Genetic variations in the DYNC2H1 gene causing SRTD3 (short-rib thoracic dysplasia 3 with or without polydactyly)

Abstract

Background and aims: Short-rib thoracic dysplasia 3 with or without polydactyly (SRTD3) represents a type of severe fetal skeletal dysplasia (SD) characterized by shortened limbs, narrow thorax with or without polydactyly, which is caused by the homozygous or compound heterozygous mutations in the DYNC2H1 gene. SRTD3 is a recessive disorder, identification of the responsible genetic variation would be beneficial to an accurate prenatal diagnosis and well-grounded counseling for the affected families. Material and methods: Two families having experienced recurrent fetal SDs were recruited and submitted to a multiplatform genetic investigation. Whole-exome sequencing (WES) was performed with samples collected from the probands. Sanger sequencing and fluorescent quantitative PCR (qPCR) were conducted as validation assays for suspected variations. Results: WES identified two compound heterozygous variations in the DYNC2H1(NM_001080463.2) gene, namely c.2386C>T (p.Arg796Trp) and c.7289T>C (p.Ile2430Thr) for one; and exon (64-83)del and c.8190G>T (p.Leu2730Phe) for the other, respectively. One variant in them, exon (64-83)del, was novelly identified. Conclusion: The study detected two compound heterozygous variation in DYNC2H1 including one novel deletion: exon (64-83) del. Our findings clarified the cause of fetal skeletal dysplasia in the subject families, provided guidance for their future pregnancies, and highlighted the value of WES in diagnosis of skeletal dysplasia with unclear prenatal indications.

Keywords: DYNC2H1 gene; prenatal diagnosis; short-rib thoracic dysplasia 3 (SRTD3); skeletal dysplasia; whole-exome sequencing.

FIGURE 1

The clinical findings and genetic variation in case 1. (A) Pedigree diagram of the family with STRD3. (B–E) Ultrasonographic and MRI indications of the fetus in case 1: the fetus had extremely short limbs and a small, narrow thorax. (F) X-ray indications of the fetus in case 1: the fetus had a narrow thorax and short limbs, but no polydactyly. (G, H) The genetic variation identified in this case: proband 1 (II-4 in Case 1) carried two missense variants, namely c.2386C>T (p.Arg796Trp) and c.7289T>C (p.Ile2430Thr). Validation with Sanger sequencing demonstrated that the variants these probands carried were all inherited from their asymptomatic heterozygous carrier parents.

FIGURE 2

The clinical findings and genetic variation in case 2. (A) Pedigree diagram of the family with STRD3. (B, C) Ultrasonographic indications of the fetus in case 2: the fetus had extremely short limbs and bowing of long bones. (D–G) The genetic variation identified in case 2: proband 2 (II-2 in Case 2) carried an exonic deletion and a missense variant: exon (64–83)del and c.8190G>T (p.Leu2730Phe). qPCR validation confirmed one copy loss of the 64-83exons in DYNC2H1of proband 2 (D). Validation with Sanger sequencing and qPCR demonstrated that the variants these probands carried were all inherited from their asymptomatic heterozygous carrier parents.

FIGURE 3

(A) Schematic diagram of the DYNC2H1 protein and the locations of the variants detected. (B) Conservation analysis of DYNC2H1 indicated that the protein at position 796, 2430 and 2730 are highly conserved in various species.

FIGURE 4

Results of structural analysis and molecular dynamics simulation of DYNC2H1: c.7289T>C (p.Ile2430Thr) variation. (A) Protein structure of DYNC2H1. (B, C) The wild-type structure of DYNC2H1: protein (DYNC2H1), and enlarged image of the segment containing Ile2430 residue. (D, E) The mutant structure of DYNC2H1, and segment containing the variant Thr2430 residue. (F) The number of hydrogen bonds formed between the target amino acid (meaning Ile2430 or Thr2430) and other residues. (G) Total number of hydrogen bonds in the wild-type model and the mutant model, respectively. (H, I) Comparison of local secondary structure data between Ile2430Thr mutant and wild-type. (J) RMSD: a numerical measurement indicating the difference between two structures. (K) RMSF: is a numerical measure similar to RMSD, but instead of indicating differences in position over time between entire structures; it calculates the flexibility of individual residues, or the extent to which a particular residue moves (fluctuates) during a simulation. (L) Gyrate: is a measure of the structural displacement of a protein atom with its common center of mass over the course of the simulation and provides comprehensive information about protein tightness over time. (M) SASA: measures the exposed surface in a protein structure accessible to solvent molecules.

FIGURE 5

Results of structural analysis and molecular dynamics simulation of DYNC2H1: c.8190G>T (p.Leu2730Phe) variation. (A, B) The wild-type structure of DYNC2H1: protein (DYNC2H1), and enlarged image of the segment containing Leu2730 residue. (C, D) The mutant structure of DYNC2H1, and segment containing the variant Phe2730 residue. (E) The number of hydrogen bonds formed between the target amino acid (meaning Leu2730 or Phe2730) and other residues. (F) Total number of hydrogen bonds in the wild-type model and the mutant model, respectively. (G, H) Comparison of local secondary structure data between Leu2730Phe mutant and wild-type. (I) RMSD: a numerical measurement indicating the difference between two structures. (J) RMSF: is a numerical measure similar to RMSD, but instead of indicating differences in position over time between entire structures; it calculates the flexibility of individual residues, or the extent to which a particular residue moves (fluctuates) during a simulation. (K) Gyrate: is a measure of the structural displacement of a protein atom with its common center of mass over the course of the simulation and provides comprehensive information about protein tightness over time. (L) SASA: measures the exposed surface in a protein structure accessible to solvent molecules.