Molecular characterization of homozygous hereditary factor I deficiency

Abstract

We have studied the molecular basis of factor I (fI) deficiency in two Brazilian sisters from a consanguineous family. By reverse transcription-polymerase chain reaction we observed that all fI cDNA amplified products from one sister had the same size as those of normal cDNA, however, they were significantly less intense. Sequencing analysis of subcloned cDNA revealed a dinucleotide insertion (AT) between positions 1204 and 1205 in the 11th exon that creates a stop codon 13 bp downstream of the insertion site. Genomic DNA sequencing and heteroduplex analysis confirmed that both probands are homozygous for this mutation, whereas their parents are heterozygous. The stop codon and the diminished amounts of fI cDNA could indicate increased fI mRNA instability, perhaps due to a mechanism of nonsense-mediated decay. This hypothesis is consistent with our observation that treatment with the translation inhibitor cycloheximide stabilized fI mRNA expression in proband's fibroblasts.

Fig 1

RT-PCR analysis of fI mRNA isolated from fibroblasts after a 24-h stimulation with LPS (1 µg/ml), IFN-γ (300 U/ml) or IL-6 (200 or 300 U/ml). Exons 11–13 were amplified with fI specific oligonucleotides 11F1233 and 13R1929 generating a 696-bp fragment. GAPDH-specific oligonucleotides were used to amplify a 337-bp fragment. NT, untreated cells; N, normal control cDNA; P, proband L.R.S. cDNA. This figure was taken from a representative experiment.

Fig 2

Comparative RT-PCR analysis between L.R.S.′s and normal's fI mRNA expression after a 24-h LPS-stimulation of fibroblasts. (a) The complete fI cDNA was amplified employing four different oligonucleotide combinations. Numbers in parentheses indicate cDNA positions of forward and reverse primers (see Material and methods). (b) Densitometry analysis of RT-PCR derived fI cDNA products normalized with respect to the amount of a segment of GAPDH cDNA (337 bp). The graph shows reduced fI mRNA expression in the proband's fibroblasts. (c) Segments of C3 (971 bp) and factor H (323 bp) cDNA were also amplified as positive controls and normalized with respect to GAPDH cDNA. N, normal control; P, proband L.R.S.

Fig 3

Sequencing analysis of the fI gene from the probands and their family. The complete L.R.S. fI cDNA was sequenced and compared with a normal fI cDNA. An AT insertion in the 11th exon between positions 1204 and 1205 was detected in the proband sequence. This mutation generates a premature stop codon (TAG) 13 nucleotides downstream of the gene. The dinucleotide (AT) insertion was confirmed in a PCR product (263 bp) amplified from genomic DNA of both L.R.S. and her deficient sister C.A.S, using primers 11F1180–11R1443. The same mutation was also detected in approximately half of father's and mother's genomic DNA clones. The healthy daughter D.S. presented only normal fI alleles.

Fig 4

Homozygous fI deficiency. (a) Pedigree of the proband's family. I.1, father; I.2, mother; II.1, healthy daughter (D.S.); II.2, fI-deficient daughter (C.A.S.); II.3, fI-deficient daughter (L.R.S.). (b) A 263-bp PCR product was amplified from genomic DNA of all family members using primers 11F1180 and 11F1443. (c) Silver-stained denaturing polyacrylamide gel of PCR product (263 bp) derived from the fI gene of the proband's family. The arrow indicates heteroduplex formation in the parents’ DNA (I.1 and I.2), and also in the mixture of the normal control and L.R.S.'s (II-3) fI DNA fragments.

Fig 5

Increased levels of nonsense fI mRNA after treatment with cycloheximide. Above: fibroblasts from fI deficient patient (L.R.S.) were treated with 100 mg/ml for 1, 2 or 3 h and the amount of fI mRNA was estimated after RT-PCR using primers 1F2- and 5R606. Below: band intensity was calculated by densitometry and normalized with respect to GAPDH. NT, cells were kept in culture medium without cycloheximide for 3 h before RNA extraction. These figures were taken from a representative experiment.