NonPatientCell: E-Coli (DH5α)

GeneFunction: GO:0006559

GeneAlteration: PAH cDNA was cloned into a pMAL-c2E expression vector encoding an N-terminal MBP tag and an enterokinase cleavage site. PAH mutants were constructed using site-directed mutagenesis and confirmed using DNA sequencing. Both the wild-type and the 10 mutant vectors were transformed into E. coli DH5α via overexpresion of fusion proteins at midexponential phase. The proteins were purified and concentration was determined.

FunctionalEvidence: Expression of the 10 mutant proteins compared to the wild-type demonstrated that several of these mutants induce protein destabilization and subsequent loss of function. Upon deconvolution analysis, five variants showed significanly distrubed oligomerization and a preponderance to misfold and form high-molecular-weight protein aggregates. Enzyme kinetic parameter analysis demonstrated moderate reduction in PAH activity and severely affected allostery in all but one mutation, indicating the strong possibility of global confirmational changes. Protein confirmation was tested by limited proteolysis with proteinase K with six out of eight tested mutants decreasing the half-life of the protein by >50% and decreased stability in five out of eight. Thermal protein denaturation was analyzed by fluorescence and demonstrated that all of the mutations led to substantial distortion of the protein's confirmation and/or loss of structural integrity compared to the wild-type, especially those in the regulatory domain. Finally, amino acid replacements were shown to disrupt local networks of amino acid interactions and alter the normal oligomeric state and therfore the function of the protein.

FAVariant1: NM_000277.3(PAH):c.1222C>T (p.Arg408Trp)

ClinVar: 577 https://www.ncbi.nlm.nih.gov/clinvar/variation/577/

FAVariant2: NM_000277.3(PAH):c.898G>T (p.Ala300Ser)

ClinVar: 92751 https://www.ncbi.nlm.nih.gov/clinvar/variation/92751/

FAVariant3: NM_000277.3(PAH):c.165T>G (p.Phe55Leu)

ClinVar: 92734 https://www.ncbi.nlm.nih.gov/clinvar/variation/92734/

FAVariant4: NM_000277.3(PAH):c.1249T>C (p.Tyr417His)

ClinVar: 102579 https://www.ncbi.nlm.nih.gov/clinvar/variation/102579/

FAVariant5: NM_000277.3(PAH):c.940C>T (p.Pro314Ser)

ClinVar: 102905 https://www.ncbi.nlm.nih.gov/clinvar/variation/102905/

PMID: 23698346

Gene: ARHGEF18

Disease: Retinitis pigmentosa

MonDO: MONDO:0019200

NonPatientCell: EFO:0002529 (INS-1), EFO:0000856 (islet of Langerhans), CL:0000182 (Hepatocyte)

GeneFunction: GO:0004353 (glutamate dehydrogenase [NAD(P)+] activity)

GeneAlteration: Recombinant adenoviruses encoding WT human GLUD1 and the S498L variant were generated via an expression system, with the human GLUD1 gene being amplified and sub-cloned into a plasmid with a promoter. Digestion using EcoRV was used to discriminate between the wild-type and the mutant with sequencing verifying each step of the cloning. This adenovirus was amplified in HEK-293 cells and INS-1E β-cells were transduced with it. Mouse and human hepatocytes were isolated and seeded before being transduced with the same adenoviruses. The transfection efficency was confirmed via immunoblotting.

FunctionalEvidence: The enzymatic activity was evaluated in both wild-type and mutant mice, with both showing very low activity without glutamate concentrations and the mutant showing higher activity when glutamate was increased. The reductive direction using α-ketoglutarate as substrate was also tested with similar increased results for the mutant. Sensitivity of both lines to the reaction activator (ADP) and inhibitor (GTP) levels were tested, showing that the mutant was more sensitive to the ADP activator and slightly reduced for the GTP inhibitor (particularly in the presence of ADP). Mitochondiral function in wid-type and mutant cells was assessed, demonstrating that glucose response from mutant and WT cells was preserved but that glutamine stimulation abnormally generates ATP in mutant cells.Calcium stimulation was also activated by both glutamine and glucose in mutant cells while only being affected by glucose in WT. Studies in mutant and WT-transfected mouse and human islets showed increased insulin and ammonia production in mutants from stimulation through this responsive glutamine sensitivity. All of this supports GLUD1's pathogenic mechanism, specifically causing a hyperctive mutant protein that responds to glutamine levels rather than simply glucose.

FAVariants: NM_005271.5(GLUD1):c.1493C>T (p.Ser498Leu)

ClinVar: 16122, https://www.ncbi.nlm.nih.gov/clinvar/variation/16122/

NonHumanModel: Vervet Monkeys (Chlorocebus pygerythrus)

GeneAlteration: PCR amplification and direct sequencing indicated that all of the vervet monkeys observed and one of the malbrouck monkeys had the TTR Ile122 allele, as opposed to the the other primates (including humans) that harbor the Met122 allele. Other than this variant, the sequence was identical to three other species of monkeys that did not demonstrate amyloidosis phenotypes including 95-98%, 93-94%, and 76-81% amino acid homologies to humans.

ModelPhenotype: HP:0031327, HP:0003216, HP:0011675, HP:0012664 (Transthyretin cardiac amyloidosis, Generalized amyloid deposition, Arrhythmia, Reduced ejection fraction)

ModelFreeText: Haphazardly arranged rigid amyloid fibrils in the heart, Increased cardiothoracic ratio, Increased plasma ANP concentrations

HumanPhenotype: HP:0001627, HP:0011034 (Abnormal heart morphology, Amyloidosis)

HumanFreeText: N/A

ModelEvidence: Aged vervet monkeys were investigated for a naturally-occuring spontaneous systemic amyloidosis and accompanying phenotypes. PCR amplification and direct sequencing of these monkeys and others showed that the vervet monkeys alone harbored the Ile122 allele, as opposed to the more common Val122 allele in other primates and humans. Histopathological investigations showed cardiac amyloidosis and rigid amyloid fibers in the heart, identical to that observed in aged humans with the allele in question. These deposits were also found in other systems such as the gastrointestinal tract, adipose tissue, lung, tongue, and vascular walls, also mirroring the multiple locations they were found in humans. Immunoblotting confirmed that amyloid proteins extracted from the cardiac specimens reacted with anti-TTR antibody. Other cardiac symptoms were observed that mirror those in humans, such as arrythmia and reduced ehection fraction. Real-time PCR showed that these monkeys and humans shared the majority of TTR expression patterns. Recombinant monkey TTRs showed a greater tendency to dissociate to monomers under the influence of urea-mediated denaturation stress than recombinant TTR of the cynomolgus monkey, which had the Val122 allele. ThT-binding assay also showed a higher number of amyloid fibers in the recombinant than the cynomolgus.

ModelVariant: NM_000371.4(TTR):c.424G>A (p.Val142Ile)

ClinVar: 13426, https://www.ncbi.nlm.nih.gov/clinvar/variation/13426/

NonHumanModel: Mouse (Mus musculus)

GeneAlteration: CRISPR-Cas9 gene editing was used to produce gene-edited mouse zygotes. This was accomplished by designing a guide RNA (gRNA) with the CRISPR design tool and microinjecting the gRNA complex along with Cas9 mRNA and a ultramer repair template including the single deletion into mouse zygotes. This directs double-strand breaks in the genome with homology directed repair causing the precise genome edit necessary. These were injected into psuedo-pregnant C57BL/6J mice. After birth, tail DNA was genotyped via PCR and Sanger seuqencing to confirm the mutation. One heterozygous 525delT mouse was crossed and the offspring backcrossed onto DBA/2J background until the F5 generation confirmed homozygous for the severe LTBP4 allele which results in a more severe disease progression. RT-PCR confirmed the presence of the deletion and loss of γ-sarcoglycan was confirmed via immunoblot analysis and immunofluorescence microscopy.

ModelPhenotype: HP:0003202, HP:0031237, HP:0030951, HP:0003713, HP:0011987, HP:0003557, HP:0003236, HP:0003323, HP:0005957 (Skeletal muscle atrophy, Internally nucleated skeletal muscle fibers, Skeletal muscle fibrosis, Muscle fiber necrosis, Ectopic ossification in muscle tissue, Increased variability in muscle fiber diameter, Progressive muscle weakness, Breathing dysregulation)

ModelFreeText: Decreased cross-sectional area of myofibers, Increased immune infiltrate, Increased dye uptake into compromised myofibers

HumanPhenotype: HP:0003202, HP:0030951, HP:0003713, HP:0003236, HP:0003323, HP:0005957 (Skeletal muscle atrophy, Skeletal muscle fibrosis, Muscle fiber necrosis, Elevated serum creatine kinase, Progressive muscle weakness, Breathing dysregulation)

HumanFreeText: N/A

ModelEvidence: This model has many positive aspects compared to the default mouse model, although it also has some challenges to overcome. In terms of the generation, CRISPR-Cas9 gene editing via HDR is a relatively simple way to modify the genome as needed and create only the variant of interest while leaving the rest untouched. This results in a knock-in with a reasonable assurance that whatever phenotypes are displayed stem solely from the change in the SGCG gene. Specifically the muscle atrophy, elevated serum creatine kinase, progressive weakness (half the strength of controls at 4 months), and signifiacntly increased muscle fibrosis/necrosis are all hallmarks of the human LGMD presentation and are recapitulated here. However, the presence of a known muscular dystrophy-modifying background raises the question of whether some of these phenotypes would not be seen in a mouse with the C57BL/6J background. Unfortunately this is not answered in the paper, so it introduces some doubt as to the total validity of the results.

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

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

NonHumanModel: Mus musculus

GeneAlteration: p.Arg386Cys the most frequently reported missense variant linked as a causal pathogenic allele in mucopolysaccharidosis IVA patients

ModelPhenotype: In addition to undetectable GALNS enzyme activity in any assayed tissue, increased keratan sulfate detection in liver tissue and blood serum, the boney deformations missing from previous models were present in this mouse model HP:0004325, MP:0001258, HP:0005736

HumanPhenotype: HP:4000197, HP:0012069, HP:0004322, HP:0005736

ModelEvidence: CRISPR/Cas9 directed site mutagenesis of murine Galns coding sequence at codon homologous to the human p.Arg388Cys mutation cited in human MPSIVA patients. Mouse model showed increased keratan sulfate accumulation in liver and blood serum, undetectable n-acetylgalactosamine-6-sulfatase activity in any body tissue assayed as well as skeletal deformities consistent with human MPSIVA patients

ModelVariant: p.Arg388Cys

WhereInPaper:

RescueModel: Rescue WT CAG::GALNS in murine model by ovexpression in a Galns variant frequently found in human's affected with MPSIVA

GeneAlteration: transduction by AAV9 of CAG promoted WT murine optimized Galns coding sequence. Widespread tissue transduction was visualized with a CAG promoted GFP cassette delivered by AAV9, subsequently GFP production was widespread and found in all relevant tissue including osteoblast rich areas.

RescueVariant: AAV9-Galns with the coding sequence under the control of a CAG promoter element

RescuePhenotype: HP:0004325, MP:0001258, HP:0005736

RescueMethod: p.Arg386Cys (MPSIVA) mice were first transducted with CAG::GFP via AAV9 to verify efficiency of delivering the coding sequence to target tissue like bone. Then a CAG::Galns (optimized WT) was transducted in to MPSIVA mice (Galns -/-) via AAV9 by 4 weeks of age to avoid the high mortality in the Galns homozygote starting at 2 months.

RescueEvidence: Early transduction of Galns -/- mice recovered higher than WT enzyme activity and WT levels of keratan sulfate detected in liver tissue. Abnormalities in the growth plate in Galns -/- mice were absent in those treated with AAV9-Galns resulting from a return to WT levels of enzyme activity and keratan sulfate in all bone tissue sampled. GALNS activity recovered in lung and heart tissue with the elevated heart rate in MPSIVA mice returned to WT.

ModelVariant: p.Arg386Cys

NonHumanModel: Mouse (Mus musculus)

GeneAlteration: A fragment covering exons 4-8 of the SLC1A3 locus was subcloned with the P290R seuqnece being inserted via PCR into the end of Exon 7. Thse clones were injected into blastocysts of C37BL/6N mice with these generatingfoster mice and chimeric mice being crossbred to excise the neomycin casette. Due to the high mortality rate, the surviving mice harboring the variant were backcrossed to 129/SvJ for a more robust mouse and higher survival rate for study.

ModelPhenotype: MP:0002064, MP:0001393, MP:0010387, MP:0008925, MP:0002182, MP:0000877, MP:0010170, MP:0014185 (Seizures, Ataxia, Abnormal Bergmann glial cell morphology, Increased cerebellar granule cell number, Abnormal astrocyte morphology, Abnormal Purkinje cell morphology, Abnormal glial cell apoptosis, Cerebellum atrophy)

ModelFreeText: Slower firing of purkinje neurons

HumanPhenotype: HP:0001251, HP:0002076, HP:0001250 (Ataxia, Migraine, Seizure)

HumanFreeText: N/A

ModelEvidence: The first set of mutant knock-in mice were subject to generalized seizures and death before the weaning period, requiring a backcross onto the 129/SvJ strain in order to reduce lethal seizure activity and delay the onset. Even so, mutant mice demonstrated significant phenotypes indicative of human Episodic Ataxia including epilepsy, ataxia, and reduced motor/gait coordination. In order to determine the pathogenc mechanism, cerebellar Bergmann glia cells were examined to find a pronounced age-dependent reduction of cell numbers between P10 and P20. Immunostaining showed decreased numbers of somata and expression, demonstrating that the EAAT1 mutants were subject to cell death. Granule cell migration was also impaired during development, coupled with invasion of reactive astrocytes into the molecular layer demonstrating attempted repair. Following from this change, Purkinje neurons were examined and found to have been reduced and the morphology altered in mutant mice. As the P290R variant is known to increase EAAT1 anion currents in heterologous expression systems, measurement of these currents in mouse Bergmann glia cells showed significantly increased current, decreased Cl- concentration, and a resulting higher TUNEL expression in these cells, indicating earlier apoptosis events of these cell types. Spiking activities and developmental acceleration is absent in these cell types, likely due to progressive deterioration. This was verified by an observation of cerebellar size, where mutant cerebellar tissue showed generalized degeneration compared to the WT mice.

ModelVariant: NM_004172.5(SLC1A3):c.869C>G (p.Pro290Arg)

ClinVar: 9441, https://www.ncbi.nlm.nih.gov/clinvar/variation/9441/

NonHumanModel: Mouse (Mus musculus)

GeneAlteration: Mutations were induced in mice germline cells at a rate near 10^-3 per locus via an alkylating agent called ethylnitrosourea. Inbred BTBR males were treated with this agent and bred to normal BTBR females. Three hundred and fifty progeny were screened for mutations that did not complement the previous model PAH_enu1 and if present bred to a previous homozygote and tested for hyperphenylalaninemia. A PHE clearance test was performed in questionable cases. For those that tested positive the carrier parents were mated to a normal BTBR partner to establish a cogenic mutant line. Western blot confirmed reduced to absent CRM in liver.

ModelPhenotype: HP:0007513, HP:0000707, HP:0005982, HP:0100610, HP:0004923, HP:0001510, HP:0000252 (Generalized hypopigmentation, Abnormality of the nervous system, Reduced phenylalanine hydroxylase activity, Maternal hyperphenylalaninemia, Hyperphenylalaninemia, Growth delay, Microcephaly)

ModelFreeText: Serum PHE concentrations were 15x wild-type for PAH_enu2 (23.1 +/- 6.9 mg/dl, 1.5 +/- 0.5 reference) and 14x wild-type for PAH_enu3 (20.3 +/- 5.9 mg/dl, 1.5 +/- 0.5 reference), PAH activity was reduced to <1% wild-type for PAH_enu2 (0 +/- 2 delta O.D. x 10^3/min/mg protein, 40 +/- 6 reference) and <10% wild-type for PAH_enu3 (5 +/- 5 delta O.D. x 10^3/min/mg protein, 40 +/- 6 reference)

HumanPhenotype: HP:0007513, HP:0000707, HP:0005982, HP:0100610, HP:0004923, HP:0000252 (Generalized hypopigmentation, Abnormality of the nervous system, Reduced phenylalanine hydroxylase activity, Maternal hyperphenylalaninemia, Hyperphenylalaninemia, Microcephaly)

HumanFreeText: N/A

ModelEvidence: PAH_enu2 has been further characterized many times and results from a missense variant in PAH (PMID: 9119379). PAH_enu3 has also been further characterized and results from a canonical splicing variant in PAH (PMID:11161825). Both models fully recapitulate the phenotypes we expect to see in phenylketonuria, including the quintessential elevated PHE levels and significantly decreased PAH activity. Also of note is the progressive hypopigmentation, microcephaly, growth delay, and neurological abnormalities which are all seen in human probands without proper intervention. Maternal hyperphenylalaninemia is also recapitulated, with no pups surviving beyond several hours with standard breeding diet when born to a mother with the homozygous genotype. Diets low in PKU resulted in less severe symptoms, similar to low protein diets in human probands.

ModelVariant1: NM_000277.3(PAH):c.788T>C (p.Phe263Ser)

ClinVar: 619150 https://www.ncbi.nlm.nih.gov/clinvar/variation/619150/

ModelVariant2: NM_000277.3(PAH):c.1199+2T>G

ClinVar: 557365 https://www.ncbi.nlm.nih.gov/clinvar/variation/557365/

ExperimentName: TAZ Function in Patient Fibroblasts

PatientCell: CL:0000057 (Fibroblast)

GeneFunction: GO:0003841 (1-acylglycerol-3-phosphate O-acyltransferase activity)

GeneAlteration: Fibroblasts were utilized from frozen tissue samples that were taken from 14 Barth Syndrome probands and compared to age-matched controls.

FunctionalEvidence: Several relevant tafazzin slice variants (full transcript, -ex5, and -ex7) were expressed in human BTHS fibroblasts and the MLCL/CL ratio was compared to age-matched controls and the previous results from the fibroblasts themselves. This ratio is important as the amount of cardiolipin (CL) conversion by tafazzin is crucial to the disease progress and observable phenotypes. Overall, the ratio observed in fibroblasts alone was significantly higher than controls (~1.1, reference 0.12). The expression of tafazzin splice variants did rescue some of the ratio, although only the preumed active variant lacking exon 5 showed significant decrease of the ratio (~0.5).

FAVariant1: NM_000116.5(TAZ):c.153C>G (p.Tyr51Ter)

ClinVar: 11101, https://www.ncbi.nlm.nih.gov/clinvar/variation/11101/

FAVariant2: NM_000116.4:c.170G>T (p.Arg57Leu)

CAID: CA415179752, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA415179752

FAVariant3: NM_000116.4:c.590G>T (p.Gly197Val)

CAID: CA415184091, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA415184091

FAVariant4: NM_000116.5(TAZ):c.280C>T (p.Arg94Cys)

ClinVar: 202092, https://www.ncbi.nlm.nih.gov/clinvar/variation/202092/

RescueModel: Mouse (Mus musculus)

GeneAlteration: PAH_enu2 mice were purchased and bred to obtain the homozygotic mice for this study. Original Generation Method: Mutations were induced in mice germline cells at a rate near 10^-3 per locus via an alkylating agent called ethylnitrosourea. Inbred BTBR males were treated with this agent and bred to normal BTBR females. Three hundred and fifty progeny were screened for mutations that did not complement the previous model PAH_enu1 and if present bred to a previous homozygote and tested for hyperphenylalaninemia. A PHE clearance test was performed in questionable cases. For those that tested positive the carrier parents were mated to a normal BTBR partner to establish a cogenic mutant line. Western blot confirmed reduced to absent CRM in liver.

RescuePhenotype: HP:0007513, HP:0004923, HP:0005982, HP:0100610 (Generalized hypopigmentation, Hyperphenylalaninemia, Reduced phenylalanine hydroxylase activity, Maternal hyperphenylalaninemia)

RescueMethod: The human PAH expression vector plasmid pAAV2-EF-hPAH-WPRE was created by subcloning a full-length human PAH cDNA into a preexisitng parent vector by substitution with eGFP cDNA. Alos included is an EF promoter, woodchuck hepatitis virus post-transcriptional element, and bovine growth hormone poly (A) signal. The rep2/cap2 plasmid or rep2/cap8 plasmid was used to package the vector and produce the final rAAV2/2-hPAH and rAAV2/8-hPAH vectors by triple plasmid transfection. Five-to-six week old PAH_enu2 mice were anesthetized with ketamine-xylazine and infused with viral particles of rAAV2/2-hPAH or rAAV2/8-hPAH through the hepatic portal vein or tail vein.

RescueEvidence: PAH activity in the livers of treated mice six weeks after treatment recovered to 58.43±5.73% and 89.25±8.79% of the wild-type activity in female and male PAH_enu2 mice respectively compared to <1% WT in untreated mice. Plasma phenylalanine concentration in female PAH_enu2 mice infused with rAAV2/8-hPAH vectors decreased markedly to 110±34.5 µM at two weeks from 2,891±336.2 µM and showed continued significant phenylalanine clearance for six month after rAAV2/8-hPAH infusion. Male PAH_enu2 showed a similar reduction, though they started at a lower base concentration of 1,800.6±233.7 µM. The fur color of treated mice also changed from greyish to black from one week after delivery with sustained fur color changes over the entire six month period. Investiation of effects on pregnant mice revealed a complete recovery of fetus length and body weight compared to the untreated mice (1.746±0.203 cm and 774±144 mg for untreated VS 2.081±0.269 cm and 916±67 mg for treated). Spontaneous abortion rate also normalized to 4.55±5.89% from 30.95±14.27%. The rAAV-hPAH genome was not found in the placentae or fetuses nor did the genome of fetuses affect the development parameters.

ModelVariant: NM_000277.3(PAH):c.788T>C (p.Phe263Ser)

ClinVar: 619150 https://www.ncbi.nlm.nih.gov/clinvar/variation/619150/

ExperimentName: PAH Variant Rescued by FokI-Cas9

RescueCellLine: Cytotoxic T cell (CL:0000910) AKA COS-7 Cell Line

GeneAlteration: A wild-type PAH cDNA was introduced into a vector via XbaI and BamHI cleavage after amplification from the plasmid. The mutation was introduced via site-directed mutagenesis and confirmed via Sanger sequencing. COS-7 cells were seeded and cultured before being transfected with either the mutant vector or the wild-type control. The resulting clone was characterized through RT-PCR and expanded.

RescueCellPhenotype: HP:0005982 (Reduced phenylalanine hydroxylase activity)

RescueMethod: The FokI-dCas9-IRES-ZsGreen1 plasmid was created by designing gRNAs to target the 1222C>T variant and synthesizing a final sgRNA expression fragment which was cloned into the base vector. Electroporation was then performed on the COS-7 cells with the expression vectors incorporated. Flow cytometry analysis confirmed that 18.4% of cells co-expressed both the FokI-dCas9 and the sgRNA.The cells were treated with the ssODN that represents the replacement sequence and confirmed via Sanger sequencing. Any off-target sites were anlyzed via Sanger sequencing and displayed no evidence of off-target cleavage.

RescueEvidence: Western blot analysis performed on cell expressing the wild-type PAH and those treated with differing levels of ODN and RS-1 showed that those treated with the replacement sequence had significantly higher PAH expression than the mutant cells that were not transfected with the vectors. Further, liquid chromatography-electrospray ionization tandem mass spectrometry was used to evaluate PAH activity after repair showing an activity of 22.08 +/- 1.67 % with the RS-1 expressed as opposed to 16.86+/- 1.31 % without the replacement sequence.

RescueCellVariant: NM_000277.3(PAH):c.1222C>T (p.Arg408Trp)

ClinVarID: 577 https://www.ncbi.nlm.nih.gov/clinvar/variation/577/

NonHumanModel: Mouse (Mus musculus)

GeneAlteration: Two approaches were used to create this model. The first imitated the slow fiber-specific expression of TPM3 by linking the coding region for αTm_slow(Met9Arg) to a human troponin I slow gene that drives expression specifically in these fibers via EcoRI-PstI digest and ligation into a linearized construct. The second, HSA–αTm_slow(Met9Arg), expressed the mutation in fast fibers by ligating a SmaI-BamHI cassette containing the intron and 3′-UTR of SV40 small t antigen directly downstream of the mutant cDNA sequence followed by blunt-end ligation of the HSA promoter into the EcoRV site upstream of the sequence before linearizing into a construct. These constructs were injected into fertilized eggs and screened by southern blot analysis to verify the successful insertion.

ModelPhenotype: HP:0003798, HP:0100303, HP:0100301, HP:0003557, HP:0001324, HP:0011807 (Nemaline bodies, Muscle fiber cytoplasmic bodies, Muscle fiber tubular aggregates, Increased variability in muscle fiber diameter, Muscle weakness, Type 1 muscle fiber atrophy)

ModelFreeText: Disorganized sarcomeres, Increased slow/fast oxidative fibers

HumanPhenotype: HP:0003798, HP:0003557, HP:0001324, HP:0011807 (Nemaline bodies, Increased variability in muscle fiber diameter, Muscle weakness, Type 1 muscle fiber atrophy)

HumanFreeText: Disorganized sarcomeres

ModelEvidence: The Met9Arg mouse models not only clearly recapitulate the phenotypes of nemaline/other myopathies but also provide evidence to suggest possible pathogenic mechanisms of the mutated allele in vivo. Mice expressing the mutated allele display nemaline rods and clear fiber-type disproportion, both of which are hallmarks for their respective diseases. They also displayed progressive muscle weakness starting around 5-6 months of age, a phenotype that matches the childhood-onset muscle weakness and other progressive symptoms common in TPM3-related congenital myopathy patients. Another classic phenotype, atrophy of Type I muscle fibers with a corresponding hypotrophy of Type II fibers was also present in the mice. This may be related to the increased number of slow/fast oxidative fibers in the mutant mice leading to the disruption of normal muscle fiber maturation. The mice also displayed some cytoplasmic bodies and tubular aggregates; these have been observed in varying functional studies and similar features have also been observed in humans.

ModelVariant: NM_152263.4(TPM3):c.26T>G (p.Met9Arg)

ClinVar: 12446 https://www.ncbi.nlm.nih.gov/clinvar/variation/12446/

NonHumanModel: Zebrafish

GeneAlteration: TPM2 complementary DNA was obtained and subcloned in-frame with GFP by PCR using the 5' CMV/SP6 promoter. Site-directed mutagenesis was performed to engineer the DNA with the p.K7del mutation. Afterwards, both types of complementary DNA were injected into stage 1 zebrafish embryos. Myofibers were isolated and analyzed from 4- and 7-day post fertilization zebrafish.

ModelPhenotype: N/A

ModelFreeText: 3x decrease in TPM2 width, Disorganized sarcomeres, Localization of TPM2 to the z-band, Decreased overall sarcomere length, Accumulations of Actinin

HumanPhenotype: N/A

HumanFreeText: Disorganized myofibrils and abnormal Z bands, Disorganized sarcomeres, Nuclear clumps

ModelEvidence: This in vivo zebrafish model reflects several of the muscular phenotypes that are displayed in many nemaline myopathy patients. Although only the myofibres were studied, which does not allow for gross muscular phenotypes to develop, there are clear similarities. The disorganized/shortened sarcomeres are commonly seen in muscle biopsies in patients, as well as actin-based nuclear clumps that accumulate. The z-bands and myofibrils can also be demonstrated in nemaline myopathy patients, although the patterns observed are somewhat dissimilar.

ModelVariant: NM_003289.3:c.14_16delAGA (p.Lys7del)

ClinVar: 140486 https://www.ncbi.nlm.nih.gov/clinvar/variation/140486/

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:

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/

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: