REEP6: Retinitis Pigmentosa PMID:

REEP6: Retinitis Pigmentosa PMID: 27889058
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REEP6: Autosomal Recessive Retinitis Pigmentosa Training Module
To complete this module, you will need: The Gene Clinical Validity Curation Process Standard Operating Procedure (Version 5) Outline of the module: Background of the disease Questions and answers on genetic evidence and experimental evidence related to the SOP Clinical validity summary Take out version 4 text? > NEXT

Background Retinitis pigmentosa (RP) is the most common inherited retinal dystrophy, resulting in more than 1 million cases of visual impairment worldwide. At least 58 genes are associated with an autosomal recessive form of RP. Proteins encoded by these genes are involved in many different functional pathways, including photoreceptor development, phototransduction, retinoid cycle, etc. RP is characterized by rod photoreceptor degeneration and subsequent cone degeneration. Patients often present with nyctalopia (night blindess) and peripheral visual field constriction. REEP6 is highly expressed in rod photoreceptor cells and encodes a putative endoplasmic reticulum shaping factor. Biallelic mutations in REEP6 were identified in 5 unrelated RP patients and are predicted to be loss-of- function variants. > NEXT

Answer the following questions in accordance with the SOP based on the information presented.
Using whole exome sequencing (WES), mutations in REEP6 were identified in an Asian male with RP from a non-consanguineous family who had tested negative for mutations in 226 genes known to be associated with inherited retinal disease. The individual was compound heterozygous for a missense variant (c.404T>C [p.Leu135Pro]) and a single-nucleotide deletion in exon 4 (c.448del [p.Ala150Profs*2]). The close proximity of the two variants (44 bp) allowed the authors to confirm the biallelic state using the Integrative Genomics Viewer (C, below). Based on this information, how would you score this proband? A. Two variants in trans and at least one de novo or a predicted/proven null variant B. Two variants (not predicted/proven null) with some evidence of gene impact in trans C. Do not score this case

A. Two variants in trans and at least one de novo or a predicted/proven null variant (Correct)
Using whole exome sequencing (WES), mutations in REEP6 were identified in an Asian male with RP from a non-consanguineous family who had tested negative for mutations in 226 genes known to be associated with inherited retinal disease. The individual was compound heterozygous for a missense variant (c.404T>C [p.Leu135Pro]) and a single-nucleotide deletion in exon 4 (c.448del [p.Ala150Profs*2]). The close proximity of the two variants (44 bp) allowed the authors to confirm the biallelic state using the Integrative Genomics Viewer (C, below). Based on this information, how would you score this proband? The single-nucleotide deletion causes a frameshift, resulting in a predicted loss of function variant. Default is 2 points. > NEXT

B. Two variants (not predicted/proven null) with some evidence of gene impact in trans (Incorrect)
Using whole exome sequencing (WES), mutations in REEP6 were identified in an Asian male with RP from a non-consanguineous family who had tested negative for mutations in 226 genes known be associated with inherited retinal disease. The individual was compound heterozygous for a missense variant (c.404T>C [p.Leu135Pro]) and a single-nucleotide deletion in exon 4 (c.448del [p.Ala150Profs*2]). The close proximity of the two variants (44 bp) allowed the authors to confirm the biallelic state using the Integrative Genomics Viewer (C, below). Based on this information, how would you score this proband? See Correct Answer

C. Do not score this case See Correct Answer
Using whole exome sequencing (WES), mutations in REEP6 were identified in an Asian male with RP from a non-consanguineous family who had tested negative for mutations in 226 genes known to be associated with inherited retinal disease. The individual was compound heterozygous for a missense variant (c.404T>C [p.Leu135Pro]) and a single-nucleotide deletion in exon 4 (c.448del [p.Ala150Profs*2]). The close proximity of the two variants (44 bp) allowed the authors to confirm the biallelic state using the Integrative Genomics Viewer (C, below). Based on this information, how would you score this proband? See Correct Answer

C. A is correct, but reduce points
WES was performed on another individual (B-II:8), one of three male siblings with RP born to consanguineous parents of African descent. This individual was negative for mutations in 176 genes known be associated with inherited retinal disease. A homozygous variant in REEP6 was identified. This variant is intronic in the canonical transcript, but it is located within exon 5 of a retinal-specific REEP6 transcript (REEP6.1, c.557dup [p.Val187Glyfs*13]). REEP6.1 includes an additional exon coding for 27 amino acid residues and is the predominant isoform (75%) present in mature optic cups. The variant is predicted to cause a frameshift leading to 12 novel amino acid residues followed by a premature stop codon that only affects REEP6.1 and is expected to undergo nonsense mediated decay (NMD). The authors transiently transfected SK-N-SH neuroblastoma cells with p.Val187Glyfs*13 REEP6.1 and observed reduced protein levels compared to wild type (red arrow, figure B). Based on this information, how would you score this proband? A. Two variants in trans and at least one de novo or a predicted/proven null variant B. Two variants (not predicted/proven null) with some evidence of gene impact in trans C. A is correct, but reduce points

A. Two variants in trans and at least one de novo or a predicted/proven null variant (Incorrect)
WES was performed on another individual (B-II:8), one of three male siblings with RP born to consanguineous parents of African descent. This individual was negative for mutations in 176 genes known be associated with inherited retinal disease. A homozygous variant in REEP6 was identified. This variant is intronic in the canonical transcript, but it is located within exon 5 of a retinal-specific REEP6 transcript (REEP6.1, c.557dup [p.Val187Glyfs*13]). REEP6.1 includes an additional exon coding for 27 amino acid residues and is the predominant isoform (75%) present in mature optic cups. The variant is predicted to cause a frameshift leading to 12 novel amino acid residues followed by a premature stop codon that only affects REEP6.1 and is expected to undergo nonsense mediated decay (NMD). The authors transiently transfected SK-N-SH neuroblastoma cells with p.Val187Glyfs*13 REEP6.1 and observed reduced protein levels compared to wild type (red arrow, figure B). Based on this information, how would you score this proband? See Correct Answer

WES was performed on another individual (B-II:8), one of three male siblings with RP born to consanguineous parents of African descent. This individual was negative for mutations in 176 genes known be associated with inherited retinal disease. A homozygous variant in REEP6 was identified. This variant is intronic in the canonical transcript, but it is located within exon 5 of a retinal-specific REEP6 transcript (REEP6.1, c.557dup [p.Val187Glyfs*13]). REEP6.1 includes an additional exon coding for 27 amino acid residues and is the predominant isoform (75%) present in mature optic cups. The variant is predicted to cause a frameshift leading to 12 novel amino acid residues followed by a premature stop codon that only affects REEP6.1 and is expected to undergo nonsense mediated decay (NMD). The authors transiently transfected SK-N-SH neuroblastoma cells with p.Val187Glyfs*13 REEP6.1 and observed reduced protein levels compared to wild type (red arrow, figure B). Based on this information, how would you score this proband? See Correct Answer

C. A is correct, but reduce points (Correct)
WES was performed on another individual (B-II:8), one of three male siblings with RP born to consanguineous parents of African descent. This individual was negative for mutations in 176 genes known be associated with inherited retinal disease. A homozygous variant in REEP6 was identified. This variant is intronic in the canonical transcript, but it is located within exon 5 of a retinal-specific REEP6 transcript (REEP6.1, c.557dup [p.Val187Glyfs*13]). REEP6.1 includes an additional exon coding for 27 amino acid residues and is the predominant isoform (75%) present in mature optic cups. The variant is predicted to cause a frameshift leading to 12 novel amino acid residues followed by a premature stop codon that only affects REEP6.1 and is expected to undergo nonsense mediated decay (NMD). The authors transiently transfected SK-N-SH neuroblastoma cells with p.Val187Glyfs*13 REEP6.1 and observed reduced protein levels compared to wild type (red arrow, figure B). Based on this information, how would you score this proband? Although the canonical transcript is unaffected, this variant is predicted to result in a truncation of the retinal isoform. Given that the disease is specific to the retina, it would still be appropriate to score this as a predicted null variant. However, the authors show that some protein is produced in SK-N-SH neuroblastoma cells expressing the variant even though the protein is expected to undergo NMD. According to the SOP, you should assign fewer points for a predicted null variant if a gene product is still made, albeit altered. Note: for cases in which the proband is homozygous for a variant, the parents do not need to be genotyped to confirm that they are in trans. > NEXT

C. A is correct, but discuss with an expert
Examination of whole genome sequencing (WGS) data from 599 unrelated individuals with inherited retinal disease revealed a large homozygous genomic rearrangement encompassing exon 1 of REEP6 in an affected individual (C-II:1) without mutations in other genes associated with retinal disease. The authors determined that there was a deletion of 6,886 bp with an insertion of an inverted 158 bp segment from the middle of the deleted region. The deletion spans exons 1-8 of PCSK4 on the reverse strand and REEP6 exon 1 on the forward strand and is predicted to abolish REEP6 and PCSK4 expression. PCSK4 is not associated with any disease in OMIM, and is predominantly expressed in placental tissue. The authors note that there were no extra-ocular findings attributable to loss of PCSK4. Based on this information, how would you score this proband? A. Two variants in trans and at least one de novo or a predicted/proven null variant B. Two variants (not predicted/proven null) with some evidence of gene impact in trans C. A is correct, but discuss with an expert

Examination of whole genome sequencing (WGS) data from 599 unrelated individuals with inherited retinal disease revealed a large homozygous genomic rearrangement encompassing exon 1 of REEP6 in an affected individual (C-II:1) without mutations in other genes associated with retinal disease. The authors determined that there was a deletion of 6,886 bp with an insertion of an inverted 158 bp segment from the middle of the deleted region. The deletion spans exons 1-8 of PCSK4 on the reverse strand and REEP6 exon 1 on the forward strand and is predicted to abolish REEP6 and PCSK4 expression. PCSK4 is not associated with any disease in OMIM, and is predominantly expressed in placental tissue. The authors note that there were no extra-ocular findings attributable to loss of PCSK4. Based on this information, how would you score this proband? See Correct Answer

C. A is correct, but discuss with an expert (Correct)
Examination of whole genome sequencing (WGS) data from 599 unrelated individuals with inherited retinal disease revealed a large homozygous genomic rearrangement encompassing exon 1 of REEP6 in an affected individual (C-II:1) without mutations in other genes associated with retinal disease. The authors determined that there was a deletion of 6,886 bp with an insertion of an inverted 158 bp segment from the middle of the deleted region. The deletion spans exons 1-8 of PCSK4 on the reverse strand and REEP6 exon 1 on the forward strand and is predicted to abolish REEP6 and PCSK4 expression. PCSK4 is not associated with any disease in OMIM, and is predominantly expressed in placental tissue. The authors note that there were no extra-ocular findings attributable to loss of PCSK4. Based on this information, how would you score this proband? The genomic rearrangement affects another gene, PCSK4, but there is no evidence to show that the loss of PCSK4 is relevant to the phenotype of the proband or that it is involved in retinal diseases. However, you should discuss with an expert whether or not to count the case or reduce points. > NEXT

Direct sequencing of all exons of REEP6 in 400 RP-affected individuals with unknown genetic etiology revealed two additional individuals (D-II:5 and E-II:1) with homozygous variants in REEP6. Individual D-II:5 was found to harbor a homozygous missense mutation in exon 4 (c.383C>T [p.Pro128Leu]), which affects a conserved residue and is predicted to be pathogenic by in silico tools. The authors transiently transfected SK-N-SH neuroblastoma cells with p.Pro128Leu REEP6 and observed reduced protein levels compared to wild type. How would you score proband D-II:5 based on this information? A. Two variants in trans and at least one de novo or a predicted/proven null variant B. Two variants (not predicted/proven null) with some evidence of gene impact in trans C. Two variants (not predicted/proven null) without sufficient evidence of impact

Direct sequencing of all exons of REEP6 in 400 RP-affected individuals with unknown genetic etiology revealed two additional individuals (D-II:5 and E-II:1) with homozygous variants in REEP6. Individual D-II:5 was found to harbor a homozygous missense mutation in exon 4 (c.383C>T [p.Pro128Leu]), which affects a conserved residue and is predicted to be pathogenic by in silico tools. The authors transiently transfected SK-N-SH neuroblastoma cells with p.Pro128Leu REEP6 and observed reduced protein levels compared to wild type. How would you score proband D-II:5 based on this information? See Correct Answer

B. Two variants (not predicted/proven null) with some evidence of gene impact in trans (Correct)
Direct sequencing of all exons of REEP6 in 400 RP-affected individuals with unknown genetic etiology revealed two additional individuals (D-II:5 and E-II:1) with homozygous variants in REEP6. Individual D-II:5 was found to harbor a homozygous missense mutation in exon 4 (c.383C>T [p.Pro128Leu]), which affects a conserved residue and is predicted to be pathogenic by in silico tools. The authors transiently transfected SK-N-SH neuroblastoma cells with p.Pro128Leu REEP6 and observed reduced protein levels compared to wild type. How would you score proband D-II:5 based on this information? Impact based on in silico predictions alone would be insufficient evidence for assigning default points for a missense variant. However, the authors provided evidence demonstrating that the missense variant impacts the protein expression. Therefore, this proband should receive the default score (1 point). > NEXT

C. Two variants (not predicted/proven null) without sufficient evidence of impact (Incorrect)
Direct sequencing of all exons of REEP6 in 400 RP-affected individuals with unknown genetic etiology revealed two additional individuals (D-II:5 and E-II:1) with homozygous variants in REEP6. Individual D-II:5 was found to harbor a homozygous missense mutation in exon 4 (c.383C>T [p.Pro128Leu]), which affects a conserved residue and is predicted to be pathogenic by in silico tools. The authors transiently transfected SK-N-SH neuroblastoma cells with p.Pro128Leu REEP6 and observed reduced protein levels compared to wild type. How would you score proband D-II:5 based on this information? See Correct Answer

Direct sequencing of all exons of REEP6 in 400 RP-affected individuals with unknown genetic etiology revealed two additional individuals (D-II:5 and E-II:1) with homozygous variants in REEP6. Individual E-II:1 was found to be homozygous for a two-nucleotide deletion in exon 3 (c.279_280del) resulting in a frameshift that extends beyond the stop codon in the canonical transcript (p.Leu94Valfs*320) and a premature termination of the retinal transcript (p.Leu94Valfs*86). How would you score proband E-II:1 based on this information? A. Two variants in trans and at least one de novo or a predicted/proven null variant B. Two variants (not predicted/proven null) with some evidence of gene impact in trans C. Do not score this case

A. Two variants in trans and at least one de novo or a predicted/proven null variant (Correct)
Direct sequencing of all exons of REEP6 in 400 RP-affected individuals with unknown genetic etiology revealed two additional individuals (D-II:5 and E-II:1) with homozygous variants in REEP6. Individual E-II:1 was found to be homozygous for a two-nucleotide deletion in exon 3 (c.279_280del) resulting in a frameshift that extends beyond the stop codon in the canonical transcript (p.Leu94Valfs*320) and a premature termination of the retinal transcript (p.Leu94Valfs*86). How would you score proband E-II:1 based on this information? Similar to individual B-II:8, this variant is predicted to result in a truncation of the retinal isoform, but has a different effect on the canonical isoform. Given that the disease is specific to the retina, it would still be appropriate to give this proband default points for a predicted null variant. > NEXT

Direct sequencing of all exons of REEP6 in 400 RP-affected individuals with unknown genetic etiology revealed two additional individuals (D-II:5 and E-II:1) with homozygous variants in REEP6. Individual E-II:1 was found to be homozygous for a two-nucleotide deletion in exon 3 (c.279_280del) resulting in a frameshift that extends beyond the stop codon in the canonical transcript (p.Leu94Valfs*320) and a premature termination of the retinal transcript (p.Leu94Valfs*86). How would you score proband E-II:1 based on this information? See Correct Answer

C. Do not score this case (Incorrect)
Direct sequencing of all exons of REEP6 in 400 RP-affected individuals with unknown genetic etiology revealed two additional individuals (D-II:5 and E-II:1) with homozygous variants in REEP6. Individual E-II:1 was found to be homozygous for a two-nucleotide deletion in exon 3 (c.279_280del) resulting in a frameshift that extends beyond the stop codon in the canonical transcript (p.Leu94Valfs*320) and a premature termination of the retinal transcript (p.Leu94Valfs*86). How would you score proband E-II:1 based on this information? See Correct Answer

Pedigrees for Families A-E are shown below. Proband II
Pedigrees for Families A-E are shown below. Proband II.1 from Family A is compound heterozygous for REEP6 variants (M1/M2), while all other affected individuals are homozygous for the respective REEP6 variants indicated (M3, M4, M5, M6). Are there enough segregations in any of the families to calculate an estimated LOD score? YES NO For recessive disorders, the estimated LOD score should be calculated using only families with at least 3 affected individuals who have been genotyped. None of the families meet this requirement, so you should not assign segregation points. > NEXT

Summary of variants > NEXT Probands Variant (HGVS nomenclature)
Type of variant gnomAD Frequency Summary of evidence Suggested points Family A, II:1 NM_ (REEP6): c.404T>C (p.Leu135Pro) and c.448del (p.Ala150Profs*2) Missense, 1 bp deletion (compound heterozygous) Not in gnomAD. 1 bp deletion is a predicted LOF. Mouse model supports pathogenicity of missense variant. 2 (default) Family B, II:8 NM_ (REEP6.1): c.557dup (p.Val187Glyfs*13) Frameshift (homozygous) Intronic in the canonical transcript, but results in a premature stop codon in retinal-specific transcript. In vitro assay shows some protein expression. 1 (reduced from default) Family C, II:1 NM_ (REEP6): c.-5835_ delins Genomic rearrangement (homozygous) Not present in in-house dataset of 4,500 WGS samples. Large deletion with an insertion of an inverted segment from the middle of the deleted region. Also spans exons 1-8 of PCSK4. Predicted LOF. 2 (default; pending discussion with expert) Family D, II:5 NM_ (REEP6): c.383C>T (p.Pro128Leu) Missense (homozygous) Transfection of the variant in SK-N-SH neuroblastoma cells causes a reduction in protein. 1 (default) Family E,II:1 NM_ (REEP6): c.279_280del (p.Leu94Valfs*320) Max MAF (East Asian) = 1.7x10-4 (3/17238), Overall MAF = 1.2x10-5 (3/245785). Extends beyond stop codon in the canonical transcript and a premature termination in the retinal transcript. > NEXT

Genetic Evidence Summary Matrix
Note: This summary only applies to the paper presented in this training module. There may be additional papers that could be relevant to the curation. Case Level Data Evidence Type Case Information Type Suggested points/case Points given Max Score Default Range Variant Evidence Autosomal Dominant OR X-Linked Disorder Variant is de novo 2 0-3 NA 12 Proband with predicted or proven null variant 1.5 0-2 10 Proband with other variant type with some evidence of gene impact 0.5 0-1.5 7 Autosomal Recessive Disease Two variants in trans and at least one de novo or a predicted/proven null variant Two variants (not predicted/proven null) with some evidence of gene impact in trans 1 Segregation Evidence Evidence of segregation in one or more families Sequencing Method 3 Total LOD Score Candidate Gene Sequencing Exome/Genome or all gene sequenced in linkage region 2-2.99 3-4.99 ≥5 Case Control Data Case-Control Study Type Case-Control Quality Criteria Suggested points/study Single Variant Analysis Variant Detection Methodology Power Bias and Confounding Factors Statistical Significance 0-6 Aggregate Variant Analysis Total Genetic Evidence Points (Maximum 12): 8 > NEXT

Functional Alteration
To examine the expression of the canonical transcript and the retina-specific transcript (REEP6.1) in human tissue, iPSC-derived 3D organoid optic cups were used. The authors used RT-PCR analysis to observe the expression of the isoforms over the course of optic cup differentiation. They found that the expression of REEP6.1 correlated with photoreceptor development (A) and was the predominant isoform present in mature optic cups. The authors also referenced another study in which the authors found that REEP6.1 is specifically expressed in rod photoreceptors of developing and mature retina in mice (Hao, 2014, PMID ). How would you curate this evidence? Click on the boxes below. (Note: for an actual curation, you should curate the evidence from Hao, 2014 directly.) Biochemical function Function Functional Alteration Protein interaction (A) Reverse-transcription PCR analysis of REEP6.1 and 6.2 isoforms in control adult fibroblasts (fibs) and control BJ optic cups during different weeks (wk) of photoreceptor differentiation. GAPDH used a loading control. Models & Rescue Expression This evidence should be counted under Function: Expression. From the SOP: “the gene is expressed in tissues relevant to the disease of interest.” Given the specificity of the REEP6.1 transcript to the retina, this should count as evidence for an involvement in retinal degeneration disease such as retinitis pigmentosa. Default is 0.5 points, but it may be appropriate to increase points to 1 due to the specificity of expression. > NEXT

Functional Alteration Cell Culture Model System
To examine the function of the p.Leu135Pro variant in vivo, a mouse knock-in model of Reep6 p.Leu135Pro was generated using CRISPR-Cas9 technology. The homozygous knock-in mice recapitulate the human disease phenotype, including progressive photoreceptor degeneration and dysfunction of rod photoreceptors. How would you curate this evidence? Click on the boxes below. (A) Histological analysis of retinal sections from Reep6+/L135P (left) and Reep6L135P/L135P (right) mice was performed at 4 months and 6 months of age. Reep6+/L135P retina shows normal layered organization compared to the Reep6L135P/L135P retina that shows moderate thinning of the outer nuclear layer (ONL) at 4 months and marked thinning at 6 months of age. “INL” indicates inner nuclear layer. Scale bars represent 40 μm. (B) TEM images of P20 and 6-month-old retina from Reep6+/L135P control retina and Reep6L135P/L135P mutant retina shows thinning of ONL, outer segment (OS), and inner segment (IS) at 6 months of age indicative of progressive retinal degeneration. Scale bars represent 2 μm (top) and 10 μm (bottom). (C) Quantitative evaluation of scotopic a-wave and b-wave and photopic b-wave amplitude data for 4-month-old Reep6+/L135P control retina and Reep6L135P/L135P mutant mice. Function Animal Model Functional Alteration Cell Culture Model System Models & Rescue Rescue Change text This evidence should be counted under Models & Rescue: Animal Model. From the SOP: “a non-human animal with a disrupted copy of the gene shows a phenotype consistent with the human disease state.” The number of points will depend on the quality of the animal model upon further analysis with an expert. > NEXT

Recommended points/ evidence
Experimental Evidence Summary Matrix Evidence Category Evidence Type Score Range Recommended points/ evidence Points Given Max Score Function Biochemical Function 0-2 0.5 NA 2 Protein Interaction Expression 1 Functional Alteration Patient cells 1 - 2 Non-patient cells ½ - 1 Models & Rescue Animal model 2 - 4 2 (TBD) 4 Cell culture model system ½ - 2 Rescue in animal model Rescue in engineered equivalent Total Final Score 3 6 Note: This summary only applies to the paper presented in this training module. There may be additional papers that could be relevant to the curation. > NEXT

12-18 AND replication over time
Assertion criteria Genetic Evidence (0-12 points) Experimental Evidence (0-6 points) Total Points (0-18) Replication Over Time (Y/N) Description Case-level, family segregation, or case-control data that support the gene-disease association Gene-level experimental evidence that support the gene-disease association Sum of Genetic & Experimental Evidence > 2 pubs w/ convincing evidence over time (>3 yrs) Assigned Points 8 3 11 N CALCULATED CLASSIFICATION LIMITED 1-6 MODERATE 7-11 STRONG 12-18 DEFINITIVE 12-18 AND replication over time Valid contradictory evidence? List PMIDs and describe evidence: CURATOR CLASSIFICATION Moderate FINAL CLASSIFICATION > NEXT Note: This summary only applies to the paper presented in this training module. There may be additional papers that could be relevant to the curation.

Email Jen McGlaughon at jen_mcglaughon@med.unc.edu
Questions or comments? Jen McGlaughon at

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