Novel hotspot detector software reveals a non-CpG hotspot of germline mutation in the factor IX gene (F9) in Latin Americans

Drost, Joni B.; Scaringe, William A.; Jaloma‐Cruz, Ana Rebeca; Li, Xuemin; Ossa, Diego F.; Kasper, Carol K.; Sommer, Steve S.

doi:10.1002/1098-1004(200009)16:3<203::aid-humu3>3.0.co;2-1

Cited by 7 publications

(5 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, it is known that mutations in F9 gene usually result in impaired hFIX secretion in patients with HB, as shown in the FIX database (FIX database: http:// www.factorix.org) and previous reports. 18,[29][30][31][32] Despite lower secretion levels of both hyperfunctional variants, they exhibited higher in vitro hFIX:C levels compared with hFIX-WT, irrespective of the method used to assess FIX:C levels, confirming previous results showing the increased specific activity of hFIX-E456H and hFIX-R384L variants. 7,26 However, our results also highlighted once again that discrepancies exist between the FIX:C levels measured by one-stage assays and chromogenic assays for different hFIX molecules (►Supplementary Fig.…”

Section: Discussionsupporting

confidence: 74%

Recombinant Adeno-Associated Viral Vectors Expressing Human Coagulation FIX-E456H Variant in Hemophilia B Mice

et al. 2019

View full text Add to dashboard Cite

Gene therapy using recombinant adeno-associated virus (AAV) has induced sustained long-term coagulation human factor IX (hFIX) levels in hemophilia B (HB) patients. However, asymptomatic transient liver toxicity was observed at high vector doses, highlighting the need to improve the potency of these vectors. We report the generation of an AAV transgene cassette containing the hyperfunctional hFIX-E456H variant showing improved binding to platelets, with a comparison to wild-type hFIX (hFIX-WT) and hFIX-R384L variant (Padua) transgenes, containing F9 truncated-intron 1 (I1). In vitro specific activity was increased by 3.2- and 4.2-fold with hFIX-E456H and hFIX-R384L variants compared with hFIX-WT, using chromogenic assay, and by 7-and 8.6-fold with hFIX-E456H and hFIX-R384L variants compared with hFIX-WT, using one-stage assay. The transgenes were packaged into single-stranded AAV2/8 vectors that were tail vein injected at 5 × 109, 2 × 1010, and 5 × 1010 vg per mouse in HB mice. Plasma FIX activity level, assessed by chromogenic assay, was up to fourfold higher for hFIX-E456H compared with hFIX-WT and was not different compared with hFIX-R384L, among the three dose cohorts. Overall, the in vivo specific activity was increased by threefold for hFIX-E456H and 4.9-fold for hFIX-R384L compared with hFIX-WT. At the lower dose of 5 × 109 vg, the blood loss was significantly lower for hFIX-E456H compared with hFIX-WT, but did not differ compared with hFIX-R384L. The results found for the hFIX-E456H variant indicate that it might be a suitable alternative for gene therapy of HB.

show abstract

Section: Discussionsupporting

confidence: 74%

Recombinant Adeno-Associated Viral Vectors Expressing Human Coagulation FIX-E456H Variant in Hemophilia B Mice

et al. 2019

View full text Add to dashboard Cite

show abstract

“…Among these, two larger entries (32 at nt 17810 and 38 at nt 31311) were associated with only two to three different haplotypes suggesting possible founder effect among the patient pool recorded in the database. Thus, as previously examined [14], we did not find any mutational hotspot involving non-CpG nucleotides in the database accumulated over the last 12 years.…”

Section: Evaluation Of Non-cpg Sequence As Hotspot Of Mutationsupporting

confidence: 71%

“…Thus, as previously examined [14], we did not find any mutational hotspot involving non-CpG nucleotides in the database accumulated over the last 12 years. Among these, two larger entries (32 at nt 17810 and 38 at nt 31311) were associated with only two to three different haplotypes suggesting possible founder effect among the patient pool recorded in the database.…”

Section: Evaluation Of Non-cpg Sequence As Hotspot Of Mutationsupporting

confidence: 69%

Analysis of haemophilia B database and strategies for identification of common point mutations in the factor IX gene

et al. 2003

View full text Add to dashboard Cite

Haemophilia B is an X-linked recessively inherited bleeding disorder caused by heterogeneous mutations spanning the entire factor IX gene. As spontaneous germ-line mutations are known to occur mostly at CpG dinucleotides in the FIX gene, control of the disease would require continuous carrier detection and mutation screening. Identification of point mutations, the most common type of mutation in FIX gene, is more challenging compared with deletion and insertion mutations. We examined the haemophilia B database to identify specific nucleotides in the FIX gene that are mutated in relatively large number of patients and the variability (if any) in the mutational hotspots at CpG dinucleotides. It was found that while mutations responsible to account for all 2348 haemophilia B patients covered 20% of the FIX cDNA, only 1% of the cDNA involving mostly CpG dinucleotides accounted for mutation in 42.41% of the patient pool. Thus, only 27 nucleotides need to be investigated to identify the common point mutations, among which 15 are predicted to undergo change in restriction sites on mutation. It is interesting to note that seven nucleotides occurring in CpG dinucleotides do not have any reported mutation despite each of those being predicted to harbour mutation as a result of transition and having mutations recorded in the database for the neighbouring nucleotides. Strikingly large number of mutation in codon 296 causing T to M change in catalytic domain originally proposed to be the result of the founder effect also contains largest number of haplotype suggesting recurrence of de novo mutation.

show abstract

“…Using cDNAs encoding FIXwt, FIXC71Y and FIXC109Y, we found that the secretion of the two mutant proteins was impaired in transiently transfected HepG2 cells. Analysis of the FIX database (http://www.kcl.ac.uk/ip/petergreen/haemBdatabase.html) showed that mutations on cysteine residues usually lead to highly reduced FIX plasma concentrations in humans (FIX:Ag <1%) [24–26]. Disulfide bond disruption would therefore play a major role in the impairment of FIX secretion.…”

Section: Discussionmentioning

confidence: 99%

Two novel mutations in EGF‐like domains of human factor IX dramatically impair intracellular processing and secretion

Enjolras¹,

Plantier²,

Rodriguez³

et al. 2004

Journal of Thrombosis and Haemostasis

View full text Add to dashboard Cite

We investigated the mechanisms responsible for severe factor IX (FIX) deficiency in two cross-reacting material (CRM)-negative hemophilia B patients with a mutation in the first and second epidermal growth factor (EGF) domains of FIX (C71Y and C109Y, respectively). We have determined the kinetics of mutant FIX biosynthesis and secretion in comparison with wild-type FIX (FIXwt). In transfected cells, FIXwt was retrieved as two intracellular molecular forms, rapidly secreted into the culture medium. One appeared to be correctly N-glycosylated, and corresponded to a form trafficking between the endoplasmic reticulum (ER) and Golgi apparatus. The other corresponded to the mature form, ready to be secreted, exhibiting correct N-glycosylation and sialylation. In contrast, the two mutants, FIXC71Y and FIXC109Y, were not secreted from the cells and did not accumulate intracellularly. Relative to FIXwt, they were retained longer in the ER and were only N-glycosylated. In addition, the intracellular concentration of the FIX mutants increased when ALLN, an inhibitor of cysteine proteases and of the proteasome degradation pathway, was added to the culture medium. Both the FIX mutants and FIXwt were associated in the ER with the 78-kDa glucose-regulated protein (GRP78/BiP) and calreticulin (CRT), though the amount of CRT associated with the two mutants was twice as strong as with FIXwt. These results strongly suggest that chaperone and lectin molecules act in concert to ensure both proper folding of FIXwt and the retention of mutant molecules.

show abstract

Novel hotspot detector software reveals a non-CpG hotspot of germline mutation in the factor IX gene (F9) in Latin Americans

Cited by 7 publications

References 28 publications

Recombinant Adeno-Associated Viral Vectors Expressing Human Coagulation FIX-E456H Variant in Hemophilia B Mice

Recombinant Adeno-Associated Viral Vectors Expressing Human Coagulation FIX-E456H Variant in Hemophilia B Mice

Analysis of haemophilia B database and strategies for identification of common point mutations in the factor IX gene

Two novel mutations in EGF‐like domains of human factor IX dramatically impair intracellular processing and secretion

Contact Info

Product

Resources

About