C. D. De Brasi

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

Summary.  Background: Inversions of F8‐intron 22 (Inv22) and F8‐intron 1 (Inv1) are responsible for 45–50% of severe hemophilia A cases. Objective: In 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: IS‐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: Diagnostic 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.

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.

Genetic testing in bleeding disorders

The aim of molecular genetic analysis in families with haemophilia is to identify the causative mutation in an affected male as this provides valuable information for the patient and his relatives. For the patient, mutation identification may highlight inhibitor development risk or discrepancy between different factor VIII assays. For female relatives, knowledge of the familial mutation can facilitate carrier status determination and prenatal diagnosis. Recent advances in understanding mutations responsible for haemophilia and methods for their detection are presented. For reporting of such mutations, participation in external quality assessment ensures that essential patient and mutation details are routinely included and that pertinent information is incorporated in the interpretation.

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).

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

Summary.  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 0.28 cM‐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.

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

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

Reliable and cost-effective approach for diagnosis of heterozygous F8/F9 large deletions by quantitative real-time PCR.

Fil: Abelleyro, Miguel Martin. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina

Identification of driver and subclonal mutations in ASXL1 and IDH1/IDH2 genes in an Argentine series of patients with myelofibrosis

Myelofibrosis (MF) is a Philadelphianegative myeloproliferative neoplasm (MPN) characterized by clonal proliferation of hematopoietic stem cells, progressive bone marrow fibrosis, abnormal cytokine expression, anemia, splenomegaly, extramedullary hematopoiesis, constitutional symptoms, cachexia, leukemic progression, and shortened survival.1 MF can be diagnosed as a primary myelofibrosis (PMF) disorder or as a complication of the evolution of polycythemia vera (MF postPV) or essential thrombocythemia (MF postET). JAK2, CALR, and MPL are considered as driver mutations being observed in 90% of patients with PMF. The JAK2V617F mutation is the most common with a frequency of ~65%,1 and its allele burden may be increased by a mitotic recombination phenomenon resulting in the transition from heterozygosity to homozygosity. Lower levels of allele burden were associated with an inferior survival in PMF.1 Substitutions in MPL, mostly affecting codon W515K/L, have been described in ~10% of PMF.1 CALR1,2 is mainly affected by insertions or deletions (indels) within exon 9 modifying the carboxylterminus, giving rise to 2 main variants: type 1, a 52bp deletion (L367fs*46), or type 2, a 5bp TTGTC insertion (K385fs*47), which are observed in ~25% of PMF. The remaining 10% of PMF with no driver mutations are considered as “triple negative” (TN) associated with an inferior survival.3 Recent studies have also identified subclonal mutations affecting genes that encode for epigenetic regulators and members of the splicing machinery. Searching these mutations may help not only in determining the clonal nature of the disease but also in identifying patients with a higher risk. Particularly, those mutations affecting the carboxylterminus planthomeodomain finger of ASXL1 have been lately associated with poor prognosis.4 Herein, we have searched for driver (JAK2V617F, MPL, and CALR) and subclonal (ASXL1, IDH1, and IDH2) mutations and described their clinical impact on a series of 39 Argentine patients with MF (28 PMF, 4 MF postPV, and 7 MF postET, according to the WHO criteria). This study was approved by the Institutional Ethical Committee, and written informed consents were obtained from all patients. In our series of PMF, driver mutations were present with a frequency of 57% (16/28) for JAK2V617F, 7% (2/28) for MPL W515K/L, and 14% (4/28) for CALR [3 cases type 1 (11%)/1 case type 2 (4%)], and 21% (6/28) were TN. The obtained data were in agreement with a recent series of Korean patients with PMF,5 with a higher frequency of TN than other published series.1 Those TN patients showed a lower Hb level (KruskalWallis test, P = .045) and shorter overall survival (KaplanMeier and logrank test, P = .009) (Table 1) in coincidence with clinical characteristics already described in TN patients3 The median JAK2V617F allele burdens, measured by an original approach developed by our group,6 were 67.8% for PMF, 97.0% for MF postPV and 51.7% for MF postET. The higher allele burdens, detected in MF postPV, confirm previous data,1 and the small size of our series may be responsible for the lack of statistical significance. (KruskalWallis test, P = .2586). ASXL1 was analyzed by a classical Sanger sequencing approach splitting exon 13 into 2 overlapping fragments. We could identify 5 risk mutations in heterozygous state, including 3 unpublished, and 4 rare missense variants with unknown clinical significance (UCS). Considering both deleterious and UCS variant, we observed a frequency of 28% (11/39) of ASXL1 mutations in our series (Table 2), being within the reported range for MPN (20%35%).4 Among the detected 5 risk mutations in ASXL1, 3 mutations were previously described: the recurrent 22bp deletion c.1900_1922del (p.E635Rfs*15) that was observed in a patient who developed acute leukemia after 2 years from diagnosis (case 1, Table 2), a missense variant c.3306G>T (p.E1102D) reported in the COSMIC database (http://cancer.sanger.ac.uk/cosmic) with a minor allele frequency TABLE 1 Hematological profile and clinical characteristics of MF patients according to driver mutations