Skip to main content
  • Loading metrics

Deciphering the genetic architecture and ethnographic distribution of IRD in three ethnic populations by whole genome sequence analysis


Patients with inherited retinal dystrophies (IRDs) were recruited from two understudied populations: Mexico and Pakistan as well as a third well-studied population of European Americans to define the genetic architecture of IRD by performing whole-genome sequencing (WGS). Whole-genome analysis was performed on 409 individuals from 108 unrelated pedigrees with IRDs. All patients underwent an ophthalmic evaluation to establish the retinal phenotype. Although the 108 pedigrees in this study had previously been examined for mutations in known IRD genes using a wide range of methodologies including targeted gene(s) or mutation(s) screening, linkage analysis and exome sequencing, the gene mutations responsible for IRD in these 108 pedigrees were not determined. WGS was performed on these pedigrees using Illumina X10 at a minimum of 30X depth. The sequence reads were mapped against hg19 followed by variant calling using GATK. The genome variants were annotated using SnpEff, PolyPhen2, and CADD score; the structural variants (SVs) were called using GenomeSTRiP and LUMPY. We identified potential causative sequence alterations in 61 pedigrees (57%), including 39 novel and 54 reported variants in IRD genes. For 57 of these pedigrees the observed genotype was consistent with the initial clinical diagnosis, the remaining 4 had the clinical diagnosis reclassified based on our findings. In seven pedigrees (12%) we observed atypical causal variants, i.e. unexpected genotype(s), including 4 pedigrees with causal variants in more than one IRD gene within all affected family members, one pedigree with intrafamilial genetic heterogeneity (different affected family members carrying causal variants in different IRD genes), one pedigree carrying a dominant causative variant present in pseudo-recessive form due to consanguinity and one pedigree with a de-novo variant in the affected family member. Combined atypical and large structural variants contributed to about 20% of cases. Among the novel mutations, 75% were detected in Mexican and 50% found in European American pedigrees and have not been reported in any other population while only 20% were detected in Pakistani pedigrees and were not previously reported. The remaining novel IRD causative variants were listed in gnomAD but were found to be very rare and population specific. Mutations in known IRD associated genes contributed to pathology in 63% Mexican, 60% Pakistani and 45% European American pedigrees analyzed. Overall, contribution of known IRD gene variants to disease pathology in these three populations was similar to that observed in other populations worldwide. This study revealed a spectrum of mutations contributing to IRD in three populations, identified a large proportion of novel potentially causative variants that are specific to the corresponding population or not reported in gnomAD and shed light on the genetic architecture of IRD in these diverse global populations.

Author summary

The study was performed to identify the underlying cause of inherited retinal degeneration (IRD) in 409 individuals from 108 families. Primarily, these families were recruited from three different geographic regions: Mexico, Pakistan and European Americans from the United States. Blood samples were collected from all individuals for genome analysis. This analysis detected causative variants in 61 out of the 108 pedigrees. A total of 93 gene variants were found in the 61 families. Among these, 54 were previously reported as causative variants and the remaining 39 have not been reported in IRD pedigrees. Interestingly, 54% of these novel variants were not listed in gnomAD. In addition to these findings, complex causative genotypes were observed in 20% of pedigrees. Overall, causative variants were detected in 63% Mexican, 60% Pakistani and 45% European American pedigrees. This study revealed the distribution of IRD causative variants in pedigrees with diverse ethnic and geographic backgrounds.


Inherited retinal degenerations (IRDs) are a group of diseases, which result in dysfunction or progressive degeneration of retinal cells causing a profound bilateral loss of vision. IRDs are relatively rare. It is currently estimated that IRDs affect 1 in 3000 individuals [1]. Significant heterogeneity has been reported in the phenotype of IRD patients with a wide variation in the age of onset, rate of progression, severity of the disease, and clinical symptoms. Variants in the same gene may also lead to marked diverse phenotypes as well as result in different patterns of inheritance. Currently, at least 271 genes are known to be associated with IRD [2].

Retinal disease genes have been identified previously by linkage analysis, homozygosity mapping, and sequencing the coding regions of several genes associated with genetic and genomic markers. The subsequent development of targeted screening panels for pathogenic variants in known IRD genes greatly improved genetic diagnosis but failed to identify novel variants and novel genes involved in IRD [35]. Gene arrays to selectively capture and sequence candidate genes are reported to result in the identification of mutations in 60%-70% of IRD patients [3, 5, 6]. Advances in whole-exome sequencing (WES) enabled the identification of causal variants associated with Mendelian diseases in known or novel genes efficiently [7, 8]. Nevertheless, about 30%-40% of cases remain unresolved. Further, while the majority of studies conducted so far focused on selected populations, the genomic architecture of IRD in certain populations remains unknown.

The affordable cost structure of whole-genome sequencing in recent years [913] has enabled the analysis of all genes including their untranslated regions and provided opportunities to identify causal variants in patients with IRDs with broad genetic and phenotypic heterogeneity. Utilizing these advances in the current study, we present the genetic analysis of IRD in 108 pedigrees. These pedigrees are mainly from three populations: the understudied populations from Pakistan (Punjab province) and Mexico as well as the well-studied European American population (individuals of European ancestry from North America). Analysis of these pedigrees revealed atypical sequence alterations and provided a glimpse of the genetic architecture of IRD in these distinctly diverse global populations.


Pedigrees analyzed

Whole-genome sequence data were obtained on 404 subjects from 108 unrelated pedigrees with a diagnosis of inherited retinal dystrophy. The study cohort included pedigrees from Mexico (35), Pakistan (15), Ashkenazi Jewish (2), India (2), and USA (European ancestry) (54).

The pattern of inheritance was observed to be recessive in 76 pedigrees, dominant in 25, and X-linked in 7. However, after completing the analysis, the pattern of inheritance was corrected in 4 pedigrees based on the causative mutations detected. One pedigree with multiple consanguineous marriages (RF.197.0113) was originally classified as recessive but determined to be dominant with a pseudo-recessive pattern of inheritance. Similarly, two pedigrees RF.VI123.0514 and RF.VI153.0216 were originally classified as dominant and recessive respectively but mutations in X-linked genes were identified as the underlying cause of the phenotype. One pedigree originally classified as dominant (RF.VI116.1215) was re-classified as recessive.

WGS sequence analysis

Analysis of sequence data identified 202 female and 202 male subjects consistent with our records and validated relationships based on identity by descent (IBD) mapping analysis. The total number of reads obtained on each individual ranged from 765 million to 1,903 million, of which 78% ~ 95% were detected as appropriately mapped reads indicating the high quality of sequence data. Analysis using GATK best practice pipeline identified 30,071,475 single nucleotide variants (SNVs) in total, including 23,409,845 single nucleotide polymorphisms (SNPs) and 6,661,630 INDELs. The number of variants in each sample ranged from 3.77 to 4.84 million SNVs. A total of 18,301,653 known and 11,769,822 novel (based on dbSNP147) SNVs were observed in 404 subjects. Among the total number of identified SNVs, 21,026,019 (70%) were identified as very rare SNVs (allele frequency < 0.001). The rare and moderate/possibly disease-causing SNVs included 186,501 (0.61%) while only 53,101 (0.18%) of them were predicted to be deleterious/probably damaging.

(i) Small variants (SNVs and small INDELs).

3.77 to 4.84 million SNVs including ~850,000 small INDELs were detected from autosomes in every individual and no outliers or plate biases were observed. Similarly, no outliers were observed in the X and Y chromosome data. The heterozygous and homozygous ratios were normal on autosomes as well as sex chromosomes in each female and male sample. Among the total SNVs observed, 112,335 (0.37%) were annotated as missense variants. These include 79,428 (71%) known and 32,907 (29%) novel variants.

(ii) Copy Number Variants (CNVs).

We observed a total of 56,299 CNVs including 25,357 deletions, 13,223 duplications, and 17,719 insertions in 404 samples. More than half of the CNVs, 29,142 (52%) were found to be common as they were found in more than 30 samples. The CNV calling software (GenomeStrip) detected CNVs with lengths greater than 1000bp. In our analysis, we identified CNVs ranging from 1000bp to 313,600bp. The CNVs were called with a quality score, and those <1 were classified as likely false positives.

(iii) ExAC Z score distribution in Retina genes.

ExAC database has constraint Z scores for 18,225 genes. In our analysis, we included 271 retinal disease-associated genes from the RetNet database [2] and 58 other possible candidate genes associated with IRD based on their expression in relevant cells and function. Among these were 311 genes listed in the ExAC database including 183 recessive, 75 dominant, 9 X-linked genes, and 44 undefined genes. Positive Z scores indicated increased variation intolerance and therefore these 311 genes had fewer variants than expected. Autosomal dominant/X-linked IRD related genes were highly conserved and sequence alterations in these genes have among the highest Z-scores. Therefore, we used Z-scores to prioritize the candidate variants for dominant and X-linked related genes but not for recessive genes.

Causative variants detected in IRD associated genes

Analysis of WGS variants identified 93 causative variants (88 SNVs and 5 CNVs) in 61 of the pedigrees (57%). 89 of these causative variants include 35 novel and 54 previously reported variants detected in 45 known IRD genes in 59 pedigrees (Tables 1, 2, 3, 4 and S1). In two additional pedigrees, 4 novel mutations were identified in 2 newly classified novel IRD associated genes, AGBL5 and IFT88 [10, 14].

(i) Novel potentially causative variants detected in known IRD genes.

In 22 pedigrees (9 Mexican, 4 Pakistani and 9 European American), 26 rare, potentially pathogenic novel (not previously reported as causative) variants in 17 different known IRD genes were identified as likely causative mutations. Seven of these pedigrees also have 8 previously reported mutations in known IRD genes. Among the variants detected, 9 were homozygous (in 8 pedigrees), 21 compound heterozygous (in 10 pedigrees), 2 dominant acting heterozygous (in 2 pedigrees), and 2 were X-linked variants (in 2 pedigrees) (Table 1). Five of these variants were nonsense, 15 missense, 9 frameshift, and 5 intronic splice altering variants. Sanger sequencing analysis of all available family members confirmed co-segregation of candidate variants with IRD (Fig 1).

Fig 1. Pedigrees with novel mutations.

The segregation analysis of 22 pedigrees showed 26 rare potentially pathogenic novel variants along with 8 previously reported mutations in 17 IRD associated genes. These include 9 homozygous, 21 compound heterozygous, two dominant acting heterozygous, and two X-linked variants. Pedigrees A-I are Mexican, J-M are Pakistani and N-V are European American. The asterisk indicates the availability of whole-genome sequencing data.

Table 1. Pedigrees with novel potentially causative variants in known IRD genes.

Pedigrees with variants of uncertain significance (VUS). Among the novel potentially causative variants observed in Table 1 and Fig 1, five variants found in RAX2 (p.Arg79Gln in RF.176.0113), PEX6 (p.Arg876Trp in PKRD038 and p.Gly862Val in RF.FO.1293), DRAM2 (p.Leu246Pro in RF.H.0506) and IFT140 (p.Arg137Gln in RF.AG.0991) are listed in CinVar as VUS.

Further analysis of the IRD pedigrees with these five VUS did not detect additional potentially causative variants in known or novel genes that are sufficient to cause disease. Future experimental evaluation of novel potentially pathogenic causative variants and VUS, and detection of these variants in additional unrelated IRD cases will provide evidence for an appropriate classification of their clinical relevance.

(ii) Previously reported mutations detected in known IRD genes.

Thirty-five previously reported mutations in 18 known IRD genes were identified in 25 pedigrees (Table 2 and Fig 2). These pedigrees include 8 Mexican, 3 Pakistani, 14 European American including two of Ashkenazi Jewish origin and one Indian. Seven frameshift, 17 missense, 7 premature stop codon mutations, and 4 splice site altering changes were observed. Ten homozygous (in 9 pedigrees), 6 dominantly acting heterozygous (in 6 pedigrees), 2 X-linked (in 2 pedigrees) and 17 compound heterozygous mutations (in 8 pedigrees) were found in these 25 pedigrees (Table 2).

Fig 2. Pedigrees with previously reported mutations.

The segregation analysis revealed 35 previously reported mutations that were identified in 25 pedigrees. There were seven frameshift, 17 missense, seven premature stop codon mutations, and four splice site altering changes. Of these ten are homozygous, six dominant heterozygous, two X-linked, and 17 compound heterozygous mutations found in these pedigrees. Pedigrees A-H are Mexican, I-K are Pakistani and L-V are European American, W-X are Ashkenazi Jewish and Y is Indian. The asterisk indicates the availability of whole-genome sequencing data.

Table 2. Pedigrees with previously reported mutations in IRD genes.

This analysis detected USH2A variants as the underlying cause of disease in eight different pedigrees. Among these, only one was a novel variant while the remaining 15 were reported previously (Tables 1 and 2). The targeted mutation screening performed prior to WGS on a subset of cases did not include all currently known IRD genes nor cover all variants in a given gene; our current WGS screening resulted in the identification of variants in known genes in this set of pedigrees.

(iii) Structural Variants detected in known IRD genes.

Five different pedigrees carried novel structural variants. These included one pedigree with dominant macular degeneration and the remaining four with recessive retinal degeneration. Of the recessive pedigrees, two had the novel structural variants in the homozygous state, one carried a previously reported nonsense mutation and one a previously reported frameshift mutation (Table 3 and Fig 3). Three of these pedigrees are European American while one each is Mexican and Pakistani.

Fig 3. Pedigrees with copy number variations (CNVs).

Five unique structural variants were identified in EYS, LCA5, CERKL, PRPH2, and CNGB3 in five different pedigrees, one with dominant macular degeneration and four with recessive retinal degeneration. (A1) 1.6Mb homozygous deletion Chr6: g.65,994,849_67,582,755del is segregating with recessive retinal degeneration in a Mexican pedigree RF.V196.0210. (A2) The schematic diagram depicting a 1.6Mb homozygous deletion Chr6: g.65,994,849_67,582,755del encompassing the exons 1 to 12 and about 1.6Mb of 5’-untranslated region of the EYS gene. (A3) PCR amplification of EYS exons 1, 6, and 11 detected the presence of expected size product in unaffected individuals (I:1, I:2, II:3) whereas the presence of PCR product was not observed in two affected individuals (II:1 and II:2). (A4) Amplification with primers flanking the deleted region followed by sequencing revealed the junction point in individual II:1 due to the 1.6Mb deletion. Examination of the sequence flanking the junction point detected paralogous repeat sequences on both sides of the deleted region (blue and yellow boxes). (B1) In a consanguineous Pakistani pedigree RF.277.0113, a 110Kb homozygous deletion in LCA5 (chr6: g.80,205,052_80,315,592del) segregated with the phenotype. (B2) The novel 110Kb homozygous deletion includes 1 to 4 exons of LCA5. (B3) PCR amplification showed the absence of exon 1 to 4 of LCA5 in both affected individuals while exon 5 is present in all family members. (B4) Amplification with primers flanking the deletion resulted in the generation of the fragment with deletion. Sequencing this PCR product revealed the presence of paralogous repeat sequences flanking the junction point in affected individuals. Sequence marked with yellow and blue rectangles represent the paralogous sequence on both sides of the deleted region. (C1) A previously reported heterozygous stop mutation CERKL p.Arg257* and a novel heterozygous large 22.8 Kb deletion (Chr2: g.182,456,422_182,479,267del) on chromosome 2, which includes exon 2 of CERKL are observed in trans configuration in the proband of RF.T.8.11. (C2) The schematic diagram shows the 22.8Kb deletion which includes ~10.3Kb of intron 1 and exon 2 (243bp) and 12.3Kb of intron 2 of CERKL gene. (C3) The WGS reads mapped to the deleted region showed decrease in read depth. (C4) Electropherogram showing the sequence of junction fragment generated by amplification with primers flanking the deletion revealed the specific boundaries of the deletion that includes exon 2 of CERKL. (D1) The segregation analysis revealed a heterozygous 33Kb deletion on chromosome 6 (Chr6: g.42,643,442_42,676,411del) segregating with the disease. (D2) A cartoon depicting the deleted region which includes two different genes: exons 39 and 40 of UBR2 and exons 2 and 3 of PRPH2 present in opposite orientation (D3) Analysis of 7 affected and 15 unaffected members using qPCR confirmed the presence of the heterozygous deletion on chromosome 6 in affected members and not in unaffected relatives. (E1) A set of compound heterozygous deletions including a novel 7Kb deletion (Chr8: g.87,616,103_87,623,431del) and a previously known 7bp deletion p.Arg274Valfs*13 in CNGB3 gene were observed in RF.M.0592 pedigree with a single affected individual. (E2) The novel 7Kb heterozygous deletion (Chr8: g.87,616,103_87,623,431del) (Pink rectangle) includes coding exon 15 of CNGB3. (E3) qPCR analysis confirmed the presence of the heterozygous deletion of CNGB3 exon 15 in II:1, which was inherited from the mother (I:2). The asterisk indicates the availability of whole-genome sequencing data.

Table 3. Pedigrees with copy number variations in IRD genes.

(A) A 1.6Mb deletion in EYS segregating with IRD. Analysis of the WGS of two affected (II:1 & II:2) and one unaffected sibling (II:3) from a Mexican pedigree RF.VI96.0210 (Fig 3A1) revealed a novel, 1.6 Mb homozygous deletion on chromosome 6 (Chr6: g.65,994,849_67,582,755del) in both affected members. This deletion was not observed in the unaffected sibling. The deleted region encompasses the exons 1 to 12 and 5’-untranslated region of the EYS gene implicated in recessive retinal degeneration (Fig 3A2). PCR amplification of exons 1 to 12 of EYS in this pedigree revealed the loss of exons in II:1 and II:2 (Fig 3A3). Amplification with primers flanking the deleted region followed by sequencing showed the overlapping of paralogous repeat sequences and deletion of in-between 1.6 Mb regions (Fig 3A4) in the affected members.

(B) LCA5 gene deletion in a pedigree. A 110Kb homozygous deletion in LCA5 (Chr6: g.80,205,052_80,315,592del) was identified in a consanguineous Pakistani pedigree RF.277.0113. This homozygous deletion includes 1 to 4 exons of LCA5. PCR amplification and sequencing of the deleted region using primers located in the flanking sequence identified specific boundaries of the deletion and its segregation with LCA in RF.277.0113 (Fig 3B).

(C) A 22.8Kb deletion in the CERKL gene. In pedigree RF.T.8.11, a previously reported nonsense mutation p.Arg257* in the CERKL gene was identified in the heterozygous state by exome sequence analysis in the proband I:1 who was adopted (53). Whole-genome sequence analysis of this individual identified a large novel heterozygous 22.8 Kb deletion (Chr2: g.182,456,422_182,479,267del) on chromosome 2 (Fig 3C). Analysis of the samples of his two offspring established the compound heterozygous nature of the nonsense variant and the large deletion in the affected individual. The 22.8 Kb chromosome 2 deletion includes the entire coding sequence of exon 2 (243 bp) and about ~10.3 Kb of intron 1 (Chr2: g.182,456,422_182,468,805del) and 12.3 Kb of intron 2 (Chr2: g.182,468,565_182,479,267del) of the CERKL gene. The nonsense change is predicted to truncate the protein or result in nonsense-mediated decay (NMD) of the transcript [53]. The deletion of 22.8 Kb sequence encompassing exon 2 of CERKL may also result in the formation of a truncated protein due to coding region frameshift or the transcript may undergo NMD. Both sequence alterations detected in the CERKL gene in this individual are predicted to lead to the loss of functional protein; null mutations in CERKL have been established as the underlying cause of IRD [55, 56].

(D) A large structural change involving two genes. Clinical evaluation of eight affected individuals in a four-generation pedigree (C790) led to the diagnosis of autosomal dominant macular degeneration (MD) with no non-ocular abnormalities co-segregating with the MD phenotype (Fig 3D1). Analysis of WGS of five affected members and seven unaffected members revealed a heterozygous 33 Kb deletion on chromosome 6 (Chr6: g.42,643,442_42,676,411del) in affected members and not in unaffected relatives. This deletion included two adjacent genes present in opposite orientation: exons 39 and 40 of UBR2 and exons 2 and 3 of PRPH2 (Fig 3D2). Segregation analysis of 7 affected and 15 unaffected members using qPCR confirmed the segregation of the chromosome 6 deletion with the phenotype (Fig 3D3). The PRPH2 gene alterations including loss of function mutations have been implicated in dominant MD [57] and other retinal dystrophies, while the UBR2 gene is not associated with IRD or any other pathological condition.

In addition to the large deletion, two affected (IV:3 & IV:6) and one unaffected (IV:5) offspring of an affected female (III:3) were observed to carry a rare heterozygous potentially pathogenic variant c.659T>G, p.Phe220Cys (Allele frequency in gnomAD = 0.00002) in the rhodopsin gene. While samples of the parents of these individuals were not available for genetic analysis, the novel rhodopsin variant c.659T>G, p.Phe220Cys was not detected in either maternal grandparents (II:1 & II:2) suggesting the possible paternal (III:4) inheritance of this variant in the three siblings (IV:3, IV:5 & IV:6). Further, this variant was not detected in the rest of the pedigree excluding the possible involvement of c.659T>G, p.Phe220Cys as the variant responsible for IRD pathology in the rest of the extended pedigree. The two affected individuals IV:3 and IV:6 have both the large deletion encompassing UBR2 and PRPH2 and the c.659T>G in the rhodopsin gene. The impact of having both sequence alterations in these individuals is unknown.

(E) Compound heterozygous deletions in CNGB3. Whole-genome sequence analysis of pedigree RF.M.0592 with a single affected individual identified a novel 7Kb heterozygous deletion (Chr8: g.87,616,103_87,623,431del) and an additional previously known 7bp heterozygous indel (c.819_826delCAGACTCC) in CNGB3 gene that results in p.Arg274Valfs*13 [54]. The frameshift mutation was inherited from the father while the large deletion was inherited from the mother (Fig 3E).

Moreover, our analysis identified deletions and sequence alterations in non-coding regions with either unknown impact or yet to be annotated. As state above, experimental and analysis approaches will need to be developed to validate this class of potential disease-causing mutations.

(iv) Atypical genotypes observed in IRD pedigrees.

Analysis of the WGS identified atypical causal variants in 7 pedigrees including four pedigrees from Mexico, one from Pakistan, one European American and one of India origin (Table 4 and Fig 4).

Fig 4. Segregation analysis of pedigrees with atypical genotypes.

A-D: Mexican pedigrees with atypical mutations. A. RF.VI13.0707 pedigree with a de-novo mutation in IMPDH1; B. RF.VI104.0514 pedigree with a homozygous nonsense mutation in C2orf71 was detected in generation II while a previously reported homozygous splice site mutation in the CLN3 gene was observed in proband in the IV generation demonstrating the involvement of two different genes in IRD pathology in different generations. C. RF.VI157.0216 pedigree with mutations in genes OPN1SW and TOPORS associated with dominant color blindness and retinitis pigmentosa; D. RF.VI111.0514 pedigree with novel heterozygous causative mutation in PRPF8 and a known mutation in PRPF31, each sufficient to cause dominant IRD, were observed in monozygotic affected twins; E. RF.197.0113 consanguineous pedigree from Pakistan with a previously known dominant acting mutation in PRPF3 segregated in a pseudo-recessive pattern; F. RF.M.1111 an European American pedigrees with causative variants in more than one known IRD genes were observed to segregate with disease. A homozygous mutation in PDE6G and a hemizygous mutation in OPN1LW were observed in Pedigree RF.M.1111. G. RF.K.0216 Indian pedigree with a heterozygous PRPH2 mutation that is sufficient to cause retinal dystrophy and an additional mutation in ROM1 that can lead to digenic RP along with the PRPH2 variant. The asterisk indicates the availability of whole-genome sequencing data.

  1. Analysis of a Mexican pedigree RF.VI13.0707 with one affected and four unaffected members available for the study detected a de-novo novel heterozygous potentially pathogenic variant c.940A>G, p.Lys314Glu in the IMPDH1 gene associated with autosomal dominant RP (Fig 4A). The clinical changes observed in the affected individual are consistent with IMPDH1 associated retinitis pigmentosa with marked macular atrophy (S1A Fig). Analysis of the WGS data of the proband, parents and the unaffected sibling using identical by descent (IBD) segment analysis established the genetic relatedness and verified the provided family structure. Examination of the haplotypes of parents and the proband that were constructed using variants in the region encompassing the IMPDH1 gene confirmed the shared haplotype between parent and offspring (Fig 5). However, the absence of the novel c.940A>G, p.Lys314Glu in the IMPDH1 variant in either parent was noted establishing the c.940A>G change as a de-novo variant (Fig 5) and suggesting it as the possible underlying cause of IRD in this patient. The maternal great grandfather (I:2) and grand uncle (II:1) of the patient were reported with vision loss but it is not known if they had a clinical phenotype of RP nor was genotyping of these individuals possible.
  2. The WGS variants data set of three affected members (II:3, II:4, and IV:1) and three unaffected members (III:1 and III:2 and IV:2) of a four-generation Mexican pedigree RF.VI104.0514 was analyzed. WGS analysis identified two potentially pathogenic variants in two separate genes in affected individuals from different generations. Two affected siblings II:3 and II:4 were observed to carry a homozygous nonsense variant c.2950C>T; p.Arg984* in C2orf71, which has previously been reported as a mutation causing recessive RP [58, 59]. However, this mutation was detected only in the heterozygous state in the affected male IV:1 as well as in his unaffected father. Further analysis of sequence variants revealed an additional previously reported homozygous splice site mutation c.125+5G>A (c.140+5G>A) in the CLN3 gene in IV:1, but not in other affected members II:3 and II:4 [60]. This variant was also observed in the heterozygous state in his unaffected parents III:1 and III:2. Both C2orf71 and CLN3 gene variants segregated with disease in separate branches of the RF.VI104.0514 pedigree (Fig 4B). Patient IV:1 was examined at the age of 10 years with a report of lipofuscinosis, which is consistent with the CLN3 mutation detected [66]. The age of onset in all three affected members IV:1, II:3, and II:4 is reported to be during early childhood (4-5yrs). But the fundus images of II:3 and II:4 at a younger age are not available. Individuals II:3 and II:4 who are currently in their 80s are likely affected with recessive RP due to the C2orf71 mutation (S1F to S1H Fig) while IV:1 has subtle macular changes due to the CLN3 variant. Best-corrected visual acuities were only 20/200 and 20/100 at the age of 10 years and the patient was noted to have major mood disturbance as well as a very serious change in personality leading to a referral to a neurologist.
  3. In the RF.VI157.0216 Mexican pedigree with a single affected member, the WGS analysis revealed the presence of two pathogenic novel heterozygous pathogenic variants in two different genes OPN1SW (c.74A>G, p.Gln25Arg; Chr 7) and TOPORS (c.2554_2557delGAGA, p.Glu852Glnfs*13; Chr 9) associated with dominant color blindness (Tritenopia) and dominant retinitis pigmentosa respectively [6769]. The age of onset of IRD in the proband (II:2) was between 6–7 years. This patient is diagnosed with Marfan syndrome and multiple sclerosis and reported color deficiency since the age of 17 years. While the color deficiency is consistent with the involvement of OPN1SW mutations, the retinal degeneration phenotype in this individual is consistent with the phenotype associated with TOPORS (Fig 4C).
  4. RF.VI111.0514 Mexican pedigree includes affected fraternal twins and an affected grandparent. WGS analysis of the affected twin sisters (III:4 & III:5) and their unaffected sibling (III:2) identified novel heterozygous damaging variant in PRPF8 (c.6949T>A; p.Phe2317Ile, Chr 17) and a known heterozygous PRPF31 (c.866_879delGGAAAGCGGCCCGG; p.Arg289Profs*30, Chr 19) mutation in affected twins and not in their unaffected sibling [32]. Sanger sequencing further confirmed these findings (Fig 4D). Either of these mutations are sufficient to cause the RP phenotype observed in affected members (S1I Fig). The presence of macular cysts and the retinal phenotype observed in affected twins is more consistent with the phenotype associated with PRPF31 than with PRPF8. The paternal grandfather (I:1) is reported to be affected with IRD, while the clinical status of the father (II:3) is unknown.
  5. A large Pakistani pedigree, RF.197.0113 with 5 consanguineous marriages, and eight affected members available for the study was analyzed. Considering recessive inheritance, the WGS data of individuals IV:6, V:1, V:5, V:8, V:9 and VI:1 for homozygous potentially damaging variants shared between affected members and not present in unaffected member did not reveal candidate causative variants segregating with the disease. Subsequently, considering the dominant inheritance, the WGS variants of six individuals were filtered for potentially damaging heterozygous variants shared by all affected members and absent in the homozygous or heterozygous state in unaffected members. The latter analysis also did not identify candidate variants segregating with the disease. Further filtering for all potentially damaging variants present in either a heterozygous or homozygous state detected a previously reported adRP-associated variant, c.1481C>T, p.Thr494Met in the PRPF3 gene in five affected members in the heterozygous state, in three in the homozygous state and none of the unaffected relatives. Analysis of all members of this pedigree for this variant revealed the segregation of the c.1481C>T PRPF3 with the disease in the pedigree RF.197.0113 in a pseudo-recessive pattern due to the consanguinity (Fig 4E).
  6. A consanguineous European American pedigree RF.M.1111 (Fig 4F) with an affected male with a diagnosis of typical retinitis pigmentosa and an unaffected brother were analyzed by WGS of the two siblings (IV:1 and IV:2) and parents (III:1 and III:2). Analysis of variants in these individuals detected a homozygous novel c.69dupC, p.Arg24Glnfs*6 variant in PDE6G segregating with retinitis pigmentosa phenotype. In addition, IV:1 carried the hemizygous variant in OPN1LW (c.814G>A, p.Val272Met) gene on the X chromosome inherited from the mother (III:2). The clinical symptoms reported in the affected individual are more consistent with severe RP phenotype associated with PDE6G mutations; color vision was not tested in this individual.
  7. In the RF.K.0216 pedigree (Fig 4G) from India, two previously known dominant mutations, one in PRPH2 (c.424C>T, p.Arg142Trp) [61, 64, 70] and another in ROM1 (c.339dupG, p.Leu114Alafs*9) [65, 71] were observed in a patient with a diagnosis of central areolar choroidal dystrophy (CACD) with onset in the 5th decade and mild central vision loss consistent with the phenotype associated with the p.Arg142Trp mutation in PRPH2 [72]. The impact on this patient of the additional ROM1 mutation p.Leu114Alafs*9 mutation is unknown.
Fig 5. Haplotype of parents and the proband constructed with variants flanking the de-novo variant detected in the IMPDH1 gene in RF.VI13.0707 pedigree (Fig 4A).

In summary, a novel de-novo causative variant c.940A>G, p.Lys314Glu in the IMPDH1 gene associated with autosomal dominant RP was observed in one pedigree (RF.VI13.0707); and a previously known dominant mutation in PRPF3 (c.1481C>T) was detected in RF.197.0113 in a pseudo-recessive pattern due to multiple consanguineous marriages. In addition, potentially pathogenic variants in two independent genes both segregating with the disease and each sufficient to cause pathology were detected in four out of the 7 pedigrees with atypical genotypes. Besides these 7 pedigrees, we previously reported the identification of mutations in two independent genes as the underlying cause of IRD in separate branches of a pedigree by WGS in a European American pedigree [11].

(v) Variants described in recently demonstrated novel IRD genes by our group.

WGS sequence analysis detected potentially pathogenic candidate causative variants in genes previously not associated with IRD segregating with disease in two pedigrees in this cohort and we have reported these findings earlier [10, 14]. Our genetic and functional evaluation of these genes established the involvement of AGBL5 and IFT88 in causing IRD in the two unrelated pedigrees [10, 14].

(vi) Classification of Clinical Phenotypes based on WGS analysis findings.

The initial clinical diagnosis of pedigrees spanned a broad spectrum including RP in 18, cone dystrophy in 1, macular dystrophy in 1, Leber congenital amaurosis (LCA) in 2, Usher syndrome in 2, with the majority (37) having unclassified retinal degeneration (S1 Table). Re-evaluation of clinical data in the context of our genetic analysis findings lead to the reclassification of clinical phenotypes in our cohort: RP in 45 pedigrees, cone dystrophy in 7, LCA in 3, congenital stationary night blindness in 1, and macular dystrophy, nephronophthisis, Ceroid lipofuscinosis, choroideremia and Usher syndrome in one family each (S1 Table).

(vii) IRD Causative mutations detected in three populations studied.

(A) Analysis of pedigrees from Mexico. In the current study, WGS analysis of 35 pedigrees with recessive retinal dystrophy excluding STGD1 detected 18 previously reported and 13 novel (42%) causative mutations in known IRD genes in 22 pedigrees (63%) leaving the remaining 13 pedigrees unresolved (Fig 6). Nine (75%) of the 12 novel mutations involving SNVs observed in cases from Mexico were not listed in the gnomAD database while the remaining are reported only in the Latino population as very rare variants (Tables 1, 2, 3, 4 and 5). Mutations in USH2A are the most frequent cause of recessive retinal degeneration in this population with four pedigrees from the current study (RF.VI148.1215, RF.VI145.1215, RF.VI129.0714, and RF.VI127.0514) and two additional pedigrees from our previous studies with causative mutations in USH2A [4]. Female carriers of RPGR mutations in two pedigrees (RF.VI123.0514 and RF.VI153.0216) developed retinal degeneration phenotype as reported earlier [73].

(B) Analysis of pedigrees from Pakistan. In this study, we have analyzed 15 consanguineous Pakistani pedigrees with multiple affected members and identified causative mutations in 9 IRD genes in 9 pedigrees (60%) while the causative mutations were not detected in 6 pedigrees (40%) (Fig 6). Among the causative mutations detected in IRD associated genes, 6 are novel (60%) and 4 are previously reported (Fig 6 and Table 5). Four of the 5 novel mutations involving SNVs detected were reported in gnomAD database as extremely rare variants in South Asians, one in Europeans and the remaining one (~20%) was not listed (Tables 1, 2, 3, 4 and 5).

Fig 6. Summary of findings and population distribution of novel mutations detected in Mexican, Pakistani and European American pedigrees.

Table 5. Summary of findings and population distribution of mutations detected in three major cohorts analyzed.

(C) Analysis of pedigrees from the United States. Fifty-four pedigrees, which comprise 50% of the total analyzed in this study are of European ancestry. Causative mutations were detected in 24 (45%) pedigrees that included 25 known mutations (~59%), 14 novel single nucleotide changes (SNVs) and 3 novel structural changes (Tables 1, 2, 3, 4 and 5). Seven (~50%) of the 14 novel SNVs in known IRD genes, were not listed in the gnomAD database (Table 5). Two variants, c.722-1G>T in GUCY2D and c.1217G>A variant in CNGB1, detected in our European American cohort were reported in the African population at low frequency (gnomAD database), while the remaining appear to be unique to the European population (Fig 6 and Table 5).

(D) Pedigrees of Indian ancestry. Two pedigrees recruited in the United States are of Indian origin in which three previously reported causative mutations were detected (Tables 2 and 4).

(E) Pedigrees of Ashkenazi Jewish ancestry. In this study a previously known homozygous p.Lys42Glu mutation in DHDDS was detected in two Ashkenazi Jewish pedigrees recruited in the United States (Table 2).

Overall, the underlying cause of IRD was identified in about 57% of pedigrees. However, the rate of causative mutation identification in Mexican (63%), Pakistani (60%) and European American (45%) pedigrees varied. The number of novel IRD causative mutations detected in each of these cohorts also varied from about 42% and 41% in both Mexican and European American pedigrees to 60% in Pakistani pedigrees. Further, among the novel IRD causative SNVs, 20% of those detected in Pakistani pedigree were not listed in gnomAD database while 50% and 75% of novel SNVs in European American and Mexican pedigrees were not in the gnomAD database (Table 5).


Analysis of the whole-genome sequence of this cohort comprised of 404 individuals from 108 pedigrees with inherited retinal degeneration identified 93 causal variants in 232 individuals in 61 (57%) pedigrees. Among the causative variants detected, 39 (42%) are novel and 54 (58%) are previously reported variants in 44 well established IRD associated genes and two IRD genes we recently reported [10, 14]. Although a majority of pedigrees underwent prior screening for mutations in known genes without success, WGS analysis identified causative variants in IRD genes. This is primarily due to the limitations in the mutation screening panels used over the past two decades that did not include many currently known IRD associated genes. Further, the early version of exome capture probes that did not cover complete coding sequences. Variants in novel genes or variants in non-coding regions of known IRD genes with unknown impact or yet to be annotated may contribute to the phenotype in the 47 pedigrees that remained unresolved in this study.

The outcomes of the analysis of 108 IRD pedigrees provided insight into the genetic architecture of IRD. Overall novel mutations were identified in genes known to be associated with IRD in 36 pedigrees while previously reported mutations were detected in 25 pedigrees. The majority of the mutations (60%) were missense mutations including stop gain variants, 23% frameshift, while only 5% were structural variants and 12% were potential splice altering variants. All the causative CNVs detected in this study were novel. Analysis of the sequence flanking these deletions revealed microhomologies suggesting potential non-homologous end-joining leading to these deletions (Fig 3). Atypical genotypes were detected in a set of pedigrees (12%). These included causative mutations in more than one gene that segregated with IRD. While causative mutation(s) in one gene is potentially sufficient to explain pathology, the impact of having an additional causative mutation in a second IRD gene is unknown due to the significant overlap in the phenotype of IRDs. Further, intrafamilial genetic heterogeneity was observed in one pedigree. Such cases reveal the need for a comprehensive analysis of all known IRD genes for molecular diagnosis, counseling, and particularly for treatment decisions. In several cases, heterozygous pathogenic variants were also detected in IRD genes in several cases in addition to the primary causative mutations. A deeper phenotype-genotype analysis on a larger cohort, in the context of additional pathogenic variants, may provide further insight into variation in the IRD phenotype and molecular pathology of IRD. The occurrence of de-novo mutations is rare in retinal disease genes [7477] and a heterozygous de-novo mutation in IMPDH1 was detected in one affected individual in our cohort. This is the first report of a de-novo variant in the IMPDH1 gene.

It is interesting to note that only a small proportion of novel causative genes were identified despite a significant proportion of our pedigrees originating from understudied populations. Further, the two novel genes observed to carry causative mutations in our cohort were detected in small pedigrees of European Americans [10, 14]. The low number of novel IRD causative genes detected is consistent with the low number of novel IRD genes reported in the literature in the past few years [2]. An exponential increase in novel IRD gene discovery occurred in two majors spurts between 2000–2005 and 2010–2015 [2]. The spurts coincided with the development of advanced genome analysis tools and consequent enhancement in our knowledge of the architecture of the genome. Continuing with this trend, recent studies revealed the contribution of atypical genomic changes in IRD genes to pathology [7880]. Our findings are consistent with the observation that the discovery of novel IRD genes is approaching a plateau phase and atypical genomic alterations in known IRD genes may contribute to about 10%-15% of cases [12, 79]. The number of unrelated pedigrees with mutations in recently identified novel IRD genes, both in our studies and in the literature is small suggesting these mutations could be more recent or private and are not major contributors to IRD. The underlying cause of pathology in 47 (43%) pedigrees that remained unresolved in our cohort after WGS may also involve atypical genotypes including alterations in non-coding sequences or in regions of the genome that are not well understood [8082]. Therefore, gaining a deeper understanding of the genome, particularly the impact of non-coding variants, may improve our understanding of the molecular architecture of IRD and help resolve the remaining cases. Further advances in genome analysis methodologies may also facilitate the detection of the molecular cause of IRD in these unresolved pedigrees.

The families analyzed in this study included families that are primarily from understudied populations from Pakistan and Mexico and a third, well-studied European American population. About a third of the pedigrees included in this study are from Mexico with a unique population in which the genetics of IRD are not well understood. Comprehensive genetic analysis of IRD in this population has been reported primarily in two publications including one of our own [4, 32, 8385]. Our previous analysis of 6 Mexican pedigrees from this region using whole-exome sequencing detected 3 novel and 6 known causative variants in IRD associated genes [4]. Zenteno et al described targeted genetic analysis of a cohort of probands with IRD and detection of mutations in 66% of cases with 48% of these mutations being novel [32]. The current analysis of 35 pedigrees using the WGS detected causative mutations in 63% of pedigrees from Mexico and 42% of these are novel. These findings are similar to the observations reported in the prior two publications and reflect the understudied nature of this population [4, 32]. Further, 75% of these novel potentially pathogenic SNVs detected in our study are not listed in the gnomAD database. Since the Mexican population is an admixture of indigenous peoples and individuals of European ancestry [86, 87]; the detection of a large proportion of novel variants not listed gnomAD may be due to their possible origin from the indigenous population in Mexico that are not well represented in gnomAD data set.

The second population included in our analysis is from the Punjab province of Pakistan. Until recently, the genetics of IRD in this population was not well studied. The structure of the Pakistani population is unique with endogamous sub-populations of multi-ethnic origin and high consanguinity in each of these populations [8890]. Our earlier studies on 208 multigenerational pedigrees from the same region with a diagnosis of recessive IRD [7, 91104] found homozygous causative mutations in 149 pedigrees (~71%). So far, mutations in novel genes were observed in only five (2.5%) unrelated Pakistani pedigrees in our cohort ASRGL1 [99], IFT43 [104], ZNF513 [105], SLC24A1 [106], and CLCC1 [93]) while the remaining resolved pedigrees (97.5%) had mutations in known IRD genes. Among the mutations detected in known genes, p.Pro363Thr in RPE65 is the most common causative mutation found in this population [7, 91, 107]; this variant was observed only in the South Asian population (gnomAD database). An independent study on a cohort of Pakistani families also reported 70% novel and 30% previously identified variants in IRD associated genes [108122]. Consistent with these findings, causative mutations were detected in 60% of pedigrees in the current study cohort with 60% of the mutations being novel. However, the majority of these novel IRD associated SNVs were listed in the South Asian population in the gnomAD database (7 out of 8) unlike the novel SNVs in the Mexican population.

Interestingly, the mutation detection rate was lower (45%) in European American pedigrees compared to the rate in Mexican and Pakistani pedigrees (63% and 60%, respectively). Despite the well-studied nature of this population, 41% of the mutations detected in this study cohort are novel. Furthermore, 50% of these novel causative SNVs are not listed in gnomAD database.

Overall, USH2A is most frequently associated with IRD followed by EYS, CERKL, CRX, IMPG1 and RPGR in the current study cohorts (Fig 7). Studies describing the genetic analysis of IRD in geographically distinct populations using a range of methods have been reported [12, 32, 123132]. These studies found USH2A as the gene frequently associated with recessive RP worldwide including the European, Mexican and Pakistani populations [12]. In addition, the involvement of selected genes including EYS, RPE65, CEP290 in IRD is reported at higher frequency in certain populations [133]. Further, the involvement of ZNF513 and INPP5E in IRD is reported only in Pakistani and European populations respectively [134]. Population specific founder mutations have also been reported [135]. Our previous studies on Pakistani population identified p.Pro363Thr variant in RPE65 that is specific to the South Asian population as the common causative mutation [7, 92]. The distribution of potentially causative variants detected in the study cohort is consistent with findings on other populations. Although the Pakistani population and some of the sub-populations in Mexico are endogamous in nature, the occurrence of causative variants at higher frequency is not observed in these populations compared to other populations.

The majority of novel mutations identified in our cohort are either not listed in the gnomAD database or observed at very low frequency in Latino (for the Mexican), South Asian (for the Pakistani), or European (for the European American) populations (Tables 1, 3, 4 and 5). It is unknown if the novel variants detected in cases from the Mexican population are more recent variants in the Latino population or have originated from the indigenous population which might not be well represented in gnomAD data. Similarly, all the novel causative variants found in the Pakistani cohort are either absent or occur at very low frequency in the South Asian population suggesting those to be unique to this population. Further, these were observed only in one or a few Pakistani pedigrees despite the endogamous nature of this population. Surprisingly, a similar trend was observed with the novel mutations detected in the well-studied American population. Eight out of 21 novel mutations detected in European American pedigrees including AGBL5 and IFT88 variants were not listed in gnomAD while the remaining are specific to European population. These findings suggest that the novel mutations detected in our cohort are possibly specific to their corresponding populations or private mutations, particularly the ones observed in European Americans. A majority of pedigrees analyzed in the current study were prescreened for mutations utilizing targeted mutation screening methodologies designed based on data predominantly from European Americans [136138]. This bias has possibly contributed to the detection of high proportion of novel causative variants, particularly in the set of European American pedigrees. Overall, the findings on geographically diverse and understudied Mexican and Pakistani populations and the well-studied Caucasian population including our own data revealed that the pattern of distribution of IRD causative mutations in this cohort was similar to the findings reported in other worldwide populations. As the number of pedigrees studied from each ethnic group is small, analysis of additional IRD cases from the understudied Pakistani and Mexican populations may provide better insight into the genetic architecture of these populations. Further, appropriate classification of the clinical relevance of novel potentially causative variants using population specific information and the impact of the corresponding gene will facilitate improved genetic diagnosis to patients from worldwide populations [139].

This study using WGS and in-depth integrated analysis of the nature and type of mutations in different populations, provided insight into the population-specific genetic architecture of IRD and enabled it’s comparison to other worldwide populations. Such information will be helpful in the design of efficient population-specific tools for molecular diagnosis, genetic counseling, and decision on the selection of therapies. Further analysis of the 47 pedigrees that remained unresolved in this study may lead to the identification of causative variants in novel genes or non-coding variants that can contribute to the phenotype by modifying enhancer-promoter interactions or other yet to be identified functions of non-coding sequences.

Fig 7. Distribution of IRD genes detected in 61 pedigrees.


Ethics statement

The study protocol adhered to the tenets of the declaration of Helsinki and was approved by the Institutional Review Boards of the University of California San Diego, USA; University of California San Francisco, USA; University of Michigan, Kellogg Eye Center, USA; Johns Hopkins University School of Medicine, USA; University of Arizona, USA; Retina and Genomics Institute, Yucatán, México; Genetics and Ophthalmology, Genelabor, Goiânia, Brazil and University of Punjab, Lahore, Pakistan. Preliminary information on the clinical history of the patients and their family members were collected for the study along with the family history. Blood samples were collected from all available family members after obtaining their written consent to participate in our study.

Pedigree selection.

Pedigrees with at least one individual with a diagnosis of non-syndromic inherited retinal degeneration (IRD) were recruited. Patients with a primary diagnosis of Stargardt (STGD1) were excluded from this study. Self-reported ethnicity information was recorded.

Patient samples.

A total of four hundred and nine individuals from 108 unrelated families were analyzed by performing whole-genome sequencing. Among these, 203 individuals were affected and 206 were unaffected with 206 females and 203 males. 15 families were recruited from Pakistan, 35 from Mexico, 2 from India, 2 were Ashkenazi Jewish, and the remaining 54 families were of European ancestry from the USA.

The 108 pedigrees in this study had previously been examined for mutations in known IRD genes using a wide range of methodologies available. A set of 31 pedigrees with 1 to 5 affected members were previously analyzed by sequencing whole-exomes of selected members using Nimblegen V1-V3 (Roche Nimblegen, Inc., Wisconsin) or Agilent V1-V5 + UTRs probes (Agilent Technologies, Santa Clara, CA) to identify disease-causing gene mutations but remained unresolved. Similarly, probands of the remaining 77 pedigrees were initially analyzed using various targeted mutation or gene screening panels that were available in the past two decades [3] including ABCA4 and recessive RP mutation panels (Asper biotechnology, Estonia), selected retinal disease gene resequencing arrays [140], targeted gene sequencing by Sanger sequencing and targeted exome capture [4] but failed to identify causative mutations.

Whole-genome sequence (WGS) Analysis.

DNA isolation was performed using standard techniques from whole blood samples of patients using the Qiagen DNeasy blood kit (Qiagen, Germantown, MD) as previously described [7]. WGS was performed on at least one affected individual, and one or more unaffected close relative from each pedigree. The Illumina HiSeqX10 (Illumina, San Diego, CA) platform was used for sequencing whole-genomes at a minimum of 30X depth. The reads were mapped against human genome 19 (hg19) with decoy sequences using BWA-MEM [141, 142]. Biobambam2 was used to mark the duplicate reads and the remaining reads were sorted by genomic coordinate [143]. Variant calling was performed using HaplotypeCaller in Genome Analysis Toolkit (GATK) following the best-practice pipeline guidance [143, 144]. The genotyping quality of single nucleotide variants (SNVs) and insertions-deletions (INDELs) was assessed using the variant quality score recalibration approach implemented in GATK. Autosomal variants from pseudo-autosomal regions of the male X chromosome (chrX, 60001–2699520 and chrX, 154931044–155260560) were treated as diploid, whereas the rest of the male X chromosome, as well as the Y chromosome, were treated as haploid. A series of quality control processes were performed to determine the sample identity and sequencing quality, which includes sex identification based on the heterozygosity rate on the X chromosome, genetic relatedness among individuals was determined using identical by descent (IBD) segment analysis and this information was verified with the provided family structure, and sample heterozygosity rate was examined to detect any potential sample contamination. The sequencing data from five individuals from five different pedigrees were eliminated because they did not pass quality control metrics.

The called variants were annotated with SnpEff v4.11 [145], PolyPhen v2.2.2 [146], and CADD v1.3 [147]. Genome STRiP (svtoolkit 2.00.1611) [148] and Lumpy [149], which are part of the SpeedSeq software [150], were used to identify copy number variations (CNVs) in patients.

ExAC Z score distribution in Retina genes

ExAC Browser (Beta) Exome Aggregation Consortium has a Z-score for each gene to evaluate its intolerance and conservation against three types of mutation; synonymous, missense and loss of function (LoF). The scores were originally developed to find disease-relevant de-novo mutations. In this study, we examined if the scores could be used to prioritize disease causative genes.

Filtering criteria.

To identify rare deleterious SNVs, insertion-deletions (INDELs) and other types of structural variants as possible candidate variants, the following filtering criteria were used: allele frequency < 0.005 in 1000Genome project, < 0.05 in our inhouse cohort and < 0.05 in our 409 samples. Further, the allele frequency was validated using the ExAC and gnomAD databases. Highly deleterious variants were assessed and scored as: SnpEff putative impact = “HIGH” or PolyPhen2 Prediction = “possibly/probably damaging” or CADD Phred Score > = 30. Relatively deleterious variants were scored as: SnpEff putative impact = “HIGH/MODERATE” or Polyphen2 Prediction = “possibly/probably damaging” or CADD Phred Score > = 20.

Following initial filtering, selected variants were further analyzed based on segregation, pattern of inheritance, status reported in Human Genome Mutation Database (HGMD professional version 2020.4;, ClinVar classification on clinical relevance using the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) guidelines, relevant population specific information and the known/reported physiological function of the corresponding gene [139].

Segregation analysis of SNVs.

Segregation analysis of potentially disease-causing variant(s) identified in the IRD families by WGS was performed by dideoxy sequencing as previously described [151].

Segregation analysis of CNVs.

Copy numbers variation of the exons of candidate genes and two reference genes ZNF80 and GPR15 were quantified using a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) as described previously [11, 123].

Control sample analysis.

A set of 95 unrelated ethnically matched Pakistani control samples were analyzed using dideoxy sequencing to validate novel variants identified in pedigrees from Pakistan, as described previously [8, 151]. A set of 768 individuals (including 422 whole-genome sequenced individuals from IRD pedigrees and 346 ethnicity matched controls) in our laboratory data set and 1000Genome data base and gnomAD database were used for the analysis of remaining variants.

qPCR analysis of structural changes.

Segregation analysis of identified large insertion and deletions were validated by quantitative polymerase chain reaction (qPCR) analysis as described before [11].

Supporting information

S1 Fig. Clinical findings of patients with atypical genotypes.

(A) Fundus image from right eye of IV:2 from RF.VI13.0707 pedigree with the heterozygous IMPDH1 c.940A>G, p.Lys314Glu variant. The image demonstrates a waxy pallor of the optic disc, retinal vascular attenuation and mottling and light pigmentation of the retina in keeping with retinitis pigmentosa (RP). In addition, to normal RP features there is marked macular atrophy, with lacquer cracks are observed in the macular region of both eyes with clusters of pigment surrounding the area of atrophy. (B to D) Fundus images of IV:1 and II:3 from pedigree RF.VI104.0514: (F) Fundus images of IV:1 with the CLN3 mutation show macula discoloration and early Bull’s eye pattern and subtle mottling of the retina at age 10. (G, H) Fundus images of II:3 show marked chorioretinal atrophy with large clusters of pigment suggestive of end-stage disease due to C2orf71. (E) Composite left eye color fundus image of RF.VI111.0514 case, III:5 with mutations in PRPF8 and PRPF31 genes. Image demonstrates way disc pallor, pigmentation of the fundus and retinal vessel attenuation suggestive of retinitis pigmentosa.


S1 Table. Clinical diagnosis of all pedigrees.



  1. 1. Bessant DA, Ali RR, Bhattacharya SS. Molecular genetics and prospects for therapy of the inherited retinal dystrophies. Curr Opin Genet Dev. 2001;11(3):307–16. pmid:11377968
  2. 2. RetNet. [Available from:
  3. 3. Alapati AN, Goetz K, Suk J, Navani M, Al-Tarouti A, Jayasundera KT, et al. Molecular Diagnostic Testing by eyeGENE(R). Analysis of patients with hereditary retinal dystrophy phenotype involving central vision loss. Invest Ophthalmol Vis Sci. 2014.
  4. 4. Villanueva A, Biswas P, Kishaba K, Suk J, Tadimeti K, Raghavendra PB, et al. Identification of the genetic determinants responsible for retinal degeneration in families of Mexican descent. Ophthalmic Genet. 2018;39(1):73–9. pmid:28945494
  5. 5. Song J, Smaoui N, Ayyagari R, Stiles D, Benhamed S, MacDonald IM, et al. High-throughput retina-array for screening 93 genes involved in inherited retinal dystrophy. Invest Ophthalmol Vis Sci. 2011;52(12):9053–60. pmid:22025579
  6. 6. Blain D, Goetz KE, Ayyagari R, Tumminia SJ. eyeGENE(R): a vision community resource facilitating patient care and paving the path for research through molecular diagnostic testing. Clin Genet. 2013;84(2):190–7. pmid:23662816
  7. 7. Maranhao B, Biswas P, Gottsch AD, Navani M, Naeem MA, Suk J, et al. Investigating the Molecular Basis of Retinal Degeneration in a Familial Cohort of Pakistani Decent by Exome Sequencing. PLoS One. 2015;10(9):e0136561. pmid:26352687
  8. 8. Biswas P, Duncan JL, Maranhao B, Kozak I, Branham K, Gabriel L, et al. Genetic analysis of 10 pedigrees with inherited retinal degeneration by exome sequencing and phenotype-genotype association. Physiol Genomics. 2017;49(4):216–29. pmid:28130426
  9. 9. Shao Q, Jiang Y, Wu J. [Whole-genome sequencing and its application in the research and diagnoses of genetic diseases]. Yi Chuan. 2014;36(11):1087–98. pmid:25567867
  10. 10. Branham K, Matsui H, Biswas P, Guru AA, Hicks M, Suk JJ, et al. Establishing the involvement of the novel gene AGBL5 in retinitis pigmentosa by whole genome sequencing. Physiol Genomics. 2016:physiolgenomics 00101 2016. pmid:27764769
  11. 11. Gustafson K, Duncan JL, Biswas P, Soto-Hermida A, Matsui H, Jakubosky D, et al. Correction: Gustafson et al., Whole Genome Sequencing Revealed Mutations in Two Independent Genes as the Underlying Cause of Retinal Degeneration in an Ashkenazi Jewish Pedigree. Genes 2017, 8, 210. Genes (Basel). 2017;8(10).
  12. 12. Pontikos N, Arno G, Jurkute N, Schiff E, Ba-Abbad R, Malka S, et al. Genetic Basis of Inherited Retinal Disease in a Molecularly Characterized Cohort of More Than 3000 Families from the United Kingdom. Ophthalmology. 2020;127(10):1384–94. pmid:32423767
  13. 13. Nishiguchi KM, Tearle RG, Liu YP, Oh EC, Miyake N, Benaglio P, et al. Whole genome sequencing in patients with retinitis pigmentosa reveals pathogenic DNA structural changes and NEK2 as a new disease gene. Proc Natl Acad Sci U S A. 2013;110(40):16139–44. pmid:24043777
  14. 14. Chekuri A, Guru AA, Biswas P, Branham K, Borooah S, Soto-Hermida A, et al. IFT88 mutations identified in individuals with non-syndromic recessive retinal degeneration result in abnormal ciliogenesis. Hum Genet. 2018;137(6–7):447–58. pmid:29978320
  15. 15. Habibi I, Chebil A, Falfoul Y, Allaman-Pillet N, Kort F, Schorderet DF, et al. Identifying mutations in Tunisian families with retinal dystrophy. Sci Rep. 2016;6:37455. pmid:27874104
  16. 16. Bujakowska K, Audo I, Mohand-Said S, Lancelot ME, Antonio A, Germain A, et al. CRB1 mutations in inherited retinal dystrophies. Hum Mutat. 2012;33(2):306–15. pmid:22065545
  17. 17. Besnard T, Garcia-Garcia G, Baux D, Vache C, Faugere V, Larrieu L, et al. Experience of targeted Usher exome sequencing as a clinical test. Molecular genetics & genomic medicine. 2014;2(1):30–43. pmid:24498627
  18. 18. Ebberink MS, Kofster J, Wanders RJ, Waterham HR. Spectrum of PEX6 mutations in Zellweger syndrome spectrum patients. Hum Mutat. 2010;31(1):E1058–70. pmid:19877282
  19. 19. van Huet RA, Pierrache LH, Meester-Smoor MA, Klaver CC, van den Born LI, Hoyng CB, et al. The efficacy of microarray screening for autosomal recessive retinitis pigmentosa in routine clinical practice. Mol Vis. 2015;21:461–76. pmid:25999674
  20. 20. Martin-Merida I, Avila-Fernandez A, Del Pozo-Valero M, Blanco-Kelly F, Zurita O, Perez-Carro R, et al. Genomic Landscape of Sporadic Retinitis Pigmentosa: Findings from 877 Spanish Cases. Ophthalmology. 2019;126(8):1181–8. pmid:30902645
  21. 21. Avila-Fernandez A, Cantalapiedra D, Aller E, Vallespin E, Aguirre-Lamban J, Blanco-Kelly F, et al. Mutation analysis of 272 Spanish families affected by autosomal recessive retinitis pigmentosa using a genotyping microarray. Mol Vis. 2010;16:2550–8. pmid:21151602
  22. 22. Weston MD, Eudy JD, Fujita S, Yao S, Usami S, Cremers C, et al. Genomic structure and identification of novel mutations in usherin, the gene responsible for Usher syndrome type IIa. Am J Hum Genet. 2000;66(4):1199–210. pmid:10729113
  23. 23. Dreyer B, Brox V, Tranebjaerg L, Rosenberg T, Sadeghi AM, Moller C, et al. Spectrum of USH2A mutations in Scandinavian patients with Usher syndrome type II. Hum Mutat. 2008;29(3):451. pmid:18273898
  24. 24. Neuhaus C, Eisenberger T, Decker C, Nagl S, Blank C, Pfister M, et al. Next-generation sequencing reveals the mutational landscape of clinically diagnosed Usher syndrome: copy number variations, phenocopies, a predominant target for translational read-through, and PEX26 mutated in Heimler syndrome. Molecular genetics & genomic medicine. 2017;5(5):531–52.
  25. 25. Nakanishi H, Ohtsubo M, Iwasaki S, Hotta Y, Usami S, Mizuta K, et al. Novel USH2A mutations in Japanese Usher syndrome type 2 patients: marked differences in the mutation spectrum between the Japanese and other populations. J Hum Genet. 2011;56(7):484–90. pmid:21593743
  26. 26. Auslender N, Bandah D, Rizel L, Behar DM, Shohat M, Banin E, et al. Four USH2A founder mutations underlie the majority of Usher syndrome type 2 cases among non-Ashkenazi Jews. Genetic testing. 2008;12(2):289–94. pmid:18452394
  27. 27. Bowne SJ, Sullivan LS, Avery CE, Sasser EM, Roorda A, Duncan JL, et al. Mutations in the small nuclear riboprotein 200 kDa gene (SNRNP200) cause 1.6% of autosomal dominant retinitis pigmentosa. Mol Vis. 2013;19:2407–17. pmid:24319334
  28. 28. Beryozkin A, Levy G, Blumenfeld A, Meyer S, Namburi P, Morad Y, et al. Genetic Analysis of the Rhodopsin Gene Identifies a Mosaic Dominant Retinitis Pigmentosa Mutation in a Healthy Individual. Invest Ophthalmol Vis Sci. 2016;57(3):940–7. pmid:26962691
  29. 29. Haim M, Grundmann K, Gal A, Rosenberg T. Novel rhodopsin mutation (M216R) in a Danish family with autosomal dominant retinitis pigmentosa. Ophthalmic Genet. 1996;17(4):193–7. pmid:9010870
  30. 30. El Shamieh S, Neuille M, Terray A, Orhan E, Condroyer C, Demontant V, et al. Whole-exome sequencing identifies KIZ as a ciliary gene associated with autosomal-recessive rod-cone dystrophy. Am J Hum Genet. 2014;94(4):625–33. pmid:24680887
  31. 31. Pelletier V, Jambou M, Delphin N, Zinovieva E, Stum M, Gigarel N, et al. Comprehensive survey of mutations in RP2 and RPGR in patients affected with distinct retinal dystrophies: genotype-phenotype correlations and impact on genetic counseling. Hum Mutat. 2007;28(1):81–91. pmid:16969763
  32. 32. Zenteno JC, Garcia-Montano LA, Cruz-Aguilar M, Ronquillo J, Rodas-Serrano A, Aguilar-Castul L, et al. Extensive genic and allelic heterogeneity underlying inherited retinal dystrophies in Mexican patients molecularly analyzed by next-generation sequencing. Molecular genetics & genomic medicine. 2020;8(1). pmid:31736247
  33. 33. Naeem MA, Gottsch AD, Ullah I, Khan SN, Husnain T, Butt NH, et al. Mutations in GRM6 identified in consanguineous Pakistani families with congenital stationary night blindness. Mol Vis. 2015;21:1261–71. pmid:26628857
  34. 34. Zeitz C, Robson AG, Audo I. Congenital stationary night blindness: an analysis and update of genotype-phenotype correlations and pathogenic mechanisms. Prog Retin Eye Res. 2015;45:58–110. pmid:25307992
  35. 35. Ramsden SC, O’Grady A, Fletcher T, O’Sullivan J, Hart-Holden N, Barton SJ, et al. A clinical molecular genetic service for United Kingdom families with choroideraemia. European journal of medical genetics. 2013;56(8):432–8. pmid:23811034
  36. 36. Bertelsen M, Zernant J, Larsen M, Duno M, Allikmets R, Rosenberg T. Generalized choriocapillaris dystrophy, a distinct phenotype in the spectrum of ABCA4-associated retinopathies. Invest Ophthalmol Vis Sci. 2014;55(4):2766–76. pmid:24713488
  37. 37. Kim MS, Joo K, Seong MW, Kim MJ, Park KH, Park SS, et al. Genetic Mutation Profiles in Korean Patients with Inherited Retinal Diseases. J Korean Med Sci. 2019;34(21):e161. pmid:31144483
  38. 38. Lenassi E, Vincent A, Li Z, Saihan Z, Coffey AJ, Steele-Stallard HB, et al. A detailed clinical and molecular survey of subjects with nonsyndromic USH2A retinopathy reveals an allelic hierarchy of disease-causing variants. Eur J Hum Genet. 2015. pmid:25649381
  39. 39. Ge Z, Bowles K, Goetz K, Scholl HP, Wang F, Wang X, et al. NGS-based Molecular diagnosis of 105 eyeGENE((R)) probands with Retinitis Pigmentosa. Sci Rep. 2015;5:18287. pmid:26667666
  40. 40. Dad S, Rendtorff ND, Tranebjaerg L, Gronskov K, Karstensen HG, Brox V, et al. Usher syndrome in Denmark: mutation spectrum and some clinical observations. Molecular genetics & genomic medicine. 2016;4(5):527–39. pmid:27957503
  41. 41. Taylor RL, Parry NRA, Barton SJ, Campbell C, Delaney CM, Ellingford JM, et al. Panel-Based Clinical Genetic Testing in 85 Children with Inherited Retinal Disease. Ophthalmology. 2017;124(7):985–91. pmid:28341476
  42. 42. Avela K, Sankila EM, Seitsonen S, Kuuluvainen L, Barton S, Gillies S, et al. A founder mutation in CERKL is a major cause of retinal dystrophy in Finland. Acta Ophthalmol. 2018;96(2):183–91. pmid:29068140
  43. 43. Parma ES, Korkko J, Hagler WS, Ala-Kokko L. Radial perivascular retinal degeneration: a key to the clinical diagnosis of an ocular variant of Stickler syndrome with minimal or no systemic manifestations. Am J Ophthalmol. 2002;134(5):728–34. pmid:12429250
  44. 44. Mackay DS, Dev Borman A, Moradi P, Henderson RH, Li Z, Wright GA, et al. RDH12 retinopathy: novel mutations and phenotypic description. Mol Vis. 2011;17:2706–16. pmid:22065924
  45. 45. Mizobuchi K, Hayashi T, Yoshitake K, Fujinami K, Tachibana T, Tsunoda K, et al. Novel homozygous CLN3 missense variant in isolated retinal dystrophy: A case report and electron microscopic findings. Molecular genetics & genomic medicine. 2020;8(8):e1308. pmid:32441891
  46. 46. Sharon D, Wimberg H, Kinarty Y, Koch KW. Genotype-functional-phenotype correlations in photoreceptor guanylate cyclase (GC-E) encoded by GUCY2D. Prog Retin Eye Res. 2018;63:69–91. pmid:29061346
  47. 47. Hanein S, Perrault I, Gerber S, Tanguy G, Barbet F, Ducroq D, et al. Leber congenital amaurosis: comprehensive survey of the genetic heterogeneity, refinement of the clinical definition, and genotype-phenotype correlations as a strategy for molecular diagnosis. Hum Mutat. 2004;23(4):306–17. pmid:15024725
  48. 48. Yi Z, Xiao X, Li S, Sun W, Zhang Q. Pathogenicity discrimination and genetic test reference for CRX variants based on genotype-phenotype analysis. Exp Eye Res. 2019;189:107846. pmid:31626798
  49. 49. Rivolta C, Peck NE, Fulton AB, Fishman GA, Berson EL, Dryja TP. Novel frameshift mutations in CRX associated with Leber congenital amaurosis. Hum Mutat. 2001;18(6):550–1. pmid:11748859
  50. 50. Li T, Feng Y, Liu Y, He C, Liu J, Chen H, et al. A novel ABHD12 nonsense variant in Usher syndrome type 3 family with genotype-phenotype spectrum review. Gene. 2019;704:113–20. pmid:30974196
  51. 51. Zelinger L, Banin E, Obolensky A, Mizrahi-Meissonnier L, Beryozkin A, Bandah-Rozenfeld D, et al. A missense mutation in DHDDS, encoding dehydrodolichyl diphosphate synthase, is associated with autosomal-recessive retinitis pigmentosa in Ashkenazi Jews. Am J Hum Genet. 2011;88(2):207–15. pmid:21295282
  52. 52. Michaelides M, Gaillard MC, Escher P, Tiab L, Bedell M, Borruat FX, et al. The PROM1 mutation p.R373C causes an autosomal dominant bull’s eye maculopathy associated with rod, rod-cone, and macular dystrophy. Invest Ophthalmol Vis Sci. 2010;51(9):4771–80. pmid:20393116
  53. 53. Tuson M, Marfany G, Gonzalez-Duarte R. Mutation of CERKL, a novel human ceramide kinase gene, causes autosomal recessive retinitis pigmentosa (RP26). Am J Hum Genet. 2004;74(1):128–38. pmid:14681825
  54. 54. Haer-Wigman L, van Zelst-Stams WA, Pfundt R, van den Born LI, Klaver CC, Verheij JB, et al. Diagnostic exome sequencing in 266 Dutch patients with visual impairment. Eur J Hum Genet. 2017;25(5):591–9. pmid:28224992
  55. 55. Garanto A, Vicente-Tejedor J, Riera M, de la Villa P, Gonzalez-Duarte R, Blanco R, et al. Targeted knockdown of Cerkl, a retinal dystrophy gene, causes mild affectation of the retinal ganglion cell layer. Biochim Biophys Acta. 2012;1822(8):1258–69. pmid:22549043
  56. 56. Yu S, Li C, Biswas L, Hu X, Liu F, Reilly J, et al. CERKL gene knockout disturbs photoreceptor outer segment phagocytosis and causes rod-cone dystrophy in zebrafish. Hum Mol Genet. 2017;26(12):2335–45. pmid:28398482
  57. 57. Khan AO, Al Rashaed S, Neuhaus C, Bergmann C, Bolz HJ. Peripherin mutations cause a distinct form of recessive Leber congenital amaurosis and dominant phenotypes in asymptomatic parents heterozygous for the mutation. Br J Ophthalmol. 2016;100(2):209–15. pmid:26061163
  58. 58. Audo I, Lancelot ME, Mohand-Said S, Antonio A, Germain A, Sahel JA, et al. Novel C2orf71 mutations account for approximately 1% of cases in a large French arRP cohort. Hum Mutat. 2011;32(4):E2091–103. pmid:21412943
  59. 59. Beryozkin A, Shevah E, Kimchi A, Mizrahi-Meissonnier L, Khateb S, Ratnapriya R, et al. Whole Exome Sequencing Reveals Mutations in Known Retinal Disease Genes in 33 out of 68 Israeli Families with Inherited Retinopathies. Sci Rep. 2015;5:13187. pmid:26306921
  60. 60. Ku CA, Hull S, Arno G, Vincent A, Carss K, Kayton R, et al. Detailed Clinical Phenotype and Molecular Genetic Findings in CLN3-Associated Isolated Retinal Degeneration. JAMA Ophthalmol. 2017;135(7):749–60. pmid:28542676
  61. 61. Sullivan LS, Bowne SJ, Reeves MJ, Blain D, Goetz K, Ndifor V, et al. Prevalence of mutations in eyeGENE probands with a diagnosis of autosomal dominant retinitis pigmentosa. Invest Ophthalmol Vis Sci. 2013;54(9):6255–61. pmid:23950152
  62. 62. Zhong Z, Yan M, Sun W, Wu Z, Han L, Zhou Z, et al. Two novel mutations in PRPF3 causing autosomal dominant retinitis pigmentosa. Sci Rep. 2016;6:37840. pmid:27886254
  63. 63. Vaclavik V, Gaillard MC, Tiab L, Schorderet DF, Munier FL. Variable phenotypic expressivity in a Swiss family with autosomal dominant retinitis pigmentosa due to a T494M mutation in the PRPF3 gene. Mol Vis. 2010;16:467–75. pmid:20309403
  64. 64. Hoyng CB, Heutink P, Testers L, Pinckers A, Deutman AF, Oostra BA. Autosomal dominant central areolar choroidal dystrophy caused by a mutation in codon 142 in the peripherin/RDS gene. Am J Ophthalmol. 1996;121(6):623–9. pmid:8644804
  65. 65. Boulanger-Scemama E, El Shamieh S, Demontant V, Condroyer C, Antonio A, Michiels C, et al. Next-generation sequencing applied to a large French cone and cone-rod dystrophy cohort: mutation spectrum and new genotype-phenotype correlation. Orphanet J Rare Dis. 2015;10:85. pmid:26103963
  66. 66. Ouseph MM, Kleinman ME, Wang QJ. Vision loss in juvenile neuronal ceroid lipofuscinosis (CLN3 disease). Ann N Y Acad Sci. 2016;1371(1):55–67. pmid:26748992
  67. 67. Kozak I, Sasik R, Freeman WR, Sprague LJ, Gomez ML, Cheng L, et al. A degenerative retinal process in HIV-associated non-infectious retinopathy. PloS one. 2013;8(9):e74712. pmid:24069333
  68. 68. Baraas RC, Hagen LA, Dees EW, Neitz M. Substitution of isoleucine for threonine at position 190 of S-opsin causes S-cone-function abnormalities. Vision Res. 2012;73:1–9. pmid:23022137
  69. 69. Coussa RG, Chakarova C, Ajlan R, Taha M, Kavalec C, Gomolin J, et al. Genotype and Phenotype Studies in Autosomal Dominant Retinitis Pigmentosa (adRP) of the French Canadian Founder Population. Invest Ophthalmol Vis Sci. 2015;56(13):8297–305. pmid:26720483
  70. 70. Essilfie JO, Sanfilippo CJ, Sarraf D. Bull’s Eye Maculopathy with Mutations in Rds/Prph2 and Rom-1. Retin Cases Brief Rep. 2018;12 Suppl 1:S55–S8. pmid:29155698
  71. 71. Kajiwara K, Berson EL, Dryja TP. Digenic retinitis pigmentosa due to mutations at the unlinked peripherin/RDS and ROM1 loci. Science. 1994;264(5165):1604–8. pmid:8202715
  72. 72. Boon CJ, Klevering BJ, Cremers FP, Zonneveld-Vrieling MN, Theelen T, Den Hollander AI, et al. Central areolar choroidal dystrophy. Ophthalmology. 2009;116(4):771–82, 82 e1. pmid:19243827
  73. 73. Churchill JD, Bowne SJ, Sullivan LS, Lewis RA, Wheaton DK, Birch DG, et al. Mutations in the X-linked retinitis pigmentosa genes RPGR and RP2 found in 8.5% of families with a provisional diagnosis of autosomal dominant retinitis pigmentosa. Invest Ophthalmol Vis Sci. 2013;54(2):1411–6. pmid:23372056
  74. 74. Strom SP, Clark MJ, Martinez A, Garcia S, Abelazeem AA, Matynia A, et al. De Novo Occurrence of a Variant in ARL3 and Apparent Autosomal Dominant Transmission of Retinitis Pigmentosa. PLoS One. 2016;11(3):e0150944. pmid:26964041
  75. 75. Apushkin MA, Fishman GA, Taylor CM, Stone EM. Novel de novo mutation in a patient with Best macular dystrophy. Arch Ophthalmol. 2006;124(6):887–9. pmid:16769844
  76. 76. Schwartz SB, Aleman TS, Cideciyan AV, Swaroop A, Jacobson SG, Stone EM. De novo mutation in the RP1 gene (Arg677ter) associated with retinitis pigmentosa. Invest Ophthalmol Vis Sci. 2003;44(8):3593–7. pmid:12882812
  77. 77. Mukherjee R, Robson AG, Holder GE, Stockman A, Egan CA, Moore AT, et al. A detailed phenotypic description of autosomal dominant cone dystrophy due to a de novo mutation in the GUCY2D gene. Eye (Lond). 2014;28(4):481–7. pmid:24480840
  78. 78. Jones KD, Wheaton DK, Bowne SJ, Sullivan LS, Birch DG, Chen R, et al. Next-generation sequencing to solve complex inherited retinal dystrophy: A case series of multiple genes contributing to disease in extended families. Mol Vis. 2017;23:470–81. pmid:28761320
  79. 79. Daiger SP, Sullivan LS, Bowne SJ. Genes and mutations causing retinitis pigmentosa. Clin Genet. 2013;84(2):132–41. pmid:23701314
  80. 80. de Bruijn SE, Fiorentino A, Ottaviani D, Fanucchi S, Melo US, Corral-Serrano JC, et al. Structural Variants Create New Topological-Associated Domains and Ectopic Retinal Enhancer-Gene Contact in Dominant Retinitis Pigmentosa. Am J Hum Genet. 2020;107(5):802–14. pmid:33022222
  81. 81. Telenti A, di Iulio J. Regulatory genome variants in human susceptibility to infection. Hum Genet. 2020;139(6–7):759–68. pmid:31807864
  82. 82. Jamshidi F, Place EM, Mehrotra S, Navarro-Gomez D, Maher M, Branham KE, et al. Contribution of noncoding pathogenic variants to RPGRIP1-mediated inherited retinal degeneration. Genet Med. 2019;21(3):694–704. pmid:30072743
  83. 83. Monroy-Jaramillo N, Ceron A, Leon E, Rivas V, Ochoa-Morales A, Arteaga-Alcaraz MG, et al. Phenotypic Variability in a Mexican Mestizo Family with Retinal Vasculopathy with Cerebral Leukodystrophy and TREX1 Mutation p.V235Gfs*6. Rev Invest Clin. 2018;70(2):68–75. pmid:29718010
  84. 84. Lopez-Rubio S, Chacon-Camacho OF, Matsui R, Guadarrama-Vallejo D, Astiazaran MC, Zenteno JC. Retinal phenotypic characterization of patients with ABCA4 retinopathydue to the homozygous p.Ala1773Val mutation. Mol Vis. 2018;24:105–14. pmid:29422768
  85. 85. Chacon-Camacho OF, Granillo-Alvarez M, Ayala-Ramirez R, Zenteno JC. ABCA4 mutational spectrum in Mexican patients with Stargardt disease: Identification of 12 novel mutations and evidence of a founder effect for the common p.A1773V mutation. Exp Eye Res. 2013;109:77–82. pmid:23419329
  86. 86. Bryc K, Velez C, Karafet T, Moreno-Estrada A, Reynolds A, Auton A, et al. Colloquium paper: genome-wide patterns of population structure and admixture among Hispanic/Latino populations. Proc Natl Acad Sci U S A. 2010;107 Suppl 2:8954–61. pmid:20445096
  87. 87. Johnson NA, Coram MA, Shriver MD, Romieu I, Barsh GS, London SJ, et al. Ancestral components of admixed genomes in a Mexican cohort. PLoS Genet. 2011;7(12):e1002410. pmid:22194699
  88. 88. Shami SA, Schmitt LH, Bittles AH. Consanguinity related prenatal and postnatal mortality of the populations of seven Pakistani Punjab cities. J Med Genet. 1989;26(4):267–71. pmid:2716036
  89. 89. Hussain R. The impact of consanguinity and inbreeding on perinatal mortality in Karachi, Pakistan. Paediatr Perinat Epidemiol. 1998;12(4):370–82. pmid:9805711
  90. 90. Hussain R, Bittles AH. The prevalence and demographic characteristics of consanguineous marriages in Pakistan. J Biosoc Sci. 1998;30(2):261–75. pmid:9746828
  91. 91. Li L, Chen Y, Jiao X, Jin C, Jiang D, Tanwar M, et al. Homozygosity Mapping and Genetic Analysis of Autosomal Recessive Retinal Dystrophies in 144 Consanguineous Pakistani Families. Invest Ophthalmol Vis Sci. 2017;58(4):2218–38. pmid:28418496
  92. 92. Kabir F, Naz S, Riazuddin SA, Naeem MA, Khan SN, Husnain T, et al. Novel mutations in RPE65 identified in consanguineous Pakistani families with retinal dystrophy. Mol Vis. 2013;19:1554–64. pmid:23878505
  93. 93. Li L, Jiao X, D’Atri I, Ono F, Nelson R, Chan CC, et al. Mutation in the intracellular chloride channel CLCC1 associated with autosomal recessive retinitis pigmentosa. PLoS Genet. 2018;14(8):e1007504. pmid:30157172
  94. 94. Ullah I, Kabir F, Iqbal M, Gottsch CB, Naeem MA, Assir MZ, et al. Pathogenic mutations in TULP1 responsible for retinitis pigmentosa identified in consanguineous familial cases. Mol Vis. 2016;22:797–815. pmid:27440997
  95. 95. Zhang Q, Zulfiqar F, Riazuddin SA, Xiao X, Ahmad Z, Riazuddin S, et al. Autosomal recessive retinitis pigmentosa in a Pakistani family mapped to CNGA1 with identification of a novel mutation. Mol Vis. 2004;10:884–9. pmid:15570217
  96. 96. Riazuddin SA, Zulfiqar F, Zhang Q, Sergeev YV, Qazi ZA, Husnain T, et al. Autosomal recessive retinitis pigmentosa is associated with mutations in RP1 in three consanguineous Pakistani families. Invest Ophthalmol Vis Sci. 2005;46(7):2264–70. pmid:15980210
  97. 97. Khan SY, Ali S, Naeem MA, Khan SN, Husnain T, Butt NH, et al. Splice-site mutations identified in PDE6A responsible for retinitis pigmentosa in consanguineous Pakistani families. Mol Vis. 2015;21:871–82. pmid:26321862
  98. 98. Ullah I, Kabir F, Gottsch CB, Naeem MA, Guru AA, Ayyagari R, et al. Mutations in phosphodiesterase 6 identified in familial cases of retinitis pigmentosa. Hum Genome Var. 2016;3:16036. pmid:27917291
  99. 99. Biswas P, Chavali VR, Agnello G, Stone E, Chakarova C, Duncan JL, et al. A missense mutation in ASRGL1 is involved in causing autosomal recessive retinal degeneration. Hum Mol Genet. 2016;25(12):2483–97. pmid:27106100
  100. 100. Li D, Jin C, Jiao X, Li L, Bushra T, Naeem MA, et al. AIPL1 implicated in the pathogenesis of two cases of autosomal recessive retinal degeneration. Mol Vis. 2014;20:1–14. pmid:24426771
  101. 101. Ali S, Riazuddin SA, Shahzadi A, Nasir IA, Khan SN, Husnain T, et al. Mutations in the beta-subunit of rod phosphodiesterase identified in consanguineous Pakistani families with autosomal recessive retinitis pigmentosa. Mol Vis. 2011;17:1373–80. pmid:21655355
  102. 102. Riazuddin SA, Zulfiqar F, Zhang Q, Yao W, Li S, Jiao X, et al. Mutations in the gene encoding the alpha-subunit of rod phosphodiesterase in consanguineous Pakistani families. Mol Vis. 2006;12:1283–91. pmid:17110911
  103. 103. Naz S, Ali S, Riazuddin SA, Farooq T, Butt NH, Zafar AU, et al. Mutations in RLBP1 associated with fundus albipunctatus in consanguineous Pakistani families. Br J Ophthalmol. 2011;95(7):1019–24. pmid:21447491
  104. 104. Biswas P, Duncan JL, Ali M, Matsui H, Naeem MA, Raghavendra PB, et al. A mutation in IFT43 causes non-syndromic recessive retinal degeneration. Hum Mol Genet. 2017;26(23):4741–51. pmid:28973684
  105. 105. Li L, Nakaya N, Chavali VR, Ma Z, Jiao X, Sieving PA, et al. A mutation in ZNF513, a putative regulator of photoreceptor development, causes autosomal-recessive retinitis pigmentosa. Am J Hum Genet. 2010;87(3):400–9. pmid:20797688
  106. 106. Riazuddin SA, Shahzadi A, Zeitz C, Ahmed ZM, Ayyagari R, Chavali VR, et al. A mutation in SLC24A1 implicated in autosomal-recessive congenital stationary night blindness. Am J Hum Genet. 2010;87(4):523–31. pmid:20850105
  107. 107. Maranhao B, Biswas P, Duncan JL, Branham KE, Silva GA, Naeem MA, et al. exomeSuite: Whole exome sequence variant filtering tool for rapid identification of putative disease causing SNVs/indels. Genomics. 2014;103(2–3):169–76. pmid:24603341
  108. 108. Ajmal M, Khan MI, Micheal S, Ahmed W, Shah A, Venselaar H, et al. Identification of recurrent and novel mutations in TULP1 in Pakistani families with early-onset retinitis pigmentosa. Mol Vis. 2012;18:1226–37. pmid:22665969
  109. 109. Ajmal M, Khan MI, Neveling K, Khan YM, Ali SH, Ahmed W, et al. Novel mutations in RDH5 cause fundus albipunctatus in two consanguineous Pakistani families. Mol Vis. 2012;18:1558–71. pmid:22736946
  110. 110. Maria M, Ajmal M, Azam M, Waheed NK, Siddiqui SN, Mustafa B, et al. Homozygosity mapping and targeted sanger sequencing reveal genetic defects underlying inherited retinal disease in families from pakistan. PLoS One. 2015;10(3):e0119806. pmid:25775262
  111. 111. Azam M, Collin RW, Khan MI, Shah ST, Qureshi N, Ajmal M, et al. A novel mutation in GRK1 causes Oguchi disease in a consanguineous Pakistani family. Mol Vis. 2009;15:1788–93. pmid:19753316
  112. 112. Azam M, Collin RW, Malik A, Khan MI, Shah ST, Shah AA, et al. Identification of novel mutations in Pakistani families with autosomal recessive retinitis pigmentosa. Arch Ophthalmol. 2011;129(10):1377–8. pmid:21987686
  113. 113. Azam M, Collin RW, Shah ST, Shah AA, Khan MI, Hussain A, et al. Novel CNGA3 and CNGB3 mutations in two Pakistani families with achromatopsia. Mol Vis. 2010;16:774–81. pmid:20454696
  114. 114. Azam M, Khan MI, Gal A, Hussain A, Shah ST, Khan MS, et al. A homozygous p.Glu150Lys mutation in the opsin gene of two Pakistani families with autosomal recessive retinitis pigmentosa. Mol Vis. 2009;15:2526–34. pmid:19960070
  115. 115. Khan MI, Collin RW, Arimadyo K, Micheal S, Azam M, Qureshi N, et al. Missense mutations at homologous positions in the fourth and fifth laminin A G-like domains of eyes shut homolog cause autosomal recessive retinitis pigmentosa. Mol Vis. 2010;16:2753–9. pmid:21179430
  116. 116. Khan MI, Kersten FF, Azam M, Collin RW, Hussain A, Shah ST, et al. CLRN1 mutations cause nonsyndromic retinitis pigmentosa. Ophthalmology. 2011;118(7):1444–8. pmid:21310491
  117. 117. Khan MI, Ajmal M, Micheal S, Azam M, Hussain A, Shahzad A, et al. Homozygosity mapping identifies genetic defects in four consanguineous families with retinal dystrophy from Pakistan. Clin Genet. 2013;84(3):290–3. pmid:23134348
  118. 118. Bandah-Rozenfeld D, Collin RW, Banin E, van den Born LI, Coene KL, Siemiatkowska AM, et al. Mutations in IMPG2, encoding interphotoreceptor matrix proteoglycan 2, cause autosomal-recessive retinitis pigmentosa. Am J Hum Genet. 2010;87(2):199–208. pmid:20673862
  119. 119. Ajmal M, Khan MI, Neveling K, Khan YM, Azam M, Waheed NK, et al. A missense mutation in the splicing factor gene DHX38 is associated with early-onset retinitis pigmentosa with macular coloboma. J Med Genet. 2014;51(7):444–8. pmid:24737827
  120. 120. El-Asrag ME, Sergouniotis PI, McKibbin M, Plagnol V, Sheridan E, Waseem N, et al. Biallelic mutations in the autophagy regulator DRAM2 cause retinal dystrophy with early macular involvement. Am J Hum Genet. 2015;96(6):948–54. pmid:25983245
  121. 121. Hull S, Owen N, Islam F, Tracey-White D, Plagnol V, Holder GE, et al. Nonsyndromic Retinal Dystrophy due to Bi-Allelic Mutations in the Ciliary Transport Gene IFT140. Invest Ophthalmol Vis Sci. 2016;57(3):1053–62. pmid:26968735
  122. 122. Shaikh RS, Reuter P, Sisk RA, Kausar T, Shahzad M, Maqsood MI, et al. Homozygous missense variant in the human CNGA3 channel causes cone-rod dystrophy. Eur J Hum Genet. 2015;23(4):473–80. pmid:25052312
  123. 123. Bujakowska KM, Fernandez-Godino R, Place E, Consugar M, Navarro-Gomez D, White J, et al. Copy-number variation is an important contributor to the genetic causality of inherited retinal degenerations. Genet Med. 2016. pmid:27735924
  124. 124. Cherry TJ, Yang MG, Harmin DA, Tao P, Timms AE, Bauwens M, et al. Mapping the cis-regulatory architecture of the human retina reveals noncoding genetic variation in disease. Proc Natl Acad Sci U S A. 2020;117(16):9001–12. pmid:32265282
  125. 125. Di Scipio M, Tavares E, Deshmukh S, Audo I, Green-Sanderson K, Zubak Y, et al. Phenotype Driven Analysis of Whole Genome Sequencing Identifies Deep Intronic Variants that Cause Retinal Dystrophies by Aberrant Exonization. Invest Ophthalmol Vis Sci. 2020;61(10):36. pmid:32881472
  126. 126. Carss KJ, Arno G, Erwood M, Stephens J, Sanchis-Juan A, Hull S, et al. Comprehensive Rare Variant Analysis via Whole-Genome Sequencing to Determine the Molecular Pathology of Inherited Retinal Disease. Am J Hum Genet. 2017;100(1):75–90. pmid:28041643
  127. 127. Monies D, Abouelhoda M, AlSayed M, Alhassnan Z, Alotaibi M, Kayyali H, et al. The landscape of genetic diseases in Saudi Arabia based on the first 1000 diagnostic panels and exomes. Hum Genet. 2017;136(8):921–39. pmid:28600779
  128. 128. Alazami AM, Patel N, Shamseldin HE, Anazi S, Al-Dosari MS, Alzahrani F, et al. Accelerating novel candidate gene discovery in neurogenetic disorders via whole-exome sequencing of prescreened multiplex consanguineous families. Cell reports. 2015;10(2):148–61. pmid:25558065
  129. 129. Sharon D, Ben-Yosef T, Goldenberg-Cohen N, Pras E, Gradstein L, Soudry S, et al. A nationwide genetic analysis of inherited retinal diseases in Israel as assessed by the Israeli inherited retinal disease consortium (IIRDC). Hum Mutat. 2020;41(1):140–9. pmid:31456290
  130. 130. Stone EM, Andorf JL, Whitmore SS, DeLuca AP, Giacalone JC, Streb LM, et al. Clinically Focused Molecular Investigation of 1000 Consecutive Families with Inherited Retinal Disease. Ophthalmology. 2017;124(9):1314–31. pmid:28559085
  131. 131. Roberts L, Rebello G, Greenberg J, Ramesar R. Update on Inherited Retinal Disease in South Africa: Encouraging Diversity in Molecular Genetics. Adv Exp Med Biol. 2019;1185:257–61. pmid:31884621
  132. 132. Huang XF, Huang F, Wu KC, Wu J, Chen J, Pang CP, et al. Genotype-phenotype correlation and mutation spectrum in a large cohort of patients with inherited retinal dystrophy revealed by next-generation sequencing. Genet Med. 2015;17(4):271–8. pmid:25356976
  133. 133. Kim YJ, Kim YN, Yoon YH, Seo EJ, Seo GH, Keum C, et al. Diverse Genetic Landscape of Suspected Retinitis Pigmentosa in a Large Korean Cohort. Genes (Basel). 2021;12(5).
  134. 134. Hanany M, Rivolta C, Sharon D. Worldwide carrier frequency and genetic prevalence of autosomal recessive inherited retinal diseases. Proc Natl Acad Sci U S A. 2020;117(5):2710–6. pmid:31964843
  135. 135. Hanany M, Allon G, Kimchi A, Blumenfeld A, Newman H, Pras E, et al. Carrier frequency analysis of mutations causing autosomal-recessive-inherited retinal diseases in the Israeli population. Eur J Hum Genet. 2018;26(8):1159–66. pmid:29706639
  136. 136. Bryant L, Lozynska O, Maguire AM, Aleman TS, Bennett J. Prescreening whole exome sequencing results from patients with retinal degeneration for variants in genes associated with retinal degeneration. Clinical ophthalmology. 2018;12:49–63. pmid:29343940
  137. 137. Wang X, Wang H, Sun V, Tuan HF, Keser V, Wang K, et al. Comprehensive molecular diagnosis of 179 Leber congenital amaurosis and juvenile retinitis pigmentosa patients by targeted next generation sequencing. J Med Genet. 2013;50(10):674–88. pmid:23847139
  138. 138. Corton M, Nishiguchi KM, Avila-Fernandez A, Nikopoulos K, Riveiro-Alvarez R, Tatu SD, et al. Exome sequencing of index patients with retinal dystrophies as a tool for molecular diagnosis. PloS one. 2013;8(6):e65574. pmid:23940504
  139. 139. Lee K, Krempely K, Roberts ME, Anderson MJ, Carneiro F, Chao E, et al. Specifications of the ACMG/AMP variant curation guidelines for the analysis of germline CDH1 sequence variants. Hum Mutat. 2018;39(11):1553–68. pmid:30311375
  140. 140. Mandal MN, Heckenlively JR, Burch T, Chen L, Vasireddy V, Koenekoop RK, et al. Sequencing arrays for screening multiple genes associated with early-onset human retinal degenerations on a high-throughput platform. Invest Ophthalmol Vis Sci. 2005;46(9):3355–62. pmid:16123440
  141. 141. Genomes Project C, Auton A, Brooks LD, Durbin RM, Garrison EP, Kang HM, et al. A global reference for human genetic variation. Nature. 2015;526(7571):68–74. pmid:26432245
  142. 142. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009;25(14):1754–60. pmid:19451168
  143. 143. Van der Auwera GA, Carneiro MO, Hartl C, Poplin R, Del Angel G, Levy-Moonshine A, et al. From FastQ data to high confidence variant calls: the Genome Analysis Toolkit best practices pipeline. Curr Protoc Bioinformatics. 2013;43:11 0 1–33. pmid:25431634
  144. 144. DePristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C, et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet. 2011;43(5):491–8. pmid:21478889
  145. 145. Cingolani P, Platts A, Wang le L, Coon M, Nguyen T, Wang L, et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin). 2012;6(2):80–92. pmid:22728672
  146. 146. Adzhubei I, Jordan DM, Sunyaev SR. Predicting functional effect of human missense mutations using PolyPhen-2. Curr Protoc Hum Genet. 2013;Chapter 7:Unit7 20. pmid:23315928
  147. 147. Kircher M, Witten DM, Jain P, O’Roak BJ, Cooper GM, Shendure J. A general framework for estimating the relative pathogenicity of human genetic variants. Nat Genet. 2014;46(3):310–5. pmid:24487276
  148. 148. Handsaker RE, Van Doren V, Berman JR, Genovese G, Kashin S, Boettger LM, et al. Large multiallelic copy number variations in humans. Nat Genet. 2015;47(3):296–303. pmid:25621458
  149. 149. Layer RM, Chiang C, Quinlan AR, Hall IM. LUMPY: a probabilistic framework for structural variant discovery. Genome Biol. 2014;15(6):R84. pmid:24970577
  150. 150. Li H. Toward better understanding of artifacts in variant calling from high-coverage samples. Bioinformatics. 2014;30(20):2843–51. pmid:24974202
  151. 151. MacDonald IM, Gudiseva HV, Villanueva A, Greve M, Caruso R, Ayyagari R. Phenotype and genotype of patients with autosomal recessive bestrophinopathy. Ophthalmic Genet.33(3):123–9. pmid:21809908