As plans move ahead to sequence the genomes of anything that moves, what exactly have people found out that’s new and different with whole-genome sequencing? James Lupski, M.D., Ph.D., a physician-scientist who has a neurological disorder called Charcot-Marie-Tooth (CMT) disease, for one, found the genetic cause of his disease after 25 years of searching by sequencing his entire genome.
While a number of human genome sequences have been published to date, Dr. Lupski's research is the first to show how whole-genome sequencing can be used to identify the genetic cause of an individual's disease. It describes the process of analyzing thousands of potentially functional gene variants to eventually find the responsible mutations.
The gene responsible for this disorder, DHODH, located on chromosome 16q22, was previously associated with CMT. It encodes dihydroorotate dehydrogenase, which catalyzes the oxidation of dihydroorotate to orotate, the fourth enzymatic step in de novo pyrimidine biosynthesis required to produce two of the component bases for nucleic acids. The protein is normally located on the outer surface of the inner mitochondrial membrane. In fruit flies, a mutation in this gene, originally described in 1910, causes wing anomalies, defective egg production, and malformed posterior legs.
David Wheeler, Ph.D., of the Human Genome Sequencing Center, Baylor College of Medicine, and his colleagues reported the DNA sequence of James Watson, Ph.D., two years ago. It was sequenced to 7.4-fold redundancy using massively parallel sequencing in picoliter-size reaction vessels. Comparison of the sequence to the reference genome led to the identification of 3.3 million SNPs, of which 10,654 cause amino-acid substitution within the coding sequence.
It took Dr. Wheeler’s team barely two months and approximately one-hundredth the cost of traditional capillary electrophoresis methods. As the first genome sequenced by next-generation technologies, the investigators said that it provides “a pilot for the future challenges of personalized genome sequencing.”
And in the first attempt ever to sequence the genomes of an entire family, the Institute for Systems Biology (ISB) partnered with Complete Genomics to sequence the genomes of a father, mother, and two children. Both children suffer from the recessive genetic disorders: Miller syndrome, a rare craniofacial disorder, and primary ciliary dyskinesia (PCD), a lung disease. By sequencing the entire family the researchers were able to reduce the number of candidate genes associated with Miller syndrome to four.
The investigators said that family-based whole-genome sequencing allowed them to delineate recombination sites precisely, identify 70% of the sequencing errors (resulting in >99.999% accuracy), and identify very rare SNPs. They also directly estimated a human intergeneration mutation rate of ~1.1 x 10-8 per position per haploid genome. This meant that by sequencing the entire family, the researchers could see how much the genome changes from one generation to the next. In this case the gene mutations from parent to child occurred at half the most widely expected rate.
David Galas, Ph.D., ISB svp of strategic partnerships, told GEN that given the sequencing technology explosion over the past five years, “the information we will be able to get will be staggering. What we don’t know how to do yet is how to interpret the data and figure out how to analyze complex genetic traits. The important thing to realize is that as the changes continue and it becomes more powerful, analysis will require a systems approach integrating biology, computation, and technological development, enabling scientists to analyze all elements in a biological system rather than one gene or protein at a time.”
He further said that “Genetics has been just looking at genotypes and phenotypes and at complex statistical correlations. We are just beginning to understand how to use the biology to interpret genetics in a powerful way.”