What is the evidence of a role for host genetics in susceptibility to influenza A/H5N1?

The apparent family clustering of avian influenza A/H5N1 has led several groups to postulate the existence of a host genetic influence on susceptibility to A/H5N1, yet the role of host factors on the risk of A/H5N1 disease has received remarkably little attention compared to the efforts focused on viral factors. We examined the epidemiological patterns of human A/H5N1 cases, their possible explanations, and the plausibility of a host genetic effect on susceptibility to A/H5N1 infection. The preponderance of familial clustering of cases and the relative lack of non-familial clusters, the occurrence of related cases separated by time and place, and the paucity of cases in some highly exposed groups such as poultry cullers, are consistent with a host genetic effect. Animal models support the biological plausibility of genetic susceptibility to A/H5N1. Although the evidence is circumstantial, host genetic factors are a parsimonious explanation for the unusual epidemiology of human A/H5N1 cases and warrant further investigation.

The hemoglobin O mutation in Indonesia: distribution and phenotypic expression.

We have investigated hemoglobin O Indonesia (HbOIna) in related ethnic populations of the Indonesian archipelago: 1725 individuals of the five ethnic populations of South Sulawesi (Bugis, Toraja, Makassar, Mandar, and Kajang) and 959 individuals of the neighboring islands, who were divided into five phylogenetic groups: (a) Batak; (b) Malay from Padang, Pakanbaru, and Palembang in the island of Sumatra; (c) Javanese-related populations (Java, Tengger, and Bali) from the islands of Java and Bali; (d) populations of the Lesser Sunda Islands of Lombok, Sumba, and Sumbawa; and (e) the Papuan-languagespeaking population of Alor Island. Nineteen individuals heterozygous for HbO(Ina) were identified from the Bugis, Toraja, Makassar, and Kajang ethnic populations, but none from the other populations. In all cases, the underlying mutation was found to be in codon 116 (GAG to AAG) of the alpha1-globin gene, resulting in the Glull6Lys amino acid change. The level of HbO in the 17 individuals plus 12 additional family members carrying the mutation was found to be 11.6 +/- 1.0%, significantly lower than the expected 17%-22%, indicating the instability of HbO.

Length variations in the COII-tRNA(Lys) intergenic region of mitochondrial DNA in Indonesian populations.

The prevalence of a 9-base-pair (bp) deletion between the mitochondrial cytochrome oxidase II (MTCOX*2) and lysine tRNA (MTTK) genes (region V) has been used to estimate the genetic relationships among Asian and Pacific populations. Many East Asian and Pacific Island populations have been examined previously, but the mitochondrial DNA (mtDNA) diversity of the intervening Indonesian archipelago has not previously been systematically examined. The 17,500 islands of Indonesia currently contain nearly 213 million people and extensive cultural, linguistic, and, presumably, genetic diversity. This study of 1091 individuals representing 15 ethnic groups is the most extensive mtDNA survey to date of the Indonesian archipelago. Six distinct length polymorphisms in region V were observed within these 15 populations. The 9-bp deletion was found in every population examined at frequencies comparable to those of previously examined East Asian populations and substantially lower than those in most Pacific Island populations. Despite the inclusion of Austronesian-speaking populations and a Papuan-speaking population, there was no statistically significant heterogeneity in the frequency of the 9-bp deletion among the 15 populations (p = 0.09). These data indicate that substantial gene flow occurred among the populations at some time in the past. Our observations of no significant correlations between genetic and geographic distances (r = -0.04, p = 0.53) coupled with the extensive cultural and linguistic differences currently within the archipelago suggest that little gene flow among neighboring populations has occurred recently.

African origin of modern humans in East Asia: a tale of 12,000 Y chromosomes.

To test the hypotheses of modern human origin in East Asia, we sampled 12,127 male individuals from 163 populations and typed for three Y chromosome biallelic markers (YAP, M89, and M130). All the individuals carried a mutation at one of the three sites. These three mutations (YAP+, M89T, and M130T) coalesce to another mutation (M168T), which originated in Africa about 35,000 to 89,000 years ago. Therefore, the data do not support even a minimal in situ hominid contribution in the origin of anatomically modern humans in East Asia.

Maori origins, Y-chromosome haplotypes and implications for human history in the Pacific.

An assessment of 28 pertinent binary genetic markers on the non-recombining portion of the Y chromosome (NRY) in New Zealand Maori and other relevant populations has revealed a diverse genetic paternal heritage of extant Maori. A maximum parsimony phylogeny was constructed in which nine of the 25 possible binary haplotypes were observed. Although approximately 40% of the samples have haplotypes of unequivocal European origin, an equivalent number of samples have a single binary haplotype that is also observed in Indonesia and New Guinea, indicative of common indigenous Melanesian ancestry. The balance of the lineages has either typical East Asian signatures or alternative compositions consistent with their affinity to Melanesia or New Guinea. Molecular analysis of mtDNA variation confirms the presence of a single predominant characteristic Southeast Asian (9-bp deletion in the Region V) lineage. The Y-chromosome results support a pattern of complex interrelationships between Southeast Asia, Melanesia, and Polynesia, in contrast to mtDNA and linguistic data, which uphold a rapid and homogeneous Austronesian expansion. The Y-chromosome data highlight a distinctive gender-modulated pattern of differential gene flow in the history of Polynesia.

Microphotometric analysis of NADH-tetrazolium reductase deficiency in fibroblasts of patients with Leber hereditary optic neuropathy.

We employed a microphotometric approach to examine whether a defect in the mitochondrial respiratory complex I expected in Leber hereditary optic neuropathy (LHON) as the consequence of a mtDNA (11778G>A) mutation in the ND4 gene coding for a subunit of the respiratory complex I can be detected at the single-cell level. Genetically stable fibroblast cell lines were established from skin biopsies of two members of a Chinese Indonesian family with LHON. The fibroblasts were homoplasmic for the 11778G>A mutation. The activity of the respiratory complex I was examined histochemically by staining for NADH-tetrazolium reductase. The histochemical staining showed a typical pattern with an apparent concentration of the activity around the nucleus, suggested as the reflection of the gradient in the thickness of the unsectioned fibroblast cells. Microphotometric quantification of the staining intensity showed that the activity is linear for at least 60 min. The activity shows a discontinuity in its Arrhenius kinetics with a break point at 13.0-13.5 degrees C (activation energy at 50-58 J/mol and 209-238 J/mol above and below the break temperature, respectively), indicating the membrane association of the NADH-tetrazolium reductase activity. Both patients showed lower fibroblast NADH-tetrazolium reductase activity, with a reduction of degrees 30%. Our results demonstrate the utility of microphotometric analysis in the study of biochemical defects associated with mutations in the mtDNA.

Mutations in the cytochrome b gene of Plasmodium berghei conferring resistance to atovaquone.

The molecular lesions which underlie the resistance of the malaria parasites to atovaquone, a coenzyme Q analogue, were investigated. Resistant clones of Plasmodium berghei ANKA strain were isolated following prolonged propagation in mice in the presence of increasing doses of the drug, and their cytochrome b gene sequenced. Three mutations were detected, T-C substitution at nt 431, G-A at nt 399 and G-T at nt 850, resulting in amino acid changes in the putative cytochrome b product at residues 133, 144 and 284. The V284F amino acid change is in the sixth transmembrane helix of the protein and was observed in all resistant clones. An additional M133I or L144S amino acid change within the Qo site at an extramembranous amphipathic helix significantly increases the resistance to atovaquone. Our results (a) provide evidence that the antimalarial activity of atovaquone indeed involves an interaction with the cytochrome b; (b) define atovaquone as an inhibitor of the ubiquinol oxidase activity of the cytochrome bc1 complex; and (c) define amino acid residues in the mammalian cytochrome b which might be critical in determining its relative resistance to atovaquone.

Phenotypic variability of Filipino beta(o)-thalassemia/HbE patients in Indonesia.

Three Indonesian patients with identical genotypes, each compound heterozygotes for Filipino beta(o)-thalassemia/HbE, expressed different clinical severities. One patient has mild disease and is transfusion independent, while the other two are severely affected and transfusion dependent. The size of the Filipino beta(o)-globin gene deletion was confirmed to be 45 kb, resolving conflicting values given in the literature. Neither ameliorating genetic factors such as alpha-globin gene deletions or the XmnI restriction site polymorphism at position -158 upstream of the (G)gamma-globin gene, nor differences in beta-globin gene haplotype, explain the phenotypic variation. These observations have implications for the development of antenatal diagnosis in Indonesia, as at present it is not possible to give an accurate prediction of severity of phenotype for this common genotype.

Meiotic breakpoint mapping of a proposed X linked visual loss susceptibility locus in Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy (LHON) is a maternally inherited degenerative disorder characterised by an acute or subacute optic nerve degeneration resulting in visual failure. Mitochondrial DNA mutations have been reported and a nuclear modifier gene(s) on the X chromosome is thought to play an important role in the onset of this disorder. We analysed a LHON family with a novel and more accurate approach using 27 X chromosomal microsatellite markers. Meiotic breakpoint mapping and two point lod score did not point to any particular area on the X chromosome which might contain the X susceptibility locus.

Developmental genetics of deleted mtDNA in mitochondrial oculomyopathy.

Heteroplasmic populations of mtDNA, consisting of normal mtDNA and mtDNA with large deletions, are found in the skeletal muscle and other tissues of certain patients with mitochondrial respiratory chain deficiencies, particularly in those with the CPEO (chronic progressive external ophthalmoplegia) phenotype. To study the developmental genetics of this mitochondrial disorder, the distribution of the deleted mtDNA in a wide range of tissues of different embryonic origins (total 34 samples from 27 tissues obtained at autopsy) was investigated in a patient with the CPEO syndrome. Three species of partially deleted mtDNA were observed, with deletions of 2.3 kb, 5.0 kb and 6.4 kb. Their tissue distribution suggests that the mtDNA deletions have occurred very early during embryonic development, prior to the differentiation events that lead to the formation of the three primary embryonic germ layers, and that the partially deleted mtDNA species were segregated during development mainly to the skeletal muscle and to tissues of the central nervous system.

Mitochondrial DNA polymorphism in disease: a possible contributor to respiratory dysfunction.

Intergenomic variation in the human mitochondrial genome was examined in 27 mtDNA sequences using a pairwise analysis technique. Analysis of 16 of these mtDNA sequences from patients with mitochondrial cytopathies indicated a wide range between different mitochondrial genes in the degree of nucleotide variation from the standard Cambridge sequence. Mean complex I polymorphic frequencies in cytopathic (CPEO, MERRF, MELAS and LHON collectively) patients and in LHON patients differed significantly from controls (P < or = 0.05, t). Total mean sequence divergence (mean number of diverging nucleotides between two sequences per 100 bp) over the entire mtDNA coding region was 0.21% for cytopathies (n = 16) as opposed to 0.18% for a control group (n = 4). Within the cytopathy group, the greatest pairwise divergence was observed in ND3 and ND6 subunits of complex I (0.46 and 0.70% respectively) and the magnitude of specific gene divergences differed considerably from those observed for the corresponding genes in the control population. The extent to which the increased variation in ND3 and ND6 is a general phenomenon applicable to all subjects rather than a finding specific to cytopathies cannot be stated with certainty given the small control group. Regardless as to which of these suggestions is correct, the possibility exists that increased nucleotide variation in certain mitochondrial ND subunits may contribute to respiratory inefficiency through a cumulative effect of a series of polymorphisms of minor individual mutagenic potential.

Heterogeneity in the phenotypic expression of the mutation in the mitochondrial tRNA(Leu) (UUR) gene generally associated with the MELAS subset of mitochondrial encephalomyopathies.

BACKGROUND: Point mutations in the mitochondrial (mt) genome underlie a number of neurological disorders. Some are well defined including the myoclonus epilepsy ragged red fibre syndrome (MERRF) and the mitochondrial encephalopathy lactic acidosis stroke like episode syndrome (MELAS). However, other clinical phenotypes are less distinctive and mitochondrial studies are often included in the workup in complex neurological syndromes of uncertain aetiology. AIMS: We investigated 27 consecutive patients with varied clinical phenotypes referred to our laboratory for mtDNA studies to determine the incidence of recognised point mutations in a patient group with a range of phenotypes including many where mt disease was possible but did not fall into a classical syndrome. METHODS: The recognised point mutations were detected by amplification of the appropriate DNA fragment by PCR followed by restriction-endonuclease digestion of the normal and mutant species. RESULTS: The A-G base substitution mutation at nucleotide (nt)3243 in the tRNA(Leu) gene of mtDNA which is present in the majority of cases of MELAS syndrome, was detected in four cases, only one of whom had typical MELAS symptoms. Their clinical manifestations ranged from mild deafness to a mixture of chronic progressive external ophthalmoplegia symptoms (CPEO) and stroke like episodes. The nt3243 mutation was also identified in one of seven mtDNA deletion negative CPEO cases. CONCLUSIONS: The presentation of the mtDNA mutation at nt3243 appears therefore to be quite variable with some mild phenotypes as well as severe phenotypes observed. In general, the chance of finding a mitochondrial point mutation in a patient with an atypical clinical phenotype is small.

Rapid and noninvasive screening of patients with mitochondrial myopathy.

In recent years, several point mutations in the mitochondrial genome have been associated with human disease. PCR Polymerase Chain Reaction/restriction endonuclease based techniques provide a reliable method for screening large numbers of specimens for many of the reported mutations. Muscle tissue usually carries the mutations and has been used in earlier studies. We describe a technique for analysis of mtDNA derived from hair follicles for a range of mutations. Both the 3243 A-->G MELAS and 8344 A-->G MERRF mutations were detected in mtDNA from hair follicles. In patients where both muscle and hair were screened, the mutation load was apparently higher in muscle. Furthermore, in patients positive for a given mutation, all the hair follicles analysed were shown to harbour the mutation, although the proportion of wild type to mutant mtDNA was found to somewhat vary. The advantages of this method are (1: six hair follicles provide sufficient mtDNA for analysis of at least 20 different mutations, and (2: specimen collection and transport to a central laboratory are easier than for other tissues. Our studies show that hair follicles constitute a reliable specimen for mitochondrial mutation screening at a diagnostic level.

A novel mtDNA deletion in an infant with Pearson syndrome.

Pearson syndrome is a multisystem mitochondrial disorder of infancy that is associated with deletions in the mitochondrial DNA (mtDNA) genome. We report a study on a male infant with Pearson syndrome. Assessment of oxidative phosphorylation activity indicated combined respiratory-chain defects in muscle, liver and fibroblasts; in particular, activity of complex I was reduced. Analysis of the patient's mtDNA identified a novel heteroplasmic 2.461 kb deletion, present at levels greater than 50% of the total mtDNA in the tissues examined. The deletion spanned nucleotides 10368 to 12828 and was flanked by a 3 bp GCC direct repeat sequence. Gene sequences affected are subunits 3, 4, 4L and 5 of complex I, and tRNAs for arginine, histidine, serine and leucine. Our findings correlate with the multiorgan involvement observed in Pearson syndrome.

Phenotypic expression of mtDNA heteroplasmy in the skeletal muscle of patients with oculomyopathy: defect in mitochondrial protein synthesis.

The biochemical consequences of mtDNA heteroplasmy, observed in patients with a range of diseases associated with the mitochondrial respiratory enzymes deficiency is of particular interest, as they might provide information with regard to the regulatory interactions which govern the expression of the human mitochondrial genome. Three patients with chronic progressive external ophthalmoplegia (CPEO) were investigated to study the consequences of mtDNA heteroplasmy on mitochondrial protein synthesis. All 3 patients exhibited partially deleted mtDNA species (varying in size from 10.5 to 14 kb) in their skeletal muscle, which co-existed with the normal 16.5 kb mtDNA. The examination of mitochondrial translation products following the incorporation of [35S]methionine by isolated mitochondria, showed grossly abnormal patterns of mitochondrial translation products, suggesting a major disturbance in the regulation of mitochondrial protein synthesis.