ExperimentName: The Cpfl1 mouse harbors loss-of-function variants in the Pde6c gene.

NonHumanModel: Mus musculus

GeneAlteration: The model animals are homozygous for two different loss-of-function variants predicted to introduce premature stop codons into the Pde6c locus. One is a 116-bp insertion between exons 4 and 5, while the other is a 1-bp deletion in exon 7.

ModelPhenotype: HP:0008020 (cone dystrophy), HP:0030465 (Undetectable light-adapted electroretinogram), normal dark-adapted ERG, rapid and progressive loss of cone photoreceptors from the photoreceptor layer

HumanPhenotype: HP:0030465 (Undetectable light-adapted electroretinogram), HP:0030534 (Abnormal best corrected visual acuity test), HP:0000551 (Color vision defect), HP:0000613 (Photophobia), HP:0000639 (Nystagmus), HP:0007401 (Macular atrophy), HP:0000545 (Myopia)

ModelEvidence: This is a naturally occurring mouse model with lack of light-adapted ERG responses and normal dark-adapted ERG responses (Figure 1). The mice also exhibit rapid and progressive loss of cone photoreceptors from retinal sections (Figure 2).

ModelVariant: This model did not introduce a disease-associated variant known from an affected human patient, but rather studied naturally occurring variants disrupting the Pde6c locus.

ExperimentName: Naturally occurring BBS7 loss-of-function primate model

NonHumanModel: Rhesus macaque

GeneAlteration: The model animals harbor a naturally occurring homozygous frameshift variant at nucleotide 160 within exon 3 of the BBS7 locus.

ModelPhenotype: HP:0000608 (Macular degeneration), HP:0030609 (Photoreceptor layer loss on macular OCT), HP:0000580 (Pigmentary retinopathy), HP:0008046 (Abnormal retinal vascular morphology), progressive kidney disease, HP:0030483 (Reduced amplitude of dark-adapted bright flash electroretinogram a-wave), HP:0030469 (Abnormal dark-adapted electroretinogram), HP:0008275 (Abnormal light-adapted electroretinogram), HP:0002171 (Gliosis), HP:0000083 (Renal insufficiency), HP:0012210 (Abnormal renal morphology), HP:0002119 (Ventriculomegaly), HP:0008734 (Decreased testicular size)

HumanPhenotype: HP:0001956 (Truncal obesity), HP:0000618 (Blindness), HP:0001249 (Intellectual disability), HP:0001631 (Atrial septal defect), HP:0001640 (Cardiomegaly), HP:0100598 (Pulmonary edema), HP:0012584 (Bilateral renal hypoplasia), HP:0004719 (Hyperechogenic kidneys), HP:0003774 (Stage 5 chronic kidney disease), HP:0000580 (Pigmentary retinopathy), HP:0000648 (Optic atrophy), HP:0008043 (Retinal arteriolar constriction), HP:0007737 (Bone spicule pigmentation of the retina), HP:0007754 (Macular dystrophy)

ModelEvidence: This is a naturally occurring Rhesus macaque model of biallelic BBS7 disruption. The mutant animals exhibit hypoechoic kidney lesions (Figure 1), retinal atrophy with lack of retinal vasculature (Figure 3A), RPE atrophy (Figure 3B), severe atrophy of retinal layers by OCT with greater severity centrally (Figure 3C), retinal gliosis (Figure 3D), Severely reduced amplitudes and delayed time courses of both dark-adapted and light-adapted flicker ERG (Figure 4), reduced brain volume (Figure 5B), dilation of the lateral ventricle (Figure 5A), cystic kidney tubules with loss of nephrons replaced with fibrosis, tubule atrophy, and ectasia (Figure 7), reduced size and histological maturity of testes (Table 1), and vision deficits (Table 1).

ModelVariant: This model did not introduce a disease-associated variant known from an affected human patient, but rather studied a naturally occurring and apparent null variant disrupting BBS7 expression.

ExperimentName: bbs1-1 Chlamydomonas have full-length flagella and normal IFT function but are nonphototactic.

NonHumanModel: Chlamydomonas reinhardtii

GeneAlteration: The bbs1-1 model animals have been identified as naturally occurring mutants with deletion of approximately half of the BBS1 gene as well as a separate gene unstream of BBS1.

ModelPhenotype: abnormal flagellar motility (inability to perform phototaxis), flagellar absence of BBSome components (specifically BBS4)

HumanPhenotype: HP:0000639 (Nystagmus), HP:0000510 (Rod-cone dystrophy), HP:0007663 (Reduced visual acuity), HP:0000662 (Nyctalopia), HP:0000518 (Cataract), HP:0008323 (Abnormal light- and dark-adapted electroretinogram), HP:0000107 (Renal cyst), HP:0001513 (Obesity), HP:0010442 (Polydactyly), HP:0002463 (Language impairment), HP:0000026 (Male hypogonadism)

ModelEvidence: The mutants lack the ability to perform phototaxis, which is common among all BBSome mutants tested (described in text but data not shown). Flagella from the mutant show loss of BBS4 localization (Figure 4C). However, flagellar length was normal (Figure 2B) and flagellar regeneration was normal as well (described in text but data not shown).

ModelVariant: This model had a naturally occurring deletion disrupting BBS1 but did not feature knock-in of a known variant from the human patient population.

ExperimentName: Zebrafish knockdown of the GPR143 ortholog OA1

NonHumanModel: Danio rerio

GeneAlteration: A zebrafish model of OA1 loss-of-function was generated by morpholino-based knockdown.

ModelPhenotype: HP:0007703 (Abnormality of retinal pigmentation)

HumanPhenotype: HP:0000639 (Nystagmus), HP:0003593 (Infantile onset), HP:0007663 (Reduced visual acuity), HP:0001361 (Nystagmus-induced head nodding), HP:0000646 (Amblyopia), HP:0000483 (Astigmatism), HP:0000613 (Photophobia), normally pigmented skin and hair, HP:0005592 (Giant melanosomes in melanocytes), HP:0007750 (Hypoplasia of the fovea), HP:0007894 (Hypopigmentation of the fundus), HP:0025551 (Optic nerve misrouting)

ModelEvidence: The zebrafish match one feature of the human patients in that they exhibit abnormalities of retinal pigmentation consistent with the ability of GPR143 loss-of-function to trigger defects in melanosome regulation (Figure 1).

ModelVariant: This model features disruption of OA1 expression by knockdown, and does not harbor a mutation equivalent to a variant from a human patient.

ExperimentName: Canine model of Adam9 loss-of-function

NonHumanModel: Canis lupus familiaris

GeneAlteration: The affected dogs have homozygous disruption of the Adam9 gene by a deletion of regions from exons 15 and 16.

ModelPhenotype: HP:0001105 (Retinal atrophy), HP:0000529 (Progressive visual loss), HP:0000580 (Pigmentary retinopathy), HP:0000662 (Nyctalopia)

HumanPhenotype: HP:0011463 (Childhood onset), HP:0000548 (Cone/cone-rod dystrophy), HP:0007663 (Reduced visual acuity), HP:0001141 (Severely reduced visual acuity), HP:0007994 (Peripheral visual field loss), HP:0000545 (Myopia), HP:0000483 (Astigmatism), HP:0000523 (Subcapsular cataract)

ModelEvidence: The affected dogs have been described as having generalized progressive retinal atrophy. Complete loss of all retinal layers was observed in affected animals (Figures 1H-1P). This matches the mode of inheritance and progressive nature of the human disease state, as well as the retinal abnormalities and visual defects.

ModelVariant: The model harbors a targeted deletion that does not have an equivalent / orthologous variant reported in human patients.

ExperimentName: CRISPR/Cas9-based disruption of mouse Impg1

NonHumanModel: Mus musculus

GeneAlteration: Model animals were subjected to CRISPR/Cas9-based deletion of 10bp from exon 2 of Impg1, and analyzed in the homozygous state. Absence of Impg1 signal was confirmed by immunofluorescent staining. Mice were studied at ages 5 months and 8 months.

ModelPhenotype: HP:0030612 (Abnormal retinal morphology, specifically mislocalization of chondroitin sulfate in the IPM, with none in the outer segment and more in the inner segment)

HumanPhenotype: HP:0007754 (Macular dystrophy), HP:0007769 (Peripheral retinal degeneration), HP:0032037 (Mildly reduced visual acuity, progressive), HP:0008179 (Decreased Arden ratio of electrooculogram)

ModelEvidence: This is the first mouse model of Impg1 disruption, with confirmed absence of Impg1 in the homozygous animals. The only observed abnormality is mislocalization of chondroitin sulfate in the IPM, with none in the outer segment and more in the inner segment (Figure 7). A mild disruption was observed in the localization of PNA, a cone-specific marker, to the outer segment (Fig. 2F). Impg2 appears to be normally localized (Figure 2C). ERG responses were normal (data not shown). Please note that an independent study generating a different model of homozygous Impg1 disruption reported different findings (PMID: 36140676).

ModelVariant: The model does not introduce a variant previously observed in human patients, but rather disrupts the expression of the Impg1 ortholog.

ExperimentName: Mouse model of homozygous Cln5 loss

NonHumanModel: Mus musculus

GeneAlteration: Mice are homozygous for Cln5 loss of function, caused by Insertion of a neomycin resistance cassette eliminating exon 3, resulting in a premature stop codon in exon 4. The presence or absence of a truncated gene product was not assessed.

ModelPhenotype: HP:0003208 (Fingerprint intracellular accumulation of autofluorescent lipopigment storage material), HP:0003205 (Curvilinear intracellular accumulation of autofluorescent lipopigment storage material), HP:0000572 (Visual loss)

HumanPhenotype: HP:0003208 (Fingerprint intracellular accumulation of autofluorescent lipopigment storage material), HP:0003205 (Curvilinear intracellular accumulation of autofluorescent lipopigment storage material), HP:0000572 (Visual loss)

ModelEvidence: The model phenotypes closely match the human patient phenotypes at the microscopic level (characteristic autofluorescent inclusion bodies ) as well as the loss of vision, but do not reproduce neurological features such as seizures, ataxia, loss of motor coordination, mental regression, and behavioral changes.

ModelVariant: While it is unclear whether the mice express a truncated form of Cln5 or whether this form is degraded, the predicted protein product does not represent a particular human variant. The authors speculate that it may be most similar to the NM_006493.4(CLN5):c.522dup (p.Trp175LeufsTer30) variant (PMID:10953198).

NonHumanModel Mus Musculus

GeneAlteration The knock-in strain was generated by homologous recombination of the targeting vector with 129Sv/J embryonic stem cells genome. A 450-bp DNA segment encompassing 5′ UTR and exon 1 of the CASQ2 was used to screen RPCI-21 PAC mouse genomic library. Positive clones were sequenced to define the structure of an 8.5-Kb region encompassing promoter, exon 1, and part of intron 1 of mouse CASQ2. This region was cloned in 3 parts and assembled into the targeting vector pFlrt (supplemental Figure I). The linearized targeting vector was electroporated into 129Sv/J embryonic stem cells. The clone selected with G418 and gancyclovir was injected into C57BL/6NCrL blastocyts and transferred to pseudopregnant CD-1 females. Genotype was determined by sequencing of DNA extracted from tail biopsy specimens (DNasy Tissue Kit, Qiagen).

ModelPhenotype HP:0004758

HumanPhenotype HP:0004758

ModelEvidence Fig. 1 ECG during stimuli shows VT in only the mutant mice

ModelVariant NM_001232.3(CASQ2):c.97C>T (p.Arg33Ter), 41043 link

NonHumanModel Mus Musculus

GeneAlteration The knock-in strain was generated by homologous recombination of the targeting vector with 129Sv/J embryonic stem cells genome. A 450-bp DNA segment encompassing 5′ UTR and exon 1 of the CASQ2 was used to screen RPCI-21 PAC mouse genomic library. Positive clones were sequenced to define the structure of an 8.5-Kb region encompassing promoter, exon 1, and part of intron 1 of mouse CASQ2. This region was cloned in 3 parts and assembled into the targeting vector pFlrt (supplemental Figure I). The linearized targeting vector was electroporated into 129Sv/J embryonic stem cells. The clone selected with G418 and gancyclovir was injected into C57BL/6NCrL blastocyts and transferred to pseudopregnant CD-1 females. Genotype was determined by sequencing of DNA extracted from tail biopsy specimens (DNasy Tissue Kit, Qiagen).

ModelPhenotype HP:0004758

HumanPhenotype HP:0004758

ModelEvidence Fig. 1 ECG during stimuli shows VT in only the mutant mice

ModelVariant NM_001232.3(CASQ2):c.97C>T (p.Arg33Ter), 41043 link

ExperimentName: Mutt mice have a FBXO11 S244L mutation

NonHumanModel: mouse (mus musculus)

GeneAlteration: ENU (ethylnitrosourea) was used to induce random mutatgeneiss in the mouse (PMID: 10932191). Animals were screened based on the development of otitis media. Sequencing of the mouse genome uncovered a single point mutation in the FBXO11 gene resulting in a Ser244Leu protein change. This mutation occurs in the region between the F-box and CASH domain. The Ser at amino acid position 244 is conserved across several species.

ModelPhenotype: HP:0000708

Also cleft palate and craniofacial abnormalities

HumanPhenotype: HP:0000708

ModelEvidence: The Mutt mutant mice have a point mutation in FBXO11, S244L. Heterozygous Mutt mice displayed reduced startle reflex at postnatal day 48. They also showed mild cranialfacila abnormalities including a shortened face. The Mutt homozygtote mice demonstrated perinatal lethality within the first day after birth in 17% of mice, but was much less severe than the Jeff mutant mice. the Jeff mice. Furthermore, that homozygotes mice had mild cleft palate and variable facial clefting, and the surviving homozygotes went on to develop craniofacial abnormalities including short face as well as reduced hearing.

ModelVariant: NM_001190274.1:c.983C>T (p. Ser328Leu) equivalent to ENST00000402508.5:c.731C>T (p. Ser244Leu)

CA ID: CA346766144

ExperimentName: Mutt mice have a FBXO11 S244L mutation

NonHumanModel: mouse (mus musculus)

GeneAlteration: ENU (ethylnitrosourea) was used to induce random mutatgeneiss in the mouse (PMID: 10932191). Animals were screened based on the development of otitis media. Sequencing of the mouse genome uncovered a single point mutation in the FBXO11 gene resulting in a Ser244Leu protein change. This mutation occurs in the region between the F-box and CASH domain. The Ser at amino acid position 244 is conserved across several species.

ModelPhenotype: HP:0000708

Also cleft palate and craniofacial abnormalities

HumanPhenotype: HP:0000708

ModelEvidence: The Mutt mutant mice have a point mutation in FBXO11, S244L. Heterozygous Mutt mice displayed reduced startle reflex at postnatal day 48. They also showed mild cranialfacila abnormalities including a shortened face. The Mutt homozygtote mice demonstrated perinatal lethality within the first day after birth in 17% of mice, but was much less severe than the Jeff mutant mice. the Jeff mice. Furthermore, that homozygotes mice had mild cleft palate and variable facial clefting, and the surviving homozygotes went on to develop craniofacial abnormalities including short face as well as reduced hearing.

ModelVariant: NM_001190274.1:c.983C>T (p. Ser328Leu) equivalent to ENST00000402508.5:c.731C>T (p. Ser244Leu)

CA ID: CA346766144

CellLine: Human Mononuclear Myoblast

CellEFO: CL:0000056

GeneFunction: GO:0007015 (Actin Filament Organization)

GeneAlteration: A wild-type C-terminal EGFP-tagged β-TM fusion gene was generated by amplifying TPM2 and cloning it into a vector while introducing XhoI and EcoRI restriction sites. All mutant constructs were formed by site-directed mutagenesis and cloning into E. coli. These constructs were sequenced to verify the correct coding sequence and introduction of the mutations. They were then introduced to human myoblast cells and mouse C2C12 cells via transfection.

FunctionalEvidence: The expression of these TPM2 mutants in cultured cells demonstrated several morphological abnormalities reminiscent of those seen in nemaline myopathy patients. In the human myoblasts/differentiated cells, these included various types of aggregates including nuclear, cytoplasmic, endogenous, and perinuclear. Large accumulations of actin along with clouds around the nucleus and cytoplasm with unorganized structure were also present. Poor incorporation of stress fibers, what appeared to be disorganized sarcomeric structure, and rod-like structures both intranuclear and in the myotubes in particular all demonstrate clear similarities to those phenotypes seen in nemaline myopathy, namely the presence of nemaline rods, disorganized sarcomeres, and z-disc abnormalities. These two groups of pathologies also seem to share the same mechanism, specifically the TPM2 mutations destabilizing the actin-myosin structures and causing accumulation and abnormal structures to form. Similarly, the modified C2C12 myoblasts/myotubes also displayed peripheral and perinuclear aggregates in both stages of development as well as diffuse cytoplasmic labeling of stress fibers. They had difficulty differentiating, as they appeared to be fused rather than elongated and multinucleated. This further supports the connection between the gene disruption and the pathologies that are demonstrated here.

CellVariant1: NM_003289.3:c.121G>A (p.Glu41Lys)

ClinVar: 12464 http://www.ncbi.nlm.nih.gov/clinvar/variation/12464

CellVariant2: NM_003289.3:c.142_144delAAG (p.Lys49del)

ClinVar: 12466 https://www.ncbi.nlm.nih.gov/clinvar/variation/12466/

CellVariant3: NM_003289.3:c.154_156dup (p.Gly52dup)

ClinVar: 140485 https://www.ncbi.nlm.nih.gov/clinvar/variation/140485/

CellVariant4: NM_003289.3:c.364G>A (p.Glu122Lys)

CAID: CA373367963 http://reg.genome.network/redmine/projects/registry/genboree_registry/by_caid?caid=CA373367963

CellVariant5: NM_003289.3:c.606C>G (p.Asn202Lys)

ClinVar: 12467 https://www.ncbi.nlm.nih.gov/clinvar/variation/12467/

CellLine: Human Mononuclear Myoblast

CellEFO: CL:0000056

GeneFunction: GO:0007015 (Actin Filament Organization)

GeneAlteration: A wild-type C-terminal EGFP-tagged β-TM fusion gene was generated by amplifying TPM2 and cloning it into a vector while introducing XhoI and EcoRI restriction sites. All mutant constructs were formed by site-directed mutagenesis and cloning into E. coli. These constructs were sequenced to verify the correct coding sequence and introduction of the mutations. They were then introduced to human myoblast cells and mouse C2C12 cells via transfection.

FunctionalEvidence: The expression of these TPM2 mutants in cultured cells demonstrated several morphological abnormalities reminiscent of those seen in nemaline myopathy patients. In the human myoblasts/differentiated cells, these included various types of aggregates including nuclear, cytoplasmic, endogenous, and perinuclear. Large accumulations of actin along with clouds around the nucleus and cytoplasm with unorganized structure were also present. Poor incorporation of stress fibers, what appeared to be disorganized sarcomeric structure, and rod-like structures both intranuclear and in the myotubes in particular all demonstrate clear similarities to those phenotypes seen in nemaline myopathy, namely the presence of nemaline rods, disorganized sarcomeres, and z-disc abnormalities. These two groups of pathologies also seem to share the same mechanism, specifically the TPM2 mutations destabilizing the actin-myosin structures and causing accumulation and abnormal structures to form. Similarly, the modified C2C12 myoblasts/myotubes also displayed peripheral and perinuclear aggregates in both stages of development as well as diffuse cytoplasmic labeling of stress fibers. They had difficulty differentiating, as they appeared to be fused rather than elongated and multinucleated. This further supports the connection between the gene disruption and the pathologies that are demonstrated here.

CellVariant1: NM_003289.3:c.121G>A (p.Glu41Lys)

ClinVar: 12464 http://www.ncbi.nlm.nih.gov/clinvar/variation/12464

CellVariant2: NM_003289.3:c.142_144delAAG (p.Lys49del)

ClinVar: 12466 https://www.ncbi.nlm.nih.gov/clinvar/variation/12466/

CellVariant3: NM_003289.3:c.154_156dup (p.Gly52dup)

ClinVar: 140485 https://www.ncbi.nlm.nih.gov/clinvar/variation/140485/

CellVariant4: NM_003289.3:c.364G>A (p.Glu122Lys)

CAID: CA373367963 http://reg.genome.network/redmine/projects/registry/genboree_registry/by_caid?caid=CA373367963

CellVariant5: NM_003289.3:c.606C>G (p.Asn202Lys)

ClinVar: 12467 https://www.ncbi.nlm.nih.gov/clinvar/variation/12467/