Abstract

Abstract 1127

Congenital defects of platelets or plasma proteins involved in haemostasis generally lead to bleeding disorders. Von Willebrand disease (VWD) and hemophilia A and B are the most common while defects in other plasma coagulation proteins and platelet factors are relatively rare with an incidence of <1: 1–2 million. Despite significant advancements in the world, state of the art care is inaccessible to a vast majority of patients in developing countries. Genetic diagnosis aimed at offering carrier detection and prenatal diagnoses thus remains an important part of the management of these patients. From the year 2000, we have systematically established cost-effective genetic diagnoses services for not only hemophilia A and B but for other rare bleeding disorders [Factor (F) 1, F2, F5F8, F7, F10, F11 and F13] and platelet disorders [Glanzmann Thrombasthenia, Bernard Soulier Syndrome, Wiskott Aldrich Syndrome] as well. Our approach for molecular diagnosis includes screening the target genes for common mutations first, followed by point mutation screening by PCR and conformation sensitive gel electrophoresis and DNA sequencing, as needed, after that. For large genes such as F8 (186kb), ITG2B and ITG3A (65kb), we employ multiplex amplification of the coding regions to simplify the mutation detection protocols. Alternately, linkage analysis has also been established for some diseases such as hemophilia A, hemophilia B and Glanzmann Thrombasthenia to assist in those families where no mutation is found. Using these validated protocols, we have compiled genetic mutations in 1246 probands from 803 families with various bleeding disorders (table 1). These does not include patients with VWD for which we have only now initiated genetic evaluation. Mutations were identified in 762 families (95%). Of these, 358 were unique mutations, of which 103 (29%) are novel. We were not able to characterize disease causing mutation in a proportion (5 %, n=41/803) of families which is similar to the published literature. The frequency and distribution of the various mutations (Table 1) for all the disorders is similar to the spectrum reported in the human genome mutation database. Based on the availability of this molecular data, we have successfully offered carrier diagnosis to 334 probands from 271 unrelated families while prenatal diagnosis was carried out on 124 probands from 113 unrelated families. The development of efficient diagnostic algorithms for each of the bleeding disorder has helped to limit the turnaround time for genetic diagnoses to an average of 3–7 days at a cost of about $100-150 per sample for reagents and disposables. In conclusion, this report is the largest single-centre molecular data from a cohort of patients with bleeding disorders. The availability of such vast amount of genetic information and robust techniques for molecular diagnosis for different disorders of hemostasis contributes significantly to the effective management of these patients with bleeding disorders in India.

Table 1.
Bleeding disorderTotal samples analyzedUnique families analyzedTotal reported mutationsNovel mutationsIntron22 inversionIntron1 inversionMissenseNonsenseFrame shiftSplice siteMajor deletionsNo mutations identified
Factor VIII deficiency 856 479 99 53 47% (n=227) 3% (n=13%) 16% (n=76) 10% (n=46) 12% (n=57) 3% (n=12) 2% (n=10) 6% (n=27) 
Factor IX deficiency 153 120 47 31   66% (n=79) 7% (n=8) 8% (n=9) 3% (n=3) 10% (n=12) 2% (n=2) 
Glanzmann Thrombasthenia 60 58 32   57 (n=34) 14% (n=8) 17% (n=10) 5% (n=3) 6.9% (n=4) 
Wiskott Aldrich Syndrome 48 29 12   10% (n=3) 24% (n=7) 24% (n=7) 14% (n=4) 3 % (n=1) 31% (n=9) 
Bernard-Soulier syndrome 28 27 11   56% (n=15) 4% (n=1) 48% (13) 
Fibrinogen deficiency 28 26 13   15% (n=4) 31% (n=8) 58% (n=15) 
Factor VII deficiency 23 14 11   50% (n=7) 29% (n=4) 14% (n=2) 14% (n=2) 
Factor XI deficiency 12 12   100% (n=12) 17% (n=2) 
Factor XIII deficiency 11   36% (n=4) 9% (n=1) 18% (n=2) 36% (n=4) 
Combined Factor V+VIII deficiency   11% (n=1) 56% (n=5) 33% (n=3) 
Factor II deficiency   88% (n=7) 25% (n=2) 
Factor × deficiency   100% (n=7) 14 (n=1) 
Factor V deficiency   40% (n=2) 60% (n=3) 
Total 1246 803 255 103   251 75 100 57 23 41 
Bleeding disorderTotal samples analyzedUnique families analyzedTotal reported mutationsNovel mutationsIntron22 inversionIntron1 inversionMissenseNonsenseFrame shiftSplice siteMajor deletionsNo mutations identified
Factor VIII deficiency 856 479 99 53 47% (n=227) 3% (n=13%) 16% (n=76) 10% (n=46) 12% (n=57) 3% (n=12) 2% (n=10) 6% (n=27) 
Factor IX deficiency 153 120 47 31   66% (n=79) 7% (n=8) 8% (n=9) 3% (n=3) 10% (n=12) 2% (n=2) 
Glanzmann Thrombasthenia 60 58 32   57 (n=34) 14% (n=8) 17% (n=10) 5% (n=3) 6.9% (n=4) 
Wiskott Aldrich Syndrome 48 29 12   10% (n=3) 24% (n=7) 24% (n=7) 14% (n=4) 3 % (n=1) 31% (n=9) 
Bernard-Soulier syndrome 28 27 11   56% (n=15) 4% (n=1) 48% (13) 
Fibrinogen deficiency 28 26 13   15% (n=4) 31% (n=8) 58% (n=15) 
Factor VII deficiency 23 14 11   50% (n=7) 29% (n=4) 14% (n=2) 14% (n=2) 
Factor XI deficiency 12 12   100% (n=12) 17% (n=2) 
Factor XIII deficiency 11   36% (n=4) 9% (n=1) 18% (n=2) 36% (n=4) 
Combined Factor V+VIII deficiency   11% (n=1) 56% (n=5) 33% (n=3) 
Factor II deficiency   88% (n=7) 25% (n=2) 
Factor × deficiency   100% (n=7) 14 (n=1) 
Factor V deficiency   40% (n=2) 60% (n=3) 
Total 1246 803 255 103   251 75 100 57 23 41 

Disclosures:

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.