1. BGN: X-linked Spondyloepimetaphyseal Dysplasia PMID: 27236923
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2. BGN: X-linked Spondyloepimetaphyseal Dysplasia (SEMD), 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
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3. Background
Spondyloepimetaphyseal dysplasias (SEMDs) comprise a heterogeneous group of autosomal dominant and autosomal recessive disorders.
SEMDs are characterized by anomalies of the spine and the epiphyses and metaphyses of the long bones, resulting in short stature.
X-linked SEMD was reported in an Italian family in 1994 (Camera et al.) and is re-analyzed in this report, along with a Korean family with radiological features of SEMD and an Indian proband with similar features.
Whole-exome sequencing (WES) was performed in 9 members of the two affected families (Korean and Italian).
No pathogenic variants were found in genes associated with similar disorders with skeletal dysplasia and short stature (ACAN, DMY, NPR2, COMP, and TRPV4).
Variants were identified in a candidate gene, BGN, which encodes biglycan.
Biglycan is highly expressed in bone and is a structural component of cartilage.
It also has a role in cell signaling, regulation of chondrocyte and pericellular matrix homeostasis.
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4. Answer the following questions in accordance with the SOP based on the information presented.
In Family A, one missense variant, c.439A>G (p.Lys147Glu) in BGN (NM_ ) was identified as a possible candidate based whole exome sequencing (WES) of 6 family members followed by Sanger sequencing. How many segregations would you count to estimate a LOD score according to the formula in the SOP? Filled boxes represent affected individuals. Circles with a dot in the middle indicate the status of a carrier. The “=“ symbol indicates the wild type allele and “0” indicates the absence of the second X chromosome in males.
4 segregations, LOD score of 1.2
5 segregations, LOD score of 1.5
6 segregations, LOD score of 1.8
Not enough segregations

5. A. 4 segregations, LOD score of 1.2 (Incorrect)
In Family A, one missense variant, c.439A>G (p.Lys147Glu) in BGN (NM_ ) was identified as a possible candidate based whole exome sequencing (WES) of 6 family members followed by Sanger sequencing. How many segregations would you count to estimate a LOD score according to the formula in the SOP? Filled boxes represent affected individuals. Circles with a dot in the middle indicate the status of a carrier. The “=“ symbol indicates the wild type allele and “0” indicates the absence of the second X chromosome in males.
See Correct Answer

6. B. 5 segregations, LOD score of 1.5 (Incorrect)
In Family A, one missense variant, c.439A>G (p.Lys147Glu) in BGN (NM_ ) was identified as a possible candidate based whole exome sequencing (WES) of 6 family members followed by Sanger sequencing. How many segregations would you count to estimate a LOD score according to the formula in the SOP? Filled boxes represent affected individuals. Circles with a dot in the middle indicate the status of a carrier. The “=“ symbol indicates the wild type allele and “0” indicates the absence of the second X chromosome in males.
See Correct Answer

7. C. 6 segregations, LOD score of 1.8 (Incorrect)
In Family A, one missense variant, c.439A>G (p.Lys147Glu) in BGN (NM_ ) was identified as a possible candidate based whole exome sequencing (WES) of 6 family members followed by Sanger sequencing. How many segregations would you count to estimate a LOD score according to the formula in the SOP? Filled boxes represent affected individuals. Circles with a dot in the middle indicate the status of a carrier. The “=“ symbol indicates the wild type allele and “0” indicates the absence of the second X chromosome in males.
See Correct Answer

8. D. Not enough segregations (Correct)
In Family A, one missense variant, c.439A>G (p.Lys147Glu) in BGN (NM_ ) was identified as a possible candidate based whole exome sequencing (WES) of 6 family members followed by Sanger sequencing. How many segregations would you count to estimate a LOD score according to the formula in the SOP? Filled boxes represent affected individuals. Circles with a dot in the middle indicate the status of a carrier. The “=“ symbol indicates the wild type allele and “0” indicates the absence of the second X chromosome in males. 1 2 3
For autosomal dominant or X-linked disorders, a LOD score should only be calculated using families with 4 or more segregations. This family has 3 segregations (see above). Individual II-1 should not be counted since he was not genotyped. Individual III-2 is a carrier between two affected individuals, but she had a 100% chance of inheriting the affected allele from her affected father, so you would not count her either. Starting from the proband, II-2, you would use 3 individuals to count segregations: II-6, IV-1, and IV-2.
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9. Another BGN missense variant, c. 776G>T (p
Another BGN missense variant, c.776G>T (p.Gly259Val), was identified as a candidate in Family B by WES and Sanger sequencing. Filled boxes represent affected individuals. Circles with a dot in the middle indicate the status of a carrier. The “=“ symbol indicates the wild type allele and “0” indicates the absence of the second X chromosome in males . Are there enough segregations in this family to estimate a LOD score according to the SOP? 3 2 4 1 5
YES NO
Starting from the proband, you could count individual V-9 as a segregation. Individual VI-2 is also genotype-positive and affected. Counting the obligate carriers between the proband and individual VI-2, (V-6, IV-6, and IV-9), there are 5 segregations. Individual V-10 is also a carrier, but you would not count her since she is unaffected and has no affected children. Using the formula from the SOP, the estimated LOD score for 5 segregations for X-linked diseases is 1.5:
Z(LOD score) = 𝑙𝑜𝑔 (0.5) 5 = 1.5
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10. The two variants identified, c. 439A>G (p. Lys147Glu) and c
The two variants identified, c.439A>G (p.Lys147Glu) and c.776G>T (p.Gly259Val), are absent from the ExAC and gnomAD databases. Both variants are located in conserved domains and are predicted by in silico tools to be damaging. Furthermore, the authors used Gromacs Molecular Dynamic (MD) simulation to visualize the altered residues on 3D structures to determine how the point mutations affect the protein stability or binding interaction with TGF-β, a known interactor of biglycan. According to the MD simulation, both variants have impacts on biglycan structure (B). The residues in which the substitutions occur are located in a region that binds to TGF-β (C). The p.Lys147Glu substitution might reduce the binding affinity by changing the polarity of the residue (D), while the p.Gly259Val substitution changes the overall biglycan structure. Based on this information, how would you score the two probands that harbor these variants? Select answer below.
Predicted/proven null variant (1.5 points default)
Other variant type with evidence of gene impact (0.5 points default)
B is correct but reduce points from default

11. A. Predicted/proven null variant (1.5 points default) (Incorrect)
The two variants identified, c.439A>G (p.Lys147Glu) and c.776G>T (p.Gly259Val), are absent from the ExAC and gnomAD databases. Both variants are located in conserved domains and are predicted by in silico tools to be damaging. Furthermore, the authors used Gromacs Molecular Dynamic (MD) simulation to visualize the altered residues on 3D structures to determine how the point mutations affect the protein stability or binding interaction with TGF-β, a known interactor of biglycan. According to the MD simulation, both variants have impacts on biglycan structure (B). The residues in which the substitutions occur are located in a region that binds to TGF-β (C). The p.Lys147Glu substitution might reduce the binding affinity by changing the polarity of the residue (D), while the p.Gly259Val substitution changes the overall biglycan structure. Based on this information, how would you score the two probands that harbor these variants? Select answer below.
See Correct Answer

12. B. Other variant type with evidence of gene impact (0
B. Other variant type with evidence of gene impact (0.5 points default) (Possible answer)
The two variants identified, c.439A>G (p.Lys147Glu) and c.776G>T (p.Gly259Val), are absent from the ExAC and gnomAD databases. Both variants are located in conserved domains and are predicted by in silico tools to be damaging. Furthermore, the authors used Gromacs Molecular Dynamic (MD) simulation to visualize the altered residues on 3D structures to determine how the point mutations affect the protein stability or binding interaction with TGF-β, a known interactor of biglycan. According to the MD simulation, both variants have impacts on biglycan structure (B). The residues in which the substitutions occur are located in a region that binds to TGF-β (C). The p.Lys147Glu substitution might reduce the binding affinity by changing the polarity of the residue (D), while the p.Gly259Val substitution changes the overall biglycan structure. Based on this information, how would you score the two probands that harbor these variants? 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.” The authors demonstrated using 3D simulations that the variant likely affects the function of the protein, in addition to in silico prediction tools. Therefore, the default 0.5 points may be given. It may be appropriate to discuss this with an expert to determine whether this 3D simulation is sufficient evidence for gene impact.
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13. See other possible answer and explanation
C. B is correct but reduce points from default (Possible answer)
The two variants identified, c.439A>G (p.Lys147Glu) and c.776G>T (p.Gly259Val), are absent from the ExAC and gnomAD databases. Both variants are located in conserved domains and are predicted by in silico tools to be damaging. Furthermore, the authors used Gromacs Molecular Dynamic (MD) simulation to visualize the altered residues on 3D structures to determine how the point mutations affect the protein stability or binding interaction with TGF-β, a known interactor of biglycan. According to the MD simulation, both variants have impacts on biglycan structure (B). The residues in which the substitutions occur are located in a region that binds to TGF-β (C). The p.Lys147Glu substitution might reduce the binding affinity by changing the polarity of the residue (D), while the p.Gly259Val substitution changes the overall biglycan structure. Based on this information, how would you score the two probands that harbor these variants? Select answer below.
See other possible answer and explanation

14. Variant Impact Experimental > NEXT
To further investigate the functional consequences of the BGN variant, the authors obtained dermal fibroblasts from Family A with the p.Lys147Glu substitution. Biglycan in fibroblasts from affected individual IV-1 was expressed at lower levels than unaffected individual III-2’s fibroblasts (A). While there was no difference in transcription between fibroblasts from the two individuals (A), biglycan appeared to more rapidly degrade in fibroblasts from the affected individual (B). Should this evidence count towards the variant impact or experimental evidence for the gene-disease relationship? Select answer below.
Variant Impact
Experimental
Reduced protein levels and more rapid degradation in the affected male compared to the unaffected female carrier demonstrates 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. You could use this to increase points for the proband harboring the p.Lys147Glu variant.
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15. The same variant found in Family A, c. 439A>G (p
The same variant found in Family A, c.439A>G (p.Lys147Glu), was also identified in an unrelated individual by Sanger sequencing. Using the information from the pedigree below, is there enough information to classify this as a de novo variant? Filled boxes represent affected individuals. Circles with a dot in the middle indicate the status of a carrier. The “=“ symbol indicates the wild type allele and “0” indicates the absence of the second X chromosome in males.
YES NO
Given the genotype of the affected individual’s parents, this can be classified as a de novo variant (2 points). If maternity is confirmed, you could increase the points from default (paternity would not need to be confirmed in this case since the affected individual is male and therefore did not inherit an X chromosome from the father).
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16. According to the SOP, LOD scores from all families meeting size requirements must be summed before awarding segregation points. Family A did not meet the size requirement for calculating a LOD score. The estimated LOD score for Family B was 1.5. Assuming there are no other families with segregation data, should you assign segregation points for this gene-disease relationship?
YES NO
In order to assign segregation points, you would need a minimum LOD score of 2 summed across all families, regardless of sequencing method. For a LOD score of 1.5, you should assign 0 points.
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17. Summary of variants > NEXT Probands Variant (HGVS nomenclature)
Type of variant
gnomAD Frequency
Summary of evidence
Suggested points
Family A, II-2
NM_ (BGN):c.439A>G (p.Lys147Glu)
Missense
Not in gnomAD
3D simulation suggests altered protein structure and decreased binding to TGF-β. Patient fibroblasts show reduced protein levels.
1 (upgraded due to variant evidence)
Family B, V-8
NM_ (BGN):c.776G>T (p.Gly259Val)
3D simulation suggests altered protein structure.
0.5
Family C, III-4
Missense, de novo
De novo variant. 3D simulation suggests altered protein structure and decreased binding to TGF-β. 2
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18. 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 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
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):
3.5
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19. While there is no experimental evidence for the gene-disease relationship in this paper, the authors reference several papers that could be curated for experimental evidence. For the following experiments, indicate how you might curate the evidence:
(Note: for actual curations, you should curate the evidence directly from the original publication.)
Function
Biochemical function
“Biglycan, a member of the class I family of small leucine-rich proteoglycans (SLRPs) in the extracellular matrix, is highly expressed in bone (Bianco et al., 1990; Ameye & Young, 2002).”
From the SOP: “the gene is expressed in tissues relevant to the disease of interest.” Given the skeletal dyplasia of individuals with the disease, expression in bone should be counted as evidence. Default 0.5 points.
Functional Alteration
Protein interaction
Models & Rescue
Expression
Function
Biochemical function
“It is an important structural component of articular cartilage and participates in the assembly of the chondrocyte extracellular matrix… and it is thought to play a critical role in the regulation of chondrocyte and pericellular matrix homeostasis (Iacob & Cs-Szabo, 2010).”
From the SOP: “the gene product performs a biochemical function shared with other known genes in the disease of interest, or is consistent with the phenotype.” Again, given the skeletal dysplasia of individuals with the disease, this function would be consistent with the phenotype. Default 0.5 points.
Functional Alteration
Protein interaction
Models & Rescue
Expression
Function
Animal model
“Bgn-deficient mice (bgn-/0) show a reduced growth rate and low bone mass that is not expressed at birth but becomes obvious with age. X-ray images show short tubular bones with decreased radiodensity compared with age-matched controls (Xu et al., 1998).”
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.
Functional Alteration
Cell culture model system
Models & Rescue
Rescue
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20. 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 2
Protein Interaction NA
Expression
Functional
Alteration
Patient cells
1 - 2 1
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.
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21. 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
3.5 3
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
Moderate
FINAL CLASSIFICATION
Although the score was between limited and moderate, a classification of moderate was assigned because there were “3 unrelated probands harboring variants with sufficient supporting evidence for disease causality” and “moderate experimental data supporting the gene-disease association.” See Figure 2 of the SOP.
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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.

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