Missense mutations and evolutionary conservation of amino acids: evidence that many of the amino acids in factor IX function as "spacer" elements.

We report 31 point mutations in the factor IX gene and explore the relationship between the level of evolutionary conservation of an amino acid and the probability of a mutation causing hemophilia B. From our total sample of 125 hemophiliacs and from those reported by others, we identify 95 independent missense mutations, 94 of which occur at amino acids that are evolutionarily conserved in the available mammalian factor IX sequences. The likelihood of a missense mutation causing hemophilia B depends on whether the residue is also conserved in the factor IX-related proteases: factor VII, factor X, and protein C. Most of the possible missense mutations in generically conserved residues (i.e., those conserved in factor IX and in all the related proteases) should cause disease. In contrast, missense mutations in factor IX-specific residues (i.e., those conserved in human, cow, dog, and mouse factor IX but not in the related proteases) are sixfold less likely to cause disease. Missense mutations at nonconserved residues are 33-fold less likely to cause disease. At least three models are compatible with these observations. A comparison of sequence alignments from four and nine species of factor IX and an examination of the missense mutations occurring at CpG residues suggest a model in which most residues fall on opposite ends of a spectrum. In about 40% of residues, virtually any missense mutation in a minority of the residues will cause disease, while virtually no missense mutations will cause disease in most of the remaining residues. Thus, many of the residues in factor IX are spacers; that is, the main chains are presumably necessary to keep other amino acid interactions in register, but the nature of the side chain is unimportant.     

Assessing the underlying pattern of human germline mutations: lessons from the factor IX gene.

Germline mutations cause or predispose to most disease. Hemophilia B is a useful model for studying the underlying pattern of recent germline mutations in humans because the observed pattern of mutation in factor IX more closely reflects the underlying pattern of mutation than the observed pattern for many other genes. In addition, it is possible to identify and correct for biases inherent in ascertaining only those mutations that cause hemophilia. Aspects of the pattern of germline mutation in the factor IX gene are becoming clear: 1) in the United States, two-thirds of mutations causing mild disease arose from three founders whereas almost all the mutations resulting in either moderate or severe disease arose independently, generally within the past 150 years; 2) direct estimates of the rates of mutation in humans indicate that transitions are more frequent than transversions, which in turn are more frequent than deletions and insertions; 3) transitions at CpG are elevated approximately 24-fold relative to transitions at non-CpG dinucleotides; 4) transversions at CpG are elevated approximately eightfold relative to transversions at non-CpG dinucleotides; 5) the sum total of the dinucleotide mutation rates produces a bias against G and C bases that would be sufficient to maintain the G+C content of the factor IX gene at its evolutionarily conserved level of 40%; and 6) the pattern of mutation is similar for Caucasians residing in the United States and for Asians residing in Asia. Two ideas emerge from this and from an analysis of the pattern of recent deleterious mutations compared with ancient neutral mutations that have been fixed during evolution into the factor IX gene. First, the bulk of germline mutations are likely to arise from endogenous processes rather than environmental mutagens. Second, the factor IX protein is composed mostly of two classes of amino acids: critical residues in which all single-base missense changes will disrupt protein function, and "spacer" residues in which the precise nature of the residue is unimportant but the peptide bond is necessary to keep the critical residues in register. More work is necessary to assess the veracity and generality of these ideas.     

Human germline mutation in the factor IX gene

The molecular epidemiology of factor IX germline mutations in patients with hemophilia B has been studied in detail because it is an advantageous model for analyzing recent germline mutations in humans. It is estimated that mutations have been defined in the majority of nucleotides that are the target for mutation. The likelihood that a factor IX missense mutation will cause disease correlates with the degree of evolutionary conservation of the amino acid. Mutation rates per base-pair have been estimated after careful consideration and correction for biases, predicting about 76 de novo mutations per generation per individual resulting in 0.3 deleterious changes. The male-to-female sex ratio of mutation varies with the type of mutation. There is evidence for a maternal age effect and an excess of non-CpG G:C to A:T transitions. The factor IX mutation pattern is similar among geographically, racially and ethnically diverse human populations. The data support primarily endogenous mechanisms of germline mutation in the factor IX gene. Mutations at splice junctions are compatible with simple rules for predicting disease causing mutations.     

3D mapping of glycogenosis-causing mutations in the large regulatory alpha subunit of phosphorylase kinase

Mutations in the liver isoform of the Phosphorylase Kinase (PhK) alpha subunit (PHKA2 gene) cause X-linked liver glycogenosis (XLG), the most frequent type of PhK deficiency (glycogen-storage disease type IX). XLG patients can be divided in two subgroups, with similar clinical features but different activity of PhK (decreased in liver and blood cells for XLG-I and low in liver but normal or enhanced in blood cells for XLG-II). Here, we show that the PHKA2 missense mutations and small in-frame deletions/insertions are concentrated into two domains of the protein, which were recently described. In the N-terminal glucoamylase domain, mutations (principally leading to XLG-II) are clustered within the predicted glycoside-binding site, suggesting that they may have a direct impact on a possible hydrolytic activity of the PhK alpha subunit, which remains to be demonstrated. In the C-terminal calcineurin B-like domain (domain D), mutations (principally leading to XLG-I) are clustered in a region predicted to interact with the regulatory region of the PhK catalytic subunit and in a region covering this interaction site. Altogether, these results show that PHKA2 missense mutations or small in-frame deletions/insertions may have a direct impact on the PhK alpha functions and provide a framework for further experimental investigation.     

DynaMut2: Assessing changes in stability and flexibility upon single and multiple point missense mutations

Predicting the effect of missense variations on protein stability and dynamics is important for understanding their role in diseases, and the link between protein structure and function. Approaches to estimate these changes have been proposed, but most only consider single‐point missense variants and a static state of the protein, with those that incorporate dynamics are computationally expensive. Here we present DynaMut2, a web server that combines Normal Mode Analysis (NMA) methods to capture protein motion and our graph‐based signatures to represent the wildtype environment to investigate the effects of single and multiple point mutations on protein stability and dynamics. DynaMut2 was able to accurately predict the effects of missense mutations on protein stability, achieving Pearson's correlation of up to 0.72 (RMSE: 1.02 kcal/mol) on a single point and 0.64 (RMSE: 1.80 kcal/mol) on multiple‐point missense mutations across 10‐fold cross‐validation and independent blind tests. For single‐point mutations, DynaMut2 achieved comparable performance with other methods when predicting variations in Gibbs Free Energy (ΔΔG) and in melting temperature (ΔT_m). We anticipate our tool to be a valuable suite for the study of protein flexibility analysis and the study of the role of variants in disease. DynaMut2 is freely available as a web server and API at http://biosig.unimelb.edu.au/dynamut2 .     

Replacement of the Y450 (c234) phenyl ring in the carboxyl-terminal region of coagulation factor IX causes pleiotropic effects on secretion and enzyme activity

The interplay between impaired protein biosynthesis and/or function caused by missense mutations, particularly in relation to specific protein regions, has been poorly investigated. As model we chose the severe p.Y450C mutation in the carboxyl-terminal region of coagulation factor IX (FIX) and, by expression of a panel of recombinant variants, demonstrated the key role of the tyrosine phenyl group for both FIX secretion and coagulant activity. Comparison among highly homologous coagulation serine proteases indicate that additive or compensatory pleiotropic effects on secretion and function by carboxyl-terminal mutations produce life-threatening or mild phenotypes in the presence of similarly reduced protein amounts.     

Molecular Basis and Therapeutic Strategies to Rescue Factor IX Variants That Affect Splicing and Protein Function

Mutations that result in amino acid changes can affect both pre-mRNA splicing and protein function. Understanding the combined effect is essential for correct diagnosis and for establishing the most appropriate therapeutic strategy at the molecular level. We have identified a series of disease-causing splicing mutations in coagulation factor IX (FIX) exon 5 that are completely recovered by a modified U1snRNP particle, through an SRSF2-dependent enhancement mechanism. We discovered that synonymous mutations and missense substitutions associated to a partial FIX secretion defect represent targets for this therapy as the resulting spliced-corrected proteins maintains normal FIX coagulant specific activity. Thus, splicing and protein alterations contribute to define at the molecular level the disease-causing effect of a number of exonic mutations in coagulation FIX exon 5. In addition, our results have a significant impact in the development of splicing-switching therapies in particular for mutations that affect both splicing and protein function where increasing the amount of a correctly spliced protein can circumvent the basic functional defects.     

Molecular Mechanisms of Disease-Causing Missense Mutations

Genetic variations resulting in a change of amino acid sequence can have a dramatic effect on stability, hydrogen bond network, conformational dynamics, activity and many other physiologically important properties of proteins. The substitutions of only one residue in a protein sequence, so-called missense mutations, can be related to many pathological conditions and may influence susceptibility to disease and drug treatment. The plausible effects of missense mutations range from affecting the macromolecular stability to perturbing macromolecular interactions and cellular localization. Here we review the individual cases and genome-wide studies that illustrate the association between missense mutations and diseases. In addition, we emphasize that the molecular mechanisms of effects of mutations should be revealed in order to understand the disease origin. Finally, we report the current state-of-the-art methodologies that predict the effects of mutations on protein stability, the hydrogen bond network, pH dependence, conformational dynamics and protein function.     

Molecular pathology of haemophilia B.

Direct sequencing of amplified genomic DNA has been used to investigate the molecular basis of haemophilia B and thus identify specific amino acids that are essential for maintenance of structure or function of factor IX. Substitution of Cys 336, Asn 120 results in loss of circulating factor IX antigen and deletion of Arg 37 in gross reduction of circulating protein and loss of activity, while substitution of Arg -4, Arg 333, Asp 64 and Pro 55 cause loss of function without marked reduction in protein serum levels. Frameshift or point mutations resulting in marked loss of coding information are found in patients who develop antibodies to administered factor IX. An enhanced rate of mutation is evident at two CpG dinucleotides in the factor IX gene, which accounts for approximately 25% of all point mutations causing haemophilia B known to date. Direct sequencing of mutations also permits, for the first time, rapid and unequivocal prenatal and carrier diagnoses, in all cases, by eliminating the need for informative segregation of markers.     

Functional characterization of human methylenetetrahydrofolate reductase in Saccharomyces cerevisiae.

Human methylenetetrahydrofolate reductase (MTHFR, EC 1.5.1.20) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. 5-Methyltetrahydrofolate is a major methyl donor in the remethylation of homocysteine to methionine. Impaired MTHFR can cause high levels of homocysteine in plasma, which is an independent risk factor for vascular disease and neural tube defects. We have functionally characterized wild-type and several mutant alleles of human MTHFR in yeast, Saccharomyces cerevisiae. We have shown that yeast MET11 is a functional homologue of human MTHFR. Expression of the human MTHFR cDNA in a yeast strain deleted for MET11 can restore the strain's MTHFR activity in vitro and complement its methionine auxotrophic phenotype in vivo. To understand the domain structure of human MTHFR, we have truncated the C terminus (50%) of the protein and demonstrated that expressing an N-terminal human MTHFR in met11(-) yeast cells rescues the growth phenotype, indicating that this region contains the catalytic domain of the enzyme. However, the truncation leads to the reduced protein levels, suggesting that the C terminus may be important for protein stabilization. We have also functionally characterized four missense mutations identified from patients with severe MTHFR deficiency and two common missense polymorphisms found at high frequency in the general population. Three of the four missense mutations are unable to complement the auxotrophic phenotype of met11(-) yeast cells and show less than 7% enzyme activity of the wild type in vitro. Both of the two common polymorphisms are able to complement the growth phenotype, although one exhibited thermolabile enzyme activity in vitro. These results shall be useful for the functional characterization of MTHFR mutations and analysis structure/function relationship of the enzyme.     

Evidence that descendants of three founders constitute about 25% of hemophilia B in the United States

In our sample of 160 consecutive Caucasian hemophiliacs, 14 (9%) had a G----A transition at bp 10,430 that substitutes serine for glycine 60 in the first EGF domain of the factor IX molecule. Each of these hemophiliacs had clinically mild disease. Haplotype data and familial pedigrees indicate that 12 of these hemophiliacs are likely to be related to a common ancestor. The 13th and 14th patients possess different haplotypes and thus represent independent origins of the mutation. In addition, we have screened these 160 hemophiliacs for the previously reported mutations resulting from founder effects at IIe397----Thr and Thr296----Met. Herein we report an additional nine hemophiliacs with the mutant Thr397 allele and five additional hemophiliacs with the mutant Met296 allele. Haplotype data for these 14 hemophiliacs support the original founder effect hypotheses for these mutations. In total, the above three mutations are found in 44 of the 160 seemingly unrelated Caucasian hemophiliacs (28%). The sample includes patients from all regions of the United States and Ontario, Canada. Descendants of these three founders comprise approximately two-thirds of the missense mutations found in our sample of Caucasian hemophiliacs with clinically mild disease.     

Identification and expression of eight novel mutations among non-Jewish patients with Canavan disease.

Canavan disease is inherited as an autosomal recessive trait that is caused by the deficiency of aspartoacylase (ASPA). The majority of patients with Canavan disease are from an Ashkenazi Jewish background. Mutations in ASPA that lead to loss of enzymatic activity have been identified, and E285A and Y231X are the two predominant mutations that account for 97% of the mutant chromosomes in Ashkenazi Jewish patients. The current study was aimed at finding the molecular basis of Canavan disease in 25 independent patients of non-Jewish background. Eight novel and three previously characterized mutations accounted for 80% (40/50) of mutant chromosomes. The A305E missense mutation accounted for 48% (24/50) of mutant chromosomes in patients of western European descent, while the two predominant Jewish mutations each accounted for a single mutant chromosome. The eight novel mutations identified included 1- and 4-bp deletions (32 deltaT and 876 deltaAGAA, respectively) and I16T, G27R, D114E, G123E, C152Y, and R168C missense mutations. The homozygous 32 deltaT deletion was identified in the only known patient of African-American origin with Canavan disease. The heterozygosity for 876 deltaAGAA mutation was identified in three independent patients from England. Six single-base changes leading to missense mutations were identified in patients from Turkey (D114E, R168C), The Netherlands (I16T), Germany (G27R), Ireland (C152Y), and Canada (G123E). A PCR-based protocol is described that was used to introduce mutations in wild-type cDNA. In vitro expression of mutant cDNA clones demonstrated that all of these mutations led to a deficiency of ASPA and should therefore result in Canavan disease.     

An Excess of G over C Nucleotides in Mutagenesis of Human Genetic Diseases

Strand asymmetries in DNA evolution, including indel and single nucleotide substitutions, were reported in prokaryotes. Recently, an excess of G>A over C>T substitutions in hemophilia B patients was recognized in our molecular diagnostic practices. Further analysis demonstrated biased point mutations between sense and antisense strands when unique changes in factor IX were counted. Similar mutation spectra of factor IX and the HGMD prompted us to speculate that the excess of G>A over C>T may be present in genes other than factor IX. Data from nine genes (each has ≥100 missense mutations) retrieved from HGMD, international factor IX database, and Dr. Sommer’s lab database in the City of Hope National Medical Center, Duarte, CA, USA were analyzed for their point mutation spectra. Similar to factor IX, all genes selected in this study have biased G>A over C>T unique mutations when nonsense mutations were excluded. The biased missense point mutations were recently convincingly documented by the statistic data of categorized missense mutation in HGMD. The consistence of the genetic observation and the genomic data from HGMD strongly indicate that biased point mutations, possibly a phenotypic selection, are more widespread than previously thought. The biased mutations have immediate clinical impact in molecular diagnostics.     

Characterization of Wiskott–Aldrich syndrome (WAS) mutants using Saccharomyces cerevisiae

Wiskott–Aldrich syndrome (WAS) is caused by alterations in the WAS protein (WASP), and 80% of the missense mutations are located in the WH1 domain, the region essential for interaction with the WASP-interacting protein (WIP). It has been suggested that loss of WASP–WIP interaction is causal to the disease. Las17p (yeast WASP) is essential for growth at 37 °C. The growth defect of the las17 Δ strain can be suppressed by the expression of human WASP together with WIP. Using the las17 Δ strain, we have analyzed 52 missense mutations in the gene encoding WASP and found that 13 of these mutant proteins were unable to suppress the growth defect of the las17 Δ strain. The majority of these 13 mutations cause the classic WAS in humans and are located within the WH1 domain, while none of the 12 mutations outside the WH1 domain abolished the activity of WASP in Saccharomyces cerevisiae cells. This suggests that some of the mutations (13 out of 40) in the WH1 domain cause the syndrome in humans by perturbing the WASP–WIP complex formation, while the rest of the mutations cause the syndrome without affecting the WASP–WIP complex formation, but may affect the activity of the complex.     

Protein stability and in vivo concentration of missense mutations in phenylalanine hydroxylase

A previous computational analysis of missense mutations linked to monogenic disease found a high proportion of missense mutations affect protein stability, rather than other aspects of protein structure and function. The purpose of this study is to relate the presence of such stability damaging missense mutations to the levels of a particular protein present under "in vivo" like conditions, and to test the reliability of the computational methods. Experimental data on a set of missense mutations of the enzyme phenylalanine hydroxylase (PAH) associated with the monogenic disease phenylketonuria (PKU) have been compared with the expected in vivo impact on protein function, obtained using SNPs3D, an in silico analysis package. A high proportion of the PAH mutations are predicted to be destabilizing. The overall agreement between predicted stability impact and experimental evidence for lower protein levels is in accordance with the estimated error rates of the methods. For these mutations, destabilization of protein three-dimensional structure is the major molecular mechanism leading to PKU, and results in a substantial reduction of in vivo PAH protein concentration. Although of limited scale, the results support the view that destabilization is the most common mechanism by which missense mutations cause monogenic disease. In turn, this conclusion suggests the general therapeutic strategy of developing drugs targeted at restoring wild type stability.     

Haemophilia B (sixth edition): a database of point mutations and short additions and deletions.

The sixth edition of the haemophilia B database lists in easily accessible form all known factor IX mutations due to small changes (base substitutions and short additions and/or deletions of <30 bp) identified in haemophilia B patients. The 1380 patient entries are ordered by the nucleotide number of their mutation. Where known, details are given on factor IX activity, factor IX antigen in circulation and origin of mutation. References to published mutations are given and the laboratories generating the data are indicated.     

Haemophilia B: database of point mutations and short additions and deletions, fifth edition, 1994.

The fifth edition of the haemophilia B database lists in easily accessible form all known factor IX mutations due to small changes (base substitutions and short additions and/or deletions of < 30bp) identified in haemophilia B patients. The 1,142 patient entries are ordered by the nucleotide number of their mutation. Where known, details are given on: factor IX activity, factor IX antigen in circulation, and origin of mutation. References to published mutations are given and the laboratories generating the data are indicated.     

Haemophilia B: database of point mutations and short additions and deletions, 7th edition.

The seventh edition of the haemophilia B database lists in easily accessible form all known factor IX mutations due to small changes (base substitutions and short additions and/or deletions of <30 bp) identified in haemophilia B patients. The 1535 patient entries are ordered by the nucleotide number of their mutation. Where known, details are given on: factor IX activity, factor IX antigen in circulation, presence of inhibitor and origin of mutation. References to published mutations are given and the laboratories generating the data are indicated.