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Abstract

Alternative splicing (AS) using a sole gene to express multiple transcripts with diverse protein coding sequences and/or RNA regulatory elements raises genomic complexities. In the nervous system, several thousand AS events play important roles in ion transportation, receptor recognition, neurotransmission, memory, and learning. Not surprisingly, AS influences human physiology, development, and disease. Many research studies have focused on aberrant AS in nervous system diseases, including Parkinson's disease (PD), the second most common progressive neurodegenerative disorder of the central nervous system. PD affects the lives of several million people globally. It is caused by protein aggregation, such as in Lewy bodies, and the loss of dopamine-containing neurons in the substantia nigra of the midbrain. To our knowledge, six genes, including PARK2, SNCAIP, LRRK2, SNCA, SRRM2, and MAPT, are involved in aberrant AS events in PD patients. In this review, we highlight the relevance of aberrant AS in PD and discuss the use of an aberrant AS profile as a potential diagnostic or prognostic marker for PD and as a possible means of applying therapy.

Keywords

Alternative splicing (AS)Parkinson's disease (PD)

Introduction

Splicing is a process of pre-mRNA maturation in the eukaryotic cell in which introns are skipped and exons are ligated. Splicing can be used to produce correct proteins through a translation mechanism. Major splicing is catalyzed by the spliceosome, a complex of five small nuclear ribonucleoprotein particles (snRNPs: U1, U2, U4, U5, and U6) and numerous protein factors. snRNPs bind to the pre-mRNA in a sequential manner. Following the binding of U4/U6.U5 tri-snRNP, the spliceosome undergoes extensive structural remodeling, leading to the release of U1 and U4 and the addition of the NineTeen complex (NTC) and activation of the spliceosome. The catalytic spliceosome undergoes two sequential transesterification reactions, thus joining the exons and releasing the intron lariat (46,54,67). After the splicing reaction is complete, the postcatalytic spliceosome dissociates, releasing the mature mRNA, and then binds to the NineTeen complex-Related proteins (NTR) complex to disassemble all components for a new cycle of splicing (63,64).

Alternative splicing (AS) is a splicing change mechanism. Exons of pre-mRNAs are ligated by AS in different orders to form a large number of transcripts with different protein coding sequences (frameshifts, insertions, and deletions) and/or RNA regulatory elements (translation efficiency, stability, and localization of RNA) (34). Five universal models of AS have been defined, including cassette exons, mutually exclusive exons, competing 5′ or 3′ splice sites, and retained introns (4,5,40). Studies show that more than 90% of human genes undergo AS, with obvious changes across tissue types and developmental stages (21,48,68). AS rarely produces “yes or no” responses to stimuli; generally AS products coexist in a single cell and give the cell the ability to manage different internal and external stimuli. Interestingly, up to one third of AS transcripts generate premature termination codons and undergo nonsense-mediated RNA decay (42).

Splicing is modulated by short helping sequences (~10 nt) that help to define real exons. These conserved sequences, named cis-regulatory elements (also called splicing codes), are located in the exonic or intronic regions of the gene. They contain the exonic and intronic splice enhancers (ESEs and ISEs, respectively), which promote exon inclusion, and conversely, exonic and intronic splice silencers (ESSs and ISSs, respectively), which promote exon exclusion (5,34,40). In early studies, splice enhancers and silencers were shown to mostly interact with trans-acting splicing factors. These splicing factors are RNA-binding proteins that contain mainly serine/arginine-rich proteins (SR proteins, which bind to ESEs), heterogeneous nuclear ribonucleoproteins (which bind to ESSs or ISSs), and polypyrimidine tract binding protein (which binds to ISSs) (5,34,40). Combinatorial binding and the relative ratio of trans-acting splicing factors determine whether a splice site is used or skipped. However, exceptions to this type of regulation have been found. Some trans-acting splicing factors can modulate exon inclusion or exclusion positively or negatively, depending on the binding regions relative to the AS exon (15,66). These studies showed that the sites of protein–RNA interactions play an important role in splicing regulation. In addition, some cis-regulatory elements may regulate AS by directly determining pre-mRNA secondary structure (6). Recent studies have indicated that epigenetic control determines not only what parts of the genome are expressed but also how they are spliced (38). The patterns of AS and the relative ratio of specific trans-acting splicing factors are dynamic and respond to intra- and extracellular signals. Chromatin modification, transcription factors, posttranscriptional modifications, and posttranslational modifications regulate trans-acting splicing factors by influencing their cellular level, activity, or localization (5,34,40).

Although numerous studies have been performed on the functions and mechanisms of AS for specific genes, the roles of AS events (RNA splicing maps) overall are only now beginning to be explored. AS microarrays with multiple probes hybridized with exon–exon junctions permit large-scale profiling of AS (5). After hybridization, the probes within the constitutive exons of the sample can be assessed for the presence or absence of the exon junction (44). Moreover, combining microarray analysis with chromatin or RNA immunoprecipitation (in vivo cross-linking and immunoprecipitation) allows identification of the populations of genes and transcripts modulated by specific trans-acting splicing factors (44). AS microarrays show that functionally related transcripts can be coregulated in splicing networks to promote specific biological functions (44).

Aberrant AS has been well known in many diseases, including cancer (11) and neurodegenerative disorders (1,22). In the nervous system, several thousand AS events play important roles in ion transportation, receptor recognition, neurotransmission, memory, and learning (1). Not surprisingly, misregulation of AS is important in related diseases (69). AS sites are affected by many mutations of cis-regulatory elements that interfere with the regulatory sequence of RNA and by mutations of trans-acting splicing factors that change the amount or activity of factors. Parkinson's disease (PD), the second most common progressive neurodegenerative disorder of the central nervous system, affects several million people worldwide (53). The three basic clinical features of PD are rigidity, resting tremor, and bradykinesia (53). PD is caused by protein aggregation, such as in Lewy bodies (LBs), and the loss of dopamine-containing neurons in the substantia nigra of the midbrain (12). Dopamine deficiency, proteasomal dysfunction, mitochondrial complex-1 activity, and markers of oxidative stress characterize PD (58). Although most cases of PD occur sporadically, genetic mutation and aging are the major factors that are unambiguously linked to this disorder. Currently, no effective therapy is available for PD, but medications, surgery, and multidisciplinary management can provide relief of symptoms. Active research directions for treatment of PD include development of a new animal model and studies of potential neuroprotective agents, gene therapy, and stem cell transplants (8,24,37,43,59,70,71). To our knowledge, six genes, including PARK2 [parkinson protein 2, E3 ubiquitin protein ligase (parkin)], SNCAIP [a-synuclein, a interacting protein (synphilin)], LRRK2 [leucine-rich repeat kinase 2 (dardarin)], SNCA (α-synuclein), SRRM2 (serine/arginine repetitive matrix 2), and MAPT (microtubule-associated protein tau), are involved in aberrant AS events in PD patients. In this review, we highlight the relevance of AS in PD. In addition, we discuss the use of the AS profile as a potential diagnostic or prognostic marker and as a means of applying therapy.

Aberrant Alternative Splicing in Pd-Related Genes

PARK2

The PARK2 gene, which encodes parkin, is mutated in autosomal recessive juvenile parkinsonism (ARJP, the absence of LBs in PD). The protein is normally expressed in many brain areas and is involved in the ubiquitin proteasome system as an E3 ubiquitin-ligase. Parkin has an ubiquitin-like domain (recognizing a specific substrate protein) in the N-terminus and two C3HC4 Really Interesting New Gene (RING) finger domains (E2 recognition sites) in the C-terminus (30,57), which are separated by an extra cysteine/histidine-rich region (in-between RING finger domain). Parkin participates in the transfer of ubiquitin to specific substrate proteins and so targets them for the proteasome degradation pathway (57). Several proteins, including synphilin-1 and α-synuclein, have been recognized as parkin substrates (27). PARK2 has at least 12 exons, which encode 465 amino acids. Seven isoforms that result from AS, including transcript variants (TVs) 1, 2, 3, 6, 7, 11, and 12, are derived from PARK2 (10). Changes in their relative expression have been linked with LB disease. The relative expression levels of TV3 (in-frame deletions lacking exons 3–5) and TV12 (in-frame deletions lacking exons 2–7) were shown to be increased in PD when compared with controls (3,27).

D'Agata and Cavallaro analyzed cDNA species encoding AS variants of parkin from adult rat and fetal human brain. AS has the potential to express hundreds of different isoforms of proteins with different amino acid compositions, posttranslational modifications, and molecular structures. These isoforms differ in the presence or absence of the ubiquitin-like domain, one or two C3HC4 ring fingers, the in-between RING fingers domain, and a thiol protease active site, which has not been previously characterized. Distinct expression profiles arise in primary cultures of neuronal and glial cells. Structural diversity of AS isoforms of parkin may have important implications for the pathogenetic mechanisms underlying ARJP (10).

Tan et al. showed an AS variant of parkin (E4SV) in the substantia nigra and leukocytes of patients with sporadic PD. The exon 4 (122 bp) deletion created a reading frameshift over the junction of exons 3–5 and a stop codon downstream from exon 3. The truncated protein was related to a total loss of the two-RING finger domain and therefore to a total loss of enzymatic activity. The expression of E4SV was increased in patients with sporadic PD compared with healthy controls. Moreover, the expression of E4SV was increased with age in PD patients, but this was not observed in the controls (62).

SNCAIP

Synphilin-1, encoded by the SNCAIP gene, is a presynaptic protein bound with synaptic terminals (52). Synphilin-1 may play a key role in the formation of LBs because coexpression of parkin, synphilin-1, and α-synuclein promotes the formation of cytoplasmic inclusions that resemble LBs (9,18). Furthermore, synphilin-1 is one of the parkin substrates (9) and it seems to connect the ubiquitin proteasome system with synaptic function. Synphilin-1 has two main functional domains: one is responsible for the interaction of parkin and the other for α-synuclein. Variable reduction of both domains is shown in the TVs. At least eight AS variants are generated from SNCAIP, but four of them present truncated proteins of the short C-terminus. Shortened isoforms of synphilin-1 could show stronger interactions than proteins of normal length. Disease-dependent changes in their differential expression have been observed in LB diseases (27). Eyal et al. and Humbert et al. have shown that synphilin-1A (lacking exons 3 and 4 and containing exon 9A) is present in PD. Overexpression of synphilin-1A causes proteasome saturation; is aggregation enhancing; and directly promotes the inclusion, formation, and neurotoxicity of proteins, which indicates that this isoform may contribute to neuronal degeneration (19,27). A further significant finding was that synphilin-1B underwent marked upregulation in PD (3).

LRRK2

The leucine-rich repeat kinase 2 (dardarin), encoded by the LRRK2 gene, is a member of the ROCO group within the Ras/GTPase superfamily. It is characterized by the existence of several conserved domains including a Roc (Ras in complex proteins) domain, a COR (C-terminus of Roc) domain, a leucine-rich repeat region, a WD40 domain, and a protein kinase catalytic domain (14). The function of LRRK2 is not yet known. It may be a cytoplasmic kinase that participates in the phosphorylation of substrates involved in PD pathogenesis (47,72). The LRRK2 gene has 51 exons and encodes a predicted protein of 2527 amino acids. Mutations in LRRK2 are the most common genetic cause of both familial late-onset parkinsonism and apparent sporadic PD (20).

Johnson et al. sequenced 51 exons of LRRK2 in 79 familial North American PD cases to identify mutations that resulted in the disease. They reported two disease-specific missense mutations, T2356I and G2019S, but the effects of these mutations are not yet known. In addition, they also observed that a 4-bp intronic deletion at the splice donor site of exon 19 (IVS20+4delGTAA) in vitro abolishes normal splicing (31). Johnson et al. indicated that mutations in LRRK2 are associated with approximately 5% of PD cases with a positive family history (31).

Di Fonzo et al. performed a wide-ranging study of LRRK2 in a large sample of families with PD that was compatible with autosomal dominant inheritance (ADPD). The full-length open reading frame and splice sites of LRRK2 were studied by genomic sequencing in 60 probands with ADPD. Three intronic variations of undecided significance were characterized. The allelic frequency of the intronic variant IVS30 +12delT was elevated in patients compared with controls, and two other intronic substitutions (IVS4-38A>G, IVS5+33T>C) were recognized in patients but not in controls (14).

SNCA

α-Synuclein (α-syn) is a presynaptic protein encoded by the SNCA gene, the first gene indicated in the pathophysiology of PD (51). A neuropathological feature of PD is the existence of LBs in surviving substantia nigra neurons, which mainly consist of α-syn (60). α-Syn has many effects on cellular function: It inhibits phospholipase D2 (50), associates with dopamine transporters, changing dopamine homeostasis (35), performs as a molecular chaperone (49), and interacts with specific proteins to alter their cellular function (61). The structure of α-syn is characterized by six repeat sequences that form five amphipathic helices on the N-terminus and by a glutamate-rich, acidic C-terminus that supports the protein in soluble form and functions as a chaperone. Accumulated evidence highlights the consequence of α-syn dosage and expression levels in the pathogenesis of PD. However, genetic variability at the 3′ region of SNCA has been frequently linked with susceptibility to sporadic PD. Studies have indicated that an acidic C-terminus of α-syn is necessary for its normal physiological function; deletion of the C-terminus allows the protein to aggregate more easily.

The gene structure of SNCA reveals the existence of seven exons, five of which correspond with a coding region. Studies of AS isoforms of SNCA have confirmed their disease-related differential expression (3). SNCA has four major isoforms: the full-length SNCA 140 and the truncated transcripts SNCA126 (in-frame deletions lacking exon 3), SNCA112 (in-frame deletions lacking exon 5), and SNCA98 (in-frame deletions lacking exon 3 and exon 5) (7,65). At the SNCA 3′ regions, an exon 5 deletion (SNCA112) predicts functional effects relevant to LB pathology. The deletion causes obvious shortening of the unstructured acidic C-terminus and has been shown to enhance α-syn aggregation, which may direct LB formation (2,26,36,60). McCarthy et al. studied the effect of PD risk-related variants at the SNCA 3′ regions on SNCA112-mRNA levels in vivo in 117 samples of brain frontal cortex from neuropathologically normal patients. Single-nucleotide polymorphisms tagging the SNCA 3′ regions showed considerable effects on the comparative levels of SNCA112-mRNA from total SNCA transcript levels. The “risk” alleles were associated with an enhanced expression ratio of SNCA112-mRNA from the total. McCarthy et al. provide evidence for the functional effects of PD-associated SNCA variants at the 3′ region, indicating that genetic modulation of SNCA AS plays an important role in the development of PD (41).

Some studies have suggested a strong correlation between proteasomal dysfunction and α-syn aggregation as one of the key pathways responsible for forming LBs and destroying dopamine neurons (12,45). Specific toxins of mitochondria or complex-1 inhibitors—1-methyl-4-phenylpyridinium (MPP⁺), a metabolic product of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and rotenone—generate symptoms similar to those of PD and amplify the expression and aggregation of α-syn (56). Using parkinsonism mimetics [MPP(+), rotenone] and related oxidants, Kalivendi et al. have identified an oxidant-generated AS of SNCA mRNA, which induces SNCA112. This AS isoform has a changed localization and strongly inhibits proteasomal function. The induction of SNCA112 was blocked by constitutively active MEK-1 [mitogen-activated protein kinase (MAPK) kinase 1] and enhanced by suppression of the extracellular signal-regulated kinase (Erk)-MAP kinase pathway. Overexpression of SNCA112 promoted cell death in human dopaminergic cells compared with full-length protein. Expression of SNCA112 and proteasomal dysfunction was also clear in the substantia nigra and to a slighter extent in the striatum, but not in the cortex, of MPTP-treated mice. Kalivendi et al. show that oxidant-generated AS of SNCA plays a central role in the mechanism of dopamine neuron cell death and thus results in PD (33). An attractive consideration is that PD-related polymorphic variants could affect AS of SNCA as a potential disease mechanism.

SRRM2

Shehadeh et al. investigated the blood microarray of PD patients and found that the AS-related gene SRRM2 (or SRm300), encoding SR repetitive matrix 2, was the only gene that was differentially upregulated among all three PD analyses (55). Two main AS variants of SRRM2 differ at their 3′ region: the full-length SRRM2 and the shortened transcript of SRRM2 (in-frame deletions lacking exons 12–15). Using a real-time PCR technique, Shehadeh et al. showed that SRRM2 in PD switches from basal transcript levels of SRRM2 to low expression of the long isoform and high expression of the short isoform in both the amygdala and substantia nigra. To confirm the results and examine the possibility of AS in PD, Shehadeh et al. performed exon microarray analyses from the peripheral blood of 17 PD patients. They found a noteworthy upregulation of the upstream (5′) exons of SRRM2 and a downregulation of the downstream exons, which caused downregulation of the long isoform. In addition, Shehadeh et al. reported that significant AS occurred in 218 genes (differential exonic expression) in PD blood. This study makes SRRM2 a strong candidate gene for PD and attracts attention to the role of AS in the disease. Further studies will be needed to understand the mechanism of AS in SRRM2 and to determine its role in the development of PD.

MAPT

The MAPT gene encodes tau protein. Tau is a microtubule (MT)-associated protein that fundamentally functions to aid MT assembly and stability. Expressed mainly in neurons of the central nervous system, it is important in axonal transport, axon polarity, and neuronal morphogenesis. MAPT mutations alter protein function or gene regulation and induce insoluble filamentous cytoplasmic inclusions in frontotemporal dementia with parkinsonism-chromosome 17 type (FTDP-17), an autosomal dominant disease (39). Some silent, missense, or deletion mutations of MAPT are in the coding region and reduce the binding activity of tau to MT and/or decrease the ability of tau to promote MT assembly when compared with normal tau. These mutations probably lead to the formation of tau filaments (23,29).

In the central nervous system of the adult human, six tau isoforms are generated from the same exons or AS of exons: exons 2, 3, and 10. Exons 9 to 12 encode four defective-repeat MT-binding domains. Exon 10 (E10), which includes a 31-amino acid repeat, gives the three isoforms with four MT-binding repeats each (4R tau). The other three tau isoforms have only three repeats each (3R tau). Tau mutations of FTDP-17 that increase inclusion of E10 induce the production of excess 4R tau, as shown in both soluble and insoluble tau from FTDP-17 autopsy samples (25,29). A normal ratio (1:1) of 3R to 4R tau isoforms is necessary for suppressing the development of tau pathology (28). The increase in the inclusion of E10 results from mutations at the 3′-end of exon 10, produced by the terminal two bases of E10 and 16 bases in the adjacent intron, which form the destabilization of the RNA stem-loop. This stem-loop generates a favorable binding site for the U1 small nuclear RNA and thus increases the splicing efficiency of E10. In healthy individuals, this loop inhibits access of the splicing machinery to E10, thus blocking its inclusion in mRNA. In addition, all reported mutations also enhance the complementarity of the 5′ splice site sequence to the 5′-end of the U1 small nuclear RNA (16). The cis-acting elements within E10 also influence the splice site choice (17).

D'Souza et al. indicate that tau mutations can increase or decrease AS of tau E10 by acting on three different cis-acting regulatory elements. These elements include an ESE that can either be strengthened (N279K) or destroyed (delK280), causing either constitutive inclusion or exclusion of E10 from tau transcripts. E10 contains a second regulatory element that is an ESS, the function of which is abolished by a silent frontotemporal dementia with parkinsonism-17 (FTDP-17) mutation (L284L), resulting in inclusion of excess E10. A third element that blocks AS of E10 is contained in the intronic sequences flanking the 5′ splice site of E10, and mutations of FTDP-17 in this element enhance the inclusion of E10 (16,17).

Alternative Splicing Signatures as Potential Diagnostic/Prognostic Indicators and Application of Therapies in Pd

Diagnostic, Prognostic, or Predictive Biomarkers

To date, the diagnosis of PD mainly occurs in clinics and is grounded in phenotypic expression. The finding of laboratory markers will improve diagnostic precision, permit preclinical detection, and track disease progression. In the previous sections, we discussed several examples that show a link between AS and PD development. It is remarkable that AS isoforms appear to be PD-specific. Moreover, evidence shows that a different set of genes undergoes shifts in AS profiles during PD development. Shehadeh et al. performed exon microarray analyses from the peripheral blood of PD patients and found that 218 genes undergo significant AS events (55). These specific AS events immediately suggested their potential use as diagnostic, prognostic, or predictive biomarkers. In additional, relationships between AS profiles and PD do not require identification, but only that these links are sufficiently dependable to be predictive. In future, the use of novel genome-wide screens will be important for analyzing AS signatures of PD and for identifying different stages and subclasses of PD.

Therapeutic Strategies

PD-specific AS variants also provide potential targets for the development of new therapeutic strategies (Fig. 1). Currently, various strategies are being studied to correct aberrant AS for the treatment of PD: (i) PD-specific AS transcripts that contain unique sequences can be targeted and degraded by using the RNA interference (RNAi) technique. However, this strategy is often associated with problems such as the delivery, toxicity, and immunogenicity of antisense oligonucleotides. The latest products of antisense oligonucleotides, which contain chemical modifications, appear to be more stable and well tolerated by patients in comparison with conventional nucleotide backbones (13). (ii) PD-related AS variants contain unique epitopes that could be used as targets for specific antibodies or peptide-mediated interference. For example, tau mutations of FTDP-17 that increase the inclusion of E10 induce the production of excess 4R tau (25). This tau isoform may be inhibited from forming insoluble filamentous cytoplasmic inclusions by using the chimera interference peptide technique. (iii) Many chemical compounds have been identified to modulate the quality and quantity of the trans-acting splicing factor. For example, posttranscriptional modifications of the splicing regulator by using RNAi and small molecules or interference peptides that block activity of the splicing regulator can be used to alter AS patterns in PD. (4) Synthetically modified oligonucleotides, such as 2′-O-methylethyl, phosphorothioate, phosphorodiamide, and morpholino, are more stable and active than regular nucleotide backbones and present low toxicity in vivo. They can target and hybridize to cis-regulatory elements of AS and suppress unsuitable exon selection by inhibiting the binding of trans-acting splicing factors. Kalbfuss et al. show that modified oligonucleotides bind to splice junctions of tau E10 and suppress the inclusion of E10. The effect is mediated by the development of a stable pre-mRNA–oligonucleotide hybrid, which blocks access of the AS machinery to the pre-mRNA. Antisense oligonucleotide-mediated exclusion of E10 has a physiological effect by increasing the ratio of 3R to 4R tau. Kalbfuss et al. demonstrate that AS defects of tau, as found in FTDP-17 patients, can be corrected by using the antisense oligonucleotide technique. These findings offer a tool for studying PD-related AS isoforms in vivo and might lead to a novel therapeutic strategy for PD (32).

Figure 1.

Various therapeutic approaches are currently being used to target alternative splicing for treatment of PD. (A) PD-specific AS transcripts that contain unique sequences can be targeted and degraded by using the RNA interference (RNAi) technique. (B) PD-related AS variants contain unique epitopes that could be used as the targets for specific antibodies or peptide-mediated interference. (C) Many chemical compounds have been identified to modulate the quality and quantity of the trans-acting splicing factor. (D) Synthetically modified oligonucleotides can target and hybridize to cis-regulatory elements of AS and suppress unsuitable exon selection by inhibiting the binding of trans-acting splicing factors.

Conclusion

Understanding AS mechanisms involved in PD programming will assist in the development of effective therapies. In future, characterization of AS mechanisms in PD will be accomplished in several ways: first, by establishing a complete data pool of AS events in PD by using bioinformatics and system biology approaches; second, by identifying cis-regulatory elements and trans-acting splicing factors of the AS mechanism involved in PD; third, by addressing the signaling pathways and regulation mechanism involved in the expression and activity of trans-acting splicing factors in PD; and fourth, by analyzing the PD-related physiological and pathological consequences resulting from the expression of various AS isoforms. Moreover, AS difference patterns and the relative ratio of specific trans-acting splicing factors may be useful as molecular markers of PD. Improved understanding of AS mechanisms will also provide an opportunity to develop pioneering strategies for therapies that target specific AS variants of PD.

Footnotes

Acknowledgments

This study is supported in part by the National Science Council (Taiwan) (NSC101-2314-B-039-010-MY2), China Medical University (CMU98-N2-01), and Taiwan Department of Health Clinical Trial and Research Center of Excellence (DOH101-TD-B-111-004). The authors declare no conflict of interest.

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Aberrant Alternative Splicing Events in Parkinson's Disease

Abstract

Keywords

Introduction

Aberrant Alternative Splicing in Pd-Related Genes

PARK2

SNCAIP

LRRK2

SNCA

SRRM2

MAPT

Alternative Splicing Signatures as Potential Diagnostic/Prognostic Indicators and Application of Therapies in Pd

Diagnostic, Prognostic, or Predictive Biomarkers

Therapeutic Strategies

Conclusion

Footnotes

Acknowledgments

References