Utility and efficiency of linked marker genes for genetic counseling. II. Identification of linkage phase by offspring phenotypes

For a linked marker locus to be useful for genetic counseling, the counselee must be heterozygous for both disease and marker loci and his or her linkage phase must be known. It is shown that when the phenotypes of the counselee's previous children for the disease and marker loci are known, the linkage phase can often be inferred with a high probability, and thus it is possible to conduct genetic counseling. To evaluate the utility of linked marker genes for genetic counseling, the accuracy of prediction of the risk for a prospective child with a given marker gene to develop the genetic disease and the proportion of families in which a particular marker locus can be used for genetic counseling are studied for X-linked recessive, autosomal dominant, and autosomal recessive diseases. In the case of X-linked genetic diseases, information from children is very useful for determining the linkage phase of the counselee and predicting the genetic disease. In the case of autosomal dominant diseases, not all children are informative, but if the number of children is large, the phenotypes of children are often more informative than the information from grandparents. In the case of autosomal recessive diseases, information from grandparents is usually useless, since they show a normal phenotype for the disease locus. If we use information on the phenotypes of children, however, the linkage phase of the counselee and the risk of a prospective child can be inferred with a high probability. The proportion of informative families depends on the dominance relationship and frequencies of marker alleles, and the number of children. In general, codominant markers are more useful than are dominant markers, and a locus with high heterozygosity is more useful than is a locus with low heterozygosity.     

Linkage relationships and gene order around the locus for X-linked retinoschisis.

X-linked recessive retinoschisis (RS) is a hereditary disorder with variable clinical features. The main symptoms are poor sight; radial, cystic macula degeneration; and peripheral superficial retinal detachment. The disease is quite common in Finland, where at least 300 hemizygous males have been diagnosed. We used nine polymorphic DNA markers to study the localization of RS on the short arm of the X chromosome in 31 families comprising 88 affected persons. Two-point linkage results confirmed close linkage of the RS gene to the marker loci DXS43, DXS16, DXS207, and DXS41 and also revealed close linkage to the marker loci DXS197 and DXS9. Only one recombination was observed between DXS43 and RS in 59 informative meioses, giving a maximum lod score of 13.87 at the recombination fraction .02. No recombinations were observed between the RS locus and DXS9 and DXS197 (lods between 3 and 4), but at neither locus was the number of informative meioses sufficient to provide reliable estimates of recombination fractions. The most likely gene order on the basis of multilocus analysis was Xpter-DXS85-(DXS207,DXS43)-RS-DXS41-DXS 164-Xcen. Because multilocus linkage analysis indicated that the most probable location of RS is proximal to DXS207 and DXS43 and distal to DXS41, these three flanking markers are the closest and most informative markers currently available for carrier detection.     

Utility and efficiency of linked marker genes for genetic counseling. III. Proportion of informative families under linkage disequilibrium.

A marker locus closely linked to a disease locus is often useful for genetic counseling provided that a counselee is heterozygous at both disease and marker loci. Furthermore, the linkage phase of these genes in the counselee must be known. When the linkage between the disease and marker loci is very close, one often finds linkage disequilibrium between the loci. To evaluate the effect of such nonrandom associations on the utility of linked marker genes for genetic counseling, the proportion of informative families is studied for X-linked recessive and autosomal dominant diseases. This proportion is higher for X-linked genes than for autosomal genes, if other factors are the same. In general, codominant markers are more useful than dominant markers. Also, under appropriate conditions, the proportion of informative families is higher when linkage disequilibrium is present. The results obtained in this paper are useful for evaluating the utility of polymorphic restriction endonuclease cleavage sites as markers in genetic counseling.     

Genetic mapping of aphicarus -- a sex-linked locus controlling a wing polymorphism in the pea aphid (Acyrthosiphon pisum)

We have initiated research to determine the genetic basis of a male wing polymorphism in the pea aphid Acyrthosiphon pisum (Hemiptera: Aphididae). Previous studies showed that this polymorphism is controlled by a single biallelic locus, which we name aphicarus (api), on the X chromosome. Our objectives were to confirm that api segregates as a polymorphism of a single gene on the X chromosome, and to obtain molecular markers flanking api that can be used as a starting point for high-resolution genetic and physical mapping of the target region, which will ultimately allow the cloning of api. We have established an F2 population segregating for api and have generated X-linked AFLP markers. The segregation pattern of api in the F2 population shows that the male wing polymorphism segregates as a polymorphism of a single gene, or set of closely linked genes on the X chromosome. Using a subset of 78 F2 males, we have constructed a linkage map of the chromosomal region encompassing api using seven AFLP markers. The map spans 74.1 cM and we have mapped api to an interval of 10 cM. In addition, we confirmed X linkage of our AFLP markers and api by using one X-linked marker developed in an earlier study. Our study presents the first mapping of a gene with known function in aphids, and the results indicate that target gene mapping in aphids is feasible.     

Bayesian analysis of linkage between genetic markers and quantitative trait loci. II. Combining prior knowledge with experimental evidence

Summary A Bayesian method was developed for identifying genetic markers linked to quantitative trait loci (QTL) by analyzing data from daughter or granddaughter designs and single markers or marker pairs. Traditional methods may yield unrealistic results because linkage tests depend on number of markers and QTL gene effects associated with selected markers are overestimated. The Bayesian or posterior probability of linkage combines information from a daughter or granddaughter design with the prior probability of linkage between a marker locus and a QTL. If the posterior probability exceeds a certain quantity, linkage is declared. Upon linkage acceptance, Bayesian estimates of marker-QTL recombination rate and QTL gene effects and frequencies are obtained. The Bayesian estimates of QTL gene effects account for different amounts of information by shrinking information from data toward the mean or mode of a prior exponential distribution of gene effects. Computation of the Bayesian analysis is feasible. Exact results are given for biallelic QTL, and extensions to multiallelic QTL are suggested.     

A gene for dominant nonspecific X-linked mental retardation is located in Xq28.

A large family (MRX48) with a nonspecific X-linked mental retardation condition is described. An X-linked semidominant inheritance is suggested by the segregation in three generations of a moderate to severe mental retardation in seven males and by a milder intellectual impairment in two females, without any specific clinical, radiological, or biological feature. Two-point linkage analysis demonstrated significant linkage between the disorder and several markers in Xq28 (maximum LOD score [Zmax] = 2.71 at recombination fraction [theta] = 0); multipoint linkage analyses confirmed the significant linkage with a Zmax of 3.3 at theta = 0, at DXS1684. A recombination event observed with the flanking marker DXS8011 delineates a locus between this marker and the telomere. The approximate length of this locus is 8-9 cM, corresponding to 5.5-6 Mb. In an attempt to explain the variable intellectual impairment in females, we examined X-chromosome inactivation in all females of the family. Inactivation patterns in lymphocytes were random or moderately skewed, and no correlation between the phenotypic status and a specific inactivation pattern was observed. The interval of assignment noted in this family overlaps with five MRX loci previously reported in Xq28.     

A strategy for constructing high-resolution genetic maps of the human genome: a genetic map of chromosome 17p, ordered with meiotic breakpoint-mapping panels.

Genetic linkage analyses with genotypic data obtained from four CEPH reference families initially assigned 24 new PCR-based markers to chromosome 17 and located the markers at specific intervals of an existing genetic map of chromosome 17p. Each marker was additionally genotyped with an ordered set of obligate, phase-known recombinant chromosomes. The breakpoint-mapping panels for each family consisted of two parents, one sib with a nonrecombinant chromosome, and one or more sibs with obligate recombinant chromosomes. The relative order of markers was determined by sorting segregation patterns of new markers and ordered anchor markers and by minimizing double-recombination events. Consistency of segregation patterns with multiple flanking loci constituted support for order. A genetic map of chromosome 17p was completed with 39 markers in 23 clusters, with an average space of 3 cM between clusters. The collection of informative genotypes was highly efficient, requiring fivefold fewer genotypes than would be collected with all the CEPH families. Given the availability of large numbers of highly informative PCR-based markers, meiotic breakpoint mapping should facilitate construction of a human genomic map with 1-cM resolution.     

The Localization of a Gene Causing X-Linked Cleft Palate and Ankyloglossia (CPX) in an Icelandic Kindred Is between DXS326 and DXYS1X

The locus responsible for X-linked, nonsyndromic cleft palate and/or ankyloglossia (CPX) has previously been mapped to the proximal long arm of the human X chromosome between Xq21.31 and q21.33 in an Icelandic kindred. We have extended these studies by analyzing an additional 14 informative markers in the family as well as including several newly investigated family members. Recombination analysis indicates that the CPX locus is more proximal than previously thought, within the interval Xq21.1-q21.31. Two recombinants place DXYS1X as the distal flanking marker, while one recombinant defines DXS326 as the proximal flanking marker, an interval of less than 5 cM. Each of the flanking markers recombines with the CPX locus, giving 2-point lod scores of Z_max = 4.16 at θ = 0.08 (DXS326) and Z_max = 5.80 at θ = 0.06 (DXYS1X).     

Interval mapping of quantitative trait loci with selective DNA pooling data

Selective DNA pooling is an efficient method to identify chromosomal regions that harbor quantitative trait loci (QTL) by comparing marker allele frequencies in pooled DNA from phenotypically extreme individuals. Currently used single marker analysis methods can detect linkage of markers to a QTL but do not provide separate estimates of QTL position and effect, nor do they utilize the joint information from multiple markers. In this study, two interval mapping methods for analysis of selective DNA pooling data were developed and evaluated. One was based on least squares regression (LS-pool) and the other on approximate maximum likelihood (ML-pool). Both methods simultaneously utilize information from multiple markers and multiple families and can be applied to different family structures (half-sib, F2 cross and backcross). The results from these two interval mapping methods were compared with results from single marker analysis by simulation. The results indicate that both LS-pool and ML-pool provided greater power to detect the QTL than single marker analysis. They also provide separate estimates of QTL location and effect. With large family sizes, both LS-pool and ML-pool provided similar power and estimates of QTL location and effect as selective genotyping. With small family sizes, however, the LS-pool method resulted in severely biased estimates of QTL location for distal QTL but this bias was reduced with the ML-pool.     

Statistical considerations for genome-wide scans: design and application of a novel software package POLYMORPHISM

OBJECTIVE: Given the cost and complexity of genome-wide scans, optimization of study design is of critical importance. Available algorithms only partially satisfy this need. We designed a software package called 'POLYMORPHISM' to meet these needs. METHODS: The program is designed to calculate linkage parameters for both 'single-point' and 'two-point' settings that are applicable also to incompletely informative microsatellite markers. In single-point analysis, the heterozygosity, polymorphism information content (PIC) and linkage information content (LIC) statistics based on marker allele frequencies are provided. In two-point analysis, joint PIC values for two markers, the conditional probability of detecting linkage phase, the frequency of double heterozygotes and the expected number of informative meioses are calculated. RESULTS: Results were obtained using S.A.G.E./DESPAIR (Design of Linkage Studies Based on Pairs of Relatives) in addition to applying this program to a Centre d'Etude du Polymorphisme pedigree-derived genotyping data set, which estimated critical parameters used in a two-stage genome scan. A single nucleotide polymorphism (SNP)-based one-stage genomic screen strategy is also considered. CONCLUSIONS: LIC values are crucial for getting accurate estimates on those parameters that are important for a two-stage genome screening study. Optimization of the cost-effectiveness of an SNP-based genomic screen strategy is possible by modeling a balance between marker information content and marker density.     

Germ-line mosaicism simulates genetic heterogeneity in Wiskott-Aldrich syndrome.

The Wiskott-Aldrich syndrome (IMD2) is an X-linked recessive immunodeficiency. Initial linkage studies mapped the disease locus on the proximal short arm of the X chromosome, a localization which was further refined to the interval framed by DXS7 and DXS14. We have recently shown that a novel hypervariable locus, DXS255, is very closely linked to the disease gene and is likely to be, at present, the marker closest to the disease gene. The analysis of one family, however, displayed conflicting linkage results, as all of the informative markers situated in the Xp11-q22 region appeared to recombine with the disease locus in two "phase-known" meioses. We have shown by X-inactivation studies that the segregation of the disease through three obligate carrier females in this family originates from a grandpaternal mosaicism, which accounts for the apparent recombinations. This shows that germ-line mosaicism can simulate genetic heterogeneity in linkage studies.     

Etiological heterogeneity in X-linked spastic paraplegia.

We describe a large family (K313) having 12 males affected with X chromosome-linked recessive hereditary spastic paraplegia (HSP). The disease phenotype in K313 is characterized by hyperreflexia and a spastic gait, but intelligence is normal. Carrier females have normal gait and unremarkable neurologic profiles. Eight widely spaced X-linked DNA markers were used to genotype 43 family members. In contrast to a published study of another family, in whom complete linkage of X-linked recessive HSP to distal chromosome Xq markers DXS15 and DXS52 was reported, we observed complete linkage with two DNA markers, pYNH3 and DXS17, located on the middle of the long arm of the X chromosome. These data have been combined with linkage data from a large reference panel of normal families to localize the new X-chromosome marker, pYNH3, and to provide evidence of significant locus heterogeneity between phenotypically distinct forms of X-linked recessive HSP.     

The use of multiple restriction fragment length polymorphisms in prenatal risk estimation. II. Conditional risk distributions

Although analytical procedures for multiple marker risk estimation are now well established, we still lack a unified optimal procedure for deciding which family members to examine and which markers to use. Towards this goal, the application of conditional risk distributions is developed, along with a suggested statistic for judging the utility of a marker. The conditional risk distribution depends on what knowledge has already been obtained about the pedigree, and indicates the expected outcome of risk estimates after another marker is examined. Population genetic aspects including haplotype frequencies, linkage disequilibrium, family size and pedigree structure and the statistical confidence in the linkage map all influence the optimal strategy for multiple marker risk estimation.     

Diagnosis of neurofibromatosis I by using tightly linked, flanking DNA markers.

We tested 132 individuals from 21 families segregating an allele for neurofibromatosis type 1 (NF-1), by using nine RFLPs tightly linked to the NF-1 locus. Family members had requested DNA testing either to determine whether "at risk" children were carrying the NF-1 allele or to determine whether their respective families would be informative for prenatal testing. Predictions about whether a child carries the NF-1 mutation were possible for all 32 at-risk offspring (greater than 98% accuracy based on the recombination estimates currently available for these DNA markers). At least one informative probe was available for all 23 matings in these 21 families; flanking markers were informative for 10 matings. Pairwise analysis showed that several of the polymorphisms were in tight linkage disequilibrium; few recombination events were observed with these markers in the families under study. We conclude that the DNA probes used in this study perform well for diagnostic testing of NF-1 in familial cases. A subset of five probe-enzyme systems (pHHH202/RsaI, p11-3C4.2/MspI, pTH17.19/Bg/II, p11-2C11.7/BamHI, and p11-2F9.8/TaqI) provide reliable linkage information for both clinical testing and prenatal diagnosis.     

Genetic heterogeneity in X-linked hydrocephalus: linkage to markers within Xq27.3.

X-linked hydrocephalus is a well-defined disorder which accounts for > or = 7% of hydrocephalus in males. Pathologically, the condition is characterized by stenosis or obliteration of the aqueduct of Sylvius. Previous genetic linkage studies have suggested the likelihood of genetic homogeneity for this condition, with close linkage to the DXS52 and F8C markers in Xq28. We have investigated a family with typical X-linked aqueductal stenosis, in which no linkage to these markers was present. In this family, close linkage was established to the DXS548 and FRAXA loci in Xq27.3. Our findings demonstrate that X-linked aqueductal stenosis may result from mutations at two different loci on the X chromosome. Caution is indicated in using linkage for the prenatal diagnosis of X-linked hydrocephalus.     

X-linked dominant cone-rod degeneration: linkage mapping of a new locus for retinitis pigmentosa (RP 15) to Xp22.13-p22.11.

Retinitis pigmentosa is the name given to a heterogeneous group of hereditary retinal degenerations characterized by progressive visual field loss, pigmentary changes of the retina, abnormal electroretinograms, and, frequently, night blindness. In this study, we investigated a family with dominant cone-rod degeneration, a variant form of retinitis pigmentosa. We used microsatellite markers to test for linkage to the disease locus and excluded all mapped autosomal loci. However, a marker from the short arm of the X chromosome, DXS989, showed 0% recombination to the disease locus, with a maximum lod (log-odds) score of 3.3. On the basis of this marker, the odds favoring X-linked dominant versus autosomal dominant inheritance are > 10(5):1. Haplotype analysis using an additional nine microsatellite markers places the disease locus in the Xp22.13-p22.11 region and excludes other X-linked disease loci causing retinal degeneration. The clinical expression of the retinal degeneration is consistent with X-linked dominant inheritance with milder, variable effects of Lyonization affecting expression in females. On the basis of these data we propose that this family has a novel form of dominant, X-linked cone-rod degeneration with the gene symbol "RP15."     

Multipoint linkage analysis. A cautionary note.

Multipoint linkage analysis is commonly used to evaluate linkage of a disease to multiple markers in a small region. Multipoint analysis is particularly powerful when the IBD relations of family members at the trait locus are ambiguous. The increased power arises because, unlike single-marker analyses, multipoint analysis uses haplotype information from several markers to infer the IBD relations. We wish to temper this advantage with a cautionary note: multipoint analysis is sensitive to power loss due to misspecification of intermarker distances. Such misspecification is especially problematic when dealing with closely spaced markers. We present computer simulations comparing the power of single-point and multipoint analyses, both when IBD relations are ambiguous, and when the intermarker distances are misspecified. We conclude that when evaluating markers in a small region to confirm or refute previous findings, a situation in which p values of modest statistical significance are important, single marker analyses may provide more reliable measures of the strength of support for linkage than multipoint statistics.     

Mapping of the gene for X-linked amelogenesis imperfecta by linkage analysis.

X-linked Amelogenesis imperfecta (AI) is a genetic disorder affecting the formation of enamel. In the present study two families, one with X-linked dominant and one with X-linked recessive AI, were studied by linkage analysis. Eleven cloned RFLP markers of known regional location were used. Evidence was obtained for linkage between the AI locus and the marker p782, defining the locus DXS85 at Xp22, by using two-point analysis. No recombination was scored between these two loci in 15 informative meioses, and a peak lod score (Zmax) of 4.45 was calculated at zero recombination fraction. Recombination was observed between the more distal locus DXS89 and AI, giving a peak lod score of 3.41 at a recombination fraction of .09. Recombination was also observed between the AI locus and the more proximal loci DXS43 and DXS41 (Zmax = 0.09 at theta max = 0.31 and Zmax = 0.61 at theta max = 0.28, respectively). Absence of linkage was observed between the AI locus and seven other loci, located proximal to DXS41 or on the long arm of the X chromosome. On the basis of two-point linkage analysis and analysis of crossover events, we propose the following order of loci at Xp22: DXS89-(AI, DXS85)-DXS43-DXS41-Xcen.     

Improved predictive test for MEN2, using flanking dinucleotide repeats and RFLPs.

Gene(s) for the autosomal dominant endocrine cancer syndromes, multiple endocrine neoplasia type 2A (MEN2A), multiple endocrine neoplasia type 2B (MEN2B), and familial medullary thyroid carcinoma (MTC1) all map to the pericentromeric region of chromosome 10. Predictive testing for the inheritance of mutant alleles in individuals at risk for these disorders has been limited by the availability of highly informative and closely linked flanking markers. We describe the development of eight new markers, including two PCR-based dinucleotide repeat polymorphisms and six RFLPs that flank the disease loci. One of the dinucleotide repeat markers (sJRH-1) derives from the RBP3 locus on 10q11.2 and has a PIC of .88. The other dinucleotide repeat (sTCL-1) defines a new locus, D10S176, that maps by in situ hybridization to 10p11.2 and has a PIC of .68. We have constructed a new genetic linkage map of the pericentromeric region of chromosome 10, on the basis of 13 polymorphisms at six loci, which places the MEN2A locus between the dinucleotide repeat markers, with odds of 5,750:1 over the next most likely position. Using this set of markers, predictive genetic testing of 130 at-risk individuals from six families segregating MEN2A revealed that 95% were jointly informative with flanking markers, representing a significant improvement in genetic testing capabilities.