Similar Pattern of Increase in FIX Levels in Female Carriers and Males with Hemophilia B Leyden
Authors: Ahmed Saad Z , O'Rourke Michelle , Jenkins Vince , Keenan Caitriona , Regan Irene Ellen , Nolan Beatrice 
Published: November 2020

Hemophilia B is a congenital X-linked recessive bleeding disorder that occurs as a result of mutations at the long (q) arm of the X chromosome at position 27. Hemophilia B affects 1 in 30,000 males; however the incidence in Ireland is more than double the international incidence and affects 1 in 12,500 male births. Hemophilia B Leyden is a sub-type of hemophilia B first recognized in 1970 in Leyden in the Netherlands. There are more than 20 genetic mutations that result in hemophilia B Leyden. These mutations cluster at three regions within the proximal promoter region of the F9 gene including mutations in nucleotides c.-34 and c.-35 (-5 and -6 legacy numbering) in almost half the cases, mutations around nucleotide c.-49 (-20) and mutations around nucleotide c.-19 (+10). Point mutations associated with hemophilia B Leyden interfere with F9 gene transcription via binding of F9 promoter to ONECUT1 in patients with c.-35 mutation, HNF4a (hepatocyte nuclear factor 4 alpha) in patients with c.-19 mutation and C/EBPa (enhancer-binding protein alpha) in patients with c.-49 mutation.
A characteristic feature of hemophilia B Leyden is progressive rise in FIX:C level from the time of puberty to reach lower normal level in young adults. This led to the widely accepted believe that the increase in FIX:C level in hemophilia B Leyden is due to the effect of the androgens surge at puberty. Androgens and anabolic steroids were used for prophylactic treatment in patients with hemophilia B Leyden in a limited number of patients. Recent evidence from animal studies using hypophysectomized hemophilia B Leyden mouse model convincingly demonstrated the pre-eminent role of growth hormone (GH) in regulating the age-dependent increase in FIX:C levels. FIX:C levels were only restored to 10% of pre-hypophysectomy levels by exogenous administration of dihydrotestosterone or oestrodiol but were restored to 80% of pre-hypophysectomy levels by exogenous administration of GH.
We investigated the pattern of the increase in FIX:C in 14 male children and four female children from seven different Irish families attending the National Haemophilia Centre at Children's Health Ireland, Crumlin. The hemizygous presence of hemophilia B Leyden associated variant c.35G&gt;A was confirmed by standard dideoxysequence analysis in all children. To evaluate the timing of increase in plasma FIX:C levels, we measured FIX:C levels using automated one stage assay. Serial measurement of FIX:C in male children with hemophilia B leyden demonstrated an increase of FIX:C from severe/moderate levels at the first year of life to mild FIX:C levels within the first 3 years of life. In all 14 male children with hemophilia B Leyden, FIX:C levels increased gradually throughout childhood with more pronounced increase in FIX:C at the time of puberty (Figure 1-A). The increase in FIX:C levels coincided with the known physiological increase in GH levels. Growth hormone level is low during infancy and increases but remains stable at low levels during early childhood, until just before puberty when GH secretion surges to the highest lifetime peak.
All four female carriers of hemophilia B Leyden showed low levels of FIX:C at diagnosis associated with variable bleeding symptoms (Figure 1-B). One female child had very low FIX:C level at diagnosis in the moderate hemophilia range (0.04 iu/ml), while all other three female children had low FIX:C levels at the mild hemophilia range at diagnosis (0.13 iu/ml, 0.27 iu/ml and 0.46 iu/ml respectively). Low FIX:C level in those female carriers is likely due to skewed inactivation of the unaffected X chromosome during embryogenesis (lyonization) as no child had compound heterozygosity for the F9 gene mutation and none of the children showed clinical features of Turner syndrome or testicular feminization syndrome. Serial measurement of FIX:C levels in those symptomatic female carriers of hemophilia B Leyden showed similar gradual increase in FIX:C levels during childhood with more steep increase at puberty. Our findings further assert that gender and sex hormones has no influence in the increase of FIX:C level in hemophilia B patients.
Figure 1

Disclosures
Nolan: Sobi: Other: personal fees; Bayer, CSL Behring, and Sanofi.: Other: sponsorship; Sanofi: Other: PI for sponsor-(Sanofi-) led clinical trial.