Residual linkage: why do linkage peaks not disappear after an association study?

Family-based candidate gene and genome-wide association studies are a logical progression from linkage studies for the identification of gene and polymorphisms underlying complex traits. An efficient way to analyse phenotypic and genotypic data is to model linkage and association simultaneously. An important result from such an analysis is whether any evidence for linkage remains after fitting polymorphisms at candidate genes (residual linkage), because this may indicate locus and allelic heterogeneity in the population and will influence subsequent molecular strategies. Here we report that substantial residual linkage is to be expected, even under genetic homogeneity and when the underlying causal polymorphisms are genotyped and fitted in the model. We simulated a powerful design to detect linkage to quantitative trait loci, with 5, 10 or 20 causal SNPs spread throughout the genome. These SNPs were responsible for all genetic variation, and hence for both linkage and association. Residual linkage at the largest linkage peak from a genome-wide scan was substantial, with mean LOD scores of 0.4, 0.7, and 1.4 for the case of 5, 10 and 20 underlying causal SNPs, respectively. For less powerful designs, the proportion of the original LOD scores that remains after association will be even larger. All cases of ‘significant’ residual linkage are false positives. The reason for the apparent paradox of detecting residual linkage after fitting causal polymorphisms is that the linkage signals at the largest peaks in a genome-scan are severely inflated, even if all peaks correspond to true linkage. Our findings are general and apply to linkage mapping of any phenotype and to any pedigree structure.     

Primordial linkage of β2-microglobulin to the MHC

β2-Microglobulin (β2M) is believed to have arisen in a basal jawed vertebrate (gnathostome) and is the essential L chain that associates with most MHC class I molecules. It contains a distinctive molecular structure called a constant-1 Ig superfamily domain, which is shared with other adaptive immune molecules including MHC class I and class II. Despite its structural similarity to class I and class II and its conserved function, β2M is encoded outside the MHC in all examined species from bony fish to mammals, but it is assumed to have translocated from its original location within the MHC early in gnathostome evolution. We screened a nurse shark bacterial artificial chromosome library and isolated clones containing β2M genes. A gene present in the MHC of all other vertebrates (ring3) was found in the bacterial artificial chromosome clone, and the close linkage of ring3 and β2M to MHC class I and class II genes was determined by single-strand conformational polymorphism and allele-specific PCR. This study satisfies the long-held conjecture that β2M was linked to the primordial MHC (Ur MHC); furthermore, the apparent stability of the shark genome may yield other genes predicted to have had a primordial association with the MHC specifically and with immunity in general.     

Genetic linkage map of a wheat×spelt cross

 We constructed a genetic map of a cross between the Swiss winter wheat (Triticum aestivum L.) variety Forno and the Swiss winter spelt (Triticum spelta L.) variety Oberkulmer. For the linkage analysis,176 polymorphic RFLP probes and nine microsatellites were tested on 204 F₅ recombinant inbred lines (RILs) of Forno×Oberkulmer revealing 242 segregating marker loci. Thirty five percent of these loci showed significant (P>0.05) deviation from a 1 : 1 segregation, and the percentage of Forno alleles ranged from 21% to 83% for individual marker loci. Linkage analysis was performed with the program MAPMAKER using the Haldane mapping function. Using a LOD threshold of 10, we obtained 37 linkage groups. After finding the best order of marker loci within linkage groups by multi-point analysis we assembled the linkage groups into 23 larger units by lowering the LOD threshold. All except one of the 23 new linkage groups could be assigned to physical chromosomes or chromosome arms according to hybridisation patterns of nulli-tetrasomic lines of Chinese Spring and published wheat maps. This resulted in a genetic map comprising 230 marker loci and spanning 2469 cM. Since the analysed population is segregating for a wide range of agronomically important traits, this genetic map is an ideal basis for the identification of quantitative trait loci (QTLs) for these traits. Received: 3 August 1998 / Accepted: 28 November 1998     

Genetic linkage studies of the human glycosphingolipid β-galactosidases

The genetic linkage relationships of the human glycosphingolipid beta-galactosidases were determined using human--mouse somatic cell hybrids. A new method was devised for the estimation of human galactosylceramide, lactosylceramide, and GMI-ganglioside beta-galactosidase activities in the presence of their mouse counterparts, which takes advantage of the reproducible specific activity of lysosomal hydrolases under a given set of culture conditions and is based on differences in both pH optima and sensitivity to chloride ion. Human and mouse chromosomes were identified by their characteristic banding patterns obtained after quinacrine staining, and the optimum glycolipid beta-galactosidase activity was determined for three different substrates. A ratio was defined for each activity which was the specific activity at the human pH optimum divided by the specific activity at the mouse pH optimum. Linear regression analysis was used to test for concordant segregation between pH ratios for each enzyme and the frequency of occurrence of different human chromosomes in the man--mouse somatic hybrid clones. The results obtained from two independent series of hybrid clones indicated that human beta-galactosidase activities consistently segregated with human chromosome 12 in these somatic cell hybrids.     

Fine mapping of multiple QTL using combined linkage and linkage disequilibrium mapping – A comparison of single QTL and multi QTL methods

Two previously described QTL mapping methods, which combine linkage analysis (LA) and linkage disequilibrium analysis (LD), were compared for their ability to detect and map multiple QTL. The methods were tested on five different simulated data sets in which the exact QTL positions were known. Every simulated data set contained two QTL, but the distances between these QTL were varied from 15 to 150 cM. The results show that the single QTL mapping method (LDLA) gave good results as long as the distance between the QTL was large (> 90 cM). When the distance between the QTL was reduced, the single QTL method had problems positioning the two QTL and tended to position only one QTL, i.e. a "ghost" QTL, in between the two real QTL positions. The multi QTL mapping method (MP-LDLA) gave good results for all evaluated distances between the QTL. For the large distances between the QTL (> 90 cM) the single QTL method more often positioned the QTL in the correct marker bracket, but considering the broader likelihood peaks of the single point method it could be argued that the multi QTL method was more precise. Since the distances were reduced the multi QTL method was clearly more accurate than the single QTL method. The two methods combine well, and together provide a good tool to position single or multiple QTL in practical situations, where the number of QTL and their positions are unknown.     

Characterization and study of a κ-casein-like chymosin-sensitive linkage

The present report is dealing with the identification, in various unrelated proteins, of protein fragments sharing local sequence and structure similarities with the chymosin-sensitive linkage surrounding the Phe-Met/Ile bond of κ-caseins. In all these proteins, this linkage is observed within an exposed β-strand-like structure, as also predicted for κ-caseins. The structure of one of these fragments, included in glutamine synthetase, particularly superimposes well with the conformation observed for a chymosin inhibitor (CP-113972) within the complex it forms with chymosin and can be similarly accommodated by specificity pockets within the enzyme substrate binding cleft. The effect of the enzyme activity of chymosin was thus tested on glutamine synthetase. Chymosin cut the latter at the Phe-Met linkage, suggesting that this system may locally resemble the κ-casein/chymosin complex.     

Genetic linkage mapping of an annual × perennial ryegrass population

Annual (Lolium multiflorum Lam.) and perennial (L. perenne L.) ryegrass are two common forage and turfgrass species grown throughout the world. Perennial ryegrass is most commonly used for turfgrass purposes, and contamination by annual ryegrass, through physical seed mixing or gene flow, can result in a significant reduction in turfgrass quality. Seed certifying agencies in the United States currently use a test called seedling root fluorescence (SRF) to detect contamination between these species. The SRF test, however, can be inaccurate and therefore, the development of additional markers for species separation is needed. Male and female molecular-marker linkage maps of an interspecific annual × perennial ryegrass mapping population were developed to determine the map location of the SRF character and to identify additional genomic regions useful for species separation. A total of 235 AFLP markers, 81 RAPD markers, 16 comparative grass RFLPs, 106 SSR markers, 2 isozyme loci and 2 morphological characteristics, 8-h flowering, and SRF were used to construct the maps. RFLP markers from oat and barley and SSR markers from tall fescue and other grasses allowed the linkage groups to be numbered, relative to the Triticeae and the International Lolium Genome Initative reference population P150/112. The three-generation population structure allowed both male and female maps to be constructed. The male and female maps each have seven linkage groups, but differ in map length with the male map being 537 cm long and the female map 712 cm long. Regions of skewed segregation were identified in both maps with linkage groups 1, 3, and 6 of the male map showing the highest percentage of skewed markers. The (SRF) character mapped to linkage group 1 in both the male and female maps, and the 8-h flowering character was also localized to this linkage group on the female map. In addition, the Sod-1 isozyme marker, which can separate annual and perennial ryegrasses, mapped to linkage group 7. These results indicate that Lolium linkage groups 1 and 7 may provide additional markers and candidate genes for use in ryegrass species separation.Communicated by C. Möllers     

Where is the weak linkage in the globin chain?

Hemoglobinopathies are important inherited disorders with high prevalence in many tropical countries. Prediction of protein nanostructure and function is a great challenge in proteomics and structural genomics. Identifying the point vulnerable to mutation is a new trend in research on disorders at the genomic and proteomic level. A bioinformatics analysis was performed to determine the positions that tend to correspond with peptide motifs in the amino acid sequence of alpha and beta globin chains. To identify the weak linkage in alpha globin and beta globin chains, a new bioinformatics tool, GlobPlot, was used. For the alpha globin chain, 22 positions were identified: the disorders were found at positions 3-8, 38-42, 46-51, and 75-79. For the beta globin chain, 46 positions were identified: the disorders were found at positions 61-146. The study showed that weak linkages in alpha globin and beta globin chains can be identified and provide good information for predicting possible new mutations that could lead to new hemoglobinopathies.     

Exploitation of pepper EST–SSRs and an SSR-based linkage map

As genome and cDNA sequencing projects progress, a tremendous amount of sequence information is becoming publicly available. These sequence resources can be exploited for gene discovery and marker development. Simple sequence repeat (SSR) markers are among the most useful because of their great variability, abundance, and ease of analysis. By in silico analysis of 10,232 non-redundant expressed sequence tags (ESTs) in pepper as a source of SSR markers, 1,201 SSRs were found, corresponding to one SSR in every 3.8 kb of the ESTs. Eighteen percent of the SSR–ESTs were dinucleotide repeats, 66.0% were trinucleotide, 7.7% tetranucleotide, and 8.2% pentanucleotide; AAG (14%) and AG (12.4%) motifs were the most abundant repeat types. Based on the flanking sequences of these 1,201 SSRs, 812 primer pairs that satisfied melting temperature conditions and PCR product sizes were designed. 513 SSRs (63.1%) were successfully amplified and 150 of them (29.2%) showed polymorphism between Capsicum annuum ‘TF68’ and C. chinense ‘Habanero’. Dinucleotide SSRs and EST–SSR markers containing AC-motifs were the most polymorphic. Polymorphism increased with repeat length and repeat number. The polymorphic EST–SSRs were mapped onto the previously generated pepper linkage map, using 107 F₂ individuals from an interspecific cross of TF68 × Habanero. One-hundred and thirtynine EST–SSRs were located on the linkage map in addition to 41 previous SSRs and 63 RFLP markers, forming 14 linkage groups (LGs) and spanning 2,201.5 cM. The EST–SSR markers were distributed over all the LGs. This SSR-based map will be useful as a reference map in Capsicum and should facilitate the use of molecular markers in pepper breeding.Gibum Yi and Je Min Lee equally contributed to this work.     

What chance did Mendel's experiments give him of noticing linkage?

The a priori probability of noticeable linkage among all conceivable experiments of the size reported by Mendel cannot reasonably be taken as greater than 24-36 per cent; and therefore, the frequently heard opinion that his chances of encountering linkage were high, approaching 99-4 per cent, appears to be mistaken.     

Why is there so little intragenic linkage disequilibrium in humans?

The efficient design of association mapping studies relies on a knowledge of the rate of decay of linkage disequilibrium with distance. This rate depends on the population recombination rate, C. An estimate of C for humans is usually obtained from a comparison of physical and genetic maps, assuming an effective population size of approximately 10(4). We demonstrate that under both a constant population size model and a model of long-term exponential growth, there is evidence for more recombination in polymorphism data than is expected from this estimate. An important contribution of gene conversion to meiotic recombination helps to explain our observation, but does not appear to be sufficient. The occurrence of multiple hits at CpG sites and the presence of population structure are not explanations.     

Amino acid–dependent stability of the acyl linkage in aminoacyl-tRNA

Aminoacyl-tRNAs are the biologically active substrates for peptide bond formation in protein synthesis. The stability of the acyl linkage in each aminoacyl-tRNA, formed through an ester bond that connects the amino acid carboxyl group with the tRNA terminal 3′-OH group, is thus important. While the ester linkage is the same for all aminoacyl-tRNAs, the stability of each is not well characterized, thus limiting insight into the fundamental process of peptide bond formation. Here, we show, by analysis of the half-lives of 12 of the 22 natural aminoacyl-tRNAs used in peptide bond formation, that the stability of the acyl linkage is effectively determined only by the chemical nature of the amino acid side chain. Even the chirality of the side chain exhibits little influence. Proline confers the lowest stability to the linkage, while isoleucine and valine confer the highest, whereas the nucleotide sequence in the tRNA provides negligible contribution to the stability. We find that, among the variables tested, the protein translation factor EF-Tu is the only one that can protect a weak acyl linkage from hydrolysis. These results suggest that each amino acid plays an active role in determining its own stability in the acyl linkage to tRNA, but that EF-Tu overrides this individuality and protects the acyl linkage stability for protein synthesis on the ribosome.     

Assignment of six enzyme loci to multipoint linkage groups in Fishes of the genusPoeciliopsis (Poeciliidae): Designation of linkage groups III–V

Three new linkage groups of enzyme loci are described usingPoeciliopsis monacha × P. viriosa-derived interspecific backcross hybrids. Comparison to known linkage groups of the confamilial genusXiphophorus shows homology betweenXiphophorus linkage group I andPoeciliopsis linkage group III,Xiphophorus linkage group II andPoeciliopsis linkage group I, andXiphophorus linkage group IV andPoeciliopsis linkage group IV. Comparison of the gene content of other fish, amphibians, and mammal syntenic groups suggests retention of plesiomorphic vertebrate gene arrangements in at least two poeciliid linkage groups. Expansion of thePoeciliopsis gene map should be of utility in the identification of tumor regulatory genes through demonstration of linkage to biochemical markers.This work was supported by NSF Grants BSR 19355 and BSR 16569 and NIH Grants CA 44303 and CA 39729 to R. S. Nairn and D.C.M. and NIEHS Grant EHS 1P50ES 0384801A1 to R.J.S.     

Unusual conformation of a 3′-thioformacetal linkage in a DNA duplex

Summary The DNA·DNA duplex ·d(GCGCAAAACGCG) (designated duplex III) containing a 3-thioformacetal (3-TFMA) linkage in the center of the sequence was characterized in detail by two- and three-dimensional homonuclear NMR spectroscopy. The NMR results were analyzed and compared with those of two duplexes of the same sequence: One is an unmodified reference sequence and the other contains a formacetal (OCH₂O) linkage at the central T^T step (designated duplex I and duplex II, respectively). In general, the NMR spectra of duplex III closely resemble those of the analogous duplexes I and II, suggesting an overall B-type structure adopted by the 3-TFMA-modified duplex III. Nonetheless, the detection of several distinct spectral features originating from the protons at the modification site is indicative of a local conformation that is clearly different from the corresponding region in duplexes I and II. The 3-thioformacetal linker, in contrast to the formacetal (FMA) linkage, cannot be accommodated in a conformation usually found in natural nucleic acid duplexes. As a consequence, the 3-TFMA-modified T6 sugar adopts an O4-endo form (an intermediate structure between the usual C2-endo and C3-endo forms). This change is accompanied by a change in the (C4–C3–S3–CH₂) dihedral angle and by subsequent adjustments of other torsion angles along the backbone. Notably, this conformational readjustment at the T6–T7 backbone linkage is localized; its collective result has negligible effect on base-base stacking of the T6 and T7 residues. A close examination of the COSY data in all three duplexes reveals a subtle variation in sugar geometry, with more S-type character adopted by the modified duplexes II and III. The results of this study illustrate that, although the difference between FMA and 3-TFMA linkages is merely in the substitution of the T6(O3) in the former by a sulfur atom in the latter, the stereoelectronic difference in a single atom can induce significant local structural distortion in an otherwise well-structured oligonucleotide duplex.Supplementary material available from the authors: One table containing J₁₂, J₁₂ and J₃₄ of duplexes I, II and III.     

Digest: The role of linkage in mimicking “magic traits”

Can divergence in a mating trait increase local adaption by increasing ecological divergence? Servedio and Bürger propose that “pseudomagic traits,” tightly linked complexes consisting of an ecological locus under divergent selection and a locus acting as a mating cue, can effectively mimic pleiotropy. Such pseudomagic traits can form even when linkage between ecological and mating loci is limited.     

Quasi-linkage: a confounding factor in linkage analysis of complex diseases?

Human linkage analysis is based on the assumption that unlinked genomic loci, particularly loci located on non-homologous chromosomes, segregate independently during meiosis. An exception to this rule is the phenomenon of quasi-linkage (QL) that describes the non-random segregation of non-homologous chromosomes, which can undermine the basic concept of linkage. Molecular mechanisms of QL are not clear; however, observations in mice and plants suggest a possible affinity between non-homologous chromosomal regions containing repetitive or like sequences. QL has not been investigated in humans. As QL may generate false linkages in genome scans of complex diseases, we sought to determine whether genomic loci detected in such genome scans exhibit QL. A number of individual markers showing linkage to schizophrenia, asthma, multiple sclerosis, inflammatory bowel disease and type-1 diabetes were tested for QL in a pairwise linkage analysis against all other markers exhibiting evidence for linkage in each specific study. The Marshfield genotype dataset of eight CEPH families was used for this purpose. The best QL lod scores generated from the analysis were within the range of the lukewarm lod scores reported in the majority of linkage studies for complex disorders. In addition, we performed a genome-wide QL analysis on the Marshfield family database which detected eight QL lod scores >6. The replication of the best Marshfield QL scores was performed using the deCODE families and although none of the eight pairs demonstrated independent evidence for QL, three pairs generated maximal lod scores of 0.11, 0.3, and 1.51. In conclusion, although complex disease relevant markers did not produce high QL lod scores, the general phenomenon of QL in humans cannot be excluded and potentially can be a confounding factor in genetic studies of complex traits.     

A first interspecific Oryza sativa×Oryza glaberrima microsatellite-based genetic linkage map

Oryza glaberrima is an endemic African cultivated rice species. To provide a tool for evaluation and utilisation of the potential of O. glaberrima in rice breeding, we developed an interspecific O. glaberrima×Oryza sativa genetic linkage map. It was based on PCR markers, essentially microsatellites and STSs. Segregation of markers was examined in a backcross (O. sativa/O. glaberrima//O. sativa) population. Several traits were measured on the BC₁ plants, and major genes and QTLs were mapped for these traits. Several of these genes correspond well to previously identified loci. The overall map length was comparable to those observed in indica×japonica crosses, indicating that recombination between the two species occurs without limitation. However, three chromosomes show discrepancies with the indica×japonica maps. The colinearity with intraspecific maps was very good, confirming previous cytological observations. A strong segregation-distortion hot spot was observed on chromosome 6 near the waxy gene, indicating the presence of s ₁₀ , a sporo-gametophytic sterility gene previously identified by Sano (1990). The main interests of such a PCR-based map for African rice breeding are discussed, including gene and QTL localisation, marker-assisted selection, and the development of interspecific introgression lines. Received: 1 June 1991 / Accepted: 22 June 1999     

Mechanical functioning of peripheral nerves: linkage with the “mushrooming” effect

Biomechanical properties of nerve have been studied extensively. All neural matrix tissues have been suggested to be the main load-bearing component. Based on the ultrastructure it has been proposed that the architecture of the epineurium allows some degree of extensibility of the nerve. A role of the perineurium could be to withstand the positive endoneurial pressure. The hypothesis is that the mechanical behaviour of nerves is dependent on an interaction between the core swelling pressure and restraint by the outer sheath. Loss of this balance will alter that behaviour. To test this, rat sciatic nerves were subjected to mechanical loading at in vivo and ex vivo tension. Retraction of nerve segments was measured after excision and after incubation at 37°C or freezing. Swelling properties of the nerve were measured by immersion in water or PBS (phosphate buffer solution) with intact or opened epineurium. Results showed a significant decrease in strength and stiffness with an increase in strain of the nerve after excision, compared to in vivo. Retraction was on average 11%. Freezing or incubation at 37°C did not alter retraction. The swelling properties of the nerve demonstrated a significant difference between intact and opened epineurium and similar results for water and PBS, indicating that epineurium is a constraint and that the nerves are underhydrated. The proposed model for the intact nerve is a continuous connective tissue tube surrounding and constraining an inner swelling pressure of the neural core. Loss of integrity of the nerve has detrimental effects on its biomechanical properties.Preliminary forms of this project were presented at the 1st UK Tissue & Cell Engineering Society meeting, Hammersmith Hospital, RPMS, London, 5 July 1999, and at the 7th FESSH, Barcelona, Spain, 22 June 2000. Support was partly given by EU grant QLK3-CT-1999-00625: NEURO-TEC     

A new technique distinguishing α2-3 sialyl linkage from α2-6 linkage in sialyllactoses and sialyl-N-acetyllactosamines by post-source decay fragmentation method of MALDI-TOF mass spectrometry

2-3 and 2-6 sialyl linkage types of sialyllactoses and sialyl-N-acetyllactosamines were analyzed by post-source decay (PSD) fragmentation method using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. A new matrix of norharmane was suited for the MALDI-TOF measurements of sialyl oligosaccharides. The fragment ions B₁ produced by the cleavage of 2-3 sialyl linkages indicate much higher intensity than those produced by the cleavage of 2-6 sialyl linkages in sialyllactoses and sialyl-N-acetyllactosamines. Thus, 2-3 sialyl linkages cleave much easier than 2-6 sialyl linkages in MALDI-PSD fragmentation method. These results suggest that the new techniques using PSD fragmentation of MALDI-TOF mass spectrometry enables us to distinguish 2-3 sialyl linkage from 2-6 linkage in sialyl oligosaccharides.     

Estimation and test for linkage between markers: a comparison of lod score and χ 2 test in a linkage study of maritime pine (Pinus pinaster Ait.)

The first step in the construction of a linkage map involves the estimation and test for linkage between all possible pairs of markers. The lod score method is used in many linkage studies for the latter purpose. In contrast with classical statistical tests, this method does not rely on the choice of a first-type error level. We thus provide a comparison between the lod score and a ² test on linkage data from a gymnosperm, the maritime pine. The lod score appears to be a very conservative test with the usual thresholds. Its severity depends on the type of data used.