PARK1 gene mutation of autosomal dominant Parkinson’s disease family＊☆

Ligang Jiang , Qiuhui Chen Ying Zhang Xinyu Hu Jia Fan Lifeng Liu, Rui Guo Yajuan Sun Yizhi Zhang Guohua Hu

1Department of Neurology, Second Hospital of Jilin University, Changchun 130041, Jilin Province, China

2Department of Neurology, Central Hospital of Jilin, Jilin 132011, Jilin Province, China

3Beihua University, Jilin 132011, Jilin Province, China

lNTRODUCTlON

Parkinson’s disease (PD) is currently poorly understood with regards to its etiology and pathogenesis, but it is shown to be associated with many factors, such as genetic factors, environmental factors, and aging of the nervous system[1-10].

There are 13 chromosomal loci thought to be involved in PD via a Mendelian inheritance pattern, named PARK1-PARK13 gene. Seven of these show autosomal dominant inheritance, four autosomal recessive, one X chromosome-linked inheritance, and one related to late-onset PD[11-16]. PARK1 gene has four exons, and two point mutations (Ala30Pro and Ala53Thr) have emerged, which are located on exons 3 and 4, respectively[17-21].

Previous studies pertaining to familial inheritance have revealed that the gene is a potential pathogenic gene for familial PD; a type of PD that is characterized by early onset age, high penetrance, and is autosomal dominant[22-23]. The PARK1 gene mutation is rare in PD patients; accounting for less than 1%. The family members carrying this gene are located in the Mediterranean region, such as Italy and Greece, as well as in other parts of Europe,such as Germany[24-25]. Little evidence is available in China regarding this mutation locus in PD patients.

This study investigated the inheritable form of PD. A total of 33 members of a family of four generations were enrolled, and a survey map was drawn. Eleven of them were selected as experimental subjects for the collection of peripheral blood and extraction of genomic DNA. The PARK1 gene exon 4 (PARK1-E4) sequence was amplified by PCR. The genetic material was sequenced and compared to find the genetic loci of the pathogenic genes for PD.

RESULTS

Drawing a survey map

There were 33 members of four generations of the PD family showing autosomal dominant inheritance. The genetic characteristics of family members were closely investigated to identify and draw the pedigree (Figure 1).

Eleven blood samples were collected from four affected cases (301, 302, 303, 304),five suspected cases (305, 306, 401, 403,405), and two non-affected cases at the fourth generation (402, 404) for the final analysis.

Figure 1 Pedigree of a Parkinson’s disease family.

PARK1-E4 gene amplification

The PARK1-E4 gene was obtained through PCR amplification. Genomic DNA extracted from 11 samples of peripheral blood served as a template. The PARK1-E4 gene-specific primers were designed for PCR reaction and amplification (Figure 2). All samples were amplified to obtain a gene fragment of 143 bp.

Figure 2 PARK1-E4 amplification products as detected by 1% agarose gel electrophoresis. 301–306, 401–405:PCR product of PARK1-E4 amplification in samples of 310–306, 401–405 members in Parkinson’s disease family taking Genomic DNA as a template; M: DNA Marker DL 2000.

Sequencing PCR products of PARK1-E4 gene

In order to clarify the PARK1-E4 gene sequences,PCR products of the amplified PARK1-E4 were sequenced. The T peak was not visible at the 166 base site(Figure 3).

Figure 3 Sequencing of PARK1-E4 gene amplification products.

PARK1-E4 gene mutation site analysis

To analyze the PARK1-E4 gene mutation location in 11 samples, the PARK1-E4 gene sequence was compared using the BioEdit software, taking the human PARK1-E4 gene sequence in GeneBank as a template. The results show that only a T base deletion mutation was found in the PARK1-E4 gene sequence from the 11 samples.

There was no mutation site in 11 samples. The deleted T base has a white mark in Figure 4.

Figure 4 PARK1-E4 gene mutation analysis. The PARK1 E4 sequence used as template was original from Genebank. The white part represented the deleted T base in 11 samples.

DlSCUSSlON

This study summarized the genetic characteristics and gene mutations in a rare PD family, the afflicted members of which exhibited early onset of PD with slow disease progression. Clinical manifestations included resting tremors, rigidity, bradykinesia. Levodopa treatment was effective, and the diagnosis was consistent with the diagnostic criteria of familial PD.

The inheritance pattern was defined as autosomal dominant inheritance, based on the following characteristics. (1) The disease occurred irrespective of gender. (2)The age of onset and clinical manifestations were similar in all patients. (3) Four generations of individuals were affected by a continuous disease, but no atavism was observed. (4) A few cases in the fourth generation were diagnosed with PD. The reason for the low incidence of PD in the fourth generation might be that they were too young to fall ill. The detailed clinical data of the family members was sorted, and the genetic maps revealed a clear genetic relationship of autosomal dominant PD in most of the affected cases.

Ten to fifteen percent of the PD patients are reported to have a family history. The present experiment for the first time confirmed that PARK1 gene mutations also occur in Asians, rather than only in European populations. The familial PD patients in Italy, Greece, and Germany that were described in the 1990s were characterized by an early onset age of the illness, shorter duration, obvious dementia, and not very typical clinical symptoms[26-28].

The present study shows that a family with 4 generations of PD varied from the above mentioned studies in several ways. (1) There was a longer duration of the disease;longer than 9 years, and a member of the family had it for 30 years and is currently alive at the age of 82 years. (2)None of the patients appeared to have any obvious mental retardation, and an 82-year-old patient had no dementia. (3) The diagnosed cases in this family exhibited typical PD clinical signs with remarkable extrapyramidal symptoms.

In this study, the point mutation in PARK1-E4 in 11 members of the family were detected. The results demonstrated a base deletion on exon 4 in 11 of the patient samples. The base deletion was located in the middle of PARK1-E4, which possibly leads to a complete change of the subsequent amino acid sequence or early termination of translation. This suggests that the mutation may cause the disease onset in most of the afflicted family members[26]. Due to the limited experimental conditions and time, the mutation on exon 3 of the PARK1 gene was not compared to that on exon 4. Such a comparison will need to be conducted in future studies.

In conclusion, the mutation on exon 4 of the PARK1 gene is an important genetic factor for familial PD.

SUBJECTS AND METHODS

Design

Genetic analysis.

Time and setting

The experiments were conducted in the Parkinson’s Disease Research Room of the Second Hospital of Jilin University, from October 2009 to March 2010.

Subjects

The family ancestry of Han nationality was from Tonghua City in Jilin Province, China. There were a total of 33 members of four generations. Thirteen members were affected (four cases died) and six suspicious cases showed autosomal dominant inheritance. PD clinical diagnosis was in accordance with Brain Bank Clinical Diagnostic Criteria of UK PD Society[29-30]. The age of onset of the family members was from 33 to 53 years,with typical PD symptoms and similar clinical manifestations, such as tremors, slowness and muscle rigidity,abnormal gait and posture, different levels of PD non-motor symptoms, and no obvious dementia. Oral levodopa treatment was effective, but the symptoms gradually worsened. According to the Administrative Regulations on Medical Institution, issued by the State Council of the People’s Republic of China[31], the patients were informed of the experimental program and the risks prior to the experiment. All individuals signed informed consent.

Members of this family were inquired of their history,and underwent physical examinations of their nervous system, routine laboratory examination and head MRI.

Intelligence scale changes were detected by Mini-Mental State Exam (MMSE), and clinical characteristics of some of members were summarized (Table 1).

Table 1 Patient demographics and traumatic brain injury measurements

Ulnar venous blood (5–10 mL) collected from 11 cases was anticoagulated with 3.8% sodium citrate. Genomic DNA was extracted using a conventional phenol/chloroform extraction method, and stored at –20 °C(supplementary Table 1 online).

Methods

Genomic DNA extraction

Fresh blood samples collected from 11 cases were placed into 1.5 mL EP tubes, then fully shaken with 3 times the volume of lysis buffer (0.25 mL whole blood plus 0.75 mL lysate). The homogenized samples were placed in 15–30 °C for 5 minutes, thus the nucleic acid protein complex was completely separated. Centrifugation was done at 4 °C, at 12 000 r/min for 10 minutes.

Then the supernatant was transferred into a new EP tube.

Lysis buffer (1 mL) was added with 0.4 mL chloroform,shaken for 15 seconds and placed at room temperature for 3 minutes. Centrifugation was done at 4 °C, at 12 000 r/min for 10–15 minutes. Genomic DNA was located mainly in the aqueous phase, and about 500 μL was transferred to a new tube. The prepared aqueous solution was added with an equal volume of isopropanol,mixed and placed at room temperature for 20–30 minutes. Centrifugation was at 4 °C at 12 000 r/min for 10 minutes; the supernatant was discarded. The samples were rinsed and precipitated with 1 mL 75%ethanol. Centrifugation was at 4 °C at 5 000 r/min for 3 minutes. The solution was poured and dried at room temperature (for about 2–3 minutes because the completely dried DNA is difficult to dissolve). The samples were repeatedly triturated and mixed by adding 30–100 μL ddH2O. DNA was fully dissolved for the detection of DNA content. DNA quality and concentration were detected by UV spectrophotometer. The ratio of A260nm/280nmat 1.8–2.0 was measured, and DNA concentration was calculated (DNA sample concentration (μg/μL) = A260nm×dilution times × 50/1 000).

PCR amplification and sequencing

Primer design: PCR amplified products were synthesized by Shanghai Sagon Company (Shanghai, China).

Primer name and sequence are as follows:

Primer name Sequence (5'–3') Product size (bp)PARK1-E4 Forward: GCA GTG GCA CAA TCT TGA C Reverse: CTT TAG GGT AGT GAT GGG AGT TAG 216

PCR reactions are as follows:

10 × PCR buffer contained Tris-HCL (pH 8.3) 100 mmol/L, KCl 500 mmol/L, MgCl2 15 mmol/L.

PCR products were amplified as follows: 94 °C denaturation for 5 minutes; 30 cycles of 94 °C denaturation for 30 seconds, 55 °C renaturation for 30 seconds, 72 °C extension for 40 seconds; 72 °C extension for additional 6 minutes. PCR products were analyzed by 2% agarose gel electrophoresis, at 110 volts for 20 minutes, then the images were collected. PARK1-E4 was determined for the PCR amplification product sequencing and gene mutation site analysis. The sequencing was repeated twice by Beijing SINO (Beijing, China) and the results were consistent.

Author contributions:Ligang Jiang was responsible for the pedigree investigation and experimental operation, providing and integrating the data, conducting data analysis, and drafting the manuscript. Qiuhui Chen, Ying Zhang, Jia Fan, Xinyu Hu,Yajuan Sun, Yizhi Zhang, and Lifeng Liu provided information and instructed the clinical works. Guohua Hu proposed and designed the study and experiment, and approved the study.

Conflicts of interest:None declared.

Funding:This study was financially sponsored by a Foundation of Science and Technology Department of Jilin Province, No.200905152.

Ethical approval:The experiment was approved by the Ethics Committee of Jilin University in China.

Supplementary information:Supplementary data associated with this article can be found, in the online version, by visiting www.nrronline.org, and entering Vol. 6, No. 5, 2011 after selecting the “NRR Current Issue” button on the page.

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