Advanced cell‐based modeling of the royal disease: characterization of the mutated F9mRNA

Abstract

Background The royal disease is a form of hemophilia B (HB) that affected many descendants of Queen Victoria in the 19th and 20th centuries. It was found to be caused by the mutation F9 c.278-3A>G. Objective To generate a physiological cell model of the disease and to study F9 expression at the RNA level. Methods Using fibroblasts from skin biopsies of a previously identified hemophilic patient bearing the F9 c.278-3A>G mutation and his mother, we generated induced pluripotent stem cells (iPSCs). Both the patient's and mother's iPSCs were differentiated into hepatocyte-like cells (HLCs) and their F9 mRNA was analyzed using next-generation sequencing (NGS). Results and Conclusion We demonstrated the previously predicted aberrant splicing of the F9 transcript as a result of an intronic nucleotide substitution leading to a frameshift and the generation of a premature termination codon (PTC). The F9 mRNA level in the patient's HLCs was significantly reduced compared with that of his mother, suggesting that mutated transcripts undergo nonsense-mediated decay (NMD), a cellular mechanism that degrades PTC-containing mRNAs. We also detected small proportions of correctly spliced transcripts in the patient's HLCs, which, combined with genetic variability in splicing and NMD machineries, could partially explain some clinical variability among affected members of the European royal families who had lifespans above the average. This work allowed the demonstration of the pathologic consequences of an intronic mutation in the F9 gene and represents the first bona fide cellular model of HB allowing the study of rare mutations at the RNA level.

In vitro recovery of FIX clotting activity as a marker of highly functional hepatocytes in a hemophilia B iPSC model

“…[ 9 ] Culture conditions mimicking liver development with the sequential addition of specific cytokines and growth factors can drive the differentiation of PSCs into hepatocyte‐like cells (HLCs) expressing specific liver markers such as hepatic nuclear factor 4α (HNF4α) or albumin and displaying hepatocyte‐specific functions. [ 10 , 11 ] To date, a few studies have reported the generation of HB patient–specific iPSCs (HB‐iPSCs) [ 12 , 13 , 14 , 15 , 16 ] and their differentiation into hepatocytes, but further characterization of the clotting FIX produced remains to be done.…”

Effects of 14 F9 synonymous codon variants on hemophilia B expression: Alteration of splicing along with protein expression

“…Recently, larger minigene construction with multiple exons has been confirmed to be more accurate to reflect the real in vivo splicing outcome of variants due to mimicking the natural genomic context (Acedo et al, 2015; Fraile‐Bethencourt et al, 2017, 2019). Third, a previous study reported an iPSC‐based model of the disease may provide a novel way to investigate the pathogenic mechanism of the splicing site variants at the RNA level (Martorell et al, 2017), especially in F9 with difficulty in ectopic transcription analysis. Fourthly, the clinical and laboratory information of cases carrying the 14 SCVs was collected from our center and F9 variant database, we have some difficulties establishing the relationship between the phenotype and genotype in some cases due to the missing data.…”

Unraveling the effect of silent, intronic and missense mutations on VWF splicing: contribution of next generation sequencing in the study of mRNA

“…However, few groups have explored its potential for analyzing splicing variants following RT-PCR. 19,20 In this new scenario, the procedure we previously described to analyze the effects of PSSM in VWF 7 has been optimized and adapted to an NGS-based technique to investigate its value in this field. Our main objective was to elucidate the true effects of 18 selected mutations (intronic, synonymous, delins, and missense) on mRNA processing and their genotype/phenotype correspondence by analysis of leukocytes and platelets from clinical samples.…”