Table 1.
Clinical, laboratory, and genetic findings of 7 families with VDDR-I.
Fig 1.
A novel deletion of 16-bp nucleotides in the human CYP27B1 gene.
(A) Sequence analysis shows a homozygous deletion of 16-bp nucleotides in exon 6 in a patient from family 1. Both of his parents carry a heterozygous deletion. (B) A schematic representation of the deletion. The deleted nucleotide sequence is underlined and the 4-bp nucleotide repeats flanking the deleted sequence are highlighted in bold.
Fig 2.
Novel splice site mutations in the human CYP27B1 gene.
(A) Sequence analysis of genomic DNA from peripheral lymphocytes. A homozygous mutation at the splice donor site of intron 7 (c.1215+2T>A) were found in a patient from family 3. A homozygous silent SNV (c.1215 T>C) at the end of exon 7 was also identified. His parents carry a heterozygous mutation at both these locations, demonstrating they are in cis and not in trans. The mutations are indicated by arrows. (B) Sequence analysis of cDNA from patient’s peripheral lymphocytes. The mutation at the c.1215+2T>A leads to skipping of exon 7, resulting in exons 6 and 8 joined together.
Fig 3.
A novel deletion of 2-bp nucleotides in the human CYP27B1 gene.
A homozygous deletion of AC nucleotides (c.934_935delAC) in exon 5 was found in a patient from family 7. A heterozygous deletion was found in both of his parents. The deletion results in a frameshift and creates a premature TGA stop codon 19 amino acids downstream from the frameshift (p.T312RfsX19).
Table 2.
CYP27B1 mutations in Turkish population.
Fig 4.
Minigene analysis of the splicing mutations.
(A) CYP27B1WT, CYP27B11215+2T>A and CYP27B11215T>C constructs were transfected into CHO cells for CYP27B1 minigene expression. RNA from transfected cells was reversed-transcribed to cDNA for RT-PCR analysis. Lane 1: CYP27B1WT, lane 2: CYP27B11215+2T>A, and lane 3: CYP27B11215T>C. (B) Sequencing analysis of cDNA fragments. Top panel: the 280 bp cDNA fragment from lane 2 lacks exon 7 (exon 7 skipping) due to 1215+2T>A mutation; Bottom panel: the 350 bp cDNA fragment contains 1215T>C silent mutation. This cDNA fragment is properly spliced containing exons 6, 7 and 8, indicating that the mutation has no effect on pre-mRNA splicing. Only exons 7 and 8 are shown together with 1215T>C silent mutation.