Vivienne M. Homer

A deep intronic mutation in FGB creates a consensus exonic splicing enhancer motif that results in afibrinogenemia caused by aberrant mRNA splicing, which can be corrected in vitro with antisense oligonucleotide treatment

We previously described a novel homozygous point mutation (FGB c.115–600A>G) located deep within intron 1 of the fibrinogen beta gene (FGB), as a likely cause of afibrinogenemia. While this was the only mutation detected, its pathological mechanism was unclear. Here we show the mutation causes the inclusion of a 50‐bp cryptic exon by creating a consensus heptad motif recognized by the spliceosome recruiting protein pre‐mRNA splicing factor (SF2)/arginine/serine‐rich alternative splicing factor (ASF) splicing factor 2/alternative splicing factor (SF2/ASF). Translation of the aberrant mRNA would result in truncation of the Bβ chain, preventing fibrinogen synthesis. Selective introduction of a second mutation into the enhancer motif abolished the SF2/ASF binding motif and re‐established normal pre‐mRNA splicing. Subsequent introduction of antisense phosphorodiamidate morpholino oligonucleotides (PMOs) into transfected cells containing the mutant construct blocked the protein‐RNA interaction and successfully restored normal splicing (∼50% at 2 µM and ∼90% at 10 µM). The molecular characterization of this case has revealed a unique disease mechanism, shown the importance of screening for deep intronic mutations, and provided evidence that antisense gene therapy is potentially practical for the treatment of diseases caused by this class of mutation. Hum Mutat 0, 1–8, 2008.

The molecular mechanisms of congenital hypofibrinogenaemia.

Congenital hypofibrinogenaemia is characterized by abnormally low levels of fibrinogen and is usually caused by heterozygous mutations in the fibrinogen chain genes (α, β and γ). However, it does not usually result in a clinically significant condition unless inherited in a homozygous or compound heterozygous state, where it results in a severe bleeding disorder, afibrinogenaemia. Various protein and expression studies have improved our understanding of how mutations causing hypo- and afibrinogenaemia affect secretion of the mature fibrinogen molecule from the hepatocyte. Some mutations can perturb chain assembly as in the γ153 Cys → Arg case, while others such as the Bβ Leu → Arg and the Bβ414 Gly → Ser mutations allow intracellular hexamer assembly but inhibit protein secretion. An interesting group of mutations, such as γ284 Gly → Arg and γ375 Arg → Trp, not only cause hypofibrinogenaemia but are also associated with liver disease. The nonexpression of these variant chains in plasma fibrinogen is due to retention in the endoplasmic reticulum, which in turn leads to hypofibrinogenaemia.

Novel Aα chain truncation (fibrinogen Perth) resulting in low expression and impaired fibrinogen polymerization

Summary.  A young woman with a history of menorrhagia and easy bruising presented with a functional fibrinogen concentration of 1.8 mg mL−1, a gravimetric concentration of 3.3 mg mL−1 and a prolonged thrombin clotting time of 32 s. Both reverse phase analysis and reducing SDS–PAGE revealed a normal profile of Aα, Bβ, and γ chains. However, non‐reducing gels revealed a broadened 340‐kDa band, while the 305‐kDa band was normal, suggesting a C‐terminal truncation of the Aα chain. DNA sequencing of all exons and intron boundaries revealed a single heterozygous cytosine deletion at nucleotide 4841 of the Aα gene predicting a frameshift and the incorporation of 23 new residues (LMKLPSSTLPQLEKHSQVSSHLC) before termination after residue 517. In agreement with a predicted mass decrease of 9953 Da, the measured mass of the AαPerth chain was 56 242 Da, while that of the normal AαA chain was 66 189 Da. Tryptic mapping of isolated Aα chains revealed a new [M + 2H] ion at 607 m z−1, corresponding to the predicted penultimate peptide LPSSTLPQLEK. The variant chain was poorly incorporated into plasma fibrinogen at a ratio of AαPerth/AαA of 0.15 : 1, suggesting the AαPerth chain might be out‐competed by normal chains during molecular assembly in the hepatocyte. Despite the low expression, polymerization curves showed a decreased Vmax and final turbidity, suggesting the fibrinogen Perth clots are composed of thinner fibers. However, the fibrinolytic rate was very similar to that of the control.

Mental retardation and ataxia due to normotriglyceridemic hypobetalipoproteinemia

A 12‐year‐old boy with mental retardation, obesity, ataxia, and visual impairment was shown to have normal fasting plasma triglyceride but low cholesterol and vitamin E levels. Investigations indicated that he was compound heterozygous for two mutations in the apolipoprotein B gene (APOB), resulting in a failure to express apolipoprotein B‐100, yet retain apolipoprotein B‐48 production. The proband therefore was able to form chylomicrons, but not a low‐density lipoprotein capable of receptor‐mediated endocytosis. This resulted in chronic vitamin E deficiency. We suggest the term normotriglyceridemic hypobetalipoproteinemia for this easily recognizable condition. Ann Neurol 2005;58:160–163

Hypofibrinogenaemia associated with common γ82Ala→Gly mutation is not mediated by altered mRNA splicing

Hypofibrinogenaemia associated with common γ82Ala→Gly mutation is not mediated by altered mRNA splicing -