Heterozygous premature stop DNA mutations in Tropomyosin 2 (TPM2) in three human males with Prune Belly Syndrome (PBS)
Thomas J. Egeland, MS, Alexandria N. Fusco, BA, Jeremy A. Mathews, MS, Brandi L. Cantarel, PhD, Nathalia G. Amado, PhD, Linda A. Baker, MD.
UT Southwestern Medical Center, Dallas, TX, USA.
BACKGROUND: PBS is a rare congenital myopathy incompletely understood at the molecular level. Proteins in thin filaments of smooth and skeletal muscle cells include actin and TPM2, regulating Ca2+-mediated actomyosin contraction/relaxation and cell morphology and adhesion. Given the significant smooth and skeletal muscle phenotype in PBS, we hypothesized that TPM2 might be deranged in PBS cases.
METHODS: With IRB-approval, PBS probands and family members were prospectively enrolled and phenotyped. Proband blood lymphocyte DNA underwent paired-end Whole Exome Sequencing (WES) using the Illumina SureSelect kit and HiSeq2500 sequencer, with data pipeline analysis. WES variants were filtered for rare minor allele frequency, predicted protein mutation impact, and type (splice site, nonsense or missense). After Sanger validation, available familial DNAs were tested for variant segregation. mRNA and protein were extracted from PBS and non-PBS control abdominal wall samples, analyzed by qPCR 2-ΔΔCt method and descriptive statistics, and western blotted (WB) for GAPDH-normalized TPM2. 3-day Insulin-differentiated C2C12 mouse cell lines were Lipofectamine 3000 co-transfected with 1ng of siRNAs (mouse TPM2 or negative control) and 500 ng of plasmid [Empty Vector (EV) GFP, TPM2-WT-Flag, or TPM2-R238*-Flag (C-terminal Flag-tagged)]. 72 hours following co-transfection, C2C12 cytoplasmic:nuclear ratio spreading assays were performed on fibronectin-coated plates at 20, 40 and 60 minutes and protein was extracted for WB analysis. Two-way ANOVA and Tukey’s multiple comparisons test were performed to detect between-group differences in spreading.
RESULTS: WES identified 2 heterozygous (het) TPM2 premature stop mutations in 3 unrelated sporadic PBS males, [c.70 C > T (p.Q24*) (n=2) and c.712 C > T (p.R238*) (n=1)]. Parental samples confirmed asymptomatic maternal inheritance in two of these males. Human abdominal wall tissue from the PBS proband carrying the het TPM2-R238* mutation revealed a 90.5% reduction in relative TPM2 transcript expression and essentially undetectable TPM2 protein (3.6% vs 203% ± 80.5 in non-PBS control abdominal wall tissues) (Fig 1A,B). Tissue was not available for PBS probands carrying the TPM2-Q24* mutation. In the C2C12 studies, 4 transfection conditions (EV+controlsiRNA, EV+TPM2siRNA, TPM2-WT+TMP2siRNA and TPM2-R238*+TPM2siRNA) interrogated the impact of TPM2 silencing and TPM2-R238* plasmid overexpression on protein expression and spreading properties. As expected, only WT-TPM2 C2C12 cells expressed any discernable Flag protein. Compared to TPM2 protein expression in EV+controlsiRNA, western blotting indicated a 50% and 89 % reduction in TPM2 for EV+TPM2siRNA and TPM2-R238*+TPM2siRNA, respectively (Fig 1C). ControlsiRNA cells exhibited decreased cell spreading across time, while siRNA-induced TPM2 depletion significantly increased cell spreading. Significant between-group differences were displayed at 60 minutes, when TPM2-WT transfection rescued the non-spreading phenotype while TPM2-R238* transfection failed to rescue the endogenous phenotype (Fig 1D).
CONCLUSIONS: Human heterozygous mutations in TPM2 cause distal arthrogryposis and CAP and nemaline myopathies. We now add to the phenotypic spectrum of TPM2 mutations, identifying that recurrent, never reported, deleterious TPM2 truncation mutations are a rare cause for PBS and reduced TPM2 protein carries functional consequences at the cellular level. 
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