NonPatientCell: EFO:0001182 (HEK293 Cells)

GeneFunction: GO:0008233 (Peptidase activity)

GeneAlteration: HEK293 cells were grown and 8 missense mutations were identified and generated by PCR-mutagenesis. The human mutant cDNAs were transfected into the cells using the calcuim-phosphate coprecipitation method and examined.

FunctionalEvidence: When HEK293 cells were transfected with wild-type and mutant human cDNA for PHEX, the resulting cells were subject to immunoblotting to determine the protein levels in both the cell lysate and the media of cells. All the variants and the wild-type showed presence in the lysate at various levels, but C85R, G579R, G579V, S711R, and A720T were all devoid of any secreted protein in the media. Ananlysis of these cells also showed possible rescue by growing transfected cells at 26 C, thus glycosylating and maturing the proteins. The endopeptidase activity of the wild-type and variants were also compared, with D237G and Y317F mutant proteins exhibiting 50–60% of wild-type activity; E581V and S711R also displayed nearly undetectable activity. This displayed that the disease causing mechanism could be related to loss of function of peptidase activity or cellular trafficking. Evidence is found under "Results".

FAVariant1: NM_000444.5:c.253T>C (p.Cys85Arg)

ClinVar: 438549 https://www.ncbi.nlm.nih.gov/clinvar/variation/438549/

FAVariant2: NM_000444.5:c.1735G>A (p.Gly579Arg)

ClinVar: 226119 https://www.ncbi.nlm.nih.gov/clinvar/variation/226119/

FAVariant3: NM_000444.5:c.1736G>T (p.Gly579Val)

ClinVar: 372777 https://www.ncbi.nlm.nih.gov/clinvar/variation/372777/

FAVariant4: NM_000444.5:c.2131A>C (p.Ser711Arg)

CAID: CA412575294 https://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA412575294

FAVariant5: NM_000444.5:c.2158G>A (p.Ala720Thr)

CAID: CA412575364 https://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA412575364

NonPatientCell: EFO:0001182 (HEK293 Cells)

GeneFunction: GO:0008233 (Peptidase activity)

GeneAlteration: HEK293 cells were grown and 8 missense mutations were identified and generated by PCR-mutagenesis. The human mutant cDNAs were transfected into the cells using the calcuim-phosphate coprecipitation method and examined.

FunctionalEvidence: When HEK293 cells were transfected with wild-type and mutant human cDNA for PHEX, the resulting cells were subject to immunoblotting to determine the protein levels in both the cell lysate and the media of cells. All the variants and the wild-type showed presence in the lysate at various levels, but C85R, G579R, G579V, S711R, and A720T were all devoid of any secreted protein in the media. Ananlysis of these cells also showed possible rescue by growing transfected cells at 26 C, thus glycosylating and maturing the proteins. The endopeptidase activity of the wild-type and variants were also compared, with D237G and Y317F mutant proteins exhibiting 50–60% of wild-type activity; E581V and S711R also displayed nearly undetectable activity. This displayed that the disease causing mechanism could be related to loss of function of peptidase activity or cellular trafficking. Evidence is found under "Results".

FAVariant1: NM_000444.5:c.253T>C (p.Cys85Arg)

ClinVar: 438549 https://www.ncbi.nlm.nih.gov/clinvar/variation/438549/

FAVariant2: NM_000444.5:c.1735G>A (p.Gly579Arg)

ClinVar: 226119 https://www.ncbi.nlm.nih.gov/clinvar/variation/226119/

FAVariant3: NM_000444.5:c.1736G>T (p.Gly579Val)

ClinVar: 372777 https://www.ncbi.nlm.nih.gov/clinvar/variation/372777/

FAVariant4: NM_000444.5:c.2131A>C (p.Ser711Arg)

CAID: CA412575294 https://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA412575294

FAVariant5: NM_000444.5:c.2158G>A (p.Ala720Thr)

CAID: CA412575364 https://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA412575364

PMID: 1522213

Gene: GALNS

Disease: Mucopolysaccharidosis IVA

MonDO: MONDO:0009659

InheritancePattern: AR

ExperimentName: Expression of variant carrying cDNA in GALNS deficient fibroblasts

FunctionalEvidence: cDNA encoding each of the 15 variants identified in the study were transfected into a GALNS deficient fibroblast cell line. 14 of the 15 altered cDNA's showed no increase in GALNS activity when the cells were assayed, confirming a lack of enzymatic activity.

HGNC:

GeneEvidence:

SharedGeneImplication:

PMID: 1522213

Gene: GALNS

Disease: Mucopolysaccharidosis IVA

MonDO: MONDO:0009659

InheritancePattern: AR

ExperimentName: Expression of variant carrying cDNA in GALNS deficient fibroblasts

FunctionalEvidence: cDNA encoding each of the 15 variants identified in the study were transfected into a GALNS deficient fibroblast cell line. 14 of the 15 altered cDNA's showed no increase in GALNS activity when the cells were assayed, confirming a lack of enzymatic activity.

HGNC:

GeneEvidence:

SharedGeneImplication:

NonHumanModel: Murine

GeneAlteration: murine Galns portions of intron 1 and exon 2 were deleted via homologous recombination of a cre-neomycin resistance-cre cassette and mated with a cre recombinase strain to remove the cassette and complete the deletion

ModelPhenotype: No outward phenotypes but biochemically there was up to a 6 fold increase in GAG excretion in urine, chondroitin-6-sulfate specifically and not keratan sulfate, consistent with the human phenotype

HumanPhenotype: in severe cases there are obvious skeletal deformations, increase of GAG's chondroitin-6-sulfate and keratan sulfate in urine and decreased or absent GALNS activity

ModelEvidence: X-rays of mice and multiple life stages, GAG assays in urine, immunohistochemistry images of tissues with labeled chondoritin-6-sulfate and keratan sulfate to show distribution in multiple tissue types and

ModelVariant: large deletion of portion of intron 1 and exon 2

WhereInPaper:

NonHumanModel: Murine

GeneAlteration: murine Galns portions of intron 1 and exon 2 were deleted via homologous recombination of a cre-neomycin resistance-cre cassette and mated with a cre recombinase strain to remove the cassette and complete the deletion

ModelPhenotype: No outward phenotypes but biochemically there was up to a 6 fold increase in GAG excretion in urine, chondroitin-6-sulfate specifically and not keratan sulfate, consistent with the human phenotype

HumanPhenotype: in severe cases there are obvious skeletal deformations, increase of GAG's chondroitin-6-sulfate and keratan sulfate in urine and decreased or absent GALNS activity

ModelEvidence: X-rays of mice and multiple life stages, GAG assays in urine, immunohistochemistry images of tissues with labeled chondoritin-6-sulfate and keratan sulfate to show distribution in multiple tissue types and

ModelVariant: large deletion of portion of intron 1 and exon 2

WhereInPaper:

PatientCell: CL:0000187 (Muscle Cell)

GeneFunction: Muscle Cell Apoptotic Process (GO:0010657)

GeneAlteration: Samples were collected from deltoid muscle biopsy of control individuals and LGMD2C individuals with the 525delT founder mutation. No further alteration necessary.

FunctionalEvidence: TUNEL staining was perfomed on muscle samples from LGMD2C patients and healthy controls to test for cell apoptosis. No positive staining was detected in any control individuals while it was commonly found in the patient muscle. This indicates that a result of SGCG disruption is increased cellular apoptosis likely contributing to the myofiber degeration and muscle atrophy seen commonly in LGMD patients.

FAVariant: NM_000231.2(SGCG):c.525del (p.Phe175fs)

ClinVarID: 189243 https://www.ncbi.nlm.nih.gov/clinvar/variation/189243/

PatientCell: CL:0000187 (Muscle Cell)

GeneFunction: Muscle Cell Apoptotic Process (GO:0010657)

GeneAlteration: Samples were collected from deltoid muscle biopsy of control individuals and LGMD2C individuals with the 525delT founder mutation. No further alteration necessary.

FunctionalEvidence: TUNEL staining was perfomed on muscle samples from LGMD2C patients and healthy controls to test for cell apoptosis. No positive staining was detected in any control individuals while it was commonly found in the patient muscle. This indicates that a result of SGCG disruption is increased cellular apoptosis likely contributing to the myofiber degeration and muscle atrophy seen commonly in LGMD patients.

FAVariant: NM_000231.2(SGCG):c.525del (p.Phe175fs)

ClinVarID: 189243 https://www.ncbi.nlm.nih.gov/clinvar/variation/189243/

ExperimentName: Accumulation of glycosaminoglycans

GOterm:

FunctionalEvidence: Patient cells assayed with artificial substrates for the N-acetylgalactosamine-6-sulfate sulfatase encoded by GALNS, depending on the severity of the coding sequence alteration, are either unable to break down the substrate or are hindered. Further, in mucopolysaccharidosis IVA patients with known variants, elevated levels of keratan sulfate and chondroitin-6-sulfate can be found in their urine where levels in unaffected individuals are much lower.

HGNC:

GeneEvidence:

SharedGeneImplication:

ExperimentName: Accumulation of glycosaminoglycans

GOterm:

FunctionalEvidence: Patient cells assayed with artificial substrates for the N-acetylgalactosamine-6-sulfate sulfatase encoded by GALNS, depending on the severity of the coding sequence alteration, are either unable to break down the substrate or are hindered. Further, in mucopolysaccharidosis IVA patients with known variants, elevated levels of keratan sulfate and chondroitin-6-sulfate can be found in their urine where levels in unaffected individuals are much lower.

HGNC:

GeneEvidence:

SharedGeneImplication: