Liliana C. Rossetti

Developing a new generation of tests for genotyping hemophilia‐causative rearrangements involving int22h and int1h hotspots in the factor VIII gene

Summary.u2002 Background:u2002Inversions of F8‐intron 22 (Inv22) and F8‐intron 1 (Inv1) are responsible for 45–50% of severe hemophilia A cases. Objective:u2002In order to improve the molecular diagnosis of Inv22 and Inv1, and to enable rapid discrimination of Inv22‐type 1 and Inv22‐type 2 patterns, int22h‐mediated deletions (Del22) and duplications (Dup22), we developed a genotyping system based on a novel inverse shifting‐polymerase chain reaction (IS‐PCR) approach. Methods:u2002IS‐PCR involved BclI restriction, followed by self‐ligation to create ‘BclI circles’, and finally PCR analysis. Three PCR analysis tests were developed: (i) Inv22‐diagnostic for a pattern‐sensitive detection of deleterious mutations (Inv22 and Del22) from non‐deleterious variants (Dup22 and normal); (ii) Inv1‐diagnostic; and (iii) Inv22‐complementary for discrimination between Inv22 and Del22, and between Dup22 and normal. For rapid molecular analysis of F8, the Inv22 and Inv1 diagnostic tests can be performed simultaneously. The optional Inv22‐complementary test need only be used for specific purposes. Results and Conclusions:u2002Diagnostic tests were validated using previously studied samples. IS‐PCR evaluated carrier mosaicisms and performed robustly over wide ranges of DNA qualities and procedural conditions. IS‐PCR improved the molecular diagnosis of hemophilia A. This genotyping strategy may potentially be adapted to virtually all known rearrangements in the human genome.

Analysis of factor VIII gene intron 1 inversion in Argentinian families with severe haemophilia A and a review of the literature.

Besides intron 22 factor VIII gene inversion (Inv22), intron 1 inversion (Inv1) has recently been reported as a further recurrent mutation that causes approximately 5% of severe haemophilia A (HA) cases. We analysed the presence of the Inv1 in a group of 64 severe HA-affected families from Argentina, and found only one positive case. This Inv1 patient has not developed a factor VIII inhibitor, and the screening for small mutations in the coding sequences of the factor VIII gene did not detect any additional defect in this case. The Inv1 genotyping was further applied to analyse the haemophilia carrier status of the probands sister. In addition, we studied the accuracy of the current polymerase chain reaction-based method to investigate the Inv1, and confirmed the absence of amplimer length polymorphisms associated to the Inv1-specific polymerase chain reaction amplifications in 101 X-chromosome haplotypes from unrelated Argentinian healthy males. In order to discuss Inv1 mutation frequency in severe HA and the risk of inhibitor formation, a review of the literature was included. Our data highlight the importance of analysis of the Inv1 in Inv22-negative severe HA cases. This will benefit both genetic counselling and the study of the relationship between genotype and inhibitor development.

Assessment of the F9 genotype-specific FIX inhibitor risks and characterisation of 10 novel severe F9 defects in the first molecular series of Argentinian patients with haemophilia B

In haemophilia B (HB) (factor IX [FIX] deficiency), F9 genotype largely determines clinical phenotype. Aimed to characterise Argentinian families with HB, this study presents F9 genotype frequencies and their specific FIX inhibitor risk and 10 novel F9 mutations. Ninety-one DNA samples from HB patients and relatives were subjected to a new scheme: a primary screen for large deletions, a secondary screen for point mutations using conformation sensitive gel electrophoresis, DNA-sequencing and bioinformatic analysis. Our unbiased HB population (N=52) (77% with severe, 11.5% moderate and 11.5% mild HB) showed 32 missense (61.5%), including three novel mutations predicting specific structural/functional defects in silico , seven nonsense (13.5%) (one novel), five large deletions, four splice including three novel mutations affecting predicted splicing scores, three indels (two novel) and one Leiden mutation. Our comprehensive HB population included five patients with long-lasting FIX inhibitors: three nonsense (p.E35* (novel), p.R75*, p.W240*) and two entire- F9 deletions. Another patient with an indel (p.A26Rfs*14) developed transient inhibitors. A case-control analysis, based on our global prevalence of 3.05% for developing inhibitors in HB revealed that missense mutations were associated with a low risk odds ratio (OR) of 0.05 and a prevalence of 0.39%, whereas nonsense and entire- F9 deletions had significantly higher risks (OR 11.0 and 32.7) and prevalence (14.3% and 44.5%, respectively). Our cost-effective practical approach enabled identification of the causative mutation in all 55 Argentine families with HB, analysis of the molecular pathology of novel F9 defects and determination of mutation-associated FIX inhibitor risks.

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

The factor VIII gene (F8) intron 22 inversion (Inv22) is a paradigmatic duplicon-mediated rearrangement, found in about one half of patients with severe hemophilia A worldwide. The identification of this prevalent cause of hemophilia was delayed for nine years after the F8 characterization in 1984. The aim of this review is to present the wide diversity of practical approaches that have been developed for genotyping the Inv22 (and related int22h rearrangements) since discovery in 1993. The sequence— Southern blot, long distance-PCR and inverse shifting-PCR—for Inv22 genotyping is an interesting example of scientific ingenuity and evolution in order to resolve challenging molecular diagnostic problems.

Novel mutational mechanism in the thyroglobulin gene: Imperfect DNA inversion as a cause for hereditary hypothyroidism

The objective of this study was to perform genetic analysis in three brothers of Turkish origin born from consanguineus parents and affected by congenital hypothyroidism, goiter and low levels of serum TG. The combination of sequencing of DNA, PCR mapping, quantitative real-time PCR, inverse-PCR (I-PCR), multiplex PCR and bioinformatics analysis were used in order to detect TG mutations. We demonstrated that the three affected siblings are homozygous for a DNA inversion of 16,962bp in the TG gene associated with two deleted regions at both sides of the inversion limits. The inversion region includes the first 9bp of exon 48, 1015bp of intron 47, 191bp of exon 47, 1523bp of intron 46, 135bp of exon 46 and the last 14,089bp of intron 45. The proximal deletion corresponds to 27bp of TG intron 45, while the distal deletion spans the last 230bp of TG exon 48 and the first 588bp of intergenic region downstream TG end. The parents were heterozygous carriers of the complex rearrangement. In conclusion, a novel large imperfect DNA inversion within the TG gene was identified by the strategy of I-PCR. This aberration was not detectable by normal sequencing of the exons and exon/intron boundaries. Remarkably, the finding represents the first description of a TG deficiency disease caused by a DNA inversion.

Factor VIII genotype characterization of haemophilia A affected patients with transient and permanent inhibitors: a comprehensive Argentine study of inhibitor risks.

Inhibitor development against exogenous factor VIII is a severe impairment of replacement therapy affecting 18% of Argentine patients with severe haemophilia A (HA). To study the molecular predisposition for inhibitor development, we genotyped 260 HA patients with and without inhibitors, countrywide. The inhibitor‐positive population (19 transients, 15 low responders, LR and 70 high responders, HR) of 104 severe‐HA patients showed 59 Inv22 (intron 22 inversions), 18 small ins/del‐frameshifts, 12 gross deletions, 12 nonsense, one splicing defect and two missense, p.Arg531Pro and p.Leu575Pro, both LR and thought to impair FVIII A2 domain secondary structure. In addition, a patient with mild HA and HR showed the missense p.Glu1704Lys associated with two neutral intronic substitutions potentially affecting the A3 domain. A case/control study (84/143) permitted estimation of F8 genotype–specific inhibitor risks [OR; prevalence (CI)] in severe‐HA patients classifying a high‐risk group including multi‐exon deletions [3.66; 55% (19–100)], Inv22 [1.8; 24% (19–100)] and nonsense in FVIII‐LCh [1.2; 21% (7–59)]; an average risk group including single‐exon deletions, indel frameshifts and nonsense‐HCh; and a low‐risk group represented by missense defects [0.14; 3% (0.6–11)]. Analysis of inhibitor concordance/discordance in related patients indicated additional genetic factors other than F8 genotype for inhibitor formation. No significant inhibitor‐predisposing factors related to FVIII product exposure were found in age‐ and F8 genotype–stratified populations of severe‐HA patients. In conclusion, the Argentine HA patient series presents similar global and mutation‐specific inhibitor risks than the HA database and other published series. This case‐specific information will help in designing fitted therapies and follow‐up protocols in Argentina.

Phenotype-genotype correlations in hemophilia A carriers are consistent with the binary role of the phase between F8 and X-chromosome inactivation.

The recessive X‐linked disorder hemophilia A (HA) is rarely expressed in female carriers, most of whom express about half of normal factor VIII activity (FVIII:C).

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

Inverse shifting PCR based prenatal diagnosis of hemophilia-causative inversions involving int22h and int1h hotspots from chorionic villus samples

., 2008). The use of this method will facilitate anal-ysis of both inversions, although side-by-side analysisof Inv22 and Inv1 from the fetus sample is restricted toexceptional circumstances (outlined below).The aim of this report is to analyze the ability ofthe method of IS-PCR for PND on DNA extractedfrom chorionic villus (CV) samples. Because humangenome sequences are openly available in the web,specific IS-PCR approaches can be easily designed forgenotyping virtually any full-defined structural variants,thus providing a powerful tool to investigate otherdisease causative rearrangements from CV samples.In 2002, a pregnant carrier of HA, mother of a severeHA patient, whose Inv22 mutation had been identifiedin our laboratory, came to our attention for PND. Duringher twelfth week of gestation, a CV sample was obtainedand gender analyzed by karyotyping, indicating that thefetus was a female. The direct mutation identificationwas then attempted by Southern blotting (Lakich

Informativeness of a novel multiallelic marker‐set comprising an F8 intron 21 and three tightly linked loci for haemophilia A carriership analysis

Summary.u2002 The extraordinary heterogeneous nature of the deleterious mutations in the F8 gene that lead to functional deficiency of clotting factor VIII in haemophilia A makes routine direct mutation profiling difficult. When direct mutation analysis cannot be performed or a causative/candidate mutation is not found, a second‐line approach to track the defective F8 gene within at‐risk families is linkage genetic analysis with, tried‐and‐tested, F8‐intragenic and/or extragenic non‐recombining multiallelic short tandem repeats (STR). Although several typing STR loci within and around F8 have been described, there is need for improving assessment, because the combined informativeness of available assays rarely reaches 100%. Here, we characterized a newly identified 0.28u2003cM‐resolution marker‐set, consisting of a dinucleotide STR located on F8 intron 21 (F8Int21; [AC]n) and three extragenic tetranucleotide STR located on GAB3 intron 1 (GAB3Int1; [TAAA]n) and TMLHE intron 1 (TMLHEInt1.1; [GAAA]n and TMLHEInt1.3; [ATTC]n). Heterozygosity rates determined in 100 unrelated females ranged from 0.25 (GAB3Int1) to 0.63 (F8Int21). The set rendered a combined informativeness of 0.91 for at least one marker and 0.60 for a minimum of two loci, with at least one F8‐intragenic. Multiallelic interlocus non‐random association analysis revealed that GAB3Int1 is not in significant gametic disequilibrium (GD) with F8Int21, F8Int9.2, TMLHEInt1.3 or TMLHEInt1.1. Gametic disequilibrium breakdown attests historical recombination between GAB3Int1 and the F8 gene. Through computational analysis of reference assembly sequence data, we note in the GD breakdown region and in the F8 gene a higher than average density of the 13‐mer CCNCCNTNNCCNC consensus motif, commonly associated with recombination hotspots.