Pedigree and molecular analysis. (A) Pedigree clearly depicts the autosomal recessive mode of inheritance. Squares and circles represent males and females. White symbols represent normal, while black symbols represent affected individuals, respectively. The double line represents the consanguineous union. (B) Picture of the affected individual (V-19) showing developmental delay and bound to a wheelchair. Posteroanterior spinal radiographs demonstrating severe scoliosis. (C) Schematic representation of the VWA8 domains (1905 amino acids). Each contains three predicted ATPase, dynein-related AAA domains (blue) with the second AAA domain-containing an ATPase binding site bounded by the Walker A (first red star) and Walker B (second red star) motifs. VWA8a has a predicted von Willebrand factor type A domain (blue star) at the C-terminus containing a metal ion-dependent adhesion site (MIDAS). (D) Representing partial amino acid sequence of the VWA8 amino acid acids, depicting the conservation of Asp316 amino acid across different species. (E,F) Structural 3D representation of VWA8^Asp316 (wild type) and VWA8^Gly316 (mutated). (G,H) Zoomed cartonic representation of the wild type (VWA8^Asp316) and mutated (VWA8^Gly316) protein structure, showing changes in the bonding and overall structure.

A schematic representation of the variant filtering and identification steps used in the present study.

(A) Human and zebrafish-conserved domain search result. (B) The mitochondrial localization signal. (C) The knockdown efficiency of each morpholino was verified by real time RT-PCR at 72 hpf. The results revealed that the antisense morpholino showed substantially reduced expression as compared to the 5 bp match control.

Zebrafish vwa8-i1e2 induced severe necrosis of embryos at the very early stage of zebrafish development. Representative micrograph of zebrafish embryos injected with either 5 base pair mismatch control morpholino (top) or with vwa8-i1e2 (bottom). Both morpholino was used at the concentration of 3 ng/embryo. The control embryos showed the sphere stage (4 hpf), whereas the zebrafish embryos injected with vwa8-i1e2 showed necrosis.

Zebrafish vwa8-i1e2 induced severe developmental delay. The figure shows representative photomicrograph taken at 24 h post-fertilization of wild-type zebrafish embryos. (A) Injected wither 5 bp mismatch i1e1 control. The control embryos developed normally up to 5 prim stage (24 hpf) and did not reveal any observable embryonic abnormalities. (B) 3 ng/embryo of vwa8-i1e2 antisense oligo morpholino. The vwa8-i1e2 morphants showed severe developmental delay by resembling spheres (4 hpf).

Knockdown of zebrafish vwa8 revealed severe scoliosis. Representative images of zebrafish embryos showing the whole embryos (top panel) and same embryos at high magnification (bottom penal) at 72 hpf. (A,A1) The control embryos developed normally and did not show any sign of toxicity and embryonic abnormality. The notochord (NC) is organized as a straight structure in control embryos shown by black lines. The vwa8-i1e2 morphants showed various degrees of scoliosis. (B,B1) Around 30% of the vwa8-i1e2 morphants (n = 250) had 90° curvature in the middle body; these embryos were also smaller than control embryos. The curvature of the notochord is more visible in the magnified image. (C,C1,D,D1) 20% of vwa8-i1e2 morphants (n = 250) showed bending of trunk region more toward posterior part, and disorganization of the notochord (NC) is quite evident in panel (C1). The morphants were also of smaller size in total length as compared to control.

The splice morpholino vwa8-e2i2 induced less severe embryonic abnormalities. Representative micro-images of live zebrafish embryos injected with 5 bp mismatch control morpholino or vwa8-e2i2 (6 ng/embryo). (A) The control embryos were at the 19 somite stage (18 hpf). (B) The vwa8-e2i2 morphants showed developmental delay and were at 50% epiboly stage (6 hpf) simultaneously. (C) Control embryos at 72 hpf showed normal development and straight body, whereas (D) the morphants showed 24 h developmental delay and the curvature at the posterior end. The embryos were overall also smaller than the control.

Knockdown of vwa8 resulted in the undeveloped posterior trunk and severe embryonic abnormalities. Representative images of zebrafish embryos (A) control (1:3 ratio of 5 base pair mismatch of vwa8-e2i2 and vwa8-i1e2 and (B–D) double knockdown 1 ng of vwa8-e2i2 and 3 ng vwa8-i1e2.