Haemophilia management: time to get personal?

The possibility of alloimmunization in patients receiving protein replacement therapy depends on (at least) three risk factors, which are necessary concomitantly but insufficient alone. The first is the degree of structural difference between the therapeutic protein and the patient's own endogenous protein, if expressed. Such differences depend on the nature of the disease mutation and the pre-mutation endogenous protein structure as well as on post-translational changes and sequence-engineered alterations in the therapeutic protein. Genetic variations in the recipients' immune systems comprise the second set of risk determinants for deleterious immune responses. For example, the limited repertoire of MHC class II isomers encoded by a given person's collection of HLA genes may or may not be able to present a 'foreign' peptide(s) produced from the therapeutic protein - following its internalization and proteolytic processing - on the surface of their antigen-presenting cells (APCs). The third (and least characterized) variable is the presence or absence of immunologic 'danger signals' during the display of foreign-peptide/MHC-complexes on APCs. A choice between existing therapeutic products or the manufacture of new proteins, which may be less immunogenic in some patients or patient populations, may require prior definition of the first two of these variables. This leads then to the possibility of developing personalized therapies for disorders due to genetic deficiencies in endogenous proteins, such as haemophilia A and B. [Correction made after online publication 11 July 2011: several critical corrections have been made to the abstract].

Genetic determinants of normal variation in coagulation factor (F) IX levels: genome-wide scan and examination of the FIX structural gene.

BACKGROUND: High-normal and elevated plasma FIX activity (FIX:C) levels are associated with increased risk for venous- and possibly arterial-thrombosis. OBJECTIVE: Because the broad normal range for FIX:C involves a substantial unknown genetic component, we sought to identify quantitative-trait loci (QTLs) for this medically important hemostasis trait. METHODS: We performed a genome-wide screen and a resequencing-based variation scan of the known functional regions of every distinct FIX gene (F9) in the genetic analysis of idiopathic thrombophilia project (GAIT), a collection of 398 Spanish-Caucasians from 21 pedigrees. RESULTS: We found no evidence for linkage (LOD scores <1.5) despite genotyping more than 540 uniformly-spaced microsatellites. We identified 27 candidate F9 polymorphisms, including three in cis-elements responsible for the increase in FIX:C that occurs with aging, but found no significant genotype-specific differences in mean FIX:C levels (P-values > or = 0.11) despite evaluating every polymorphism in GAIT by marginal multicovariate measured-genotype association analysis. CONCLUSIONS: The heritable component of interindividual FIX:C variability likely involves a collection of QTLs with modest effects that may reside in genes other than F9. Nevertheless, because the alleles of these 27 polymorphisms exhibited a low overall degree of linkage disequilibrium, we are currently defining their haplotypes to interrogate several highly-conserved non-exonic sequences and other F9 segments not examined here.

A patient homozygous for a mutation in the prothrombin gene 3'-untranslated region associated with massive thrombosis.

We describe the first reported case of a thrombophilia patient genetically homozygous for a recently described polymorphism in the 3'-UTR (untranslated region) of the prothrombin gene. It has previously been demonstrated that this genetic variant due to a G to A transition at nucleotide 20210 is common and associated with an almost threefold increased risk of venous thrombosis. This polymorphism was also shown to be associated with elevated plasma prothrombin (factor II) levels, which in itself was found to be a risk factor for venous thrombosis. The patient was a healthy young Mexican male who presented with a myocardial infarction and subsequent ileofemoral venous thrombosis and massive saddle pulmonary embolus. Testing done during his initial hospitalization suggested a congenital protein C deficiency. The patient was found to be homozygous for the prothrombin gene polymorphism as well as a carrier for factor V Leiden. This case strongly implies a clinically significant role for the factor II gene mutation in both arterial and venous thrombosis and demonstrates the need to perform diagnostic clotting based assays after resolution of acute thrombotic events. These findings further support the 'double hit' theory for thrombophilia in young patients.

The critical role of tissue angiotensin-converting enzyme as revealed by gene targeting in mice.

Angiotensin-converting enzyme (ACE) generates the vasoconstrictor angiotensin II, which plays a critical role in maintenance of blood pressure in mammals. Although significant ACE activity is found in plasma, the majority of the enzyme is bound to tissues such as the vascular endothelium. We used targeted homologous recombination to create mice expressing a form of ACE that lacks the COOH-terminal half of the molecule. This modified ACE protein is catalytically active but entirely secreted from cells. Mice that express only this modified ACE have significant plasma ACE activity but no tissue-bound enzyme. These animals have low blood pressure, renal vascular thickening, and a urine concentrating defect. The phenotype is very similar to that of completely ACE-deficient mice previously reported, except that the renal pathology is less severe. These studies strongly support the concept that the tissue-bound ACE is essential to the control of blood pressure and the structure and function of the kidney.

Mice lacking angiotensin-converting enzyme have low blood pressure, renal pathology, and reduced male fertility.

Mammals produce two isozymes of angiotensin-converting enzyme (ACE). Somatic ACE plays an important role in the control of blood pressure. The function of testis ACE, produced by male and germ cells, is not known. To examine the roles of these isozymes, we used targeted homologous recombination to introduce a modified ACE allele into a mouse line. Mice homozygous for this mutant allele lack both ACE isozymes and have markedly reduced blood pressures. Contrary to a previous report, we found heterozygous male mice to have normal blood pressures. Homozygous mutant mice also have severe renal disease. The renal papilla is markedly reduced, and the intrarenal arteries exhibit vascular hyperplasia associated with a perivascular inflammatory infiltrate. These animals cannot effectively concentrate urine. They also have an abnormally low urinary sodium to potassium ratio despite reduced levels of aldosterone. Homozygous mutant male mice sire significantly smaller litters than wild-type male mice; however, no defect in sperm number, morphology, or motility was detected. ACE-deficient animals demonstrate the role of this enzyme in systemic blood pressure, renal development and function, and male fertility.

Tissue specific expression of angiotensin converting enzyme.

Angiotensin converting enzyme (ACE) is a component of the renin-angiotensin system and is critical in the homeostatic control of systemic blood pressure. There are two isozymes of ACE that result from two distinct promoter regions with the single ACE gene. In this article, we discuss the biochemistry of tissue specific promoter recognition as exemplified by the ACE gene.

Transgenic mice demonstrate a testis-specific promoter for angiotensin-converting enzyme.

There are two isozymes of angiotensin-converting enzyme (ACE), one produced by somatic tissues and a smaller protein synthesized by developing spermatozoa (testis ACE). To investigate the molecular control of testis ACE, we generated mice transgenic for a construct containing a putative testis-specific ACE promoter linked to the Escherichia coli reporter gene encoding beta-galactosidase. The transgenic mice express beta-galactosidase protein and RNA only within the testis. Histochemical analysis of the transgenic mice shows co-localization of beta-galactosidase protein and endogenous ACE within elongating spermatozoa. These studies demonstrate that transcription of testis ACE is controlled by a strong intragenic testis-specific promoter that is contained within a 698-base pair fragment immediately upstream from the transcription start site of testis ACE. Characterization of the testis ACE promoter may provide insights into the molecular mechanisms controlling cell stage-specific gene expression in the male germ line.

Transcription of testicular angiotensin-converting enzyme (ACE) is initiated within the 12th intron of the somatic ACE gene.

Angiotensin-converting enzyme (ACE) is a zinc-containing dipeptidyl carboxypeptidase that catalyzes the conversion of angiotensin I to the potent vasoconstrictor angiotensin II. By analyzing cDNA and genomic DNA, we have constructed a consensus sequence encoding the testis isozyme of mouse ACE. Testis ACE cDNA contains 2,435 base pairs and encodes a protein of 732 amino acids. The N-terminal 66 amino acids are unique to the testis isozyme, while the remaining 666 are identical to the carboxyl half of mouse somatic ACE. The overall conservation of amino acid sequence between the testis isozymes of the mouse, rabbit, and human is 78 to 84%. The conservation of amino acids for the N-terminal domain uniquely expressed within the testis is 63 to 67% between these species. Primer extension and RNase protection experiments show that RNA transcription of the testis ACE isozyme begins 16 or 17 bases upstream from the translation start site. A sequence element resembling a TATA box is found 25 bases 5' of the transcription start site. To create its unique isozyme of ACE, the testis begins mRNA transcription in the middle of the exonic-intronic structure of somatic ACE, within a sequence treated as an intron by somatic tissues. Testis ACE is not the result of alternative RNA splicing but seems due to the start of transcription at a unique site within the ACE gene.