Molecular Genetic Analysis of the Autosomal Recessive Non-Syndromic Inherited Retinitis Pigmentosa

Introduction: 90% of visually impaired people live in developing countries. There are various types of vision impairment, but the focus of the current study is retinitis pigmentosa (RP). Up to now, 150 mutations have been reported that are linked with RP. Methodology: Healthy and affected members from two Pakistani families (RP01 and RP02) segregating autosomal recessive RP were selected for DNA extraction. PCR was conducted, and the amplified PCR products were analyzed using Polyacrylamide Gel Electrophoresis (PAGE) and visualized in the Gel Doc system for linkage analysis. The Gene Hunter 2.1r5 tool in the Simple Linkage v5.052 beta software suite was used to conduct multipoint parametric linkage analysis on the two consanguineous families examined on the 6K Illumina array. Exons and intron-exon borders of all known arRP genes found in homozygous areas were sequenced in the matching probands using a 3130 automated sequencer and the Big Dye Terminator Cycle Sequencing Kit v3.1. The mutation study was carried out using the AlaMut 1.5 program. Results: In both families, linkage analysis was performed using microsatellite marker DIS422 for gene crumbs homolog 1 (CRB1) and microsatellite marker D8S2332 for gene Retinitis Pigmentosa 1 (RP1). Multipoint linkage analysis identifies genomic regions that could potentially contain the genetic defect. In family RP01, only a single peak with a maximal multipoint LOD score of 3.00 was identified on chromosome 1, whereas in family RP02, multiple peaks with multipoint LOD scores of 1.80 were identified on chromosome 8. Analysis of the CRB1 gene revealed a homozygous substitution of glycine for valine (c.1152T>G; p.V243G), whereas the RP1 gene demonstrated that leucine was substituted for proline as a result of cytosine to thymine transfer (c.3419C>T; p. P1035L). Conclusion: Homozygosity mapping is a powerful method for finding genetic abnormalities that are both precise and comprehensive for identifying harmful variations in consanguineous families. This method is invaluable for providing accurate clinical diagnostic and genetic advice in remote regions of Pakistan while also increasing knowledge about autosomal recessive diseases and the dangers of mixing.


Introduction
Dr. Donders coined the term "retinitis pigmentosa" (RP) in 1857 [1,2], which identifies a group of hereditary retinal illnesses characterized by a steady decline of rods and cones (light-sensing cells) [2,3], which can result in severe vision impairment or blindness [4,5]. RP affects 1 in every 4000 people in the United States and approximately 1 in 5000 people worldwide [6], making it one of the most common genetic causes of vision impairment worldwide. RP is heterogeneous clinically and genetically and is categorized into nonsyndromic, syndromic, and systemic [7,8].
The inheritance patterns of non-syndromic RP may be autosomal recessive, which is 50%-60% (the most prevalently inherited RP worldwide), an autosomal dominant pattern in about 30%-40% of cases, or X-linked patterns, which are almost 5%-15% prevalent [7]. Sporadic cases of the mitochondrial or digenic mode of inheritance are also reported [9]. Usher syndrome (the most prevalent cause of collective blindness and deafness) and Bardet-Biedl syndrome (in this condition, RP is accompanied by polydactyly, obesity, renal 1 2 3 4 5 6 7 8 9 10 abnormalities, hypogenitalism, and mental retardation) are the most common forms of syndromic RP [9,10].
More than 50 known genes cause retinitis pigmentosa, which constitutes only 50% of the prevailing disorder, while 22 genes are associated with autosomal dominant RP (adRP), 40 genes are associated with autosomal recessive RP (arRP), and five genes are associated with X-linked RP [5,[7][8][9]. This research was carried out to identify the gene mutation that causes RP in Pakistani families using homozygosity mapping.

Ethical statement
Research approval was granted by the Research Ethical Committee of the International Islamic University, Islamabad, Pakistan, under the No. IIUI/BIOTECH/ASRB2016. All participants, including those who were impacted, their parents, and other regular family members of the afflicted households, provided written consent.    Age  70  65  37  35  33  30  27  31  11  09  07  04 Speech

Sample collection
Five milliliters (5ml) of a peripheral blood sample from each individual was collected by sterile syringe, using the venipuncture technique, in an EDTA vacutainer. Collected samples were stored at 4° C in a laboratory refrigerator till the extraction of genomic DNA.

Linkage analysis and homozygosity mapping
The genomic DNA of participants was extracted from peripheral blood using an organic technique (phenolchloroform), and the isolated DNA samples were examined using a 1% agarose gel, as reported in our previously published article [8]. Following that, PCR was conducted, and the amplified PCR products were analyzed using polyacrylamide gel electrophoresis (PAGE) and visualized in the Gel Doc system (BioRad, Italy) for linkage analysis. The Gene Hunter 2.1r5 tool in the Simple Linkage v5.052 beta software suite was used to conduct multipoint parametric linkage analysis on the two consanguineous families examined on the 6K Illumina array. Exons and intron-exon borders of all known arRP genes found in homozygous areas were sequenced in the matching probands using a 3130 automated sequencer and the Big Dye Terminator Cycle Sequencing Kit v3.1. The mutation study was carried out using the Alamut 1.5 program.   However, linkage was found using the microsatellite marker D8S2332 for gene RP1, and all non-affected members were heterozygous for D8S2332 ( Figure 4).

8%.
The Illumina 6K array and multipoint linkage analysis were used to search for potential chromosomal areas containing the genetic flaw in two consanguineous households with impacted members. Only one peak on chromosome 1 was found in the RP01 family, and its highest multipoint LOD value was 3.00 ( Figure 5). Multiple peaks on chromosome 8 with multipoint LOD values of 1.80 were found to be at risk for carrying the genetic abnormality in family RP02 ( Figure 6). The crumbs homolog 1 (CRB1) gene was found on chromosome 1 and was shared by two afflicted siblings in the RP01 family. The mutant study of CRB1 revealed a glycine for a valine (c.1152T>G; p.V243G) substitution in a homozygous state. In family RP02, however, all four afflicted members shared only three copies of the big homozygous area on chromosome 8 that contains the RP1 gene, and sequencing showed a cytosine to thymine shift that causes a proline to leucine mutation (c.3419C>T; p. P1035L) (

Discussion
RP is a heterogeneous group of hereditary retinopathies with a large number of known mutations [11], several disease-causing genes, and extremely diverse clinical outcomes [7,9]. In the past two decades, remarkable progress has been achieved in locating the genes responsible for hereditary retinal conditions, including RP. The link between genes, mutations, and clinical data has unavoidably grown quite complicated [12]. Greater knowledge of the biological foundation of vision and insights into the mechanisms involved in retinal pathology are just a couple of the numerous effects that might result from successfully identifying the origins of hereditary retinal illnesses [13][14][15]. Finding the genes and mutations linked with RP requires both gene discovery and mutation screening in affected people and their families. Our aim was to identify the gene mutation that causes RP in Pakistani families using homozygosity mapping, which will advance the search for remedies.
Studies have identified several genes associated with RP in Pakistani families, including RP1, RP2, RP9, DHX38, and PRPF31 [9,16,17]. Mutations in these genes account for a significant proportion of cases of autosomal recessive RP in Pakistan. Ali et al. [9] found linkage analysis, but most of these families contain compound heterozygous variants in either a known arRP gene or an as-yet-undiscovered arRP gene [13,15,16]. To identify chromosomal areas that might house the underlying genetic flaw, homozygosity mapping and mutation analysis were conducted in this research.
In our study, all members of the RP02 family who were not impacted by the linkage were heterozygous, whereas those who were affected were homozygous, according to the microsatellite marker D8S2332, which was used to identify the linkage in the RP1 gene. Additionally, the CRB1 gene linkage in the RP01 family was found using the microsatellite marker DIS422, and all unaffected people showed a heterozygous pattern for this marker, while afflicted members were homozygous. In the study by Umm-e-Aiman et al. [18] on the autosomal recessive RP family RP01, there was one dead person and three afflicted members. In the autosomal recessive RP family RP02, there was one dead person and one afflicted person. Due to homozygosity for the genes ZNF513, TULP1, RP1, and MERTK in both affected and unaffected members of the two RP families, no linkage was detected in these families. However, few pieces of research had linked these genes to RP families [19,20].
The genetic flaw was sought by performing a multipoint linkage analysis to locate possible hotspots in the genome. A single peak on chromosome 1 was found in the RP01 family, with the highest multipoint LOD value of 3.00. Multiple points on chromosome 8 were found to have multipoint LOD values of 1.80, suggesting that they may be the site of the genetic abnormality in family RP02. Leucine was replaced for a proline residue as a consequence of the cytosine to thymine transition in the RP1 gene analysis (c.3419C>T; p. P1035L), and glycine was used in place of valine in the CRB1 mutation research (c.1152T>G; p.V243G). In this study by Latif et al. [21], 11 members of a large consanguineous family were selected from Azad Jammu and Kashmir, where they underwent linkage mapping analysis and Sanger sequencing confirmation. This family's non-syndromic autosomal recessive RP was caused by homozygous c.2536G>A mutations in the CRB1 gene.
The RP genetics of Pakistani families are complex and entail numerous gene mutations. These mutations are essential for genetic counseling and targeted treatments for this dreadful disease. We provide additional information regarding the phenotypic and mutational spectrum of this variant of RP. These findings help explain the molecular genetic factors underlying inheritable RP in Pakistan, which impact genetic counseling, detection, prognosis, and possibly the selection of patients eligible for genetic therapies to halt its progression.

Limitation
This research was confined to families with retinitis pigmentosa patients. Due to a shortage of resources, only five genes (CRB1, RP1, TULP1, RPE65, and ABCA4) were randomly selected.

Conclusions
In conclusion, our research supports the efficacy of homozygosity mapping, which is accurate and thorough while being reasonably priced, for locating pathogenic mutations in consanguineous families with arRP. This method is important for providing accurate clinical diagnoses and genetic counseling in remote parts of Pakistan while also promoting public knowledge of arRP and the dangers of consanguineous marriage.

Additional Information Disclosures
Human subjects: Consent was obtained or waived by all participants in this study. Research Ethical Committee of the International Islamic University, Islamabad, Pakistan issued approval IIUI/BIOTECH/ASRB2016. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.