The presence of ancient human T-cell lymphotropic virus type I provirus DNA in an Andean mummy.

The worldwide geographic and ethnic clustering of patients with diseases related to human T-cell lymphotropic virus type I (HTLV-I) may be explained by the natural history of HTLV-I infection. The genetic characteristics of indigenous people in the Andes are similar to those of the Japanese, and HTLV-I is generally detected in both groups. To clarify the common origin of HTLV-I in Asia and the Andes, we analyzed HTLV-I provirus DNA from Andean mummies about 1,500 years old. Two of 104 mummy bone marrow specimens yielded a band of human beta-globin gene DNA 110 base pairs in length, and one of these two produced bands of HTLV-I-pX (open reading frame encoding p40x, p27x) and HTLV-I-LTR (long terminal repeat) gene DNA 159 base pairs and 157 base pairs in length, respectively. The nucleotide sequences of ancient HTLV-I-pX and HTLV-I-LTR clones isolated from mummy bone marrow were similar to those in contemporary Andeans and Japanese, although there was microheterogeneity in the sequences of some mummy DNA clones. This result provides evidence that HTLV-I was carried with ancient Mongoloids to the Andes before the Colonial era. Analysis of ancient HTLV-I sequences could be a useful tool for studying the history of human retroviral infection as well as human prehistoric migration.

A new mtDNA mutation associated with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS).

In 3 of 40 MELAS patients, a new common mutation, a T-to-C transition at nucleotide position 3271 in the mitochondrial tRNA(Leu(UUR] gene was recognized and was very near to the most common mutation site at 3243. With a simple detection method using polymerase chain reaction with a mismatch primer, none of 46 patients with other mitochondrial diseases and 50 controls had this mutation.

Specific amplification of deleted mitochondrial DNA from a myopathic patient and analysis of deleted region with S1 nuclease.

Heteroplasmy of the normal-sized and the deleted mitochondrial genome has been observed in mitochondrial myopathy. The deleted region of the genome in the skeletal muscle of a patient was analyzed both by the conventional Southern blot method and by the novel method of employing the combination of polymerase chain reaction and S1 nuclease digestion. The results obtained by these methods were compared. Southern hybridization using various mitochondrial DNA fragments localized the deletion from at least position 9020 to 14,955, but regions of uncertainty of 1 kb remained on both ends of the deletion. Using the polymerase chain reaction, a fragment from the deleted genome was specifically amplified by choosing a pair of primers surrounding the deletion, and two fragments adjacent to the starting and end of the deletion were amplified from the normal-sized genome. S1 nuclease analysis of the heteroduplexes formed among these fragments demonstrated that the deletion extended from positions 8650 +/- 50 to 15,660 +/- 60. This method does not require radioisotopes and, moreover, can determine the deleted region within 5 h, in contrast to the 2 days required by the conventional Southern blot analysis. These results indicate that the novel method is faster and more accurate than the conventional method for the determination of the deleted region of genome.

The geographic distribution of human Y chromosome variation.

We examined variation on the nonrecombining portion of the human Y chromosome to investigate human evolution during the last 200,000 years. The Y-specific polymorphic sites included the Y Alu insertional polymorphism or "YAP" element (DYS287), the poly(A) tail associated with the YAP element, three point mutations in close association with the YAP insertion site, an A-G polymorphic transition (DYS271), and a tetranucleotide microsatellite (DYS19). Global variation at the five bi-allelic sites (DYS271, DYS287, and the three point mutations) gave rise to five "YAP haplotypes" in 60 populations from Africa, Europe, Asia, Australasia, and the New World (n = 1500). Combining the multi-allelic variation at the microsatellite loci (poly(A) tail and DYS19) with the YAP haplotypes resulted in a total of 27 "combination haplotypes". All five of the YAP haplotypes and 21 of the 27 combination haplotypes were found in African populations, which had greater haplotype diversity than did populations from other geographical locations. Only subsets of the five YAP haplotypes were found outside of Africa. Patterns of observed variation were compatible with a variety of hypotheses, including multiple human migrations and range expansions.

Phylogenetic analysis of mitochondrial DNA in Japanese pedigrees of sensorineural hearing loss associated with the A1555G mutation.

Thirteen Japanese families (ten of which were from the northern part of Japan), with sensorineural hearing loss associated with the 1555 A to G (A1555G) mitochondrial mutation, a known cause of non-syndromic hearing loss, were phylogenetically analysed using data obtained by restriction fragment length polymorphism (RFLP) and D-loop sequencing of mitochondrial DNA (mtDNA). Various types of mtDNA polymorphism were detected by restriction enzymes and D-loop sequence. No common polymorphic pattern throughout the 13 families was found, though three families exhibited the same restriction patterns and the same sequence substitution in the D-loop. To find where each of the 13 families are situated in the phylogenetic tree, the 482-bp of D-loop sequence were compared with those of 62 normal Japanese subjects. Despite the three families mentioned above appearing to be clustered, the remaining 10 families were scattered along the phylogenetic tree. This indicates that there was no common ancestor for the 13 Japanese families bearing the A1555G mutation except three families, and that the A1555G mutation occurred sporadically and multiplied through evolution of the mtDNA in Japan. The present results showed that the common pathogenicity (hearing loss associated with the A1555G mutation) can occur sporadically in families which have different genetic backgrounds, even in the Japanese population.

Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS): a correlative study of the clinical features and mitochondrial DNA mutation.

We studied 40 MELAS patients (21 male and 19 female) to characterize the clinical features and biochemical and muscle biopsy findings related to the mtDNA mutation at the nucleotide position of 3,243, the most common genetic defect in MELAS. The most frequent symptom was episodic sudden headache with vomiting and convulsions, which commonly affected patients aged 5 to 15 years (80%). Biochemical defects in the muscle were variable; 13 patients had complex I, seven complex IV, and four complexes I + IV deficiencies. In four muscle biopsies without ragged-red fibers or any enzyme defect, we based the diagnosis on the identification of strongly SDH-reactive blood vessels, which occurred in 87.5% of the biopsies. The mtDNA mutation was present in 32 of 40 patients (80%). We conclude that there are no clinical and pathologic differences between the patients with and without this mtDNA mutation.

Confirmation that a T-to-C mutation at 9176 in mitochondrial DNA is an additional candidate mutation for Leigh's syndrome.

Among 80 patients with the clinical and brain imaging characteristics of Leigh's syndrome, 11 patients had a well-known mutation at nucleotide position (nt) 8993 in mitochondrial DNA. In addition, three patients had a T-to-C mutation at nt 9176 which had been described previously in only two brothers with bilateral striatal necrosis and one patient with Leigh's syndrome. In our three patients, one had the typical clinical characteristics of Leigh's syndrome from early infancy, and two had the later onset of neurological deficits. All had a slowly progressive course and basal ganglia abnormalities by neuroimaging. As nt 8993 and 9176 are located in the ATPase 6 coding region, altered ATPase function may be one of the enzyme abnormalities in Leigh's syndrome and other similar conditions with bilateral striatal necrosis.

Detection of platelet antibodies by enzyme-linked immunosorbent assay (ELISA) on artificial monolayers of platelets.

For the detection of platelet antibodies we developed an enzyme-linked immunosorbent assay (ELISA) on artificial monolayers of platelets in wells of micro-titre-U-trays. This simple and reproducible test can not only detect the presence and the specificity of platelet antibodies but also the class and subclass of the antibodies involved.

Detection of platelet antibodies by enzyme-linked immunosorbent assay (ELISA). Comparative studies with the indirect immunofluorescence assay.

A newly developed enzyme-linked immunosorbent assay (ELISA) for the detection of platelet antibodies was compared with the platelet immunofluorescence test (PIF). A good correlation was found between both assays. However, ELISA seems to be more sensitive than PIF. Some sera reacted only in ELISA whereas no sera were found that were negative in ELISA and positive in PIF. When comparing the antibody titres, ELISA is at least 8 times more sensitive than PIF.

Cytotoxic T lymphocytes directed against HLA-Bw 35-linked target determinants show differences in sensitivity toward antibiotics during sensitization period.

Recently, we demonstrated that the outcome of human CML was influenced by the presence of antibiotics in the culture medium, In earlier studies we had found that cytotoxic T lymphocytes could recognize HLA-Bw 35-linked target determinants. These so-called Bw35 a and b determinants showed mendelian segregation. We describe in this article a difference between the Bw35 a and b cytotoxic T cells in their sensitivity toward antibiotics. The lysis against the Bw35 b cytotoxic determinant was not influenced by either the presence or the absence of antibiotics during the sensitization period, whereas the lysis against the Bw35 a cytotoxic determinant was drastically diminished when the effector cells were cultured in the absence of antibiotics during the sensitization period.

Polymorphism of the HLA-DRB1 locus in Colombian, Ecuadorian, and Chilean Amerinds.

We have characterized the DRB1 genotypes in a sample of 64 South American Indians drawn from populations in Chile, Colombia, and Ecuador. No novel DRB1 alleles were found in the total of 17 different alleles characterized, indicating that rapid allelic generation does not occur at the DRB1 loci, in contrast to HLA-B. Comparison between Chilean and Colombian/Ecuadorian samples revealed no major differences in their allelic frequencies. In the combined Amerind sample the HLA-DRB1*0407 and HLA-DRB1*1402 alleles occurred in the highest frequencies (38% and 22%, respectively). Genetic distance measurement showed the HLA-DRB1 frequencies reported here to agree with findings in other Amerind groups. The high frequencies of both HLA-DRB1*0407 and HLA-DRB1*1602 alleles, in conjunction with their absence in Siberian samples, suggest that migratory groups other than Siberians may have been involved in the peopling of the Americas.

Seroepidemiological survey of human T-lymphotropic retrovirus among indigenous populations in Taiwan.

The prevalence of human T-lymphotropic retrovirus (HTLV) was examined in Taiwan's indigenous populations. In all, 797 healthy subjects in Taiwan including Han Chinese and nine indigenous populations (Ami, Atayal, Bunun, Saisiat, Paiwan, Puyuma, Rukai, Tsuo, and Yami) were examined for the presence of antibodies to HTLV by particle agglutination, indirect immunofluorescence and Western blot test. Two seropositive cases were found in this screening. One Saisiat male and a Han Chinese female were seropositive for HTLV. The Western blot profile indicated the virus was type-1 HTLV.

Mitochondrial DNA mutations at nucleotide positions 3243 and 3271 in mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes: a comparative study.

Of 50 patients with the clinical characteristics of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), 38 had a point mutation at nucleotide position (nt) 3243 in the tRNA(Leu(UUR)) region in mitochondrial DNA and 6 at nt 3271 in the same tRNA(Leu(UUR)) gene. Except for the later onset of the disease in the patients with the 3271 mutation, there were no clinical, biochemical and pathological differences between the two groups. Since the nt 3271 region is not located in the binding site for mitochondrial transcription termination (mTERM) factor, which has been proposed to be defective in the 3243 mutation, a functional defect in tRNA itself might be responsible for the enzyme defects in MELAS patients; however the mechanism by which the defective tRNA(Leu(UUR)) induces the stroke-like episodes remains to be clarified.

Chronic progressive external ophthalmoplegia: a correlative study of mitochondrial DNA deletions and their phenotypic expression in muscle biopsies.

Deleted mitochondrial DNA (mtDNA) has been shown to coexist with normal mtDNA (heteroplasmy) in muscles from chronic progressive external ophthalmoplegia, including Kearns-Sayre syndrome. In this study, we correlated heteroplasmic mtDNA abnormality with clinical, biochemical and histological findings with the following results: (1) large deletions ranging from 1.8 to 8.8 kb in 22 muscle specimens from 28 patients who had ophthalmoplegia clinically and focal cytochrome c oxidase (CCO) deficiency by histochemistry, (2) no difference in clinical and biochemical findings between patients with and without mtDNA deletions, (3) no relationship between the size, site or populations of deleted mtDNA and respiratory chain enzyme activities in muscles, (4) positive correlation between the number of CCO-deficient fibers and the populations of deleted mtDNA, and (5) higher incidence of CCO-negative fibers in patients with deleted mtDNA than in those with no deletion of mtDNA. These results suggest that deleted mtDNA is, at least in part, responsible for focal CCO deficiency as a phenotypic expression and that the investigation on pathogenetic mechanism of focal CCO deficiency may provide a clue to understanding the underlying pathophysiology in this disorder.

Is Parkinson's disease a mitochondrial disorder?

Parkinson's disease (PD) is a common degenerative disease, but its etiology is still unknown. However, since the discovery of MPTP, many investigators have been interested in the mitochondrial function in PD. We investigated mitochondrial functions in PD patients using the methods which have successfully been applied to mitochondrial myopathies (MM), i.e. assay of lactate and pyruvate, measurement of muscle mitochondrial respiratory enzyme activities and Southern blot analysis of muscle mitochondrial DNA. Parkinson's disease patients did not differ from controls in the mean blood and CSF (cerebrospinal fluid) lactate and pyruvate levels at the basal resting state or during an aerobic exercise. But mitochondrial complex I activity of the skeletal muscle was significantly decreased in PD. In the Southern blot analysis, we could not find major deletions or insertions of mitochondrial DNA in PD. Our studies disclosed a differential mitochondrial impairment between PD and MM. We discuss the implication of our observation.

Point mutations in mitochondrial tRNA genes: sequence analysis of chronic progressive external ophthalmoplegia (CPEO)

We have sequenced all mitochondrial tRNA genes from 9 Japanese patients with chronic progressive external ophthalmoplegia (CPEO) who had no detectable large mtDNA deletions nor mutations previously reported, and identified 6 different base substitutions in 6 patients. Since 5 of the 6 substitutions were homoplasmic in distribution and recognizable in some normal controls, they were thought to be polymorphisms in normal individuals. One mutation at nucleotide (nt) 12311 in the tRNA(Leu(CUN)) gene was not present in 90 normal controls nor in 103 patients with other mitochondrial myopathies. This mutation was in a heteroplasmic state, and the mutated site was conserved among other species during evolution, suggesting a disease-related mutation. However, the significance of this mutation has to be studied further. In Japanese CPEO patients without large deletions, a point mutation in the mitochondrial tRNA gene is not likely to be a frequent cause.

Pleiotropic molecular defects in energy-transducing complexes in mitochondrial encephalomyopathy (MELAS).

The extent of molecular defects in the mitochondrial energy-transducing system was examined in autopsied tissues of a 14-year-old male with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) in order to elucidate the underlying molecular and genetic abnormalities. The patient also had other multiorganic disorders: hypertrophic cardiomyopathy, nephrotic syndrome, and pseudohypoparathyroidism. Enzymic activities of complex I and IV were severely decreased, and those of complex III and V were mildly decreased in the mitochondria isolated from various tissues, but the severity of the deficiencies varied from tissue to tissue. In contrast, complex II and citrate synthase activities were normal or were decreased to a lesser extent than the enzymic activities of other complexes in all the tissues examined. These results suggest that the energy-transducing complexes, namely complexes, I, III, IV, and V, that contain mitochondrially synthesized subunits, were selectively affected. Immunoblot analysis demonstrated that the decreased enzymic activities were based on decreased contents of subunits in these complexes. The multiorganic manifestation of the disorder may result from wide and uneven distribution of abnormal mitochondria that have pleiotropic molecular defects in the energy-transducing complexes among the organs of the patient.

A T-to-G mutation at nucleotide pair 8993 in mitochondrial DNA in a patient with Leigh's syndrome.

We studied a patient with Leigh's syndrome using neurophysiologic, radiologic, enzymatic, biochemical, and molecular analysis. Her clinical course had started with acute encephalopathic symptoms at 7 months of age. With repeated remission and exacerbation, she developed hypotonia and symptoms of brainstem dysfunction, such as irregular respiration and swallowing difficulty. These symptoms were followed by epileptic seizures, including simple partial seizures and tonic spasms. Both serum lactate and serum pyruvate levels were elevated, and deficient activity was detected in cytochrome c oxidase in her quadriceps femoris muscle. From the early stages, we noted an abnormality in the auditory brainstem response and visual evoked potentials, and an abnormal symmetrical low-density area in the basal ganglia on the computed tomographic scan. We found a mitochondrial DNA point mutation at 8993 in blood samples from both the patient and her mother using a simple polymerase chain reaction method. The ratio of wild and mutant mitochondrial DNA calculated densitometrically on polymerase chain reaction products was 56.6% in the patient's blood cells and 8.4% in her mother's. This patient's disorder was thought to be maternally inherited Leigh's syndrome. Her brother had died of the identical clinical features at 1 year 9 months of age.

Evolution and the origins of man: clues from complete sequences of hominoid mitochondrial DNA.

Dating the origins of Homo sapiens sapiens is a central problem in human population genetics and anthropology. Do we descend from a single recent ancestral population in Africa, or from multiple ancestral populations in various regions of the world which one million years ago simultaneously began developing into H.s.sapiens? The high substitution rate of mitochondrial DNA (mtDNA) makes this molecule suitable for genealogical and chronological research on closely related hominoid species. We have analyzed the complete mtDNA sequences of three humans (African, European and Japanese) and two African apes (common chimpanzee and gorilla) in an attempt to estimate more accurately the substitution rates and divergence times of hominoid mtDNAs. Nonsynonymous substitutions and substitutions in RNA genes have accumulated at an approximately constant rate. Under the assumption, supported by the fossil record, that the orangutan and African apes diverged 13 million years ago, we have previously obtained 4.7 million years as the divergence time between humans and chimpanzees. Using this date, we calibrated the substitution rates at synonymous sites and in the displacement-loop region as 4.03 and 7.25 x 10(-8)/site/year, respectively. Based on these rates together with the observation that the African sequence presented here is most diverged from all other human sequences, we inferred the age of the last common ancestor of the human mtDNAs as 140,000 +/- 18,000 years. The result strongly supports the recent African origin of modern humans, H.s. sapiens.

[Genetic relationship among four Yunnan populations revealed by sequence of mtDNA D-loop].

mtDNA D-loop noncoding region 16048-16569 and the following 1-41 (563 bp) in 99 individuals of four Yunnan ethnic minorities (Dai, Wa, Lahu and Tibetan) were sequenced and then a phylogenetic tree was reconstructed by Neighbor-Joining method. These 99 mtDNA lineages were classified into 3 genotype groups in the tree. All lineages with 9 bp deletion in the COII/tRAN(Lys) intergenic region were clustered in group I, some individuals of Dai, Lahu, Wa and only 2 Tibetan individuals clustered in group II, individuals of all four populations were included in group III. A phylogenetic tree of the four populations was constructed by NJ method on the basis of estimate of net genetic distance among them. Our results showed, the genetic distance among Dai, Wa and Lahu is very close, but far from Tibetan, their genetic distance is similar to their geographic distance. Although both as descendants of ancient Di-Qiang tribe in history and speaking similar language, Lahu and Tibetan are not closely related. This result indicates that there are different origins of these two populations.