Use of DNA fingerprints for the detection of major genes for quantitative traits in domestic species

The detection of marker loci linked to major genes or quantitative trait loci (QTL) of large effect in farm animal populations is of great potential value, both because it allows the easy manipulation of the major genes and because it provides a possible route to their ultimate isolation. At present the number of markers available is limited in farm animals. DNA fingerprints provide a promising source of informative marker loci and have the advantage that several loci can be detected on a single Southern hybridization. The disadvantage of DNA fingerprints is the difficulty in determining allelism of DNA fingerprint bands in different pedigrees and the fact that not all potentially resolvable loci can be resolved in a single pedigree. With probes capable of detecting 50 randomly distributed loci, about 50% of the genome of a typical domestic mammal might be expected to be closely linked to a marker (at a distance of 0.2 Morgans or less). If a proportion of DNA fingerprint loci prove to be clustered near chromosomal telomeres or elsewhere in the genome, coverage will be less. In order to detect linkage to a major gene, sires known or suspected to be heterozygous are used to produce large half-sibships, all animals in the pedigree are DNA fingerprinted and the phenotypes of the offspring are recorded. Where several heterozygous sires are available, sires can be selected in an attempt to maximize the number of marker loci resolved. The optimum number of sires needed to produce pedigrees will depend upon the size of the major gene, the number of DNA fingerprint probes available and the characteristics of the DNA fingerprints produced, but often one or two pedigrees will be optimum. Monte Carlo simulation was used to explore the power of detection of linkage between a major gene and a marker locus in a backcross. Maximum likelihood and analysis of variance of mean differences between marker genotypes were of similar power, but maximum likelihood provided reasonable estimates of the major gene effect and its linkage to the marker under some circumstances. One hundred offspring informative for the segregation of a marker would provide reasonable power for the detection of a gene causing a difference between the heterozygote and the homozygote of at least one within-sire, within-genotype standard deviation when linkage was very close (0.05 or less).(ABSTRACT TRUNCATED AT 400 WORDS)     

Transmission test for linkage disequilibrium: the insulin gene region and insulin-dependent diabetes mellitus (IDDM).

A population association has consistently been observed between insulin-dependent diabetes mellitus (IDDM) and the "class 1" alleles of the region of tandem-repeat DNA (5'' flanking polymorphism [5''FP]) adjacent to the insulin gene on chromosome 11p. This finding suggests that the insulin gene region contains a gene or genes contributing to IDDM susceptibility. However, several studies that have sought to show linkage with IDDM by testing for cosegregation in affected sib pairs have failed to find evidence for linkage. As means for identifying genes for complex diseases, both the association and the affected-sib-pairs approaches have limitations. It is well known that population association between a disease and a genetic marker can arise as an artifact of population structure, even in the absence of linkage. On the other hand, linkage studies with modest numbers of affected sib pairs may fail to detect linkage, especially if there is linkage heterogeneity. We consider an alternative method to test for linkage with a genetic marker when population association has been found. Using data from families with at least one affected child, we evaluate the transmission of the associated marker allele from a heterozygous parent to an affected offspring. This approach has been used by several investigators, but the statistical properties of the method as a test for linkage have not been investigated. In the present paper we describe the statistical basis for this "transmission test for linkage disequilibrium" (transmission/disequilibrium test [TDT]). We then show the relationship of this test to tests of cosegregation that are based on the proportion of haplotypes or genes identical by descent in affected sibs. The TDT provides strong evidence for linkage between the 5''FP and susceptibility to IDDM. The conclusions from this analysis apply in general to the study of disease associations, where genetic markers are usually closely linked to candidate genes. When a disease is found to be associated with such a marker, the TDT may detect linkage even when haplotype-sharing tests do not.     

The detection of linkage and heterogeneity in nuclear families for complex disorders: one versus two marker loci.

Using exact expected likelihoods, we have computed the average number of phase-unknown nuclear families needed to detect linkage and heterogeneity. We have examined the case of both dominant and recessive inheritance with reduced penetrance and phenocopies. Most of our calculations have been carried out under the assumption that 50% of families are linked to a marker locus. We have varied both the number of offspring per family and the sampling scheme. We have also investigated the increased power when the disease locus is midway between two marker loci 10 cM apart. For recessive inheritance, both linkage and heterogeneity can be detected in clinically feasible sample sizes. For dominant inheritance, linkage can be detected but heterogeneity cannot be detected unless larger sibships (four offspring) are sampled or two linked markers are available. As expected, if penetrance is reduced, sampling families with all sibs affected is most efficient. Our results provide a basis for estimating the amount of resources needed to find genes for complex disorders under conditions of heterogeneity.     

Segregation of HLA A,B haplotypes and the distribution of antigen mismatches between mother and offspring in Hutterite families

Segregation of HLA haplotypes and offspring genotype distributions were analyzed in families from an inbred Caucasoid population, the Dariusleut Hutterite Brethren. Both parents and from one to 12 offspring were typed for HLA-A and -B antigens in 108 families. Segregation of paternal haplotypes was analyzed conditional on sibship size in 95 sibships (a total of 547 offspring), and segregation of maternal haplotypes, in 90 sibships (a total of 515 offspring). The distribution of the number of different genotypes among the offspring was analyzed conditional on sibship size in 90 families (515 offspring) where four equiprobable genotypes were expected. The distribution of the number of antigenic differences or mismatches for broad specificities between mother and offspring was analyzed in pooled family data consisting of a total of 377 offspring comprising 68 families. Compared with the multinomial distribution of segregation classes of haplotypes, there was no significant departure (probability .05 or less) from the expected segregation ratio for either paternal or maternal haplotypes. Compared with the multinomial distribution of the number of genotypes among the offspring, only two of the 11 sibship sizes had configurations that exceeded the 5% level of significance. Given the number of statistical tests performed, it is likely that these results could be explained by chance variation. Finally, there was no relative deficiency of offspring who were less mismatched with their mother for HLA-A and -B broad specificities. Therefore, if HLA-A,B region variation does have a major effect on the differential survival of fetuses in some families, it is an uncommon factor among fertile couples from this inbred population.     

Testing for segregation distortion in genetic scoring data from backcross or doubled haploid populations

It is important that breeders have the means to assess genetic scoring data for segregation distortion because of its probable effect on the design of efficient breeding strategies. Scoring data is usually assessed for segregation distortion by separate nonindependent chi2 tests at each locus in a set of marker loci. This analysis gives the loci most affected by selection if it exists, but it cannot give a statistically correct test for the presence or absence of selection in a linkage group as a whole. I have used a combined test based on the statistic, which is the most significant P-value from the above tests, called the single locus test. I have also derived mathematically a new combined statistical test, the overall test, for segregation distortion that requires genetic scoring data for a single linkage group. This test also takes genetic linkage into account. Using a range of marker densities and population sizes, simulations were carried out, to compare the power of these two statistical tests to detect the effect of selection at one or two loci. The single locus test was always found to be more powerful than the overall test, but the single locus test required a more complicated P-value correction. For the single locus test, approximate correction factors for the P-values are given for a range of marker densities and genetic lengths.     

DSLINK: a computer program for gene-centromere linkage analysis in families with a trisomic offspring.

Trisomic individuals provide information for gene-centromere mapping, since two of the four chromatids in a meiotic tetrad can be recovered. When centromeric markers are available, linkage analysis between the centromere and any marker locus can be performed in nuclear families having one or more trisomic offspring. Since conventional linkage programs consider only disomic individuals, we have written a FORTRAN computer program, DSLINK, that performs gene-centromere linkage analysis on the basis of information on trisomic and disomic offspring. This program makes it possible to study the relationship between recombination and chromosome segregation.     

Genetic markers linked to quantitative traits in poultry

This study utilized DNA fingerprints and crosses of two genetically distinct lines of layer-type chickens to identify genetic markers linked to quantitative trait loci (QTL). In phase I, back-cross (BC¹) hens were separately ranked for each of eight traits and then blood pools were produced in groups along each phenotypic distribution. The DNA was isolated from the blood pools and used in a gradient analysis to screen for DNA fingerprint bands that exhibited intensity gradients associated with the phenotypic traits. To identify linkage of bands with QTL and to estimate band effects, F₂ progeny were produced in phase II from the phase I BC, population. A single-trait animal model was used for analysis of associations of all individual DNA fingerprint bands of sires and their progeny phenotypic performance. Twenty fingerprint bands, only two of which had shown trait-associated gradients in phase I, were identified by the animal model analysis of the progeny test as QTL linked (P≤005) to specific traits of growth, reproduction and egg quality. These 20 bands warrant further study as potentially valuable molecular markers for QTL.     

Reconstructing parental genotypes when testing for linkage in the presence of association

Various family-based association methods have recently been proposed that allow testing for linkage in the presence of linkage disequilibrium between a marker and a disease even if there is only incomplete parental-genotype information. For some families, it may be possible to reconstruct missing parental genotypes from the genotypes of their offspring. Treating such a reconstructed family as if parental genotypes have been typed, however, can introduce bias. The reconstruction-combined transmission/disequilibrium test (RC-TDT) and its X-chromosomal counterpart, XRC-TDT, employ parental-genotype reconstruction and correct for the biases involved in this reconstruction without relying on population marker allele frequencies. For the two tests, exact P values can be obtained by numerically calculating the convolution of the null distributions corresponding to the families in the sample.     

Transmission/Disequilibrium Tests Incorporating Unaffected Offspring

We propose a new method for family-based tests of association and linkage called transmission/disequilibrium tests incorporating unaffected offspring (TDTU). This new approach, constructed based on transmission/disequilibrium tests for quantitative traits (QTDT), provides a natural extension of the transmission/disequilibrium test (TDT) to utilize transmission information from heterozygous parents to their unaffected offspring as well as the affected offspring from ascertained nuclear families. TDTU can be used in various study designs and can accommodate all types of independent nuclear families with at least one affected offspring. When the study sample contains only case-parent trios, the TDTU is equivalent to TDT. Informative-transmission disequilibrium test (i-TDT) and generalized disequilibrium test(GDT) are another two methods that can use information of both unaffected offspring and affected offspring. In contract to i-TDT and GDT, the test statistic of TDTU is simpler and more explicit, and can be implemented more easily. Through computer simulations, we demonstrate that power of the TDTU is slightly higher compared to i-TDT and GDT. All the three methods are more powerful than method that uses affected offspring only, suggesting that unaffected siblings also provide information about linkage and association.     

DNA fingerprinting analysis of Booroola pedigrees: a search for linkage to the Booroola gene

Seven minisatellite probes from a variety of sources were used to analyse 11 paternal half-sib families in which the Booroola gene was segregating. A total of 402 bands that showed segregation in the pedigrees were examined for linkage to the Booroola gene. None of the bands showed segregation with the Booroola gene. The most likely evidence for a linked band was produced by the HaRas HVR probe in Family 902 (=0.0; LOD 2.3). The conclusion, however, is that the minisatellite probes used in this study could not be used as markers for the Booroola gene. The study highlighted problems associated with the use of minisatellite probes in linkage studies in half-sib families. The complex banding patterns found on fingerprinting gels was a major source of scoring error. In a few cases both of the sire's alleles could be identified at a particular locus, but in most cases only one of the alleles could be identified. For the most part, the bands had to be treated as dominant alleles. The contribution of dam alleles to the banding pattern could only be estimated. There was an indication that minisatellite loci in sheep are clustered in particular regions of the sheep genome as the rate at which bands segregated with each other was higher than one would expect from loci randomly distributed throughout the genome.     

A multistage testing strategy for detection of quantitative trait Loci affecting disease resistance in Atlantic salmon

A multistage testing strategy to detect QTL for resistance to infectious salmon anemia (ISA) in Atlantic salmon is proposed. First, genotyping of amplified fragment length polymorphisms (AFLP) and a transmission disequilibrium test (TDT) were carried out using dead offspring from a disease resistance challenge test. Second, AFLP genotyping among survivors followed by a Mendelian segregation test was performed. Third, within-family survival analyses using all offspring were developed and applied to significant TDT markers with Mendelian inheritance. Maximum-likelihood methodology was developed for TDT with dominant markers to exploit linkage disequilibrium within families. The strategy was tested with two full-sib families of Atlantic salmon sired by the same male and consisting of 79 offspring in total. All dead offspring from the two families were typed for 64 primer combinations, resulting in 340 scored markers. There were 26 significant results out of 401 TDTs using dead offspring. In the second stage, only 17 marker families showed Mendelian segregation and were tested in survival analysis. A permutation test was performed for all survival analyses to compute experimentwise P-values. Two markers, aaccac356 and agccta150, were significant at P < 0.05 when accounting for multiple testing in the survival analyses. The proposed strategy might be more powerful than current mapping strategies because it reduces the number of tests to be performed in the last testing stage.     

The detection of linkage disequilibrium between closely linked markers: RFLPs at the AI-CIII apolipoprotein genes.

Study of very closely linked DNA variants at various loci has frequently shown linkage disequilibrium. We studied three closely linked RFLPs at the apolipoprotein AI-CIII locus. Two variants detected by MspI and SstI were in strong linkage disequilibrium; but when conventional statistical tests were used, a third variant (PstI), located between the MspI and SstI markers, appeared to be in linkage equilibrium with these two "outside" markers. Similar discrepancies from the expected monotone relationship between physical distance and linkage disequilibrium have been reported by others. To investigate these discrepancies, the power to detect linkage disequilibrium was calculated. It could be shown that, for the gene frequencies encountered, very large sample sizes would be required to demonstrate negative (i.e., repulsion-phase) linkage disequilibrium. Such numbers are usually very difficult to attain in human studies. Failure to demonstrate linkage disequilibrium by conventional methods therefore does not imply its absence. Appropriate nomograms and tables are provided.     

Linkage analyses of five chromosome 4 markers localizes the facioscapulohumeral muscular dystrophy (FSHD) gene to distal 4q35

The genetic locus for facioscapulohumeral muscular dystrophy (FSHD) has been mapped to chromosome 4. We have examined linkage to five chromosome 4q DNA markers in 22 multigenerational FSHD families. Multipoint linkage analyses of the segregation of four markers in the FSHD families and in 40 multigenerational mapping families from the Centre d'Etude du Polymorphisme Humaine enabled these loci and FSHD to be placed in the following order: cen-D4S171-factor XI-D4S163-D4S139-FSHD-qter. One interval, D4S171-FSHD, showed significant sex-specific differences in recombination. Homogeneity tests supported linkage of FSHD to these 4q DNA markers in all of the families we studied. The position of FSHD is consistent with that generated by other groups as members of an international FSHD consortium.     

Minisatellite DNA markers in the chicken genome

This paper reports the detailed characterization of multilocus minisatellite DNA fingerprints in the chicken. Results are presented of DNA fingerprint segregation analyses carried out in three chicken pedigrees, calculating the number of detected loci, testing for Mendelian inheritance, and cosegregation among fingerprint bands. Two pedigrees (families 1 and 2) were analysed using the Jeffreys probes 33.6 and 33.15 only, and one pedigree (family 3) was analysed using 33.6, 33.15. 3′α-globin HVR and M13 protein III gene repeat. Mean band transmission frequencies in families 1 and 2 were near to the Mendelian expectation of 0.5 and no mutations were observed. Family 3 showed transmission frequencies slightly exceeding 0.5. Linkage among bands was higher than observed in some other avian species, with each allele represented by a mean of 1.48 HaeIII fragments. Cosegregation of heterozygous parental fragments representing distinguishable loci followed the expected binomial distribution. The number of minisatellites detectable by the four probes was estimated to be 217. The pattern of cosegregation among those minisatellite loci was tested against that expected for different levels of recombination through the use of a simulation model. We conclude that most minisatellites are unlinked and probably widely dispersed in the chicken genome.     

Detecting linkage for genetically heterogeneous diseases and detecting heterogeneity with linkage data.

Interest in searching for genetic linkage between diseases and marker loci has been greatly increased by the recent introduction of DNA polymorphisms. However, even for the most well-behaved Mendelian disorders, those with clear-cut mode of inheritance, complete penetrance, and no phenocopies, genetic heterogeneity may exist; that is, in the population there may be more than one locus that can determine the disease, and these loci may not be linked. In such cases, two questions arise: (1) What sample size is necessary to detect linkage for a genetically heterogeneous disease? (2) What sample size is necessary to detect heterogeneity given linkage between a disease and a marker locus? We have answered these questions for the most important types of matings under specified conditions: linkage phase known or unknown, number of alleles involved in the cross at the marker locus, and different numbers of affected and unaffected children. In general, the presence of heterogeneity increases the recombination value at which lod scores peak, by an amount that increases with the degree of heterogeneity. There is a corresponding increase in the number of families necessary to establish linkage. For the specific case of backcrosses between disease and marker loci with two alleles, linkage can be detected at recombination fractions up to 20% with reasonable numbers of families, even if only half the families carry the disease locus linked to the marker. The task is easier if more than two informative children are available or if phase is known. For recessive diseases, highly polymorphic markers with four different alleles in the parents greatly reduce the number of families required.     

The impact of transmission-ratio distortion on allele sharing in affected sibling pairs

There have been recent reports of transmission-ratio distortion (TRD) or segregation distortion in families not selected for genetic disease. If TRD exists but is ignored, linkage studies searching for disease genes in affected relatives may be misinterpreted. We show that the identical-by-descent sharing patterns for affected sib pairs are strongly affected by TRD and, further, that the estimated statistical significance of a sib-pair linkage study may be extremely biased. However, we also show that, if TRD is suspected during the planning stage of a study, the planned sample size of the study needs to be increased by only a small amount to maintain the desired power.     

Testing quantitative traits for association and linkage in the presence or absence of parental data

Zhu and Elston developed a transmission disequilibrium test for quantitative traits by defining a linear transformation to condition out founder information. The method tests the null hypothesis of no linkage or association and can be applied to general pedigree structures. However, this method requires both genotype and phenotype parental information, which may be difficult to obtain. In this paper, we describe parametric and non-parametric methods to relax this requirement when only nuclear families are sampled. We show that neither method is affected by population stratification in the absence of linkage. The statistical power and validity of the tests are investigated by simulation. A simple simulation method to calculate the power of the nonparametric method is also discussed. In practice, the data may have some families with parental phenotype and genotype information available and some without. We briefly discuss how all the data may be analyzed jointly.     

A test for linkage and association in general pedigrees: the pedigree disequilibrium test

Family-based tests of linkage disequilibrium typically are based on nuclear-family data including affected individuals and their parents or their unaffected siblings. A limitation of such tests is that they generally are not valid tests of association when data from related nuclear families from larger pedigrees are used. Standard methods require selection of a single nuclear family from any extended pedigrees when testing for linkage disequilibrium. Often data are available for larger pedigrees, and it would be desirable to have a valid test of linkage disequilibrium that can use all potentially informative data. In this study, we present the pedigree disequilibrium test (PDT) for analysis of linkage disequilibrium in general pedigrees. The PDT can use data from related nuclear families from extended pedigrees and is valid even when there is population substructure. Using computer simulations, we demonstrated validity of the test when the asymptotic distribution is used to assess the significance, and examined statistical power. Power simulations demonstrate that, when extended pedigree data are available, substantial gains in power can be attained by use of the PDT rather than existing methods that use only a subset of the data. Furthermore, the PDT remains more powerful even when there is misclassification of unaffected individuals. Our simulations suggest that there may be advantages to using the PDT even if the data consist of independent families without extended family information. Thus, the PDT provides a general test of linkage disequilibrium that can be widely applied to different data structures.     

DNA Fingerprinting and Analysis of Population Structure in the Chestnut Blight Fungus, Cryphonectria Parasitica

We analyzed DNA fingerprints in the chestnut blight fungus, Cryphonectria parasitica, for stability, inheritance, linkage and variability in a natural population. DNA fingerprints resulting from hybridization with a dispersed moderately repetitive DNA sequence of C. parasitica in plasmid pMS5.1 hybridized to 6-17 restriction fragments per individual isolate. In a laboratory cross and from progeny from a single perithecium collected from a field population, the presence/absence of 11 fragments in the laboratory cross and 12 fragments in the field progeny set segregated in 1:1 ratios. Two fragments in each progeny set cosegregated; no other linkage was detected among the segregating fragments. Mutations, identified by missing bands, were detected for only one fragment in which 4 of 43 progeny lacked a band present in both parents; no novel fragments were detected in any progeny. All other fragments appeared to be stably inherited. Hybridization patterns did not change during vegetative growth or sporulation. However, fingerprint patterns of single conidial isolates of strains EP155 and EP67 were found to be heterogenous due to mutations that occurred during culturing in the laboratory since these strains were first isolated in 1976-1977. In a population sample of 39 C. parasitica isolates, we found 33 different fingerprint patterns with pMS5.1. Most isolates differed from all other isolates by the presence or absence of several fragments. Six fingerprint patterns each occurred twice. Isolates with identical fingerprints occurred in cankers on the same chestnut stems three times; isolates within the other three pairs were isolated from cankers more than 5 m apart. The null hypothesis of random mating in this population could not be rejected if the six putative clones were removed from the analysis. Thus, a rough estimate of the clonal fraction of this population is 6 in 39 isolates (15.4%).     

Accounting for linkage in family-based tests of association with missing parental genotypes

In studies of complex diseases, a common paradigm is to conduct association analysis at markers in regions identified by linkage analysis, to attempt to narrow the region of interest. Family-based tests for association based on parental transmissions to affected offspring are often used in fine-mapping studies. However, for diseases with late onset, parental genotypes are often missing. Without parental genotypes, family-based tests either compare allele frequencies in affected individuals with those in their unaffected siblings or use siblings to infer missing parental genotypes. An example of the latter approach is the score test implemented in the computer program TRANSMIT. The inference of missing parental genotypes in TRANSMIT assumes that transmissions from parents to affected siblings are independent, which is appropriate when there is no linkage. However, using computer simulations, we show that, when the marker and disease locus are linked and the data set consists of families with multiple affected siblings, this assumption leads to a bias in the score statistic under the null hypothesis of no association between the marker and disease alleles. This bias leads to an inflated type I error rate for the score test in regions of linkage. We present a novel test for association in the presence of linkage (APL) that correctly infers missing parental genotypes in regions of linkage by estimating identity-by-descent parameters, to adjust for correlation between parental transmissions to affected siblings. In simulated data, we demonstrate the validity of the APL test under the null hypothesis of no association and show that the test can be more powerful than the pedigree disequilibrium test and family-based association test. As an example, we compare the performance of the tests in a candidate-gene study in families with Parkinson disease.