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M. Guipponi et al.
tories. One lesson learned from this study is that amplify- ing larger overlapping portions of the genes of interest may allow the identification of similar mutations involving large insertions and duplications. In our case, amplifying for example FGG exons 7 to 10, around 4,400 bp in the normal sequence, and allowing sufficient elongation time to amplify the larger mutant band would have enabled the identification of the mutation sooner. Recent technology such as Nanopore DNA sequencing allowing sequencing of DNA fragments up to a few Mbp may be useful to iden- tify such mutations, allowing for example the sequencing of the entire fibrinogen gene cluster with haplotype phas- ing of variant sites along the sequence.
The duplication is only one of five mutations involving rearrangements of more than 100 bp of the fibrinogen genes, and the first identified in the FGG gene. As previ- ously mentioned four deletions of several kilobases have been identified in FGA including the recurrent deletion we identified in a Swiss family.8,13-15 In addition, an in- frame duplication of 117 bp “Fibrinogen Champagne au Mont d’Or” leads to duplication of 39 amino acids within a repetitive sequence of 13 amino acids in the connector portion of the aC domain23 a mutation predicted to cause an extension of the coiled coil. In FGB, while fibrinogen New York24 is described as a deletion of the amino acids encoded by FGB exon 2 the mutation is not characterized at the DNA level and as mentioned by the authors is most probably due to a splice-site mutation leading to exon 2 skipping rather than deletion of exon 2.
It is likely that many more mutations of this sort have remained elusive even for laboratories specialized in mutation identification. We hypothesized that the same duplication was likely to be found in other afibrinogene- mic patients and their family members from the same geographical region or ethnic group. We therefore screened 10 unrelated patients from Turkey by PCR amplification and identified one additional afibrinogene- mic patient who was homozygous for the same duplica- tion. His parents, his grandmother and his two sisters were all heterozygous (data not shown). Enquiries into the geographical origin of this family revealed that they were originally from Kumbet village, Ortaköy, Aksaray, i.e., the same region as the first family.
While the nature of the mutation and the clear associ- ation with the phenotype i.e., complete fibrinogen defi- ciency with a homozygous genotype and partial fibrino- gen deficiency with a heterozygous phenotype did not allow any reasonable doubt that we had identified the causative mutation we wished to identify the underly- ing molecular mechanism. Since the mutation duplicates the donor splice site of intron 8 we predicted that the mutation would impact FGG transcript splicing, of both
the major g transcript and the minor g’ transcript. Analysis of RNA produced by cells transiently transfect- ed with normal or duplicated minigene constructs showed that in this model system the duplication causes production of several different aberrant transcripts of both isoforms leading to frameshifts and premature truncating codons.
Regarding the clinical significance of our findings, geno- typing patients with fibrinogen disorders is now recom- mended in guidelines from the International Society of Thrombosis and Hemostasis.25 In quantitative fibrinogen disorders, the identification of the causative mutation(s) can help to distinguish between afibrinogenemia and severe hypofibrinogenemia. Providing an accurate diag- nosis for these patients is important since in specific clin- ical settings such as pregnancy or surgery, patient man- agement could be different.
In conclusion, we have identified a large duplication of several hundreds of basepairs at the FGG exon 8-intron 8 junction accounting for congenital afibrinogenemia in a large consanguineous Turkish family. The nature and size of the duplication can explain why this mutation was not identified using a standard PCR approach. It is highly likely that other patients with inherited quantitative fib- rinogen disorders for whom no causative mutation has been identified harbor similar rearrangements.
Disclosures
MG, FM, FS-B, MM, NÖ, BM and MN-A have no conflicts of interest to disclose; FP declares that she is a speaker at educa- tional meetings and a member of advisory boards for Roche, Sanofi, SOBI and Takeda; AC declares grants and fees paid to his institution from Takeda and travel support from SOBI.
Contributions
MG, FM, FSB and CDS performed genetic experiments and interpreted the results; NO, FP, MM, AC and BM collected patient samples and clinical information and performed fibrino- gen measurements; MNA directed the study and wrote the first draft of the manuscript. All authors contributed to writing and editing the final manuscript.
Acknowledgements
The authors thank Dr. Cédric Howald and Dr. Keith Harshman at the Health 2030 Genome Centre at Campus Biotech, Geneva for whole exome sequencing and initial process- ing of the data and Dr. Nermin Keni and Dr. Ekrem Unal for providing patient samples from the second Turkish family.
Funding
This study was funded by a grant from the Swiss National Science Foundation (grant # 31003A_172864) to MNA.
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