-linked entity with mutations in as many as 32 distinct genes identified so far [17].Gill and colleagues have reported that 62.2% cases of recessively inherited CD/CRD are linked to mutations in ABCA4 gene.Similarly, of the dominantly inherited CD/CRD, 34.6% cases are attributed to mutations in GUCY2D gene.Lastly, of the X-linked inherited CD/CRD, 73.0% cases are due to mutations in RPGR gene [18].

Congenital stationary night blindness (CSNB)
As the name indicate, congenital stationary night blindness (CSNB) is a form of non-progressive inherited retinal dystrophy that appears at birth, however identified in childhood [19].Visual symptoms include night blindness, nystagmus and reduced visual acuity [20].ERG findings show normal cone response; however, reduced or abolished rod response is detected on ERG in CSNB.Similarly, fundoscopy mostly remains unremarkable [21].CSNB is divided into four distinct types namely, Schubert-Borstein type, Riggs type, Oguchi disease, and fundus albipunctatus [22].Though patients with Schubert-Borstein or Riggs type both have normal fundi, the two types can be distinguished from each other with the help of full-field electroretinography (ff-ERG) [23].Oguchi disease and fundus albipunctatus shows fundus abnormalities.For instance, Oguchi disease is characterized by a gray-white metallic sheen that disappears after dark adaptation, a feature called the Mizuo-Nakamura phenomenon.Fundus albipunctatus is characterized by small white dots scattered across the posterior pole sparing the fovea.In scotopic settings, Riggs type has flat a-wave in dim flash and reduced a-and b-wave with a strong single flash.In contrast, the Schubert-Bornschein type has normal a-wave and severely reduced b-wave, classically described as an electronegative waveform.In photopic settings, Riggs type has a normal photopic response, the Schubert-Bornschein type has abnormal photopic findings [23][24][25].To our knowledge, at least 18 genes are known to cause CSNB, including 13 genes for AR-CSNB, 3 genes for AD-CSNB, and 2 genes for XL-CSNB [20].

Leber congenital amaurosis (LCA)
Leber's congenital amaurosis (LCA) is a rare, and one of the most clinically severe form of IRDs with a worldwide prevalence of 1:80,000 [26].LCA is, typically, associated with early onset vision loss, nystagmus, and amaurotic pupils [27].Clinical pattern includes pigmentory retinopathy, reduced or absent ERG response, poor central vision or complete blindness at birth [28].LCA predominately follows autosomal recessive inheritance pattern except for CRX, IMPDH1, and OTX2 genes that results in autosomal dominant LCA [29].LCA account for approximately 5% of the total IRDs cases [30], and ~ 20% of childhood blindness [27].So far, pathogenic variants in 38 genes are known to cause LCA [27].Some of the frequently mutated genes in LCA include AIPL1, CEP290, CRB1 and GUCY2D [31].

Macular degeneration (MD)
Macular degeneration (MD) is a heterogeneous group of progressive eye disorders that are clinically characterized by bilateral symmetrical macular abnormalities and macular flecks.Visual complaints include, reduced visual acuity, central vision loss, photophobia, slow dark adaptation, and nystagmus [32].Stargardt disease 1 (STGD1) disease is the most common form of macular dystrophy with a prevalence rate of 1 in 8000-10000 [33].STGD1 follows an autosomal recessive mode of inheritance with ABCA4 as the leading cause of the disease that accounts for 95% STGD1 cases [34].Of the total 18 genes known to cause MD, 14 genes are responsible for AD-MD while the remaining 4 genes results in AR-MD [RetNet-Retinal Information Network (uth.edu), accessed on March 6, 2023].

Retinitis pigmentosa (RP)
Retinitis pigmentosa (RP), with a worldwide incidence rate of 1:3000 to 1:4000, appears as the most highly frequent type of IRDs [35].Though symptoms and onset of RP is largely variable, it usually starts with night blindness during first or second decade of life due to the degeneration of rod photoreceptor cells.This is followed by visual field constriction (tunnel vision) and degeneration of cone photoreceptors, finally leading to complete blindness by late third or early fourth decade of life.Auxiliary symptoms may include photophobia, nystagmus and reduced visual acuity [35].Around 70%-80% of RP cases are non-syndromic (isolated) while the remaining 20-30% cases are associated with non-ocular manifestations, and thus classified as syndromic RP [36].RP may be inherited as an autosomal dominant (15-25%), autosomal recessive (5-20%), X-linked (5-15%) [37,38], or a di-genic entity [39] with associated mutations reported in over 132 different genes [RetNet-Retinal Information Network (uth.edu)].Mutations in RPGR gene is the leading cause of X-linked RP [40].

Usher syndrome (USH)
Usher syndrome (USH) is a rare form syndromic IRDs, presenting deafness in conjunction with blindness, that affect people with a worldwide prevalence of 4-17 in 100000 [48] [49].Chief ocular complaints are night blindness, progressive vision loss, nystagmus whereas non-ocular symptoms include hearing difficulty or sensorineural hearing loss of variable degree [50].USH is subdivided into different types, for example, USH1, USH2 and USH3, based upon clinical presentation of the disease.USH2 is the predominant type among all sub-types [51].USH is passed in an autosomal recessive pattern [52], and di-genic [53] with mutation in 10 genes thus far known to cause the disease.While mutations in USH2A gene account for roughly 80% of USH2 cases, MYO7A gene mutations are responsible for over 50% of USH1 cases [54].

Joubert syndrome
Joubert syndrome (JBTS) is an infrequent genetic ciliopathy characterized by the involvement of multiple systems and organs, including the brain, kidneys, liver, and eyes.The clinical presentation of JBTS typically involve mild to moderate mental retardation, macrocephaly, retinal dystrophy, nystagmus, coloboma, visual impairment, hepatic and renal anomalies, and skeletal deformities with characteristic "molar tooth sign" on MRI.Given its rarity as a genetic ciliopathy, JBTS has a global occurrence rate of 1 in 80,000 to 1 in 100,000 live births [55].JBTS is predominantly inherited as an autosomal recessive disorder [56].However, there have been reports of X-linked JBTS due to mutations in the OFD1 gene [55].Pathogenic sequence variations in at least 40 genes have been reported to cause JBTS so far [57].Mutations in AIH1 and CEP290 genes collectively account for approximately 38% of genetically diagnosed JBTS patients [55].

Methods
This review was carried out between November-2022 and April-2023 by following PRISMA guidelines as mentioned in Supplementary Table S1.Briefly, the inclusion criteria for considering genetic studies comprised families of Pakistani descent, exhibiting both syndromic and non-syndromic inherited retinal disorders, and articles published during the period spanning from 1999 to April-2023.A thorough literature survey was performed for this purpose using PubMed (NCBI), Google Scholar, and Web of Science.Different key words such as inherited retinal degenerations/dystrophies, IRDs, retinitis pigmentosa (syndromic/non-syndromic), Stargardt disease, cone-and/or cone-rod dystrophy, macular degeneration, congenital stationary night blindness, leber congenital amaurosis, achromatopsia, color blindness, mutations, Pakistan, etc. were used for retrieving relevant literature.All articles that appeared during our search were further shortlisted and papers that did not fall under the purview of this review were excluded.This included articles presenting findings related to non-retinal phenotypes like cataract, age related macular dystrophy, microphthalmia, anophthalmia, aniridia, anterior segment dysgenesis, and sporadic cases.Data pertaining to genetic and clinical aspects of the families were extracted from all papers and recorded (and tabulated) in Microsoft (MS) excel sheet.This followed manual curation of the data using different options of the MS excel tool.Genomic nomenclature of all variants reported in this study were validated using VariantValidator [https:// varia ntval idator.org].Additional features of the variants such as impact of the variations on cDNA and protein level, gene symbols, protein and transcript ID, genomic coordinates of the variation, inheritance pattern, and allele frequencies were determined using major genomic data bases (Ensemble, UCSC, HGNC, OMIM, gnomAD).All identified variants were queried in different online databases such as ClinVar, HGMD and Varsome to check their clinical significance.ACMG verdicts about the variants were retrieved from Varsome database.Retinal information network (RetNet) was used to calculate number of genes associated with each IRDs type.All missense variants were evaluated for their predicted pathogenicity using different in-silico tools, namely, CADD, DANN, LRT, Mutation Assessor, Mutation Taster, Mutpred, PolyPhen-2, PROVEAN, and SIFT.

Demographic and clinical features of IRDs
This review paper focused on a total of 126 published research articles, including 1 case series, 34 case reports, and 91 cohort papers, all documenting IRDs in families of Pakistani descent between 1999 and April-2023.Excluding a retrospective case series study (n = 1), major diagnostic methods utilized in the remaining 125 articles included linkage analysis and/or homozygosity mapping coupled with targeted Sanger sequencing (n = 62), targeted Sanger sequencing (n = 24), and next-generation sequencing (panel-based sequencing, whole exome sequencing, and whole genome sequencing) (n = 39) (Fig. 1, Table S3).
All articles were thoroughly checked to infer different features such as ethnic affiliation of the patients, parental consanguinity, zygosity of the identified alleles, mutation types /molecular impact of the alleles, inheritance pattern, and disease type.Collectively, highest number of IRDs cases were reported from Punjab province (32.21%), followed by Khyber Pakhtunkhwa (13.73%),Sindh (1.12%), Baluchistan (0.84%), and Kashmir (< 1%), while a subset (1.12%) of studies documented IRDs in Pakistani families living abroad (UK/Canada).In the remaining 50.70% cases, no ethnic affiliation could be inferred from the published reports.Analysis of the retrieved articles for presence/absence of parental consanguinity revealed that parents were consanguineously married in 70.03% of the cases, 2.24% non-consanguineously married while no information about parental consanguinity were available in 27.73% cases.Interestingly, 95.50% IRDs cases were found to follow autosomal recessive (AR) inheritance pattern followed by autosomal dominant (AD) (1.70%), X-Linked (1.10%) pattern (Table 1).In the remaining 1.70% cases, information about inheritance pattern was not known.The authors, however, reported homozygous pathogenic variants in genes which can cause both AD and AR inheritance pattern (as per OMIM database).
While ethnic affiliations of index cases carrying c.1151delC allele were not provided in five out of seven studies, c.1151delC allele was independently reported in two unrelated families from KPK and Punjab provinces.The second most recurrent allele was a missense substitution (c.1138A > G:p.Thr380Ala) in the TULP1 (NM_003322.6)gene which was reported by five independent studies.Of them, two families belonged to ethnically matched KPK population, and thus possibly related.However, one family carrying c.1138A > G allele belonged to Punjab province.No data on the ethnic affiliation was available in the remaining two reports [88-90, 92, 93].In addition to the likelihood that these alleles constitute hotspot mutations, frequent occurrence of these recurrent alleles in multiple and ethnically matched Pakistani families might indicate a founder effect in the society.
Our findings, of RP as the most leading IRD type in Pakistani families (41%), align with several previous studies suggesting RP as the most frequently reported form of IRDs in world populations [94][95][96][97][98]. Also, we have found that RP is the most genetically heterogeneous disorder among IRDs in Pakistani population as mutations in 37 different RP-associated genes were identified.Nonetheless, Pakistani families somehow present unique genetic architecture of IRDs.For example, unlike ABCA4 and/or USH2A gene mutations which are considered as a major etiology of the IRDs cases worldwide [96][97][98], we have found that PDE6A gene mutations are the leading cause of IRDs in Pakistani families.While disease-causing mutations in SNRNP200 gene are known to cause adRP [99][100][101], SNRNP200 gene mutation correlated with arRP in a Pakistani IRD family [102].Lastly, detection of rare forms of IRDs notably STX3associated intestinal-retinal syndrome [103], VPS13Bassociated Cohen syndrome [104], and SLC6A6-associated taurine transporter (TauT) deficiency disorders [105] in Pakistani families not only points towards the distinctive genetic nature of this population but it also highlights its potential in medical research.
Our CADD score analysis of all missense variants that are described in this report revealed two alleles with lowest CADD-PHRED scores.These included RP1 (c.2005G > A; p.Ala669Thr), and RAX2 (c.374G > A; p.Gly125Glu) with a CADD-PHRED scores of 1.173 and 1.497, respectively.Though our in-silico findings about these alleles are not final, we recommend further researcher to further investigate these two missense variants in order to fully characterize their impact on protein structure and/or function.

Conclusions
This study provides a comprehensive overview of IRDs in Pakistani families over a period of 25 years (1999-2023).Our analysis reaffirms the fact that majority of the prevalent IRDs cases in Pakistan are recessively inherited, and that they mostly appeared due to the bi-allelic inheritance of rare pathogenic mutations from both parents.Undoubtedly, RP was the most frequently occurring IRD in Pakistan followed by LCA.Overall, PDE6A gene mutations was the leading cause of IRDs in Pakistani families followed by mutations in TULP1 gene.Altogether, marked genetic and allelic and heterogeneity was observed in the Pakistani IRDs families.In summary, Pakistani families are notable in expressing both common and rare Mendelian disorders such as Cohen syndrome, intestinal-retinal syndrome, and taurine transporter deficiency possibly due to the traditional practice of endogamy in the society.

Limitations
Since the data presented in this study were all retrieved from published reports, and further validated/curated using online databases, we foresee certain limitations in our study.For example, we do not claim causality of variants (if any) presented in this report as it does not fall under the purview of our study.Despite our own efforts, we do see the possibility of overlooking certain relevant literature on the subject.Incomplete information, inconsistencies or in some cases errors seen in the retrieved data may have skewed our own analysis.It is pertinent to mention here that all calculations about genomic variants were based on the total number of alleles (n = 277) reported in the 126 research articles.We were unfortunately unable to assess objectively the total number of families and/or patients affected by these 277 alleles due to the inadequate information provided in the literature.Nevertheless, published reports emerged mostly from two provinces of Pakistan i.e.Punjab and Khyber Pakhtunkhwa.Therefore, we recommend taking care while extrapolating our findings to other ethnic Pakistani populations like Sindh, Baluchistan and Gilgit-Baltistan.

ACMG
The

Fig. 2
Fig. 2 Pie chart showing frequency distribution of IRDs types reported in Pakistani families

Fig. 3
Fig. 3 Bar chart showing frequently mutated IRDs-associated genes in Pakistani families

Table 1
Demographic attributes of the index cases and genomic features of the observed alleles in Pakistani IRDs families between 1999 and April 2023 Types of single nucleotide variants (SNVs) reported (n = 170)

Table 2
In-silico predictions of the identified 116 missense variants reported in Pakistani families

Table 3
List of missense variants classified as 'benign or Likely benign' by the ACMG guidelines