Haemophilia management: time to get personal?

Summary. 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].     

Familial homozygous hypobetalipoproteinemia

An apparently new form of abetalipoproteinemia, homozygous hypobetalipoproteinemia, was studied in progeny of a mating of two parents each heterozygous for familial hypobetalipoproteinemia, which was characterized by three-generation vertical transmission on both maternal and paternal sides of the family. The two children, with an apparent homozygous form of hypobetalipoproteinemia, had, in addition to abetalipoproteinemia, acanthocytosis, intestinal etpithelial and hepatic steatosis and steatorrhea. No low desity lipoprotein (LDL) was detected by immunodiffusion with antisera to LDL or apoLDL. The defects in apolipoproteins in these two children were the same as those reported in children with abetalipoproteinemia inherited as an autosomal recessive trait. The genetic defect of hypobetalipoproteinemia, when homozygous, can lead to all of the known clinical and biochemical features of abetalipoproteinemia.     

Synonymous Mutations in RNASEH2A Create Cryptic Splice Sites Impairing RNase H2 Enzyme Function in Aicardi–Goutières Syndrome

Aicardi–Goutières syndrome is an inflammatory disorder resulting from mutations in TREX1, RNASEH2A/2B/2C, SAMHD1, or ADAR1. Here, we provide molecular, biochemical, and cellular evidence for the pathogenicity of two synonymous variants in RNASEH2A. Firstly, the c.69G>A (p.Val23Val) mutation causes the formation of a splice donor site within exon 1, resulting in an out of frame deletion at the end of exon 1, leading to reduced RNase H2 protein levels. The second mutation, c.75C>T (p.Arg25Arg), also introduces a splice donor site within exon 1, and the internal deletion of 18 amino acids. The truncated protein still forms a heterotrimeric RNase H2 complex, but lacks catalytic activity. However, as a likely result of leaky splicing, a small amount of full‐length active protein is apparently produced in an individual homozygous for this mutation. Recognition of the disease causing status of these variants allows for diagnostic testing in relevant families.     

Intraspecific DNA variation in nuclear genes of the mosquito Aedes aegypti

Single nucleotide polymorphisms (SNPs) are an abundant source of genetic variation among individual organisms. To assess the usefulness of SNPs for genome analysis in the yellow fever mosquito, Aedes aegypti, we sequenced 25 nuclear genes in each of three strains and analysed nucleotide diversity. The average frequency of nucleotide variation was 12 SNPs per kilobase, indicating that nucleotide variation in Ae. aegypti is similar to that in other organisms, including Drosophila and the malaria vector Anopheles gambiae. Transition polymorphisms outnumbered transversion polymorphisms, at a ratio of about 2:1. We examined codon usage and confirmed that mutational bias favours G and C ending codons. Codon bias was most pronounced in highly expressed genes. Nucleotide diversity estimates indicated that substitution rates are positively correlated in coding and non-coding regions. Nucleotide diversity varied from one gene to another. The unequal distribution of SNPs among Ae. aegypti nuclear genes suggests that single base variations are non-neutral and are subject to selective constraints. Our analysis showed that ubiquitously expressed genes have lower polymorphism rates and are likely under strong purifying selection, whereas tissue specific genes and genes with a putative role in parasite defence exhibit higher levels of polymorphism that may be associated with diversifying selection.     

Methods for the Detection of Non-Random Base Substitution in Virus Genes: Models of Synonymous Nucleotide Substitution in Picornavirus Genes

A substantial fraction of phylogenetic divergence between closely related RNA virus genes is generally accounted for by synonymous (non-amino acid changing) point mutation. Viral evolution may be a complicated phenomena, governed by many different processes. However in this study we ask whether there are any properties in the patterns of synonymous nucleotide substitutions in three different Picornavirus genes that permit the process of accumulation of synonymous point mutation in these genes to be distinguished from some of the simplest most basic evolutionary models. We conclude that while the observed patterns in the occurrence of synonymous point substitution are consistent with those predicted by a model in which base mutation is equi-probable along a gene, and the probability of synonymous substitution determined only by local codon usage, some patterns in the actual nucleotides exchanged remain to be explained. This revised version was published online in July 2006 with corrections to the Cover Date.     

Dynamic programming procedure for searching optimal models to estimate substitution rates based on the maximum-likelihood method

The substitution rate in a gene can provide valuable information for understanding its functionality and evolution. A widely used method to estimate substitution rates is the maximum-likelihood method implemented in the CODEML program in the PAML package. A limited number of branch models, chosen based on a priori information or an interest in a particular lineage(s), are tested, whereas a large number of potential models are neglected. A complementary approach is also needed to test all or a large number of possible models to search for the globally optional model(s) of maximum likelihood. However, the computational time for this search even in a small number of sequences becomes impractically long. Thus, it is desirable to explore the most probable spaces to search for the optimal models. Using dynamic programming techniques, we developed a simple computational method for searching the most probable optimal branch-specific models in a practically feasible computational time. We propose three search methods to find the optimal models, which explored O(n) (method 1) to O(n(2)) (method 2 and method 3) models when the given phylogeny has n branches. In addition, we derived a formula to calculate the number of all possible models, revealing the complexity of finding the optimal branch-specific model. We show that in a reanalysis of over 50 previously published studies, the vast majority obtained better models with significantly higher likelihoods than the conventional hypothesis model methods.     

Characterization of VWF gene conversions causing von Willebrand disease

We previously reported that von Willebrand Factor gene (VWF) conversions are a relatively frequent cause of von Willebrand disease (VWD), however, their molecular pathomechanisms resulting in variant phenotypes is largely unknown. Here, we characterized VWF conversions harbouring missense and synonymous mutations, through generating a series of mutant constructs followed by transient expression in 293 cells, and qualitative and quantitative analysis of recombinant VWF (rVWF). The characterization of mutant rVWF showed the critical roles of synonymous variants in the pathogenicity of VWF conversions. The gene conversion variants p.Val1229Gly, p.Asn1231Thr, p.Asn1231Ser and p.Ala1464Pro in the absence of synonymous p.Ser1263= and p.Gln1449= showed minimal effect on rVWF synthesis and activity. Interestingly, a construct including the synonymous variants displayed significantly low rVWF expression and activity. The variant p.Pro1266Leu showed gain of rVWF function toward glycoprotein Ibα; surprisingly, this function was significantly abolished in the presence of gene conversion variants p.Val1229Gly-p.Asn1231Thr. Taken together, our expression studies suggest that synonymous variants in the combination of other gene conversion variants suppress the protein expression, possibly due to defective primary mRNA structure or processing. The variants p.Val1229Gly-p.Asn1231Thr affected the VWF gain of function caused by variant p.Pro1266Leu, probably due to conformational changes in VWF.