Case4: Female, 74 yrs report

CasePresentingHPOs: HP:0002751, HP:0002540, HP:0100753, HP:0003236, HP:0003089, HP:0003798, HP:0031237, HP:0003555, HP:0012785 (Kyphoscoliosis, Inability to walk, Schizophrenia, Elevated serum creatine phosphokinase, Hamstring contracture, Nemaline bodies, Internally nucleated skeletal muscle fibers, Muscle fiber splitting, Flexion contracture of finger)

HPOsFreeText: Jaw contractures, Pectoral contractures, Quad contractures, Disorganized sarccomeres, Nuclear clumps

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Mother of Case 5 and 6, Proband for Family C

CasePreviousTesting: N/A

CaseGenotypingMethod: The TPM2 gene was sequenced and variants identified. No other information is provided.

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

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

gnomAD: Not in gnomAD

Case6: Male, 40 yrs report

CasePresentingHPOs: N/A

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Son of Case 4, Sibling of Case 5

CasePreviousTesting: N/A

CaseGenotypingMethod: The TPM2 gene was sequenced and variants identified. No other information is provided.

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

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

gnomAD: Not in gnomAD

Case3: Male, 41 yrs report

CasePresentingHPOs: HP:0012432, HP:0003738, HP:0003236, HP:0003273, HP:0003044, HP:0012785, HP:0006466, HP:0005997, HP:0003798, HP:0100295, HP:0031237, HP:0003201, HP:0012548 (Chronic fatigue, Exercise-induced myalgia, Elevated serum creatine phosphokinase, Hip contracture, Shoulder flexion contracture, Flexion contracture of finger, Ankle contracture, Neck flexion contracture, Nemaline bodies, Muscle fiber atrophy, Internally nucleated skeletal muscle fibers, Rhabdomyolysis, Skeletal muscle fatty infiltration)

HPOsFreeText: Jaw contractures, Core-like regions

CaseNotHPOs: HP:0001627 (Cardiac abnormality)

NotHPOsFreeText: N/A

CaseAddInfo: Variant is confirmed de novo via parental testing

CasePreviousTesting: ACTA1 and TPM3 were sequenced and returned negative.

CaseGenotypingMethod: The TPM2 gene was sequenced and variants identified. No other information is provided.

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

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

gnomAD: Not in gnomAD

Case2: Male, 14 yrs report

CasePresentingHPOs: HP:0002355, HP:0002527, HP:0000023, HP:0001763, HP:0002942, HP:0001276, HP:0006466, HP:0003089 (Difficulty walking, Falls, Inguinal hernia, Pes planus, Thoracic kyphosis, Hypertonia, Ankle contracture, hamstring contracture)

HPOsFreeText: In-toe gait, Tendo-Achilles release performed

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Son of Case 1, Variant is de novo in mother

CasePreviousTesting: N/A

CaseGenotypingMethod: The TPM2 gene was sequenced and variants identified. No other information is provided.

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

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

gnomAD: Not in gnomAD

Case5: Female, 50 yrs report

CasePresentingHPOs: HP:0002355, HP:0100753, HP:0003089, HP:0003798, HP:0031237, HP:0031318 (Difficulty walking, Schizophrenia, Hamstring contracture, Nemaline bodies, Internally nucleated skeletal muscle fibers, Myofiber disarray)

HPOsFreeText: Jaw contractures, Pectoral contractures, Ankle dorsiflexor contractures, Quad contractures, Increased oxidative staining

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Daughter of Case 4, Sibling of Case 6

CasePreviousTesting: N/A

CaseGenotypingMethod: The TPM2 gene was sequenced and variants identified. No other information is provided.

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

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

gnomAD: Not in gnomAD

Case8: Female, 65 yrs report

CasePresentingHPOs: HP:0001263, HP:0030051, HP:0003326, HP:0003731, HP:0002540, HP:0003236, HP:0006466, HP:0005830, HP:0012785, HP:0009600, HP:0003798, HP:0031237, HP:0003555, HP:0001761 (Delayed milestones, Tip-toe gait, Myalgia, Quadriceps weakness, Non-ambulatory, Elevated serum creatine phosphokinase, Ankle contracture, Toe contractures, Flexion contracture of finger, Contracture of thumb, Nemaline bodies, Internally nucleated skeletal muscle fibers, Muscle fiber splitting, Pes cavus)

HPOsFreeText: Tendo-achilles release performed, NCPAP from 54 yrs, Jaw contractures, Nuclei clumps, Fiber type grouping, Required non-invasive nocturnal ventilatory support at age 54 years

CaseNotHPOs: HP:0001627 (Cardiac abnormality)

NotHPOsFreeText: N/A

CaseAddInfo: Parental testing not performed

CasePreviousTesting: ACTA1 was sequenced and returned negative.

CaseGenotypingMethod: The TPM2 gene was sequenced and variants identified. No other information is provided.

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

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

gnomAD: Not in gnomAD

Case1: Female, 45 yrs report

CasePresentingHPOs: HP:0008994, HP:0002015, HP:0003236, HP:0003273, HP:0031237, HP:0003555, HP:0100295, HP:0012548 (Proximal muscle weakness in lower limbs, Dysphagia, Elevated serum creatine phosphokinase, Hip contracture, Internally nucleated skeletal muscle fibers, Fiber splitting, Muscle fiber atrophy, Skeletal muscle fatty infiltration)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Proband of Family 1, Mother of Case 2, Variant is de novo

CasePreviousTesting: N/A

CaseGenotypingMethod: The TPM2 gene was sequenced and variants identified. No other information is provided.

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

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

gnomAD: Not in gnomAD

Case7: Male, 13 yrs report

CasePresentingHPOs: HP:0001288, HP:0003701, HP:0009053, HP:0000765, HP:0002650, HP:0001250, HP:0006466, HP:0003798, HP:0003803 (Abnormal gait, Proximal limb weakness, Distal lower limb muscle weakness, Abnormality of the chest, Scoliosis, Epilepsy, Ankle contracture, Nemaline bodies, Type 1 muscle fiber predominance)

HPOsFreeText: Jaw contracture

CaseNotHPOs: HP:0001627 (Cardiac abnormality)

NotHPOsFreeText: N/A

CaseAddInfo: Parental testing not performed

CasePreviousTesting: ACTA1 was sequenced and returned negative.

CaseGenotypingMethod: The TPM2 gene was sequenced and variants identified. No other information is provided.

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

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

gnomAD: Not in gnomAD

Case3: Female, Birth onset

CasePresentingHPOs: HP:0001558, HP:0008947, HP:0002643, HP:0011470, HP:0001270, HP:0001349, HP:0001488, HP:0007941, HP:0010628, HP:0003327, HP:0003722, HP:0003391, HP:0030051, HP:0002938, HP:0002943, HP:0008081, HP:0003273, HP:0012785, HP:0012432, HP:0010300, HP:0001611, HP:0002792, HP:0003458, HP:0003557, HP:0003803, HP:0003715 (Reduced fetal movements, Infantile hypotonia, Neonatal respiratory distress, Nasogastric tube feeding in infancy, Motor delay, Facial diplegia, Bilateral ptosis, Limited extraocular movement, Facial muscle weakness, Axial muscle weakness, Neck flexor weakness, Gowers sign, Tip-toe gait, Lumbar hyperlordosis, Thoracic scoliosis, Pes valgus, Hip contracture, Flexion contracture of finger, Chronic fatigue, Abnormally low-pitched voice, Nasal voice, Reduced vital capacity, EMG: myopathy, Increased fiber size variation, Type 1 muscle fiber predominance, Myofibrillar myopathy)

HPOsFreeText: Nocturnal ventilation started at 2 yrs, Tracheostomy performed, Hypermobility of the upper extremities, Spinal contracture, Intermyofibrillar network was coarse and irregular, Cap structures, Jagged z-lines

CaseNotHPOs: HP:0001627, HP:0040081, HP:0040129 (Cardiac abnormality, Abnormal levels of creatine kinase in blood, Abnormal nerve conduction)

NotHPOsFreeText: 24-hour Holter-EKG recording and cardiac ultrasound were normal

CaseAddInfo: Healthy parents and 3 healthy siblings, Mutation is de novo

CasePreviousTesting: N/A

CaseGenotypingMethod: PCR and sequencing analysis were performed on genomic DNA, specifically the entire coding sequence of TPM2. TPM2 was sequenced in both parents and all siblings with negative results.

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

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

gnomAD: Not in gnomAD

Case2: Male, 6 yrs report

CasePresentingHPOs: HP:0001252, HP:0002194, HP:0001324, HP:0002355, HP:0009046, HP:0008376, HP:0002788, HP:0004396, HP:0001824, HP:0002058, HP:0003691, HP:0002938, HP:0001763, HP:0001290, HP:0001284, HP:0003722, HP:0003391, HP:0003557, HP:0003803, HP:0003715 (Muscular hypotonia, Delayed gross motor development, Muscle weakness, Difficulty walking, Difficulty running, Nasal and dysarthic speech, Recurrent upper respiratory tract infections, Poor appetite, Weight loss, Myopathic facies, Scapular winging, increased lumbar lordosis, Pes planus, Decreased muscle tone, Areflexia, Neck flexor weakness, Gowers sign, Increased fiber size variation, Type 1 muscle fiber predominance, Myofibrillar myopathy)

HPOsFreeText: Walk unsupported at 17 months, Abnormal, partly irregular, and coarse-meshed intermyofibrillar network, Cap structures, Jagged z-lines

CaseNotHPOs: HP:0001263, HP:0010862, HP:0001371, HP:0002650, HP:0003115, HP:0002877, HP:0002792, HP:0003798 (Global developmental delay, Delayed fine motor development, Joint contracture, Scoliosis, Abnormal EKG/ECG, Nocturnal hypoventilation, Reduced vital capacity, Nemaline bodies)

NotHPOsFreeText: N/A

CaseAddInfo: Healthy parents, Normal pregnancy, delivery, and neonatal period, Variant is de novo

CasePreviousTesting: Laboratory investigations, including muscle enzymes, have been normal, MYH1, ACTA1, MYH2, MYH7, and TPM3 were all sequenced with negative results.

CaseGenotypingMethod: PCR and sequencing analysis were performed on genomic DNA, specifically the entire coding sequence of TPM2. A PCR fragment of exon 2 of TPM2 was sequenced in both parents with negative results.

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

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

gnomAD: Not in gnomAD

Case1: Male, 42 yrs report

CasePresentingHPOs: HP:0002359, HP:0003324, HP:0002515, HP:0003391, HP:0003273, HP:0000098, HP:0002751, HP:0003691, HP:0000465, HP:0002792, HP:0001620, HP:0001284, HP:0003458, HP:0003557, HP:0003803, HP:0003715 (Frequent falls, Diffuse muscle weakness, Waddling gait, Gowers sign, Hip contracture, Tall stature, Kyphoscoliosis, Scapular winging, Webbed neck, Reduced vital capacity, High-pitched voice, Areflexia, EMG: myopathy, Increased fiber size variation, Type 1 muscle fiber predominance, Myofibrillar myopathy)

HPOsFreeText: Uncomplicated pregnancy and delivery, Difficulty sitting at 8 months, Inability to walk longer distances and must use a wheelchair outdoors at 15 yrs, Extreme gracility of the limb and trunk muscles, Partly irregular and abnormally coarse-meshed pattern of the intermyofibrillar network, Cap structures, Loss of thick filaments, Thickened fragments of Z-bands

CaseNotHPOs: HP:0008947, HP:0010628, HP:0040081, HP:0011675, HP:0003798 (Infantile muscular hypotonia, Facial muscle weakness, Abnormal levels of creatine kinase in blood, Arrhythmia, Nemaline bodies)

NotHPOsFreeText: N/A

CaseAddInfo: Healthy parents and two healthy brothers, Mutation is de novo

CasePreviousTesting: N/A

CaseGenotypingMethod: PCR and sequencing analysis were performed on genomic DNA, specifically the entire coding sequence of TPM2. A PCR fragment of exon 2 of TPM2 was sequenced in both parents and a sibling with negative results.

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

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

gnomAD: Not in gnomAD

CURATE AS FAMILY

Case2: Male, Birth onset, Bosnia

CasePresentingHPOs: HP:0008872, HP:0006829, HP:0001270, HP:0010628, HP:0006466, HP:0000486, HP:0012630, HP:0012240 (Feeding difficulties in infancy, Severe muscular hypotonia, Motor delay, Facial muscle weakness, Ankle contracture, Squint, Abnormality of the trabecular meshwork, Increased muscle lipid droplets)

HPOsFreeText: Predominance of Type 1 muscle fibres, Broad z-band

CaseNotHPOs: HP:0002795, HP:0002540, HP:0003798 (Abnormal respiration, Inability to walk, Nemaline bodies)

NotHPOsFreeText: N/A

CaseAddInfo: Affected Mother had myopathic facies and high-arched palate, weak neck flexors, and asymmetric limb involvement, Healthy grandparents and the father did not have any mutations present

CasePreviousTesting: N/A

CaseGenotypingMethod: Genomic DNA and RNA was extracted from blood or lymphoblastoid cell lines. SSCP was used to analyze all 10 exons of TPM2. After locating the mutations, RT-PCR was used to confirm the mutated allele expression and haplotype analysis was performed on the probands and their family.

Variant: NM_003289.3:c.349G>A (p.Glu117Lys)

ClinVar: 12462 https://www.ncbi.nlm.nih.gov/clinvar/variation/12462/

gnomAD: Not in gnomAD

Case1: Female, 12 yrs onset, Dutch

CasePresentingHPOs: HP:0002093, HP:0001288, HP:0003690, HP:0010628, HP:0003722, HP:0003202, HP:0003798 (Respiratory insufficiency, Gait difficulties, Limb muscle weakness, Facial muscle weakness, Neck flexor weakness, Muscle hypotrophy, Nemaline rods)

HPOsFreeText: Mild form of nemaline myopathy, Wheelchair from the age of 48, Selective hypotrophy of Type 1 muscle fibres

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: No family history of neuromuscular disorders, Variant was not detected in healthy mother or 7 unaffected siblings, Father was deceased so no sample was available

CasePreviousTesting: N/A

CaseGenotypingMethod: Genomic DNA and RNA was extracted from blood or lymphoblastoid cell lines. SSCP was used to analyze all 10 exons of TPM2. After locating the mutations, RT-PCR was used to confirm the mutated allele expression and haplotype analysis was performed on the probands and their family.

Variant: NM_003289.3:c.440A>C (p.Gln147Pro)

ClinVar: 12461 https://www.ncbi.nlm.nih.gov/clinvar/variation/12461/

gnomAD: Not in gnomAD

Case2: Female, Birth onset

CasePresentingHPOs: HP:0002093, HP:0002938, HP:0002015, HP:0003798, HP:0003200, HP:0003548 (Respiratory insufficiency, Lumbar hyperlordosis, Dysphagia, Nemaline bodies, Ragged-red fibers, Subsarcolemmal accumulations of abnormally shaped mitochondria)

HPOsFreeText: N/A

CaseNotHPOs: N/A

NotHPOsFreeText: N/A

CaseAddInfo: Mother of proband

CasePreviousTesting: N/A

CaseGenotypingMethod: See Family

Variant: NM_003289.3:c.397C>T (p.Arg133Trp)

ClinVar: 12463 https://www.ncbi.nlm.nih.gov/clinvar/variation/12463/

gnomAD: Not in gnomAD

Case1: Female, Birth onset

CasePresentingHPOs: HP:0008947, HP:0008872, HP:0002033, HP:0030043, HP:0001349, HP:0001488, HP:0010804, HP:0002650 (Infantile hypotonia, Feeding difficulties in infancy, Poor suck, Hip subluxation, Facial diplegia, Bilateral ptosis, Tented mouth, Scoliosis)

HPOsFreeText:Disorganized myofibrils and thickened Z bands

CaseNotHPOs: HP:0003798 (Nemaline bodies)

NotHPOsFreeText: N/A

CaseAddInfo: Proband of Family 1

CasePreviousTesting: N/A

CaseGenotypingMethod: See Family

Variant: NM_003289.3:c.397C>T (p.Arg133Trp)

ClinVar: 12463 https://www.ncbi.nlm.nih.gov/clinvar/variation/12463/

gnomAD: Not in gnomAD

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/

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/