F8 Intron Research Articles

Whole F8 gene sequencing identified pathogenic structural variants in the remaining unsolved patients with severe hemophilia A

BackgroundNo F8 genetic abnormality is detected in approximately 1% to 2% of patients with severe hemophilia A (HA) using conventional genetic approaches. In these patients, deep intronic variation or F8 disrupting genomic rearrangement could be causal. ObjectivesThe study aimed to identify the causal variation in families with a history of severe HA for whom genetic investigations failed. MethodsWe performed whole F8 gene sequencing in 8 propositi. Genomic rearrangements were confirmed by Sanger sequencing of breakpoint junctions and/or quantitative polymerase chain reaction. ResultsA structural variant disrupting F8 was found in each propositus, so that all the 815 families with a history of severe HA registered in our laboratory received a conclusive genetic diagnosis. These structural variants consisted of 3 balanced inversions, 3 large insertions of gained regions, and 1 retrotransposition of a mobile element. The 3 inversions were 105 Mb, 1.97 Mb, and 0.362 Mb in size. Among the insertions of gained regions, one corresponded to the insertion of a 34 kb gained region from chromosome 6q27 in F8 intron 6, another was the insertion of a 447 kb duplicated region from chromosome 9p22.1 in F8 intron 14, and the last one was the insertion of an Xq28 349 kb gained in F8 intron 5. ConclusionAll the genetically unsolved cases of severe HA in this cohort were due to structural variants disrupting F8. This study highlights the effectiveness of whole F8 sequencing to improve the molecular diagnosis of HA when the conventional approach fails.

Correction of F8 intron 1 inversion in hemophilia A patient-specific iPSCs by CRISPR/Cas9 mediated gene editing.

Introduction: Hemophilia A (HA) is the most common genetic bleeding disorder caused by mutations in the F8 gene encoding coagulation factor VIII (FVIII). As the second predominant pathogenic mutation in hemophilia A severe patients, F8 Intron one inversion (Inv1) completely splits the F8 gene into two parts and disrupts the F8 transcription, resulting in no FVIII protein production. The part which contains exon 2-exon 26 covers 98% of F8 coding region. Methods: We hypothesized that in situ genetic manipulation of F8 to add a promoter and exon one before the exon two could restore the F8 expression. The donor plasmid included human alpha 1-antitrypsin (hAAT) promoter, exon one and splicing donor site (SD) based on homology-mediated end joining (HMEJ) strategy was targeted addition in hemophilia A patient-derived induced pluripotent stem cell (HA-iPSCs) using CRISPR/Cas9. The iPSCs were differentiated into hepatocyte-like cells (HPLCs). Results: The hAAT promoter and exon one were targeted addition in HA-iPSCs with a high efficiency of 10.19% via HMEJ. The FVIII expression, secretion, and activity were detected in HPLCs derived from gene-targeted iPSCs. Discussion: Thus, we firstly rescued the 140kb reversion mutation by gene addition of a 975bp fragment in the HA-iPSCs with Inv1 mutation, providing a promising gene correction strategy for genetic disease with large sequence variants.

Long-Read Sequencing Resolves Complex Structural Variants and Identifies Missing Disease-Causing Variants in Unsolved Cases of Hemophilia

Introduction: Despite recent advances in genetic testing, many individuals with inherited bleeding disorders remain unsolved after a complete clinical evaluation. In some cases, no variant is found in a particular gene or genes of interest, while in other cases a large structural variant (SV) is found or suspected that cannot be resolved using existing methods. In the large U.S. hemophilia genotyping initiative MyLifeOurFuture, ~2% of participants with hemophilia A had no F8 variant identified despite exhaustive investigation. Additionally, large SVs were common in severe hemophilia and associated with inhibitor risk but were incompletely characterized in nearly all cases. We hypothesized that long-read sequencing (LRS) would identify missing disease-causing variants and fully characterize SVs associated with hemophilia in these individuals. Methods: We performed targeted or whole-genome LRS on the Oxford Nanopore platform of 25 hemophilia cases with FVIII levels <14% in which prior testing identified no pathogenic variant or identified a suspected SV impacting F8. After basecalling with guppy and alignment to GRCh38 using minimap2, single nucleotide and insertion or deletion variants were identified with Clair3. All variant calls were refined using population frequency and pathogenicity predictions. SVs were called with Sniffles2, SVIM, and CuteSV. We performed de novo assembly of SVs and called methylation in all samples. Methylation differences were identified using an in-house developed pipeline. Results: We identified candidate pathogenic variants and previously undetected SVs in 10/10 cases with no variant found on clinical testing. Candidate intronic variants predicted to affect splicing were validated by exon trap assays. SVs identified included deletions and large inversions. Among 11 cases with suspected SVs, LRS resolved the candidate SV providing detailed genomic structure, orientation, and predicted impact of the variant. Whole genome LRS resolved SVs involving the large segmental duplication within F8 intron 22 (Int22h1), revealing variants that have not previously been observed including unsuspected orientations in the F8 intron 22 positive control sample. We analyzed four females with unexplained low FVIII levels and identified skewed methylation profiles that may help explain their phenotype. Conclusion: In a single genetic test LRS can identify disease-causing variants missed by prior genetic testing, resolve complex SVs, and identify methylation differences that explain hemophilia phenotypes. These comprehensive methods are necessary to fully understand causes of hemophilia and complications such as inhibitor development. We expect this approach will similarly resolve "missing” genetic variation in other inherited disorders. Supported and funded by the BBI at UW, WACBD, HTRS MCRA (Genentech), and NHLBI TOPMed.

Comprehensive analysis of F8 large deletions: Characterization of full breakpoint junctions and description of a possible DNA breakage hotspot in intron 6

BackgroundLarge F8 deletions represent 3–5% of the variations found in severe hemophilia A patients, but only a few deletion breakpoints have been characterized precisely. ObjectivesResolving at the nucleotide level 24 F8 large deletions to provide new data on the mechanisms involved in these rearrangements. MethodsBreakpoint junctions of 24 F8 large deletions were characterized using a combination of long‐range polymerase chain reaction, whole F8 NGS sequencing, and Sanger sequencing. Repeat elements, non‐B DNA, and secondary structures were analyzed around the breakpoints. ResultsDeletions ranged from 1.667 kb to 0.5 Mb in size. Nine involved F8 neighboring genes. Simple blunt ends and 2–4 bp microhomologies were identified at the breakpoint junctions of 10 (42%) and 8 (33%) deletions, respectively. Five (21%) deletions resulted from homeologous recombination between two Alu elements. The remaining case corresponded to a more complex rearrangement with an insertion of a 19 bp‐inverted sequence at the junction. Four different breakpoints were located in a 562‐bp region in F8 intron 6. This finding suggested that this region, composed of two Alu elements, is a DNA breakage hotspot. Non‐B DNA and secondary structures were identified in the junction regions and may contribute to DNA breakage. ConclusionMolecular characterization of deletion breakpoints revealed that non‐homologous non‐replicative DNA repair mechanisms and replication‐based mechanisms seemed to be the main causative mechanisms of F8 large deletions. Moreover, we identified a possible F8 DNA breakage hotspot involved in non‐recurrent rearrangements.

Risk factors for inhibitors in hemophilia A based on RNA‐seq and DNA methylation

BackgroundThe development of factor VIII (FVIII) inhibitor is a severe complication during replacement therapy for hemophilia A patients. ObjectivesWe investigated the potential risk factors for FVIII inhibitor formation based on genome‐wide RNA‐sequencing and whole‐genome bisulfite sequencing analysis. MethodsRNA‐sequencing and whole‐genome bisulfite sequencing analysis were applied on 17 blood samples with F8 intron 22 inversion, including seven with inhibitors and 10 without. ResultsAltogether, 344 mRNA transcripts and 20 long noncoding RNAs (lncRNA) transcripts were differentially expressed. Among the differentially expressed transcripts, 200 mRNAs and 12 lncRNAs were upregulated, and 144 mRNAs and eight lncRNAs were downregulated. Gene ontology enrichment analysis of differentially expressed mRNAs showed that genes involved in immune stimulation, especially those for T‐cell activation, were upregulated, whereas genes involved in negative immune response regulation were downregulated. Coexpression analysis revealed that the targeted upregulated genes of differentially expressed lncRNA were similarly closely related to immune activation, especially T‐cell activation. Methylation analysis showed inhibitor patients exhibited a slightly lower methylation status in the CpG islands, 5′ untranslated region, and exon regions (p < 0.01). Genes with differentially methylated regions were also related to T‐cell activation. ConclusionsThere is an upregulation of genes involved in activation of the immune system in hemophilia A patients with inhibitors. The lncRNA and methylation modifications may play important roles in inhibitor production. These findings are potentially to reveal novel therapeutic targets for prevention and treatment of inhibitors.

CRISPR/Cas9-Mediated in vivo Genetic Correction in a Mouse Model of Hemophilia A.

Hemophilia A (HA), a common bleeding disorder caused by a deficiency of coagulation factor VIII (FVIII), has long been considered an attractive target for gene therapy studies. However, full-length F8 cDNA cannot be packaged efficiently by adeno-associated virus (AAV) vectors. As the second most prevalent mutation causing severe HA, F8 intron 1 inversion (Inv1) is caused by an intrachromosomal recombination, leaving the majority of F8 (exons 2–26) untranscribed. In theory, the truncated gene could be rescued by integrating a promoter and the coding sequence of exon 1. To test this strategy in vivo, we generated an HA mouse model by deleting the promoter region and exon 1 of F8. Donor DNA and CRISPR/SaCas9 were packaged into AAV vectors and injected into HA mice intravenously. After treatment, F8 expression was restored and activated partial thromboplastin time (aPTT) was shortened. We also compared two liver-specific promoters and two types of integrating donor vectors. When an active promoter was used, all of the treated mice survived the tail-clip challenge. This is the first report of an in vivo gene repair strategy with the potential to treat a recurrent mutation in HA patients.

Novel Severe Hemophilia A Mouse Model with Factor VIII Intron 22 Inversion.

Simple SummaryRecently, innovative gene therapy has been developing toward functional restoration by gain of function or gene correction. Hemophilia is a representative genetic disorder with many human patients and is considered a candidate disease for gene therapy. The most frequent severe hemophilia A is caused by inversion mediated structural variation of the human F8 gene. Nevertheless, a mouse model with F8 intron 22 inversion is not developed yet. This study presents a novel hemophilia A mouse model with 319 kb inversion and severe coagulation disorder and could be utilized in future gene correction preclinical trials.Hemophilia A (HA) is an X-linked recessive blood coagulation disorder, and approximately 50% of severe HA patients are caused by F8 intron 22 inversion (F8I22I). However, the F8I22I mouse model has not been developed despite being a necessary model to challenge pre-clinical study. A mouse model similar to human F8I22I was developed through consequent inversion by CRISPR/Cas9-based dual double-stranded breakage (DSB) formation, and clinical symptoms of severe hemophilia were confirmed. The F8I22I mouse showed inversion of a 391 kb segment and truncation of mRNA transcription at the F8 gene. Furthermore, the F8I22I mouse showed a deficiency of FVIII activity (10.9 vs. 0 ng/mL in WT and F8I22I, p < 0.0001) and severe coagulation disorder phenotype in the activated partial thromboplastin time (38 vs. 480 s, p < 0.0001), in vivo bleeding test (blood loss/body weight; 0.4 vs. 2.1%, p < 0.0001), and calibrated automated thrombogram assays (Thrombin generation peak, 183 vs. 21.5 nM, p = 0.0012). Moreover, histological changes related to spontaneous bleeding were observed in the liver, spleen, and lungs. We present a novel HA mouse model mimicking human F8I22I. With a structural similarity with human F8I22I, the F8I22I mouse model will be applicable to the evaluation of general hemophilia drugs and the development of gene-editing-based therapy research.

Partial F8 gene duplication (factor VIII Padua) associated with high factor VIII levels and familial thrombophilia

AbstractHigh coagulation factor VIII (FVIII) levels comprise a common risk factor for venous thromboembolism (VTE), but the underlying genetic determinants are largely unknown. We investigated the molecular bases of high FVIII levels in 2 Italian families with severe thrombophilia. The proband of the first family had a history of recurrent VTE before age 50 years, with extremely and persistently elevated FVIII antigen and activity levels (>400%) as the only thrombophilic defects. Genetic analysis revealed a 23.4-kb tandem duplication of the proximal portion of the F8 gene (promoter, exon 1, and a large part of intron 1), which cosegregated with high FVIII levels in the family and was absent in 103 normal controls. Targeted screening of 50 unrelated VTE patients with FVIII levels ≥250% identified a second thrombophilic family with the same F8 rearrangement on the same genetic background, suggesting a founder effect. Carriers of the duplication from both families showed a twofold or greater upregulation of F8 messenger RNA, consistent with the presence of open chromatin signatures and enhancer elements within the duplicated region. Testing of these sequences in a luciferase reporter assay pinpointed a 927-bp region of F8 intron 1 associated with >45-fold increased reporter activity in endothelial cells, potentially mediating the F8 transcriptional enhancement observed in carriers of the duplication. In summary, we report the first thrombophilic defect in the F8 gene (designated FVIII Padua) associated with markedly elevated FVIII levels and severe thrombophilia in 2 Italian families.

Rapid genotyping of F8 intron 22 inversion by nested PCR based on long-distance PCR.

F8 intron 22 inversion (Inv22) accounts for about 40% of severe hemophilia A (HA) cases and is mainly genotyped by long-distance PCR (LD-PCR) or inverse-PCR (I-PCR). These methods require long separation times or enzymatic digestion. We aimed to shorten the separation time of LD-PCR. Long-read sequencing was applied for LD-PCR products from 20 Inv22 patients and 4 controls to validate the differences between products generated using P-Q and P-B primer pairs in LD-PCR. We then confirmed two unique regions (chrX: 154879481-154880814, chrX: 155376388-155376505, GRCh38) in the PCR products from P-Q and P-B primer pairs, respectively. The nested PCR P1, Q1, and B1 primers were located near the homologous sequence and two unique regions, respectively. The P1-Q1 and P1-B1 primer pairs generated 1621bp and 540bp fragments, respectively, and the Inv22 carriers produced both fragments. In total, 228 previously diagnosed subjects including 39 Inv22 carriers, 52 Inv22 patients, 82 Inv22 negative males, and 55 Inv22 negative females were genotyped using nested PCR, and the results revealed excellent sensitivity and specificity (100 and 100%, respectively). The separation time was shortened from 5 to 0.5h. Therefore, we present a rapid genotyping method for F8 Inv22 by nested PCR based on LD-PCR.

Severe hemophilia A caused by an unbalanced chromosomal rearrangement identified using nanopore sequencing

Unlabelled Box BackgroundNo F8 genetic abnormality is detected in about 2% of severe hemophilia A patients using conventional genetic approaches. In these patients, deep intronic variation or F8 disrupting genomic rearrangement could be causal. ObjectiveTo characterize, in a genetically unresolved severe hemophilia A patient, a new Xq28 rearrangement disrupting F8 using comprehensive molecular techniques including nanopore sequencing. ResultsLong‐range polymerase chain reaction (PCR) performed throughout F8 identified a nonamplifiable region in intron 25 indicating the presence of a genomic rearrangement. F8messanger ribonucleic acid (mRNA) analysis including 3′rapid amplification of complementary deoxyribonucleic acid (cDNA) ends and nanopore sequencing found the presence of a F8 fusion transcript in which F8 exon 26 was replaced by a 742‐bp pseudoexon corresponding to a noncoding region located at the beginning of the long arm of chromosome X (Xq12; chrX: 66 310 352‐66 311 093, GRCh37/hg19). Cytogenetic microarray analysis found the presence of a Xq11.1q12 gain of 3.8 Mb. The PCR amplification of junction fragments and fluorescent in situ hybridization (FISH) analysis found that the Xq11q12 duplicated region was inserted in the F8 intron 25 genomic region. ConclusionWe characterized a novel genomic rearrangement in which a 3.8‐Mb Xq11.1q12 gain inserted in the F8 intron 25 led to an aberrant fusion transcript in a patient with severe hemophilia A (HA), using comprehensive molecular techniques. This study highlights the value of single‐molecule long‐read sequencing technologies for molecular diagnosis of HA especially when conventional genetic approaches have failed.

Novel Structural Variants Originating in F8 Non-Coding Regions in Previously Unresolved Cases of Severe Hemophilia A

Background. Hemophilia A is a rare X-linked bleeding disorder resulting from deficiency in coagulation factor VIII. Numerous genetic variants (>2000) affecting the F8 gene have been implicated as causative of hemophilia A, including structural variants (SVs) such as copy number variants (CNVs) and large intra-chromosomal inversions caused by recombination between distant regions with high homology to sequences within F8 intron 1 or intron 22. SVs detected in patients with hemophilia are associated with more severe disease, and different types of SVs may inform inhibitor risk. For the vast majority of patients, causative variants can be identified using targeted DNA sequencing of F8 coding regions and/or the use of methods which detect known SVs (e.g. inverse shifting PCR, long-range PCR, MLPA). However, these approaches fail to explain 1-3% of hemophilia A cases. We hypothesized that a dedicated structural variant analysis at the F8 locus using whole genome sequencing data could identify previously undetected deleterious F8 gene variants in unsolved cases of hemophilia A.Methods. Cases were selected from the My Life, Our Future (MLOF) hemophilia study cohort recently whole genome sequenced by the NHLBI TOPMed program. In this study, we performed a custom SV analyses using whole genome sequencing (WGS) data from 11 cases of severe hemophilia A (factor VIII activity level < 1%) that remained genetically unexplained after exhausting all available laboratory testing methods. Two of the eleven unsolved severe hemophilia A cases (18%) were reported to have had an inhibitor.Results. SV analyses of the F8 genomic region revealed previously undetected deletions and inversions in 6 out of the 11 cases. In these 6 samples, SV calls were supported by multiple sequencing reads (> 25 reads) and multiple types of read evidence (read depth, paired-end and/or split read evidence). Two deletions within intron 6 were detected in a single hemophilia A case, a finding which suggests F8 intron 6 may contain one or more regulatory elements critical for F8 expression. Three distinct large inversions predicted to disrupt the F8 structural gene were detected in five other cases; a 720Kb inversion with breakpoints in F8 intron 6 and SPRY3 intron 1 (n=1), a 20Mb inversion with breakpoints in F8 intron 1 and INTS6L intron 8 (n=1), and a 7.4Kb inversion with breakpoints in F8 intron 25 and the SMIM9 intron 1 (n=3). These events are novel in hemophilia and were also not present in the larger, sequenced My Life, Our Future dataset (N=2186), supporting these SVs as likely causative of severe hemophilia A. Both cases with inhibitors had the F8 intron 25-SMIM9 inversion.Conclusions. This work demonstrates that dedicated analyses of WGS for SVs originating in non-coding regions can identify novel variants in previously unsolved cases of hemophilia A. We conclude that any genetic studies of diseases caused by loss-of-function variants should consider dedicated analyses for SVs. We predict additional deleterious SVs remain to be discovered in rare unexplained cases of hemophilia. DisclosuresKonkle:BioMarin: Consultancy; Bioverativ: Research Funding; CSL Behring: Consultancy; Genentech: Consultancy; Spark: Consultancy, Research Funding; Pfizer: Research Funding; Gilead: Consultancy; Sangamo: Research Funding; Shire: Research Funding. Johnsen:CSL Behring: Consultancy; Octapharma: Consultancy.

The Highly Prevalent Deletions in F8 Intron 13 Found in French Mild Haemophilia a Patients Result of Both Founder Effect and Recurrent De Novo Events

Background: Genetic variations that are found at a relatively high frequency can be the consequence of a founder effect or repeated de novo events. Recently, our group has identified an intronic deletion, c.2113+461_2113+473del [DEL13T], in the F8 intron 13, in two mild haemophilia A patients. This deletion removed a part of the poly(T)-tail from the right arm of antisens AluY element and led to AluY exonisation. Then, deletions in the poly(T)-tail of AluY in F8 intron 13, from 10 to 14 pb in size, were found in approximately 6% of all cases of mild haemophilia in France.Aim: In this study, we determined whether these highly prevalent deletions are the result of recurrent molecular mechanism or of a founder effect.Methods: Haplotype reconstruction was performed after analysis of F8 extragenic and intragenic polymorphic markers in 71 unrelated French mild haemophilia A patients carrying a deletion in the poly(T)-tail of AluY in F8 intron 13 (c.2113+460_2113+473del [DEL14T] n=1; DEL13T, n=62; c.2113+462_2113+473del [DEL12T], n=2; c.2113+463_2113+473del [DEL11T], n=5 and c.2113+464_2113+473del, n=1 [DEL10T]) and in 50 non-haemophilia A subjects. The ESTIAGE tool was used to estimate the age of the DEL13T. Nineteen genetically unresolved mild Haemophilia A patients from Queen's University, Canada, were also included in the study. All patients and controls gave informed consent for genetic studies.Results: Two intragenic (STR13 and STR22) and 3 extragenic (DXS8061, ST14 and DF2) microsatellites were investigated in the DNA of the 71 mild haemophilia A patients. This set of polymorphisms markers covered a genomic region of 2,779,113 nucleotides (4.17 cM).Among the 62 patients carrying the DEL13T, 60 patients had the same allele for the marker directly flanking the deletion on the centromeric side (STR13, genetic distance 0.0062 cM). Two of them differed from the others at the next centromeric maker STR22 (genetic distance 0.2047 cM) and 20 patients differed from the others at ST14 (genetic distance 3.4237 cM). On the telomeric side, only 8 patients differed from the others at DF2 (genetic distance 0.0292 cM). None of control individuals shared such haplotypes with these 60 patients. These results provided evidence that the founder effect hypothesis was very plausible for the variant DEL13T. The ESTIAGE tool estimated that the DEL13T occurred about 61 generations ago (95% CI : 51-74 generations). Assuming that a generation spanned 25 years, the French common ancestor carrying the c.2113+461_2113+473del was estimated to have lived between the 2th and the 8th century.However, two patients carrying the DEL13T and 9 patients carrying the other deletions (DEL10T, DEL11T, DEL12T and DEL14T) had a different haplotype suggesting that these deletions arose independently. In order to support the hypothesis of a recurrent molecular mechanism, we have investigated the presence of these deletions in other geographies. F8 intron 13 deletions were found in 3/19 Canadian patients included in this study (DEL13T, n=2 and DEL10T, n=1). Haplotype analysis performed in these three patients suggested a de novo mechanism for two of them.Conclusion: This study supports both a founder effect for the c.2113+461_2113+473del in the French mild haemophilia A patients and a recurrent molecular mechanism leading to deletion in the poly(T)-tail of AluY in F8 intron 13. We recommended that these deletions be specifically investigated in all mild haemophilia A patients in whom no genetic abnormality has been detected by standard genetic analysis. Finally, these results suggest that large poly(T)-tail of inverted Alu elements may be a mutational hot spot and such deletions leading to alu-exonization, could occur in other genes. DisclosuresNegrier:Sobi/Bioverativ: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Alnylam: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; LFB: Honoraria, Membership on an entity's Board of Directors or advisory committees; Baxalta/Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Honoraria, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Mutational Profiles of F8 and F9 in a Cohort of Haemophilia A and Haemophilia B Patients in the Multi-ethnic Malaysian Population.

BackgroundHaemophilia A (HA) and Haemophilia B (HB) are X-linked blood disorders that are caused by various mutations in the factor VIII (F8) and factor IX (F9) genes respectively. Identification of mutations is essential as some of the mutations are associated with the development of inhibitors. This study is the first comprehensive study of the F8 mutational profile in Malaysia.Materials and methodsWe analysed 100 unrelated HA and 15 unrelated HB patients for genetic alterations in the F8 and F9 genes by using the long-range PCR, DNA sequencing, and the multiplex-ligation-dependent probe amplification assays. The prediction software was used to confirm the effects of these mutations on factor VIII and IX proteins.Results44 (53%) of the severe HA patients were positive for F8 intron 22 inversion, and three (3.6%) were positive for intron one inversion. There were 22 novel mutations in F8, including missense (8), frameshift (9), splice site (3), large deletion (1) and nonsense (1) mutations. In HB patients, four novel mutations were identified including the splice site (1), small deletion (1), large deletion (1) and missense (1) mutation.DiscussionThe mutational spectrum of F8 in Malaysian patients is heterogeneous, with a slightly higher frequency of intron 22 inversion in these severe HA patients when compared to other Asian populations. Identification of these mutational profiles in F8 and F9 genes among Malaysian patients will provide a useful reference for the early detection and diagnosis of HA and HB in the Malaysian population.

Review of molecular mechanisms at distal Xq28 leading to balanced or unbalanced genomic rearrangements and their phenotypic impacts on hemophilia.

The distal Xq28 region is very gene-rich, comprising a relatively large number of low-copy repeats (LCRs) predisposing to genomic rearrangements. The best-known rearrangement at this locus is the F8 intron 22 inversion, responsible for up to 45% of severe hemophilia A (HA) cases. An additional inversion of intron 1 of F8 has more recently been described, affecting 2%-5% of patients with severe HA. These "balanced" rearrangements are mediated by intrachromosomal homologous recombination between inversely oriented LCRs located in intron 1 or 22 and other extragenic copies positioned more telomerically outside the F8 gene. The successive innovations of semi-quantitative technologies like multiplex ligation-dependent probe amplification (MLPA) and array comparative genomic hybridization (array CGH) have rendered it possible to highlight a significant number of "unbalanced" rearrangements associated or not with these inversions. Some rearrangements are generated by the non-allelic homologous recombination (NAHR) pathway between directly oriented LCRs. Others are probably the result of unequal crossing-over or U-loop exchanges during female meiosis. This review sought to provide an overview of the mechanisms underlying rearrangements at the distal Xq28 locus and discuss their clinical impacts other than HA, such as risks of developing high inhibitor levels and spontaneous abortion, as well as other pathologies like cardiovascular disease or potentially X-linked intellectual disease.

Reccurrent F8 Intronic Deletion Found in Mild Hemophilia A Causes Alu Exonization

Incorporation of distant intronic sequences in mature mRNA is an underappreciated cause of genetic disease. Several disease-causing pseudoexons have been found to contain repetitive elements such as Alu elements. This study describes an original pathological mechanism by which a small intronic deletion leads to Alu exonization. We identified an intronic deletion, c.2113+461_2113+473del, in the F8 intron 13, in two individuals with mild hemophilia A. Invivo and invitro transcript analysis found an aberrant transcript, with an insertion of a 122-bp intronic fragment (c.2113_2114ins2113+477_2113+598) at the exon 13-14 junction. This out-of-frame insertion is predicted to lead to truncated protein (p.Gly705Aspfs∗37). DNA sequencing analysis found that the pseudoexon corresponds to antisense AluY element and the deletion removed a part of the poly(T)-tail from the right arm of these AluY. The heterogenous nuclear riboprotein C1/C2 (hnRNP C) is an important antisense Alu-derived cryptic exon silencer and binds to poly(T)-tracts. Disruption of the hnRNP C binding site in AluY T-tract by mutagenesis or hnRNP C knockdown using siRNA in HeLa cells reproduced the effect of c.2113+461_2113+473del. The screening of 114 unrelated families with mild hemophilia A in whom no genetic event was previously identified found a deletion in the poly(T)-tail of AluY in intron 13 in 54% of case subjects (n = 61/114). In conclusion, this study describes a deletion leading to Alu exonization found in 6.1% of families with mild hemophila A in France.

Genetic analysis of bleeding disorders.

Molecular genetic analysis of inherited bleeding disorders has been practised for over 30years. Technological changes have enabled advances, from analyses using extragenic linked markers to next-generation DNA sequencing and microarray analysis. Two approaches for genetic analysis are described, each suiting their environment. The Christian Medical Centre in Vellore, India, uses conformation-sensitive gel electrophoresis mutation screening of multiplexed PCR products to identify candidate mutations, followed by Sanger sequencing confirmation of variants identified. Specific analyses for F8 intron 1 and 22 inversions are also undertaken. The MyLifeOurFuture US project between the American Thrombosis and Hemostasis Network, the National Hemophilia Foundation, Bloodworks Northwest and Biogen uses molecular inversion probes (MIP) to capture target exons, splice sites plus 5' and 3' sequences and to detect F8 intron 1 and 22 inversions. This allows screening for all F8 and F9 variants in one sequencing run of multiple samples (196 or 392). Sequence variants identified are subsequently confirmed by a diagnostic laboratory. After having identified variants in genes of interest through these processes, a systematic procedure determining their likely pathogenicity should be applied. Several scientific societies have prepared guidelines. Systematic analysis of the available evidence facilitates reproducible scoring of likely pathogenicity. Documentation of frequency in population databases of variant prevalence and in locus-specific mutation databases can provide initial information on likely pathogenicity. Whereas null mutations are often pathogenic, missense and splice site variants often require in silico analyses to predict likely pathogenicity and using an accepted suite of tools can help standardize their documentation.

Genotyping of Intron Inversions and Point Mutations in Exon 14 of the FVIII Gene in Iranian Azeri Turkish Families with Hemophilia A.

Hemophilia A (HA) is an inherited X-linked bleeding disorder caused by a variety of mutations that are distributed throughout the large FVIII gene (F8). The most common mutations in studied populations with severe HA are introns 22 and 1 inversions, gross exon deletions and point mutations in exon 14. The aim of this study was to define the frequency of these common mutations in Iranian population of Azeri Turkish in North West of Iran. Fifty patients with severe HA and forty-three female potential carriers were genotyped by inverse shifting polymerase chain reaction (IS-PCR), long-range PCR, multiplex PCR, and sequencing methods for the detection of Intron 22 and 1 inversions, gross exon deletions, and exon 14 point mutations, respectively. F8 intron 22 inversion was detected in 22 (44%) out of 50 patients. Moreover, we detected one intron 1 inversion (2%), and one point mutation in exon 14 (2%). In this population, 52% of the patients with hemophilia A did not show to carry a mutation in the analyzed regions by three mentioned methods. F8 intron 22 inversion was the major causative mutation in nearly 50% of severe HA cases in an Azerbaijani Turkish population, which is similar to the incidence of other populations. IS-PCR is a robust, rapid, efficient, and cost-effective method for the genetic analysis of patients with severe HA and for HA carrier detection, especially in developing countries.

Principles of genetic variations and molecular diseases: applications in hemophilia A.

DNA structure alterations are the ultimate source of genetic variations. Without them, evolution would be impossible. While they are essential for DNA diversity, defects in DNA synthesis can lead to numerous genetic diseases. Due to increasingly innovative technologies, our knowledge of the human genome and genetic diseases has grown considerably over the last few years, allowing us to detect another class of variants affecting the chromosomal structure. DNA sequence can be altered in multiple ways: DNA sequence changes by substitution, deletion, or duplication of some nucleotides; chromosomal structure alterations by deletion, duplication, translocation, and inversion, ranging in size from kilobases to mega bases; changes in the cell's genome size. If the alteration is located within a gene and sufficiently deleterious, it can cause genetic disorders. Due to the F8 gene's high rate of new small mutations and its location at the tip of X chromosome, containing high repetitive sequences, a wide variety of genetic variants has been described as the cause of hemophilia A (HA). In addition to the F8 intron 22 repeat inversion, HA can also result from point mutations, other inversions, complex rearrangements, such as duplications or deletions, and transposon insertions causing phenotypes of variable severity characterized by complete or partial deficiency of circulating FVIII. This review aims to present the origins, mechanisms, and consequences of F8 alterations. A sound understanding of the multiple genetic mechanisms responsible for HA is essential to determine the appropriate strategy for molecular diagnosis and detected each type of genetic variant.

In situ genetic correction of F8 intron 22 inversion in hemophilia A patient-specific iPSCs.

Nearly half of severe Hemophilia A (HA) cases are caused by F8 intron 22 inversion (Inv22). This 0.6-Mb inversion splits the 186-kb F8 into two parts with opposite transcription directions. The inverted 5′ part (141 kb) preserves the first 22 exons that are driven by the intrinsic F8 promoter, leading to a truncated F8 transcript due to the lack of the last 627 bp coding sequence of exons 23–26. Here we describe an in situ genetic correction of Inv22 in patient-specific induced pluripotent stem cells (iPSCs). By using TALENs, the 627 bp sequence plus a polyA signal was precisely targeted at the junction of exon 22 and intron 22 via homologous recombination (HR) with high targeting efficiencies of 62.5% and 52.9%. The gene-corrected iPSCs retained a normal karyotype following removal of drug selection cassette using a Cre-LoxP system. Importantly, both F8 transcription and FVIII secretion were rescued in the candidate cell types for HA gene therapy including endothelial cells (ECs) and mesenchymal stem cells (MSCs) derived from the gene-corrected iPSCs. This is the first report of an efficient in situ genetic correction of the large inversion mutation using a strategy of targeted gene addition.

F8 intron 22 inversions and SNP rs73563631 in unrelated families with severe haemophilia A: clinical features and gene testing implications.

Supplementary Material to this article is available online at www.thrombosis-online.com.