Inverse-shifting PCR Research Articles

Relationship between mutations in severe hemophilia A and risk of inhibitor development: A large single-center study

BackgroundOne of the major problems for patients with severe hemophilia A (HA) is the development of neutralizing antibodies against factor VIII. This study aimed to analyze the molecular and clinical profiles of patients with severe HA and to determine if certain genetic variants predispose to inhibitor development in these patients. MethodsA single-center study was conducted among patients with severe HA between March 20, 2000, and June 31, 2023. Demographic data and laboratory results of patients were collected. The inverse-shifting PCR (IS-PCR) technique was initially used to screen patients for intron 22 and 1 inversions (Inv-22 and Inv-1). ResultsA total of 480 patients with severe HA (408 without inhibitors and 72 with inhibitors) were enrolled in this study. The median age of the patients at the time of diagnosis was 6 months (IQR: 3 months to 18 months). Inv-22 was observed in 199 (41.5 %) of the cases. Among those patients who developed inhibitors, 53 (73.6 %) were classified as high-titer and 19 (26.4 %) as low-titer. Inv-22, positive family history of inhibitor formation, and history of intense injections revealed a statistically significant association with the risk of inhibitor development. ConclusionThe results of this study confirm the important role of different genetic variants, family history of inhibitor formation, and history of intense injections for the formation of inhibitors in patients with severe HA. This would allow us to stratify the patients which can have important clinical implications, especially in terms of their management and outcome.

Genomic alterations in the F8 gene correlating with severe hemophilia A in Egyptian patients.

BackgroundHemophilia A (HA) is an inherited X‐linked recessive coagulation disorder caused by factor VIII (F8) deficiency. F8 rearrangements involving intron 22 (int22) and intron 1 (int1) account for almost half of severe HA phenotype also a hotspot exon 14 provides numerous mutational patterns. This study aims to identify F8 gene mutations among Egyptian HA patients.MethodsDNA samples from 60 HA patients were screened for int22 and int1 rearrangements using simplified inverse shifting PCR (IS‐PCR) followed by exon 14 sequencing. Also, four uncharacterized patients were studied by targeted exome sequencing.ResultsIn 33.3% of the studied patients, we identified three int22 rearrangements, three exon 14 mutations (two frameshift; one novel (NM_000132.3:c.2734_2735delAA, p.(N912Ffs*6)), a second reported mutation (NM_000132.3:c.3091_3094delAGAA, p.(K1031Lfs*9)), and one nonsense mutation (NM_000132.3:c.2440C>T, p.(R814*)). All identified mutations were detected in patients with severe HA phenotype. Targeted exome sequencing could not detect any known pathogenic variants.ConclusionIntron 22 rearrangement and exon 14 mutations correlate with most severe hemophilia A Egyptian patients.

Factor VIII mutation spectrum in haemophilia A patients in the population of Henan, China.

: Defects in the coagulation factor VIII gene cause haemophilia A, which is the most common X-linked recessive bleeding disorder. In total, 45 affected families were investigated to elucidate the factor VIII gene mutation spectrum. The families were subjected to clinical, biochemical, and molecular analyses. Inverse-shifting PCR was first applied to severe haemophilia A patients to identify inversions in introns 22 and 1. Then, next-generation sequencing was performed to detect mutations in inversion-negative patients with severe haemophilia A and moderate-mild haemophilia A patients. Finally, multiplex ligation-dependent probe amplification was utilized to identify rare cases with large fragment duplications or deletions in the factor VIII gene. In total, 41 mutations were identified, 19 of which (c.24C>A, c.49T>C, c.170_171delTT, c.533T>C, c.1126delG, c.1495delA, c.1660A>C, c.1736A>G, c.2711-2712insAATCT, c.3077C>G, c.3846delA, c.4238C>G, c.4349delG, c.4828G>C, c.5821A>C, c.6190C>T, c.6656T>C, c.6902T>G, and c.1904-2A>T) were novel and 80% (44/55) of the pathogenic mutations fell into the categories of missense (43.6%), nonsense (16.36%), frameshift (14.55%), and splice (5.45%) mutations. Additionally, 10 (18.18%) patients displayed inversions in intron 22 or 1 and one case (1.82%) exhibited a 3059-bp large fragment deletion in factor VIII. This study aimed to provide insight contributing to the genetic diagnosis of haemophilia A and to fill gaps in the factor VIII mutation spectrum in northern China.

An Accurate, Simple, and Inexpensive Assay to Diagnose F8 Gene Inversion Mutations in Hemophilia a Patients and Carriers

The most frequent mutations resulting in hemophilia A are an intron 22 or intron 1 F8 gene inversion, which together result in ~50% of severe hemophilia A cases. We report simple and accurate RNA- and DNA-based assays to detect these mutations in patients and carrier mothers. They do not require specialized equipment or expensive reagents, and therefore they may provide useful and economic protocols that could be standardized for central laboratory testing.RNA is purified from a whole-blood RNA-stabilized sample or from peripheral blood mononuclear cells, and a two-step nested PCR (RT-PCR followed by PCR) reaction is carried out to amplify complementary DNA (cDNA) fragments with F8 sequences spanning the exon 22-23 splice site (intron-22 inversion test) or the exon 1-2 splice site (intron-1 inversion test). These sequences will be amplified from F8 mRNA without an intron-22 or intron-1 inversion mutation, respectively. Additional RT-PCR reactions are carried out to amplify a unique intron 22 inverted-gene mRNA sequence extending from F8 exon 19 to the first in-frame stop codon within intron 22, or a chimeric intron 1 inverted-gene transcript containing sequences from F8 exon 1 and the adjacent VBP1 gene. These latter two DNA products will be amplified only from F8 mRNA isolated from patients with an intron-22 or an intron-1 inversion mutation, respectively. Amplification of a FVIII C2 domain sequence serves as an internal control for the RT-PCR reactions, as this product will be generated from samples with and without an inversion mutation. Primers were designed to amplify PCR products having different lengths (between 200 and 600 bp) that could be clearly distinguished following electrophoresis on agarose gels. Samples from carrier mothers amplified the normal control as well as the inversion-mutation-specific bands, as expected.A nested PCR protocol reliably produced interpretable results from low template mRNA concentrations, while a one-step multiplexed RT-PCR version of the assay produced the expected bands, with fewer experimental steps, from higher template mRNA concentrations.The standard DNA-based inverse-shifting PCR (IS-PCR) reaction may be used for primary diagnosis and/or to classify intron 22 inversions as type 1 or type 2 (reflecting the specific homologous recombination site). We utilized a commercially available Bcl I enzyme that efficiently cleaves DNA in 5 minutes, a substantial improvement over previous IS-PCR protocols that required overnight DNA digests.These rapid, accurate protocols can economically detect or rule F8 inversion mutations via same-day testing of patient samples, providing timely information that is useful to families and in clinical evaluations. DisclosuresDutta:Grifols, Inc: Research Funding. Ragni:OPKO: Research Funding; Vascular Medicine Institute: Research Funding; Tacere Benitec: Consultancy; SPARK: Research Funding; Shire: Consultancy; Novo Nordisk: Research Funding; Genentech: Research Funding; CSL Behring: Research Funding; Biomarin: Consultancy; Biogen: Consultancy, Research Funding; Baxalta: Research Funding; Alnylam Pharmaceuticals: Consultancy, Research Funding.

FVIII intron 22 homologous region mediated recombinations and genotyping methods of hemophilia A

The intron 22 inversion is caused by intrachromosomal homologous recombination of the Int22h (intron 22 homologous region) repeats in opposite orientation, accounting for almost 45%-50% of all cases of severe hemophilia A. By contrast, recombinations between similarly oriented int22h repeats in the same chromosome, homologous chromosomes or chromatids may cause deletion or duplication of the intermediate region between the two int22h copies, resulting in false-positive or false-negative possibility that obscure the characterization of intron 22 inversion. The modified long distance PCR(LD-PCR) and inverse shifting PCR(IS-PCR) would distinguish all the possible int22h-mediated rearrangements.

Identification of intron 1 and intron 22 inversions of factor VIII gene in Serbian patients with hemophilia A

Hemophilia A (HA) is a common X-linked recessive bleeding disease caused by mutations of FVIII gene. Inversion of intron 1 (inv1) and intron 22 (inv22) are recurrent mutations in severe HA, causing 50% of cases. Inv1 has been reported to occur in 2-5% and inv 22 in 45% of severe HA patients. Our objective was to determine, for the first time in Serbia, the frequency of inv1 and inv22 in a group of severe HA patients and to compare these data with those from other countries. Study subjects were 50 HA patients, diagnosed and treated from April 2009 to June 2012 at Mother and Child Health Care Institute of Serbia ?Dr Vukan Cupic? (IHS) and Institute for Child and Youth Health Care of Vojvodina (IHV).The presence of inv1 and inv22 was analyzed using Inverse shifting PCR (IS-PCR). Our results revealed that the frequencies of inv1 and inv22 in the cohort of Serbian patients were 6 % and 42% (34% of inv22 type I and 8% of inv22 type II) respectively . These frequencies were in line with those found in other populations. Carrier status analyses of 65 family members (mothers and sisters) showed the de novo inversion of intron 22 in one patient. Genetic Counseling Units of IHS and IHV provide the adequate genetic advice to all HA affected patients and their family members.

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

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. 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. 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. 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.

Are int22h-mediated deletions a common cause of hemophilia?

Dear Editor, Hemophilia A (HA) (OMIM 306700) is an X-linked inherited bleeding disorder caused by deleterious mutations in the coagulation factor VIII gene (F8). Even though there is a broad diversity of HA-causative mutations, an uncommon type of rearrangement—a large DNA inversion involving F8 intron 22 (Inv22)—accounts for approximately one half of severely affected patients. Inv22 was formerly described by Lakich et al. [1] and Naylor et al. [2]. A collaborative international effort estimated that Inv22 is the cause of 43% (35%, 7%, and 1% for Inv22 type I, type II, and rare types, respectively) of severe HA cases worldwide with minor geographical or ethnical differences [3], in close agreement with our corresponding Argentinean series (42% of Inv22, and 34% and 7% for type I and type II, respectively) [4]. Naylor et al. [5] indicated that Inv22 originates by homologous recombination between well-defined duplicons (int22h) of 9.5 kb located one copy within F8 intron 22 (int22h-1, h1) and the other, inversely oriented, from a group of two extragenic copies (int22h-3, h3 for Inv22 type I and int22h-2, h2 for type II). It was formerly believed that h2 and h3 were equally oriented (i.e., head to tail). However, Ross et al. [6] determined that h2 and h3 are inversely oriented (i.e., head to head), both embedded in the arms of a large imperfect palindrome (Fig. 1). This finding prompted Bagnall et al. [7] to hypothesize recombination between these arms interchanging the location of the extragenic int22h copies and generating a non-deleterious inversion polymorphism in Xq28, i.e., h123 and h132. In this scenario, Inv22 type I may be generated from intrachromosomal recombination between h1 and h3 on the most frequent variant h123 whereas Inv22 type II may be generated between h1 and h2 on the least frequent h132 (Fig. 1). Moreover, on each of these normal structural variants of the X chromosome, recombination between h1 with either equally oriented copies (h2 or h3) may generate deletions (Del22) or duplications (Dup22) but not inversions [7]. More precisely, Del22 type I would be generated by recombination between h1 and h3 on variant h132 whereas Del22 type II by recombination between h1 and h2 on variant h123 [8] (Fig. 1). Notwithstanding these theoretical speculations, until recently, no such Del22 mutation has been unequivocally reported in the literature, and Del22 has been suspected to be extremely deleterious, even to compromise the viability of hemizygous males [8]. Unexpectedly, a recent paper by Abou-Elew et al. [9] detected three cases of Del22 (two Del22 type II and one type I) by the use of inverse-shifting PCR (IS-PCR) [10] in a group of 13 Egyptian patients with severe HA. In this scenario and previous beliefs on the phenotype of int22h-mediated deletions (Del22), the aims of this scientific letter are, first, to report the lack of Del22 in a series of int22h-mediated rearrangements from Argentina and, second, to provide a simple practical approach to Electronic supplementary material The online version of this article (doi:10.1007/s00277-011-1295-z) contains supplementary material, which is available to authorized users. M. M. Abelleyro : L. C. Rossetti : C. P. Radic : I. B. Larripa : C. D. De Brasi (*) Departamento de Genetica, Instituto de Investigaciones Hematologicas, Academia Nacional de Medicina de Buenos Aires, Pacheco de Melo 3081, Buenos Aires 1425, Argentina e-mail: cdebrasi@hematologia.anm.edu.ar

Genotyping of intron 22-related rearrangements of F8 by inverse-shifting PCR in Egyptian hemophilia A patients

Hemophilia A (HA) is the most common severe bleeding disorder in humans, affecting one in 5,000 male births. In severe HA, intron 22 inversion of F8 is the most prevalent mutation, accounting for 40-50% of all mutations; however, little is known about the disease-causing mutations among Egyptian hemophiliacs. We aimed at genotyping all possible known DNA rearrangements of intron 22 of F8 in Egyptian HA patients. Peripheral blood samples were collected from 30 Egyptian HA patients (13 severe, ten moderate, and seven mild cases). Genotyping of F8 intron 22 rearrangements was performed by inverse-shifting PCR (IS-PCR). Our study revealed that seven patients (23.3%) had inversion 22, three patients (10%) had deletion 22, and 20 patients (66.7%) carried the wild-type allele. No intron 22 duplication was detected. The relative proportion of inversion 22-type 1 to inversion 22-type 2 was 85.7% and 14.3%, respectively, whereas the relative proportion of deletion 22-type 1 to deletion 22-type 2 was 33.3% and 66.7%, respectively. A statistically highly significant relation was found between disease severity and F8 intron 22 rearrangements (p = 0.008). Among severe cases, 46.1% had inversion 22, 23.1% had deletion 22, and 30.8% carried the wild-type allele. We conclude that F8 intron 22 inversion/deletion is responsible for about one third of disease-causing mutations among Egyptian hemophiliacs and for nearly 70% in severe cases. In addition, F8 intron 22 inversion/deletion by IS-PCR has proven to be a rapid and robust technique and might be the recommended tool for genetic analysis of HA patients specially with severe cases in developing countries.