Page 57 - Haematologica May 2022

FGG mutation in congenital afibrinogenemia
erodimeric or homodimeric form. Normal plasma fibrino- gen levels vary between 2 and 4 g L-1. Variation in fibrino- gen levels is a complex trait, influenced by both the envi- ronment and genotype.
Inherited disorders of fibrinogen include Type I disor- ders (afibrinogenemia and hypofibrinogenemia) which affect the quantity of fibrinogen in circulation and Type II disorders (dysfibrinogenemia and hypodysfibrinogene- mia) which affect the quality of circulating fibrinogen.3,4 Congenital afibrinogenemia is the most severe disorder, characterized by undetectable fibrinogen in circulation.5,6
While the first dysfibrinogenemia mutation was identi- fied as early as 19687 the molecular basis of afibrinogene- mia was elucidated much later.8 This disorder is charac- terized by autosomal recessive inheritance and the com- plete absence of fibrinogen in plasma. In populations where consanguineous marriages are common, the preva- lence of afibrinogenemia is increased.9
We identified the first causative mutation for congeni- tal afibrinogenemia, a large, recurrent deletion in FGA in 19998 identified in homozygosity in four members of a Swiss family. Since then, the underlying molecular patho- physiology of numerous causative mutations leading to fibrinogen deficiency has been determined by our group and many others (reviewed in4).10-12
Causative mutations can be divided into two main classes: null mutations with no protein production at all and missense mutations producing abnormal protein chains that are retained inside the cell. The vast majority of cases are due to single base pair mutations or small insertions of deletions in the coding regions or intron-exon junctions of FGB, FGA and FGG. These can easily be iden- tified by polymerase chain reaction (PCR) amplification followed by Sanger sequencing or by next-generation sequencing in particular whole exome sequencing (WES). Only a few large rearrangements have been described. In addition to the recurrent deletion we identified with breakpoints in FGA intron 1 and the FGA–FGB intergenic region, while three other large deletions in the fibrinogen gene cluster have been reported by others, all involving part of the FGA gene. These are a deletion of 1.2 kb elim- inating the entire FGA exon 4 in a Japanese patient;13 a deletion of 15 kb, with breakpoints situated in FGA intron 4 and in the FGA–FGB intergenic region in a Thai patient;14 and a 4.1-kb deletion encompassing FGA exon 1 in an Italian patient.15 All patients were homozygous for the identified deletions except for the Thai patient, for whom complete maternal uniparental disomy was confirmed for the deleted chromosome 4.14
Rearrangements of this type cannot be identified by simple PCR analysis of coding regions. Other techniques such as array comparative genomic hybridization (CGH) can be useful in some cases, however the resolution of commercial arrays limits the discovery of rearrangements i.e., deletions, duplications greater than 15 kb. Consequently mutations less than 15 kb will escape detection using this technique in most diagnostic settings.
Here we report the characterization of a 403 bp dupli- cation of the FGG exon 8-intron 8 junction accounting for congenital afibrinogenemia in a large consanguineous family from Turkey. This mutation, which had escaped detection by Sanger sequencing of short PCR amplicons of coding sequences and splice sites, was identified by studying multiple alignments of reads obtained from WES of a heterozygous individual followed by PCR
amplification and sequencing of à larger portion of FGG. The mutation duplicates the donor splice site of intron 8 which leads to aberrant splicing of both the major g tran- script and the minor g’ transcript.
Methods
Patient samples
This study was performed with Institutional Review Board approval and with written informed consent from all patients, in accordance with the Declaration of Helsinki. Platelet-poor plas- ma samples were obtained from citrated venous blood and ana- lyzed as described in the Online Supplementary Appendix.
Polymerase chain reaction and Sanger sequencing
Genomic DNA was extracted from whole blood-EDTA according to standard protocols. PCR amplifications of all FGB, FGA and FGG coding regions and intron-exon junctions were performed as previously described.16 Standard primer sequences and PCR protocols are available on demand. Specific primer sequences for this study are available in the Online Supplementary Appendix. Sanger sequencing of purified PCR products was per- formed by Fasteris AG, Geneva, Switzerland.
Array comparative genomic hybridization analysis
The array CGH analysis was performed using Human Genome CGH Microarray Kit G3 1 M (Agilent Technologies, Palo Alto, USA) with ~2.4 kb overall median probe spacing according to protocols provided by the manufacturers. Copy number variant analysis was done using the Agilent Genomic Workbench Software 7.0.4.0. and UCSC Genome Browser Human Genome GRCh37/hg19.
Next-generation sequencing
WES was performed at the Health 2030 Genome Center at Campus Biotech, Geneva using IDT Research Exome reagents. Read mapping and variant calling were performed using BWA 0.7.13, Picard 2.9.0, GATK HaplotypeCaller 3.7, aligned to the GRCh37/hg19 reference genome and annotated with Annovar 2017/07/17 and UCSC RefSeq (refGene) downloaded on 2018/08/10.
Minigene constructs and transfections
PCR products including intronic and exonic sequences from FGG intron 7 to FGG exon 10 were amplified from the genomic DNA of one homozygous affected individual and one normal individual and cloned into the pcDNA3.1 V5His TOPO-TA eukaryotic expression vector (Invitrogen) to obtain mutant and normal minigenes. The presence of the 403 bp duplicated frag- ment in the mutant clone was confirmed by Sanger sequencing. Transient transfections of HEK-293T cells (105 cells/ condition) were performed in 6-well plates using Lipofectamine 2000 (Invitrogen) in OptiMEM (Gibco Invitrogen) and 2 μg of normal or mutant construct. Two days post-transfection, cells were lyzed in Trizol for RNA extraction using the Turbo DNA free kit (Invitrogen). Reverse transcription and PCR amplification of cDNA for analysis of splicing variants are described in the Online Supplementary Appendix.
Results
The members of the large consanguineous family (Figure 1) all originate from a village in Turkey which
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