Positive selection of a pre-expansion CAG repeat of the human SCA2 gene

A region of approximately one megabase of human Chromosome 12 shows extensive linkage disequilibrium in Utah residents with ancestry from northern and western Europe. This strikingly large linkage disequilibrium block was analyzed with statistical and experimental methods to determine whether natural selection could be implicated in shaping the current genome structure. Extended Haplotype Homozygosity and Relative Extended Haplotype Homozygosity analyses on this region mapped a core region of the strongest conserved haplotype to the exon 1 of the Spinocerebellar ataxia type 2 gene (SCA2). Direct DNA sequencing of this region of the SCA2 gene revealed a significant association between a pre-expanded allele [(CAG)₈CAA(CAG)₄CAA(CAG)₈] of CAG repeats within exon 1 and the selected haplotype of the SCA2 gene. A significantly negative Tajima's D value (−2.20, p < 0.01) on this site consistently suggested selection on the CAG repeat. This region was also investigated in the three other populations, none of which showed signs of selection. These results suggest that a recent positive selection of the pre-expansion SCA2 CAG repeat has occurred in Utah residents with European ancestry.     

A prevalent POLG CAG microsatellite length allele in humans and African great apes

The human nuclear gene for the catalytic subunit of mitochondrial DNA polymerase (POLG) contains within its coding region a CAG microsatellite encoding a polyglutamine repeat. Previous studies demonstrated an association between length variation at this repeat and male infertility, suggesting a mechanism whereby the prevalent (CAG)₁₀ allele, which occurs at a frequency of >80% in different populations, could be maintained by selection. Sequence analysis of the POLG CAG microsatellite region of more than 1000 human chromosomes reveals that virtually all allelic variation at the locus is accounted for by length variation of the CAG repeat. Analysis of POLG from African great apes shows that a prevalent length allele is present in each species, although its exact length is species-specific. In common chimpanzee (Pan troglodytes) a number of different sequence variants contribute to the prevalent length allele, strongly supporting the idea that the length of the POLG microsatellite region, rather than its exact nucleotide or amino acid sequence, is what is maintained. Analysis of POLG in other primates indicates that the repeat has expanded from a shorter, glutamine-rich sequence, present in the common ancestor of Old and New World monkeys.     

Somatic expansion of the (CAG) n repeat in Huntington disease brains

The mutation causing Huntington disease (HD) has been identified as an expansion of a polymorphic (CAG) _n repeat in the 5 part of the huntingtin gene. The specific neuropathology of HD, viz. selective neuronal loss in the caudate nucleus and putamen, cannot be explained by the widespread expression of the gene. Since somatic expansion is observed in affected tissue in myotonic dystrophy, we have studied the length of the (CAG) _n repeat in various regions of the brain. Although we have not found clear differences when comparing severely and mildly affected regions, we have observed a minor increase in repeat length upon comparison of affected brain samples with cerebellum or peripheral blood. Hence, although further somatic amplification seems to occur in affected areas of the brain, the differences between affected and unaffected regions are too small to make this mechanism an obvious candidate for the cause of differential neuronal degeneration in HD.     

Genomic Structure of the Human Gene for Spinocerebellar Ataxia Type 2 (SCA2) on Chromosome 12q24.1

Spinocerebellar ataxia type 2 (SCA2) is a member of a group of neurodegenerative diseases that are caused by instability of a DNA CAG repeat. We report the genomic structure of theSCA2gene. Its 25 exons, encompassing approximately 130 kb of genomic DNA, were mapped onto the physical map of the region. Exonic sizes varied from 37 to 890 bp, and intronic sizes ranged from 323 bp to more than 15 kb. The CAG repeat was contained in the 5′ coding region of the gene in exon 1. Determination of the splice junction sequences indicated the presence of only one deviation from the GT-AG rule at the donor splice site of intron 9, which contained a GC instead of a GT dinucleotide. Exon 10, immediately downstream from this rare splice donor site, was alternatively spliced. Alternative splicing does not affect the reading frame and is predicted to encode an isoform containing 70 amino acids less.     

Codon-usage variants in the polymorphic (GGN) n trinucleotide repeat of the human androgen receptor gene

The human androgen receptor gene (hAR) has a long, polymorphic trinucleotide (GGN; glycine) _n repeat in the 3′ portion of its first exon, with n = 10–31. Owing to technical difficulties that have precluded routine sequencing of this region, it is widely unknown that N represents T, G or C, and that the usual sense codon sequence of the GGN tract is (GGT)₃GGG(GGT)₂(GGC)_4–25. Furthermore, on 4 of 61 X chromosomes, we observed that the internal GGT sequence was present three or four times instead of twice. Strikingly, each of the three alleles with an internal (GGT)₃, and only these three, also had a (GGC)₂₀ repeat. The size or composition of a (GGN) _n repeat was not correlated with the length of the accompanying (CAG) _n CAA repeat in the 5′ portion of exon one. Hence, codon-usage variants of the GGN tract may be used to seek associations with particular diseases, as diagnostic aids in families with androgen insensitivity whose AR mutations have not yet been identified, or as internal controls for observations on intergenerational contractions or expansions of the (CAG) _n CAA tract in a given hAR allele. Received: 28 May 1997 / Accepted: 22 July 1997     

Mapping of the interleukin 5 receptor gene to human Chromosome 3 p25–p26 and to mouse Chromosome 6 close to the Raf-1 locus with polymorphic tandem repeat sequences

Simple-sequence tandem repeat sequences in the 3 UTR of interleukin 5 (IL5)-receptor gene of human and mouse are polymorphic in their length among humans and different strains of mice. In 20 different human Epstein-Barr virus (EBV)-transformed cell lines, six alleles of IL5R could be distinguished. In the mouse, three different alleles are found. With the human-specific IL5R tandem repeat marker in human-rodent somatic cell hybrids, the IL5R gene was mapped to human Chromosome (Chr) 3 p25–p26. With the mouse-specific IL5R tandem repeat sequence in recombinant inbred strains of mice, the Il5r gene was mapped to the distal part of mouse Chr 6 close to the Raf-1 locus.     

Impact of the Regulatory Regions of Retrotransposon copia on the Level of Its Expression in Testes of Drosophila melanogaster

Expression of the lacZ reporter gene under the control of five deletion derivatives of the copia regulatory region including the 5 long terminal repeat (LTR) and the 5 untranslated region (UTR) was assayed in the testes of transgenic Drosophila melanogaster males (larvae and imago). The full-length copia regulatory region (LTR + UTR) ensured expression of the reporter gene in testes of both larvae and adult males. Deletion of UTR or 3 end of LTR increased lacZ expression in the testes, whereas deletion of the 5 end of LTR increased it. This indicated that a positive regulator ofcopia expression is at the 5 end of LTR and that negative regulators are at the 3 end of LTR and in UTR. The effects of the fragments of the copia regulatory region on reporter gene expression in the testes in vivo did not completely coincide with the effects observed earlier in cultured cells. We suggest that this difference is due to different regulation of expression of the fusion constructs integrated into chromatin as compared to their transient expression.     

The bovine interleukin-4 gene: genomic organization,localization, and evolution

Interleukin-4 (IL4) is involved in the immune response to certain parasites and possibly in the development of some atopic diseases since it triggers the T helper 2 lymphocyte response. Therefore, IL4 is a candidate gene, for example, for disease association studies and gene mapping. We isolated bovine IL4 cosmids and determined the genomic organization. Fragments carrying the exons as well as 725 base pairs (bp) from the 5 flanking and 190 bp from the 3 flanking region were cloned and sequenced. The first 481 base pairs of the 5 flanking region, including the putative promoter sequences, are surprisingly similar (92%) between cattle and human. In addition, we cloned and sequenced a mixed [(t/g)a]_m(ca)_n repeat located approximately 35 kilobases upstream from the IL4 gene. It showed seven repeat length alleles in a limited number of animals. The IL4 gene has been assigned to 7q15-q21 by fluorescence in situ hybridization in cattle. Evolutionary aspects are discussed on the basis of sequence data as well as interspecies chromosomal homologies.     

Mitotic and meiotic instability of the CAG trinucleotide repeat in spinocerebellar ataxia type 1

Spinocerebellar ataxia type 1 (SCA1) is an autosomal, dominantly inherited neurodegenerative disease caused by an unstable CAG trinucleotide repeat expansion in the ataxin-1 gene located on chromosome 6p22-p23. The expanded CAG repeat is unstable during transmission, and a variation in the CAG repeat length has been found in different tissues, including sperm samples from affected males. In order further to examine the mitotic and meiotic instability of the (CAG)_n stretch we have performed single sperm and low-copy genome analysis in SCA1 patients and asymptomatic carriers. A pronounced variation in the size of the expanded allele was found in sperm cells and peripheral blood leucocytes, with a higher degree of instability seen in the sperm cells, where an allele with 50 repeat units was contracted in 11.8%, further expanded in 63.5% and unchanged in 24.6% of the single sperm analysed. We found a low instability of the normal alleles; the normal alleles from the individuals carrying a CAG repeat expansion were significantly more unstable than the normal alleles from the control individuals (P<0.001), indicating an interallelic interaction between the expanded and the normal alleles. Received: 8 June 1998 / Accepted: 10 September 1998     

Initiation of 8-oxoguanine base excision repair within trinucleotide tandem repeats

Trinucleotide repeat expansion provides a molecular basis for several devastating neurodegenerative diseases. In particular, expansion of a CAG run in the human HTT gene causes Huntington’s disease. One of the main reasons for triplet repeat expansion in somatic cells is base excision repair (BER), involving damaged base excision and repair DNA synthesis that may be accompanied by expansion of the repaired strand due to formation of noncanonical DNA structures. We have analyzed the kinetics of excision of a ubiquitously found oxidized purine base, 8-oxoguanine (oxoG), by DNA glycosylase OGG1 from the substrates containing a CAG run flanked by AT-rich sequences. The values of k ₂ rate constant for the removal of oxoG from triplets in the middle of the run were higher than for oxoG at the flanks of the run. The value of k ₃ rate constant dropped starting from the third CAG-triplet in the run and remained stable until the 3′-terminal triplet, where it decreased even more. In nuclear extracts, the profile of oxoG removal rate along the run resembled the profile of k ₂ constant, suggesting that the reaction rate in the extracts is limited by base excision. The fully reconstituted BER was efficient with all substrates unless oxoG was near the 3′-flank of the run, interfering with the initiation of the repair. DNA polymerase β was able to perform a strand-displacement DNA synthesis, which may be important for CAG run expansion initiated by BER.     

Mapping of the Gene Encoding Human β-Defensin-2 (DEFB2) to Chromosome Region 8p22–p23.1

We recently reported the isolation of human β-defensin-2 (hBD-2), a novel epithelia-derived peptide antibiotic belonging to the β-defensin family. hBD-2 is expressed in skin and epithelia of the airway system, where it is believed to contribute to its antimicrobial defense. By fluorescencein situhybridization using a hBD-2 genomic DNA probe and subsequent fluorescence R-banding, the hBD-2 gene (HGMW-approved symbol DEFB2) was assigned to human chromosome region 8p22–p23.1. PCR with a set of CEPH YAC clones spanning this chromosomal region revealed CEPH YACs 773G4, 920D12, and 820B4 to contain the hBD-2 gene. Relying on the preexisting physical maps of 8p22–p23.1, the hBD-2 gene was mapped in close proximity to D8S1993 (WI-9956) within the interval flanked by D8S552 and D8S1130 (CHLC.GATA25C10). The fact that all currently described genes encoding defensins map to chromosome 8p21–pter suggests that a gene cluster in this chromosomal region may play a major role in antimicrobial defense.     

The HumanHNRPDLocus Maps to 4q21 and Encodes a Highly Conserved Protein

The hnRNP D protein interacts with nucleic acids bothin vivoandin vitro.Like many other proteins that interact with RNA, it contains RBD (or “RRM”) domains and arg-gly-gly (RGG) motifs. We have examined the organization and localization of the human and murine genes that encode the hnRNP D protein. Comparison of the predicted sequences of the hnRNP D proteins in human and mouse shows that they are 96.9% identical (98.9% similar). This very high level of conservation suggests a critical function for hnRNP D. Sequence analysis of the humanHNRPDgene shows that the protein is encoded by eight exons and that two additional exons specify sequences in the 3′ UTR. Use of two of the coding exons is determined by alternative splicing of theHNRPDmRNA. The humanHNRPDgene maps to 4q21. The mouseHnrpdgene maps to the F region of chromosome 3, which is syntenic with the human 4q21 region.     

CAG repeat length in an infertile male population of Irish origin

The androgen receptor (AR) gene, located on the X chromosome, is an important regulator of human spermatogenesis. In the past decade, the link between the CAG polyglutamine tract, situated on exon one of the AR gene, and reduced spermatogenesis has become a controversial one. Alterations in the length of the CAG polyglutamine tract have been associated with prostate cancer at a reduced intrinsic length and neuromuscular diseases at a CAG repeat length of 40. Minimal intermediate increases have been linked with depressed spermatogenesis in infertile males. Asian and Australian groups have published an association between increased CAG repeat length and reduced spermatogenesis while many European studies have found no such association. The aim of this study was to document the association between increased CAG repeat length and reduced spermatogenesis in a group of Irish infertile males and controls known to have fathered at least one child. The study employed the ABI 377 DNA sequencer to size the CAG repeat region of exon one of the AR gene in each group. Statistical analysis revealed no actual link between the length of the CAG tract and a reduction of spermatogenesis in a cohort of infertile patients (n = 66) of Irish ethnic origin when compared to a fertile control group (n = 77) (p = 0.599).     

Polymorphisms in the human surfactant protein-D (SFTPD) gene: strong evidence that serum levels of surfactant protein-D (SP-D) are genetically influenced

The collectin surfactant protein-D (SP-D) plays a significant role in innate immunity. Epidemiological studies described associations between single nucleotide polymorphisms (SNPs) of the human gene coding surfactant protein-D (SFTPD) and infectious pulmonary diseases. Studies on twins indicated very strong genetic dependence for serum levels of SP-D. The aim of this study was to determine the genetic influence of sequence variations within the SFTPD gene on the constitutional serum SP-D levels. We sequenced the 5 untranslated region (5UTR), the coding region and the 3 region of the SFTPD gene of 32 randomly selected blood donors. Six validated SNPs were genotyped with sequence-specific probes (TaqMan 7000) in 290 German blood donors. Serum SP-D levels were analysed by ELISA, and the association of SFTPD haplotype estimates with the quantitative phenotype serum SP-D level was determined. One single SFTPD haplotype (allele frequency 13.53%) revealed a negative association with serum SP-D levels (P<0.0001). This was confirmed in a second prospectively collected group of blood donors (n=160, P=0.0034). The discovery of a frequent negative variant of the SFTPD gene provides a basis for genetic analysis of the function of SP-D in the resistance against pulmonary infections and inflammatory disorders in humans.