A germline variant affects putative mi<scp>RNA</scp>‐binding sites at the <i>F8</i> 3′<scp>UTR</scp> and acts as a potential haemophilia A phenotype modifier in Southern Brazilian patients

Rosset, Clévia; Vieira, Igor Araújo; Salzano, Francisco M.; Bandinelli, Eliane

doi:10.1111/hae.12953

Cited by 12 publications

(14 citation statements)

References 6 publications

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“…Hemophilia A (HA) is an X chromosome-linked bleeding disorder that is associated with mutations in the Factor 8 ( F8 ) gene leading to either reduced expression or production of a dysfunctional FVIII protein. However, several studies indicate that in about 1% of severe and about 3% of mild or moderate HA patients, no mutations were detected in the F8 gene ( El-Maarri et al, 2005 ; Johnsen et al, 2017 ), suggesting that there are other molecular mechanisms in addition to mutations in F8 that regulate FVIII expression ( Rosset et al, 2016 ; Jankowska et al, 2019 ).…”

Section: Introductionmentioning

confidence: 99%

Further Evidence That MicroRNAs Can Play a Role in Hemophilia A Disease Manifestation: F8 Gene Downregulation by miR-19b-3p and miR-186-5p

Jankowska

McGill

Pezeshkpoor³

et al. 2020

Front. Cell Dev. Biol.

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Hemophilia A (HA) is a F8 gene mutational disorder resulting in deficiency or dysfunctional FVIII protein. However, surprisingly, in few cases, HA is manifested even without mutations in F8 . To understand this anomaly, we recently sequenced microRNAs (miRNAs) of two patients with mild and moderate HA with no F8 gene mutations and selected two highly expressing miRNAs, miR-374b-5p and miR-30c-5p, from the pool to explain the FVIII deficiency that could be mediated by miRNA-based F8 /FVIII suppression. In this report, an established orthogonal in vivo RNA-affinity purification approach was utilized to directly identify a group of F8 -interacting miRNAs and we tested them for F8 /FVIII suppression. From this pool, two miRNAs, miR-19b-3p and miR-186-5p, were found to be upregulated in a severe HA patient with a mutation in the F8 coding sequence and two HA patients without mutations in the F8 coding sequence were selected to demonstrate their role in F8 gene expression regulation in mammalian cells. Overall, these results provide further evidence for the hypothesis that by targeting the 3′UTR of F8 , miRNAs can modulate FVIII protein levels. This mechanism could either be the primary cause of HA in patients who lack F8 mutations or control the severity of the disease in patients with F8 mutations.

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Section: Introductionmentioning

confidence: 99%

Further Evidence That MicroRNAs Can Play a Role in Hemophilia A Disease Manifestation: F8 Gene Downregulation by miR-19b-3p and miR-186-5p

Jankowska

McGill

Pezeshkpoor³

et al. 2020

Front. Cell Dev. Biol.

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“…Differential profiling of samples from 15 patients with HA with unknown F8 mutations were used in the study . In an in vitro study, we identified single‐nucleotide polymorphisms that affect putative miRNA‐binding sites at the F8 3′ untranslated region (UTR) and act as potential HA phenotype modifiers . A large body of evidence demonstrates the importance of miRNAs in biology and associations between miRNA dysregulation and human diseases, mediated by miRNA binding to either 3′ UTR or 5′ UTR of target mRNA, resulting in gene regulation …”

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confidence: 99%

Clinical manifestation of hemophilia A in the absence of mutations in the F8 gene that encodes FVIII: role of microRNAs

Jankowska

McGill

Pezeshkpoor³

et al. 2019

Transfusion

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BACKGROUND: Hemophilia A (HA) is associated with mutations in the F8 gene that expresses factor VIII (FVIII). Unexpectedly, HA also manifests in a small subset of individuals with no mutations (exonic or intronic) in their F8 gene. MicroRNAs (miRNAs) cause translational interference, affecting protein quality and stoichiometry. Here, by analyzing miRNAs of two patients from this subset, we evaluated miRNA-based FVIII suppression as a testable hypothesis to explain FVIII deficiency in patients with HA with no F8 gene mutations. STUDY DESIGN AND METHODS:To test the hypothesis, miRNA sequencing from two patients with mild and moderate HA with no mutations in their F8 gene, followed by experimental verification, was used to identify a group of upregulated miRNAs in patients with HA compared to normal controls; with binding sites in the 3 0 untranslated region (UTR) of F8 messenger RNA (mRNA), a prerequisite for miRNA-based gene regulation. From this pool, miR-374b-5p and miR-30c-5p, known to be expressed in human liver, where FVIII is expressed, were subjected to extensive characterization. RESULTS:In two cell lines that constitutively express FVIII, we demonstrated that overexpression of miR-374b or miR-30c decreased FVIII expression, while an miR-30c inhibitor partially restored FVIII expression.CONCLUSION: These data support a role for microRNAs in fine-tuning F8 gene regulation. Based on our findings, our current model suggests that in HA cases where the F8 gene is normal and is predicted to express normal levels of FVIII, F8 mRNA 3 0 UTR targeting miRNAs may be responsible for a FVIIIdeficiency phenotype clinically manifesting as HA. ABBREVIATIONS: AT = antithrombin; cDNA = complementary DNA; DMEM = Dulbecco's Modified Essential Medium; eGFP = enhanced green fluorescent protein; FBS = fetal bovine serum; FV = factor V; FVII = factor VII; FVIII = factor VIII; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; HA = hemophilia A; HUVEC = human umbilical vein endothelial cells; LCL = lymphoblastoid cell line; MEM = Minimum Essential Medium; miRNAs = MicroRNAs; MS1 = Mile Seven 1; PCR = polymerase chain reaction; UTR = untranslated region; VWF = von Willebrand factor.

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“…Another hypothesis for our 4 severe patients could be that a variant in a regulatory RNA such as miRNA located in a distant genomic region could modulate the stability of the F8 mRNA or negatively regulate the gene expression, aggravating the consequence of the variant by increasing the degradation of the F8 mRNA in severe cases. Recently, Rosset et al discovered that the c.8728A>G SNP (rs1050705, c*1672A>G) located in the 3′UTR of F8 in HA patients altered FVIII expression and acted as a phenotype modifier in HA patients . Alternatively, variants in loci that influence FVIII levels, including the VWF gene and ABO blood group locus could modify the expressivity of the F8 variant in this subgroup of severe HA patients .…”

Section: Discussionmentioning

confidence: 99%

“…The occurrence of de novo variants at this position was consistent with the fact that c.835G>A occurred within a CpG dinucleotide, a sequence prone to variants in F9 6,10,14 and F8. 15,16 A few other cases were described in different countries in Europe, [17][18][19] India, 20 29 Alternatively, variants in loci that influence FVIII levels, including the VWF gene and ABO blood group locus could modify the expressivity of the F8 variant in this subgroup of severe HA patients. 30 In conclusion, we identified two early haemophilia founder variants that occurred in two distinct locations in France.…”

Section: Discussionmentioning

confidence: 99%

Recurrent F8 and F9 gene variants result from a founder effect in two large French haemophilia cohorts

et al. 2018

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Introduction Haemophilia A (HA) and haemophilia B (HB) are X‐linked recessive diseases, caused by a large number of pathogenic variants in the F8 and F9 genes. With the exception of introns 22 and 1 inversions which are frequent in severe HA cases, about 2000 unique variants in F8 and 1000 in F9 have been described in databases and their recurrence remains limited. Aim and methods During routine analysis, we identified two recurrent missense variants, the F8 gene c.1244C>T, p.Ala415Val variant in 27 HA patients and the F9 gene c.835G>A, p.Ala279Thr variant in 34 HB patients, in two groups of haemophiliac patients from two different regions of France. We aimed to identify whether these variants result from a founder effect. We performed haplotype reconstruction after analysis of extragenic and intragenic polymorphic markers. The ESTIAGE programme was used to estimate the age of the variant. Results We identified a common ancestral haplotype HA1, in all the HA patients sharing the p.Ala415Val variant, and HB1 for 22 of 34 HB patients sharing the p.Ala279Thr variant. The estimated time of occurrence of the founder variant was between the 13th and 17th century (95% CI: 16 to 29 generations) for the F8 variant and between the 3rd and the 11th century for the F9 variant (95% CI: 44 to 72 generations). Conclusion This study supports a founder effect for these two variants in the two largest reported cohorts of haemophilia patients with an identical variant. These pathogenic variants are among the three most early reported variants in haemophilia.

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A germline variant affects putative miRNA‐binding sites at the F8 3′UTR and acts as a potential haemophilia A phenotype modifier in Southern Brazilian patients

Cited by 12 publications

References 6 publications

Further Evidence That MicroRNAs Can Play a Role in Hemophilia A Disease Manifestation: F8 Gene Downregulation by miR-19b-3p and miR-186-5p

Further Evidence That MicroRNAs Can Play a Role in Hemophilia A Disease Manifestation: F8 Gene Downregulation by miR-19b-3p and miR-186-5p

Clinical manifestation of hemophilia A in the absence of mutations in the F8 gene that encodes FVIII: role of microRNAs

Recurrent F8 and F9 gene variants result from a founder effect in two large French haemophilia cohorts

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