1. KLHL24: Generalized epidermolysis bullosa simplex (skin fragility)
PMID:
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2. 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: )
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
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3. 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).
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4. 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.

5. 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

6. 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).
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7. 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

8. 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

9. 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.
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10. 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.

11. 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.
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12. 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

13. 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

14. 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

15. Click here to see entries
Summary of variants
Variant
Probands
Segregation
ExAC frequency
ClinVar assertion
Prediction
Default points
NM_ :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_ : 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
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16. 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*)
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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.

17. 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.
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18. 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
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Expression

19. 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…”
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20. Curate this evidence? Yes or no. Functional Alteration
3A, B 3D 3C
DIII 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
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Note: If there had been direct evidence (in this paper or elsewhere) that KLHL24 ubiquitinates keratin 14, you could count that under “Biochemical Function”.

21. 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
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22. 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.
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23. 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.
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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.

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