Case1: Male, United States, Birth onset

CasePresentingHPOs: HP:0001558, HP:0003552, HP:0002804, HP:0001762, HP:0001188, HP:0000023, HP:0001385, HP:0006380, HP:0004482, HP:0004322, HP:0003782, HP:0011220, HP:0000160, HP:0000470, HP:0000301, HP:0002751, HP:0003306, HP:0001270, HP:0003808, HP:0003044, HP:0012453, HP:0001193, HP:0012785, HP:0003273, HP:0012894, HP:0002174, HP:0004370, HP:0002362, HP:0002942, HP:0012418, HP:0002789, HP:0002205, HP:0002870, HP:0003458, HP:0040129, HP:0003236, HP:0003388, HP:0002015, HP:0003557, HP:0003803, HP:0003715, HP:0012240, HP:0100295 (Decreased fetal movement, Muscle stiffness, Arthrogryposis multiplex congenita, Talipes equinovarus, Hand clenching, Inguinal hernia, Hip dysplasia, Knee flexion contracture, Relative macrocephaly, Short stature, Eunuchoid habitus, Prominent forehead, Small mouth, Short neck, Abnormality of facial musculature, Kyphoscoliosis, Spinal rigidity, Motor delay, Abnormal muscle tone, Shoulder flexion contracture, Bilateral wrist flexion contracture, Ulnar deviation of the hand or of fingers of the hand, Flexion contracture of finger, Hip contracture, Paraspinal muscle hypertrophy, Postural tremor, Abnormality of temperature regulation, Shuffling gait, Thoracic kyphosis, Hypoxemia, Tachypnea, Recurrent respiratory infections, Obstructive sleep apnea, EMG: myopathic abnormalities, Abnormal nerve conduction velocity, Elevated serum creatine phosphokinase, Easy fatigability, Dysphagia, Increased variability in muscle fiber diameter, Type 1 muscle fiber predominance, Myofibrillar changes, Increased intramyocellular lipid droplets, Muscle fiber atrophy)

HPOsFreeText: Jaw contracture, Heel contracture, Strong bilateral grasp, Occasional grunting, Supplemental oxygen required, Ultrastructural evidence of accumulation of mitochondria, Focal Z-line streaming and broadening

CaseNotHPOs: HP:0100543, HP:0001315, HP:0001324, HP:0002486, HP:0100288, HP:0002353, HP:0002363, HP:0003116, HP:0003798 (Cognitive impairment, Reduced/absent deep tendon reflexes, Muscle weakness, Myotonia, EMG: myokymic discharges, EEG abnormality, Abnormality of brainstem morphology, Abnormal echocardiogram, Nemaline bodies)

NotHPOsFreeText: N/A

CaseAddInfo: Case is de novo and confirmed via parental testing

CasePreviousTesting: Negative Results from WES: ACTA1, MYH3, TPM2, HSPG2, TNNT3, TNNI2, FKRP, CRYAB, and microarray.

CaseGenotypingMethod: DNA was obtained from blood and WES was performed on the samples using Illumina technology. Mutations were analyzed and filtered.

Variant: NM_152263.4(TPM3):c.670_672GAA[1] (p.Glu225del)

ClinVar: 235143 https://www.ncbi.nlm.nih.gov/clinvar/variation/235143/

gnomAD: Not in gnomAD

Case2: Female, Birth onset, Germany

CasePresentingHPOs: HP:0003552, HP:0001558, HP:0002375, HP:0002747, HP:0004887, HP:0001385, HP:0001537, HP:0000023, HP:0012418, HP:0000278, HP:0000347, HP:0000470, HP:0001276, HP:0002808, HP:0001376, HP:0002063, HP:0011968, HP:0000975, HP:0003236, HP:0000938, HP:0009803, HP:0030084, HP:0001561, HP:0002828, HP:0005949, HP:0001284, HP:0003198, HP:0003557, HP:0003687, HP:0010602 (Muscle stiffness, Decreased fetal movement, Hypokinesia, Respiratory insufficiency due to muscle weakness, Respiratory failure requiring assisted ventilation, Hip dysplasia, Umbilical hernia, Inguinal hernia, Hypoxemia, Retrognathia, Micrognathia, Short neck, Hypertonia, Kyphosis, Limited joint motion, Rigidity, Feeding difficulties, Profuse sweating, Elevated serum creatine phosphokinase, Osteopenia, Shortened phalanges, Clinodactyly, Polyhydramnios, Multiple joint contractures, Apneic episodes in infancy, Areflexia, Myopathic changes, Increased variability in muscle fiber diameter, Centrally nucleated skeletal muscle fibers, Type 2 muscle fiber predominance)

HPOsFreeText: Tracheostomy performed, Bladder catheter required, Inability to sit or roll, Increased echogenicity on muscle ultrasound, Treatment with carbamazepine, benzodiazepines, and baclofen reportedly provided no substantial benefit, Difficulty sitting due to hyperactivity of hip extensors, Mini rods and z-band broadening were seen in atrophic fibers

CaseNotHPOs: HP:0100543, HP:0003730, HP:0002486, HP:0002353, HP:0002363, HP:0000496, HP:0003202, HP:0003798 (Cognitive impairment, EMG: myotonic runs, Myotonia, EEG abnormality, Abnormality of brainstem morphology, Abnormality of eye movement, Skeletal muscle atrophy, Nemaline bodies)

NotHPOsFreeText: Abnormal metabolic screening, Muscle fiber type grouping

CaseAddInfo: Case is de novo and confirmed via parental testing

CasePreviousTesting: Negative ACTA1 and CRYAB

CaseGenotypingMethod: Based on the Patient 1 results, the same region of TPM3 (Exon 6) was PCR amplified and sequenced in the proband and her parents via bidirectional Sanger sequencing.

Variant: NM_152263.4(TPM3):c.654_656AGA[1] (p.Glu219del)

ClinVar: 235144 https://www.ncbi.nlm.nih.gov/clinvar/variation/235144/

gnomAD: Not in gnomAD

Family6: West Indies

FamilyPresentingHPOs: HP:0008947, HP:0003701, HP:0002650, HP:0003803, HP:0003755 (Infantile muscular hypotonia, Proximal muscle weakness, Scoliosis, Type 1 muscle fiber predominance, Type 1 fibers relatively smaller than type 2 fibers)

HPOsFreeText: N/A

FamilyNotHPOs: HP:0000508, HP:0001249, HP:0030177 (Ptosis, Intellectual disability, Abnormal nerve conduction study)

NotHPOsFreeText: N/A

FamilyPreviousTesting: Patients were diagnosed with CFTD via clinical notes and muscle biopsies at pathology centers throughout Australia and several international centers.

FamilyGenotypingMethod: Two methods were used for genotyping. In 16 patients, the 10 exons spliced onto the muscle-specific isoform of TPM3 were bidirectionally sequenced from genomic DNA. For the other 7 patients, complementary DNA was generated from patient muscle and isoform-specific primers were used to sequence the transcript. Paternity was confirmed in isolated patients using microsatellite markers.

FamilyAddInfo: Father is diagnosed with CFTD and daughter is diagnosed with nemaline myopathy resulting from the same variant

FamilyPMIDs: N/A

AddSegregationInfo: Not enough data to count segregation, Pedigree in Figure 1

ProbandLabel: Case 11

Variant: NM_152263.4(TPM3):c.503G>A (p.Arg168His)

ClinVar: 12450 https://www.ncbi.nlm.nih.gov/clinvar/variation/12450/

gnomAD: Not in gnomAD

Case9: Female, 32 yrs report, France

CasePresentingHPOs: HP:0002421, HP:0002205, HP:0003551, HP:0003701, HP:0002460, HP:0000467, HP:0003391, HP:0001488, HP:0002751, HP:0003202, HP:0003782, HP:0005997, HP:0003803, HP:0003755 (Poor head control, Recurrent respiratory infections, Difficulty climbing stairs, Proximal muscle weakness, Distal muscle weakness, Neck muscle weakness, Gowers sign, Bilateral ptosis, Kyphoscoliosis, Skeletal muscle atrophy, Eunuchoid habitus, Neck flexion contracture, Type 1 muscle fiber predominance, Type 1 fibers relatively smaller than type 2 fibers)

HPOsFreeText: Noninvasive BiPAP from 19 yrs, Type 2B fibers absent

CaseNotHPOs: HP:0003798, HP:0030177 (Nemaline bodies, Abnormal nerve conduction study)

NotHPOsFreeText: N/A

CaseAddInfo: Variant is de novo with parental testing confirmation

CasePreviousTesting: Patients were diagnosed with CFTD via clinical notes and muscle biopsies at pathology centers throughout Australia and several international centers.

CaseGenotypingMethod: Two methods were used for genotyping. In 16 patients, the 10 exons spliced onto the muscle-specific isoform of TPM3 were bidirectionally sequenced from genomic DNA. For the other 7 patients, complementary DNA was generated from patient muscle and isoform-specific primers were used to sequence the transcript. Paternity was confirmed in isolated patients using microsatellite markers.

Variant: NM_152263.4(TPM3):c.502C>T (p.Arg168Cys)

ClinVar: 12454 https://www.ncbi.nlm.nih.gov/clinvar/variation/12454/

gnomAD: Not in gnomAD

Case3: Male, 8 yrs onset, Australia

CasePresentingHPOs: HP:0009046, HP:0003701, HP:0003722, HP:0001488, HP:0002938, HP:0010628, HP:0030877, HP:0003803, HP:0003755 (Difficulty running, Proximal muscle weakness, Neck flexor weakness, Bilateral ptosis, Lumbar hyperlordosis, Facial palsy, Obstructive deficit on pulmonary function testing, Type 1 muscle fiber predominance, Type 1 fibers relatively smaller than type 2 fibers)

HPOsFreeText: Type 2B fibers absent

CaseNotHPOs: HP:0003798, HP:0030177, HP:0003391 (Nemaline bodies, Abnormal nerve conduction study, Gowers sign)

NotHPOsFreeText: N/A

CaseAddInfo: Variant is likely de novo, but there is no parental testing to confirm

CasePreviousTesting: Patients were diagnosed with CFTD via clinical notes and muscle biopsies at pathology centers throughout Australia and several international centers.

CaseGenotypingMethod: Two methods were used for genotyping. In 16 patients, the 10 exons spliced onto the muscle-specific isoform of TPM3 were bidirectionally sequenced from genomic DNA. For the other 7 patients, complementary DNA was generated from patient muscle and isoform-specific primers were used to sequence the transcript. Paternity was confirmed in isolated patients using microsatellite markers.

Variant: NM_152263.4(TPM3):c.502C>G (p.Arg168Gly)

ClinVar: 12453 https://www.ncbi.nlm.nih.gov/clinvar/variation/12453/

gnomAD: Not in gnomAD

Case1: Male, 3 months onset, Australia

CasePresentingHPOs: HP:0002878, HP:0002460, HP:0001270, HP:0003701, HP:0000467, HP:0001488, HP:0002938, HP:0003691, HP:0003391, HP:0003803, HP:0003755 (Respiratory failure, Distal muscle weakness, Motor delay, Proximal muscle weakness, Neck muscle weakness, Bilateral ptosis, Lumbar hyperlordosis, Scapular winging, Gowers sign, Type 1 muscle fiber predominance, Type 1 fibers relatively smaller than type 2 fibers)

HPOsFreeText: BiPAP from 3.5 yrs, Type 2B fibers absent

CaseNotHPOs: HP:0003798, HP:0030177, HP:0031237 (Nemaline bodies, Abnormal nerve conduction study, Internally nucleated skeletal muscle fibers)

NotHPOsFreeText: N/A

CaseAddInfo: Variant is de novo with parental testing confirmed

CasePreviousTesting: Patients were diagnosed with CFTD via clinical notes and muscle biopsies at pathology centers throughout Australia and several international centers.

CaseGenotypingMethod: Two methods were used for genotyping. In 16 patients, the 10 exons spliced onto the muscle-specific isoform of TPM3 were bidirectionally sequenced from genomic DNA. For the other 7 patients, complementary DNA was generated from patient muscle and isoform-specific primers were used to sequence the transcript. Paternity was confirmed in isolated patients using microsatellite markers.

Variant: NM_152263.4(TPM3):c.733A>G (p.Arg245Gly)

ClinVar: 42117 https://www.ncbi.nlm.nih.gov/clinvar/variation/42117/

gnomAD: Not in gnomAD

Case2: Male, Birth onset, Australia

CasePresentingHPOs: HP:0008947, HP:0002460, HP:0001270, HP:0003701, HP:0000467, HP:0001488, HP:0002938, HP:0010628, HP:0003391, HP:0002942, HP:0003803, HP:0003755 (Infantile muscular hypotonia, Distal muscle weakness, Motor delay, Proximal muscle weakness, Neck muscle weakness, Bilateral ptosis, Lumbar hyperlordosis, Facial palsy, Gowers sign, Thoracic kyphosis, Type 1 muscle fiber predominance, Type 1 fibers relatively smaller than type 2 fibers)

HPOsFreeText: Type 2B fibers absent

CaseNotHPOs: HP:0003798, HP:0030177, HP:0030877, HP:0031237 (Nemaline bodies, Abnormal nerve conduction study, Obstructive deficit on pulmonary function testing, Internally nucleated skeletal muscle fibers)

NotHPOsFreeText: N/A

CaseAddInfo: Variant is de novo with parental testing confirmation

CasePreviousTesting: Patients were diagnosed with CFTD via clinical notes and muscle biopsies at pathology centers throughout Australia and several international centers.

CaseGenotypingMethod: Two methods were used for genotyping. In 16 patients, the 10 exons spliced onto the muscle-specific isoform of TPM3 were bidirectionally sequenced from genomic DNA. For the other 7 patients, complementary DNA was generated from patient muscle and isoform-specific primers were used to sequence the transcript. Paternity was confirmed in isolated patients using microsatellite markers.

Variant: NM_152263.4(TPM3):c.505A>G (p.Lys169Glu)

ClinVar: 42115 https://www.ncbi.nlm.nih.gov/clinvar/variation/42115/

gnomAD: Not in gnomAD

Family4: Australia

FamilyPresentingHPOs: HP:0002460, HP:0003701, HP:0000508, HP:0003803, HP:0003755 (Distal muscle weakness, Proximal muscle weakness, Ptosis, Type 1 muscle fiber predominance, Type 1 fibers relatively smaller than type 2 fibers)

HPOsFreeText: Type 2B fibers absent

FamilyNotHPOs: HP:0001249, HP:0030177, HP:0003798 (Intellectual disability, Abnormal nerve conduction study, Nemaline bodies)

NotHPOsFreeText: N/A

FamilyPreviousTesting: Patients were diagnosed with CFTD via clinical notes and muscle biopsies at pathology centers throughout Australia and several international centers.

FamilyGenotypingMethod: Two methods were used for genotyping. In 16 patients, the 10 exons spliced onto the muscle-specific isoform of TPM3 were bidirectionally sequenced from genomic DNA. For the other 7 patients, complementary DNA was generated from patient muscle and isoform-specific primers were used to sequence the transcript. In this family, 14 further DNA samples were sequenced and a LOD score analysis was performed.

FamilyAddInfo: Affected members of this family seem to have different symptoms based on age

FamilyPMIDs: N/A

Affected: 5

Segregations: 6

PublishedLOD: 3.01 at θ = 0

AddSegregationInfo: Candidate Gene Sequencing

ProbandLabel: Case 5

Variant: NM_152263.4(TPM3):c.298C>A (p.Leu100Met)

ClinVar: 12452 https://www.ncbi.nlm.nih.gov/clinvar/variation/12452/

gnomAD: Not in gnomAD

Family1: N/A

FamilyPresentingHPOs: HP:0009027, HP:0031374, HP:0008963, HP:0001761, HP:0012515, HP:0008944, HP:0008948, HP:0003484, HP:0001265, HP:0003458 (Foot dorsiflexor weakness, Ankle weakness, Tibialis muscle weakness, Pes cavus, Hip flexor weakness, Distal lower limb amyotrophy, Proximal upper limb amyotrophy, Upper limb muscle weakness, Hyporeflexia, EMG: myopathic abnormalities)

HPOsFreeText: N/A

FamilyNotHPOs: HP:0009130, HP:0003474, HP:0003445 (Hand muscle atrophy, Sensory impairment, EMG: neuropathic changes)

NotHPOsFreeText: N/A

FamilyPreviousTesting: Genomic DNA was extracted and RFLP was performed on all family members. Fragments were separated, hybridized, and subject to linkage analysis.

FamilyGenotypingMethod: A refined linkage analysis was performed with the deaths of some family members and additions of others, allowing for the narrowing of the genomic region. Afterwards, RNA was extracted from a frozen sample of the proband and cDNA was sequenced. PCR amplification and direct sequencing was used to confirm the mutation in the proband. SSCP was used to screen the family members and other NM probands for the mutation.

FamilyAddInfo: Pedigree in Figure 1

FamilyPMIDs: 1347195 (Family Characterization)

Affected: 9 Alive, 2 Now deceased

Unaffected: 11

Segregations: 10

PublishedLOD: 4.52 at zero recombination with candidate gene sequencing

AddSegregationInfo: Annotate with other pertinent information as needed.

ProbandLabel: IV-3

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

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

gnomAD: European (Non-Finnish) 8.828e-6, Overall 4.009e-6 https://gnomad.broadinstitute.org/variant/1-154164469-AT-A

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/

Case1: Male, Birth onset

CasePresentingHPOs: HP:0008936, HP:0002058, HP:0200096, HP:0000218, HP:0000774, HP:0005957, HP:0001290, HP:0001284, HP:0002938, HP:0003324, HP:0009046, HP:0008994, HP:0009027, HP:0003722, HP:0002650, HP:0000278, HP:0001724, HP:0002792, HP:0002877, HP:0003557, HP:0100299, HP:0003803 (Muscular hypotonia of the trunk, Myopathic facies, Triangular-shaped open mouth, High-arched palate, Narrow chest, Abnormal breathing, Generalized hypotonia, Areflexia, Exaggerated lumbar lordosis, Generalized muscle weakness, Difficulty running, Proximal muscle weakness in lower limbs, Foot dorsiflexor weakness, Neck flexor weakness, Scoliosis, Retrognathia, Aortic dilatation, Reduced vital capacity, Nocturnal hypoventilation, Increased variation in fiber size, Muscle fiber inclusion bodies, Type 1 muscle fiber predominance)

HPOsFreeText: Crawled at age 10 months and walked independently at age 15 months, Inability to jump, PVC was 37% of predicted, BiPAP commenced at age 20, Cap-like structures

CaseNotHPOs: HP:0003115, HP:0003798 (Abnormal ECG, Nemaline bodies)

NotHPOsFreeText: N/A

CaseAddInfo: No family history of muscle weakness, Variant is de novo and confirmed absent in the parents

CasePreviousTesting: Tested caps appeared pale on all myosin ATPase stains and dark on Gomori Trichrome and NADH stains and were strongly reactive for troponin T, α-actinin-2, myotilin, tropomyosin, actin and desmin by immunoperoxidase, No positive staining for neonatal or embryonic myosin isoforms, Large regions of disorganised thin filament structures containing Z band remnants were present in subsarcolemmal positions

CaseGenotypingMethod: Bidirectional sequencing on TPM2 and TPM3 for all exons and exon-intron boundaries was performed using genomic DNA.

Variant: NM_152263.4(TPM3):c.502C>T (p.Arg168Cys)

ClinVar: 12454 https://www.ncbi.nlm.nih.gov/clinvar/variation/12454/

gnomAD: Not in gnomAD