Mitochondrial Genetic Defects Associated with NBIA

Neema Patel 11/1/2014 Biology 303 H01 Mitochondrial Genetic Defects associated with NBIA

Neurodegeneration with brain iron accumulation (NBIA) is associated with brain iron overload that is genetically heterogeneous with progressive extrapyramidal signs and neurological deterioration (Dusi et. al. 2014). Some common characteristics of NBIA are neuromuscular symptoms, muscle cramping, jerky movements, stiffness, and seizures (NBIA disorder association, 2014). The iron accumulates in the basal ganglia, the region in the brain that is responsible for controlling involuntary movements. This is a common feature in all NBIA cases. Figure 1C depicts this iron deposition, with a slight brown pigmentation, as well as the hyper-intensity in the globus pallidus of a PKAN affected individual. PKAN, panthothenate kinase-associated neurodegeneration, is one of the most common NBIA cases, which is caused by a mutation in the PANK2 gene (Campanella, A. et al. 2012). Some of the more recently identified genetic defects causing NBIA are in the COASY gene or in C19orf12. The PANK2 gene is involved in the production of pantothenate kinase, an enzyme that catalyzes the phosphorylation of vitamin B5, which is the first step of the CoA biosynthetic pathway (Leonardi et al. 2005). On the other hand, COASY codes for CoA Synthase that catalyzes the last few steps in the synthesis of CoA. CoA (Coenzyme A) is important for the synthesis and oxidation of fatty acids, as well as the oxidation of pyruvate in the citric acid cycle. The C19orf12 produces mitochondrial proteins, but the exact function of them is unknown. The latter two genetic defects are the most recently discovered by Dusi et al. (2014) and Hartig et al. (2011), respectively. They based their research off the already known mutated PANK2 gene to help them locate and understand other causes of NBIA. Dusi et al. (2014), Hartig, M. et al. (2011), and Campanella et al. (2012) were all able to link NBIA to genes that code for some mitochondrial protein, as well as understand the link between a genetic defect and iron accumulation. Neema Patel 11/1/2014 Biology 303 H01 Research conducted by Dusi et al. (2014) discovered that a mutation in the CoA Synthase was a contributing factor in NBIA. The COASY gene produces CoA Synthase, which is a bifunctional enzyme that possesses the 4’PP adenyltrasferase (PPAT) and dephospho-CoA kinase (DPCK) activities (Aghajanian and Worrall 2002). The researchers found that the mutations previously associated with NBIA were not found in every patient with this disease. For this reason, they conducted an exome sequence on two patients that presented clinical symptoms of NBIA, but did not have any of the mutations in previously known genes. From the exome sequencing analysis on the first subject who was born to consanguineous parents, Dusi et al. (2014) identified 12 mutant genes that were potentially relevant to the disease (See Figure 2). However, they didn’t investigate all of these genes because most of the variants were either associated with other clinical phenotypes or were not compatible with the NBIA clinical symptoms. For instance, many of the variations found in the patients were also found in the healthy family members, showing that those particular variants may have nothing to do with NBIA. Polymorphisms in the FBXO47 gene were excluded because the gene is expressed mainly in liver, kidney, and pancreas, and the remaining polymorphisms were not present among the 56 NBIA affected individuals. In contrast, the COASY mutation was a good candidate, because of its similarity to the PANK2 gene, which is involved in encoding proteins for CoA synthesis as well. So COASY was considered potentially relevant to NBIA.

Figure 2: This table represents the candidate genes found in subject-II- 3. Link to see a clear image http://www.cell.com/cms/attachment/2010525437/2032585575/mmc1.pdf

Sanger sequencing confirmed the presence of a missense mutation in the COASY gene, a c.1495C>T transition causing an amino acid change to p.Arg499Cys in the DPCK domain of Neema Patel 11/1/2014 Biology 303 H01 the dephospho-CoA kinase, which is a part of the CoA synthase that catalyzes the very last step for the synthesis of CoA. This discovery prompted them to perform a Sanger sequence analysis on the nine exons of the COASY gene in a larger group of people with NBIA. Interestingly, Dusi et al. (2014) identified a second Italian subject with the same mutation except he was heterozygote, as he also had a mutation in the c.175C>T transition, which “resulted in a premature pGln59* stop codon” in the N terminus regulatory domain. The figure below (3B) shows the variations present in subject-II-3 and subject-II-2, where the disease came from two different alleles, one from the mother and one from the father (Dusi et al. 2014).

Figure 3: Pedigree of family 1 and 2. Subject-II-3 is from family 1 where the heterozygous mutation is indicated by -/- and the parents have a +/- to indicate they are carriers. Subject-II-2 is from family 2. To understand the impact of these mutations beyond the neurological deterioration, Dusi et al. (2014) reverse transcribed mRNA from the fibroblasts of each individual and analyzed it through a qPCR. The major result that they saw was a 50% decrease in COASY transcript in individual-II-2, the second Italian subject, compared to the control group (Figure 4A), which most likely indicated RNA decay. This is because individual-II-2 contains a premature stop codon that promotes nonsense mediated RNA decay. The researchers further analyzed the protein levels, using an immunoblot and detected a significant reduction of the protein level in fibroblasts of subject-II-2, which correlates to the low COASY transcription. From these results, they were able to decipher that the p.Arg499Cys mutation is associated with instability or accelerated degradation of the protein, as a minimally detectable immunoreactive band was observed for subject-II-3 (see Figure 4B) who was carrying the homozygous mutation. Dusi et al. (2014) also found that the DPCK—pArg499Cys mutation abolishes the CoA biosynthesis, because they noticed that the mutant gene did not produce the enzymatic activity to completely convert dephospho-CoA into CoA (Figure 4C). Hence, if the DPCK is defective, the CoA Neema Patel 11/1/2014 Biology 303 H01 synthase will be too and will fail to synthesize CoA. These mutations in COASY reveal the importance and the role of CoA biosynthetic pathway for the development and functioning of the nervous system.

Figure 4: (A) Quantification of COASY mRNA levels. The amount of COASY transcript is reduced in subject-II-2 versus control samples. (B) Immunoblot analysis of COASY in fibroblasts. (C) Chromatogram showing the peak corresponding to the reaction product (green) of wild-type DPCK and mutant DPCK. Similarly, Hartig et al. (2011) conducted a study that identified an additional genetic variation associated with NBIA—MPAN. MPAN, mitochondrial membrane protein associated neurodegeneration, is caused by C19orf12 mutations. C19orf12 proteins are predominantly located in the mitochondria and hence they termed the genetic defect as MPAN. This study was also built from previously known genetic defects in the PANK2, PLA2G6, FTL, and CP. PANK2 and PLA2G6 are both genes that code for mitochondrial proteins. The mutations in CP and FTL are defects in the copper binding involved in iron transport and iron storage, respectively. Neema Patel 11/1/2014 Biology 303 H01 Hartig et al. (2011) used homozygosity mapping on 52 individuals from Poland with a case of NBIA and essentially conducted a genetic sequence analysis for variants in the PANK2, PLA2G6, FTL, and CP genes. Among the 52 only 28 individuals carried a mutation in the PANK2 gene, whereas 24 of them lacked this mutation. A candidate gene sequencing of DNA from the 24 individuals revealed a family that contained three members with a novel single homozygous mutation, c.204_214del11 (Gly69ArgfsX10), in the orphan gene C19orf12 (Hartig et al. 2011). An orphan gene is a gene that lacks a common descent due to undetectable similarity of the genes to other species (Wissler et al. 2013). This 11 bp (base pair) deletion in the C19orf12 gene causes a frameshift with a premature stop codon, causing the loss of more than half of the amino acid sequence. Thus Hartig et al. (2011) proposed that this loss of C19orf12 function results in the gradual degeneration of the neuronal tissue. Hartig et al. (2011) also found other missense mutations, p.Gly65Glu, p.Gly53Arg, p.Thr11Met, pLys142Glu, and Tyr11Met, in the C19orf12 genes of other patients. Figure 5 shows the position of these mutations in the C19orf12 gene and its two isoforms, and the variations between the two protein coding isoforms that are affected by the splice variant. The three missense mutations, p.Gly65Glu, p.Gly53Arg, and p.Gly69Arg, change conserved glycines to charged amino acids, whereas the p.Lys142Glu changes a lysine residue to a charged glutamate. Any two combination of these mutations were presented as homozygous in 18/24 individuals, where most of them showed speech and gait difficulties. These individuals also showed much earlier signs of neurodegeneration compared to the ones who only had one C19orf12 missense mutation. However, both cases revealed motor axonal neuropathy, which is paralysis or loss of reflexes, and optic atrophy. This particular genetic defect showed hypointensities in the globus pallidus and substantia nigra in all affected individuals as well. From these results, Hartig et al. (2011) concluded that a considerable proportion of NBIA cases worldwide are due to mutations in the C19orf12 gene, as there were a number of different disease alleles found on this gene. Even though the sample sizes in this study were considerably small, which might overestimate the proportion of NBIA cases with this defect. Neema Patel 11/1/2014 Biology 303 H01

Figure 5: Shows the gene structure of the two isoforms of C19orf12 with the identified mutation.

Factors other than genetic causes have been looked at as well to get a better understanding of the disease itself, beyond the genetic deficiency. Campanella et al. (2012) wanted to understand the relationship between the iron accumulation and neurodegenerative diseases, specifically PKAN. So Campanella et al. (2012) approached their study by identifying iron metabolism alterations in PKAN, panthothenate kinase-associated neurodegeneration. Out of all the different forms of NBIA, those with mutations in the PANK2 gene have the most severe brain iron overload, although the actual mechanism that leads to this iron overload is still enigmatic. So they hypothesized that genetic defects related to CoA may indirectly lead to alterations in iron homeostasis and to oxidative stress due to negative effects on membrane synthesis. Three PKAN patients and three healthy patients were used as subjects in this study. Their skin fibroblasts were analyzed for oxidative status and iron homeostasis. Oxidative stress is basically when there is an imbalance between the reactive oxygen species and the biological system’s ability to detoxify its intermediate, causing tissue damage and such (DJ 2000). Of the three PKAN patients tested, one was homozygous for a single amino acid substitution located on the protein surface and two of them were homozygous for a frameshift mutation that affects the catalytic region of the enzyme and leads to premature termination. All three of these affected Neema Patel 11/1/2014 Biology 303 H01 patients showed high amounts of carbonylated proteins, which indicates oxidative damage and loss of protein function, with respect to the control fibroblasts (Figure 6). This shows that polymorphisms in PANK2 gene induce an alteration in cellular oxidative status (Campanella et al. 2012). Next, they wanted to see the impact of iron and iron homeostasis. They analyzed this by incorporating 55Fe into the control and PKAN fibroblasts (Campanella et al. 2012). Iron is usually bound to ferritin proteins. They found that the PKAN fibroblasts stored the least amount of iron in ferritins, meaning that most of the iron was free floating and not stored. This indicated that the little amount of Fe found in ferritin was due to low ferritin protein levels and not to reduced enzymatic activity. Hence, it is possible that patients’ fibroblasts could have a high amount of potentially toxic ferritin-free iron. This was verified through an iron-sensitive fluorescent probe Calcein-AM. Long-term iron supplementation caused cells to respond by up- regulating ferritins and down-regulating TfR1 proteins, which deliver iron to the cell. If this regulation is damaged, free iron increases and induces the oxidative stress. Another aspect Campanella et al. (2012) had to consider was the iron regulatory protein (IRP) in homeostasis. The IRP regulates protein expression when it is bound to the iron response elements of mRNAs (mRNA-bound IRP complex). This complex was found in low amounts among the PKAN patients compared to the control, and when iron was supplemented there was still a low amount. For the control group, the level of the mRNA-bound IRP complex decreased when iron was supplemented. This shows that whenever iron is in excess, the IRP complex and thus protein expression is reduced. Because of this reduction, the iron storage and delivery systems are defective in PKAN patients, and this leads to an overall increase in free iron and further damage in the cell (Figure 7). However, where the excess iron comes from is still unclear. Also, Campanella et al. (2012) realized that even though the patients varied in the type of mutations, the overall influence was the same, such as alteration of iron homeostasis. This research primarily focused on PKAN and that defects in PANK2 gene promotes an increased oxidative status by the addition of iron, which causes neuronal damage. Neema Patel 11/1/2014 Biology 303 H01

Figure 6: (A) shows the carbonylated protein levels in fibroblasts of PKAN individuals, who are labeled as 1527, 1535, and 1265.

Figure 7: Molecular mechanism of iron role in PKAN. The scheme shows the various structural conformations of IRP1 after iron addition in control (left) and in PKAN (right) cells. The mRNA-bound IRP is lower in PKAN than in controls, likely as a consequence of oxidative status. Together these studies evaluated three different genetic defects involved in NBIA: COASY, C19orf12, and PANK2, as well as the impact of iron accumulation. All of these genes are related in the sense that they code for mitochondrial proteins. However, it is important to keep in mind that there are still many unknown aspects of NBIA, and so there can be other regions in the body or defects that might play a role in the disease and not just excluded to Neema Patel 11/1/2014 Biology 303 H01 mitochondrial DNAs. Campanella et al. (2012) helped understand the influence a genetic defect has on neurodegeneration and the role of iron in the disease. Some of the genetic defects, specifically PANK2, cause certain proteins/enzyme failure, especially the iron storage and delivery system. This impacts the individual by agitating the oxidative status and prompting neuron damage. Dusi et al. (2014) and Hartig et al. (2011) specifically found different genes that NBIA patients may have defects in, the COASY gene and C19orf12 gene. However, this only accounts for a small population and there may be other genetic mutations that differ from other NBIA patients. Overall, the clinical presentations of the patients were quite similar. The information provided by Dusi et al. (2014), Hartig et al. (2011), and Campanella et al. (2012) can be useful in the near future to help cure NBIA.

References:

1. Dusi S, Valletta L, Haack T, Tsuchiya Y, Venco P, Pasqualato S, Goffrini P et al. (2014) Exome Sequence Reveals Mutations in CoA Synthase as Cause of Neurodegeneration with Brain Iron Accumulation. The American Journal of Human Genetics, 94, 11-22. Link: http://www.cell.com/ajhg/fulltext/S0002-9297(13)00523-5 Neema Patel 11/1/2014 Biology 303 H01 2. Hartig M, Iuso A, Haack T, Kmiec T, Jurkiewicz E, Heim K, Roeber S et al. (2011) Absence of an Orphan Mitochondrial Protein, C19orf12, Causes Distinct Clinical Subtype of Neurodegeneration with Brain Iron Accumulation. The American Journal of Human Genetics 89, 543-550. Link: http://www.sciencedirect.com/science/article/pii/S0002929711003971

3. Campanella A, Privitera D, Guaraldo M, Rovelli E, Barzaghi C, Garavaglia B, Santambrogio P et al. (2012) Skin Fibroblasts from pantothenate kinase-associate neurodegeneration patients show altered cellular oxidative status and have defective iron handling properties. Human Molecular Genetics 21, 18, 4049-4059 doi:10.1093/hmg/dds229 Link: http://hmg.oxfordjournals.org/content/21/18/4049.full?sid=0f483706-347b-427a-8f03- 81884554eca7#ref-26

4. Aghajanian S and Worrall D (2002) Identification and characterization of the gene encoding the human phosphopatetheine adenylyltransferase and dephospho-CoA kinase bifunctional enzyme (CoA synthase). Biochem J. 365 (13-18) doi:10.1042/BJ20020569 Link: http://www.biochemj.org/bj/365/0013/bj3650013.htm 5. Leonardi R, Zhang Y.M, Rock C.O, and Jackowski S (2005) Coenzyme A: back in action. Prog. Lipid. Res., 44, 125 – 153.

6. Wissler L, Gadau J, Simola DF, Helmakampf M, and Bornberg-Baur E (2013) Mechanisms and Dynamics of Orphan Gene Emergence in Insect Genomes. Genome Biol Evol (2013) 5 (2):439-455.doi: 10.1093/gbe/evt009 Link: http://gbe.oxfordjournals.org/content/5/2/439.full 7. DJ, B. (2000) What is Oxidative Stress? Metabolism 49, 3-8. Link: http://www.ncbi.nlm.nih.gov/pubmed/10693912 8. NBIA Disorders Association http://www.nbiadisorders.org/