Figure 1; The c.471+2 C>A mutation in exon 1 of the CRTAP gene
Interestingly, upon viewing the human CRTAP genomic sequence, we noticed that the splice donor site of intron 1 does not start with GT. Comparison with the transcript (ENST00000320954) showed that the first four bases of exon 2 are identical with the first four bases of intron 1. A more likely splice donor site is the GT four bases into the proposed intron (figure 1). However, this would require the use of an unlikely splice acceptor site (AT). In the latter case the mutation c.471+2 C>A would be in the coding sequence and result in a missense: c.473C>A; p.Ala158Glu. Based on the genomic and cDNA sequences it is impossible to determine which splice donor and acceptor sites are actually being used.
Figure 2: the effect of the c.471+2C>A mutation
In affected individuals from family 3 a c.198 C>A nonsense mutation resulting in p.Y66X in combination with a presumed splice site mutation c.471+2 C>A and 2 heterozygous polymorphisms in exon 5 were found. Amplification of gDNA and cDNA across the mutations and polymorphisms with a forward 3’ primer of exon 1 confirmed loss of heterozygosity at the cDNA level suggesting a null allele. This null allele is indeed caused by the c.471+2 C>A mutation since the c.198 C>A mutation on the other allele appears homozygous on cDNA sequencing as opposed to heterozygous if the null allele would have been caused by the nonsense mutation c.198 C>A. The observation that the c.471+2 C>A allele causes a null allele is more likely to be the result of a splice error than of a missense mutation.
Figure 3: radiological features of VI:3 of family 1
A. No rib fractures. Mild platyspondyly vertrebrae 4-10.
B. Normal mineralization for gestational age.
C. No abnormalities.
D. No abnormalities.
E. Fractures of both femora (arrows) with minor loss of modeling.
Figure 4: radiological images of VI:4 from family 1
A. Normal chest radiograph - rib fractures.
B. Slight flattening of the skull (arrow head).
C. Normal aspect of the right arm.
D. Possible fracture of the proximal left ulna (see insert)
E. Two fractures in left and in right femora (arrows), minor loss of femoral shape. Irregularity of the distal tibiae (arrow heads).
Figure 5
A Radiographs of the lower extremities show multiple fractures for which surgical intervention has been performed. No popcorn epiphyses are observed. Multiple growth acceleration lines are visible due to intravenous biphosphonate treatment (arrow).
B) Radiograph of the left arm shows normal epiphyses, with a broad metaphysic of the distal humerus. Note middiaphyseal fractures of the radius and ulna (arrow). Dislocation of the radial head is observed (arrow head)
C) AP Spine shows platypondyly (arrow) and scoliosis
Figure 6: new and previously described pathogenic CRTAP mutations
The gene for human Cartilage Associated Protein (CRTAP) consists of 1.203 bp and is localized at 3p22. The 7 exons of the CRTAP gene are shown as boxes, with the exact localization of the mutations in the exons and the connecting introns not to scale. The previously reported mutations were described by Morello et al. [1], Barnes et al. [2] and Baldridge et al. [3].
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Table 1: clinical features of probands affected with CRTAP mutations
Family(pedigree nr) / Sex / First appearance of features of OI / Gestational age / Head circumference
(cm) at birth / Length (cm) / Weight (g) / Status / Age at death
1 (VI:3) / M / 21 weeks / 23+2 / 20 / 28 / 548 / TAB
1 (VI:4) / M / 21 weeks / 23+2 / 20,5 / 29,5 / 571 / TAB
1 (VI:5) / F / 19 weeks / 39 / 3245 / LB / alive
2 (IV:1) / M / Third trimester / 42+2 / 2570 / LB / 1 month
2 (IV:4) / F / 20 weeks / 38+3 / 34 / 2725 / LB / 24 days
3 (II: 1) / F / birth / 40+6 / 24 / 2710 / LB / 2,5 month
3 (II: 2) / M / 18+4 / 20+2 / 16,5 / 275 / TAB
3 (II: 5) / M / TAB
4 (II: 1) / M / 20 / 39+4 / LB / 5 days
5 (II: 1) / M / 23 / 40+4 / 35 (at 3 weeks) / 3110 / LB / alive
TAB= Therapeutic abortion
LB= Life birth
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