When genomics pioneer Dr. James Lupski found the first gene for the disorder called Charcot-Marie-Tooth disease in 1991, he also uncovered a new kind of disorder – copy number variation or the duplication of genetic material that contained the gene of interest.
In a new report in the American Journal of Human Genetics, the vice chair of molecular and human genetics at Baylor College of Medicine (and a host of colleagues from Baylor and around the world demonstrate that sometimes the problem is not a duplication. Instead, it’s a triplication – three repeats of part of the genome that contains the gene for Charcot-Marie-Tooth 1A (CMT1A). This results in an overabundance of the protein associated with the gene and a severe form of the disorder.
The existence of duplications and triplications was first posited by the iconic fly geneticist Calvin Bridges in the 1930s, when he found that reduction of the size of the eye in fruit flies (Drosophila melanogaster) was due to a duplication on one of the chromosomes. He even noted an apparent triplication in another fly with an even smaller eye.
One step further
Lupski took the science a step further, in a different model organism – Homo Sapiens – in 1991 when he discovered the genomic duplication that causes CMT1A.
“We knew there is considerable clinical variability among individuals with the disorder,” said Dr. Pengfei Liu, now a clinical genetics fellow at Baylor and first author of the report. “Sometimes, in a family, the mother or father is affected and one of the children is affected with more severe symptoms of the disease. We thought it was a normal variation. But when we looked at it carefully, we found that the parent had a duplication and the child had a triplication.”
There are many forms of Charcot-Marie-Tooth disease (CMT), one of the most common inherited neurological disorders.
Charcot-Marie-Tooth 1A is dominantly inherited, which means that if a child has the extra gene copy or copies, he or she has the disorder. The gene carries instructions for a critical protein that is part of the nerve covering called the myelin sheath. Two copies means there is too much protein and the myelin sheath is abnormal, resulting in weakness and deterioration of the muscles of the lower legs.
When Liu, Lupski and their colleagues studied two families, they found that a parent had duplication of the area of chromosome that contained the gene, and their more severely affected children had a triplication (three copies).
Currently, said Liu, most people with duplications are diagnosed using a technique called multiplex-ligation-dependent probe amplification (MLPA). He said a more sensitive technique, such as chromosome microarray analysis (CMA), might be better in families in which children have a more severe form of the disease.
How do triplications occur?
That finding begged the question: How often do such triplications occur?
“The duplication goes to triplication,” said Liu. “We believe the triplication is formed by a mechanism similar to the one that results in duplication – non-allelic homologous combination. We can say that the presence of duplication predisposes the chromosome to triplication. Duplication makes the chromosome unstable.”
Non-allelic homologous replication occurs during meiosis (sexual cell division) in which a DNA molecule from the mother and one from the father pair up with the appropriate genetic sequences aligned, creating the genome of their child. However, sometimes this pairing goes wrong and the recombination results in duplication or deletion of part of the chromosome.
To find out how often the triplications occurred, Liu, Lupski and their colleagues looked at a large genetic database of people with CMT1A. They found that the rate of duplication to triplication was 100-fold higher than the rate of normal to duplication. That does not mean there are more triplications than duplications because a duplication has to exist before the next generation can have a triplication. However, it does give credence to the notion that duplication makes the chromosome unstable and makes it easier for a triplication to occur in the next generation.
Greater amplifications possible
Liu emphasized that they think the principles in this study could mean that greater amplifications could exist, with four or more copies of the genome resulting. These principles should potentially apply to many other disorders caused by duplications, and triplications for those conditions can be under-diagnosed.
Others who took part in this work include: Violet Gelowani and Erin Roney, both of Baylor; Feng Zhang of Fudan University in Shanghai, China; Vivian E. Drory , Shay Ben Schachar and Avi Orr Urtreger of Sourasky Medical Center in Tel Aviv, Israel; Adam C. Medeiros, Rebecca J. Moore, Christina DiVincenzo, and Joseph J. Higgins of Quest Diagnostics, Inc., Athena Diagnostics of Worcester, Mass.; William B. Burnette and Jun Li of Vanderbilt University in Nashville, Tenn.
Funding for this work came from: National Institutes of Health (NIH) National Institute of Neurological Disorders and Stroke (Grant R01NS058529, Grant R01NS066927; Texas Children’s Hospital General Clinical Research Center (Grant M01RR00188); Intellectual and Developmental Disabilities Research Centers (Grant P30HD024064); Vanderbilt Institute for Clinical and Translational Research fund (Grant VR1687) and Vanderbilt National Center for Advancing Translational Sciences award (Grant UL1TR000445).