PatientCell CL:0000057 (Fibroblast)

GeneFunction GO:0032006, (regulation of TOR signaling)

GeneAlteration n/a

FunctionalEvidence Western blot of PS6 isoforms, a product of mTOR signalling, were 74% reduced in patient 2-1 fibroblasts compared to control (Fig 3).

Western blot of PS6 in subject 1-1&1-2 fibroblasts also demonstrated 78% reduced PS6 compared to controls (Fig 2b).

As S6 is phosphorylated during normal mTOR activation, reduced PS6 suggests reduced mTOR signalling.

29283439 (fig 3) suggests that the p.Arg126Ter variant in patient fibroblasts causes increased autophagy compared to controls due to the increase in autophagiosomes. (fig 5) the same variant also leads to excess oligosaccharides as a downstream effect of increased autophagy as show using mass spec.

FAVariants (Fig 3) NM_001163435.2(TBCK):c.831_832insTA (p.Pro278Tyrfs), ClinVar: 225238, (Fig 2) NM_033115.4(TBCK):c.1708+1G>A, ClinVar: 183338

ExperimentName: Reduced E2-lipoylation in LIPT2 patient cells

PatientCell: Fibroblast (not specified where it was derived)

GeneFunction: GO:0009249

GeneAlteration: Patient fibroblasts from P1 harboring the LIPT2 c.89T>C(p.Leu30Pro) and c.377T>G(Leu126Arg) and fibrobalsts from P2 harboring the c.377T>G(Leu126Arg) and c.314T>G(p.Leu105Pro) were used in this assay.

FunctionalEvidence: Patient fibroblasts both from P1 and P2, both with compound heterozygous mutation in LIPT2 that represent 3 different variants (one overlaps), were assayed for E2 lipoylation in PDHc and alpha-KGDHc (Figure 3), compared to control cells. The assay indicates reduced lipoylation of both PDHc and alpha KGSHc compared to controls both by western blot (Figure 3A) and immunocytochemistry (figure 3B). the reduction in lipoylation was consistent with that observed in patients with LIPT1 mutations, indicating a role for LIPT2 in lipoylation.

FAVariants:<br /> 1) CA224979286 (NM_001144869.1:c.377T>G(p. Leu126Arg))

2) CA224979330 (NM_001144869.1:c.89T>C(p.Leu30Pro))

3) CA381976642 (NM_001144869.1:c.314T>C(p.Leu105Pro)

ExperimentName: Ojala 2016 hiPSC-derived CMs

PatientCell: CL_0002620 (skin fibroblasts)

GeneFunction: GO:0008016 (regulation of heart contraction)

GeneAlteration: hiPSC-derived cardiomyocytes (CMs) generated from skin fibroblasts collected from patients carrying a TPM1-Asp175Asn mutation

FunctionalEvidence: Figure 2 shows enlarged cardiomyocytes (CMs) (a-b), an increase in multinucleated CMs (c), and calcium handling abnormalities (d) for the HCM-TPM1 variant CMs compared to WT CMs.

FAVariants: NM_001018005.2(TPM1):c.523G>A (p.Asp175Asn), CA018106

ExperimentName: Functional characterization of patient-derived hiPSC-RPE cells

PatientCell: CL:0002586, or retinal pigment epithelial cell (retinal pigmented epithelium generated from patient-derived induced pluripotent stem cells, or hiPSC-RPE)

GeneFunction: GO:0008191 (metalloendopeptidase inhibitor activity), GO:0051045 (negative regulation of membrane protein ectodomain proteolysis), GO:0016525 (negative regulation of angiogenesis)

GeneAlteration:** Patient cells were heterozygous carriers of the NM_000362.5(TIMP3):c.610A>T p.Ser204Cys variant, or unaffected controls that were homozygous for the non-variant allele.

FunctionalEvidence: hiPSC-RPE cells from SFD patients exhibit altered function relative to equivalent cells from unaffected individuals. They secrete a decreased quantity of TIMP3 (Figure 4C), indicating accumulation and retention of TIMP3 either within the cells or in the associated extracellular matrix. Finally, they express abnormal proteomic profiles with elevated levels of the known TIMP3 binding partner EFEMP1 and the known drusen component apolipoprotein E (Figure 5). The epithelial layer formed by these patient-derived cells exhibits reduced transepithelial electrical resistance (Figure 2C). Other known functions like metalloproteinase inhibition are retained by the variant-harboring cells (Figure 4D). These findings are consistent with a gain-of-function mechanism.

FAVariants: This experiment studied patient cells harboring the NM_000362.5(TIMP3):c.610A>T p.Ser204Cys variant (ClinVarID 12676).

ExperimentName: Altered pattern of splicing speckles in patient fibroblasts harboring a heterozygous p.Pro315Leu variant in PRPF4

PatientCell: CL:0000057 (fibroblast)

GeneFunction: GO:0000398 (mRNA splicing, via spliceosome)

GeneAlteration: Cells harbor a heterozygous p.Pro315Leu variant in PRPF4.

FunctionalEvidence: Patient cells harboring the variant exhibit a much higher level of endogenous marker of splicing speckles (SC-35) with a diffused distribution rather than a pattern of distinct speckles (Figures 4B and 4C). RT-PCR showed increased expression of SRSF1 and SRSF2 as well (Figure 4A). Collectively, the patient cells harboring the variant show molecular evidence of disrupted splicing.

FAVariants: The model harbors the NM_001244926.2(PRPF4):c.941C>T (p.Pro314Leu) variant in the heterozygous state (ClinVar ID: 143057).

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/