The fibrinogen locus is comprised of single copies of three genes coding for fibrinogen gamma (FGG
), alpha (FGA
), and beta (FGB
), clustered in a region of ~50 kb on chromosome 4q28–q31 (7
). We studied this region in a Swiss family with two pairs of affected brothers (Fig. a
) using microsatellite analysis, PCR amplification, and Southern blotting. Despite intensive genealogical investigation, no consanguinity was identified in the family. The diagnosis of afibrinogenemia for all the affected individuals was made in the neonatal period, after bleeding from the umbilical cord. All four patients (now 30–38 years old) were first treated with purified fibrinogen only after trauma or before operative surgery, a strategy replaced in early adulthood by a prophylactic protocol consisting of a fibrinogen infusion every two to four weeks. Anti-fibrinogen antibodies have never been detected in any of the patients. All coagulation tests performed (activated partial thromboplastin time, thrombin time, prothrombin time, fibrinogen Clauss, and reptilase) were compatible with the complete absence of active fibrinogen, the blood being uncoagulable. Furthermore, no fibrinogen was detectable by Laurell electroimmunoassay in plasma from affected individuals.
Figure 1 (a) Pedigree of the Swiss congenital afibrinogenemia family and haplotypes of the region surrounding the fibrinogen locus. The markers (from top to bottom) and genetic distances were: D4S1625 – (13%) – D4S2962 – (2%) (more ...)
Figure shows the family tree, together with haplotype data obtained for five microsatellite markers (D4S1625, D4S2962, FGAi3, D4S2934, D4S1629) covering a region of 15–20 cM around the fibrinogen gene cluster. One of these, FGAi3, a (TCTT)n polymorphic marker situated in intron 3 of the FGA gene, was homozygously deleted in all four affected individuals and was hemizygous in the obligate carriers, providing the first identification of the causative mutation.
The haplotypes of the mutated chromosomes were determined by studying four further polymorphic genetic markers, two to each side of the fibrinogen gene cluster. Three of these four flanking markers were very closely linked to fibrinogen: the genetic distances were D4S1625 – (13%) – D4S2962 – (2%) – fibrinogen – (2%) – D4S2934 – (2%) – D4S1629. The haplotype data reveal that the deletions were present on three different chromosomes, with the haplotypes 3-3-del-3-2, 2-2-del-2-4, and 1-1-del-2-2 (the last being shared by the two fathers of the affected patients; Fig. ). This implies either that the deletions occurred as separate historical events on three different ancestral chromosomes, or that the mutation occurred originally on a single founder chromosome that produced the three observed haplotypes by repeated recombination. In our opinion, the hypothesis of a common molecular mechanism leading to recurrent mutation is more likely, given the number of recombination events between closely linked markers that would be necessary to generate the observed haplotypes.
To characterize the extent of the deletions, we performed PCR amplification of portions of the three fibrinogen genes. Bands of the predicted sizes were obtained for all individuals for FGG
exons 1, 2, and 7, FGA
exon 1, and FGB
exons 2 and 7 (details of the primers are in Methods). PCR amplification of FGA
exons 2, 3, and 5 resulted in bands of the expected size from heterozygous carriers, but in contrast, no amplification was obtained from DNA from all four patients (Fig. , and not shown). These results indicate that the centromeric breakpoint of all the deletions is within FGA
intron 1. Consequently, the deleted gene could at best direct the production of a severely truncated FGA polypeptide, containing just the 18 amino acids encoded by exon 1 (the normal protein has 625 amino acids). It is, therefore, unsurprising that sensitive immunoassay was unable to detect fibrinogen protein in the plasma of affected individuals in this family. Interestingly, a number of less severely truncated fibrinogen alpha chains have been described in hypofibrinogenemic patients, the smallest of which was due to a single base pair insertion in codon 268 (10
Figure 2 Representative PCR analyses of the extent of the fibrinogen locus deletion. DNA samples in lanes 1–3 were from obligate carriers (individuals 3–5 in Fig. ) and in lanes 4–7 were from patients (individuals 6–9). (more ...)
To estimate the size of the deletions and to establish if they were, in fact, identical on all three mutant alleles, we performed Southern blotting analysis. Genomic DNA samples from all individuals from the affected family and from four normal controls were digested with BamHI and hybridized with a cloned probe pAFS1 generated by PCR amplification from the intergenic region between FGG and FGA (Fig. ). The expected band of 13 kb was found for the control samples (Fig. , lanes 1–4), as well as for the obligate carriers (lanes 5–8). In addition, a larger band (~17 kb) was obtained in these same carriers. Only the larger fragment was observed in the affected individuals (lanes 9–12). This abnormal fragment represents a deletion of ~11 kb (which includes two BamHI sites and thus generates a band of paradoxically increased size). All three deletions were identical within the limits of the resolution of the technique.
Figure 4 Schematic representation of the human fibrinogen gene cluster (after ref. 7). The arrows represent the gamma-, alpha-, and beta-chain genes and their 5′→3′ orientation. The extent of the deletion is shown by the black bar. The (more ...)
Figure 3 Southern blot analysis of the FGA deletion. The deletion appears as a band of paradoxically increased size because two BamHI sites are eliminated (see schematic in Fig. ). Lanes 1–4, normal controls; lanes 5–8, obligate (more ...)
Figure depicts the genomic organization and Bam
HI restriction map of the fibrinogen gene cluster on chromosome 4 and summarizes the PCR and Southern data. The position of the telomeric end of the deletion is defined by the size of the deletion and by the Bam
HI restriction map (7
). We conclude that the genetic defect in this family was a recurrent deletion of ~11 kb of DNA that eliminates the majority of the FGA
gene and thus leads to an absence of functional fibrinogen.