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Biology 303 H01Mitochondrial 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.

Figure 1: (A) shows an MRI image of a healthy individual and (B) is of a PKAN individual with hyperintensity. (C) shows the iron deposition in the globus pallidus.

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Biology 303 H01Research 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

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Biology 303 H01 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

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

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Biology 303 H01fibroblasts 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

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.

Neema Patel 11/1/2014

Biology 303 H01Similarly, 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.

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.

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Biology 303 H01From 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.

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.

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Biology 303 H01Three 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

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

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Biology 303 H01mutations, 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.

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

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Biology 303 H01mRNA-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

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.

Neema Patel 11/1/2014

Biology 303 H01

References:

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Exome Sequence Reveals Mutations in CoA Synthase as Cause of Neurodegeneration

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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

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P et al. (2012) Skin Fibroblasts from pantothenate kinase-associate neurodegeneration

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Biology 303 H017. DJ, B. (2000) What is Oxidative Stress? Metabolism 49, 3-8.

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8. NBIA Disorders Association http://www.nbiadisorders.org/