Analysis of Polymorphism of the Number of Tandem Repeats in the Aggrecan Gene Exon G3 in the Families with Idiopathic Scoliosis

In our previous study we showed that the inheritance of pronounced forms of idiopathic scoliosis was described by an autosomal-dominant major gene model assuming incomplete sex- and age-dependent penetrance. In the present study a search for the major gene was carried out by means of testing candidate genes. The aggrecan gene with known polymorphism of the number of tandem repeats in exon G3 was considered to be one of these candidate genes. Various alleles of this gene provide attachment of different number of chondroitin sulfate chains to a proteoglycan core protein, thereby changing functional properties of cartilage. Using the TDT analysis of 33 unrelated families consisting of a proband and his/her parents, we examined the existence of associations between the aggrecan alleles and the disease. Among nine alleles identified, three alleles with tandem repeats numbers of 25, 26, and 27 prevailed. We did not reveal preferable transmission of any of these alleles to the proband (TDT-statistics for different alleles varied from 0 to 0.71). There was also no correlation between the number of tandem repeats and the disease severity. Thus, either the polymorphism of the number of tandem repeats is not the direct reason for development of idiopathic scoliosis in the families tested, or its effect is too low to be detected using the samples examined.     

Short aggrecan gene repetitive alleles associated with lumbar degenerative disc disease in Turkish patients

We investigated a possible association between aggrecan gene polymorphism and lumbar degenerative disc disease in Turkish patients. One hundred 20-30-year-old patients with or without low back pain were selected for the study. Lumbar magnetic resonance imaging was performed on all patients. The patient group had low back pain clinically and degenerative disc disease radiographically. The control group included patients with and without low back pain: all were negative radiographically for degenerative disc disease. Genomic DNA was extracted from all participants. A PCR assay were used to evaluate variable number of tandem repeat polymorphism of aggrecan gene alleles to determine if there was any correlation with degenerative disc disease. Significant associations were found between short repeated alleles of the aggrecan gene and severe disc degeneration. A significant association was also found between short repeated alleles of the aggrecan gene and multilevel disc herniation as well as extrusion and sequestration types of disc herniation. In Turkish population, short repeated alleles of the aggrecan gene are associated with increased disc degeneration and disc herniation.     

Expansion of mouse involucrin by intra-allelic repeat addition

Involucrin, loricrin and the small proline-rich proteins (SPRRs) are precursors of the cornified envelope of terminally differentiated keratinocytes. The genes for these proteins are closely linked on mouse chromosome 3. Each of the proteins is encoded by a single exon and is largely composed of a segment of short tandem repeats. No size polymorphism of either loricrin or the SPRRs was observed. In contrast, involucrin was found in at least eight polymorphic forms of different size with molecular weights ranging from 51 to 82kDa. Two classes of involucrin alleles were identified. Size polymorphism of involucrin has resulted from the recent expansion of the segment of repeats in one class of alleles, but not in the other. In expanding alleles, repeats were added at a precise location within the segment of repeats, in a 5'-to-3' direction. A study of a large number of allele-specific markers, located on both sides of the site of repeat addition, revealed no evidence for recombination between any of the alleles examined. Expansion of the segment of repeats of the gene for mouse involucrin must result from an intra-allelic process controlled by a cis-acting element, active in one class of alleles, and inactive in the other.     

Association of variable number of tandem repeats polymorphism in the IL-4 gene with end-stage renal disease in Malaysian patients

Variable number of tandem repeats (VNTR) polymorphism in the interleukin 4 (IL-4) gene has been associated with end-stage renal disease (ESRD) subjects in many different populations, although with conflicting results. We determined the 70 bp of VNTR polymorphism at intron 3 of the IL-4 gene in Malaysian ESRD subjects. Buccal cells were collected from 160 case and 160 control subjects; genomic DNA was amplified using PCR, followed by agarose gel electrophoresis. There were significant differences in genotypes and alleles of the IL-4 gene. We conclude that VNTR polymorphism of the IL-4 gene is a risk factor for the development of ESRD among Malaysians.     

Evolution of the cystatin B gene: implications for the origin of its variable dodecamer tandem repeat in humans

The human cystatin B gene contains a variable number of 12-bp tandem repeats in its promoter region, of which the common alleles contain two or three copies and unusual expansion causes progressive myoclonus epilepsy of the Unverricht-Lundborg type. We undertook a comprehensive analysis of the genomic sequence to address the evolutionary events of this variable repeat. By examination of a contiguous genome sequence spanning 5.0 kb and linkage analysis of detected polymorphic changes, we identified six major intragenic haplotypes in unrelated Japanese subjects. The number of normal repeats was closely correlated with these alleles, indicating that changes in the array should be comparatively rare events during human evolution. To examine the origin of the repeat array further, we also analyzed five primate genomes. Repetitive polymorphism was unlikely in hominoids, and the array originated with the dodecamer itself in the course of primate evolution. The variability conceivably developed after the separation to humans.     

Molecular cloning of two novel mucin-like genes in the disease-susceptibility locus for diffuse panbronchiolitis

Diffuse panbronchiolitis (DPB) is a rare complex genetic disease affecting East Asians and is strongly associated with the class I human leukocyte antigens (HLA)-B54 in Japanese and HLA-A11 in Koreans. We recently showed that an HLA-associated major susceptibility gene for DPB is probably located within the 200?kb in the class I region 300?kb telomeric of the HLA-B locus on the chromosome 6p21.3. We cloned two novel mucin-like genes designated panbronchiolitis related mucin-like 1 and 2 (PBMUCL1 and PBMUCL2) in the candidate region, which form a mucin-like gene cluster together with two adjacent genes, MUC21 and DPCR1. PBMUCL1 gene expression was remarkably upregulated by polyinosine-polycytidylic acid [poly(I:C)] stimulation in normal human bronchial epithelial cells redifferentiated at the air-liquid interface. We found genetic polymorphisms in PBMUCL1 gene which were associated with DPB: the A-allele of the PBMUCL1 intron 2 single nucleotide polymorphism (SNP) was positively associated and variable numbers of tandem repeats (VNTR) polymorphism in exon 3 (1,890-base pair deletion) was negatively associated. Despite a strong association with HLA-B in the Japanese, the mucin-like gene PBMUCL1 is also one of the candidate genes of DPB susceptibility.     

Analysis of promoter methylation and polymorphic minisatellites of BORIS and lack of association with gastric cancer

BORIS is a member of the cancer-testis gene family that comprises genes normally expressed only in testis but abnormally activated in different malignancies. In this study, we examined the relation between BORIS expression and gastric cancer, which is the most common cancer in Korea. Abnormal BORIS expression in the patient's gastric cancer tissues was observed. We checked the methylation status of the gene in gastric cancer tissue, because the regulation by methylation in its CpG islands is well known for BORIS. However, there was no correlation between the methylation status and gene expression. Then, we focused on the minisatellites (variable number of tandem repeats) of BORIS as another possible regulator for this abnormal expression. Previously, we reported the characterization of BORIS-MS2 and determined the frequency of alleles in cancer patients. A case-control study was performed using DNA from 774 controls and 496 patients with gastric cancer. There was no significant difference observed in the overall distribution of minisatellite alleles. These results suggest that additional different regulators for the abnormal BORIS expression in gastric cancer may exist. Additionally, we performed a segregation analysis of BORIS-MS2 with genomic DNA obtained from two generations of five families and from three generations of two families. BORIS-MS2 alleles were transmitted through meiosis following Mendelian inheritance, which suggests that this polymorphic minisatellite could be a useful marker for paternity mapping and DNA fingerprinting.     

Analysis of Allelic Association: Estimation of the Power of the TDT

Analysis of allelic associations is an increasingly more widely used approach to fine mapping of genes of various diseases. To interpret the results correctly, it is necessary to estimate the power of the statistical test used. The principle of the analysis of associations and testing of hypothesis are described, and analytically obtained estimates of the power of the transmission disequilibrium test (TDT), one of the most popular methods of analysis of allelic associations, are presented. These estimates are applicable to arbitrary models of inheritance formulated in terms of genotypic relative risk. The proposed method is illustrated by analysis of the associations of idiopathic scoliosis and aggrecan gene alleles.     

Prion protein gene polymorphisms in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae genome encodes several proteins that, in laboratory strains, can take up a stable, transmissible prion form. In each case, this requires the Asn/Gln-rich prion-forming domain (PrD) of the protein to be intact. In order to further understand the evolutionary significance of this unusual property, we have examined four different prion genes and their corresponding PrDs, from a number of naturally occurring strains of S. cerevisiae. In 4 of the 16 strains studied we identified a new allele of the SUP35 gene (SUP35delta19) that contains a 19-amino-acid deletion within the N-terminal PrD, a deletion that eliminates the prion property of Sup35p. In these strains a second prion gene, RNQ1, was found to be highly polymorphic, with eight different RNQ1 alleles detected in the six diploid strains studied. In contrast, for one other prion gene (URE2) and the sequence of the NEW1 gene encoding a PrD, no significant degree of DNA polymorphism was detected. Analysis of the naturally occurring alleles of RNQ1 and SUP35 indicated that the various polymorphisms identified were associated with DNA tandem repeats (6, 12, 33, 42 or 57 bp) within the coding sequences. The expansion and contraction of DNA repeats within the RNQ1 gene may provide an evolutionary mechanism that can ensure rapid change between the [PRION+] and [prion-] states.     

Analysis of allelic association: estimation of the criteria power of the TDT

Analysis of allelic associations is an increasingly more widely used approach to fine mapping of genes of various diseases. To interpret the results correctly, it is necessary to estimate the power of the statistical test used. The principle of the analysis of associations and testing of hypothesis are described, and analytically obtained estimates of the power of the transmission disequilibrium test (TDT), one of the most popular methods of analysis of allelic associations, are presented. These estimates are applicable to arbitrary models of inheritance formulated in terms of relative genotypic risk. The proposed method is illustrated by analysis of the associations of idiopathic scoliosis and aggrecan gene alleles.     

The genomic organization of dispersed tRNA and 5 S RNA genes in Xenopus laevis

The 5 S DNAs and several tDNAs of Xenopus laevis reside primarily in large clusters of tandem repeating units. We have discovered that a substantial number of these genes, along with portions of their adjacent spacer sequences, are also located in dispersed genomic locations apart from the major clusters. This was accomplished by "null-digesting" total genomic DNA with restriction enzymes that do not cut within the X. laevis tDNA or 5 S DNA major repeats. The tDNA and 5 S DNA main clusters therefore remain intact and can be easily separated on gels from the dispersed tDNAs and 5 S DNAs present as low molecular weight restriction fragments. Probing these smaller fragments with different portions of the major repeats has revealed that many of the dispersed genes are organized differently from the corresponding tDNAs and 5 S DNAs of the primary clusters. Some of the fragments containing dispersed genes are actually present in multiple copies. In addition, many tDNA null-digestion fragments contain more than one type of tRNA coding region. One set of "dispersed" tDNAs actually comprises a tandemly arranged minor tDNA family which has retained the same repeat length (3.18 kb) as the major tDNA family, but has a substantially different organization. There is significant population polymorphism in the organization of the dispersed tDNAs and 5 S DNAs. Dispersed genes that appear to be derived from clusters of tandem repeats ("orphons") have been described for several gene families in invertebrates. The occurrence of this phenomenon in vertebrates as well, suggests that such dispersed genes may be a general feature of all eukaryotic genomes.     

Association of polymorphisms in the dopamine D4 receptor gene and the activity-impulsivity endophenotype in dogs

A variable number of tandem repeats (VNTR) polymorphism in exon 3 of the human dopamine D4 receptor gene ( DRD4 ) has been associated with attention deficit hyperactivity disorder (ADHD). Rodents possess no analogous repeat sequence, whereas a similar tandem repeat polymorphism of the DRD4 gene was identified in dogs, horses and chimpanzees. Here, we present a genetic association study of the DRD4 VNTR and the activity-impulsivity dimension of the recently validated dog-ADHD Rating Scale. To avoid false positives arising from population stratification, a single breed of dogs (German shepherd) was studied. Two DRD4 alleles (referred to as 2 and 3a ) were detected in this breed, and genotype frequencies were in Hardy–Weinberg equilibrium. For modelling distinct environmental conditions, 'pet' and 'police' German shepherds were characterized. Police German shepherds possessing at least one 3a allele showed significantly higher scores in the activity-impulsivity dimension of the dog-ADHD Rating Scale than dogs without this allele ( P  = 0.0180). This difference was not significant in pet German shepherds. To the best of our knowledge, this is the first report of an association between a candidate gene and a behaviour trait in dogs, and it reinforces the functional role of DRD4 exon 3 polymorphism.     

A high frequency of length polymorphisms in repeated sequences adjacent to Alu sequences

We describe a new class of DNA length polymorphism that is due to a variation in the number of tandem repeats associated with Alu sequences (Alu sequence-related polymorphisms). The polymerase chain reaction was used to selectively amplify a (TTA)n repeat identified in the 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase gene from genomic DNA of 41 human subjects, and the size of the amplified products was determined by gel electrophoresis. Seven alleles were found that differed in size by integrals of three nucleotides. The allele frequencies ranged from 1.5% to 52%, and the overall heterozygosity index was 62%. The polymorphic TTA repeat was located adjacent to a repetitive sequence of the Alu family. A homology search of human genomic DNA sequences for the trinucleotide TTA (at least five members in length) revealed tandem repeats in six other genes. Three of the six (TTA)n repeats were located adjacent to Alu sequences, and two of the three (in the genes for beta-tubulin and interleukin-1 alpha) were found to be polymorphic in length. Tandemly repetitive sequences found in association with Alu sequences may be frequent sites of length polymorphism that can be used as genetic markers for gene mapping or linkage analysis.     

Population and Family Studies of the CAG Repeats in the IT-15Gene

A simple and effective method for typing of CAG repeats in the IT-15gene has been suggested. This method was applied for examination of the CAG allele distribution in Huntington's disease (HD) patients in five different populations from the Commonwealth of Independent States. A total of 21 normal alleles with the sizes ranging from 9 to 32 triplet repeats units were revealed. Moreover, alleles with the sizes ranging from 16 to 20 repeats predominated constituting from 54.4 to 74.6% of all alleles in different populations. The number of repeats in one allele in HD patients exceeded 38 units (43 triplets on average). In two families an increase in the CAG repeat units number in the mutant allele upon its paternal transmission was recorded.     

Extended MHC haplotypes and CYP21/C4 gene organisation in Irish 21-hydroxylase deficiency families

Summary We have analysed fifteen classical 21-hydroxylase deficiency families from throughout Southern Ireland and report the serologically defined HLA-A, HLA-B, HLA-Cw, HLA-DR, C4A and C4B polymorphisms that characterize the inferred disease haplotypes. Additionally, we have used a combination of short and long range restriction mapping procedures in order to characterize the CYP21/C4 gene organization associated with individual serologically defined haplotypes. The results obtained indicate that disease haplotypes are characterized by a high frequency (33%) of CYP21B gene deletion and 8 out of 10 such deletion haplotypes are represented by the extended haplotype HLA-DR1, C4BQo, C4A3, HLA-B40(w60), HLA-Cw3, HLA-A3. Large scale length polymorphism in the CYP21/C4 gene cluster was found to conform strictly to a variable number of tandem repeats model with 4 alleles being detected. Disease haplotypes in which defective CYP21B gene expression is inferred to result from pathological point mutations show extensive diversity of associated HLA markers and include two examples of the extended HLA haplotype HLA-DR3, B8, Cw7, A1 haplotype, which has previously been reported to be negatively associated with 21-hydroxylase deficiency. One unusual disease haplotype has two CYP21 + C4 units, both of which appear to contain CYP21B-like genes.     

Analysis of ALS5 and ALS6 allelic variability in a geographically diverse collection of Candida albicans isolates

The Candida albicans ALS (agglutinin-like sequence) gene family encodes eight cell-surface glycoproteins, some of which function in adhesion to host surfaces. ALS genes have a central tandem repeat-encoding domain comprised entirely of head-to-tail copies of a conserved 108-bp sequence. The number of copies of the tandemly repeated sequence varies between C. albicans strains and often between alleles within the same strain. Because ALS alleles can encode different-sized proteins that may have different functional characteristics, defining the range of allelic variability is important. Genomic DNA from C. albicans strains representing the major genetic clades was PCR amplified to determine the number of tandemly repeated sequence copies within the ALS5 and ALS6 central domain. ALS5 alleles had 2-10 tandem repeat sequence copies (mean=4.82 copies) while ALS6 alleles had 2-8 copies (mean=4.00 copies). Despite this variability, tandem repeat copy number was stable in C. albicans strains passaged for 3000 generations. Prevalent alleles and allelic distributions varied among the clades for ALS5 and ALS6. Overall, ALS6 exhibited less variability than ALS5. ALS5 deletions can occur naturally in C. albicans via direct repeats flanking the ALS5 locus. Deletion of both ALS5 alleles was associated particularly with clades III and SA. ALS5 exhibited allelic polymorphisms in the coding region 5' of the tandem repeats; some alleles resembled ALS1, suggesting recombination between these contiguous loci. Natural deletion of ALS5 and the sequence variation within its coding region suggest relaxed selective pressure on this locus, and that Als5p function may be dispensable in C. albicans or redundant within the Als family.     

Use of short sequence repeat DNA polymorphisms after PCR amplification to detect the parental origin of the additional chromosome 21 in Down syndrome.

The origin of nondisjunction in trisomy 21 has so far been studied using cytogenetic heteromorphisms and DNA polymorphisms using Southern blot analysis. Short sequence repeats have recently been described as an abundant class of DNA polymorphisms in the human genome, which can be typed using the polymerase chain reaction (PCR) amplification. We describe the usage of such markers on chromosome 21 in the study of parental origin of the additional chromosome 21 in 87 cases of Down syndrome. The polymorphisms studied were (a) two (GT)n repeats and a poly(A) tract of an Alu sequence within the HMG14 gene and (b) a (GT)n repeat of locus D21S156. The parental origin was determined in 68 cases by studying the segregation of polymorphic alleles in the nuclear families (either by scoring three different alleles in the proband or by dosage comparison of two different alleles in the proband). Our results demonstrate the usefulness of highly informative PCR markers for the study of nondisjunction in Down syndrome.     

Complex segregation analysis of Parkinson's disease in the Finnish population

The risk of Parkinson's disease (PD) is higher among relatives of affected individuals than among other members of the population, and most family studies have suggested autosomal dominant inheritance, although both autosomal dominant and recessive susceptibility genes have recently been identified. We carried out a complex segregation analysis with POINTER to assess the mode of inheritance of PD in the population of northern Finland. Nuclear families (n=265) were identified through a proband with idiopathic PD. The analysis was first carried out for the total data set, and then the heterogeneity between early-onset (proband under 55 years at onset) and late-onset families was examined. Finally, families with more than one affected individual were analyzed separately. The sporadic model was rejected (P<0.0001). Significant heterogeneity was found between the early-onset and late-onset families, suggesting that major genes have a greater role in early-onset PD than in late-onset PD and that the etiology of idiopathic PD is heterogeneous, even in the Finnish population, which has evolved from a small group of founders. The analysis of familial PD supported the hypothesis that a major locus was present in this subset, but it was not possible to distinguish between a recessive model with a high penetrance and a dominant model with lower penetrance.