LRP6: Non-syndromic Oligodontia PMID: 26387593 > START

LRP6: Non-syndromic Oligodontia, Training Module To complete this module, you will need: The Gene Clinical Validity Curation Process Standard Operating Procedure, Version 4 Outline of the module: Background of the disease Questions and answers on genetic evidence and experimental evidence related to the SOP. Clinical validity summary > NEXT

Background Staal and Clevers, 2005 Oligodontia is defined as the absence of 6 or more permanent teeth and can be isolated or part of a syndrome. Variants in WNT10A, which is part of the canonical Wnt/β-catenin signaling pathway, were recently identified as a major contributor to non- syndromic oligodontia (van den Boogaard et al., 2012). In this study, whole-exome sequencing (WES) was performed on a cohort of 20 non-syndromic oligodontia cases to identify new candidate genes. Variants were excluded in known non-syndromic oligodontia genes prior to WES: WTN10A, MSX1, PAX1, IRF6, EDA, and AXIN2. The authors identified 4 unrelated individuals with variants in LRP6, which encodes a transmembrane cell-surface protein that functions as a co- receptor with Frizzled proteins in the Wnt/β-catenin signaling pathway. Inheritance follows an autosomal dominant pattern. > NEXT

Answer the following questions in accordance with the SOP based on the information presented. Whole-exome sequencing was performed on 20 unrelated individuals with non-syndromic oligodontia. An additional 8 patients without causative variants in non-syndromic oligodontia genes were analyzed by Sanger sequencing. Four heterozygous variants in LRP6 were identified in four unrelated individuals. All four variants were absent from variation databases: dbSNP, 1000 Genomes, NHLBI Exome Project, and ExAC. A nonsense variant (c.1779dupT [p.Glu594*]) and two insertion variants (c.2224_225dupTT [p.Leu742Phefs*7], c.1144_1145dupAG [p.Ala383Glyfs*8]) in individuals from familes F2, F3 and F4, respectively, result in a premature stop codon in the middle of the protein. The resulting transcripts likely undergo nonsense-mediated mRNA decay. How would you score these 3 cases? Select answer below. Proband with predicted/proven null variant (1.5 default points each) Proband with other variant type with some evidence of gene impact (0.5 default points each) Functional evidence is required to score default points

A. Proband with predicted/proven null variant (1 A. Proband with predicted/proven null variant (1.5 default points each) (Correct) Whole-exome sequencing was performed on 20 unrelated individuals with non-syndromic oligodontia. An additional 8 patients without causative variants in non-syndromic oligodontia genes were analyzed by Sanger sequencing. Four heterozygous variants in LRP6 were identified in four unrelated individuals. All four variants were absent from variation databases: dbSNP, 1000 Genomes, NHLBI Exome Project, and ExAC. A nonsense variant (c.1779dupT [p.Glu594*]) and two insertion variants (c.2224_225dupTT [p.Leu742Phefs*7], c.1144_1145dupAG [p.Ala383Glyfs*8]) in individuals from familes F2, F3 and F4, respectively, result in a premature stop codon in the middle of the protein. The resulting transcripts likely undergo nonsense-mediated mRNA decay. How would you score these 3 cases? Select answer below. Since all three variants result in a premature stop codon and a transcript that undergoes nonsense-mediated decay, these cases can be categorized as probands with a predicted or proven null variant. The default points are 1.5 each, for a total of 4.5 points. > NEXT

B. Proband with other variant type with some evidence of gene impact (0.5 default points each) (Incorrect) Whole-exome sequencing was performed on 20 unrelated individuals with non-syndromic oligodontia. An additional 8 patients without causative variants in non-syndromic oligodontia genes were analyzed by Sanger sequencing. Four heterozygous variants in LRP6 were identified in four unrelated individuals. All four variants were absent from variation databases: dbSNP, 1000 Genomes, NHLBI Exome Project, and ExAC. A nonsense variant (c.1779dupT [p.Glu594*]) and two insertion variants (c.2224_225dupTT [p.Leu742Phefs*7], c.1144_1145dupAG [p.Ala383Glyfs*8]) in individuals from familes F2, F3 and F4, respectively, result in a premature stop codon in the middle of the protein. The resulting transcripts likely undergo nonsense-mediated mRNA decay. How would you score these 3 cases? Select answer below. See Correct Answer

C. Functional evidence is required to score default points (Incorrect) Whole-exome sequencing was performed on 20 unrelated individuals with non-syndromic oligodontia. An additional 8 patients without causative variants in non-syndromic oligodontia genes were analyzed by Sanger sequencing. Four heterozygous variants in LRP6 were identified in four unrelated individuals. All four variants were absent from variation databases: dbSNP, 1000 Genomes, NHLBI Exome Project, and ExAC. A nonsense variant (c.1779dupT [p.Glu594*]) and two insertion variants (c.2224_225dupTT [p.Leu742Phefs*7], c.1144_1145dupAG [p.Ala383Glyfs*8]) in individuals from familes F2, F3 and F4, respectively, result in a premature stop codon in the middle of the protein. The resulting transcripts likely undergo nonsense-mediated mRNA decay. How would you score these 3 cases? Select answer below. See Correct Answer

Segregation analysis for the 3 probands with predicted null variants showed a family history of oligodontia. Incomplete penetrance for oligodontia with an LRP6 variant was shown only in family 4 (F4), indicated by the proband’s genotype-positive, unaffected mother. Reduced penetrance and variable expression for this disease has been observed previously (Nieminen, 2009, PMID: 19219933). Can you count segregation points for any of these families? Select answer below. Note: taurodontism was found in one third of affected individuals. Taurodontism is a developmental disturbance of the tooth in which the body of the tooth and pulp chamber is enlarged at the expense of the roots. Taurodontism is also associated with WNT10A mutations. -recommendation from low penetrance group- ask Bryce YES NO In order to calculate an estimated LOD score using the formula in the SOP for dominant disorders, 4 or more segregations must be present in the family. The families here have only 1 or 2 segregations. > NEXT

In addition to the 3 patients with loss-of-function variants in LRP6, the authors identified a fourth patient with a missense variant (c.56C>T [p.Ala19Val]) located at the cleavage site of the signal peptide of the protein. The SignalP 4.1 server predicts that this amino acid change results in the loss of the cleavage site (see figures below). The variant is also absent from variation databases: dbSNP, 1000 Genomes, NHLBI Exome Project, and ExAC. Without any additional information about this missense variant, how would you score the proband? Select answer below. Proband with predicted/proven null variant (1.5 default points) Proband with other variant type with some evidence of gene impact (0.5 default points) B is correct but reduce points.

A. Proband with predicted/proven null variant (1 A. Proband with predicted/proven null variant (1.5 default points) (Incorrect) In addition to the 3 patients with loss-of-function variants in LRP6, the authors identified a fourth patient with a missense variant (c.56C>T [p.Ala19Val]) located at the cleavage site of the signal peptide of the protein. The SignalP 4.1 server predicts that this amino acid change results in the loss of the cleavage site (see figures below). The variant is also absent from variation databases: dbSNP, 1000 Genomes, NHLBI Exome Project, and ExAC. Without any additional information about this missense variant, how would you score the proband? Select answer below. See Correct Answer

B. Proband with other variant type with some evidence of gene impact (0.5 default points) (Incorrect) In addition to the 3 patients with loss-of-function variants in LRP6, the authors identified a fourth patient with a missense variant (c.56C>T [p.Ala19Val]) located at the cleavage site of the signal peptide of the protein. The SignalP 4.1 server predicts that this amino acid change results in the loss of the cleavage site (see figures below). The variant is also absent from variation databases: dbSNP, 1000 Genomes, NHLBI Exome Project, and ExAC. Without any additional information about this missense variant, how would you score the proband? Select answer below. See Correct Answer

C. B is correct but reduce points (Correct) In addition to the 3 patients with loss-of-function variants in LRP6, the authors identified a fourth patient with a missense variant (c.56C>T [p.Ala19Val]) located at the cleavage site of the signal peptide of the protein. The SignalP 4.1 server predicts that this amino acid change results in the loss of the cleavage site (see figures below). The variant is also absent from variation databases: dbSNP, 1000 Genomes, NHLBI Exome Project, and ExAC. Without any additional information about this missense variant, how would you score the proband? Select answer below. The SOP states that for variants other than null, “at least some impact to gene function must be demonstrated for the case to count. Thus, impact based on predictions only would score less than the default.” If the authors do not show functional data for the variant impact, reduce points from the default. > NEXT

Segregation analysis for the proband with the missense variant also showed a family history of oligodontia. Using the pedigree below, can you count segregation points for this family? Select answer below. 1 2 3 YES NO In order to calculate an estimated LOD score using the formula in the SOP for dominant disorders, 4 or more segregations must be present in the family. The family in this case only shows 3 segregations (see above). If one the individuals in the first generation (I:1 or I:2) had been tested and genotype-positive, you could count another segregation. However, since those individuals were not tested, you cannot exclude the possibility of mosaicism. > NEXT

Variant Impact Experimental > NEXT In order to gain insight into the molecular mechanisms by which the missense variant (c.56C>T [p.Ala19Val]) affects the function of LRP6, the authors performed a series of experiments. For each of the experiments described, indicate whether you would count it towards variant-level evidence (and therefore upgrade the points assigned to the proband) or experimental evidence for the gene-disease relationship. Experiment 1: HEK293T cells were transfected with increasing amounts of Lrp6-GFP or Lrp6 p.Ala19Val-GFP and analyzed by immunoblotting. Wild type Lrp6-GFP was processed to the fully glycosylated mature form, while Lrp6 p.Ala19Val-GFP displayed only a single band of lower molecular weight, likely representing the immature high-mannose precursor that is generated in the endoplasmic reticulum (ER). Also, the amount of Lrp6 p.Ala19Val-GFP was significantly lower than wild type Lrp6-GFP. Altered processing and reduced levels of Lrp6 demonstrate that the variant has some impact on the protein’s normal function. It does not necessarily relate to the gene-disease relationship and should not be counted under experimental evidence. Variant Impact Experimental > NEXT

Variant Impact Experimental > NEXT In order to gain insight into the molecular mechanisms by which the missense variant (c.56C>T [p.Ala19Val]) affects the function of LRP6, the authors performed a series of experiments. For each of the experiments described, indicate whether you would count it towards variant-level evidence (and therefore upgrade the points assigned to the proband) or experimental evidence for the gene-disease relationship. Experiment 2: To determine whether Lrp6 p.Ala19Val is retained in the ER, the authors determined its sensitivity to Endoglycosidase H (EndoH) activity, which cleaves high-mannose glycans that are added to nascent proteins in the ER. When proteins enter the Golgi, glycan modifications occur which render them resistant to EndoH. HEK293T cells were transfected with Lrp6-GFP or Lrp6 p.Ala19Val-GFP, incubated with EndoH, and analyzed by immunoblotting. Low molecular weight protein bands of both wild type and Lrp6 p.Ala19Val were sensitive to EndoH, whereas the mature form of wild type Lrp6 remained unaffected (B). This indicates that the missense variant causes retention of the nascent Lrp6 in the ER. Using confocal microscopy, they demonstrated that while the wild type Lrp6 protein is found at the cell surface, Lrp6 p.Ala19Val does not reach the plasma membrane and co-localizes with KDEL, an ER marker (C). Retention of Lrp6 in the ER demonstrates that the variant has some impact on the protein’s normal function and/or localization. It does not necessarily relate to the gene-disease relationship and should not be counted under experimental evidence. Variant Impact Experimental > NEXT

Variant Impact Experimental > NEXT In order to gain insight into the molecular mechanisms by which the missense variant (c.56C>T [p.Ala19Val]) affects the function of LRP6, the authors performed a series of experiments. For each of the experiments described, indicate whether you would count it towards variant-level evidence (and therefore upgrade the points assigned to the proband) or experimental evidence for the gene-disease relationship. Experiment 3: Like WNT10A, which has been implicated in non-syndromic oligodondia, Lrp6 is a component of the canonical Wnt pathway. Therefore, the authors determined whether the missense variant altered its ability to activate the Wnt pathway using the TOPFlash luciferase assay. HEK293T cells were transfected with increasing amounts of Lrp6-GFP or Lrp6 p.Ala19Val. Unlike wild type, altered Lrp6 failed to initiate β-catenin-mediated transcription, either alone or in the presence of Fzd5 (D), which is known to act downstream of Lrp6 in the Wnt pathway, indicating an inability of Lrp6 p.Ala19Val to activate the Wnt pathway. Similarly, Lrp6 p.Ala19Val-GFP expressing cells were unable to induce enhanced Wnt pathway activation upon stimulation with exogenous Wnt3a (E). *additional experiments showing similar results in the paper, consider upgrading Since the Wnt pathway has been directly linked to the disorder, and the authors are showing that an alteration in Lrp6 leads to aberrant Wnt signaling, this evidence strengthens the gene-disease relationship and should be counted under experimental evidence (functional alteration: non-patient cells). Also, since the authors thoroughly demonstrated that Wnt signaling is altered and cannot be rescued by adding the ligand or co-receptor, consider increasing points from the default to 1. Variant Impact Experimental > NEXT

Summary of variants > NEXT Affected Individuals Variant (HGVS nomenclature) Segregation gnomAD Frequency ClinVar Assertion Summary Suggested Points F1, III:1 NM_002336.2(LRP6):c.56C>T (p.Ala19Val) 3 seg, 0 points Total: 1/245,098 (4.08e-6) Although present in a control database, it is at a low enough frequency to be scored as possibly disease-causing. Pathogenic, no assertion criteria Missense variant, protein is retained in the ER in immature form 1 F2, II:1 NM_002336.2(LRP6):c.1779dupT (p.Glu594Terfs) 1 seg, 0 points Not in gnomAD Nonsense variant 1.5 F3, II:2 NM_002336.2(LRP6):c.2224_2225dupTT (p.Leu742Phefs 2 seg, 0 points Insertion, premature stop codon F4, II:1 NM_002336.2(LRP6):c.1144_1145dupAG p.Ala383Glyfs*8 0 seg, 0 points Not in ClinVar *1.5 (see note below) Note: Although family 4 showed incomplete penetrance, you should still count this proband. Incomplete penetrance is consistent with previous reports on this disease. It may also be beneficial to consult a disease expert. > NEXT

Genetic Evidence Summary Matrix 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 4.5 10 Proband with other variant type with some evidence of gene impact 0.5 0-1.5 1 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 Segregation Evidence Evidence of segregation in one or more families LOD Score Examples 3 5 0-7 4 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): 5.5 > 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.

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 Functional Alteration Patient cells 1 - 2 1 Non-patient cells ½ - 1 Models & Rescue Animal model 2 - 4 4 Cell culture model system ½ - 2 Rescue in animal model Rescue in engineered equivalent Total Final Score 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 5.5 1 6.5 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 Limited to 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? Email Jen McGlaughon at jen_mcglaughon@med.unc.edu