Severe haemophilia B due to a 6 kb factor IX gene deletion including exon 4: non-homologous recombination associated with a shortened transcript from whole blood.
In genotyping a severe hemophilia B subject, exons 1-3 and 5-8 were normal. Exon 4 did not amplify, suggesting a partial gene deletion. Previously, a French family with an exon 4 deletion had severe haemophilia B with a circulating, dysfunctional factor IX protein missing its first growth factor-like domain; breakpoints were not analyzed. Using a 5' primer for exon 3 and a 3' primer for exon 5 fragments, the subject's factor IX gene amplified a 5 kb fragment whereas 11 kb was predicted, indicating a 6 kb deletion. Restriction endonucleases localized the 3' intron 4 deletion breakpoint to 1.2 kb 5' to exon 5. Sequencing through the breakpoints revealed a 5,969 bp deletion that included exon 4 and was accompanied by a 13 bp duplication inserted near the 3' breakpoint site. Haemophilia was familial; on testing, his mother was confirmed as a heterozygous carrier, whereas his sister was homozygous for the normal, larger fragments. As exons 4 and 5 of the factor IX gene are in frame, this deletion should produce a shortened transcript, missing 114 bp (38 codons from the first growth factor-like domain). Reverse transcription of mRNA prepared from whole blood and PCR identified the shorter cDNA fragment. Western blotting demonstrated a smaller factor IX protein.
Hsu
Ting-Chang
TC
Puget Sound Blood Center, 921 Terry Ave, Seattle, WA 98104-1256, USA.
Nakaya
Shelley M
SM
Thompson
Arthur R
AR
D000293:Adolescent; D001483:Base Sequence; D005091:Exons / Q000235:genetics*; D005164:Factor IX / Q000235:genetics* / Q000378:metabolism; D017353:Gene Deletion; D005838:Genotype; D002836:Hemophilia B / Q000097:blood / Q000235:genetics*; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D010375:Pedigree; D012333:RNA, Messenger / Q000097:blood*; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012720:Severity of Illness Index
D012333:RNA, Messenger; D005164:Factor IX