AccuCopy quantification combined with pre-amplification of long-distance PCR for fast analysis of intron 22 inversion in haemophilia A

SummarySevere haemophilia A was diagnosed postpartum in a newborn. The mother was known as a conductor (intron 22 inversion) and an uncle had a persistently high titer inhibitor after failed ITI.Due to a cephalhaematoma, a high-dose pdFVIII substitution was given within the first days after birth. At the age of six month a severe cerebral haemorrhage occurred, making a high-dose pdFVIII substitution and neurosurgical intervention necessary. Several days later a porth-a-cath-system was implanted. The development of a high titer inhibitor occured six days later, an ITI was started according to the Bonn Protocol. Initially rFVIIa was given in addition to the pdFVIII substitution. Seven days after the beginning of treatment the inhibitor was no longer detectable. At monthly intervals the FVIII dosage was reduced until the dosage complied with a prophylaxis in severe haemophilia A.The duration of the ITI was nine months. A total of 30 mg rFVIIa and 276 000 IU pdFVIII were used; costs in total: 280 173.60 Euro.

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Long-Distance PCR-based Screening for Large Rearrangements of the LDL Receptor Gene in Korean Patients with Familial Hypercholesterolemia

Clinical Chemistry ◽

10.1093/clinchem/45.9.1424 ◽

1999 ◽

Vol 45 (9) ◽

pp. 1424-1430 ◽

Cited By ~ 17

Author(s):

Sung Han Kim ◽

Ji Hyun Bae ◽

Jae Jin Chae ◽

Un Kyung Kim ◽

Seong-Joon Choe ◽

...

Keyword(s):

Sequence Analysis ◽

Familial Hypercholesterolemia ◽

Receptor Gene ◽

Blot Hybridization ◽

Screening Method ◽

Ldl Receptor ◽

Southern Blot Hybridization ◽

Long Distance ◽

Large Rearrangements ◽

Long Distance Pcr

Abstract Background: The LDL receptor is a cell-surface protein that regulates plasma cholesterol by specific uptake of LDL particles from the blood circulation. Familial hypercholesterolemia (FH) results from defective catabolism of LDL, which is caused by mutations in the LDL-receptor gene. Methods: For the rapid and reliable detection of large rearrangements in the LDL-receptor gene, we established a screening method based on long-distance PCR as an alternative to Southern-blot hybridization. Using long-distance PCR, 45 unrelated Korean subjects heterozygous for FH were screened to assess the frequency and nature of major structural rearrangements in the LDL-receptor gene. Results: Two different deletion mutations, FH6 (same type as FH3 and FH311) and FH 32, were detected in four families by long-distance PCR. Detailed restriction mapping and sequence analysis showed that FH6 was a 5.71-kb deletion extending from intron 8 to intron 12 and that FH32 was a 2-kb deletion extending from intron 6 to intron 7. Sequence analysis for the breakpoints of all deletions detected in Korean FH patients showed that only the left arms of the Alu repetitive sequences were involved in the deletion event. Conclusions: The screening method based on long-distance PCR provides a powerful strategy for the detection of large rearrangements in the LDL-receptor gene and is a rapid and reliable screening alternative to Southern-blot hybridization.

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Mutation Profiling in Haemophilia A

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615636 ◽

2001 ◽

Vol 85 (04) ◽

pp. 577-579 ◽

Cited By ~ 13

Author(s):

J. Oldenburg

Keyword(s):

Denaturing Gradient Gel Electrophoresis ◽

Mutation Screening ◽

Screening Method ◽

Coagulation Factor ◽

Causative Mutation ◽

Haemophilia A ◽

Coding Region ◽

Systematic Analysis ◽

Intron 22 Inversion ◽

Gradient Gel Electrophoresis

SummaryHaemophilia A is a X-linked recessive bleeding disorder caused by deficiency or absence of coagulation factor VIII (FVIII) due to heterogeneous defects in the FVIII gene. The large size of the FVIII gene (26 exons spanning 186 kb) has hampered mutation analysis for many years. In 1991 the first systematic analysis of the complete coding region of the FVIII gene was performed by Higuchi et al. using Denaturing Gradient Gel Electrophoresis (DGGE) as a mutation screening method (1, 2). Notably, the causative mutation was not found in about half of the severely affected patients (1). This mystery was solved in 1993, when the intron 22 inversion was discovered (3, 4) that accounts for about 50% of the severe haemophilia A cases. The inversion mutation can be easily detected by Southern Blot. A recently described PCR-based method is more sophisticated, however once established, it allows rapid and convenient detection of the intron 22 inversion (5).

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