We collected one DA1 family who came to the Noor Gene Genetic lab, Ahvaz, Iran (Fig. 1). The proband, a 5-year-old boy was diagnosed with DA1. Based on clinical evaluations, the proband had congenital ulnar bilateral and symmetric arthrogryposes of 5th fingers without other malformation. In addition, this family reported that I:1, II:2, II:5, II:8, III:3, and III:6 had DA, but I:2, II:1, II:3, II:4, II:6, II:7, II:9, II:10, II:11, II:12, III:1, III:2, III:4, III:5, III:7, III:8, III:9, and IV:1 were phenotypically normal. Written informed consent was obtained from all participants, and the study was carried out according to the guidelines of the Declaration of Helsinki.
Peripheral blood samples were collected from the patient and other members of his family, and genomic DNA was isolated from blood samples of all participants by using standard salting-out protocol. NanoDrop 1000 spectrophotometer was used to measure the concentration of DNA samples.
Next, WES was only performed on the proband to find out the pathogenic variant. The DNA sequence analysis of the genes involved in the pathogenesis of DA1 showed a heterozygous mutation in TPM2 gene and no other potential disease-causing variants were identified. This mutation is a novel and rare missense heterozygous mutation (c.456G>C), located in exon 4 (NM_003289.4) and results in substitution of Lysine to Asparagine at codon 152.
The international publicly available mutation and polymorphism databases (1000 Genomes Project, ExAC browser) were used for the analysis and interpretation of sequencing data. Only low-frequency of detected variants (less than 1%) were selected. This variant of uncertain significance has not been reported. Three bioinformatics tools for analyzing protein functions, SIFT, PolyPhen2, and MutationTaster, predicted that K152N variant is probably damaging, deleterious, and disease-causing.
Sanger sequencing was carried out in the patient and family members to confirm the true positive of the identified TPM2 variant. The genomic DNA samples were amplified by polymerase chain reaction (PCR) and then the PCR products were directly sequenced on the automated genetic analyzer (ABI3130, Applied Biosystems, USA). The results showed heterozygous change in the proband and his affected father, but the normal members in the family were lack of K152N mutation (Fig. 2A). The alignment of TPM2 from seven diverse species is shown in Fig. 2B. This result revealed that this variant occurred in a conserved region of the protein. Therefore, it is recommended that p.K152N residue is important for protein organization and function.