Dwarfism and age-associated spinal degeneration of heterozygote cmd mice defective in aggrecan

Abstract

Mouse cartilage matrix deficiency (cmd) is an autosomal recessive disorder caused by a genetic defect of aggrecan, a large chondroitin sulfate proteoglycan in cartilage. The homozygotes (-/-) are characterized by cleft palate and short limbs, tail, and snout. They die just after birth because of respiratory failure, and the heterozygotes (+/-) appear normal at birth. Here we report that the heterozygotes show dwarfism and develop spinal misalignment with age. Within 19 months of age, they exhibit spastic gait caused by misalignment of the cervical spine and die because of starvation. Histological examination revealed a high incidence of herniation and degeneration of vertebral discs. Electron microscopy showed a degeneration of disc chondrocytes in the heterozygotes. These findings may facilitate the identification of mutations in humans predisposed to spinal degeneration.

Figure 1

Analysis of genotypes by genomic PCR. Genomic DNA was prepared from the tails or livers of mice using the QIAamp tissue kit. PCRs were performed using a set of primers, W74, and W33 with genomic DNA as a template. The reaction products were electrophoresed on 5% or 8% polyacrylamide gels. (A) Schematic diagram of a part of the aggrecan gene containing exon 5. The expected sizes of the PCR products are indicated. (B) The gel electrophoresis patterns of the PCR products with DNA from a wild-type mouse (1), heterozygote (2), or homozygote (3). (C) The patterns of the gel electrophoresis of the PCR products after digestion by BpmI.

Figure 2

Quantitative RT-PCR analyses of mRNA for aggrecan (A) and type II collagen (Col2a1) (B) in the cmd mice. mRNA was prepared from cartilaginous tissue of day 15 embryonic limbs of cmd homozygote, cmd heterozygote, and wild-type mice. Quantitative RT-PCR analysis was performed using the PCR MIMIC Construction kit (CLONTECH). The RT-PCR mixture contained a constant amount of cDNA, various amounts of the neutral mimic DNA as a competitor, and two oligonucleotide primers for aggrecan or Col2a1. The competitor DNA fragment (mimic DNA) was unrelated to the aggrecan gene or to Col2a1 except for containing the same short sequences for aggrecan or Col2a1 at both ends. mRNA levels for wild-type mice (Top), cmd heterozygotes (Middle), and cmd homozygotes (Bottom) were determined. Mimic DNA added was 10 or 80 attomoles (amol) (1 amol = 10⁻¹⁸ mol) (lane 1); 5 or 40 amol (lane 2); 2.5 or 20 amol (lane 3); 1.25 or 10 amol (lane 4); 0.63 or 5 amol (lane 5); 0.31 or 2.5 amol (lane 6); and 0.15 or 1.5 amol (lane 7) for aggrecan or Col2a1, respectively. Open arrowheads indicate mimic PCR product. Closed arrowheads indicate PCR product for aggrecan or Col2a. (Lower graphs) Ratios of cDNA for aggrecan or Col2a1 to PCR mimic vs. input mimic DNA amounts are shown.

Figure 3

Survival rate of the heterozygotes and wild-type mice. Survival rate of the heterozygotes (n = 17, closed circle) is significantly less than that of the wild-type mice (n = 20, open circle).

Figure 4

Radiographs of 1-year-old wild-type and heterozygous cmd mice. Head and spine were taken from mice killed with CO₂ gas and were fixed by 10% buffered formalin. (Top) Control wild-type mouse. (Middle and Bottom) Two different heterozygous cmd mice. Arrow in the Middle indicates misalignment at C7∼Th1. In the lower panel, vertebral bodies show deformation.

Figure 5

Histological analysis of the spine from 1-year-old wild-type and heterozygous cmd mice. Hematoxylin/eosin-stained samples from the wild type (A) and heterozygotes (B and C), toluidine blue-stained sample from a heterozygote (D), and Alcian blue-stained samples from the wild type (E) and heterozygotes (F) are shown. Spines of heterozygotes show protrusion of discs [Th3∼4 (B) and Th4∼5 (C)] or marked herniation at C4∼5 (D). Note disappearance of apophyseal cartilage and deformation of vertebral bodies in heterozygotes (B-D) while the spines of the wild type show no remarkable changes (A). Note staining around chondrocytes in the cartilage from heterozygotes (F) while diffuse staining patterns are observed in that from the wild type (E). (A–C, ×10; D, ×25; E and F, ×125.)

Figure 6

Transmission electron micrographs of vertebral discs from 1-year old wild type (A) and age-matched heterozygote (B), stained by uranyl acetate. The chondrocytes of the heterozygote show degeneration and are packed together. The extracellular matrix shows concentric dense bundle patterns whereas that of the wild type shows fine diffuse patterns (×4500).