The A1 allele of TaqI A restriction fragment length polymorphism (RFLP) in the D2 receptor (DRD2) gene locus has been suggested to be associated with low D2 receptor density in man. Striatal dopamine transporter (DAT) densities were studied with [(123)I]2-beta-carbometoxy-3beta(4-iodophenyl)tropane and single-photon emission tomography in 29 detoxified alcoholics, who were also genotyped for the two alleles of TaqI A RFLP at the DRD2 receptor gene locus. Alcoholics with the A1/A2 genotypes (n = 10) had statistically significantly higher DAT densities than subjects with the A2/A2 genotypes [n = 19; 8.0 +/- 1.2 (mean +/- SD) vs 6.9 +/- 1.1, P = 0.035]. We suggest that the TaqI A RFLP is in linkage disequilibrium with a gene variant modifying DAT density in alcoholics.
The results of DNA analysis of deletion in exons 7 and 8 of SMN gene, and exon 5 of NAIP gene in 24 SMA-families from Ukraine are presented. Deletions of SMN exons 7 and 8, or 7 were found in 46 (97.9%) of 47 SMA-chromosomes. A homozygous deletion of NAIP exon 5 was demonstrated in 4 (19%) of 21 SMA-families. The authors have demonstrated that in 2 SMA patients with homozygous deletion SMN exon 7 only, the remaining SMN exon 8 was a part of a chimeric CBCD41/SMN gene.
DNA was extracted from specimens derived from the calcaneus of the Tyrolean Ice Man under sterile conditions in a laboratory, where no DNA extractions and PCR experiments had been performed before. Agarose gel electrophoresis and ethidium bromide staining did not reveal any evidence of genomic DNA in the preparation obtained, indicating a high degree of DNA degradation. Nevertheless, we performed PCR amplifications with this sample using primer pairs specific for HLA class II alleles. HLA-DRB and DQB1 alleles were amplified in a nested PCR approach. In one of the reactions, we observed a distinct amplification product, which we directly sequenced. By comparing the obtained nucleotide sequence with a database of HLA alleles we assigned the HLA-DRB1*1402 type to the amplified sample. None of the investigators involved possesses this allele, indicating that no contamination with modern DNA had occurred. The HLA-DRB1*1402 allele is extremely rare in Europe, but is common in Inuits and South American Indians and has previously only once been identified in the laboratory.
Smoking-induced damage to the cardiovascular system has been shown in many studies; however, the degree of damage varies from individual to individual. We hypothesized that the -930A/G CYBA gene polymorphism in the NADPH oxidase influences the association between cigarette smoking and carotid intima-media thickness (IMT) in young healthy adults.
Cross-sectional data obtained in 2001 for the Cardiovascular Risk in Young Finns Study were used. IMT was measured with ultrasound. The genotyping was performed using a 5'-nuclease assay. A linear regression model was used to test whether the interaction between smoking and the genotypes was associated with IMT. The magnitude of the interaction effect was further examined by performing a stratified analysis according to smoking habits.
In the entire population, the mean and maxima IMT were higher in smokers than nonsmokers (P = 0.005 and 0.008, respectively). The differences were most significant in subjects with the GG genotype, borderline significant for the GA genotype, and nonsignificant for the AA genotype. The interaction of genotypes with smoking was associated with mean and maximal IMT (P = 0.042 and 0.022). Among smokers, subjects with the GG genotype had a higher mean and maximal IMT compared with carriers of the A allele (P = 0.021 and 0.012). In contrast, the mean and maximal IMT were lower for G allele carriers than subjects with the AA genotype among nonsmokers (P = 0.022 and 0.026). All results had been adjusted for potential risk factors related to IMT.
The -930A/G polymorphism modifies the association between cigarette smoking and IMT in young healthy adults.
Brachydactyly type A1 (BDA1; MIM 112500) is characterized by shortness or absence of the middle phalanx of the hands and feet. The condition is caused by heterozygous mutations in the Indian hedgehog (IHH) gene or a yet unidentified gene on chromosome 5p13. We investigated six affected members of a large Swedish family segregating autosomal dominant brachymesophalangia. Affected individuals show hypoplasia of the ulnar styloid processes, ulna minus, osteoarthritis, normal length of all distal phalanges and shortening or absence of the middle phalanges. Stationary ossicles or sesamoid bones were observed at the metacarpal heads in all patients. Genetic analysis of the family showed that the IHH-gene was linked to the disease (Z(max) 3.42 at theta 0.00) and sequence analysis of IHH revealed a novel c.472C > T transition in all affected family members. The mutation results in a p.158Arg > Cys substitution located in the highly conserved amino-terminal domain of IHH. This domain is of importance for the interaction between IHH and the Patched receptor. Our combined findings add radiological findings to the BDA1 phenotype and confirm a critical functional domain of IHH.
Catching the fish with the worm: a case study on eDNA detection of the monogenean parasite Gyrodactylus salaris and two of its hosts, Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss).
Environmental DNA (eDNA) monitoring is growing increasingly popular in aquatic systems as a valuable complementary method to conventional monitoring. However, such tools have not yet been extensively applied for metazoan fish parasite monitoring. The fish ectoparasite Gyrodactylus salaris, introduced into Norway in 1975, has caused severe damage to Atlantic salmon populations and fisheries. Successful eradication of the parasite has been carried out in several river systems in Norway, and Atlantic salmon remain infected in only seven rivers, including three in the Drammen region. In this particular infection region, a prerequisite for treatment is to establish whether G. salaris is also present on rainbow trout upstream of the salmon migration barrier. Here, we developed and tested eDNA approaches to complement conventional surveillance methods.
Water samples (2 × 5 l) were filtered on-site through glass fibre filters from nine locations in the Drammen watercourse, and DNA was extracted with a CTAB protocol. We developed a qPCR assay for G. salaris targeting the nuclear ribosomal ITS1 region, and we implemented published assays targeting the mitochondrial cytochrome-b and NADH-regions for Atlantic salmon and rainbow trout, respectively. All assays were transferred successfully to droplet digital PCR (ddPCR).
All qPCR/ddPCR assays performed well both on tissue samples and on field samples, demonstrating the applicability of eDNA detection for G. salaris, rainbow trout and Atlantic salmon in natural water systems. With ddPCR we eliminated a low cross-amplification of Gyrodactylus derjavinoides observed using qPCR, thus increasing specificity and sensitivity substantially. Duplex ddPCR for G. salaris and Atlantic salmon was successfully implemented and can be used as a method in future surveillance programs. The presence of G. salaris eDNA in the infected River Lierelva was documented, while not elsewhere. Rainbow trout eDNA was only detected at localities where the positives could be attributed to eDNA release from upstream land-based rainbow trout farms. Electrofishing supported the absence of rainbow trout in all of the localities.
We provide a reliable field and laboratory protocol for eDNA detection of G. salaris, Atlantic salmon and rainbow trout, that can complement conventional surveillance programs and substantially reduce the sacrifice of live fish. We also show that ddPCR outperforms qPCR with respect to the specific detection of G. salaris.
The intron 16 insertion/deletion (I/D) polymorphism in the angiotensin-converting enzyme (ACE) gene has been associated with rupture of intracranial aneurysms, but the effect of haplotypes within ACE has not been studied. This study investigated whether ACE haplotypes including the I/D polymorphism are associated with aneurysmal subarachnoid hemorrhage.
The hypothesis was tested with a case-control design in 176 patients with aneurysmal subarachnoid hemorrhage and with 498 hospital controls. Through the pairwise tagging principle, single nucleotide polymorphisms (rs4291 A/T, rs4295 C/G, rs4305 C/T, rs4311 C/T, rs4331 T/C, rs4343 C/T) in the ACE gene were genotyped along with the I/D polymorphism. Haplotypes were estimated using the PHASE software.
Fifty-five haplotypes were identified with 3 of these having a frequency above 5%: ACCCCIT (41.6±0.4%), TGTTTDC (32.1±0.5%), and ACCTTDC (9.5±0.2%). No significant difference in distribution of alleles, genotypes, haplotypes, or haplotype pairs between the 2 populations was found. Specifically, we could not reproduce previously reported associations between the ACE I genotype and intracranial aneurysms. When subdivided into groups of aneurysm location, we found a trend toward an association between homozygotes of the ACCCCIT haplotype and middle cerebral artery aneurysms, odds ratio=2.9 (1.0 to 7.6), which however proved insignificant (P=0.22) after correction for multiple testing.
In this Danish population, ACE haplotypes and the I/D polymorphism did not contribute significantly to the overall risk of intracranial aneurysm rupture. Larger studies are needed to delineate the association between ACE polymorphism and ruptured middle cerebral artery aneurysms.
We have determined the primary structure of the alpha 1(IV)-chain of human type IV collagen by nucleotide sequencing of overlapping cDNA clones that were isolated from a human placental cDNA library. The present data provide the sequence of 295 amino acids not previously determined. Altogether, the alpha 1(IV)-chain contains 1642 amino acids and has a molecular mass of 157625 Da. There are 1413 residues in the collagenous domain and 229 amino acids in the carboxy-terminal globular domain. The human alpha 1(IV)-chain contains a total of 21 interruptions in the collagenous Gly-X-Y repeat sequence. These interruptions vary in length between two and eleven residues. The alpha 1(IV)-chain contains four cysteine residues in the triple-helical domain, four cysteines in the 15-residue long noncollagenous sequence at the amino-terminus and 12 cysteines in the carboxy-terminal NC-domain.
We have isolated and characterized overlapping cDNA clones which code for a previously unidentified human collagen chain. Although the cDNA-derived primary structure of this new polypeptide is very similar to the basement membrane collagen alpha 1(IV) and alpha 2(IV) chains, the carboxyl-terminal collagenous/non-collagenous junction sequence does not correspond to the junction sequence in either of the newly described alpha 3(IV) or alpha 4(IV) chains (Butkowski, R.J., Langeveld, J.P.M., Wieslander, J., Hamilton, J., and Hudson, B. G. (1987) J. Biol. Chem. 262, 7874-7877). Thus the protein presented here has been designated the alpha 5 chain of type IV collagen. Four clones encode an open reading frame of 1602 amino acids that cover about 95% of the entire chain including half of the amino-terminal 7S domain and all of the central triple-helical region and carboxyl-terminal NC1 domain. The collagenous region of the alpha 5(IV) chain contains 22 interruptions which are in most cases identical in distribution to those in both the alpha 1(IV) and alpha 2(IV) chains. Despite the relatively low degree of conservation among the amino acids in the triple-helical region of the three type IV collagen chains, analysis of the sequences clearly showed that alpha 5(IV) is more related to alpha 1(IV) than to alpha 2(IV). This similarity between the alpha 5(IV) and alpha 1(IV) chains is particularly evident in the NC1 domains where the two polypeptides are 83% identical in contrast to the alpha 5(IV) and alpha 2(IV) identity of 63%. In addition to greatly increasing the complexity of basement membranes, the alpha 5 chain of type IV collagen may be responsible for specialized functions of some of these extracellular matrices. In this regard, it is important to note that we have recently assigned the alpha 5(IV) gene to the region of the X chromosome containing the locus for a familial type of hereditary nephritis known as Alport syndrome (Myers, J.C., Jones, T.A., Pohjalainen, E.-R., Kadri, A.S., Goddard, A.D., Sheer, D., Solomon, E., and Pihlajaniemi, T. (1990) Am. J. Hum. Genet. 46, 1024-1033). Consequently, the newly discovered alpha 5(IV) collagen chain may have a critical role in inherited diseases of connective tissue.
The European land leech Xerobdella lecomtei was discovered in 1868 and is one of the rarest animals on Earth. During the 1960s, several individuals of these approx. 40 mm long, cold-adapted terrestrial annelids that inhabit the moist soils of birch forests around Graz, Austria, were investigated. Only one original research paper has been published on the biology of this species. Between 2001 and 2005, we re-investigated the morphology of preserved specimens and searched for living individuals in their natural habitat that appeared to be intact. We found only one juvenile individual (length approx. 10 mm), indicating that this local leech population became largely extinct over the past four decades. The feeding behaviour of our 'lonesome George of the annelids' was studied and is described here in detail. After its death, the Xerobdella individual was used for chemical extraction and molecular studies (deoxyribonucleic acid [DNA] barcoding, based on one gene, the mitochondrial cytochrome c oxidase subunit I). In addition, novel DNA barcodes for a land leech from Madagascar and a recently discovered species from Europe were obtained. Our phylogenetic tree shows that X. lecomtei is not a member of the tropical land leeches (family Haemadipsidae), as previously thought, but represents a separate line of descent (family Xerobdellidae). The decline of the local leech population around Graz correlates with a rise in average summer temperatures of +3 degrees C between 1961 and 2004. This warming led to a drastic reduction in the moisture content of the soil where X. lecomtei lives. We suggest that human-induced climate change without apparent habitat destruction can lead to the extinction of populations of cold-adapted species that have a low colonization ability.