Familial aggregation of stroke amongst young patients in Lund Stroke Register.

BACKGROUND AND PURPOSE: The known monogenic forms of stroke are rare. The aim of this study was to analyze pedigrees of young stroke patients regarding possible monogenic cerebrovascular disease and to evaluate the possibility of genetic stroke in these families. This may contribute to a better understanding of disease mechanism in stroke. METHODS: Lund Stroke Register includes consecutive patients with first-ever stroke from a defined geographical area in southern Sweden. Early-onset (≤55 years) stroke patients were systematically screened with regard to family history (FHx), and families with stroke aggregation were compiled. Participants provided information in a questionnaire on occurrence of stroke or transient ischaemic attack (TIA) in their families. Information on cardiovascular risk factors (VRFs) and clinical stroke subtype was collected. FHx for stroke was considered positive when the patient reported either ≥1 first-degree relative with stroke/TIA, or no first-degree relative but ≥3 second- or third-degree relatives with stroke/TIA in a distribution compatible with monogenic inheritance. RESULTS: Of 4103 stroke patients registered, 426 (10%) had first-ever stroke at ≤55 years and 338 (79%) of these answered the questionnaire. Of them, 159 (47%) reported a positive FHx. Twenty-eight (18%) of the probands with positive FHx had no known VRFs. Thirty-two families with ≥4 members with stroke were identified. In all these larger families the affected individuals with stroke were present in more than one generation. CONCLUSION: Aggregation of stroke in families of early-onset stroke patients is not uncommon. Genetic factors with impact on stroke risk, including monogenic causes, need to be evaluated in future stroke studies.

Quantitative trait loci mapping for conjugated linoleic acid, vaccenic acid and ∆(9) -desaturase in Italian Brown Swiss dairy cattle using selective DNA pooling.

A selective DNA pooling approach was applied to identify QTL for conjugated linoleic acid (CLA), vaccenic acid (VA) and Δ(9) -desaturase (D9D) milk content in Italian Brown Swiss dairy cattle. Milk samples from 60 animals with higher values (after correction for environmental factors) and 60 animals with lower values for each of these traits from each of five half-sib families were pooled separately. The pools were genotyped using the Illumina BovineSNP50 BeadChip. Sire allele frequencies were compared between high and low tails at the sire and marker level for SNPs for which the sires were heterozygous. An r procedure was implemented to perform data analysis in a selective DNA pooling design. A correction for multiple tests was applied using the proportion of false positives among all test results. BTA 19 showed the largest number of markers in association with CLA. Associations between SNPs and the VA and Δ(9) -desaturase traits were found on several chromosomes. A bioinformatics survey identified genes with an important role in pathways for milk fat and fatty acids metabolism within 1 Mb of SNP markers associated with fatty acids contents.

Body composition and reproductive performance at entry into lay of anno 1980 versus anno 2000 broiler breeder females under fast and slow release from feed restriction.

During the 1990s, various disturbances arose affecting broiler breeder females at entry into lay. These disturbances were associated with even slight overfeeding during release of feed restriction in this critical maturation period. The present experiment was carried out to gain some insight into the causes of these disturbances by comparing the effect of fast (FF) and slow (SF) release from feed restriction at entry into lay in 2 broiler breeder populations: B1980, representing the genetic level of 1980, and B2000, the genetic level of 2000. Under the FF treatment, B1980 entered lay 19.2 d earlier than B2000; this increased to 37.4 d earlier under SF. The B1980 population entered lay at virtually the same mean age for SF and FF, whereas B2000 entered lay 15.7 d earlier under the FF. Body weight at first egg were 2,621 g for the B1980 and 3,591 g for B2000. Differences in BW at first egg between feeding treatments within lines were minor. As a percentage of BW, ovary, oviduct, and follicle weights were the same for B1980 and B2000; breast weight was 14.9% for B1980 and 21.2% for B2000; abdominal fat pad weight was 5.37% for B1980 and 2.67% for B2000. Follicle weight and absolute difference in weight between successive follicles was greater in B2000 than in B1980. It is concluded that body fat content does not limit entry into lay, and that threshold BW for onset of sexual maturity of broiler breeder hens increased by about 1,000 g between 1980 and 2000, indicating a tight association between juvenile growth rate and threshold BW for onset of sexual maturity. It is also concluded that disturbances at entry into lay due to overfeeding are not due to smaller differences between successive follicles in B2000 compared with B1980. There are hints, however, that overfeeding may contribute to these disturbances by decreasing differences between successive follicles.

Localization of genes controlling resistance to trypanosomiasis in mice.

Tsetse fly-transmitted trypanosomes (Trypanosoma spp.) cause "sleeping sickness' in man and have a serious impact on livestock-based agriculture in large areas of Africa. Multigene control of variation in susceptibility to trypanosomiasis is known to occur in mice, where the C57BI/6 (B6) strain is relatively resistant and the A/J (A) and Balb/c (B) strains are susceptible. Such resistance is also well described among several types of west African cattle. We report here the results of genome-wide scans for genes controlling this trait in the B6 mouse using crosses with two different susceptible strains. Regions on mouse chromosomes 5 and 17 were found to be important in determining resistance in both crosses while an additional region on chromosome 1 showed evidence of involvement in only one cross. We confirmed the size of the effect due to chromosome 17 in F3 intercross populations fixed for alternative parental chromosomes. The three loci are of large effect and account for most of the genetic variation in both F2 populations. We propose that they be designated Tir1, Tir2 and Tir3.

Variation in the ovocalyxin-32 gene in commercial egg-laying chickens and its relationship with egg production and egg quality traits.

Avian eggshell quality is an important trait for commercial egg production, as the eggshell is the primary packaging material and antimicrobial barrier for the internal food resource. Strong eggshells are essential to ensure that eggs can reach their final destination without damage. Ovocalyxin-32 (OCX32) is a matrix protein found within the outer layers of the eggshell and in the cuticle. Numerous reports in the literature have identified association between variants in the gene encoding this protein, OCX32, and various eggshell quality traits. Thus, OCX32 is a candidate gene for selection for eggshell traits in commercial poultry populations. Sequencing of exons 2-6 of the OCX32 gene in eight elite brown and white eggshell commercial egg-laying lines revealed 28 SNPs and one SNP/indel. Eighteen of these SNPs were predicted to alter the amino acid sequence of the protein. Clusters of SNPs in complete linkage disequilibrium were found in both exons 2 and 6. A total of 19 different versions or protein-sequence haplotypes of the OCX32 protein were inferred, revealing considerable variation within commercial lines. Genotypes for 13 of the SNPs were determined for 330-1819 individuals per line. Trait association studies revealed a significant effect of OCX32 on shell color in white egg lines and line-specific significant effects on albumen height, early egg weight, puncture score, and yolk weight. Three of the lines showed a significant change in OCX32 frequency over time, indicating selection pressure for certain variants of this gene during the breeding program.

A whole genome scan for QTL affecting milk protein percentage in Italian Holstein cattle, applying selective milk DNA pooling and multiple marker mapping in a daughter design.

We report on a complete genome scan for quantitative trait loci (QTL) affecting milk protein percentage (PP) in the Italian Holstein-Friesian cattle population, applying a selective DNA pooling strategy in a daughter design. Ten Holstein-Friesian sires were chosen, and for each sire, about 200 daughters, each from the high and low tails of estimated breeding value for PP, were used to construct milk DNA pools. Sires and pools were genotyped for 181 dinucleotide microsatellites covering all cattle autosomes. Sire marker allele frequencies in the pools were obtained by shadow correction of peak height in the electropherograms. After quality control, pool data from eight sires were used for all subsequent analyses. The QTL heterozygosity estimate was lower than that of similar studies in other cattle populations. Multiple marker mapping identified 19 QTL located on 14 chromosomes (BTA1, 2, 3, 4, 5, 6, 8, 9, 12, 14, 17, 20, 23 and 27). The sires were also genotyped for seven polymorphic sites in six candidate genes (ABCG2, SPP1, casein kappa, DGAT1, GHR and PRLR) located within QTL regions of BTA6, 14 and 20 found in this study. The results confirmed or excluded the involvement of some of the analysed markers as the causative polymorphic sites of the identified QTL. The QTL identified, combined with genotype data of these candidate genes, will help to identify other quantitative trait genes and clarify the complex QTL patterns observed for a few chromosomes. Overall, the results are consistent with the Italian Holstein population having been under long-term selection for high PP.

Extensive long-range and nonsyntenic linkage disequilibrium in livestock populations: deconstruction of a conundrum.

Great interest was aroused by reports, based on microsatellite markers, of high levels of statistically significant long-range and nonsyntenic linkage disequilibrium (LD) in livestock. Simulation studies showed that this could result from population family structure. In contrast, recent SNP-based studies of livestock populations report much lower levels of LD. In this study we show, on the basis of microsatellite data from four cattle populations, that high levels of long-range LD are indeed obtained when using the multi-allelic D' measure of LD. Long-range and nonsyntenic LD are exceedingly low, however, when evaluated by the standardized chi-square measure of LD, which stands in relation to the predictive ability of LD. Furthermore, specially constructed study populations provided no evidence for appreciable LD resulting from family structure at the grandparent level. We propose that the high statistical significance and family structure effects observed in the earlier studies are due to the use of large sample sizes, which accord high statistical significance to even slight deviations from asymptotic expectations under the null hypothesis. Nevertheless, even after taking sample size into account, our results indicate that microsatellites testify to the presence of usable LD at considerably wider separation distances than SNPs, suggesting that use of SNP haplotypes may considerably increase the usefulness of a given fixed SNP array.

A genome scan for quantitative trait loci affecting milk somatic cell score in Israeli and Italian Holstein cows by means of selective DNA pooling with single- and multiple-marker mapping.

Mastitis is an important and common dairy cattle disease affecting milk yield, quality, and consumer safety as well as cheese yields and quality. Animal welfare and residues of the antibiotics used to treat mastitis cause public concern. Considerable genetic variation may allow selection for increased resistance to mastitis. Because of high genetic correlation to milk somatic cell score (SCS), SCS can serve as a surrogate trait for mastitis resistance. The present study intended to identify quantitative trait loci (QTL) affecting SCS in Israeli and Italian Holstein dairy cattle (IsH and ItH, respectively), using selective DNA pooling with single and multiple marker mapping. Milk samples of 4,788 daughters of 6 IsH and 7 ItH sires were used to construct sire-family high- and low-tail pools, which were genotyped at 123 (IsH) and 133 (ItH) microsatellite markers. Shadow correction was used to obtain pool allele frequency estimates. Frequency difference between the tails and empirical standard error of D, SE(D), were used to obtain P-values. All markers significant by single marker mapping were also significant by multiple marker mapping, but not vice versa. Combining both populations, 22 QTL on 21 chromosomes were identified; all corresponded to previous reports in the literature. Confidence intervals were set by chi-squared drop method. Heterozygosity of QTL was estimated at 44.2%. Allele substitution effects ranged from 1,782 to 4,930 cells/mL in estimated breeding value somatic cell count units. Most (80%) of the observed variation in estimated breeding value somatic cell score could be explained by the QTL identified under the stringent criteria. The results found here can be used as a basis for further genome-wide association studies for the same trait.

Fine mapping and association analysis of a quantitative trait locus for milk production traits on Bos taurus autosome 4.

To fine map a quantitative trait locus (QTL) affecting milk production traits previously associated with microsatellite RM188, we implemented an interval mapping analysis by using microsatellite markers in a large Israeli Holstein half-sib sire family, and linkage disequilibrium (LD) mapping in a large set of US Holstein bulls. Interval mapping located the target QTL to the near vicinity of RM188. For the LD mapping, we identified 42 single nucleotide polymorphisms (SNP) in 15 genes in a 12-Mb region on bovine chromosome 4. A total of 24 tag SNP were genotyped in 882 bulls belonging to the University of California Davis archival collection of Holstein bull DNA samples with predicted transmitted ability phenotypes. Marker-to-marker LD analysis revealed 2 LD blocks, with intrablock r(2) values of 0.10 and 0.46, respectively; outside the blocks, r(2) values ranged from 0.002 to 0.23. A standard additive/dominance model using the generalized linear model procedure of SAS and the regression module of HelixTree software were used to test marker-trait associations. Single nucleotide polymorphism 9 on ARL4A, SNP10 on XR_027435.1, SNP12 on ETV1, SNP21 on SNX13, and SNP24 were significantly associated with milk production traits. We propose the interval encompassing ARL4A and SNX13 genes as a candidate region in bovine chromosome 4 for a concordant QTL related to milk protein traits in dairy cattle. Functional studies are needed to confirm this result.

Fractioned DNA pooling: a new cost-effective strategy for fine mapping of quantitative trait loci.

Selective DNA pooling (SDP) is a cost-effective means for an initial scan for linkage between marker and quantitative trait loci (QTL) in suitable populations. The method is based on scoring marker allele frequencies in DNA pools from the tails of the population trait distribution. Various analytical approaches have been proposed for QTL detection using data on multiple families with SDP analysis. This article presents a new experimental procedure, fractioned-pool design (FPD), aimed to increase the reliability of SDP mapping results, by "fractioning" the tails of the population distribution into independent subpools. FPD is a conceptual and structural modification of SDP that allows for the first time the use of permutation tests for QTL detection rather than relying on presumed asymptotic distributions of the test statistics. For situations of family and cross mapping design we propose a spectrum of new tools for QTL mapping in FPD that were previously possible only with individual genotyping. These include: joint analysis of multiple families and multiple markers across a chromosome, even when the marker loci are only partly shared among families; detection of families segregating (heterozygous) for the QTL; estimation of confidence intervals for the QTL position; and analysis of multiple-linked QTL. These new advantages are of special importance for pooling analysis with SNP chips. Combining SNP microarray analysis with DNA pooling can dramatically reduce the cost of screening large numbers of SNPs on large samples, making chip technology readily applicable for genomewide association mapping in humans and farm animals. This extension, however, will require additional, nontrivial, development of FPD analytical tools.

Mapping quantitative trait loci affecting susceptibility to Marek's disease virus in a backcross population of layer chickens.

Marek's disease (MD), caused by the oncogenic MD avian herpes virus (MDV), is a major source of economic losses to the poultry industry. A reciprocal backcross (BC) population (total 2052 individuals) was generated by crossing two partially inbred commercial Leghorn layer lines known to differ in MDV resistance, measured as survival time after challenge with a (vv+) MDV. QTL affecting resistance were identified by selective DNA pooling using a panel of 198 microsatellite markers covering two-thirds of the chicken genome. Data for each BC were analyzed separately, and as a combined data set. Markers showing significant association with resistance generally appeared in blocks of two or three, separated by blocks of nonsignificant markers. Defined this way, 15 chromosomal regions (QTLR) affecting MDV resistance, distributed among 10 chromosomes (GGA 1, 2, 3, 4, 5, 7, 8, 9, 15, and Z), were identified. The identified QTLR include one gene and three QTL associated with resistance in previous studies of other lines, and three additional QTL associated with resistance in previous studies of the present lines. These QTL could be used in marker-assisted selection (MAS) programs for MDV resistance and as a platform for high-resolution mapping and positional cloning of the resistance genes.

CHD7 mutation spectrum in 28 Swedish patients diagnosed with CHARGE syndrome.

CHARGE syndrome is a disorder characterized by Coloboma, Heart defect, Atresia choanae, Retarded growth and/or development, Genital hypoplasia and Ear anomalies. Heterozygous mutations in the chromodomain helicase DNA-binding protein 7 (CHD7) gene have been identified in about 60% of individuals diagnosed with CHARGE syndrome. We performed a CHD7 mutation screening by direct exon sequencing in 28 index patients (26 sporadic cases, 1 familial case consisting of a brother and sister and 1 case consisting of monozygotic twins) diagnosed with CHARGE syndrome in order to determine the mutations in a cohort of Swedish CHARGE syndrome patients. The patients without a detectable CHD7 mutation, or with a missense mutation, were further investigated by multiplex ligation-dependent probe amplification (MLPA) in order to search for intragenic deletions or duplications. Thirteen novel mutations and five previously reported mutations were detected. The mutations were scattered throughout the gene and included nonsense, frameshift and missense mutations as well as intragenic deletions. In conclusion, CHD7 mutations were detected in a large proportion (64%) of cases diagnosed with CHARGE syndrome. Screening for intragenic deletions with MLPA is recommended in cases where mutations are not found by sequencing. In addition, a CDH7 mutation was found in an individual without temporal bone malformation.

Using integrative genomics to elucidate genetic resistance to Marek's disease in chickens.

While rearing birds in confinement and at high density are very successful practices for producing poultry meat and eggs, these conditions may promote the spread of infectious diseases. Consequently, the poultry industry places greatemphasis on disease control measures, primarily at the animal husbandry level. The field of genomics offers great promise to complement these current control measures by providing information on the molecular basis for disease, disease resistance, and vaccinal immunity. This briefly summarizes some of our efforts to apply several genomic and functional genomics approaches to identify genes and pathways that confer genetic resistance to Marek's disease (MD), a herpesvirus-induced T cell lymphoma of chickens. By utilizing the "top-down" approach of QTL to identify genomics regions, and integrating it with "bottom-up" approaches of transcript profiling and Marek's disease virus (MDV)-chicken protein-protein interactions, three genes that confer resistance to MD are revealed, plus a number of other positional candidate genes of high confidence. These genes can be further evaluated in poultry breeding programmes to determine if they confer genetic resistance to MD. This integrative genomics strategy can be applied to other infectious diseases. The impact of the genome sequence and other technological advancements are also discussed.

Expected effects on protein yield of marker-assisted selection at quantitative trait loci affecting milk yield and milk protein percentage.

Protein yield (PY) is currently the major economic product of the dairy herd. Genome-wide scans for quantitative trait loci (QTL) affecting milk yield (MY) and milk protein percentage (PP) suggest that of the loci affecting the 2 traits, about 1/4 exclusively affect MY, 1/4 exclusively affect PP, and half affect both traits. Because PY is the product of MY and PP, it is of interest to evaluate the expected effects on PY of marker-assisted selection (MAS) applied to these 3 classes of QTL. It is clear that selection for the appropriate allele at QTL exclusively affecting MY or PP will have a positive effect on PY. The question arises as to the effect of MAS directed at QTL affecting both MY and PP. Because the observed genetic correlation of about -0.5 between MY and PP must be generated by these loci, and because they comprise about half the total number of loci affecting the 2 traits, it can be inferred that the genetic correlation between MY and PP at loci affecting both traits is close to -1.0. This seems to imply that generally such loci would be neutral in their effects on PY. In the present study, biometrical expressions originally developed to describe the relationships of MY, fat percentage, and fat yield were adapted to describe the relationships of MY, PP, and PY. The resultant expressions were validated by showing that they correctly predicted the observed phenotypic standard deviation and heritability of PY, and the vastly different genetic correlations of PY with MY (very high positive) and of PY with PP (very low positive). Contrary to initial impressions, further biometrical analysis of the projected effects on PY of MAS at the loci affecting both traits, showed that even under the assumption that the genetic correlation between MY and PP at these loci is -1.0, selection for the allele favoring MY will have a strong positive effect on PY, whereas selection for the allele favoring PP will have an equal but opposite negative effect on PY. These diametrically opposed effects are due to the lower genetic coefficient variation of PP compared with MY. It is speculated that the reduced coefficient of variation of PP may be because of more stringent homeostatic buffering of milk composition compared with milk yield.

Effect of quantitative trait loci for milk protein percentage on milk protein yield and milk yield in Israeli Holstein dairy cattle.

Although numerous quantitative trait loci (QTL) mapping studies involving milk protein percent (PP), milk yield (MY), and protein yield (PY) have been carried out, there has not been any systematic evaluation of the effects of individual QTL on these 3 interrelated traits. Consequently, the aim of the present study was to investigate the effects on MY and PY of QTL for PP previously mapped in various laboratories. The study, based on selective DNA pooling of milk samples, included 10 Israeli Holstein artificial insemination bulls, each the sire of 1,800 or more milk-recorded daughters. For each sire-trait combination across the 10 sires, milk samples of the highest and lowest daughters with respect to estimated breeding values for PP, PY, and MY were collected for pooling. A total of 134 dinucleotide microsatellites distributed over 25 bovine autosomes were used. An empirical standard error for marker-QTL linkage testing was calculated based on the variation among split samples within the same tail. Threshold comparison-wise error rate P-values were set to control proportion of false positives at P = 0.10 level for declaring significant effects at the marker-trait level. Estimates of the number of true null hypotheses for each trait were obtained from the histogram of marker comparison-wise error rate P-values. Based on these estimates, effective power of the experiment at the marker-trait level was estimated as 0.75, 0.41, and 0.73 for PP, PY, and MY. The proportion of heterozygosity at the QTL was estimated as 0.46, 0.39, and 0.40, respectively. After correcting for incomplete power and proportion of false positives, it was estimated that 38.7 and 37.5% of the markers affecting PP and MY, respectively, also affected PY. Of the markers affecting PY, 68.9 and 76.5%, respectively, also affected PP and MY. Apparently, none of the significant markers affected PY exclusively, and only 6.5 and 16.0%, respectively, affected PP or MY exclusively. Thus, almost all significant markers, and by inference almost all QTL, had effects on at least 2 of the 3 traits.

Multiple-marker mapping for selective DNA pooling within large families.

Selective DNA pooling is a very powerful method for quantitative trait loci (QTL) mapping. It considerably reduces genotyping costs while maintaining high statistical power. Applied to a daughter design, milk samples of offspring with extreme phenotypic values for a trait of interest are assigned to high and low groups, respectively, and within each group the pooled DNA is used for densitometric estimation of allele frequencies in the 2 groups. A single-marker test for linkage between marker and QTL considers marker allele frequency differences between high and low groups. Single-marker across-sire test statistics are strongly affected by the number of sires that are heterozygous for a given marker and the QTL status (homozygous or heterozygous) of these sires, which decreases the accuracy of QTL mapping. Here we propose a simple method to deal with this problem by taking information from multiple linked markers into account. In particular, given the single-marker test statistics, a multiple-marker method was developed to predict test statistics for markers for which a sire was homozygous (or at any other location on the chromosome). Power and map resolution of the proposed method were assessed by simulation, and we show that for the same data set, multiple-marker mapping performed better than the commonly used single-marker analyses.

Quantitative trait loci affecting milk yield and protein percentage in a three-country Brown Swiss population.

Quantitative trait loci (QTL) mapping projects have been implemented mainly in the Holstein dairy cattle breed for several traits. The aim of this study is to map QTL for milk yield (MY) and milk protein percent (PP) in the Brown Swiss cattle populations of Austria, Germany, and Italy, considered in this study as a single population. A selective DNA pooling approach using milk samples was applied to map QTL in 10 paternal half-sib daughter families with offspring spanning from 1,000 to 3,600 individuals per family. Three families were sampled in Germany, 3 in Italy, 1 in Austria and 3 jointly in Austria and Italy. The pools comprised the 200 highest and 200 lowest performing daughters, ranked by dam-corrected estimated breeding value for each sire-trait combination. For each tail, 2 independent pools, each of 100 randomly chosen daughters, were constructed. Sire marker allele frequencies were obtained by densitometry and shadow correction analyses of 172 genome-wide allocated autosomal markers. Particular emphasis was placed on Bos taurus chromosomes 3, 6, 14, and 20. Marker association for MY and PP with a 10% false discovery rate resulted in nominal P-values of 0.071 and 0.073 for MY and PP, respectively. Sire marker association tested at a 20% false discovery rate (within significant markers) yielded nominal P-values of 0.031 and 0.036 for MY and PP, respectively. There were a total of 36 significant markers for MY, 33 for PP, and 24 for both traits; 75 markers were not significant for any of the traits. Of the 43 QTL regions found in the present study, 10 affected PP only, 8 affected MY only, and 25 affected MY and PP. Remarkably, all 8 QTL regions that affected only MY in the Brown Swiss, also affected MY in research reported in 3 Web-based QTL maps used for comparison with the findings of this study (http://www.vetsci.usyd.edu.au/reprogen/QTL_Map/; http://www.animalgenome.org/QTLdb/cattle.html; http://bovineqtl.tamu.edu/). Similarly, all 10 QTL regions in the Brown Swiss that affected PP only, affected only PP in the databases. Thus, many QTL appear to be common to Brown Swiss and other breeds in the databases (mainly Holstein), and an appreciable fraction of QTL appears to affect MY or PP primarily or exclusively, with little or no effect on the other trait. Although QTL information available today in the Brown Swiss population can be utilized only in a within family marker-assisted selection approach, knowledge of QTL segregating in the whole population should boost gene identification and ultimately the implementation and efficiency of an individual genomic program.

Simple sequence repeats as a source of quantitative genetic variation.

Most traits in biological populations appear to be under stabilizing selection, which acts to eliminate quantitative genetic variation. Yet, virtually all measured traits in biological populations continue to show significant quantitative genetic variation. The paradox can be resolved by postulating the existence of an abundant, though unspecified, source of mutations that has quantitative effects on phenotype, but does not reduce fitness. Does such a source actually exist? We propose that it does, in the form of repeat-number variation in SSRs (simple sequence repeats, of which the triplet repeats of human neurodegenerative diseases are a special case). Viewing SSRs as a major source of quantitative mutation has broad implications for understanding molecular processes of evolutionary adaptation, including the evolutionary control of the mutation process itself.

The neuron-enriched splicing pattern of Drosophila erect wing is dependent on the presence of ELAV protein.

Although the Drosophila melanogaster erect wing (ewg) gene is broadly transcribed in adults, an unusual posttranscriptional regulation involving alternative and inefficient splicing generates a 116-kDa EWG protein in neurons, while protein expression elsewhere or of other isoforms is below detection at this stage. This posttranscriptional control is important, as broad expression of EWG can be lethal. In this paper, we show that ELAV, a neuron-specific RNA binding protein, is necessary to regulate EWG protein expression in ELAV-null eye imaginal disc clones and that ELAV is sufficient for EWG expression in wing disc imaginal tissue after ectopic expression. Further, analysis of EWG expression elicited from intron-containing genomic transgenes and cDNA minitransgenes in ELAV-deficient eye discs shows that this regulation is dependent on the presence of ewg introns. Analyses of the ewg splicing patterns in wild-type and ELAV-deficient eye imaginal discs and in wild-type and ectopic ELAV-expressing wing imaginal discs, show that certain neuronal splice isoforms correspond to ELAV levels. The data presented in this paper are consistent with a mechanism in which ELAV increases the splicing efficiency of ewg transcripts in alternatively spliced regions rather than with a mechanism in which stability of specific splice forms is enhanced by ELAV. Additionally, we report that ELAV promotes a neuron-enriched splice isoform of Drosophila armadillo transcript. ELAV, however, is not involved in all neuron-enriched splice events.

Differential and inefficient splicing of a broadly expressed Drosophila erect wing transcript results in tissue-specific enrichment of the vital EWG protein isoform.

In this report, we document an unusual mode of tissue-enriched gene expression that is primarily mediated by alternative and inefficient splicing. We have analyzed posttranscriptional regulation of the Drosophila erect wing gene, which provides a vital neuronal function and is essential for the formation of certain muscles. Its predominant protein product, the 116-kDa EWG protein, a putative transcriptional regulator, can provide all known erect wing-associated functions. Moreover, consistent with its function, the 116-kDa protein is highly enriched in neurons and is also observed transiently in migrating myoblasts. In contrast to the protein distribution, we observed that erect wing transcripts are present in comparable levels in neuron-enriched heads and neuron-poor bodies of adult Drosophila. Our analyses shows that erect wing transcript consists of 10 exons and is alternatively spliced and that a subset of introns are inefficiently spliced. We also show that the 116-kDa EWG protein-encoding splice isoform is head enriched. In contrast, bodies have lower levels of transcripts that can encode the 116-kDa protein and greater amounts of unprocessed erect wing RNA. Thus, the enrichment of the 116-kDa protein in heads is ensured by tissue-specific alternative and inefficient splicing and not by transcriptional regulation. Furthermore, this regulation is biologically important, as an increased level of the 116-kDa protein outside the nervous system is lethal.