Mitochondrial mutation rate revisited: hot spots and polymorphism.
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DNA, Mitochondrial↗
Biomedical subjects
U Gyllensten
Publications and source records attributed to U Gyllensten.
Recent origin of HLA-DRB1 alleles and implications for human evolution.
The HLA class I and class II loci are the most highly polymorphic coding regions in the human genome. Based on the similarity of the coding sequences of alleles between species, it has been claimed that the HLA polymorphism is ancient and predates the separation of human (Homo) and chimpanzee (Pan), 4-7.4 Myr ago. Analysis of intron sequences, however, provides support for a more recent origin and for rapid generation of alleles at the HLA class II DRB1 locus. The human DRB1 alleles can be divided into groups (allelic lineages); most of these lineages have diverged from each other before the separation of Homo and Pan. Alleles within such a lineage, however, appear to be, on average, 250,000 years old, implying that the vast majority (greater than 90%) of the more than 135 contemporary human DRB1 alleles have been generated after the separation of Homo and Pan. The coalescence time of alleles within allelic lineages indicates that the effective population size (Ne) for early hominids (over the last 1 Myr) was approximately 10(4) individuals, similar to estimates based on other nuclear loci and mitochondrial DNA. With a single exception, the genetic mechanisms (gene conversion and point mutation) that have diversified the exon-2 sequences do not appear to extend into the adjacent intron sequences. The part of exon 2 encoding the beta-sheet evolves in concert with the surrounding introns, while the alpha-helix appears to have been subjected to gene conversion-like events, suggesting that such exchange events are highly localised and occur over extremely short sequence tracts.
Alleles↗
High resolution genetic typing of the class II HLA-DRB1 locus using group-specific amplification and SSO-hybridisation in microplates.
The HLA-DRB1 locus is one of the most polymorphic HLA class II loci and rapid and accurate typing of this polymorphism is important both in bone-marrow transplantation, analysis of disease association and in forensic medicine. The allelic variation at DRB1 is characterized by combinations of a limited number of amino-acid motifs, reducing the resolution of a typing strategy based on a single PCR and subsequent analysis of polymorphic motifs. In the present paper we describe a strategy for typing of DRB1 based on eight allele-specific PCRs followed by sandwich hybridization to immobilized probes in a microplate format. The combined approach results in a rapid typing system with very high resolution. Using a rapid DNA extraction protocol, a complete HLA-DRB1 typing can be performed in less than a day.
Alleles↗
Intron based radiation hybrid mapping of 15 complex I genes of the human electron transport chain.
At least 34 complex I subunits of the electron transport chain are encoded by the nuclear genome, but only 14 of these have been mapped in the human. To rapidly map additional subunits, we have performed a combination of database mining and direct "wet" experimentation to identify intron and/or 5' upstream genomic DNA regions for 16 complex I genes. Wet experimentation was applied to 5 genes, and involved direct PCR amplification of introns by inter-exon PCR or splinkerette based PCR walking. Database mining was applied to 11 genes, and entailed the identification of incompletely spliced mRNAs and genomic CpG island clone sequences. This data was in files that carried no documentary description of the non-exon regions. Non-exon sequences were thus derived for 15 complex I genes and used to design functional gene specific PCR assays. Radiation hybrid mapping of these PCRs located 15 complex I genes to chromosomes l, 4, 5 (2 genes), 7 (2 genes), 8, 9 (2 genes), 11, 14, 16 (2 genes), 18, and 19.
Chromosome Mapping↗
Rapid DNA purification from agarose gels using 96-well format, centrifuge-driven filtration.
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Biotechnology↗
Mitochondrial DNA sequencing of shed hairs and saliva on robbery caps: sensitivity and matching probabilities.
Sequencing of mitochondrial DNA (mtDNA) has been used for human identification based on teeth and skeletal remains. Here, we describe an amplification system for the mtDNA control region (D-loop) suited for the analysis of shed hair, which constitutes the most common biological evidence material in forensic investigations. The success rate was over 90% when applied to evidence materials such as shed hair, saliva stains and saliva on stamps. The analysis of evidence materials collected from three similar robberies revealed the presence of mtDNA sequences identical to those of the suspects in the three crimes. The use of mtDNA control region sequences for individual identification was evaluated. The probability of identity by chance for the mtDNA types of the suspects in the robberies was found to vary between Pr = 0.017 - < 0.0017, depending on the reference population used, emphasizing the need for large population databases to obtain the appropriate estimate.
Base Sequence↗
HLA sequence polymorphism and the origin of humans.
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Africa↗
Human brain contains high levels of heteroplasmy in the noncoding regions of mitochondrial DNA.
We have analyzed the level of intraindividual sequence variability (heteroplasmy) of mtDNA in human brain by denaturing gradient gel electrophoresis and sequencing. Single base substitutions, as well as insertions or deletions of single bases, were numerous in the noncoding control region (D-loop), and 35-45% of the molecules from a single tissue showed sequence differences. By contrast, heteroplasmy in coding regions was not detected. The lower level of heteroplasmy in the coding regions is indicative of selection against deleterious mutations. Similar levels of heteroplasmy were found in two brain regions from the same individual, while no heteroplasmy was detected in blood. Thus, heteroplasmy seems to be more frequent in nonmitotic tissues. We observed a 7.7-fold increase in the frequency of deletions/insertions and a 2.2-fold increase in the overall frequency of heteroplasmic mutations in two individuals aged 96 and 99, relative to an individual aged 28. Our results show that intraindividual sequence variability occurs at a high frequency in the noncoding regions of normal human brain and indicate that small insertions and deletions might accumulate with age at a lower rate than large rearrangements.
Aging↗
Low diversity and biased substitution patterns in the mitochondrial DNA control region of sperm whales: implications for estimates of time since common ancestry.
The mitochondrial DNA (mtDNA) control region was sequenced in 37 sperm whales from a large part of the global range of the species. Nucleotide diversity was several-fold lower than that reported for control regions of abundant and outbred mammals, but similar to that for populations known to have experienced bottlenecks. Relative neck tests did not suggest that the low diversity is due to a lower substitution rate in sperm whale mtDNA. Rather, it is more likely that demographic factors have reduced diversity. The pattern of nucleotide substitutions was examined by cladistic methods, facilitated by the apparent monophyly of lineages from the Southern Hemisphere, as defined by a single base pair deletion. Substitutions were nonrandom in nature, confined to a few "hot spots," and parallel substitutions constituted a majority of the inferred changes. The substitution pattern fitted a negative binomial distribution better than a Poisson distribution, and the bias in number of substitutions among sites was considerably higher than previously reported for the mtDNA control region of any species. A novel method of estimating time since common ancestry was developed, which utilizes the transition/transversion ratio R and the number of substitutions inferred from a parsimony analysis. Using this method, we estimated the age of sperm whale mtDNA diversity to be about 6,000-25,000 years, and when the uncertainty of R was accounted for, a range of about 1,000-100,000 years was obtained.
Animals↗
Rapid allelic diversification and intensified selection at antigen recognition sites of the Mhc class II DPB1 locus during hominoid evolution.
The evolution of polymorphism at the Mhc class II DPB1 locus was studied by comparison of chimpanzee (Pan troglodytes), pygmy chimpanzee (Pan paniscus), gorilla (Gorilla gorilla) and human DPB1 alleles. Extensive polymorphism was found in all hominoids. The clustering of sequences in the phylogenetic tree is consistent with rapid generation of the DPB1 polymorphism. Analysis of the substitution pattern for human alleles shows an excess of non-synonymous changes to synonymous changes at antigen recognition sites, indicating that the amino acid polymorphism at these sites is being maintained by selection. By contrast, no excess of nonsynonymous changes was found at the antigen recognition sites of nonhuman hominoid species. Thus, it appears that diversifying selection on the DPB1 polymorphism has intensified in the lineage leading to humans. No evidence was found for the existence of ancient allelic lineages predating the divergence of the hominoid species. The number of synonymous differences among DPB1 alleles is lower than among DQB1 and DRB1 alleles, indicative of a more recent origin for the DPB1 polymorphism and consistent with the more rapid evolution suggested by the phylogenetic tree.
Alleles↗
HLA DQ-DR haplotype and susceptibility to cervical carcinoma: indications of increased risk for development of cervical carcinoma in individuals infected with HPV 18.
The association of HLA class II DQB1 and DRB1 alleles with the development of cervical carcinoma was studied in 150 Swedish patients using PCR-based HPV and HLA typing. The association of cervical carcinoma with alleles encoding the DQ3 antigen, previously found among German and Norwegian patients, was not observed in the Swedish patients. Five DQ-DR haplotypes were indicated to be positively associated with development of cervical carcinoma in the Swedish patients. Two of these HLA associations were specific for HPV 18 infected patients, suggesting that the ability of the oncogenic HPV 18 to cause more rapid-transit tumors than other high risk HPV types may be due to a deficiency in antigen presentation by the HLA molecules encoded by carried on these haplotypes.
Biopsy↗
Analysis of loss of heterozygosity in microdissected tumor cells from cervical carcinoma using fluorescent dUTP labeling of PCR products.
The use of fluorescently labeled nucleotides (F-dUTP) in the PCR for genetic typing of microsatellite polymorphisms was investigated. Microdissected tumor cells from cervical squamous cancer biopsies were compared with cells from surrounding normal tissue for loss of heterozygosity (LOH) at the mismatch repair gene hMLH1. Removal of unincorporated F-dUTP before analysis is necessary to reduce background fluorescence; ethanol-precipitation was found to be as efficient as the use of a spin column for this purpose. The gel resolution was sufficient to distinguish alleles differing by about four nucleotides. Alleles differing by only one dinucleotide repeat were possible to identify, but the ratio of alleles was difficult to assert with any reliability due to the wide peaks obtained. Single primer-pair and nested amplification systems were shown in reconstitution experiments to reliably quantitate the ratio of alleles. Of 36 cervical cancer biopsies, amplification and typing of the hMLH1 microsatellite marker was successful in 20 cases. Among 9 informative (heterozygous) biopsies, 2 (22%) were found to show LOH.
Alleles↗
Decreased cytochrome-c oxidase activity and lack of age-related accumulation of mitochondrial DNA deletions in the brains of schizophrenics.
Defects in mitochondrial energy production have been implicated in several neurodegenerative disorders, such as Parkinson disease and amyotrophic lateral sclerosis. To study the contribution of mitochondrial defects to Alzheimer disease and schizophrenia, cytochrome-c oxidase (COX) activity and levels of the mtDNA4977 deletion in postmortem brain tissue specimens of patients were compared with those of asymptomatic age-matched controls. No difference in COX activity was observed between Alzheimer patients and controls in any of five brain regions investigated. In contrast, schizophrenic patients had a 63% reduction of the COX activity in the nucleus caudatus (P < 0.0001) and a 43% reduction in the cortex gyrus frontalis (P < 0.05) as compared to controls. The average levels of the mtDNA4977 deletion did not differ significantly between Alzheimer patients and controls, and the deletion followed similar modes of accumulation with age in the two groups. In contrast, no age-related accumulation of mtDNA deletions was found in schizophrenic patients. The reduction in COX activity in schizophrenic patients did not correlate with changes in the total amount of mtDNA or levels of the mtDNA4977 deletion. The lack of age-related accumulation of the mtDNA4977 deletion and reduction in COX activity suggest that a mitochondrial dysfunction may be involved in the pathogenesis of schizophrenia.
Aged↗
Evolution of Mhc class II polymorphism: the rise and fall of class II gene function in primates.
The substitution rate at the codons implicated at ARS of Mhc class II genes has previously been shown to be heavily biased towards nonsynonymous substitutions, indicative of positive selection for polymorphism. Based on our analysis of the number of synonymous changes at codons outside putative ARS in primates, the average age of the polymorphism at class II loci was found to increase in the following order: DPB1, DRB3, DRB5, DRB1, DRB4, DQB1, DQA1. For DRB loci, nonsynonymous changes were found to exceed synonymous changes at HLA-DRB1, DRB3 and DRB5, while no evidence of deviations from equal rates of synonymous and nonsynonymous substitutions were found for DRB6. The pattern of substitutions at the DRB loci of most Catarrhini species indicates constant positive selection at ARS codons over the evolutionary period examined. An exception to the relatively stable selection pattern between species exhibited by most loci is the appearance of polymorphism under positive selection at DRB4 only in the regular chimpanzee. The ds/dn ratios for DQA1 and DQB1 alleles are lower than for the most polymorphic DRB genes. Since the dn/ds ratio of ARS codons may be positively correlated to the ds for non-ARS codons, at least for DQB1, caution must be exercised in interpreting the low ratio for the DQ genes as an indication of weaker selection. The DQA1 allelic lineages show different dn/ds ratios, consistent with the hypothesis that the lineages are constrained from evolving in relation to the diversity of the interacting DQB1 alleles. In contrast to all other class II loci, DPB1 appears to have been subjected to strong positive selection only in the human lineage, and may represent the most conspicuous example of an Mhc locus acquiring an altered function in antigen presentation.
Animals↗
Analysis of HLA-class II DQA1, DQB1, DRB1 and DPB1 in Italian multiple sclerosis patients.
We studied the allelic constitution at the HLA class II DQA1, DQB1, DRB1 and DPB1 in 94 Italian multiple sclerosis (MS) patients and 98 controls. No significant increase in the frequency of DR2 alleles was detected among MS patients, as previously observed both in European and some Italian studies. A slight increase was found for the DQA1*0301 and DQB1*0602 alleles in the MS patients. No significant association was found with the glutamine residue at position 34 of the DQ alpha chain, which was noted previously in MS patients from northern Europe.
Alleles↗
Detection of genital human papillomavirus by single-tube nested PCR and type-specific oligonucleotide hybridization.
Cervical cancer is, on a global scale, the second most common form of cancer in women. Development of cervical carcinoma is strongly associated with infection by certain types of human papillomavirus (HPV). To facilitate the detection and molecular typing of HPV in clinical samples, nested-PCR amplification systems were developed for regions of the E1 and L1 genes. The nested amplifications were performed in a single reaction tube, and shifting between inner and outer primer pairs was achieved by a two-phase amplification with different annealing temperatures. This method eliminates cross-contamination between samples during transfer from the first to the second amplification step. A set of type-specific oligonucleotide probes were designed for the E1 system and used to distinguish 19 genital HPV types. The sensitivities of our amplification systems compare favorably with that for the L1 system on the basis of the MY09-MY11 primer pair (M.M. Manos, Y. Ting, D. K. Wright, A. J. Lewis, T. R. Broker, and S. M. Wolinsky, Cancer Cells 7:209-214, 1989) and our systems can be used on materials such as HPV-infected cell lines, cytobrush samples, cancer biopsies, and recent as well as archival Papanicolaou (Pap) smears. The high sensitivity coupled with the effective elimination of contamination in the transfer between the two amplification steps of the nested PCR makes these systems suitable for research as well as clinical analyses.
Base Sequence↗
Allele-specific HLA-DRB1 amplification of forensic evidence samples with mixed genotypes.
A major problem in analyzing forensic casework samples is the presence of genetic material from more than one individual in the material evidence. For instance, in sexual assault cases the evidence (vaginal swabs) usually contains a majority of vaginal epithelial cells and varying amounts of sperm cells from the perpetrator. Samples with mixed genotypes are also common among other biological evidence materials such as nail scrapes and mixed bloodstains. We have developed an allele-specific amplification system for the highly polymorphic HLA class II DRB1 locus that permits the detection of individual alleles in a sample with mixed genotypes, independent of the initial frequency of the alleles. Using a set of eight allele-specific amplification primers and typing the amplified fragments with sequence-specific probes, most of the 60 DRB1 alleles can be resolved. The method is highly specific and sensitive, with the potential for amplifying 15 copies of a particular allele in a background of 3 x 10(5) copies of other alleles. The method was successfully applied to three forensic cases, where the material evidence consisted of sperm stains on panties, nail scrapes and bloodstains on skin. Thus the DRB1 allele-specific amplification system can be employed for the unambiguous determination of the presence of individual alleles in materials suspected to contain mixed genotypes, even when the alleles of interest constitute only a small fraction of the total DNA.
Alleles↗
Fluorescent-based typing of the two short tandem repeat loci HUMTH01 and HUMACTBP2: reproducibility of size measurements and genetic variation in the Swedish population.
The aim of this study was to investigate the reproducibility of genetic typing of two tetrameric short tandem repeat (STR) loci and the extent of genetic variation in the Swedish population. An automated, fluorescent-based Applied Biosystems 373A sequencer was used for typing of the HUMTH01 and HUMACTBP2 loci (also named SE33). The former locus has seven alleles in the size range of 154-174 bp, while the latter is a complex locus with more than 32 alleles in the range of 227-316 bp. Using different fluorescent dyes, polymerase chain reaction (PCR) products from the two STR loci were sized in one lane using an internal size standard. In order to compare within- and between-gel reproducibility of fragment size estimates, a control sample was typed three times on each of 20 gels. Within the gel, the standard deviation (SD) of fragment size variability was less than 0.1 bp for four fragment sizes between 158-291 bp. Standard deviations between gels were slightly higher for the two shorter fragment sizes (HUMTH01), while the larger fragments varied between 0.3 and 0.4 bp (HUMACTBP2). The amount of genetic variation was investigated in samples from three Swedish cities (n = 301). Seven alleles were found at HUMTH01 and the observed heterozygosity was 0.77. At the HUMACTBP2 locus more than thirty alleles were found and the observed heterozygosity was 0.96. The observed genotype frequencies at HUMTH01 and HUMACTBP2 did not deviate significantly from Hardy-Weinberg expectations. No indication of a significant excess of homozygotes was found at any of the loci. We conclude that both HUMTH01 and HUMACTBP2 can be reliably typed using the method described. However, the latter locus requires an allelic ladder to be run on each gel.
Alleles↗