ABSTRACT Inherited retinal dystrophies are phenotypically and genetically heterogeneous. This extensive heterogeneity poses a challenge when performing molecular diagnosis of patients,

especially in developing countries. In this study, we applied homozygosity mapping as a tool to reduce the complexity given by genetic heterogeneity and identify disease-causing variants in

consanguineous Pakistani pedigrees. DNA samples from eight families with autosomal recessive retinal dystrophies were subjected to genome wide homozygosity mapping (seven by SNP arrays and

one by STR markers) and genes comprised within the detected homozygous regions were analyzed by Sanger sequencing. All families displayed consistent autozygous genomic regions. Sequence

termination codon. In addition to better delineating the genetic landscape of inherited retinal dystrophies in Pakistan, our data confirm that combining homozygosity mapping and candidate

gene sequencing is a powerful approach for mutation identification in populations where consanguineous unions are common. SIMILAR CONTENT BEING VIEWED BY OTHERS WHOLE EXOME SEQUENCING AND

HOMOZYGOSITY MAPPING REVEALS GENETIC DEFECTS IN CONSANGUINEOUS IRANIAN FAMILIES WITH INHERITED RETINAL DYSTROPHIES Article Open access 10 November 2020 WHOLE EXOME SEQUENCING IN 17

CONSANGUINEOUS IRANIAN PEDIGREES EXPANDS THE MUTATIONAL SPECTRUM OF INHERITED RETINAL DYSTROPHIES Article Open access 29 September 2021 GENETIC SPECTRUM OF RETINAL DYSTROPHIES IN TUNISIA

Article Open access 08 July 2020 INTRODUCTION Inherited retinal dystrophies (IRDs) are a group of rare genetic disorders for which mutations in causative genes result either in the

degeneration or the dysfunction of retinal cells. In the majority of cases, they are progressive conditions that can lead to legal or complete blindness1. IRD phenotypes are rather

heterogeneous in terms of onset, progression and severity of the disease. Symptoms and signs may be mild and stationary, such as for example in congenital stationary night blindness and

achromatopsia, or progressive and severe, such as in retinitis pigmentosa (RP) and cone and cone-rod dystrophies2. Genetically, IRDs are also heterogeneous, with more than 190 responsible

genes reported to date3. Furthermore, IRDs are inherited as an autosomal recessive, autosomal dominant, or X-linked trait, with autosomal recessive being the most prominent one3.

Homozygosity mapping is an efficient tool to map regions harboring either novel or known recessive mutations4 and is particularly effective in consanguineous families or in populations that

are geographically isolated and are prone to result in a high rate of endogamy5. Although homozygosity mapping is not a recent technique, introduction of SNP-based genotyping microarrays has

provided a fast and effective means of analysis, as demonstrated by a number of studies already5,6,7,8. It is also particularly effective for detecting IRDs mutations because of the rather

elevated frequency of heterozygous recessive IRD variants in the general population9. IRD diagnosis and genotype-phenotype correlations can be extremely daunting tasks, especially in

developing countries. This study was designed to apply homozygosity mapping in consanguineous Pakistani families segregating IRDs that were minimally characterized from a clinical standpoint

with the aims of (i) identifying the causative genetic agents of the disease and (ii) helping clinical diagnosis of patients. MATERIALS AND METHODS ETHICS STATEMENT This study was designed

in compliance with the tenets of the Declaration of Helsinki and carried out according to protocols that were approved by the Institutional Review Boards of Quaid-i-Azam University,

University of Lausanne and Tohoku University. Written informed consent for providing medical information and blood samples was obtained from each participant. FAMILIES AND PREPARATION OF

SAMPLES Families with two or more affected individuals were ascertained by physicians and scientists from the Quaid-i-Azam University, who visited them at their places of residence. Out of

eight pedigrees, six (MA25, MA69, MA94, MA117, MA123 and MA132) were enrolled from rural areas of the Punjab province, while two (MA62 and MA97) were from the Sindh province. Pedigrees were

drawn (Fig. 1) and a standard questionnaire was used to collect information including: family history, visual complaints, pattern of disease inheritance and assessment of additional

non-ocular clinical signs such as polydactyly, male infertility, renal and hearing impairment. Further fundoscopic examination and electroretinography (ERG) was performed when available.

Blood samples of affected and unaffected individuals from each family were collected on site. DNA purification was carried out using standard organic phenol-chloroform extraction methods.

For low-volume samples, the NucleoSpin blood extraction kit (Macherey-Nagel, Bethlehem, PA) was used. HOMOZYGOSITY MAPPING Seven families (MA62, MA69, MA94, MA97, MA117, MA123 and MA132)

were genotyped by using the Illumina HumanCytoSNP-12v2.1 SNPs array (Illumina, Santa Clara, CA, USA), containing ~300,000 markers, at the NCCR Genomics Platform of the University of Geneva,

Switzerland. Arrays were processed according to manufacturer’s protocols. The SNP data were analyzed by using HomozygosityMapper10 and homozygous regions shared by affected individuals of

each family were further assessed to explore involvement of genes known to be implicated in IRDs’ molecular pathology. An additional family, MA25, was genotyped by using highly polymorphic

microsatellite markers encompassing known achromatopsia candidate genes, as described previously11. MOLECULAR ANALYSIS The entire open reading frame (ORF) and exon-intron boundaries of

candidate genes were screened by means of PCR-amplification and Sanger sequencing in probands of each family. Primers (Supplementary Table S1) were designed by using the Primer 3 software12

and PCR amplification was performed under standard conditions, with an annealing step at 57 °C for 30 seconds that was common to all primer pairs. PCR products were purified by treatment

with the ExoSAP-it reagent (Affymetrix, Santa Clara, CA) and sequenced using the Big Dye Terminator Cycling Sequencing Kit v3.1 (Applied Biosystem, Foster City, CA) by an ABI 3130xl Genetic

Analyzer (Applied Biosystems). Sequencing data were analyzed using the CLC Bio software (Qiagen, Boston, MA) and compared with the corresponding human reference sequence (build hg19).

Co-segregation analysis of all mutations was done in all families. Novel DNA variations were compared with data from public databases (Exome Variant Server, EVS13 and 1000 genomes14) and

with information obtained by sequencing 200 healthy controls from different ethnic groups from all provinces of Pakistan. In order to evaluate the putative pathological nature of the novel

missense variants reported in this study, we used three in silico tools, namely Polymorphism Phenotyping v2 (Polyphen-2)15, Sorting Intolerant from Tolerant (SIFT)16 and Mutation Taster17.

RPGRIP1 protein sequences from different species including human (_H. sapiens_, NP_065099.3) macaque (_M. mulatta_, XP_002808500.1) mouse (_M. musculus_, NP_076368.1), cow (_B. taurus_,

NP_851377.1), dog (_C. lupus familiaris_, XP_851597.2), Xenopus (_X. tropicalis_, XP_002933948.2) and zebrafish (_D. rerio,_ ENSDARP00000118806) were aligned using the CLC Genomics Workbench

(Qiagen) in order to check the evolutionary conservation of the substituted amino acid in RPGRIP1. The same procedure was applied to CNGA3 protein sequences (human, NP_001289.1; macaque,

XP_001101944.2; mouse, NP_001268939.1; cow, NP_776704.1; dog, XP_538462.3; Xenopus, XP_002931690.2; zebrafish, XP_005166141.1). SPLICING VARIANT ANALYSIS To predict the putative impact of

the identified splice site variation c.413-1G > A in _CNGB1_, in silico analysis was done using MutPred Splice (v1.3.2)18, Human Splice Finder (v2.4.1)19 and SKIPPY20. In silico results

were experimentally validated by means of a minigene assay. More specifically, a genomic DNA region spanning introns 4 to 8 of _CNGB1_ from patient MA97/IV-1 and one healthy control

individual was PCR-amplified using the High Fidelity Phusion polymerase (Thermo Scientific, Pittsburgh, PA) for which a distinct primer pair (forward: 5′-AAGGTACCGGGGAGACAGTGGTTTAGGA-3′ and

reverse: 5′-AATCTAGAACAGTCACTCCTCCCCATAGA-3′) was designed. The resulting PCR-products were subsequently cloned into the pcDNA 3.1/V5-His TOPO vector using the TOPO TA Cloning according to

manufacturer’s protocol (Life Technologies, Carlsbad, CA). Plasmids were analyzed by direct Sanger sequencing and then transfected into HeLa cells. Total RNA was extracted using the

Nucleospin RNA extraction kit (Macherey-Nagel), retrotranscribed with the Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA) and the resulting cDNA was PCR-amplified by using

primers lying within exon 5 (forward: 5′-AGGGTACTGACCTGGCTCAT-3′) and exon 8 (reverse: 5′-CAGATTCTGCTCCAGCCACA-3′) of the gene. The amplified products were separated by electrophoresis on a

2% agarose gel and were subsequently analyzed by Sanger sequencing. RESULTS CLINICAL EXAMINATION Due to the diversity of geographical origins of patients and the scarcity of diagnostic means

available in rural Pakistan, the extent of clinical examination varied greatly across individuals (Table 1). The clinical presentation of patients in families MA25, MA69 and MA94 was

consistent with that of achromatopsia, i.e. early onset of symptoms (<6 months of age) including photophobia, nystagmus and a complete absence of color discrimination. Fundus examination

of individual IV-12 (aged 15 years) of MA69 showed essentially a normal fundus except for the loss of foveal reflex, indicating the presence of a modest maculopathy or a foveal hypoplasia

(Fig. 2a). In MA117 and MA123, affected members had also photophobia and color vision loss, but the onset of their symptoms was reported during the second decade of life, suggestive of cone

or cone-rod dystrophy. Patient IV-2 (aged 47 years) of MA117 also had fundus examination, showing macular degeneration as well as a widespread retinal degeneration accompanied by vascular

attenuation and waxy pallor of the optic nerve head (Fig. 2b). None of the affected individuals from these families experienced night blindness. In contrast, the clinical picture of families

MA62, MA97 and MA132 showed the presence of RP-like symptoms, for which affected individuals initially experienced night blindness with eventual progressive visual loss. Specifically,

affected members of family MA97 reported significant vision loss between 15 to 17 years of age, resulting in the end in legal blindness. Funduscopy of patient IV-1 (24 years old) revealed

retinal degeneration with diffuse atrophy of the retinal pigment epithelium with macular involvement, scattered retinal pigment depositions vascular attenuation and a modest pallor of the

optic nerve head (Fig. 2c). This patient has self-reported myopia. The fundus appearance of patient IV-3 from family MA132 (18 years old) showed attenuated vessels, modest disc pallor and

diffuse atrophy of the retina and the retinal pigment epithelium with occasional pigment deposits sparing the macular area (Fig. 2d). HOMOZYGOSITY MAPPING AND MUTATION ANALYSIS DNA samples

of all available affected and healthy members of the families studied were subject to whole genome SNP genotyping and homozygosity mapping (Supplementary Table S2), except for family MA25

(see below). Our analysis revealed several large homozygous regions that were shared among the affected members within the same family (Supplementary Table S3). In particular, regions

containing more than 300 consecutive homozygous SNPs, on average corresponding to a genomic size of 1 Mb or larger, were prioritized. Family MA62 had a single homozygous peak, while others

displayed two (MA94 and MA123) or multiple peaks (MA69, MA97, MA117 and MA132) (Fig. 3). In families where more than one peak of homozygous regions were observed, regions encompassing known

IRD genes were selected for direct Sanger sequencing. All of the DNA variants identified by sequencing, described below, were identified in homozygous state and co-segregated perfectly with

affected individuals within the respective families, as expected. Details are reported in Table 2. In family MA62, a single peak on chromosome 3 that comprised the Rhodopsin gene

(_RHO/_NM_000539) was detected and sequencing revealed a known missense mutation c.448G > A (p.E150K)21. Similarly, in family MA94 there were two homozygous stretches on chromosomes 7 and

8, spanning the genomic regions where known IRD genes _IMPDH1_ and _CNGB3_, respectively, are located. Sequencing of the coding region of _CNGB3_ (NM_019098) showed the presence of the

previously-reported mutation c.646C > T (p.R216X)22. In MA132, a homozygous region on chromosome 5 contained the known RP gene _PDE6A_ (NM_000440) and sequencing revealed the presence of

the described mutation c.1408-2A > G (IVS10-2A > G)23. In MA123, one of the two detected homozygous peaks (on chromosome 16) comprised _KCNV2_, which is associated with cone-rod

dystrophy24. However, screening of the ORF and exon-intron boundaries failed to identify any putative pathogenic variants, indicating that the culprit for IRD in this family is either a

novel disease gene or a mutation in _KCNV2_ that was not detectable by the methodology used in our analysis (e.g. a large structural variation, a mutation in deep intronic sequences, etc.).

Homozygosity mapping of family MA25 was done using highly polymorphic microsatellite markers spanning known achromatopsia loci. Analyses revealed a homozygous region between microsatellites

D2S2333 and D2S1343, containing _CNGA3_ (NM_001298). Sanger sequencing of the gene’s ORF revealed the known missense mutation c.1306C > T (p.R436W) in exon 825,26. In MA69, multiple peaks

were observed, but a 18.7 Mb region on chromosome 2 contained _CNGA3_. Sanger sequencing identified a novel homozygous missense change, c.991G > C (p.G331R) (Fig. 4). Similarly, in

family MA117, a homozygous stretch on chromosome 14 between markers rs7148898 to rs12892350 harboring the known IRD gene _RPGRIP1_ (NM_020366), was identified. Sequencing of _RPGRIP1_

revealed a novel missense variation c.2656C > T (p.L886F) in exon 17 (Fig. 4). Neither c.991G > C in _CNGA3_ nor c.2656C > T in _RPGRIP1_ were present in any public databases,

including the EVS and the 1000 genomes project. Both changes affect fully conserved amino acids from human to fish (Fig. 4). Furthermore, all in silico tools for the prediction of missense

variants pathogenicity, namely Polyphen, SIFT and Mutation Taster, predicted the changes to be probably damaging, deleterious and disease-causing, respectively. For family MA97, our

attention was caught by a homozygous peak on chromosome 13 that contained _CNGB1_, a gene linked with RP. Sequencing revealed the splice site variation c.413-1G > A (IVS6-1G > A)

affecting the invariant acceptor site of intron 6, which was never previously linked to disease (Fig. 4). Interestingly, this nucleotide change was present in dbSNP (rs189234741) but was

observed only once in 5,000 chromosomes. Furthermore, it was detected in the framework of the 1,000 Genomes project, genotyping phase 1 (low coverage sequencing), indicating a possible

technical artifact. Indeed, in silico analyses clearly confirmed that this variant should have a strong impact on the normal splicing pattern of the gene, as expected for DNA changes

involving the -1 and -2 bases of intron acceptor sequences (scores: 0.394, 0.74 and “site broken” by Skippy, MutPred Splice and Human Splice Finder, respectively). Transfection of HeLa cells

with minigene constructs bearing this change and its wild-type counterpart revealed that IVS6-1G > A did affect the canonical splicing of exon 7 by knocking down its natural 5’ splice

site and eliciting the use of a cryptic splice site, just one base pair downstream of intron 6’s acceptor sequence (Fig. 5). This event led to the loss of the first nucleotide of exon 7,

producing a -1 frameshift and a premature stop codon 413 bases downstream of it (p.C139AfsX138/NP_001288.3). Further screening of probands from 50 additional Pakistani pedigrees with IRD for

all these mutations failed to reveal any additional positive subject (not shown). DISCUSSION Despite recent technological advances, molecular diagnosis of IRDs remains a challenging task,

because of their high genetic and phenotypic heterogeneity. This is particularly true for laboratories that have no access to next-generation sequencing platforms and large-scale screening

systems and therefore have to assess candidate IRD genes one by one. Luckily, geographic isolation, consanguinity, or endogamy may increase the prevalence of particular mutations in selected

populations, which can be pinpointed by homozygosity mapping. The rationale for this approach is that unaffected parents who have some degree of relatedness, are from a geographical

isolate, or belong to an ethnic group for which endogamy is frequent, could be heterozygotes for the same recessive mutation from a common ancestor. This mutation, which at the population

level may even be very rare, can be brought to homozygosity because of consanguinity and cause disease in these parents’ offspring. Since meiotic recombination affects chromosomes at a

megabase scale, the mutation is co-inherited with large surrounding haplotypic blocks that are homozygous in patients and easily recognizable via the analysis of SNP alleles. In this study

we take advantage of the power of homozygosity mapping to identify mutations in Pakistani families with IRDs, originating from different geographical regions of the country and displaying

consanguinity. In agreement with other studies27,28,29 our results indicate that indeed genomic regions harboring IRD genes can be efficiently highlighted by this technique and that very few

candidate sequences have to be screened to reach molecular diagnosis. Consanguinity, however, is a key factor. Other cohort studies in consanguineous populations (e.g. Saudi Arabia), have

shown that mutations in known retinal dystrophy genes were identified in >75% of the patient tested30,31. In outbred populations, where consanguinity is not common (e.g. the Netherlands),

this success rate was considerably lower (in the range of 10–15%)32. Nonetheless, it should be noted that autozygome-guided mutation analysis has some limitations, since only homoallelic

mutations are identified by this method. Thus, compound heterozygosity for recessive mutations, heterozygosity for dominant mutations, as well as hemizygosity for X-linked mutations are

generally missed. Concerning the variants identified, the change c.991G > C (p.G331R) in _CNGA3_ detected in family MA69 affects a highly conserved glycine residue and, as the majority of

previously-identified mutations, is a missense33. _CNGA3_ encodes a channel protein which consists of six transmembrane helices, a pore region, a C-linker and cyclic nucleotide-binding

domain34. It is highly conserved in different species and mutations in this gene have been linked to achromatopsia35 a disease that causes symptoms that are compatible with those described

in members of this family. The facts that (i) family MA69 displayed homozygosity for the _CNGA3_ region, (ii) the DNA change identified involved a conserved amino acid residue and was absent

in healthy controls and, despite we could not perform a detailed clinical examination, (iii) symptoms of patients corresponded to those of achromatopsia, which (iv) is an extremely rare

disease, all strongly suggest that p.G331R is a pathogenic mutation. The same arguments can be made for _RPGRIP1_ c.2656C > T (p.L886F), detected in family MA117. RPGRIP1 is a ciliary

protein composed of three different regions: an N-terminal coiled-coil domain, a central part containing two protein kinase C conserved region 2 (C2) motif and a C-terminal region having a

RPGR interacting domain36. The p.L886F missense change is located in the second C2 domain of RPGRIP1, which also harbors the majority of previously reported missense mutations36. Signs and

symptoms of affected members are also compatible with those of cone-rod dystrophy, which can indeed be caused by _RPGRP1_ mutations. The variant c.413-1G > A (IVS6-1A > G) of _CNGB1_

identified in family MA97 was predicted in silico and verified in vitro to alter the splicing pattern of this gene, inducing the use of a cryptic splice site and finally producing a

homozygous frameshift that leads to a premature termination codon. Since this acquired stop codon occurs before the last exon, mutant mRNA likely undergoes nonsense-mediated decay and

therefore produces no or very little protein37. Once again, the clinical picture of the affected members of family MA97 is compatible with RP and is similar to that of patients with

previously-identified _CNGB1_ mutations38,39. Two families (MA62 and MA132) with mutations in _RHO_ and _PDE6A_, respectively, also showed typical symptoms of RP. The c.448G > A/p.E150K

mutation in _RHO_ identified in MA62 was initially found in an Indian family21 but later Azam and colleagues also reported it in two separate Pakistani families40. Genotype analysis of these

three families showed a common, disease-associated haplotype. Our findings further support the notion that this mutation has probably common ancestral origins in this area. Similarly,

c.1408-2A > G (IVS10-2A > G) in _PDE6A_ was reported in Pakistani individuals with recessive RP23. The relatively high prevalence of these mutations indicates once more an ancestral

origin and provides a rather strong element for performing targeted molecular diagnosis of IRDs in this region. The _CNGA3_ mutation c.1306C > T (p.R436W) identified in family MA25 is

relatively frequent in achromatopsia cases33. This variation has mostly been recorded in a compound heterozygous state with other mutations25 and was initially thought to be a variant that

was limited to German patients33. Subsequent studies have shown that this mutation is present in other populations41, as we also report in this work and therefore represent a common cause of

achromatopsia worldwide. In conclusion, our study confirms the power of homozygosity mapping for identifying pathogenic variants in consanguineous families with IRDs. This approach is

precious to provide correct clinical diagnosis and genetic counseling in isolated areas of Pakistan, raising at the same time awareness about IRDs and the risks of intermarriage. ADDITIONAL

INFORMATION HOW TO CITE THIS ARTICLE: Saqib, M. A. N. _et al._ Homozygosity mapping reveals novel and known mutations in Pakistani families with inherited retinal dystrophies. _Sci. Rep._ 5,

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Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS This work was funded in part by the University Research Fund of Quaid-I-Azam University, Islamabad, Pakistan and the Swiss

National Science Foundation, Switzerland (Grant 310030_138346). Muhammad Arif Nadeem Saqib was supported by the Indigenous Fellowships and the International Research Support Program (IRSIP)

from the Higher Education Commission (HEC) of Pakistan. We would like to thank the iGE3 Genomics Platform at University of Geneva and in particular all members of the families that

participated in this study for their invaluable participation and cooperation. AUTHOR INFORMATION Author notes * Rivolta Carlo contributed equally to this work. AUTHORS AND AFFILIATIONS *

Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, 45320, Pakistan Muhammad Arif Nadeem Saqib, Ehsan Ullah, Falak Sher Khan, Jamila Iqbal, Rabia

Bibi, Afeefa Jarral, Sundus Sajid & Muhammad Ansar * Department of Medical Genetics, University of Lausanne, Lausanne, 1005, Switzerland Muhammad Arif Nadeem Saqib, Konstantinos

Nikopoulos, Giulia Venturini, Muhammad Ansar & Carlo Rivolta * Department of Ophthalmology, Tohoku University Graduate School of Medicine, Sendai, 980-8574, Miyagi, Japan Koji M.

Nishiguchi * Pakistan Medical Research Council, Islamabad, 44000, Pakistan Muhammad Arif Nadeem Saqib Authors * Muhammad Arif Nadeem Saqib View author publications You can also search for

this author inPubMed Google Scholar * Konstantinos Nikopoulos View author publications You can also search for this author inPubMed Google Scholar * Ehsan Ullah View author publications You

can also search for this author inPubMed Google Scholar * Falak Sher Khan View author publications You can also search for this author inPubMed Google Scholar * Jamila Iqbal View author

publications You can also search for this author inPubMed Google Scholar * Rabia Bibi View author publications You can also search for this author inPubMed Google Scholar * Afeefa Jarral

View author publications You can also search for this author inPubMed Google Scholar * Sundus Sajid View author publications You can also search for this author inPubMed Google Scholar *

Koji M. Nishiguchi View author publications You can also search for this author inPubMed Google Scholar * Giulia Venturini View author publications You can also search for this author

inPubMed Google Scholar * Muhammad Ansar View author publications You can also search for this author inPubMed Google Scholar * Carlo Rivolta View author publications You can also search for

this author inPubMed Google Scholar CONTRIBUTIONS M.A.N.S., K.N., K.M.N., M.A. and C.R. wrote the manuscript; M.A.N.S., K.N., G.V., M.A. and C.R. designed the study; M.A.N.S., K.N., E.U.,

F.S.K., J.I., R.B., A.J., S.S. and G.V. performed data acquisition. All authors analyzed the data and reviewed the manuscript. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no

competing financial interests. ELECTRONIC SUPPLEMENTARY MATERIAL SUPPORTING INFORMATION Supplementary Tables S1-S3 RIGHTS AND PERMISSIONS This work is licensed under a Creative Commons

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this license, visit http://creativecommons.org/licenses/by/4.0/ Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Saqib, M., Nikopoulos, K., Ullah, E. _et al._ Homozygosity

mapping reveals novel and known mutations in Pakistani families with inherited retinal dystrophies. _Sci Rep_ 5, 9965 (2015). https://doi.org/10.1038/srep09965 Download citation * Received:

14 November 2014 * Accepted: 25 March 2015 * Published: 06 May 2015 * DOI: https://doi.org/10.1038/srep09965 SHARE THIS ARTICLE Anyone you share the following link with will be able to read

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