ExperimentName: Co-IP showing interaction of RPGR with CEP290

InteractingGene: CEP290, which is implicated in Leber congenital amaurosis 10 and multiple syndromic phenotypes (Joubert syndrome, Meckel syndrome, and Senior-Loken syndrome).

InteractionType: Physical

InteractionMethod: Co-immunoprecipitation was performed with a positive result, while yeast-2-hybrid (Y2H) was performed with a negative result.

InteractionEvidence: Co-IP with an RPGR antibody (Figure 4A) or CEP290 antibody (Figure 4B) successfully immunoprecipitates both proteins from wild-type mouse retinal extracts. The two (endogenously expressed) proteins also co-localize in IMDC-3 cells (Figure 4C). However, yeast 2-hybrid experiments do not confirm this interaction as direct (data not shown).

Selection B - Gene function is consistent with phenotype.

GOterm: GO:0032391 (photoreceptor connecting cilium)

FunctionalEvidence: This study uses a RPGR antibody to stain mouse retinal sections, showing localization of RPGR to the region between the outer segment and inner segment associated with the connecting cilia of rod and cone photoreceptors (Figure 2A). A similar experiment was performed in earlier studies with similar results (PMID: 11104772 Figure 4A, PMID: 10725384 Figure 2D upper). Within mouse airway epithelium tissue sections, RPGR antibody stained a narrow band across the apical domains of multi-ciliated cells (Figure 7A), corresponding with the basal parts of the motile cilia adjacent to the basal bodies (Figures 7B, 7C). This transitional zone of the motile cilia is the structural equivalent of the connecting cilia of photoreceptors, consistent with a biochemical role for RPGR in regulating ciliary organization and/or transport in both the retina and the respiratory tract.

FunctionalHPO: HP:0007642 (Congenital stationary night blindness), HP:0007994 (Peripheral visual field loss), HP:0000529 (Progressive visual loss), HP:0000365 (Hearing impairment), HP:0002205 (Recurrent respiratory infections), HP:0011109 (Chronic sinusitis), HP:0000388 (Otitis media), HP:0002837 (Recurrent bronchitis), HP:0012263 (Immotile cilia), HP:0005938 (Abnormal respiratory motile cilium morphology)

PhenotypeEvidence: This identification of RPGR as a potential regulator of ciliary organization and/or transport in multiple tissues is consistent with the diverse phenotypes associated with RPGR loss-of-function, ranging from visual defects associated with rod and cone dysfunction to respiratory defects associated with dysfunction of the respiratory motile cilia.

Case#: Meindl_1996_Family_F47_Proband, male, age not specified, family origin either German or British

CasePresentingHPOs: HP:0000510 (Rod-cone dystrophy)

CaseHPOFreeText: The proband and affected family members have been diagnosed with retinitis pigmentosa, with an X-linked pattern of inheritance. Disease onset was reported as early.

CaseNotHPOs: N/A

CaseNotHPOFreeText: N/A

CasePreviousTesting: The family members previously underwent linkage analysis to map the underlying genetic cause to the region on chromosome Xp21.1 between the CYBB and OTC genes.

CaseMethod1: Hybridization analysis of amplified cDNA fragments from the RPGR locus to look for deletions, and SSCP analysis to look for point mutations

CaseMethod2: Sanger sequencing of the RPGR locus

CaseGenotypingMethod: The family members previously underwent linkage analysis to map the underlying genetic cause to the region on chromosome Xp21.1 between the CYBB and OTC genes. Amplified cDNA fragments from the RPGR locus were then screened by hybridization analysis to look for deletions and SSCP analysis to look for point mutations. The presence of the variant was confirmed by Sanger sequencing of the RPGR locus.

Variant: The affected family members harbor the NM_001034853.2(RPGR):c.294C>A (p.His98Gln) variant in the hemizygous state.

ClinVar: 98775

gnomAD: The variant was not found in gnomAD v.2.1.1.

VariantEvidence: NM_001034853.2(RPGR):c.294C>A (p.His98Gln) is a missense variant and does not have associated evidence of impact on RPGR function.

CaseAddInfo: The proband is a member of Family 47.

CasePMIDs: N/A

Case#: van_Dorp_1992_Family_P9145_patient_IV-20, male, age not reported, family origin reported to be Dutch

CasePresentingHPOs: HP:0012263 (Immotile cilia), HP:0002205 (Recurrent respiratory infections), HP:0012261 (Abnormal respiratory motile cilium physiology), HP:0012255 (Dynein arm defect of respiratory motile cilia), HP:0012260 (Abnormal central microtubular pair morphology of respiratory motile cilia), HP:0012864 (Abnormal sperm morphology),

CaseHPOFreeText: The proband has been diagnosed with retinitis pigmentosa in combination with recurrent respiratory infections. The family exhibits an X-linked pattern of inheritance. Inner dynein arms of respiratory cilia were described as deficient. Cilia were described as disoriented.

CaseNotHPOs: N/A

CaseNotHPOFreeText: N/A

CasePreviousTesting: The patient and his family members were subjected to linkage analysis using X chromosomal markers, followed by a chromosomal analysis of the patient that revealed no abnormalities (such as a translocation involving the X chromosome).

CaseMethod1: Linkage analysis (PMID: 1733835)

CaseMethod2: Sanger sequencing of PCR products from the RPGR locus (PMID: 10094550)

CaseGenotypingMethod: The patient and family members were subjected to linkage analysis using X chromosomal markers, and then the patient DNA underwent chromosomal analysis that revealed no abnormalities (such as a translocation involving the X chromosome). The family members were then genotyped by Sanger sequencing of PCR products from the RPGR locus (PMID: 10094550).

Variant: The patient harbors the NM_001034853.2(RPGR):c.469+1G>T variant in the hemizygous state.

ClinVar: 9912

gnomAD: The variant was not found in gnomAD v.2.1.1.

VariantEvidence: NM_001034853.2(RPGR):c.469+1G>T is a canonical splice site variant in the final exon (exon 15) that disrupts the 5' splice site of intron 5. Functional evidence confirming the expected effect on splicing is not available.

CaseAddInfo: The proband is a member of Family P9145 (shown in Figure 1), which includes four other affected genotype-positive males and as well as three carrier females who showed no detectable abnormality. The detailed genotype for the affected and carrier individuals was later published in PMID: 10094550.

CasePMIDs: PMID: 10094550

ExperimentName: Rescue of Rpgr-deficient mice with human RPGR

RescueModel: Mus musculus

GeneAlteration: The Rpgr−/y mouse model was constructed with targeted hemizygous (male) disruption of the mouse Rpgr gene by insertion of a neomycin resistance cassette to replace exons 4 through 6 and generate a null variant (PMID: 10725384).

RescueVariant: Rescue was performed with wild-type human RPGR.

RescuePhenotype: HP:0030612 (Abnormal retinal morphology on macular OCT), partial mislocalization of blue and green cone opsins to the inner segment, reduced rhodopsin content in the outer segment, HP:0000546 (Retinal degeneration), HP:0030609 (Photoreceptor layer loss on macular OCT), HP:0030610 (Photoreceptor outer segment loss on macular OCT), disorganization of disk membranes, normal connecting cilia morphology, HP:0030483 (Reduced amplitude of dark-adapted bright flash electroretinogram a-wave), HP:0008275 (Abnormal light-adapted electroretinogram)

RescueMethod: Rescue was performed by subretinal injection of AAV8 particles delivering wild-type human RPGR (Figure 5).

RescueEvidence: ERG analyses show significant rescue of rod function and a rescue of cone function that is trending towards statistical significance (Figures 5B and 5C).

ModelVariant: The model does not harbor a variant equivalent to one from the human patients, but rather has targeted disruption of Rpgr leading to full loss-of-function.