Genetic Analysis of Patients with Spinal Defects and CANT1 Gene Mutations

1. a. b. c. figure s1 The arrows point to the index patients. a., b. and c. are case one to three, respectively. g.w.= gestational week s.a. = spontaneos abortion i.a. = induced abortion

2. A B C figure s2: X-ray of the spine showing sagittal clefts of vertebral bodies in case 1 (A) and 2 (B) and more defective ossification in case 3 (C).

3. I I A A E E L L D D (c). a a t t c c g g c c a a g a a c t g g a c (a.) I I A A D D L L D D c a t c g c a g a c t g g a c (b). • Supplementary figure 3. • Sequencing Chromatogram of exon 2 of CANT1-gene of the patient. The arrow points to the homozygous missense mutation. In capital on the top is the corresponding amino acids' sequence (in red is the changed amino acid). b. Sequence of a control DNA. The arrow points to the wild type cytosine. c. multiple alignment of the corresponding sequence containing the relevant aspartic acid at position 367 that show its very high conservation up to vegetable organisms.

4. forward reverse fetus fetus mother control father parents Supplementary figure 4. Sequencing Chromatogram of exon 2 of CANT1-gene of the fetus 2 and its parents. The sequence of the fetus shows a frameshift. The comparison with the DNA of the parents unveiled the presence of two overlapping frameshift causing mutations in the same exon: the c.277_278delCT and the c.228_229insC. Both mutations were then amplified from the fetal DNA separately without interference using tailored PCRs and confirmed by sequencing. Supplementary figure 5. Sequencing Chromatogram of exon 2 of CANT1-gene of the fetus 3 with the c.228_229insC mutation, control and parents. The chromatogram of the fetus shows a homozygous insertion of a cytosin in comparison to the control sequence. Both parents are heterozygous carriers of the corresponding mutation.