WFH2016 Poster Presented at: National study of prevalence of the introns 1 and 22 inversions in F8 gene and its relation to inhibitors development to factor VIII in Mexican severe hemophilia A patients Hilda LunaZáizar 1 , José Ángel González-Alcázar 1 , María Cecilia Aguayo-Ramos 1 , Sandra Luz Ruíz-Quezada 1 , Sara Marisela Ponce-Castellanos 1 , Ana Rebeca Jaloma-Cruz 2 . 1 CUCEI, Universidad de Guadalajara, Guadalajara, Jalisco, México. 2 División de Genética, CIBO, IMSS, Guadalajara, Jalisco, México. E-mail: [email protected] We investigated prevalence of Inv1, and Inv22-1/2, and its association as risk factors for inhibitors development to FVIII, in Mexican severe hemophilia A (HA) patients. Intra-chromosomal/chromatid homologous recombination yielding an inverted palindrome configuration. [1] Results Methods Introduction and objectives Figure 1. Flowchart of Inverse shifting-Polymerase Chain Reaction (IS-PCR). [2] Circles gDNA digested with BclI Self-ligation of fragmented DNA BclI – circles Analysis of IS–PCR products via 1.5% agarose gel electrophoresis Wild allele int22h.1-3 – Inv22-1 int22h.1-2 – Inv22-2 PCR int1h.1-2 – Inv1 Wild allele Inv1 Diagnostic test Inv22 Diagnostic test We studied a cohort of 197 patients from 167 unrelated severe HA families, countrywide, for the detection of Inv1 and Inv22-1/2. All participants signed written consent. Figure 3. Inv22 and Inv1 products by IS-PCR on agarose gel electrophoresis. L1: Inv1 carrier; L2 and L4: non-Inv1 HA; L3: Inv1 HA. L6: Inv22-1 HA; L7: HA Inv22-2; L8–L10: non-Inv22 HA; L11: Inv22-1 HA. M: 100 bp. Inv22-1 39% Inv-22-2 10% No-Inv22 51% 80,2% 19,8% Inv1 2,3% No-Inv1 97,7% Figure 2. Inv22 and Inv1 prevalence in 167 unrelated Mexican families with severe HA. A prevalence of 48.5% for Inv22: 80.2% Inv22-1 and 19.8% Inv22-2, and 2.3% for Inv1 genotype. Inv22-1, 38.9% Inv22-2, 9.6% Non-Inv22, 51.5% Non-Inv1, 97.7% Conclusions Prevalence of Inv22 (48.5%) and Inv1 (1.2%) was similar to other populations. Inv1 was found for the first time in Mexican population in two independent families [3]. Inv22-1/2 or Inv1 positive patients showed lower risk for developing inhibitors with respect to patients without these severe mutations, but differences were not significant. Nevertheless, familial aggregation and inhibitor concordance in related patients indicate a significant genetic component to be explored in Mexican patients with severe HA. References 1. Bagnall et al., J Thromb Haemost 2006;4:591–8. 2. Rossetti et al., J Thromb Haemost 2008;6:830–6. 3. Mantilla et al., Am J Hematol 2007;82:283–7 The common palindrome configuration; above the X chromosome sequence (nucleotide number at relevant positions). [1] Median age of patients was 13 (1 – 58) years. 63.5% of cases were familial and the other 36.5% were sporadic. In two cases, Inv22-1 occurred de novo (non-carrier mother). Related patients shown familial concordance (78%) to develop or not develop inhibitors. Genotype Positive inhibitor Negative inhibitor Total Inv22-1 26 52 78 Inv22-2 1 12 13 Inv1 2 3 5 Inv22-1/2, Inv1 29 (30.2%) 67 (69.8%) 96 Non-Inv22, Non-Inv1 34 (34.0%) 67 (66.0%) 101 Total of patients 63 (32.0%) 134 (68.0%) 197 Figure 4. Inhibitors to FVIII:C according to genotype in Mexican patients with severe HA. Only 29/96 (30.2%) patients with Inv22 or Inv1 genotype developed inhibitors (P = 0.357, Fisher exact test. RR: 0.897 CI 95%: 0.596–1.351). 45--PO-M Jose Angel DOI: 10.3252/pso.eu.WFH2016.2016 Genetics of bleeding disorders
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WFH2016
Post
er
Pre
sen
ted
at:
National study of prevalence of the introns 1 and 22 inversions in F8 gene and its relation to inhibitors development to factor VIII in Mexican severe hemophilia A patients
Hilda LunaZáizar1, José Ángel González-Alcázar1, María Cecilia Aguayo-Ramos1, Sandra Luz Ruíz-Quezada1, Sara Marisela Ponce-Castellanos1, Ana Rebeca Jaloma-Cruz2.1CUCEI, Universidad de Guadalajara, Guadalajara, Jalisco, México. 2División de Genética, CIBO, IMSS, Guadalajara, Jalisco, México. E-mail: [email protected]
We investigated prevalence of Inv1, and Inv22-1/2, and its association as risk factors forinhibitors development to FVIII, in Mexican severe hemophilia A (HA) patients.
Intra-chromosomal/chromatid homologous recombinationyielding an inverted palindrome configuration. [1]
Results
MethodsIntroduction and objectives
Figure 1. Flowchart of Inverse shifting-Polymerase Chain Reaction (IS-PCR). [2]
Circles
gDNA digestedwith BclI
Self-ligation of fragmented DNA BclI – circles
Analysis of IS–PCR products via 1.5% agarose gel electrophoresis
Wild allele int22h.1-3 – Inv22-1int22h.1-2 – Inv22-2
PCR
int1h.1-2 – Inv1Wild allele
Inv1 Diagnostic testInv22 Diagnostic test
We studied a cohort of 197 patients from 167 unrelated severe HA families,countrywide, for the detection of Inv1 and Inv22-1/2. All participants signedwritten consent.
Figure 3. Inv22 and Inv1 products by IS-PCR on agarose gelelectrophoresis. L1: Inv1 carrier; L2 and L4: non-Inv1 HA; L3:Inv1 HA. L6: Inv22-1 HA; L7: HA Inv22-2; L8–L10: non-Inv22HA; L11: Inv22-1 HA. M: 100 bp.
Inv22-1 39%
Inv-22-2 10%No-Inv22
51%
80,2%
19,8%
Inv12,3%
No-Inv197,7%
Figure 2. Inv22 and Inv1 prevalence in 167unrelated Mexican families with severe HA. Aprevalence of 48.5% for Inv22: 80.2% Inv22-1and 19.8% Inv22-2, and 2.3% for Inv1 genotype.
Inv22-1,38.9%
Inv22-2,9.6%
Non-Inv22,51.5%
Non-Inv1,97.7%
Conclusions
Prevalence of Inv22 (48.5%) and Inv1 (1.2%) was similar to other populations. Inv1 was found
for the first time in Mexican population in two independent families [3]. Inv22-1/2 or Inv1
positive patients showed lower risk for developing inhibitors with respect to patients without
these severe mutations, but differences were not significant. Nevertheless, familial
aggregation and inhibitor concordance in related patients indicate a significant genetic
component to be explored in Mexican patients with severe HA.
References1. Bagnall et al., J Thromb Haemost 2006;4:591–8.2. Rossetti et al., J Thromb Haemost 2008;6:830–6.3. Mantilla et al., Am J Hematol 2007;82:283–7
The common palindrome configuration; above the Xchromosome sequence (nucleotide number at relevantpositions). [1]
Median age of patients was 13 (1 – 58) years. 63.5% of cases were familial and the other36.5% were sporadic. In two cases, Inv22-1 occurred de novo (non-carrier mother).Related patients shown familial concordance (78%) to develop or not develop inhibitors.
GenotypePositiveinhibitor
Negativeinhibitor
Total
Inv22-1 26 52 78
Inv22-2 1 12 13
Inv1 2 3 5
Inv22-1/2, Inv1 29 (30.2%) 67 (69.8%) 96
Non-Inv22, Non-Inv1 34 (34.0%) 67 (66.0%) 101
Total of patients 63 (32.0%) 134 (68.0%) 197
Figure 4. Inhibitors to FVIII:Caccording to genotype inMexican patients withsevere HA. Only 29/96(30.2%) patients with Inv22or Inv1 genotype developedinhibitors (P = 0.357, Fisherexact test. RR: 0.897 CI 95%:0.596–1.351).
45--P
O-M
Jose
Ang
elDO
I: 10.3
252/p
so.eu
.WFH
2016
.2016
Gene
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f blee
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