Analysis of the Polymorphic Markers within the CFTR Gene in Cystic Fibrosis Patients and Healthy Donors from Bashkortostan

The differences in the polymorphic allele frequency distribution patterns of the biallelic (M470V and TUB20) and microsatellite (IVS6aGATT, IVS8CA, and IVS17CA) markers within the CFTR gene between normal and delF508 chromosomes have been established. For most of the marker loci similar distribution of the allele frequencies on normal and mutant chromosomes without delF508 was demonstrated. Certain polymorphic alleles displayed substantial linkage disequilibrium with the delF508 mutation. Analysis of the IVS6aGATT-IVS8CA-M470V-IVS17CA-TUB20 haplotypes association on normal and mutant chromosomes provided identification of the delF508 ancestral haplotype. It was suggested that delF508 mutant chromosomes were introduced into the modern Bashkir gene pool as a result of Slavic migrations from the Eastern Europe. The IVS6aGATT-IVS8CA-M470V-IVS17CA-TUB20major haplotype (77272) revealed was statistically significantly most frequently found on the mutant chromosomes without the delF508 mutation. This finding suggests that the Bashkir cystic fibrosis patients, mostly belonging to the Turkic-speaking families, possessed specific CFTR gene defect associated with the given haplotype.     

Complex HTR2C linkage disequilibrium and promoter associations with body mass index and serum leptin

The occurrence of obesity, eating disorders, and related diseases has increased in many parts of the world. Given that few strong genetic factors have been found, it is clear that these are complex multi-factorial diseases. The serotonin receptor 2C, a member of the 5-HTergic system, has been implicated in the control of phagia and obesity. We report a detailed investigation of linkage disequilibrium (LD) within and between the HTR2C promoter and the flanking sequences around a commonly utilized marker in the second coding exon of HTR2C. We suggest that inconsistent associations between HTR2C and several phenotypes, including obesity, may be due to the LD pattern across the gene in which recombination and gene conversion have been influential. The nucleotide and haplotype distribution is consistent with that of the neutral mutation model. The number of haplotypes suggests demographic influences or over dominant selection that may have a function in HTR2C expression. Using the fine LD pattern, we describe a possible association with promoter haplotypes and diplotypes, including a GT microsatellite, and body mass index (BMI) 30 kgm^–2 (P<0.0001). SNP –995G>A heterozygotes, as well as promoter diplotypes, were found to marginally influence higher serum leptin corrected for percentage body fat (P=0.01), which might suggest that these subjects are leptin resistant. Our results complement previous studies of HTR2C in both mice and humans, and suggest the importance of genetic variation and elucidating the fine LD structure in uncovering the genetic factors of obesity.Electronic Supplementary Material Supplementary material is available for this article at     

Lipoprotein Lipase Gene is Linked and Associated with Hypertension in Taiwan Young-onset Hypertension Genetic Study

Summary Hypertriglyceridemia has been extensively associated with hypertension. However, the mechanism behind it is poorly understood. A positive linkage signal between Lipoprotein lipase (LPL) and young-onset hypertension has been identified by us as the strongest among 18 candidate genes. Here we report our fine mapping works with seven microsatellite markers flanking LPL, sequencing results for its promoter and exons, and an extended association study with the identified single nucleotide polymorphisms(SNP). First, using data from 213 individuals in 59 nuclear families of young-onset hypertension, multipoint analysis revealed a NPL score of 3.02 for the LPL (GZ-14/GZ-15) marker in intron 6. LPL marker (p<10⁻¹²) and the haplotypes containing its allele 1 (p<0.0001) were also significantly associated with young hypertension by transmission disequilibrium test. In-depth sequencing revealed no mutation in promoter and exon regions, except two cSNP: 7754C→ A (C/A: 0.91/0.09), a silent mutation in exon 8 and S447X (C/G: 0.92/0.08), a stop codon mutation in exon 9. Other 11 cSNPs documented in NCBI GenBank are absent in our sample. Constructed from the above 2 cSNPs, haplotype AC showed a moderate TDT association with elevated triglyceride (p=0.02) and with hypertension and elevated triglyceride combined (p=0.06). Again, in an extended case-control study, a significant association was found between S447X and patients with persistent hypertension and elevated triglyceride (p=0.02). We conclude that LPL variants may play a causal role in the development of hypertension in Taiwan Han Chinese. The moderate association with SNP haplotype suggests that other regulatory LPL variant may exist.     

Genetic Variation in the Leptin Receptor Gene,Leptin, and Weight Gain in Young Dutch Adults

Objective: To investigate the association between leptin levels, polymorphisms in the leptin receptor (LEPR) gene, and weight gain. Research Methods and Procedures: From two large prospective cohorts in The Netherlands (n = 17, 500), we compared the baseline leptin of 259 subjects who had gained an average of 12.6 kg (range 5.5 to 33 kg) with 277 subjects who kept stable weight (range ?2.6 to 3.1 kg) after a mean follow‐up of 6.8 years. Three polymorphisms in the LEPR gene (Lys109Arg, Gln223Arg, and Lys656Asn) were determined. Results: Weight gainers had significantly higher baseline leptin levels than those who kept stable weight (odds ratio = 1.27, 95% confidence interval 1.1 to 1.5, per SD increase in log_e‐transformed leptin). Weight gainers with the Arg109 or the Arg223 alleles had higher leptin levels compared with the noncarriers of these alleles. Only among men, the association between leptin and weight gain tended to be stronger among those with an Arg223 allele compared with those without this mutation. Discussion: Relatively high leptin levels predict weight gain, suggesting that leptin resistance plays a role in the development of obesity in the general population. Higher leptin levels for those with a Lys109Arg or Gln223Arg mutation (or a linked other marker) may imply that these subjects have a modified functional leptin receptor. However, the role of these mutations on weight gain is limited.     

New haplotypes in the Bedlington terrier indicate complexity in copper toxicosis

Copper toxicosis (CT) is an autosomal recessive disorder common in Bedlington terriers. Previously, the CT locus was mapped to canine Chromosome (Chr) 10q26 through linkage to marker C04107. Diagnosis, traditionally based on liver biopsy, has recently shifted to interpretation of the C04107 microsatellite alleles where allele 2 segregates with the disease with 90–95% accuracy. Recently, CT has been attributed to a deletion of exon 2 in the MURR1 gene. We also identified a deletion of exon 2 of MURR1 in our collection of 2-2 homozygous affected terriers. However, our collection also included affected 1-1 homozygotes and 1-2 heterozygotes, and these dogs did not have the homozygous deletion. In addition to C04107, we analyzed an adjacent microsatellite (C04107B), and two novel SNPs, all within intron 1 of MURR1, and sequenced all exons and their intronic boundaries. Pedigree analysis indicates that there are two typical haplotypes, one normal and one affected, maintaining complete linkage disequilibrium between C04107 allele 2 and the deletion in most pedigrees. Most importantly, we identified a recombinant haplotype present in a North American pedigree, where allele 2 is not linked with the deletion, and a fourth haplotype containing a splice site variant. Although the splice site alteration appears to be a normal variant, it is present in two affected dogs, which do not carry homozygous deletions of MURR1.     

Analysis of intragenic polymorphic markers of the CFTR gene in cystic fibrosis patients and health donors from Bashkorostan

The differences in the polymorphic allele frequency distribution patterns of the biallelic (M470 and TUB20) and microsatellite (IVS6aGATT, IVS8CA, and IVS17CA) markers within the CFTR gene between normal and delF508 chromosomes have been established. For most of the marker loci similar distribution of the allele frequencies on normal and mutant chromosomes without delF508 was demonstrated. Certain polymorphic alleles displayed substantial linkage disequilibrium with the delF508 mutation. Analysis of the IVS6aGATT-IVS8CA-M470-IVS17CA-TUB20 haplotypes association on normal and mutant chromosomes provided identification of the delF508 ancestral haplotype. It was suggested that delF508 mutant chromosomes were introduced into the modern Bashkir gene pool as a result of Slavic migrations from the Eastern Europe. The IVS6aGATT-IVS8CA-M470-IVS17CA-TUB20 major haplotype (77272) revealed was statistically significantly most frequently found on the mutant chromosomes without the delF508 mutation. This finding suggests that the Bashkir cystic fibrosis patients, mostly belonging to the Turkic-speaking families, possessed specific CF gene defect associated with the given haplotype.     

Data on the CGG repeat at the fragile X site in the non-retarded Japanese population and family suggest the presence of a subgroup of normal alleles predisposing to mutate

The fragile X mutation is the result of amplification in the repeat number of p(CGG) _n in FMR-1; alleles with more than 52 repeats have been shown to be so unstable as to mutate in the repeat number in almost every transmission. To improve our understanding of mutations in normal alleles of FMR-1, the following studies were carried out in the Japanese population: a study on length variation in the repeat to determine the allele distribution of the repeat length in a non-retarded population, family studies to observe new mutations in normal allele, and haplotype analyses with microsatellite markers flanking the repeat to confirm estimated mutation rates and founder chromosomes in the fragile X syndrome. Analysis of the p(CGG) _n in 370 unrelated males detected 24 distinct alleles with repeats of 18–44. A comparison with previously reported data suggests the presence of racial/ethnic differences in the allele distribution. No premutation allele was found in 824 unrelated X chromosomes examined by the polymerase chain reaction and Southern blot analysis. Family studies detected one new mutation in a total of 303 meioses. However, the mutation rate was not in accordance with the expected or observed heterozygosities in the population or with linkage disequilibrium observed between the repeat numbers and the haplotypes of the markers flanking the CGG. The haplotype in the chromosome in which the new mutation was found was the same as that frequently found in the Japanese fragile X chromosomes, and the variance in the CGG repeat number was wider in chromosomes with the haplotypes frequently found in the fragile X chromosome than in those with the other haplotypes. These observations suggest that a subgroup is present in normal alleles and that this subgroup is more liable to mutate than others.     

Evolution of Microsatellite Alleles in Four Species of Mice (Genus Apodemus)

Microsatellite length variation was investigated at a highly variable microsatellite locus in four species of Apodemus. Information obtained from microsatellite allele sequences was contrasted with allele sizes, which included 18 electromorphs. Additional analysis of a 400-bp unique sequence in the flanking region identified 26 different haplotype sequences or ``true' alleles in the sample. Three molecular mechanisms, namely, (1) addition/deletion of repeats, (2) substitutions and indels in the flanking region, and (3) mutations interrupting the repeat, contributed to the generation of allelic variation. Size homoplasy can be inferred for alleles within populations, from different populations of the same species, and from different species. We propose that microsatellite flanking sequences may be informative markers for investigating mutation processes in microsatellite repeats as well as phylogenetic relationships among alleles, populations, and species. Received: 3 November 1999 / Accepted: 2 May 2000     

Sequence tagged microsatellites for the Xgwm533 locus provide new diagnostic markers to select for the presence of stem rust resistance gene Sr2 in bread wheat ( Triticum aestivum L.)

The stem rust resistance gene Sr2 has provided durable broad-spectrum, adult-plant resistance to the fungal pathogen Puccinia graminis Pers. f. sp. tritici throughout wheat-growing regions of the world for more than 50 years. The ability to select for Sr2 in wheat breeding programs was recently improved by the identification of a tightly linked microsatellite marker gwm533. This marker typically amplifies a 120-bp polymerase chain reaction fragment from wheat lines carrying Sr2. In instances where the 120-bp fragment is not associated with the presence of Sr2, DNA sequence analysis has shown that a second allele was amplified, differing in the structure of the microsatellite repeat. To discriminate this allelic homoplasy (alleles identical in size, but not identical by descent), sequence-tagged microsatellites (STM) markers were developed for the Xgwm533 locus. These markers were shown to be diagnostic for the presence of Sr2 in a wide range of germplasm, representative of all major wheat varieties historically grown in Australia. The STMs will be particularly useful for marker-assisted selection in Southern Australian breeding programs, where the use of the marker gwm533 is often precluded by the presence of the non-Sr2-associated 120-bp allele in the pedigree of current breeding germplasm. The STMs also revealed a high incidence of previously undetected allelic homoplasy at the Xgwm533 locus and may have broader utility in genetic research and breeding, as this locus is also reported to be strongly associated with a major gene conferring resistance to Fusarium head blight.     

Correlation between grass carp (Ctenopharyngodon idella) resistance to grass carp reovirus and the genetic insert-deletion polymorphisms in promoter and intron of RIG-I gene

RIG-I (retinoic acid-inducible gene I) is an essential cytosolic pathogen recognition receptor that binds to a variety of viral RNA or DNA to induce type I interferons. In the present study, insert–deletion polymorphisms in promoter and introns of CiRIG-I (Ctenopharyngodon idella RIG-I) were explored, their associations with resistance/susceptibility to grass carp reovirus (GCRV) were analyzed. To this end, genomic sequence of CiRIG-I gene was obtained, and twenty pairs of primers were prepared for the detection of insert–deletion polymorphisms. Five insert–deletion mutations were found, a 2-bp mutation and an 8-bp mutation existed in the promoter and other three sizes in 74 bp, 146 bp and 53 bp were sited in the intron 8. After a challenge experiment, only the genotype and allele of − 740 insert–deletion mutation in the promoter and allele of 6804 insert–deletion mutation were significantly associated with resistance/susceptibility to GCRV among the five mutations (P < 0.05). To further identify this correlation, another independent challenge test was carried out. The result revealed that the cumulative mortality in ins/ins genotype individuals (43.75%) at − 740 insert–deletion mutation was significantly lower than that in ins/del (72.09%) and del/del (74.19%) genotypes (P < 0.05). Linkage disequilibrium and haplotype analysis showed 6610 insert–deletion mutation and 6804 insert–deletion mutation were linkage disequilibrium. The haplotype ins–ins (6610ins–6804ins) was significantly susceptible to GCRV, and ins–del (6610ins–6804del) was significantly resistant to GCRV (P < 0.05). Those could be potential gene markers for the future molecular selection of strains that are resistant to GCRV.     

Identification of a missense phenylketonuria mutation at codon 408 in Chinese

Summary A single base transition of G to A at codon 408 of the phenylalanine hydroxylase gene is identified. This missense mutation results in the substitution of Arg⁴⁰⁸ for Gln⁴⁰⁸ (R408Q) and accounts for about 5% of phenylketonuria (PKU) chromosomes among Chinese. This mutation is in linkage disequilibrium with restriction fragment length polymorphism haplotype 4. In addition, another mutation (R408W), at the same codon and prevalent on haplotype 2 PKU chromosomes in Caucasians, is identified in a PKU allele of haplotype 41. Previously, this mutation has been observed on a haplotype 44 background in Chinese PKU patients.     

Sequence variants of the brain-derived neurotrophic factor (BDNF) gene are strongly associated with obsessive-compulsive disorder

We evaluated a possible association between the brain-derived neurotrophic factor (BDNF) gene and susceptibility to obsessive-compulsive disorder (OCD) by genotyping a number of single-nucleotide polymorphisms (SNPs) and one microsatellite marker from the extended BDNF locus in 164 triads with OCD. Extensive background linkage disequilibrium was observed at this locus. Single-locus transmission-distortion tests revealed significant evidence of association with the disease for all the BDNF gene markers tested, including a Val66Met variation affecting the sequence of the proBDNF protein. Analysis of multi-SNP haplotypes provided similar results. Haplotype transmission comparisons in this and previous studies point to a functionally distinct BDNF haplotype uniquely marked by the rare Met66 allele, which is undertransmitted and likely confers a protective effect in OCD and other psychiatric disorders.     

Genetic association analysis of KCNQ3 and juvenile myoclonic epilepsy in a South Indian population

Juvenile myoclonic epilepsy (JME) is a common subtype of idiopathic generalized epilepsy that shows a complex pattern of inheritance. We have tested the association between JME phenotype and an intragenic marker in KCNQ3 by using the transmission disequilibrium test in 119 probands and their parents. Mutations in KCNQ3 are known to cause benign familial neonatal convulsions and are involved in the physiologically important M current in neurons. Our results provide suggestive evidence of allelic association between JME and KCNQ3 (P-value=0.008) and raise an interesting possibility of a genetic contribution to JME, viz., of a gene that causes a monogenic form of human epilepsy.     

The age of human mutation: genealogical and linkage disequilibrium analysis of the CLN5 mutation in the Finnish population.

Variant late infantile neuronal ceroid lipofuscinosis (vLINCL) is an autosomal recessive progressive encephalopathy of childhood enriched in the western part of Finland, with a local incidence of 1 in 1500. We recently assigned the locus for vLINCL, CLN5, to 13q21.1-q32. In the present study, the haplotype analysis of Finnish CLN5 chromosomes provides evidence that one single mutation causes vLINCL in the Finnish population. Eight microsatellite markers closely linked to the CLN5 gene on chromosome 13q were analyzed, to study identity by descent by shared haplotype analysis. One single haplotype formed by flanking markers D13S160 and D13S162 in strong linkage disequilibrium (P < .0001) was present in 81% of disease-bearing chromosomes. Allele 4 at the marker locus D13S162 was detected in 94% of disease-bearing chromosomes. To evaluate the age of the CLN5 mutation by virtue of its restricted geographical distribution, church records were used to identify the common ancestors for 18 vLINCL families diagnosed in Finland. The pedigrees of the vLINCL ancestors merged on many occasions, which also supports a single founder mutation that obviously happened 20 to 30 generations ago (i.e., approximately 500 years ago) in this isolated population. Linkage disequilibrium was detected with seven markers covering an extended genetic distance of 11 cM, which further supports the young age of the CLN5 mutation. When the results of genealogical and linkage disequilibrium studies were combined, the CLN5 gene was predicted to lie approximately 200 - 400 kb (total range 30 - 1360 kb) from the closest marker D13S162.     

RFLP haplotyping and mutation analysis of the phenylalanine hydroxylase gene in Dutch phenylketonuria families

Restriction fragment length polymorphism haplotyping of mutated and normal phenylalanine hydroxylase (PAH) alleles in 49 Dutch phenylketonuria (PKU) families was performed. All mutant PAH chromosomes identified by haplotyping (n = 98) were screened for eight of the most predominant mutations. Compound heterozygosity was proven in 40 kindreds. Homozygosity was found for the IVS12nt1 mutation in 5 families, and for the R158Q and IVS10nt546 mutations in one family each. All patients from these families suffer from severe PKU, providing additional proof that these mutations are deleterious for the PAH gene. Genotypical heterogeneity was evident for mutant haplotype 1 (n = 27) carrying the mutations R261Q (n = 12), E280K (n = 4), P281L (n = 1) and unknown (n = 10), and likewise for mutant haplotype 4 (n = 30) carrying the mutations R158Q (n = 13), Y414C (n = 1) and unknown (n = 16). Mutant haplotype 3 (n = 20), in tight association with mutation IVS12nt1, appeared to be in strong linkage disequilibrium (LDE) with its normal counterpart allele (n = 4). Mutant haplotype 6 (n = 4), in tight association with the IVS10nt546 mutation, showed moderate LDE with its counterpart allele (n = I). The distribution of the mutant PAH haplotypes 1, 3 and 4 among the Dutch PKU population resembles that in other Northern and Western European countries, but it is striking that mutant haplotype 2 and its associated mutation R408W is nearly absent in The Netherlands, in strong contrast to its neighbouring countries.     

The experience of brace treatment in children/adolescents with scoliosis

Background

In a previous study, a number of genes, associated with spine musculoskeletal deformity phenotypes in mouse and in synteny between mouse and man, were identified as candidate genes for IS. Among these genes, MATN 1, which carries a polymorphic microsatellite marker within its sequence, was selected for a linkage analysis. MATN1 is localised at 1p35 and is mainly expressed in cartilage. The objective of this study was to assess a linkage disequilibrium between the matrilin-1 (MATN 1) gene and the idiopathic scoliosis (IS).

Methods

The genetic study was conducted on a population of 81 trios, each consistent of a daughter/son affected by idiopathic scoliosis (IS) and both parents. In all trios components, the region of MATN1 gene containing the microsatellite marker was amplified by a polymerase chain reaction. The amplicons were analysed by a DNA sequencer-genotyper. The statistical linkage analysis was performed using the extended transmission/disequilibrium test.

Results

Three microsatellite polymorphisms, respectively consisting of 103 bp, 101 bp and 99 bp, were identified. ETDT evidenced a significant preferential transmission for the 103 bp allele (Chi-square = 5.058, df = 1, P = 0.024)

Conclusion

The results suggest that the familial idiopathic scoliosis is associated to the MATN1 gene.     

Bacterial artificial chromosome-derived molecular markers for early bolting in sugar beet

Early bolting in sugar beet (Beta vulgaris L.) is controlled by the dominant gene B. From an incomplete physical map around the B gene, 18 bacterial artificial chromosomes (BACs) were selected for marker development. Three BACs were shotgun-sequenced, and 61 open reading frames (ORFs) were identified. Together with 104 BAC ends from 54 BACs, a total number of 55,464 nucleotides were sequenced. Of these, 37 BAC ends and 12 ORFs were selected for marker development. Thirty-one percent of the sequences were found to be single copy and 24%, low copy. From these sequences, 15 markers from ten different BACs were developed. Ten polymorphisms were determined by simple agarose gel electrophoresis of either restricted or non-restricted PCR products. Another five markers were determined by tetra-primer amplification refractory mutation system-PCR. In order to select candidate BACs for cloning the gene, genetic linkage between seven markers and the bolting gene was calculated using 1,617 plants from an F₂ population segregating for early bolting. The recombination values ranged between 0.0033 and 0.0201. In addition, a set of 41 wild and cultivated Beta accessions differing in their early bolting character was genotyped with seven markers. A common haplotype encompassing two marker loci and the b allele was found in all sugar beet varieties, indicating complete linkage disequilibrium between these loci. This suggests that the bolting gene is located in close vicinity to these markers, and the corresponding BACs can be used for cloning the gene.