**2.** *SNCA* **triplication and duplication in hereditary PD**

PD is the second most frequent neurodegenerative disorder following Alzheimer disease in the elderly. The main symptoms of PD are tremor, bradykinesia, and gait disturbance. PD genetics is categorized into two groups; one is sporadic PD and the other is familial PD. Familial PD has two forms; autosomal dominant heredity (ADPD) and autosomal recessive heredity (ARPD). ADPD has been observed to be caused by mutations in *SNCA* and *LRRK2*. ARPD is caused by homozygous or compound heterozygous mutations in *PARKIN*, *PINK1*, and *DJ-1*(Farrer, 2006). This review will focus on *SNCA* which is located on chromosome 4q21-22 and encodes the 140 amino acid alpha-synuclein protein. *SNCA* has three point mutations; c.88G>C (Ala30Pro), c.188G>A (Glu46Lys) and c.209G>A (Ala53Thr) (Kruger et al., 1998, Zarranz et al., 2004), but they are very rare.

*SNCA* duplications and triplications have also been identified as a genetic cause of ADPD. Duplication has two *SNCA* copies on one allele (50% dose increase) and triplication has three, 100 percent dose increase (Figure 1). Rarely, compound heterozygote forms (two duplication alleles) are seen as *SNCA* triplication events (Ikeuchi et al., 2008). These multiplications generate higher *SNCA* expression of mRNA and protein, the so called gene

*SNCA* Gene Multiplication: A Model Mechanism of Parkinson Disease 375

Swedish-American family was also reported as a *SNCA* triplication family and the patients had DLB-type prognosis and pathological findings. Their expression level of messenger RNA and protein of alpha-synuclein was also double the dosage of normal subjects in

These findings provided two novel insights regarding the underlying mechanisms of PD; (1) over-expression of alpha-synuclein may cause a more severe form of Parkinsonism such as DLB and (2) the gene dosage of alpha-synuclein may directly correlate with the clinical features of PD. Furthermore, these findings give us hints into the development of potential therapeutic avenues of treatment for PD by decreasing the expression of alpha-synuclein. The suppression of alpha-synuclein by siRNA approaches has proven successful in decreasing the levels of LB pathology in animal models (Lewis et al., 2008, McCormack et

After the detection of *SNCA* locus triplication, *SNCA* duplications were also reported in two families from France and Italy (Chartier-Harlin et al., 2004, Ibanez et al., 2004). Chartier-Harlin et al detected one duplication family among nine ADPD. Ibanez et al detected two families with *SNCA* duplication among 119 ADPD by 250K Affymetrix microarray and semi-quantitative PCR method(Ibanez et al., 2004). The symptoms of the patients with *SNCA* duplication are milder than that of *SNCA* triplication, younger age at onset and have

Following these findings, we started screening for *SNCA* multiplications within 216 ADPD and 271 sporadic PD patients originating from Japan (Nishioka et al., 2006, Nishioka et al., 2009). We found six ADPD families and one sporadic case with *SNCA* duplication. Haplotype analysis showed these seven patients are derived from four common founders. Interestingly, the clinical manifestations of these patients were quite diverse such as sporadic PD, DLB-type and also many elderly asymptomatic carriers. The estimated penetrance ratio is about 30-40%. One patient presented with a severe clinical course with no efficacy for levodopa therapy. He progressed to Hoehn and Yahr stage V in a few years

In addition, we also detected five asymptomatic carriers over 65 years old. We therefore focused our work on the reasons why the asymptomatic carriers at later ages do not present any clinical features of parkinsonism. We started to assess Brain MRI, PET study with [18F] labeled 2-fluoro-2-deoxy-D-glucose (FDG) and [11C]-labeled 2β-carbomethoxy-3β-(4 flurophenyl)-tropane (CFT), polysomnography, and Sniffin' sticks to explore the differences between the patients and the asymptomatic carriers with *SNCA* duplication. Against our expectation, the assessments for asymptomatic carriers did not show any abnormal results in our studies, with similar results as those obtained for normal individuals (Nishioka et al., 2009). It is an area of intense research why the asymptomatic carriers do not develop Parkinsonism at these late ages and its resolution will be a key in the puzzle regarding the

Apart from our cases, other teams have reported two families from Japan with *SNCA* duplication (Ikeuchi et al., 2008, Uchiyama et al., 2008). Interestingly, one family has both heterozygote and homozygote duplication (producing a pseudo-triplication) (Ikeuchi et al., 2008). The clinical features of the individual with the *SNCA* homozygote duplication showed severe Parkinsonism similar to that of a triplication carrier. These findings also confirm the gene dosage effect within the same family. Earlier studies had reported that the

a good efficacy for levodopa therapy, similar to sporadic PD.

and at autopsy demonstrated features similar to DLB (Obi et al., 2008).

frontal cortex (Farrer et al., 2004).

al., 2010).

late-onset of PD.

dosage effect. Increasing the levels of protein appears to influence the clinical manifestations of PD patients. A subtle increase in alpha-synuclein expression may increase the risk of developing typical sporadic PD, whereas higher expression may cause severe forms of Parkinsonism similar to DLB. Pathologically, the burden of LB correlates with a PD or DLB clinical diagnosis, and it is still unclear whether PD and DLB are a continuum within the disease spectrum.

Fig. 1. A model for gene dosage of deletion, duplication and triplication

Singleton et al. first reported *SNCA* gene triplication within a large family with PD and dementia family (the Iowa kindred) (Singleton et al., 2003). The family members had been followed both clinically and pathologically by Mayo Clinic doctors for 100 years (Muenter et al., 1998, Gwinn-Hardy et al., 2000). The pattern of inheritance is autosomal dominant. The patients show a severe clinical course, early age at onset, complicating with dementia, or Parkinsonism. The pathological features are similar to DLB; (1) widespread LB pathology in the brain, (2) neuronal loss in the CA2/3, and (3) neuronal loss in the substantia nigra (Muenter et al., 1998). *SNCA* triplication was confirmed with quantitative PCR and FISH methodology. The size of the triplication region was over 2.0Mb. The expression values of messenger RNA and protein in peripheral blood and brain were twice the amount that is present in control subjects as predicted (Miller et al., 2004). Apart from the Iowan family, a

dosage effect. Increasing the levels of protein appears to influence the clinical manifestations of PD patients. A subtle increase in alpha-synuclein expression may increase the risk of developing typical sporadic PD, whereas higher expression may cause severe forms of Parkinsonism similar to DLB. Pathologically, the burden of LB correlates with a PD or DLB clinical diagnosis, and it is still unclear whether PD and DLB are a continuum within the

Fig. 1. A model for gene dosage of deletion, duplication and triplication

Singleton et al. first reported *SNCA* gene triplication within a large family with PD and dementia family (the Iowa kindred) (Singleton et al., 2003). The family members had been followed both clinically and pathologically by Mayo Clinic doctors for 100 years (Muenter et al., 1998, Gwinn-Hardy et al., 2000). The pattern of inheritance is autosomal dominant. The patients show a severe clinical course, early age at onset, complicating with dementia, or Parkinsonism. The pathological features are similar to DLB; (1) widespread LB pathology in the brain, (2) neuronal loss in the CA2/3, and (3) neuronal loss in the substantia nigra (Muenter et al., 1998). *SNCA* triplication was confirmed with quantitative PCR and FISH methodology. The size of the triplication region was over 2.0Mb. The expression values of messenger RNA and protein in peripheral blood and brain were twice the amount that is present in control subjects as predicted (Miller et al., 2004). Apart from the Iowan family, a

disease spectrum.

Swedish-American family was also reported as a *SNCA* triplication family and the patients had DLB-type prognosis and pathological findings. Their expression level of messenger RNA and protein of alpha-synuclein was also double the dosage of normal subjects in frontal cortex (Farrer et al., 2004).

These findings provided two novel insights regarding the underlying mechanisms of PD; (1) over-expression of alpha-synuclein may cause a more severe form of Parkinsonism such as DLB and (2) the gene dosage of alpha-synuclein may directly correlate with the clinical features of PD. Furthermore, these findings give us hints into the development of potential therapeutic avenues of treatment for PD by decreasing the expression of alpha-synuclein. The suppression of alpha-synuclein by siRNA approaches has proven successful in decreasing the levels of LB pathology in animal models (Lewis et al., 2008, McCormack et al., 2010).

After the detection of *SNCA* locus triplication, *SNCA* duplications were also reported in two families from France and Italy (Chartier-Harlin et al., 2004, Ibanez et al., 2004). Chartier-Harlin et al detected one duplication family among nine ADPD. Ibanez et al detected two families with *SNCA* duplication among 119 ADPD by 250K Affymetrix microarray and semi-quantitative PCR method(Ibanez et al., 2004). The symptoms of the patients with *SNCA* duplication are milder than that of *SNCA* triplication, younger age at onset and have a good efficacy for levodopa therapy, similar to sporadic PD.

Following these findings, we started screening for *SNCA* multiplications within 216 ADPD and 271 sporadic PD patients originating from Japan (Nishioka et al., 2006, Nishioka et al., 2009). We found six ADPD families and one sporadic case with *SNCA* duplication. Haplotype analysis showed these seven patients are derived from four common founders. Interestingly, the clinical manifestations of these patients were quite diverse such as sporadic PD, DLB-type and also many elderly asymptomatic carriers. The estimated penetrance ratio is about 30-40%. One patient presented with a severe clinical course with no efficacy for levodopa therapy. He progressed to Hoehn and Yahr stage V in a few years and at autopsy demonstrated features similar to DLB (Obi et al., 2008).

In addition, we also detected five asymptomatic carriers over 65 years old. We therefore focused our work on the reasons why the asymptomatic carriers at later ages do not present any clinical features of parkinsonism. We started to assess Brain MRI, PET study with [18F] labeled 2-fluoro-2-deoxy-D-glucose (FDG) and [11C]-labeled 2β-carbomethoxy-3β-(4 flurophenyl)-tropane (CFT), polysomnography, and Sniffin' sticks to explore the differences between the patients and the asymptomatic carriers with *SNCA* duplication. Against our expectation, the assessments for asymptomatic carriers did not show any abnormal results in our studies, with similar results as those obtained for normal individuals (Nishioka et al., 2009). It is an area of intense research why the asymptomatic carriers do not develop Parkinsonism at these late ages and its resolution will be a key in the puzzle regarding the late-onset of PD.

Apart from our cases, other teams have reported two families from Japan with *SNCA* duplication (Ikeuchi et al., 2008, Uchiyama et al., 2008). Interestingly, one family has both heterozygote and homozygote duplication (producing a pseudo-triplication) (Ikeuchi et al., 2008). The clinical features of the individual with the *SNCA* homozygote duplication showed severe Parkinsonism similar to that of a triplication carrier. These findings also confirm the gene dosage effect within the same family. Earlier studies had reported that the

*SNCA* Gene Multiplication: A Model Mechanism of Parkinson Disease 377

**The number of screening samples**

6 487 1.2 48.2

4 286 1.4 43.6

1 57

1 60

1 71

1 487 0.2 31

1 37

1 31

1 28

France 1 9 11 50.8

Germany 1 403 0.3 36

**Frequency (%)** 

 **Average ot age at onset** 

**of pedigrees**

Sironi et al. 2009 Italy 1 144 0.7 41

Ahn et al. 2007 Korea 1 906 0.1 40

total average 51.5

Nuytemans et al.2009 Belgium 1 219 0.5 71

Troiano et al. 2008 France 1 101 1 35 Ahn et al. 2007 Korea 2 906 0.2 57.5 total average 46.1

Ibanez et al. 2009 France 1 286 0.3 42

1

Singlton et al. 2003 Iowa 1 33.2

total average 32.9 Table 1. The clinical manifestations and prevalence of *SNCA* duplication and triplication

**Country The number**

Japan (Juntendo)

France and Italy

(Niigata)

(Niigata)

(Lister complex)

(Juntendo)

(Juntendo)

(Lister complex)

American

(Niigata)

**SNCA duplication in** 

Nishioka et al. 2006

Ibanez et al. 2004 and

Chartier-Harliln et al.

**SNCA duplication in sporadic cases** 

Brueggemann et al.

**SNCA triplication**

**SNCA homozygote duplication** 

Nishioka et al. 2009 Japan

Sekine et al. 2010 Japan

Fuchs et al. 2007 Sweden

Farrer et al. 2004 Swedish-

Ikeuchi et al. 2008 Japan

Ikeuchi et al. 2008 Japan

Uchiyama et al. 2008 Japan

Fuchs et al. 2007 Sweden

**AD cases** 

and 2009

2009

2004

2008

Swedish family named the "Lister family complex" has both *SNCA* duplication and triplication patients within different branches of the pedigree suggesting a primary duplication event followed later by another resulting in the triplication (Fuchs et al., 2007). In this family also the patient with *SNCA* triplication presented with more severe symptoms than the patients with duplication. Recently one small pedigree with SNCA triplication was detected in Japan (Sekine et al., 2010).

The breakpoint of *SNCA* multiplication is different in each family. The largest multiplication about 4.9Mb is detected within a French family. The smallest one about 0.2 Mb is in a Japanese family (Nishioka et al., 2009). The size and gene make-up of each multiplication region does not seem to influence the clinical presentation of the carrier. The single common determining factor that appears between all patients with *SNCA* multiplication is the presence of the entire *SNCA* gene. To conclude, it is clear that *SNCA* multiplication alone is sufficient to result in the parkinsonian phenotype.
