A nine-year-old male child born from a non-consanguineous marriage, presented with excessive bleeding from upper gum after minor trauma and repeated major history of knee swellings after trauma (Figure 1). Physical examination revealed ecchymotic patches. He had history of easy bruisability since 6 months of age and spontaneous as well as post traumatic bleeding since then. There was no history of jaundice and other hematological illness in the family. He had been transfused thrice with fresh frozen plasma.

Coagulation screening

The initial coagulation screening in the laboratory showed prolonged activated partial thromboplastin time (aPTT) of 78.6 sec (reference value 25-32 sec), normal prothrombin time (PT), and fibrinogen levels. In the diagnostic work-up, on mixing with normal pooled plasma (1:1) the aPTT was corrected to 35 seconds. The mixing study was suggestive of a factor deficiency. Factor VIII assay was found to be <1% and confirmed factor VIII deficiency. Von Willebrand antigen levels were normal.

Molecular Studies

Peripheral blood of the patients and family members are collected in EDTA vial for genomic DNA extraction. and was extracted by phenol chloroform method. As patient was factor VIII deficient he was screened for the most common Intron 22 inversion (Inv22) using inverse shift PCR (IS-PCR) method as describes by Rossetii et al. Amplified products were resolved in 2% agarose in 0.5 X TBE buffer and visualized under UV. He was hemizygous for the inversion 22 and his mother was found to be carrier for the same variant (Figure 2). However, there was no history of HA in mother’s family. This family was offered prenatal diagnosis in the next pregnancy and the fetus was found to be affected (Figure 2).

Learning Points

Ø  Hemophilia-A (HA) or classic hemophilia is an X-linked recessive disorder and is caused by the deficiency of factor VIII.

Ø  Females with Hemophilia A gene (carrier) will transmit the gene to 50% of their male offsprings and may have lower Factor VIII but may not manifest symptoms.

Ø  Male Hemophilia A patients do not transmit hemophilia to their sons, but all their daughters will be obligate carriers.

Ø  Diagnosis of HA requires clinical criteria, coagulation tests and molecular confirmation to establish the severity of the disease as well as provide valuable information to the clinicians in treatment, counseling for antenatal and carrier screening in female relatives.

Ø  Intron 22 and 1 inversion (Inv22 and Inv1) are the most frequent molecular alterations found in severe HA patients with a frequency of 45-50% and 0.5- 5%, respectively.

Ø  Before doing further molecular characterization like conformation sensitive gel electrophoresis(CSGE), Multiple Ligation Probe Amplification (MLPA) and Next Generation Sequencing (NGS) it is required to rule out these intrachromosomal recombination events which results in Inv22 and Inv1.