Eighteen Years of Molecular Genotyping the Hemophilia Inversion Hotspot: From Southern Blot to Inverse Shifting-PCR

Rossetti, Liliana Carmen; Radic, Claudia Pamela; Abelleyro, Miguel Martín; Larripa, Irene; Brasi, Carlos Daniel de

doi:10.3390/ijms12107271

Cited by 23 publications

(29 citation statements)

References 47 publications

(90 reference statements)

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“…Regular or long-range PCR [20], [25], [27], [38], [39] are limited by the size of the fragments to amplify and work poorly for fragments above 10 kb. Therefore, their applicability is reduced to inversions generated by simple breaks or small IRs at their breakpoints.…”

Section: Introductionmentioning

confidence: 99%

Validation and Genotyping of Multiple Human Polymorphic Inversions Mediated by Inverted Repeats Reveals a High Degree of Recurrence

et al. 2014

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In recent years different types of structural variants (SVs) have been discovered in the human genome and their functional impact has become increasingly clear. Inversions, however, are poorly characterized and more difficult to study, especially those mediated by inverted repeats or segmental duplications. Here, we describe the results of a simple and fast inverse PCR (iPCR) protocol for high-throughput genotyping of a wide variety of inversions using a small amount of DNA. In particular, we analyzed 22 inversions predicted in humans ranging from 5.1 kb to 226 kb and mediated by inverted repeat sequences of 1.6–24 kb. First, we validated 17 of the 22 inversions in a panel of nine HapMap individuals from different populations, and we genotyped them in 68 additional individuals of European origin, with correct genetic transmission in ∼12 mother-father-child trios. Global inversion minor allele frequency varied between 1% and 49% and inversion genotypes were consistent with Hardy-Weinberg equilibrium. By analyzing the nucleotide variation and the haplotypes in these regions, we found that only four inversions have linked tag-SNPs and that in many cases there are multiple shared SNPs between standard and inverted chromosomes, suggesting an unexpected high degree of inversion recurrence during human evolution. iPCR was also used to check 16 of these inversions in four chimpanzees and two gorillas, and 10 showed both orientations either within or between species, providing additional support for their multiple origin. Finally, we have identified several inversions that include genes in the inverted or breakpoint regions, and at least one disrupts a potential coding gene. Thus, these results represent a significant advance in our understanding of inversion polymorphism in human populations and challenge the common view of a single origin of inversions, with important implications for inversion analysis in SNP-based studies.

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Section: Introductionmentioning

confidence: 99%

Validation and Genotyping of Multiple Human Polymorphic Inversions Mediated by Inverted Repeats Reveals a High Degree of Recurrence

et al. 2014

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“…The sequence of the abnormal 5‐kb fragment contained a breakpoint located at nt 154 241 636 in F8 intron 1. The adjacent telomere side sequence was located at nt 154 625 305 between the palindromic regions of int22h ‐2 and ‐3 in centromere direction in the Inv22 type II X chromosome; however, it was in telomere direction in the WT F8 variant X chromosome, int22h ‐132 (according to the Xq‐>Xtel orientation of int22h ‐1, ‐2, and ‐3 sequences) [16]. There were no bases of microhomology at the junction (Fig.…”

Section: Resultsmentioning

confidence: 99%

A possible mechanism for Inv22‐related F8 large deletions in severe hemophilia A patients with high responding factor VIII inhibitors

Fujita

Miyawaki

Suzuki

et al. 2012

Journal of Thrombosis and Haemostasis

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Summary. Background: Intron 22 inversion (Inv22) of the coagulation factor (F)VIII gene (F8) is a frequent cause of severe hemophilia A. In addition to Inv22, a variety of F8 mutations (1492 unique mutations) causing hemophilia A have been reported, of which 171 involve deletions of over 50 bp (HAMSTeRs database; http://hadb.org.uk/). However, only 10% of these large deletions have been fully characterized at the nucleotide level. Patients and methods: We investigated gene abnormalities in three unrelated severe hemophilia A patients with high titer FVIII inhibitors. They had previously been shown to carry large deletions of the F8, but the precise gene abnormalities remain to be elucidated. Results: Inverse shifting-PCR (IS-PCR) Inv22 diagnostic tests revealed that these patients carried either type I or II Inv22. However, they showed a wild-type (WT) pattern in the IS-PCR Inv22 complementary tests. We further analyzed their X chromosomes to account for the puzzling results, and found that they had different centromeric breakpoints in the Inv22 X chromosomes, adjacent to the palindromic regions containing int22h-2 or -3, and their spacer region, respectively. The connections appeared to be shifted towards the telomere of the WT F8 Xq28, resulting in a new telomere with an additional intact int22h copy. Conclusions: These gene rearrangements might result from double-strand breaks in the most distal regions of the long arms of the Inv22 X chromosomes, followed by DNA restorations using the WT F8 Xq28 by non-homologous end joining or break-induced replication; thus leading to large F8 deletions in severe hemophilia A patients.

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“…The participants were 40 men with severe hemophilia A caused by Inv22, classified by inhibitor status in subjects positive for inhibitors (n = 20), including index cases with high response (+HR, > 5 UB/mL), and low response (+LR, 5 UB/mL), and individuals who were negative (-) for inhibitors (n = 20), including negative cases (< 0.5UB/mL), and transients whose inhibitor titers disappear before a six-month period (Supplemental Table S1). The patients had been genotyped earlier as positive for either the Inv22-1 or Inv22-2 causative mutations by inverse shifting-PCR (Rossetti et al, 2005; Radic et al, 2009; Rossetti et al, 2011). We also included a group of healthy males (n = 20) from the same ethnic background than the patients from the case and control groups (Supplemental Table S2).…”

Section: Methodsmentioning

confidence: 99%

“…One of the most frequent mutations that causes hemophilia A is the inversion of the F8 intron 22 (Inv22) (Ghosh and Shetty, 2009). This mutation occurs in about 45% of cases (Antonarakis et al, 1995; Rossetti et al, 2011). Among the other mutations are deletions, insertions and point mutations that result in stop-codons or amino acid exchanges (Margaglione et al, 2008; Oldenburg et al, 2010; Rallapalli et al, 2013; Rydz et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

A Differentially Methylated CpG Site in theIL4Gene Associated with Anti-FVIII Inhibitor Antibody Development in Hemophilia A

Souza

Ferreira

et al. 2019

Preprint

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40Hemophilia A is the most common clotting disorder in humans. It affects one in five 41 thousand live-born children. Mutations in the X-chromosome linked F8 gene lead to the 42 deficiency of circulating factor VIII (FVIII). The defect is characterized by severe 43 bleeding. The standard therapy is to replace the deficient factor intravenously. The main 44 adverse event of the therapy is the development of anti-FVIII inhibitor antibodies that 45 impair coagulation and result in increased complications and risk of death. Several risk 46 factors have been described for the development of inhibitor antibodies, among them 47 age, type of FVIII administered, ethnicity, and variant alleles in immune response 48 genes. Epigenetic risks factors have not yet been explored. This work aimed to evaluate 49 2 the methylation statuses at thirteen CpG sites (5meCpG) in regulatory regions of the 50 IL1B, IL2, IL4, IL6, IL10, TNF, IFNG, CTLA4, CD28, and CST7 immune regulation 51 genes in hemophilia A affected males on replacement therapy who develop or do not 52 develop inhibitor antibodies. At each of the thirteen specific CpG sites, we observed one 53 of three possible statuses: hypermethylated, hypomethylated or intermediate 54 methylated. We found a statistically significant (p = 0.04) decrease in the methylation 55 level at one CpG site in the IL4 intron 1 (CpG-3) in the affected group of patients 56 presenting with anti-FVIII inhibitors as compared with the group of patients without 57 inhibitors. The differential 5meCpG-3 maps within a predicted enhancer region in IL4 58 intron 1 that overlaps DNase I hypersensitive chromatin region of the Th2 IL5, IL13, 59 and IL4 cytokine gene cluster and, therefore, permissive for gene expression. Six-bp 60 upstream of the differentially 5meCpG-3 is the rs2227282 cis expression quantitative 61 trait locus that influences the transcript levels of the PDLIM4, SLC22A4, SLC22A5, 62 RAD50, IL4, KIF3A, SEPT8 genes. We consider the IL4 (CpG-3) site a promising lead 63 epigenetic mark, the potential value of which must be appraised in a larger group of 64 patients. The methodology employed also allowed to evaluate the distribution of the IL6 65 rs35081782 insertion/deletion variant, associated with white blood cell count traits in 66 genome-wide association studies, and which showed no difference in distribution 67 between the groups of patients. 68 69 90 91Risk factors for inhibitor development are classified into non-genetic and genetic. 92Among non-genetic risks, the age at the replacement therapy, the type of hemorrhage 93 and the factor administered have been described (Ragni et al., 2009). Among the genetic 94 risk factors, the most well-described factor is the type of causative mutation. Mutations 95 that associate with a higher risk of developing inhibitors are linked to significant 96 functional alterations or complete absence of FVIII. About 40% of patients with large 97 deletions develop inhibitors, while nonsense mutations account for up to 30% of 98

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Eighteen Years of Molecular Genotyping the Hemophilia Inversion Hotspot: From Southern Blot to Inverse Shifting-PCR

Cited by 23 publications

References 47 publications

Validation and Genotyping of Multiple Human Polymorphic Inversions Mediated by Inverted Repeats Reveals a High Degree of Recurrence

Validation and Genotyping of Multiple Human Polymorphic Inversions Mediated by Inverted Repeats Reveals a High Degree of Recurrence

A possible mechanism for Inv22‐related F8 large deletions in severe hemophilia A patients with high responding factor VIII inhibitors

A Differentially Methylated CpG Site in theIL4Gene Associated with Anti-FVIII Inhibitor Antibody Development in Hemophilia A

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