five Int22h Homologous Copies in Association with Intron22 Inversion Type 3

Abstract Introduction F8 intron22 inversion is the causative gene defect in up to 45% of severe hemophilia A (HA) patients mediated by recombination between three highly homologous copies located in intron 22 (int22h-1) and two other extragenic copies (int22h-2 and int22h-3) positioned more telomerically outside the gene. Intrachromosomal rearrangement between int22h-1 and int22h-3 provide the F8 type 1 inversion while the F8 type 2 inversion could be explained by the participation of the int22-h2 sequence in the homologous recombination. However, a third type, known since 1993*, was explained by the existence of a duplicated copy of int22h-3 or int22h-2 in case of intron22 inversion type 3A or 3B respectively. Methods Three unrelated HA patients with intron22 type 3A/3B were identified by southern blotting. To appreciate the length of the int22h extragenic duplicated, genomic hybridisation was performed using Affimetrix CytoScan High-Density array. Results Breakpoint analyses by CGH-array in all patients show same duplication of approximately 180kb delimited between intron 1 of CLIC2 gene and distal repeat int22h-3reflecting the presence of five genomic int22 copies. A fourth non-hemophiliac case was added since analysis by CGH technique identified duplication delineated by same boundaries. Conclusion This study suggests the existence of genotypes harboring five int22h copies mediated by 180 kb duplication at Xq28 locus probably not associated with HA. We propose that this duplication has happened by tandem inversed duplication. Such genotype is to be considered as polymorphism and could be associated with the two kinds of F8 intron22 inversion type 3 when intrachromosomal recombination has occurred between homologous copies. *Antonarakis SE and the international consortium study. Factor VIII gene inversions in severe hemophilia A: results of an international consortium study. Blood 1995; 86: 2206-2212 Disclosures No relevant conflicts of interest to declare. </jats:sec

Differential diagnosis of neonatal alloimmune thrombocytopenia: Type 2B von Willebrand disease

At birth, severe thrombocytopenia without context of infection should mainly suggest neonatal alloimmune thrombocytopenia (NAIT), especially in case of a platelet count below 20 GL−1. We report two cases of severe neonatal thrombocytopenia, first suspected as being NAIT. Both had a platelet count below 20 GL−1 with platelet clumps. The absence of alloantibodies and failure of platelet transfusion and intravenous immunoglobulins to improve the platelet count led to question the diagnosis and to evoke inherited bleeding disorders. Measurements of Von Willebrand factor (VWF) levels showed a marked reduction of VWF:RCo and a normal VWF:Ag, suggesting a type 2B Von Willebrand disease (VWD2B). Ristocetin-induced platelet aggregation could not be performed because of the very low platelet count. In the first case, after sequencing VWF exon 28, a heterozygous p.Leu1460Pro mutation was found consistent with VWD2B. In the second case, the genetic analysis of VWF exon 28 identified a homozygous mutation: p.Pro1337Leu confirming type VWD2B and also the p.Arg854Gln homozygous mutation in exon 20 confirming type 2N (ratio FVIII/VWF:Ag <0.5). The two cases underline that, even if NAIT remains the most common diagnosis in severe neonatal thrombocytopenia, it should be challenged in the absence of documented incompatibility, chronic evolution, or treatment failure. Diagnosis of VWD2B should be considered in early thrombocytopenia, even without familial history. In the cases presented, genotyping confirmed the subtype of VWD and helped to guide the therapeutic management of bleeding episodes

Molecular analysis of eight severe FV‐deficient patients in Pakistan: A large series of homozygous for frameshift mutations

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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

Abstract 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. Disclosures Negrier: 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. </jats:sec

Whole F9 gene sequencing identified deep intronic variations in genetically unresolved hemophilia B patients

International audienceBackground: The disease-causative variant remains unidentified in approximately 0.5% to 2% of hemophilia B patients using conventional genetic investigations, and F9 deep intronic variations could be responsible for these phenotypes.Objectives: This study aimed to characterize deep intronic variants in hemophilia B patients for whom genetic investigations failed.Methods: We performed whole F9 sequencing in 17 genetically unsolved hemophilia B patients. The pathogenic impact of the candidate variants identified was studied using both in silico analysis (MaxEntScan and spliceAI) and minigene assay.Results: In total, 9 candidate variants were identified in 15 patients; 7 were deep intronic substitutions and 2 corresponded to insertions of mobile elements. The most frequent variants found were c.278-1806A>C and the association of c.278-1244A>G and c.392-864T>G, identified in 4 and 6 unrelated individuals, respectively. In silico analysis predicted splicing impact for 4 substitutions (c.278-1806A>C, c.392-864T>G, c.724-2385G>T, c.723+4297T>A). Minigene assay showed a deleterious splicing impact for these 4 substitutions and also for the c.278-1786_278-1785insLINE. In the end, 5 variants were classified as likely pathogenic using the American College of Medical Genetics and Genomics guidelines, and 4 as of unknown significance. Thus, the hemophilia B-causing variant was identified in 13/17 (76%) families.Conclusion: We elucidated the causing defect in three-quarters of the families included in this study, and we reported new F9 deep intronic variants that can cause hemophilia B

High-Throughput Genotyping of Haemophilia A and B Using Next-Generation Sequencing Technology in Lille University Hospital

Abstract Introduction The identification of molecular defects in haemophilia is essential for the optimization of patient treatment and the formal characterization of female carriers. The Sanger method is the gold standard for sequencing F8 and F9 genes but is time-consuming and expensive. We aimed to develop a high-throughput method to genotype haemophilia A (HA) and B (HB) patients using the Next-Generation Sequencing (NGS) technology for an exhaustive and less expensive analysis of F8 and F9 genes. Material &amp; Methods We developed a small panel containing F8 and F9 for exons and introns/exons junctions sequencing. We used two different methods for library preparation (AmpliSeq™, Life Technologies™ and HaloPlex™, Agilent™), performed in the same PCR emulsion system (Ion One Touch 2™, Life Technologies™) and sequenced with a Ion 316™ chip in a PGM™ Ion Torrent sequencer, or a Ion PI™ chip in a Proton™ sequencer (Life Technologies™) respectively. The promoter and 3' regions of F8 and F9 were always studied by Sanger. NGS analysis was first performed in 62 samples (HA: n= 42; HB: n=13; carriers: n=7) previously characterized for F8 or F9 mutations by Sanger method or Multiplex Ligation-Probe Amplification (MLPA). All types of mutations were studied (nonsense, missense, splice, small insertion/deletion and exons deletion/duplication) and were distributed in all exons of F8 and F9. NGS analysis was further performed in 42 haemophilia patients (HA: n=31, HB: n=11; of which 36 presented a mild phenotype), with unknown mutation status. All patients were included by the local Comprehensive Care Haemophilia Center of Lille University Hospital after written informed consent. Data were analyzed with SeqNext™ software (JCI Medical System™). A Normalized Reads Depth (NRD) ratio was used to detect exons deletion/duplication. Results All exons were well covered by AmpliSeq™ (average number of reads, ANR = 300) and overall by HaloPlex™ (ANR = 15000, except small parts of exons 14 and 19 in F8). The average quality value for mutation detection was 60 (risk of false result &lt;0.0001%). In previously-genotyped patients, 92% (57/62) of F8 and F9 mutations were detected by AmpliSeq™ and 85% (53/62) by HaloPlex™. The detection rate of small insertion/deletion in homopolymers of exon 14 in F8 was only 20% (1/5) with both methods and 71% (5/7) in other exons of F8. Four deletions and one duplication of exons accounting for a severe haemophilia phenotype were identified (3 HA, 1 HB and 1 HA-carrier). In 5 uncharacterized patients by Sanger method, neither AmpliSeq™ nor HaloPlex™ were able to find a mutation suggesting that the molecular defect is located in introns of F8 or F9. In never-genotyped patients, a mutation was detected in 90% (38/42) of cases (including 34 missense, 2 nonsense and 2 splice mutations in both F8 and F9). A duplication of exons 10 to 14 was also detected in a severe HA patient and was confirmed by MLPA. In NGS negative patients, no mutation was found in promoter or 3' regions. Of the ten candidate mutations identified in our cohort, seven were predicted to be deleterious by in silico analysis and/or co-segregation studies. No mutation was found in 10% (4/42) of never-genotyped patients with mild haemophilia A, in consistence with the available data for the mild phenotype. The technical development and laboratory protocol was easier and less expensive ($530 vs$602 including reagents and technical/medical staff) with AmpliSeq™ than HaloPlex™. Conclusion We confirmed that NGS is able to detect the main types of mutations in F8 and F9 genes, albeit with a lower mutation detection rate with HaloPlex™ compared to AmpliSeq™. These detections were associated with an important depth of reads and high Quality Values, except for exons 14 and 19 in F8 with HaloPlex™. AmpliSeq™ seems also an interesting screening method for the detection of exons deletion/duplication using the NRD ratio. However, both strategies fail to detect small insertion/deletion located in homopolymers of exon 14 in F8, whom identification will still rely on Sanger sequencing. AmpliSeq™ protocol performed in the PGM™ sequencer appears as a new interesting tool in genotyping of HA and HB patients of the Lille University Hospital. Disclosures Zawadzki: Pfizer Pharmaceutical Company: Research Funding. </jats:sec

Abnormal bleeding phenotype for mild haemophilia B patients with the p.Ile112Thr variation on the gene for factor IX

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