Syndromic forms of inherited retinal dystrophies: a comprehensive molecular diagnosis of consanguineous Pakistani families using capture panel sequencing

• Introduction
• Methods
• Results
• Discussion

Abstract

Background: Inherited retinal dystrophies (IRDs) represent a clinically and genetically heterogeneous group of genetic disorders that involve photoreceptors and/or retinal pigment epithelium degeneration. IRDs may occur as an isolated condition or may represent an ocular manifestation of a multisystemic disorder referred as syndromic IRD. To increase the understanding of the molecular determinants of syndromic IRD-related genes in the Pakistani population, we revealed the genetic profile of 13 consanguineous Pakistani families using capture panel sequencing.

Methods: We performed comprehensive molecular testing on 72 IRD segregating Pakistani families using targeted capture panel sequencing of 344 known genes. The pathogenicity of candidate variants was assessed using American College of Medical Genetics and Genomics guidelines, followed by Sanger sequencing for segregation analysis.

Results: Causative variants in previously reported syndromic IRDs genes were detected in 13/72 (18%) IRD families, including 5/72 (6.94%), 4/72 (5.55%), 2/72 (2.8%), 1/72(1.38%) and 1/72 (1.38%) in Usher syndrome, Bardet–Biedl syndrome, Batten disease, retinitis pigmentosa with situs inversus and Stickler syndrome segregated families, respectively. Disease-causing variants included nine previously reported and six novel homozygous variants, i.e., c.1143G>C in USH2A, c.470G>A in MYO7A, c.877–2A>G in PCDH15, c.347C>T in ARL6, c.581C>T in CLN5 and c.100+1G>T in ARL2BP gene segregation with disease phenotype in eight families. Two heterozygous variants of the USH2A gene, i.e., c.12093C>A and c.9815C>T, were segregated in a compound heterozygous form in family RP243. Furthermore, RP151 showed segregation of a heterozygous variant c.247G>A in a Stickler syndrome gene, i.e., COL2A1, in an autosomal dominant manner.

Conclusions: This study reaffirms the clinical and genetic heterogeneity of syndromic IRD-associated genes and confirms the usefulness of molecular methods in advancing our understanding of these conditions in consanguineous populations. The most commonly mutated Bardet–Biedl syndrome gene was ARL6 (75%) and the most commonly mutated Usher syndrome genes were USH2A (40%) and MYO7A (40%). Our data could serve as a reference for future studies and the development of treatment modalities for affected families of Pakistani origin.

Inherited retinal dystrophies (IRDs) are a group of genetic retinal dystrophies characterized by photoreceptor and retinal pigment epithelium abnormalities that lead to progressive retinal degeneration, atrophy and vision loss [1,2]. IRDs are subdivided into nonsyndromic IRDs, without systemic abnormalities, and syndromic IRDs, in which retinal degeneration is associated with other symptoms due to systemic diseases [2–4]. Syndromic IRDs are classified into four main categories, comprising ciliopathies, Usher syndrome, inborn errors of metabolism (IEMs) and mitochondrial disorders [4,5]. Management of syndromic IRD cases requires definitive diagnosis and a thorough understanding of these conditions.

Ciliopathies, which result from primary cilium dysfunction, often exhibit pleiotropic effects, causing abnormalities in the eyes as well as urinary, central nervous and skeletal systems [3,5]. Variants in protein-encoding genes related to ciliary structure or function are known to cause approximately 36% of genetically diagnosed cases of retinal degeneration [5]. Photoreceptors are modified primary cilia that are susceptible to ciliopathy-related dysfunction, leading to the retinitis pigmentosa (RP) phenotype, as observed in Bardet–Biedl syndrome (BBS), Senior-Løken syndrome and Alstrom syndrome [5–7]. BBS shows heterogeneous clinical manifestations encompassing primary features of the disease: rod–cone dystrophy, polydactyly, obesity, genital abnormalities, renal defects and learning difficulties, and secondary features such as developmental delay, speech deficit, brachydactyly/syndactyly, dental defects, ataxia/poor coordination, olfactory deficit, diabetes mellitus, congenital heart disease, and so on [8]. However, most of these characteristics often appear and progressively get worse in the first and second decades of life [9]. To confirm the diagnosis of BBS, it is recommended that the patient bears four out of six primary features of the disease. If only three primary features are detected, two secondary features are required to confirm the BBS diagnosis. However, these criteria focus on BBS mostly as a clinical entity and ignore attenuated forms of the disease and the possible gene-specific manifestations [10,11].

In case of BBS, more than 20 genes (BBS1, BBS2, ARL6, BBS4, BBS5, BBS6/MKKS, BBS7, TTC8, BBS9, BBS10, TRIM32, BBS12, MKS1, CEP290, WDPCP, SDCCAG8, LZTFL1, BBIP, IFT27, IFT74, C8orf37, SCLT1, NPHP1, SCAPER) have already been identified [12–14]. BBS is a rare condition with differing frequencies among various geographic areas. It ranges between 1:120,000 and 1: 160,000 in North America and Europe. However, the rate is higher in some isolated areas where consanguineous marriages are common. For example, the prevalence rate is far higher among the mixed Arab population in Kuwait (1:36,000), among Bedouins (1:13,500), in the Jahra district (1:6900), in Newfoundland (1:18,000) and in the Faroe Islands (1:3700) [15].

Usher syndrome accounts for the most prevalent cause of deaf-blindness world, with a prevalence of 4–17 per 100,000 people. It causes simultaneous loss of vision and hearing with vestibular dysfunction in some cases [16]. It is inherited in an autosomal recessive manner [17]. Usher syndrome is primarily categorized into 3 clinical types: USH1, USH, and USH3, which are further subcategorized into 14 subtypes. Each subtype is caused by a mutation in a distinct gene. Nine specific genes have been discovered causing Usher syndrome, among which MYO7A, USH1C, CDH23, PCDH15 and USH1G are associated with USH type 1; USH2A, ADGR1 and WHRN contribute to USH type 2, and CLRN1 contributes to USH type 3 [18]. USH-involved mutations disrupt structural proteins in the auditory, visual and vestibular systems. The impairment of sensory cells expressing actin filaments is seen to be constrained, explaining the impairment of eyes and ears in affected individuals [19].

In Usher syndrome, sensorineural hearing loss alongside progressive RP is evident. At the same time, the onset and severity of hearing loss, together with the presence of vestibular dysfunction, determine the type of syndrome. Within each subtype of Usher syndrome, vast clinical heterogeneity facilitates an atypical presentation [16]. The prevalence data for Usher syndrome is ambiguous with large differences observed internationally, probably due to the lack of proper diagnostic and rehabilitation facilities [18]. However, the global prevalence approximation lies at 1 in 30,000 individuals, and some alternate investigations have revealed the aforementioned wide occurrence range from 4 to 17 in every 100,000 people [16].

In IEMs, neurologic symptoms are common. As the retina develops from the embryonic forebrain thus, neurodegeneration resulting from IEMs often involves retinal degeneration. Many syndromic IRDs are as a result of IEMs, including neuronal ceroid lipofuscinoses (CLNs), and types 1 and 2 Stickler syndrome (SS) [4]. CLNs are autosomal recessive conditions caused by mutations in genes that lead to endo-lysosomal dysfunction. CLNs, also known as Batten disease, are neurodegenerative disorders with a common presentation of early vision impairment and progressive neuronal loss [20]. To date, mutations in at least 14 genes are known to cause CLN phenotypes [21]. SS is a subtype of collagenopathy that was initially named “hereditary progressive arthro-ophthalmopathy” by Gunnar B. Stickler, who first studied and reported the findings of this syndrome in 1965 [22]. SS can be inherited as an autosomal dominant condition due to heterozygous disease-causing variants in the COL2A1, COL11A1 and COL11A2 genes, or as an autosomal recessive condition due to biallelic variants in the COL9A1, COL9A2 and COL9A3 genes [23]. SS is a group of connective tissue disorders that is associated with short sight and a very high risk of blindness from retinal detachment. Other features include cleft palate, conductive or sensorineural hearing loss, orofacial anomalies and premature arthritis [24,25]. SS is clinically and genetically heterogeneous and shows variable expression; therefore, mild forms of the disease often remain unrecognized, necessitating the use of molecular genetic techniques in the diagnosis of suspected cases for patient management and identification of at-risk family members [26]. In an ongoing effort to study the spectrum and frequency of disease-causing variants in a large-scale Pakistani IRD cohort using next-generation sequencing techniques, we identified disease-causing variants in syndromic IRD genes in 13 families, explaining the clinical and genetic heterogeneity of these conditions.

We recruited a cohort of 72 multi-generational consanguineous families, each with the segregating retinal phenotype, through Al-Shifa Trust Eye Hospital, Rawalpindi, Pakistan. Most of the enrolled families had multiple affected individuals. Upon molecular genetic analysis, 13 families were identified with disease-causing variants in syndromic IRD-related genes. Clinical data of the affected individuals of these syndromic families obtained at the time of enrollment are detailed in Table 1. Usher syndrome family data revealed that family RP057 had three, RP094 had two, RP182 had one, RP220 had seven and RP243 had six affected family members (Figure 1 and Appendix 1). The phenotypic onset in the proband of enrolled families ranged from birth to the second decade of life (Table 1). Among the families, the RP094 and RP182 probands (individuals III.I and IV.III) were affected by birth, individual IV.II in family RP057 had disease onset in the first decade of life, while individual V.XI in family RP220 and IV.IX in family RP243 were diagnosed in the second decade of life (Figure 1 and Appendix 1). In addition to the retinal phenotypes shared by the affected individuals of all families, such as night blindness, poor day vision and photosensitivity, affected individuals of Usher families also experienced progressive hearing loss. Astigmatism was observed in the affected individuals of family RP094, and strabismus and maculopathy were also observed in the RP243 family (Table 1). Fundus photographs of an affected individual of RP 243 (IV.IX) presenting waxy bone spicule with vascular attenuation are shown in Appendix 2. Brainstem-evoked response audiometry and auditory brainstem response testing of the proband (IV.II) of RP057 revealed bilateral severe-to-profound hearing impairment (Appendix 2).

In BBS families, each RP043, RP157 and RP164 family had three affected members, whereas RP173 had five affected cases. The proband (IV.V) of family RP043 and individual IV.II of RP173 were affected by birth (Table 1 and Appendix 3), while individuals III.XI of RP157 and V.III of RP164 were diagnosed in the first decade of life (Figure 2 and Appendix 3). BBS families showed the primary features of BBS such as retinal pigmentosa and polydactyly. A fundus image of the right and left eyes showing RP signs (Appendix 4) and photographs of the hand and foot showing postaxial polydactyly in affected individual IV.II of RP173 are shown in Appendix 4. Secondary features of BBS—macular dystrophy, low intellect and intellectual disability—were noted in family RP164. Renal dysfunction was reported in affected members of family RP173.

Two affected members of family RD002 and three cases of RP155 had characteristics features of Batten disease/CLN including retinal degeneration, speech problems, seizures, loss of motor skills, cognitive decline, and behavioral and learning problems. The affected member V.II of RD002 had folded hands and weak bones (Figure 3). The brain magnetic resonance imaging scan of individual V.II of RD002 revealed generalized cerebral and cerebellar atrophic changes with associated volume loss not consistent with the age of patient, i.e., 17 years. The affected member IV.II of family RP155 has intellectual disability and remains mostly in an unconscious state (Figure 3). Medical records of all affected cases of RP067 revealed RP with situs inversus, and there are three affected males and one affected female (IV.XX) in RP067 (Figure 4). The chest X-rays of the affected individuals of RP067 revealed a reversed position of the heart and aorta. In families, the RP067 and RP155 probands (individuals IV.VII and IV.II) were affected by birth, and in family RD002, individual V.II had disease onset within the first decade of life. All affected members of family RP151 had onset of retinal phenotype by birth. They also had myopia, premature arthritis, aggressive behavior and slowly progressive hearing loss, and one affected member—IV.I—had a speaking defect (Table 1).

Capture panel sequencing led to the identification of 15 disease-causing variants, among which 9 were previously reported while the remaining 6 were novel (Table 2). Consistent with the mode of phenotypic inheritance, all Usher, BBS, CLNs and RP variants with situs inversus genes except c.12093C>A and c.9815C>T of USH2A were homozygous in affected cases. Variants c.12093C>A and c.9815C>T of USH2A segregated in a compound heterozygous form in family RP243 (Appendix 1). Furthermore, a heterozygous disease-causing variant c.247G>A of the COL2A1 gene segregated in an autosomal dominant form with a disease phenotype in one family, i.e., RP151 (Appendix 5). All of the novel variants identified and previously reported, likely pathogenic/variant of uncertain significance/benign variants, were curated per ACMG guidelines and are listed in Table 2 and Table 3.

Disease-causing variants in Usher syndrome genes were found in the USH2A, MYO7A and PCDH15 genes in five families (Table 2, Figure 1 and Appendix 1). USH2A gene variants were associated with disease phenotype in two unrelated families (RP220 and RP243; Figure 1 and Appendix 1). A novel homozygous substitution, c.1143G>C, was found to be segregating disease phenotype in five affected and four unaffected individuals of the RP220 family (Figure 1C). This variant substitutes glutamine to histidine at position 381 in the protein, i.e., p.(Gln381His; Figures 1C and Figure 3C). This variant is absent from gnomAD, ClinVar and dbSNP but present in LOVD. Two heterozygous pathogenic variants in the USH2A gene segregated in RP243, with one a truncating transversion, i.e., c.12093C>A causing p.(Tyr4031Ter), which is listed as pathogenic in ClinVar and dbSNP (rs55921307); and the other is a known missense pathogenic variant c.9815C>T:p.(Pro3272Leu) listed in ClinVar and dbSNP (rs104893791). Segregation testing for the five affected and two unaffected family members confirmed the perfect segregation of both USH2A variants in a compound heterozygous manner with the disease phenotype (Appendix 1). Interestingly, a nonsyndromic RP-affected female (III.I) of family RP243 did not carry USH2A variants found in the other five affected cases; instead she was homozygous for a previously reported missense variant, i.e., c.448G>A; p. (Glu150Lys) of the RHO gene (Appendix 1). This variant is reported in the gnomAD (v.2.1.1) database only within the South Asian population with an estimated frequency of 0.00004772 and is listed in ClinVar (variation ID: 13,046) as pathogenic by multiple submitters for the autosomal recessive RP phenotype.

Two homozygous missense disease-causing variants in the MYO7A gene, i.e., c.3508G>A: p.(Glu1170Lys) and c.470G>A: p.(Ser157Asn), were found in families RP094 and RP182, respectively (Figure 1 and Appendix 1). Consistent with the molecular diagnosis, patients from both families exhibited RP and hearing loss. The p.(Glu1170Lys) variant identified in RP094 is reported in ClinVar as a pathogenic variant with a minor allele frequency of 0.00002012 in gnomAD (v.2.1.1). Segregation testing was performed on the two affected and two unaffected family members, and perfect segregation between mutation and disease phenotype was observed (Appendix 1). Similarly, one affected case of RP182 was homozygous for the c.470G>A variant while controls were either homozygous for the wild-type allele or heterozygous carriers of the disease allele (Figure 1B). The c.470G>A: p.(Ser157Asn) variant identified in RP182 is a novel report, as this is absent from gnomAD (v.2.1.1) and other databases including ClinVar, dbSNP and LOVD.

The PCDH15 gene, another Usher syndrome gene, was mutated in family RP057. All patients of family RP057 had speaking and hearing defects (Table 1). Capture panel sequencing data analysis revealed a novel homozygous missense variant, c.877–2A>G, in chromosome 10—55996693T>C in intron 8 of the PCDH15 gene, which is predicted to cause splice site defects (Table 2). Similarly, segregation testing in two affected and one unaffected family member indicated the segregation of identified variants with the Usher syndrome phenotype in RP057 (Figure 1A). This variant is absent from gnomAD (v.2.1.1) and all other databases, including ClinVar, and is likely to affect splicing, with SpliceAI scores for donor loss (delta score of 0.50, −110 bp), acceptor gain (delta score of 0.26, −6 bp) and acceptor loss (delta score of 0.1, −2 bp).

Among the BBS genes, three disease-causing variants in the ARL6 gene were found to be segregated in families RP043, RP164 and RP173, respectively (Table 2, Figure 2 and Appendix 3). A previously reported pathogenic missense homozygous variant, i.e., c.281C>T: p.(ILe94Thr) with a minor allele frequency of 0.00003186 in gnomAD (v.2.1.1) was shown to be segregated in RP043 (Appendix 3). This variant is reported in ClinVar (variation ID: 438,186) as pathogenic for the BBS3 phenotype and likely pathogenic for the RP phenotype. A novel homozygous substitution in exon 5 of the ARL6 gene, i.e., c.347C>T: p.(Gln352Pro) was found in family RP164 (Figure 2). This variant is absent from the gnomAD (v.2.1.1), ClinVar, LOVD and dbSNP databases. Segregation of this variant with disease was observed by genotyping two affected and two unaffected family members, where affected members were homozygous for the variant, while unaffected members were carriers of the mutant allele (Figure 2). In RP173, a homozygous substitution, i.e., c.534A>G: p.(Gln178=) in exon 7 of the ARL6 gene segregated with the phenotype (Appendix 3). This variant is reported in gnomAD (v.2.1.1) with a minor allele frequency of 0.000007960 and as pathogenic in ClinVar (variation ID: 1,805,423) for the BBS3 phenotype and is predicted to affect the splicing of pre-mRNA transcripts, as suggested by SpliceAI scores for acceptor loss (delta score of 0.71, −54 bp) and donor loss (delta score of 0.87, −1 bp). Sanger sequencing confirmed five affected family members as homozygous and four unaffected family members as heterozygous carriers of mutant allele, further supporting the pathogenicity of this variant. The affected individuals of all three families with ARL6 variants had severe phenotypes, as all affected family members recorded RP and polydactyly at birth. The proband of RP173 was 16 years old at the time of enrollment for this study and had rectal dysfunction with low intellect (Table 1).

In family RP157, homozygous missense deleterious variant c.1055A>C: p.(Gln352Pro) was detected in exon 2 of the BBS12 gene (Appendix 3). This variant is reported in gnomAD (v.2.1.1) with a minor allele frequency of 0.00004775 and in ClinVar (variation ID: 560,429) with conflicting interpretations of pathogenicity, i.e., pathogenic, likely pathogenic and variant of uncertain significance by multiple submitters for the BBS12 phenotype. In this study, we detected perfect segregation of this variant with phenotype in two affected and one control member of family RP157, adding to the evidence of pathogenicity per ACMG guidelines. Affected members of RP157 had astigmatism and polydactyly with initial complaints of retinal detachment and blurred vision (Table 1).

Two families, RD002 and RP155, showed segregating missense variants in Batten disease genes, i.e., CLN5 and MFSD8/CLN7, respectively (Table 2 and Figure 3). In RD002, a homozygous single-base transition, i.e., c.581C>T leading to p.(Thr194Ile), missense change was found segregating in two affected and two phenotypically unaffected members. This variant is absent from gnomAD (v.2.1.1) and other databases including ClinVar. Similarly, in RP155, a single-base substitution, i.e., c.826T>G causing p.(Phe276Val) in MFSD8/CLN7, was segregated as an autosomal recessive variant with the Batten disease phenotype. This variant is reported in gnomAD (v.2.1.1) as a single allele of South Asian ancestry with a minor allele frequency of 0.000003978 and in ClinVar (variation ID: 3,294,568) as a variant of uncertain significance by a single submitter for inborn genetic disease. In family RP067, a novel splice site variant of the ARL2BP gene was found to be segregated with the RP and situs inversus phenotype in a recessive manner (Figure 4). This variant is likely to affect splicing, with Splice AI scores of 0.96 for acceptors loss and 0.99 for donor loss. This variant is absent from gnomAD (v.2.1.1) and other databases including ClinVar.

Finally, in family RP151, a disease-causing single-nucleotide transition, c.247G>A, leading to glycine to arginine substitution, i.e., p.(Gly83Arg) in the COL2A1 gene, was segregated in an autosomal dominant manner with an ocular phenotype (Table 2 and Appendix 4). This variant is found in gnomAD (v.2.1.1) with a minor allele frequency of 0.00002003 and is listed in ClinVar (variation ID: 964324) as likely to be benign by a single submitter of unknown disease status. However, segregation testing in the present study confirmed perfect segregation of this hemizygous variant with the SS phenotype in three affected males, whereas a phenotypically normal female was homozygous for the wild-type allele, as shown in (Appendix 3; Appendix 5), thereby providing evidence of pathogenicity (Table 3).

In conclusion, the six disease-causing variants (USH2A: c.1143G>C, p.(Gln381His); MYO7A: c.470G>A, p.(Ser157Asn); PCDH15: c.877–2A>G, ARL6: c.347C>T, p.(Pro116Leu), CLN5: c.581C>T, p.(Thr194Ile) and ARL2BP: c.100+1G>T) identified in our study cohort are novel findings. Segregation results as well as chromatograms of all variants are given along the family tree of each family in Figure 1, Figure 2, Figure 3 and Figure 4 and Appendix 1, Appendix 2 and Appendix 3. Clustal W analysis of four novel amino acid substitutions identified in ARL6, i.e., p.(Pro116Leu), MYO7A, i.e., p.(Ser157Asn), USH2A, i.e., p.(Gln381His) and CLN5, i.e., p.(Thr194Ile) revealed evolutionary conservation across species (Figure 5A-D).

Developing therapeutic measures for inherited eye diseases requires a detailed understanding of the disease pathways through the identification of genetic mutations [27,30–32]. Current therapeutic strategies include delivering a wild-type (nonmutated) copy of the affected gene [33] or precisely modified nucleotide sequences within the genome through genome editing technologies [34]. However, due to genetic heterogeneity, i.e., the involvement of multiple genes in a disease phenotype, mutations within the same gene causing different phenotypes and mutations in different genes causing similar clinical phenotypes, it is important to understand the precise genetic defects to develop suitable treatment strategies for these blinding conditions [35,36]. All three modes of inheritance—dominant, recessive and X-linked—have been reported for IRDs [27,32]. However, recessive forms are more common in populations where endogamy is a social preference [32,37], including Pakistan [27,32,38,39]. Studies have revealed several genetic mutations causing IRDs from Pakistan, but these data are too limited compared with the high prevalence of these disorders in our population [27,32,38]. Thus, here we used a cost-effective panel sequencing approach followed by Sanger sequencing for segregation testing to reveal the mutation spectrum of 72 IRD-affected Pakistani families. Our results identified disease-causing variants of syndromic IRD genes in 13 analyzed families (13/72; 18%) with significant genetic and phenotypic heterogeneity.

Interesting observations from this study include the presence of compound heterozygous disease-causing variants of USH2A and a homozygous missense pathogenic variant of RHO in five affected cases and one affected case, respectively, in the same family RP243 (Appendix 1). Pathogenic variant c.448G>A: p.(Glu150Lys) of the RHO gene was present in one nonsyndromic RP patient (individual III.I; Appendix 1) and it was also previously reported in three consanguineous Pakistani families and an Indian family with segregated autosomal recessive RP [40–42]. A heterozygous pathogenic variant of the USH2A gene, i.e., c.12093C>A, was previously reported in compound heterozygous form along with a heterozygous splice site variant, i.e., 12,295–3T>A in a male European Usher patient [43]. Similarly, the other USH2A heterozygous variant, c.9815C>T, identified in RP243, was also previously detected as a heterozygous allele in an Italian autosomal recessive RP patient, a Dutch Usher II patient and a Chinese Usher II patient [44–46]. Furthermore, the c.1143G>C transversion leading to a glutamine-to-histidine substitution at the 381 position of the Usherin protein identified as a homozygous variant in affected cases of family RP220 in this study was previously present as a heterozygous allele segregating in a compound heterozygous manner with another heterozygous splice site substitution, i.e., c.8559‐2A>G, in a sporadic Chinese Usher patient of Han ethnicity [47]. However, in family RP220, segregation of this variant in autosomal recessive form with disease phenotype was confirmed by genotyping five affected and four unaffected individuals, elevating evidence of the pathogenicity of this variant per ACMG criteria (Figure 1C and Table 3).

USH1 is the most severe form of Usher syndrome and is reported to account for approximately 25%–44% of all cases of Usher syndrome globally [48]; however, in this study disease-causing variants in Usher type 1 (USH1)–related genes, i.e., PCDH15 and MYO7A, were found in three out of five (60%) Usher-segregating families, highlighting the unique mutation spectrum of this disorder in the Pakistani population. Furthermore, in the current study we identified pathogenic variants of the BBS3 (ARL6) gene in three out of four (75%) BBS families, which contrasts with previous reports indicating BBS6 as a frequently mutated BBS gene in families from Pakistan [39,49]. However, this variability may be due to the different ethnic lineages of families enrolled in previous studies as well as this study.

Identification of a novel pathogenic variant of the ARL6 gene, i.e., c.347C>T causing p.(Pro116Leu), inherited recessively in three patients of family RP164 reaffirms the genetic heterogeneity of BBS3 in the Pakistani population. Initial clinical assessment of the proband of RP164 indicated nonsyndromic RP; however, examination of all other affected individuals showed intellectual disability in one patient (V.III; Figure 2). The phenotypic variability across different affected cases may be due to the pleiotropic nature of BBS, as a wide range of clinical variability has been observed both within and between families [6]. Previously, nonsyndromic RP was reported in Saudi Arabian patients carrying a homozygous missense variant (p.Ala89Val) in the ARL6 gene [50]. The occurrence of different clinical phenotypes due to the same mutation suggests a role of the complex genetic background of each patient in these heterogeneous clinical outcomes. Furthermore, our molecular genetic testing assisted with the diagnosis of RP164 (Table 1 and Figure 2) and suggested monitoring of all affected cases of this family for the risk of cardiac and kidney issues for the timely management of patients.

In this study, we identified disease-causing variants in CLN (Batten disease) proteins that localized to lysosomes, i.e., CLN5 and MFSD8/CLN7, segregating with progressive retinal degeneration along with cognitive and motor regression, as well as epileptic abnormalities in family RD002 and RP155, respectively (Table 2 and Figure 3) [51]. To date, a total of three disease-causing mutations (c.1072_1073delTT [p.Leu358AlafsX4], c.925_926del [p.Leu309AlafsTer4] and c.477T>C [p.Cys159Arg]) in exon 4 of the CLN5 gene have been reported in Batten disease cases of Pakistani decent [52,53]. Identification of a likely novel pathogenic variant in exon 4 of the CLN5 gene causing a missense change, i.e., p.Thr194Ile in family RD002 in the present study, highlights exon 4 as a hot-spot exon for the molecular genetic analysis of Batten disease cases of Pakistani origin. However, the missense variant, i.e., p.(Phe276Val) of MFSD8/CLN7 segregating in family RP155 has been reported in ClinVar (variation ID: 3,294,568) for inborn genetic disease with no further details of clinical significance/inheritance; however, our study provides the first evidence of the inheritance of this variant with the Batten disease phenotype in a Pashtun sibship in Pakistan (Figure 3).

In this study, the main limitation/s explaining genetic variants to phenotypic diversity is the unavailability of detailed clinical reports including electroretinography, audiometry of all Usher syndrome cases, and other necessary clinical tests to confirm BBS and SS phenotypes. In family RP151, all affected cases had vision and hearing impairments, as well as premature joint pain, which are symptoms of SS (Table 1 and Appendix 5), a connective tissue disorder [25]. However, the family was not willing to participate in detailed clinical examination to rule out the type of hearing loss; i.e., conductive or sensorineural assessment, as well as testing for degenerative joint disease. However, in this context, genetic screening in this study assisted with diagnosis. Our study highlights the need to invest in better diagnostic equipment, laboratory capabilities and electronic health record systems in underserved communities to capture more comprehensive clinical data for future research for a complete understanding of disease mechanisms and potential therapeutic targets. To the best of our knowledge, this is the first report of genetic screening for MFSD8/CLN7, ARL2BP and COL2A1 in Pakistani families inheriting Batten disease (RP155), RP along with situs inversus (RP067) and Stickler syndrome (RP151), respectively. Our data support genetic testing of patients with inherited disorders in Pakistan for accurate diagnosis and early interventions to improve the quality of life of affected families.