Michael Malan and Dr. Scott R. Woodward, Molecular Biology
Advances in recombinant DNA technology have greatly assisted the study of human population history by helping to shed light on human origins and relationships. Study of the extra nuclear mitochondrial genome (mtDNA) has been a most significant tool used in these investigations. Numerous studies designed with the purpose of documenting world wide genetic diversity and distribution have used the mitochondrial genome as their primary focus. Most significant to this research are those which focus on the mtDNA of New World peoples. Past research has identified and confirmed the existence of 4 major mitochondrial haplotypes (A, B, C, D) present in modern populations (1). How these types are defined is explained later in the discussion.
This research involves the distribution of mtDNA lineages within populations of a specific region of Peru, the Lake Titicaca Basin, also known as the Altiplano. Analysis reveals the predominance of the B haplotype, possessing a frequency in excess of 77%. Of additional interest is that the percentage of the B haplotype found in the population gradually decreases as distance from the lake increases. From this information we have identified the B haplotype as the founding lineage of the area. One possibility for the haplotype gradient is that the B type demonstrates a positive selection by possessing a superior adaptation for resilience to the harsh conditions of this environment (i.e. altitude), and with such, a greater ability for survival. This Andean location is situated at >3800m. Torroni postulates that in light of the growing number of pathologic mutations detected in the mitochondrial genome, the possibility exists for the appearance of an advantageous mutation, one that would be specifically suited to a particular environment (2). We report here on the possible correlation between mitochondrial genotype and selective mortality at elevation in this Andean population and speculate that evidence for such an advantageous mutation may exist.
From the maternity wards of the two major hospitals of the Lake Titicaca Basin, in the cities of Juliaca and Puno, a sample size of 74 mothers was collected. Samples consisted of three hairs taken from the scalp that included the root. It is from the root that we extract the DNA for analysis. Also included in the data collection was a reproductive history for each mother in addition to birth weight, sex, and delivery observations for each infant. The purpose of this approach is to classify each mother according to her mtDNA type and then compare this information with the health and survival rate of her offspring. By this way we hope to see if there exists a relationship that sheds light on the B haplotype preponderance in the area.
Once in the lab, mtDNA was isolated using Chelex 100® as described by (3). Using the polymerase chain reaction (PCR) multiple copies of the mtDNA template are produced. A 6-10 μl aliquot of the mtDNA extract is used as a template in a 25 μl PCR-based reaction. The reaction is performed on a Perkins Elmer 9600 GeneAmp system using different primer sets necessary to amplify specific regions of the mitochondrial genome where defining polymorphic activity of the 4 New World lineages occurs. The 25 μl reaction is subject to 40 cycles of PCR that consisted of denaturing at 93qC for 30s, annealing at 48qC for 90s, and elongating at 72qC for 30s. The B haplotype is characterized by a 9-bp intergenic deletion located between the cytochrome oxidase II and lysine tRNA genes2 o0f7 Region V. Its presence is noticed by a length difference when exposed to electrophoretic fragment separation on an agarose gel. The other three lineages are obtained by the use of restriction enzyme digests. Again, digested fragments are separated for analysis using gel electrophoresis.
The 74 samples used for this study are representative of the Altiplano as a whole due to the fact that the patients who go to the two major hospitals come from outer lying areas. The results of the mtDNA typing of these 74 individuals are consistent with previous results showing a clear predominance of the B haplotype. 52/74 (70%) are of the B haplotype, 3/74 (4%) are of the A haplotype, 12/74 (16%) are of the C haplotype, and 7/74 (9%) are of the D haplotype.
Coupling these results with a careful examination of the mothers’ reproductive histories reveals some interesting trends (Myres et al in preparation). The patient histories indicate that for all women 133 known conceptions had occurred which resulted either in spontaneous abortion, a still, or a live birth. Of the total number of conceptions, B females accounted for 76 (57.1%) compared to 57 (42.9%) for non-Bs. Sixty three births were attributable to sixty two women, the B haplotype accounting for 41 (65.1%) while non-Bs made up 22 (34.9%) of the total. The increase in percentage of B births, and conversely the decrease in percentage of non-B births, compared to the percentage of total conceptions is due to a greater proportion of non-B lethal events occurring between conception and birth. Also of note is the fact that non-B mothers recorded a greater proportion of spontaneous abortions (3/57), and also still births (3/57) as compared to B mothers, with (1/76) and (4/76) respectively.
Further evidence to support the possibility of an increased survival rate for the B genotype is reflected in the data of neonate survival. Of live births, 2/71 Bs and 6/56 non Bs had expired before 3 months, with one instance of paternal child abuse being responsible for a B infant death thus decreasing the B total to 1/70. A comparison of the number of conceptions per mother indicates non-Bs to be considerably higher than Bs 2.59 to 1.9. However, taking into account the number of infants alive at 3 months narrows this disparity with non-Bs at 2.05 and Bs at 1.75. In all, B infants showed a 92.1% (70/76) survival rate from conception to 3 months compared to 78.9% (45/57) for non-B infants.
From this data we see two existing trends. First, non-B mothers show a lesser probability of taking an infant to term than do B mothers, and second, B infants show a considerably greater ability for survival through the first 3 months of life. This information provides strong support to the hypothesis that the B haplotype possesses a selective advantage over the non-B Haplotype for this particular environment. The knowledge that certain genotypes at elevation are at a disadvantage to survive infancy makes preventative measures possible. Thus, potential mothers and health care workers aware of this circumstance can be better informed to know how to reduce the risk of infant mortality on all fronts.
References
- Horai, S., R. Kondo, Y. Nakagawa-Hattorri, S. Hayashi, S. Sonoda, K. Tajima. (1993). Peopling of the Americas, Founed by four Major Lineages of mitochondrial DNA. Mol Biol Evol 10: 23- 47.
- Torroni A., D. C. Wallace. (1994) Mitochondrial DNA Variation in Human Populations and Implications for Detection of Mitochondrial DNA Mutations of Pathological Significance. J Bioenergetic Biomem 26:261-271.
- Walsh, P. S., D. A. Metzger, R. Higuchi. (1991). Chelex 100® as a Medium for Simple Extraction of DNA for PCR-Based Typing from Forensic Material. Biotechniques 4:506- 13.