What's in a name
Daphne's disease has many names, none of which are particularly enlightening at first blush. Leukoencephalopathy with thalamus and brainstem involvement with high lactate (LTBL). Combined oxidative phosphorylation deficiency 12 (COXPD12). Glutamyl-tRNA synthetase deficiency. EARS2 mutation. This etymological diversity in large part reflects the historical development of the diagnosis of the disease, but it also serves to illuminate the disease's many layers. Each name in fact describes one particular step in the pathology from genetic mutation to MRI presentation.
1 in 16,000,000
A typical cell has 2 distinct genomes, both act as repositories for the instructions for building proteins, written in the language of DNA. The nuclear genome contains the vast majority of these instructions. There are 2 copies of each gene in the nucleus, one copy inherited from each parent. The mitochondrial genome contains instructions for building a relatively small number of proteins, all involved in cellular respiration (see below). The mitochondrial DNA is inherited only from the mother.
Daphne’s disease is caused by a mutation in the nuclear genome known as the EARS2 gene. The inheritance pattern is recessive, meaning that two mutated copies are necessary for the disease to present. The frequency of carriers in the general population is about 1 in 2,000. The odds then of a child having both a mother and a father who carry the mutated gene are 1 in 4,000,000. The odds of that child inheriting a mutated copy from each parent is 1 in 4. So, all together, 1 in 16,000,000. This is roughly the frequency of Daphne's disease in the general population.
Gene to protein
The EARS2 gene codes for an enzyme called glutamyl-tRNA synthetase. This enzyme is involved in translation, the process of reading DNA and stringing together amino acids to assemble proteins. This process, and the role of the tRNA synthetases, is most easily understood with a picture.
Figure 1 shows how proteins are built. The instructions in the DNA are converted into messenger RNA and fed through the ribosome. The ribosome matches each 3 letter sequence (aka codon) with the complementary tRNA molecule. Each tRNA molecule has attached to it one amino acid – the next amino acid in the chain. When the stop codon is reached, the chain terminates and the protein folds into its functional form.
There are 21 amino acids. One of those is called glutamic acid (abbreviated Glu or E). The glu-tRNA synthetase enzyme attaches free glutamic acid to the glu-tRNA molecule. Without that enzyme, the glutamic acid remains free and the glu-tRNA remains empty. In the picture above, a codon calling for glutamic acid would be made to wait for a rare “charged” glu-tRNA molecule to come along, and the protein being assembled would not be produced at a sufficient rate.
The process of protein translation occurs both in the nucleus of the cell (translating DNA into proteins) and in the mitochondria (translating mitochondrial DNA (mtDNA) into proteins). Daphne’s tRNA synthetase deficiency occurs inside her mitochondria. This is the “2” in EARS2. (The gene that encodes for the glu-tRNA synthetase that operates in the nucleus is called EARS1.) The mtDNA contains the bulk of the instructions for building the proteins involved in cellular respiration.
On a cellular level, the fundamental unit of energy is called ATP. This fuel molecule can be broken apart to build things or make things move. And a lot of it is needed. A working muscle, for example, requires 10 million molecules of ATP per second. The high energy demand of the body is met with efficient large scale production - an adult human typically cycles through 140 lbs of ATP a day. This all occurs in the mitochondria, the power plant of the cell.
Glucose and oxygen are the inputs to a complex triad of linked metabolic pathways, glycolysis, the Krebb’s cycle and oxidative phosphorylation (oxphos or the electron transport chain). The first process, glycolosis, cleaves glucose into pyruvate. Pyruvate then enters the 10-step Krebb’s cycle that produces a number of molecules including NADH and succinate. These molecules then become the inputs to the third and final pathway, the electron transport chain.
There are 5 complexes involved in the electron transport chain, illustrated in Figure 2. The fundamental role of each of the first four is to push a positively charged proton (H+ in Figure 2) from the mitochondrial matrix into the intermembrane space. This creates a charge imbalance and an electric potential across the membrane. In the last step, the protons slide down this electrical potential, through the axis of the water wheel-like 5th complex, ATP synthase, which spins to convert that kinetic energy into chemical energy by attaching a third phosphate group to ADP, creating the cell's universal energy currency, ATP.
The detailed chemical reactions performed by each complex are beyond the scope of this summary. But I would like to point out the enzyme labeled Q in Figure 2. This is Coenzyme Q10, which is converted from ubiquinone into ubiquinol by accepting eletrons from Complexes 1 or 2 and converted back to ubiquinone by donating those electrons to Complex 3. It is also a standard nutritional supplement and part of the “mito cocktail” of many mitochondrial patients, including Daphne.
In addition to the energy producing processed of the Krebb’s cycle and electron transport chain, as described above, the cells have access to an additional source, a sort of nitro boost that is only good for about 90 seconds before lactic acid begins to build up and the reaction stops – anaerobic respiration: the production of ATP through glycolysis with no oxygen necessary. The effect of that energy boost can be clearly seen in Figure 3 of speed vs. distance for world records of human running. There is a line connecting the points at longer distances, and then a bump for shorter distances. Runners have access to large stores of anaerobic respiration during those short races that run out during the long ones. Marathons must be run aerobically.
Because Daphne doesn’t charge her glu-tRNA fast enough, there aren’t enough electron transport chain complexes. Because there aren’t enough electron transport chain complexes, she cannot produce enough ATP for the demands of her body. The last ‘L’ in LTBL stands for high Lactic acid. Daphne’s body tries to compensate for her low aerobic output with anaerobic respiration. Her lactic acid levels are constantly elevated. She is constantly sprinting. Constantly redlined.
The most devastating aspect of Daphne’s disease is neurological. But surprisingly, while the genetics and the biochemistry are all well known and understood, how this particular enzyme, which appears to be fundamental to all cellular processes, plays such a disproportionate role in the very specific process of myelination, is not.
Had Daphne been born in the middle of the 20th century, she likely would have been diagnosed with leukodystophy, a disease in which the white (leuko-) matter of the brain is damaged though any of a number of mechanisms. White matter in this case refers to myelin, the insulating sheath on the conducting axons. It’s a high lipid content molecule, one that requires a lot ATP to produce. When the myelin coverage is incomplete, or the myelin itself is of poor quality (in Daphne’s case, low in lipids, high in water), the ability of neurons to communicate is drastically slowed. (Other more well known white matter diseases are cerebral palsy and multiple sclerosis.) Because Daphne’s version has a (known) mitochondrial cause, it is called Leukoencephalopathy, the distinction without much difference being that the white matter disease is not fundamental. I’ve found that something like “leukodystrophy, similar to cerebral palsy” is often the most concise way to convey the syndrome of Daphne’s condition to people hearing about if for the first time.
There are two commonly used MRI imaging techniques, T1-weighted and T2-weighted. In T1-weighting, regions of dysmelination appear dark. In T2-weighting, they appear bright. By comparing Daphne’s MRIs to normal MRIs, as in Figure 3, the extent of the dysmelination can be clearly seen. Daphne’s leukoencephalopathy is significant, but not severe by LTBL standards. (See paper by Steenweg in the Research Library [link to research library].)
The last two pieces of the neurology of the name LTBL are the ‘T’ and the ‘B,’ the thalamus and the brainstem. Both are key components of the primordial brain. The thalamus acts like a hub for incoming sensory and motor signals from the body, assigned with the switchboard-esque task of transferring those signals to their proper destination. It’s visible in the MRI as the two small egg-shaped masses straddling the very center of the image. The brainstem regulates the heartbeat, breathing, and sleep.
The process of myelination continues through the first 12 years of life. Dysmyelination events in LTBL patients are discrete. The child experiences periods of normal neurological development punctuated with regressions. Regressions have triggers, events that tax metabolism like infection, fasting, stress, or exhaustion. If the triggers can be minimized, regressions might be avoided, and the child often shows recovery from the neurological injury. At this point, this is our most realistic hope for Daphne.
The preceding is a description of Daphne’s disease, to the best of my understanding, written for the Daphne’s Lamp webpage. It is an attempt to succinctly convey the fundamental components of a complex disease. It also in no way captures my daily experience with Daphne or our relationship to her disease.
Figure 1 The process of protein synthesis (from the Wikipedia article, Transfer RNA).
Figure 2. The Electron Transport Chain (by OpenStax College [CC BY 3.0 ( via Wikimedia Commons).
Figure 3. The bump visible on the above curves at short distance is due to anaerobic respiration (M. Denny, Journal of Experimental Biology 2008 211: 3836-3849; doi: 10.1242/jeb.024968).
Figure 4. A comparison of a normal 1 yr old's T1 MRI (top left) and a normal 10 mo old's T2 MRI (bottom left) to Daphne's T1 and T2 MRIs at 11 mo (top right and bottom right, respectively.) (Normal T1 from: Human Brain Development, Wikimedia Commons. Normal T2 from: A. Prof Frank Gaillard, Radiopaedia.org, case 6812 (