Case1: Female, Italy, Birth onset

CasePresentingHPOs: HP:0003798, HP:0002033, HP:0100703, HP:0000218, HP:0000689, HP:0000297, HP:0003325 (Nemaline bodies, Poor suck, Tongue thrusting, High-arched palate, Dental malocclusion, Facial hypotonia, Limb-girdle muscle weakness)

HPOsFreeText: N/A

CaseNotHPOs: HP:0003560, HP:0003715, HP:0001557, HP:0001270, HP:0002093, HP:0001627, HP:0000602, HP:0001283, HP:0002650, HP:0003306 (Muscular dystrophy, Myofibrillar myopathy, Prenatal movement abnormality, Motor delay, Respiratory impairment, Cardiac abnormality, Ophthalmoplegia, Bulbar palsy, Scoliosis, Spinal rigidity)

NotHPOsFreeText: Congenital myasthenic syndromes

CaseAddInfo: Typical NM presentation, Parental segregation was confirmed

CasePreviousTesting: ACTA1, TPM2 and TPM3 mutations were ruled out via amplification and direct sequencing

CaseGenotypingMethod: Genomic DNA was extracted from peripheral blood leukocytes or fibroblasts. 183 exons of NEB, the relative introns, and the full promoter region was captured via litigation-mediated PCR and high-throughput sequencing of resulting fragments. These were then aligned and filtered for inheritance and pathogenicity. Variants were evaluated for amino acid conservation and confirmed by Sanger sequencing.

Variant1: NM_001164507.1:c.8899A>C (p.Thr2967Pro)

CAID: CA348807784 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvsOrDescriptor=NM_001271208.2%3Ac.8899A%3EC

gnomAD: Not in gnomAD

Variant2: NM_001164507.1:c.23803_23806del (p.Arg7935Serfs*48)

CAID: CA758865527 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvsOrDescriptor=NM_001271208.2%3Ac.23908_23911del

gnomAD: Not in gnomAD

Case3: Female, Italy, 7 yrs report

CasePresentingHPOs: HP:0003798, HP:0003325, HP:0001270, HP:0002093, HP:0002650, HP:0002355, HP:0003324, HP:0001283, HP:0003306 (Nemaline bodies, Limb-girdle muscle weakness, Motor delay, Respiratory impairment, Scoliosis, Difficulty walking, Generalized muscle weakness, Bulbar palsy, Spinal rigidity)

HPOsFreeText: Semi-solid diet

CaseNotHPOs: HP:0003560, HP:0003715, HP:0001627, HP:0000602, HP:0001557, HP:0001290 (Muscular dystrophy, Myofibrillar myopathy, Cardiac abnormality, Ophthalmoplegia, Prenatal movement abnormality, Hypotonia)

NotHPOsFreeText: Congenital myasthenic syndromes

CaseAddInfo: Intermediate NM presentation, Parental segregation was confirmed, Variant 2 creates a new splicing donor site and activates an upstream acceptor site which partially expresses intron 125 and causes a premature termination codon

CasePreviousTesting: ACTA1, TPM2 and TPM3 mutations were ruled out via amplification and direct sequencing

CaseGenotypingMethod: Genomic DNA was extracted from peripheral blood leukocytes or fibroblasts. 183 exons of NEB, the relative introns, and the full promoter region was captured via litigation-mediated PCR and high-throughput sequencing of resulting fragments. These were then aligned and filtered for inheritance and pathogenicity. Variants were evaluated for amino acid conservation and confirmed by Sanger sequencing.

Variant1: NM_001271208.2:c.24559C>T (p.Arg8187Ter)

ClinVar: 496135 https://www.ncbi.nlm.nih.gov/clinvar/variation/496135/

gnomAD: Not in gnomAD

Variant2: NM_001271208.1:c.19429-381_19429-379delinsA (p.=)

CAID: CA913203565 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA913203565

gnomAD: Not in gnomAD

Case2: Male, Italy, Birth onset

CasePresentingHPOs: HP:0003798, HP:0002033, HP:0003325, HP:0001558, HP:0001270, HP:0002093, HP:0000194, HP:0006830, HP:0003701, HP:0002650, HP:0003458 (Nemaline bodies, Poor suck, Limb-girdle muscle weakness, Decreased fetal movement, Motor delay, Respiratory impairment, Open mouth, Congenital hypotonia, Proximal muscle weakness, Scoliosis, EMG: myopathic abnormalities)

HPOsFreeText: Wheelchair dependent

CaseNotHPOs: HP:0003560, HP:0003715, HP:0001627, HP:0000602, HP:0001283, HP:0003306 (Muscular dystrophy, Myofibrillar myopathy, Cardiac abnormality, Ophthalmoplegia, Bulbar palsy, Spinal rigidity)

NotHPOsFreeText: Congenital myasthenic syndromes

CaseAddInfo: Intermediate NM presentation, Parental segregation was confirmed

CasePreviousTesting: ACTA1, TPM2 and TPM3 mutations were ruled out via amplification and direct sequencing

CaseGenotypingMethod: Genomic DNA was extracted from peripheral blood leukocytes or fibroblasts. 183 exons of NEB, the relative introns, and the full promoter region was captured via litigation-mediated PCR and high-throughput sequencing of resulting fragments. These were then aligned and filtered for inheritance and pathogenicity. Variants were evaluated for amino acid conservation and confirmed by Sanger sequencing.

Variant1: NM_001164507.1:c.17737-2A>T (p.=)

CAID: CA348804839 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvsOrDescriptor=NM_001271208.2%3Ac.17737-2A%3ET

gnomAD: Not in gnomAD

Variant2: NM_001271208.1:c.21423del (p.Lys7141Asnfs*23)

CAID: CA913203564 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA913203564

gnomAD: Not in gnomAD

Case4: Male, Spain, Post-birth onset

CasePresentingHPOs: HP:0003798, HP:0001324, HP:0000297, HP:0003701, HP:0001252, HP:0001284, HP:0002307, HP:0000218, HP:0001382 (Nemaline bodies, Muscle weakness, Facial hypotonia, Proximal muscle weakness, Muscular hypotonia, Areflexia, Drooling, High palate, Joint hypermobility)

HPOsFreeText: Cephalic support, Sedestation

CaseNotHPOs: HP:0000602, HP:0001249, HP:0001627, HP:0002093, HP:0002307, HP:0003722, HP:0002460, HP:0001283, HP:0002650, HP:0002540, HP:0006466, HP:0011471 (Ophthalmoplegia, Intellectual disability, cardiac abnormality, Ankle contracture, Drooling, Neck flexor weakness, Distal muscle weakness, Bulbar palsy, Respiratory insufficiency, Inability to walk, Scoliosis, PEG-fed in infancy)

NotHPOsFreeText: Bimodal communication, Wheelchair dependence, Mechanic ventilation

CaseAddInfo: Typical variant, Paternity/maternity was confirmed, cDNA analysis confirmed that Variant 1 results in a splice-site donor mutation which skips exon 24 which was confirmed by Sanger sequencing and leads to this deletion: p.His738_Asp770del (c.2212_2310del)

CasePreviousTesting: N/A

CaseGenotypingMethod: A research panel with 751 genes relating to neurological disorders, including the coding and intronic flanking regions of ACTA1, CFL2, KBTBD13, NEB, TNNT1, TNNT3, TPM2 and TPM3, was used to locate any variants in the proband. These variants were then prioritized and confirmed by Sanger sequencing in the probands and their relatives.

Variant1: NM_001164507.1:c.2310+5G>A (p.=)

CAID: CA913203562 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA913203562

gnomAD: Not in gnomAD

Variant2: NM_001164507.1:c.17779_17780del (p.Tyr5927Hisfs*17)

CAID: CA913203561 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA913203561

gnomAD: Not in gnomAD

Case1: Male, Spain, Birth onset

CasePresentingHPOs: HP:0003722, HP:0001324, HP:0000297, HP:0003701, HP:0001252, HP:0001284, HP:0001283, HP:0002540, HP:0002307, HP:0000218, HP:0011471 (Nemaline bodies, Muscle weakness, Facial hypotonia, Proximal muscle weakness, Muscular hypotonia, Areflexia, Bulbar palsy, Inability to walk, Drooling, High palate, PEG-fed in infancy)

HPOsFreeText: Bimodal communication, Wheelchair dependence, Cephalic support, Sedestation

CaseNotHPOs: HP:0002460, HP:0000602, HP:0001249, HP:0001627, HP:0002093, HP:0002650, HP:0006466, HP:0001382, HP:0003798 (Neck flexor weakness, Distal muscle weakness, Ophthalmoplegia, Intellectual disability, cardiac abnormality, Respiratory insufficiency, Scoliosis, Ankle contracture, Joint hypermobility)

NotHPOsFreeText: Mechanic ventilation

CaseAddInfo: Intermediate variant, Paternity/ maternity was confirmed

CasePreviousTesting: N/A

CaseGenotypingMethod: A research panel with 751 genes relating to neurological disorders, including the coding and intronic flanking regions of ACTA1, CFL2, KBTBD13, NEB, TNNT1, TNNT3, TPM2 and TPM3, was used to locate any variants in the proband. These variants were then prioritized and confirmed by Sanger sequencing in the probands and their relatives.

Variant1: NM_001164507.1:c.17779_17780del (p.Tyr5927Hisfs*17)

CAID: CA913203561 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA913203561

gnomAD: Not in gnomAD

Variant2: NM_001164507.1:c.11086A>C (p.Thr3696Pro)

CAID: CA348785269 http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/allele?hgvsOrDescriptor=NM_001271208.2%3Ac.11086A%3EC

gnomAD: Not in gnomAD

Case2: Male, Spain, Birth onset

CasePresentingHPOs: HP:0003722, HP:0001324, HP:0000297, HP:0003701, HP:0001252, HP:0001284, HP:0001283, HP:0002540, HP:0002307, HP:0000218, HP:0011471, HP:0002460, HP:0002650, HP:0006466 (Neck flexor weakness, Nemaline bodies, Muscle weakness, Facial hypotonia, Proximal muscle weakness, Muscular hypotonia, Areflexia, Bulbar palsy, Inability to walk, Drooling, High palate, PEG-fed in infancy, Distal muscle weakness, Respiratory insufficiency, Scoliosis)

HPOsFreeText: Bimodal communication, Wheelchair dependence, Mechanic ventilation

CaseNotHPOs: HP:0001249, HP:0001627, HP:0002093, HP:0001382, HP:0003798, HP:0002307 (Ophthalmoplegia, Intellectual disability, cardiac abnormality, Ankle contracture, Joint hypermobility, Drooling)

NotHPOsFreeText: Cephalic support, Sedestation

CaseAddInfo: Severe variant, Paternity/maternity was confirmed, Variant 2 is predicted to be likely benign due to the population frequency and it not being located in any binding sites however no other mutations were found (including CNVs)

CasePreviousTesting: N/A

CaseGenotypingMethod: A research panel with 751 genes relating to neurological disorders, including the coding and intronic flanking regions of ACTA1, CFL2, KBTBD13, NEB, TNNT1, TNNT3, TPM2 and TPM3, was used to locate any variants in the proband. These variants were then prioritized and confirmed by Sanger sequencing in the probands and their relatives.

Variant1: NM_001271208.2:c.21076C>T (p.Arg7026Ter)

ClinVar: 190457 https://www.ncbi.nlm.nih.gov/clinvar/variation/190457/

gnomAD: European (non-Finnish) 5.343e-5, Overall 3.233e-5 https://gnomad.broadinstitute.org/variant/2-152394412-G-A

Variant2: NM_001271208.2:c.23372T>C (p.Met7791Thr)

ClinVar: 167328 https://www.ncbi.nlm.nih.gov/clinvar/variation/167328/

gnomAD: Ashkenazi Jewish 0.01744, Overall 1.605e-3 https://gnomad.broadinstitute.org/variant/2-152369326-A-G