MITOCHONDRIAL VARIATION

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. There is a high degree of genetic variation in human populations. For nuclear DNA (nDNA) about 90% of this variation is distributed among all populations while about 10% is population specific. For the mitochondrial DNA (mtDNA) over 30% of the variation is population specific. Cataloging this variation is essential for understanding the interrelationships between populations and thus human origins (molecular anthropology) and for defining the "normal" genetic variation so that it can be distinguished from the pathogenic variation to be found in patients with disease. Naturally occurring genetic variation of use for studying human origins can be found in both the nDNA and mtDNA by a variety of techniques. Nuclear DNA variation can be identified as restriction site polymorphisms (restriction fragment length polymorphisms, RFLPs), microsatellite repeat variation, variable number of tandem repeat (VNTR) polymorphisms, rearrangements, present or absence of SINE and LINE repeat elements, nucleotide base substitutions, and direct gene sequence analysis. Mitochondrial DNA variation can be analyzed by RFLPs, control region (D-loop) sequencing, nucleotide substitution analysis, and direct sequencing of genes and/or the genome. These types of information can be used to reconstruct population affinities and thus to deduce human origins by a variety of analytic tools. These include genetic distance analyses, population substructure studies, and phylogenetic comparisons. These data, together with estimates of the sequence evolution rate and geographic distribution of the samples provide insights into human origins. This information is also vital for disease and aging studies. All DNA is polymorphic in sequence. Most sequence variants are neutral (innocuous). These have to be identified before the interspersed disease mutations can be identified. This is true for nDNA, but is particularly true for mtDNA. The maternally-inherited mtDNA's sequence is extraordinarily variable, with much of the variation having arisen as women moved out of Africa and occupied all continents of the world. Hence, is the mtDNA sequence from a patient whose ancestors were derived from one continent is compared to the mtDNA of a "control" of a different continental origin, approximately 20 to 80 nucleotide differences will be found, any of which could be the clinically relevant mutation. To overcome this problem, patient mtDNAs sequences must be compared to closely related mtDNAs, those sharing a similar haplotype. In such a comparison, the normal variants are shared between the patient and control. Generally only the recent pathologic mutation is different and hence can be identified (Wallace, 1999 Gene 238: 211-230). Thus, it is essential for all genetic research to have well defined populations representing normal genetic variation for controls in disease studies. As a specific example, we have used this information to identify the disease mutation of a large Hispanic pedigree in which both Leber's Hereditary Optic Neuropathy (LHON) and generalized dystonia occurred along the maternal lineage (Novotny et al., 1986 Neurology 36:1053-60). We sequenced the mtDNA of one of the dystonia patients, and compared it to the "standard" Cambridget sequence. This revealed 40 nucleotide differences, several of which could be argued to be the pathogenic mutation, with no criteria to distinguish between them. Consequently, this project stalled. Concurrently, we were studying the mtDNA variation in Native Americans and discovered that virtually all Native American mtDNAs were derived from four founder haplogroups, A, B, C, and D (Wallace, 1999 Gene 238: 211-230). Subsequent comparison of the dystonia patient's mtDNA with the Native American mtDNAs revealed the unexpected result that this Hispanic family harbored a Native American mtDNA belonging to haplogroup D. We then compared all of the patient's sequence variants to those of other Native American haplogroup D mtNDAs, and we found that all but two of the sequence variants were naturally occurring variants of the haplogroup D lineage and hence non-pathogenic. Of the two remaining nucleotide changes, one was a neutral third codon change and the other was a mssense mutation at nucleotide pair (np) 14459 in the MTND6 gene. This mutation changed a highly conserved alanine to a valine and was heteroplasmic. Hence, this was the probable cause of the disease (Jun et al., 1994 Proceeding Academic of Sciences U S A 91:6206-10). Subsequent screening of other LHON and dystonia patients for this mutation revealed two additional families with the np 14459 mutation, one with LHON and the other with generalized dystonia. The LHON family mutation occured on an African haplogroup L mtDNA while the dystonia patient mutation occurred on a European haplogroup I mtDNA, and all three families were heteroplasmic. Hence, each of these families must be due to independent mutations (Shoffner et al., 1995 Annals of Neurology 38:163-9). Subsequently, biochemical and somatic cell genetic studies linked the np 14459 mutation to a mitochondrial respiratory Complex I defect in all three families (Jun et al., 1996 Molecular Cell Biology 16:771-7). Hence, this study shows how a knowledge of naturally occurring mtDNA variation is essential to identifying and characterizing putative patient disease mutations. This same logic applies to nuclear gene variants as well. We have been collecting and analyzing samples of various human populations for nearly 20 years. Without these resulting reference populations, we would not have been able to identify many of the mtDNA disease mutations which are now routinely used in medical diagnostics (Wallace, 2001 Scriver et al The Metabolic and Molecular Basis of Inherited Disease, 2425-2509). Moreover, the human population, and as a result the immigrant U.S. population in general and Southern Californian population in particular, is highly polymorphic. Hence, we must continue to collect and characterize additional reference populations from various ages and ethnic backgrounds if we are to understand the genetic basis of the common disease processes of the North American population. Objectives: The question being asked by these studies is what is the nature and extent of human genetic variation? Once obtained, this information will be used to explore the origin and prehistory of our species and also provide background information necessary for identifying pathogenic mutations in patients with genetic disease and possible involvement of certain genetic variants in the aging process.