KLHL24: Generalized epidermolysis bullosa simplex (skin fragility) PMID: 27889062 > START

KLHL24: Skin Fragility, Training Module To complete this module, you will need: The Gene Clinical Validity Curation Process Standard Operating Procedure, Version 4 He et al., 2016 (PMID: 27889062) Outline of the module: Genetic evidence: description of individuals with variants in KLHL24 (case-level data) Experimental evidence: functional assays that implicate the role of KLHL24 in EB simplex Clinical validity summary > NEXT

Genetic Evidence: Methods Whole- exome sequencing identified monoallelic variants in KLHL24 in 14 individuals from 10 families with genetically unsolved EB simplex. Variants validated by Sanger sequencing. Genes involved in EB were sequenced in index patients; no pathogenic variants were found. c.1A>G (p.Met1Val) was identified in 9 families; c.2T>C (p.Met1Thr) identified in 1 family. Both variants were absent in healthy controls from dbSNP, 1000 Genomes and ExAC. The variants were predicted to shift the start codon, resulting in a 28 amino acid truncation. Recombinant expression of normal and mutant KLHL24 cDNA in HEK293 cells confirmed the truncation, suggesting Met29 was used for initiation of translation (p.Val2_Met29del). > NEXT

c.1A>G (p.Met1Val): In which of these families would the variant be considered de novo? Click on answer below. Black-filled symbols indicate affected individuals, all with a monoallelic KLHL24 variant. Individuals marked with “-” lack the variant. Individuals marked with “nt” were not tested. Check this. All of the above. Families A, B, F, G, I, and J. Families G, H, and I. None of the above.

A. All of the above (Incorrect) Black-filled symbols indicate affected individuals, all with a monoallelic KLHL24 variant. Individuals marked with “-” lack the variant. Individuals marked with “nt” were not tested. See correct answer

B. Families A, B, F, G, I, and J (Correct) Black-filled symbols indicate affected individuals, all with a monoallelic KLHL24 variant. Individuals marked with “-” lack the variant. Individuals marked with “nt” were not tested. From the SOP: “In order for a variant to be considered truly de novo, both parents must be sequenced”. In families C, D, and H, one parent of each proband was not tested. A paper may claim a variant is de novo, but for curation purposes, it can only be counted as de novo if both parents are genotyped (default is 2 points). > NEXT

C. Families G, H, and I (Incorrect) Black-filled symbols indicate affected individuals, all with a monoallelic KLHL24 variant. Individuals marked with “-” lack the variant. Individuals marked with “nt” were not tested. See correct answer

D. None of the above (Incorrect) Black-filled symbols indicate affected individuals, all with a monoallelic KLHL24 variant. Individuals marked with “-” lack the variant. Individuals marked with “nt” were not tested. See correct answer

According to the SOP, you may increase the score for a de novo variant if the maternity and paternity of the proband have been confirmed. Was maternity and paternity confirmed for any of the probands in this paper? Yes or no. YES NO The paper states that WES was employed in families A (affected mother AII.2 and daughter AIII.1; and unaffected parents AI.1 and AI.2) and E (affected EII.1 and unaffected parents). This can be used as confirmation of maternity and paternity. Consider increasing the score from the default of 2. > NEXT

c.1A>G (p.Met1Val): How would you score segregation data for this variant? Click on answer below. Black-filled symbols indicate affected individuals, all with a monoallelic KLHL24 variant. Individuals marked with “-” lack the variant. Individuals marked with “nt” were not tested. Check this. Do not score segregation data. Add segregations across families A, B and J. Count segregations in families A, B and J separately. Count the number of affected and unaffected individuals.

A. Do not score segregation data (Correct) Black-filled symbols indicate affected individuals, all with a monoallelic KLHL24 variant. Individuals marked with “-” lack the variant. Individuals marked with “nt” were not tested. Check From the SOP: “For dominant or X-linked disorders, the estimated LOD score should be calculated using ONLY families with 4 or more segregations present.” Families A, B, and J each have fewer than 4 segregations, so they cannot be counted at all (separately or added together). Finally, you would not count the affected and unaffected individuals since this is an autosomal dominant condition. > NEXT

B. Add segregations across families A, B and J (Incorrect) Black-filled symbols indicate affected individuals, all with a monoallelic KLHL24 variant. Individuals marked with “-” lack the variant. Individuals marked with “nt” were not tested. See correct answer

C. Count segregations in families A, B and J separately (Incorrect) Black-filled symbols indicate affected individuals, all with a monoallelic KLHL24 variant. Individuals marked with “-” lack the variant. Individuals marked with “nt” were not tested. See correct answer

D. Count the number of affected and unaffected individuals (Incorrect) Black-filled symbols indicate affected individuals, all with a monoallelic KLHL24 variant. Individuals marked with “-” lack the variant. Individuals marked with “nt” were not tested. See correct answer

Click here to see entries Summary of variants Variant Probands Segregation ExAC frequency ClinVar assertion Prediction Default points NM_017644.3:c.1A>G (p.Met1Val) AII-2 (dn), BII2 (dn), CII-2, DIII-1, FIII-2 (dn), GII-1(dn), HII-1, III-1(dn), JII-2 (dn) dn= de novo N/A. See pedigrees. Not in ExAC. Constraint scores for KLHL24: Missense; z=2.83 LoF; pLI=0.25 264648: Pathogenic (criteria provided, Clinical Genetics Lab of Dermatology, Peking) Mutation Taster: disease causing. 2 points for de novo* (12 total), 0.5 points for non-LOF (1.5 total). *Consider increasing proband AII-2’s points for confirming maternity and paternity NM_017644.3: c.2T>C (p.Met1Thr) EII-1 (dn) N/A. 370042: Pathogenic (no assertion criteria). 2 points for de novo* *Consider increasing points for confirming maternity and paternity Click here to see entries > 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 12 Proband with predicted or proven null variant 1.5 0-2 NA 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 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): 12 (13.5*) > 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.

Experimental Evidence: Methods Expression of KLHL24 in skin and skin cells using culture human keratinocytes, dermal fibroblasts, and melanocytes. Generation of cell lines from primary cells of individuals DIII.1, EII.1 and controls. KLHL24 variant effects on ubiquitination and degradation of keratin 14 using immunostaining, heat stress induction, and overexpression. > NEXT

Curate this evidence? Yes or no. Functional Alteration Figure S3A. RT-PCR of KLHL24 shows expression in all main skin types: keratinocytes, dermal fibroblasts, and melanocytes. Figure S3B and C. Immunofluorescence staining performed on skin cryosections and keratinocytes shows KLHL24 localized intracellularly and at the cell periphery, where it colocalizes with desmoplakin and E-cadherin. Curate this evidence? Yes or no. YES NO Evidence should be scored under Function: Expression if it shows that “the gene is expressed in tissues relevant to the disease of interest and/or is altered in expression in patients who have the disease.” The authors show here that KLHL24 is expressed in all skin types, which is relevant to the disease. 0.5 points default. Function Functional Alteration Models & Rescue Biochemical Function Protein Interaction > NEXT Expression

Curate under experimental evidence or count towards variant impact? Figure 2F. A cell line was generated from primary cells of individual DIII.1(c.1A>G). Immunostaining of KLHL24 in mutant keratinocytes shows a reduction of the signal in the cytoplasm and at the cell periphery compared to normal human keratinocytes (NHK). A similar reduction in KLHL24 is seen in keratinocytes treated with KLHL24 specific siRNA. Curate under experimental evidence or count towards variant impact? Experimental Variant Impact A reduction in KLHL24 from the patient demonstrates that the variant has some impact on the gene function. From the SOP: “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 0.5 points and impact based on functional validation can score 0.5 or above…” > NEXT

Curate this evidence? Yes or no. Functional Alteration 3A, B 3D 3C DIII.1 NHK Figure 2H. In keratinocytes of patient DIII.1, keratin 14 staining appeared disorganized. Figure 2I. The amount of keratin14 was 2-fold higher in whole-cell lysates of KLHL24 mutant keratinocytes vs controls. Figure 3A and 3B. After heat stress, the abnormalities of keratin 14 in patient DIII.1 were even more pronounced, whereas control cells changed very little. Figure 3C. The overall abundance of ubiquitinated proteins increased in control cells after heat stress but not in mutant cells. Figure 3D. Ubiquitin co-localizes with keratin 14 in controls cells, but not in mutant cells. Curate this evidence? Yes or no. YES NO Evidence should be scored under Functional Alteration: Patient Cells if “the gene and/or gene product function is demonstrably altered in cell culture models and/or patients carrying candidate mutations.” In cultured skin cells from patients carrying the mutations, the ubiquitination and subsequent degradation of keratin 14 is altered. Alterations in keratin 14 is a known disease mechanism. 1 point default. Function Functional Alteration Models & Rescue Patient Cells Non-Patient Cells > NEXT Note: If there had been direct evidence (in this paper or elsewhere) that KLHL24 ubiquitinates keratin 14, you could count that under “Biochemical Function”.

Curate this evidence? Yes or no. Functional Alteration Figures 3E and F. Assessed keratin 14 by immunofluorescence staining after recombinant overexpression of GFP-tagged normal and mutant KLHL24 cDNAs in HaCaT keratinocytes. Consistent with the role of KLHL24 in promoting IF degradation, 72% of the cells were negative for keratin 14 compared to 59% of the mutant cells. Curate this evidence? Yes or no. YES NO Evidence should be scored under Functional Alteration: Non-Patient Cells if “the gene and/or gene product function is demonstrably altered in cell culture models and/or patients carrying candidate mutations.” In HaCaT keratinocytes carrying the mutations, the degradation of keratin 14 is altered compared to controls. 0.5 points default. Could this be biochemical function instead? Going by the interface, it makes more sense under functional alteration. Function Functional Alteration Models & Rescue Patient Cells Non-Patient Cells > 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 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 Clinical Validity Summary Matrix 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 12 2 14 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 Strong FINAL CLASSIFICATION Note: In the SOP, Figure 2, it states that in order to receive a “strong” classification, “the role of this gene in disease has been independently demonstrated in at least two separate studies...” However, if the expert determines that the evidence from one paper satisfies the requirements for the “strong” category, that classification may be used. > 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