**4. Autosomal recessive PD**

The strategies of linkage mapping and positional cloning can also be used to identify loci in genes responsible for autosomal-recessive monogenic diseases. This mode of inheritance is characterized typically by the occurrence of the disease in siblings while the parents are obligatory heterozygous mutation carriers and usually remain healthy. Autosomal-recessive PD has clinically been first recognized and characterized in Japan (Ishikawa and Tsuji, 1996). Sibling pairs with PD often have much earlier age of onset compared with patients with the sporadic disease, which is why the term 'autosomal-recessive juvenile parkinsonism'(AR-JP): has been coined. Since families with a recessive disease are usually much smaller than multigenerational dominant pedigrees, linkage analysis is only successful if several families mapping to the same locus are included into a study.

So far, mutations in three genes have been identified in clinically 'pure' forms of autosomal recessive PD: *PARKIN* (PRKN, or PARK2), *PINK1* (PARK6) and *DJ-1*(PARK7). *PRKN* encodes for parkin, a cytoplasmatic protein which functions in the cellular ubiquitination/protein degradation pathway as an ubiquitin ligase. *DJ-1* and *PINK1* encode for mitochondrial proteins. Genetics mutations in these genes cause PD through the loss of the wild-type protein neuroprotective function, leading to oxidative stress, iron accumulation and mitochondrial dysfunction.

They cause PD with earlier onset (<45 years) and slower progression compared to idiopatic PD. Three additional recessive genes have been added to the list more recently-*ATP13A2*, *PLA2G6* and *FBXO7*- which all also cause, when mutated, an early-onset disease with parkinsonism, but also with additional features such as dystonia, which often is seen as the first symptom, dementia, oculomotor disturbances and spasticity.

#### **4.1 PARKIN (PARK2, 6q25.2-q27)**

Juvenile cases of parkinsonism in siblings were first recognized in Japan. The first genetic locus for autosomal- recessive juvenile parkinsonism (AR-JP), as this form of PD was called, was mapped to chromosome 6.

Mutations were then identified in a large gene in that region that was called parkin. Clinically, these patients suffer from L-dopa-responsive parkinsonism and often develop early and severe L-dopa-induced motor fluctuations and dyskinesias. Some show diurnal fluctuations, with symptoms becoming worse later in the day. Dystonia at onset of the disease is common. *Parkin* mutations turned out to be a common cause of parkinsonism

proteasomal degradation, but also via lysin63 (K63), which may play a role intracellular signaling processes and also in Lewy body formation. A recent study revealed a decreased abundance of a number of proteins involved in mitochondrial function or oxidative stress, accompanied by a reduction in respiratory capacity of striatal mitochondria, a decreased serum antioxidant capacity and increased protein and lipid peroxidation These novel findings indicate that proteasomal dysfunction, although supported by several lines of evidence, might not be the sole mechanism contributing to neurodegeneration in *parkin*-related disease. Whatever the mechanism, increasing evidence suggests an important role of *parkin* for dopamine neuron survival. Overexpression of wildtype rat parkin could protect against the toxicity of mutated human A30P α-*SYN* in a rat lentiviral model of PD. The *parkin* mediated neuroprotection was associated with an increase in hyperphosphorylated α-*SYN* inclusions, suggesting a key role for parkin in the genesis of Lewy bodies. Recently, two biochemical modifications of Parkin (*S-*nitrosylation and dopamine quinine-adduct formation) were identified in cellular studies and human brain specimens. These data indicated that reduced E3-ligase activity of the wild-type Parkin protein (rather than an autosomal recessive mutation in the two *Parkin* alleles) could also occur as a result of the principal pathogenetic process that

The new locus was identified in a large Italian family and it causes familial recessive PD in 1-9% of the cases. The phenotype was similar to that seen with *PRKN* mutations and characterised by early-onset parkinsonism (range 32 to 48 years), with slow progression and sustained response to L-dopa. In this and two other consanguineous PARK6- linked families, two different mutations in the gene *PINK1* (encoding PTEN-induced putative kinase 1) were identified. Several studies confirmed the presence of *PINK1* mutations in patients with early-onset PD. Most mutations were missense mutations in conserved regions, but whole-gene deletions have also been described. Almost all described patients with *PINK1* mutations have slow disease progression and a good response to L-dopa. As in *PRKN* related disease, except for the earlier average age of onset, no single feature can separate *PINK1*-related disease from idiopathic PD. There are some indications that *PINK1* mutated patients have a higher prevalence of psychiatric disturbances, particularly anxiety and depression, which is only relatively rarely observed in *PRKN*-related cases . Wild-type PINK1 is thought to function as a protein kinase with possible activity inside the mitochondria, thereby strengthening the hypothesized link between mitochondrial

The third locus for AR-JP, PARK7, was mapped also to chromosome 1p36, in a Dutch family, and the gene was identified as the oncogene *DJ-1*. Again, the phenotype closely resembles that found in patients with *PRKN* and *PINK1* mutations, but this statement is based on a small number of identified patients.Mutations in the gene are responsible for 1- 2% of the AR-JP. However, one recessive family, which carries two homozygous mutations in the *DJ-1* gene, has been described with early-onset parkinsonism, dementia and amyotrophic lateral sclerosis, suggesting that the clinical phenotype associated with

is responsible for the development of sporadic PD.

dysfunction and oxidative stress in PD pathogenesis.

mutations in this gene, although rare, may be rather wide.

**4.3 DJ-1 (PARK7, chr.1p36)** 

**4.2 PINK1 (PARK6, chr.1p35-1p36)** 

with early onset, particularly in individuals with evidence of recessive inheritance. Nearly 50% of families from a population of sibling pairs with PD had *parkin* mutations. Also, *parkin*  mutations are responsible for the majority of sporadic cases with very early onset (before age 20), and are still common (25%) when onset is between 20 and 35. Prevalence is almost certainly well below 5% in those with onset later than 45. Several studies have described the clinical spectrum of *parkin*-associated parkinsonism. Mean age at onset in a European population was 32 years; progression of the disease was usually relatively slow, but L-dopaassociated fluctuations and dyskinesias occurred frequently. Dystonia (usually in a lower extremity) at disease onset was found in about 40% of patients, and brisk reflexes of the lower limbs were present in 44%. Psychiatric abnormalities have been recognized in PD patients with *parkin* mutations but there are no systematic studies to determine whether this is a characteristic feature associated with *parkin*-mutations. Phenotype– genotype studies implicate that the type of mutation may influence the clinical phenotype to a certain degree: patients with at least one missense mutation showed a faster progression of the disease with a higher UPDRS (United Parkinson's Disease Rating Scale) motor score than carriers of truncating mutations. Missense mutations in functional domains of the *parkin* gene resulted in earlier onset. It is still controversial whether heterozygous mutations in the *parkin* gene can cause parkinsonism or can confer an increased susceptibility for typical late-onset PD. There is evidence from imaging studies that heterozygous carriers of *parkin* mutations have reduced uptake of fluorodopa in the basal ganglia. Furthermore, families with heterozygous mutation carriers manifesting symptoms of PD have been described. On the other hand, the frequency of heterozygous mutations in the *parkin* gene was found to be similar in elderly healthy individuals, as compared to a cohort with late-onset typical PD and in a large family reported recently, 12 heterozygous carriers of a particular *parkin* mutation (ex3delta40) were asymptomatic. Also, in a group of families with PD showing anticipation (late-onset PD in the parent generation and early-onset PD in the offspring) genotyping results did not support the explanation that the presence of single or compound heterozygous *parkin*mutations contribute to this phenomenon. Therefore, at present the data are still insufficient to confidently judge the role of single heterozygous *parkin* mutations in the development of PD. Knowledge on the neuropathology of molecularly confirmed cases of AR-JP is still based on only a few cases. Severe and rather selective degeneration of neurons in the substantia nigra and the locus coeruleus, usually with absence of Lewy bodies, has been described.

As mutations in *parkin* cause parkinsonism, in all likelihood by a loss-of-function mechanism, the study of the normal function of parkin provides insight into the molecular pathogenesis of the disorder. Several groups have now shown that parkin, a protein found in the cytosol but also associated with membranes, functions in the cellular ubiquitination/protein degradation pathway as a ubiquitin ligase. It has been hypothesized that the loss of parkin function may lead to the accumulation of a nonubiquitinated substrate that is deleterious to the dopaminergic cell but, due to its nonubiquitinated nature, does not accumulate in typical Lewy bodies. Several proteins have been shown to interact with parkin. However, the putative toxic protein, which has been hypothesized to accumulate due to the lack of *parkin* in patients (or in knock-out animals) has not yet been identified. However, novel functions of *parkin* are being identified, and it is possible that they may be of equal or even greater relevance to the pathogenesis of PD. For example, it has been shown that parkin does not only mediate the well-studied ubiquitinylation via lysin48 (K48), which directs ubiquitinylated proteins for

with early onset, particularly in individuals with evidence of recessive inheritance. Nearly 50% of families from a population of sibling pairs with PD had *parkin* mutations. Also, *parkin*  mutations are responsible for the majority of sporadic cases with very early onset (before age 20), and are still common (25%) when onset is between 20 and 35. Prevalence is almost certainly well below 5% in those with onset later than 45. Several studies have described the clinical spectrum of *parkin*-associated parkinsonism. Mean age at onset in a European population was 32 years; progression of the disease was usually relatively slow, but L-dopaassociated fluctuations and dyskinesias occurred frequently. Dystonia (usually in a lower extremity) at disease onset was found in about 40% of patients, and brisk reflexes of the lower limbs were present in 44%. Psychiatric abnormalities have been recognized in PD patients with *parkin* mutations but there are no systematic studies to determine whether this is a characteristic feature associated with *parkin*-mutations. Phenotype– genotype studies implicate that the type of mutation may influence the clinical phenotype to a certain degree: patients with at least one missense mutation showed a faster progression of the disease with a higher UPDRS (United Parkinson's Disease Rating Scale) motor score than carriers of truncating mutations. Missense mutations in functional domains of the *parkin* gene resulted in earlier onset. It is still controversial whether heterozygous mutations in the *parkin* gene can cause parkinsonism or can confer an increased susceptibility for typical late-onset PD. There is evidence from imaging studies that heterozygous carriers of *parkin* mutations have reduced uptake of fluorodopa in the basal ganglia. Furthermore, families with heterozygous mutation carriers manifesting symptoms of PD have been described. On the other hand, the frequency of heterozygous mutations in the *parkin* gene was found to be similar in elderly healthy individuals, as compared to a cohort with late-onset typical PD and in a large family reported recently, 12 heterozygous carriers of a particular *parkin* mutation (ex3delta40) were asymptomatic. Also, in a group of families with PD showing anticipation (late-onset PD in the parent generation and early-onset PD in the offspring) genotyping results did not support the explanation that the presence of single or compound heterozygous *parkin*mutations contribute to this phenomenon. Therefore, at present the data are still insufficient to confidently judge the role of single heterozygous *parkin* mutations in the development of PD. Knowledge on the neuropathology of molecularly confirmed cases of AR-JP is still based on only a few cases. Severe and rather selective degeneration of neurons in the substantia nigra and the locus coeruleus, usually with absence of Lewy bodies, has been

As mutations in *parkin* cause parkinsonism, in all likelihood by a loss-of-function mechanism, the study of the normal function of parkin provides insight into the molecular pathogenesis of the disorder. Several groups have now shown that parkin, a protein found in the cytosol but also associated with membranes, functions in the cellular ubiquitination/protein degradation pathway as a ubiquitin ligase. It has been hypothesized that the loss of parkin function may lead to the accumulation of a nonubiquitinated substrate that is deleterious to the dopaminergic cell but, due to its nonubiquitinated nature, does not accumulate in typical Lewy bodies. Several proteins have been shown to interact with parkin. However, the putative toxic protein, which has been hypothesized to accumulate due to the lack of *parkin* in patients (or in knock-out animals) has not yet been identified. However, novel functions of *parkin* are being identified, and it is possible that they may be of equal or even greater relevance to the pathogenesis of PD. For example, it has been shown that parkin does not only mediate the well-studied ubiquitinylation via lysin48 (K48), which directs ubiquitinylated proteins for

described.

proteasomal degradation, but also via lysin63 (K63), which may play a role intracellular signaling processes and also in Lewy body formation. A recent study revealed a decreased abundance of a number of proteins involved in mitochondrial function or oxidative stress, accompanied by a reduction in respiratory capacity of striatal mitochondria, a decreased serum antioxidant capacity and increased protein and lipid peroxidation These novel findings indicate that proteasomal dysfunction, although supported by several lines of evidence, might not be the sole mechanism contributing to neurodegeneration in *parkin*-related disease. Whatever the mechanism, increasing evidence suggests an important role of *parkin* for dopamine neuron survival. Overexpression of wildtype rat parkin could protect against the toxicity of mutated human A30P α-*SYN* in a rat lentiviral model of PD. The *parkin* mediated neuroprotection was associated with an increase in hyperphosphorylated α-*SYN* inclusions, suggesting a key role for parkin in the genesis of Lewy bodies. Recently, two biochemical modifications of Parkin (*S-*nitrosylation and dopamine quinine-adduct formation) were identified in cellular studies and human brain specimens. These data indicated that reduced E3-ligase activity of the wild-type Parkin protein (rather than an autosomal recessive mutation in the two *Parkin* alleles) could also occur as a result of the principal pathogenetic process that is responsible for the development of sporadic PD.

#### **4.2 PINK1 (PARK6, chr.1p35-1p36)**

The new locus was identified in a large Italian family and it causes familial recessive PD in 1-9% of the cases. The phenotype was similar to that seen with *PRKN* mutations and characterised by early-onset parkinsonism (range 32 to 48 years), with slow progression and sustained response to L-dopa. In this and two other consanguineous PARK6- linked families, two different mutations in the gene *PINK1* (encoding PTEN-induced putative kinase 1) were identified. Several studies confirmed the presence of *PINK1* mutations in patients with early-onset PD. Most mutations were missense mutations in conserved regions, but whole-gene deletions have also been described. Almost all described patients with *PINK1* mutations have slow disease progression and a good response to L-dopa. As in *PRKN* related disease, except for the earlier average age of onset, no single feature can separate *PINK1*-related disease from idiopathic PD. There are some indications that *PINK1* mutated patients have a higher prevalence of psychiatric disturbances, particularly anxiety and depression, which is only relatively rarely observed in *PRKN*-related cases . Wild-type PINK1 is thought to function as a protein kinase with possible activity inside the mitochondria, thereby strengthening the hypothesized link between mitochondrial dysfunction and oxidative stress in PD pathogenesis.

#### **4.3 DJ-1 (PARK7, chr.1p36)**

The third locus for AR-JP, PARK7, was mapped also to chromosome 1p36, in a Dutch family, and the gene was identified as the oncogene *DJ-1*. Again, the phenotype closely resembles that found in patients with *PRKN* and *PINK1* mutations, but this statement is based on a small number of identified patients.Mutations in the gene are responsible for 1- 2% of the AR-JP. However, one recessive family, which carries two homozygous mutations in the *DJ-1* gene, has been described with early-onset parkinsonism, dementia and amyotrophic lateral sclerosis, suggesting that the clinical phenotype associated with mutations in this gene, although rare, may be rather wide.

iron accumulation (which is also found in PD proper), a-synuclein aggregation, and neurodegeneration with parkinsonian symptoms.While the 'classic' phenotype of NBIA types 1 and 2 is that of a young-onset progressive extrapyramidal-pyramidal syndrome with visual disturbance through optic atrophy or pigmentary retinopathy, mutations in both genes can be associated with a parkinsonian syndrome of later onset. Recently, *PLA2G6* mutations have been identified in patients with adult-onset L-dopa-responsive dystonia– parkinsonism, pyramidal signs and cognitive/psychiatric features, and cerebral and cerebellar atrophy on magnetic resonance imaging but lack of iron in the basal ganglia.

Finally, mutations in a novel and still poorly characterised gene, *FBXO7*, have been found in members of two families with early-onset, progressive parkinsonism and pyramidal tract signs, a phenotype that had been described clinically as the pallidopyramidal syndrome. Loss of function mutations in *FBX07* appear to give a phenotype which resembles *PRKN* mutation associated phenotype but the disease is generally less benign and has a reduced life expectancy, pyramidal signs and late cognitive problems. This overlap of phenotypes related to *FBX07* and *PRKN* mutations is consistent with the related functions of these two genes and their likely common disease pathway. Like PRKN, F-box proteins, such as FBXO7, are components of the modular E3 ubiquitin protein ligases. The genetic

A considerable percentage of patients with PD was shown to carry a single heterozygous mutation in the *Parkin*, *DJ1* or *PINK1* genes, raising the intriguing question of whether the much more frequent heterozygous mutations in 'recessive' genes might act as susceptibility factors for PD. Several ways lead to explore the potential role of these mutations. First, the frequency of single heterozygous mutations in ethnically matched PD cases and controls could be compared. According to recent reports, heterozygosity for *Parkin* mutations was similar between patients and controls, whereas heterozygous *PINK1* mutations were rarer in controls. Lincoln *et al*. indicated that there was no elevation in PD risk for people who carry a single mutant *Parkin* allele. In most studies, however, healthy controls are not subjected to detailed neurological and neuroimaging examinations, leaving open the possibility that mild clinical (or preclinical) changes could have been present but were not screened for. As recently shown for *Parkin* and *PINK1* families, subtle, but unequivocal, clinical signs of possible or probable PD can be found on careful motor examination in a considerable number of the heterozygous mutation carriers who consider themselves asymptomatic. Furthermore, it could be argued that at least some of the controls had not yet reached the age of their disease onset. Second, the heterozygous offspring of homozygous or compound heterozygous mutation carriers could be examined in a prospective manner, an approach that is currently being used in several cohorts. The probability that a second mutation might have been overlooked in these carriers is much lower than the probability of a mutation being missed in sporadic cases of PD. Last, further functional studies of the affected allele carriers would be highly valuable. Haploinsufficiency, leading to a functional loss of heterozygosity or a dominant-negative effect of some mutant alleles, could explain why a second mutation cannot (and need not) be found for some mutations in the above-

heterogeneity was surprising given their initially common clinical features

**5. Role of heterozygous mutations in 'recessive' genes** 

**4.6 FBXO7 (PARK15, 22q12-q13)** 

There are now eight recessive loci, which can lead to EOPD syndromes. These are the classical recessive loci, *PRKN* (PARK2), *PINK1* (PARK6), *DJ-1* (PARK7). Loss of function mutations at *PRKN*, *PINK1*, and *DJ-1* nearly always give rise to a pure parkinsonian phenotype which has an early onset, a benign course, sleep benefit and a good and prolonged response to L-dopa. The lifespan of mutation carriers is only marginally reduced and there have been no reports of brain iron accumulation. All three proteins have functions related to mitochondrial biology and *PRKN* mutations are usually not associated with Lewy bodies. More recently, five other genes, *ATP13A2* (PARK9), *PLA2G6* (PARK14), *FBX07* (PARK15) and *SPG11* and the *PANK2*, have been identified that cause early-onset forms of parkinsonism associated with a variety of other signs and symptoms, including, in variable combinations, dystonia, ataxia, spasticity and dementia.

#### **4.4 ATP13A2 (PARK9, 1p36)**

It was the first of these genes to be characterized, encoding a predominantly neuronal Ptype ATPase, in a recessively inherited early-onset parkinsonian syndrome described as 'Kufor- Rakeb syndrome' . Patients with this disease have rapidly progressive parkinsonism, spasticity, vertical upgaze palsy and dementia. The substrate and function of the protein is unknown. The wild-type protein was found to be located in the lysosome of transiently transfected cells, while the unstable truncated mutant proteins were retained in the endoplasmic reticulum and degraded by the proteasome. It can be speculated that either overload of the proteasomal protein degradation machinery or lysosomal dysfunction due to the absence of sufficient levels of ATP13A2 protein might lead to neurodegeneration. In fact, there is increasing evidence for an important role of the lysosome in the aetiology of PD: α-synuclein is degraded by chaperone mediated autophagy, and mutations in the gene encoding lysosomal glucocerebrosidase are an important cause of PD. Nevertheless, a direct involvement of the lysosome in the neurodegenerative process in Kufor-Rakeb syndrome and its potential bearing for PD remains speculative at this time

#### **4.5 PLA2G6 (PARK14, 22q13.1)**

Another interesting addition to the recessive genes causing parkinsonian syndromes that may give important insight into underlying pathogenetic processes is the gene for phospholipase A2 group VI (*PLA2G6*). Mutations in this gene have been identified in two recessive childhood-onset disorders: infantile neuroaxonal dystrophy (INAD) and neurodegeneration with brain iron accumulation (NBIA). There is brain iron accumulation in some patients with INAD, so when gene mapping identified a common locus in families with these disorders on chromosome 22, it was reasonable to suspect that the disorders may be allelic. In fact, a large number of mutations were identified, including missense changes, small deletions with and without frameshift, nonsense mutations and large deletions. As patients with two null mutations tended to have the most severe phenotype, a loss of function mechanism can be assumed. Interestingly, in addition to axonal swellings throughout the cortex, striatum, cerebellum, brainstem and spinal cord, the pathological picture also includes a-synuclein positive Lewy bodies, and thus these disorders share this important pathological feature with PD. Lewy bodies are also found in another form of neurodegeneration with brain iron accumulation (NBIA type 1; formerly called Hallervorden–Spatz disease), caused by mutations in the gene for pantothenate kinase 2 (*PANK2*). Therefore, there seems to be an interesting but still little understood link between

There are now eight recessive loci, which can lead to EOPD syndromes. These are the classical recessive loci, *PRKN* (PARK2), *PINK1* (PARK6), *DJ-1* (PARK7). Loss of function mutations at *PRKN*, *PINK1*, and *DJ-1* nearly always give rise to a pure parkinsonian phenotype which has an early onset, a benign course, sleep benefit and a good and prolonged response to L-dopa. The lifespan of mutation carriers is only marginally reduced and there have been no reports of brain iron accumulation. All three proteins have functions related to mitochondrial biology and *PRKN* mutations are usually not associated with Lewy bodies. More recently, five other genes, *ATP13A2* (PARK9), *PLA2G6* (PARK14), *FBX07* (PARK15) and *SPG11* and the *PANK2*, have been identified that cause early-onset forms of parkinsonism associated with a variety of other signs and symptoms, including, in variable

It was the first of these genes to be characterized, encoding a predominantly neuronal Ptype ATPase, in a recessively inherited early-onset parkinsonian syndrome described as 'Kufor- Rakeb syndrome' . Patients with this disease have rapidly progressive parkinsonism, spasticity, vertical upgaze palsy and dementia. The substrate and function of the protein is unknown. The wild-type protein was found to be located in the lysosome of transiently transfected cells, while the unstable truncated mutant proteins were retained in the endoplasmic reticulum and degraded by the proteasome. It can be speculated that either overload of the proteasomal protein degradation machinery or lysosomal dysfunction due to the absence of sufficient levels of ATP13A2 protein might lead to neurodegeneration. In fact, there is increasing evidence for an important role of the lysosome in the aetiology of PD: α-synuclein is degraded by chaperone mediated autophagy, and mutations in the gene encoding lysosomal glucocerebrosidase are an important cause of PD. Nevertheless, a direct involvement of the lysosome in the neurodegenerative process in Kufor-Rakeb syndrome

Another interesting addition to the recessive genes causing parkinsonian syndromes that may give important insight into underlying pathogenetic processes is the gene for phospholipase A2 group VI (*PLA2G6*). Mutations in this gene have been identified in two recessive childhood-onset disorders: infantile neuroaxonal dystrophy (INAD) and neurodegeneration with brain iron accumulation (NBIA). There is brain iron accumulation in some patients with INAD, so when gene mapping identified a common locus in families with these disorders on chromosome 22, it was reasonable to suspect that the disorders may be allelic. In fact, a large number of mutations were identified, including missense changes, small deletions with and without frameshift, nonsense mutations and large deletions. As patients with two null mutations tended to have the most severe phenotype, a loss of function mechanism can be assumed. Interestingly, in addition to axonal swellings throughout the cortex, striatum, cerebellum, brainstem and spinal cord, the pathological picture also includes a-synuclein positive Lewy bodies, and thus these disorders share this important pathological feature with PD. Lewy bodies are also found in another form of neurodegeneration with brain iron accumulation (NBIA type 1; formerly called Hallervorden–Spatz disease), caused by mutations in the gene for pantothenate kinase 2 (*PANK2*). Therefore, there seems to be an interesting but still little understood link between

combinations, dystonia, ataxia, spasticity and dementia.

and its potential bearing for PD remains speculative at this time

**4.4 ATP13A2 (PARK9, 1p36)** 

**4.5 PLA2G6 (PARK14, 22q13.1)** 

iron accumulation (which is also found in PD proper), a-synuclein aggregation, and neurodegeneration with parkinsonian symptoms.While the 'classic' phenotype of NBIA types 1 and 2 is that of a young-onset progressive extrapyramidal-pyramidal syndrome with visual disturbance through optic atrophy or pigmentary retinopathy, mutations in both genes can be associated with a parkinsonian syndrome of later onset. Recently, *PLA2G6* mutations have been identified in patients with adult-onset L-dopa-responsive dystonia– parkinsonism, pyramidal signs and cognitive/psychiatric features, and cerebral and cerebellar atrophy on magnetic resonance imaging but lack of iron in the basal ganglia.

#### **4.6 FBXO7 (PARK15, 22q12-q13)**

Finally, mutations in a novel and still poorly characterised gene, *FBXO7*, have been found in members of two families with early-onset, progressive parkinsonism and pyramidal tract signs, a phenotype that had been described clinically as the pallidopyramidal syndrome. Loss of function mutations in *FBX07* appear to give a phenotype which resembles *PRKN* mutation associated phenotype but the disease is generally less benign and has a reduced life expectancy, pyramidal signs and late cognitive problems. This overlap of phenotypes related to *FBX07* and *PRKN* mutations is consistent with the related functions of these two genes and their likely common disease pathway. Like PRKN, F-box proteins, such as FBXO7, are components of the modular E3 ubiquitin protein ligases. The genetic heterogeneity was surprising given their initially common clinical features

#### **5. Role of heterozygous mutations in 'recessive' genes**

A considerable percentage of patients with PD was shown to carry a single heterozygous mutation in the *Parkin*, *DJ1* or *PINK1* genes, raising the intriguing question of whether the much more frequent heterozygous mutations in 'recessive' genes might act as susceptibility factors for PD. Several ways lead to explore the potential role of these mutations. First, the frequency of single heterozygous mutations in ethnically matched PD cases and controls could be compared. According to recent reports, heterozygosity for *Parkin* mutations was similar between patients and controls, whereas heterozygous *PINK1* mutations were rarer in controls. Lincoln *et al*. indicated that there was no elevation in PD risk for people who carry a single mutant *Parkin* allele. In most studies, however, healthy controls are not subjected to detailed neurological and neuroimaging examinations, leaving open the possibility that mild clinical (or preclinical) changes could have been present but were not screened for. As recently shown for *Parkin* and *PINK1* families, subtle, but unequivocal, clinical signs of possible or probable PD can be found on careful motor examination in a considerable number of the heterozygous mutation carriers who consider themselves asymptomatic. Furthermore, it could be argued that at least some of the controls had not yet reached the age of their disease onset. Second, the heterozygous offspring of homozygous or compound heterozygous mutation carriers could be examined in a prospective manner, an approach that is currently being used in several cohorts. The probability that a second mutation might have been overlooked in these carriers is much lower than the probability of a mutation being missed in sporadic cases of PD. Last, further functional studies of the affected allele carriers would be highly valuable. Haploinsufficiency, leading to a functional loss of heterozygosity or a dominant-negative effect of some mutant alleles, could explain why a second mutation cannot (and need not) be found for some mutations in the above-

Common genetic variants in *SNCA* can increase the susceptibility for idiopatic PD, while the *LRRK2* p.G2019S mutation can cause sporadic PD. Recently GWAS discovered new genes associated with PD: *GBA*, *MAPT*, *GAK, BST1, HLA, LONGO1*and *LONGO2, PARK16,* 

Previous studies have found associations between Parkinson's disease and polymorphisms located within both the α-synuclein gene promoter and other gene regions. Particularly interesting is a complex polymorphic dinucleotide repeat polymorphism (NACP-REP1), located 10 kb upstream of the transcriptional start site of *SNCA*. In this case the number of dinucleotide repeats are directly linked to an higher risk to develop PD. Indeed the 263

The *SNCA* gene consists in two haplotype blocks (genetic regions usually inherited as a single block, with a high degree of Linkage disequilibrium). The first block ranges from the promoter region to intron 4 while the second block includes exons 5 and 6 and the 3' untraslated region. The second block gives the strongest association signal with PD. Becasuse the SNP variability is noncoding and not within a region of species-conserved sequence identity or a miRNA binding site, the biologic mechanism remain unclear. Alternative splicing, phosporilation, expression modification or even linkage disequilibrium with another functional variant within the gene have been hypothesized as possible mechanisms. Gene expression may also be influenced by epigenetic interactions, including methylation, recently implicated in the downregulation of *SNCA* gene expression, which

Even if mutation in *LRRK2* are the most common cause of familial autosomal dominant PD, the p.G2019S substitution is of special significance as it is frequently identified not only in autosomal dominant, but also sporadic PD. Thus, being the most common cause of PD. The mutation is particularly frequent in PD patients residing in, or having genealogical ties to North Africa or the Middle East. This phenomenon can be explained by the fact that most

P.G2019S is located in the mitogen-activated protein kinase (MAP) domain of the LRRK2 protein. The identification of p.G2019S substitutions as the most common cause of both familial and sporadic PD has been a major breakthrough. The frequency of p.G2019S substitutions differ remarkably throughout the world. This is due to a common founder for most p.G2019S carriers, originating from the Middle East or North Africa. Two large studies on Lrrk2 p.G2019S parkinsonism conclude that the phenotype of Lrrk2 p.G2019S can not be distinguished from idiopathic PD. There are some indications of a more benign course of p.G2019S parkinsonism compared to idiopathic PD with a slower disease progression and less cognitive impairment. However, methodological issues may have contributed to these observations. The penetrance of Lrrk2 p.G2019S has been much debated over the last years. Hulihan et al. investigated sporadic PD in Tunisia and found a lifetime penetrance of 45% (95% CI: 20–100%) for p.G2019S substitution carriers. Healy et al. additionally included hereditary patients and estimated that 74% had PD by age 79 years. Interestingly,

*LRRK2* p.G2019S substitution carriers originate from a common founder.

**6.2.1 The LRRK2 c.6055G>A (p.G2019S) mutation** 

haplotype is associated to the disease while the 259 one has a protective effect.

*SYNPHILIN.* 

**6.1 SNCA (PARK1-4, 4q21)** 

may warrant further investigation.

**6.2 LRRK2 (PARK8,12q12)** 

mentioned recessive genes. Although the role of heterozygous mutations in the development of clinical signs currently remains a matter for debate, there is growing evidence that they are associated with pre clinical changes. PET studies have revealed reduced [18F]fluoro-dopa uptake by nerve terminals in the striatum of heterozygotes; there are also structural neuroimaging changes that indicate an increased deposition of metals in the substantia nigra, and there is reorganization of striatocortical motor loops with detectable changes in connectivity patterns.These collective data have important implications. Some carriers of heterozygous mutations might be in the preclinical period of PD, thereby affording unique opportunities to examine the relative risk associated with the affected allele and to study the natural history of the disease. This group also represents an ideal study population to be used not only to investigate compensatory mechanisms, facilitating the development of a sensitive surrogate marker, but also to detect the earliest PD-specific changes, allowing the development of urgently needed clinical biomarkers. Finally, these individuals could provide a small, but important, target population in which to evaluate the 'proof of principle' of a therapeutic intervention in future neuroprotection trials.
