Proteolipid protein 1 gene sequencing of hereditary spastic paraplegia.

PCR amplification and sequencing of whole blood DNA from an individual with hereditary spastic paraplegia, as well as family members, revealed a fragment of proteolipid protein 1 (PLP1) gene exon 1, which excluded the possibility of isomer 1 expression for this family. The fragment sequence of exon 3 and exon 5 was consistent with the proteolipid protein 1 sequence at NCBI. In the proband samples, a PLP1 point mutation in exon 4 was detected at the basic group of position 844, T→C, phenylalanine→leucine. In proband samples from a male cousin, the basic group at position 844 was C, but gene sequencing signals revealed mixed signals of T and C, indicating possible mutation at this locus. Results demonstrated that changes in PLP1 exon 4 amino acids were associated with onset of hereditary spastic paraplegia.

INTRODUCTION

Hereditary spastic paraplegia (HSP) is a hereditary disease of the nervous system with clinical and genetic heterogeneity, which is primarily manifested by progressively decreased extremity weakness and spastic paraplegia. A total of 46 pathogenic gene loci have been identified today and are termed SPG1-SPG46; in addition, 20 HSP pathogenic genes have been cloned[12]. SPG2 is X-linked recessive and hereditary, and the pathogenic gene is at Xq21-22 encoding proteolipid protein 1 (PLP1). Kobayashi et al[3] detected an isoleucine-to-threonine (T) mutation at position 186 in the PLP gene, which was termed SPG2. No specific treatment has been discovered for HSP to date[4]. Long-term follow-up and early diagnosis for high-risk positive families is crucial for increasing the entire quality of one family population[5]. Prenatal diagnosis could help to prevent birth of infants with hereditary disease. Therefore, rapid and simple gene sequencing could provide reliable and early diagnosis for the treatment of HSP.

The present study detected gene defects in an individual with hereditary spastic paraplegia, as well as family members, to provide evidence for gene diagnosis.

RESULTS

Quantitative analysis of participants and gene mapping

One HSP family, comprising 10 individuals from Yanbian, Jilin Province, China, was enrolled in the present study. HSP had been transmitted to two generations of this family. All subjects were included in the final analysis, and the family map is shown in Figure 1.

Figure 1

Family map of patient with hereditary spastic paraplegia.

Subject numbers 1–10, aged 52, 55, 50, 46, 29, 42, 58, 56, 7, and 25 years, respectively.

Acquisition of PLP1 exon 1, exon 3, exon 4, and exon 5 fragments

PLP1 gene exon 1–5 fragments were amplified. Using extracted blood genome as the template, sense and anti-sense primers of PLP1 gene exon 1, exon 3, exon 4, and exon 5 fragments were amplified. The predicted PCR product size for exon 1 was 486 bp, exon 3 was 493 bp, exon 4 was 345 bp, and exon 5 was 541 bp. The bands were observed and detected by 0.8% agarose gel electrophoresis (Figures 2, 3).

Figure 2

Electrophoretic analysis of proteolipid protein 1 gene fragment PCR amplification.

M: Marker (DL 2000); lanes 1–4: proband exon 1, 3, 4, 5 gene fragments (molecular weight 486, 493, 493, 541 bp, respectively).

Figure 3

Expression of PCR product and T vector plasmid extract (exon 1 PCR product and T vector plasmid ligation product).

M: Marker, band with maximum expression was 2 500 bp (plasmid: ring-shaped; marker: line-shaped; electrophoresis rate was different);

Lanes 1–2: proband exon 1 PCR product (486 bp) and T vector plasmid extract;

Lanes 3–4: proband exon 5 PCR product (541 bp) and T vector plasmid extract.

Sequencing of PLP1 exon 1, exon 3, exon 4, and exon 5 fragments

PCR products and vector plasmids were ligated, followed by PCR amplification. Exon 1 was amplified and effective fragment was obtained; exon 3 and 5 fragment sequences were consistent with the PLP1 sequence in NCBI. In exon 4, a T-to-C mutation was detected at position 844 (sample number 10, Figure 1), which resulted in a phenylalanine-to-leucine mutation. The basic group at position 844 was C in sample number 5 (Figure 1), but gene sequencing revealed mixed signals of T and C, indicating a possible mutation at this locus (Figure 4).

Figure 4

Sequencing of proteolipid protein 1 (PLP1) gene exon 4 fragment.

(A–C) Sequencing of PLP1 gene exon 4 fragment from subject number 10 (proband), number 5 (male cousin of proband), and normal subject in Figure 1.

(D) Amino acid alignment results of PLP1 gene exon 4 expression products; black represents the same sequence, and the center represents the variant amino acid.

Plp.pro: normal exon4 amino acid sequence; Plpv.pro: amino acid sequence from subject number 10 sample exon 4; consensus: consensus sequence. Arrows: Variant basic group.

DISCUSSION

PCR methodology, in combination with recombinant Escherichia coli-transfected DNA, has been developed to rapidly and accurately detect genes involved in hereditary diseases. The pathogenic gene for Pelizaeus Merzbacher disease was located at Xq22.2 PLP1 gene[6]. The PLP1 gene is 17 kb, contains seven exons, and encodes the PLP1 protein, which comprises 276 amino acids and the splicing isomer DM20. PLP1 is a major component of the myelin sheath in the central nervous system, accounting for 50% of the entire myelin protein[7]. PLP1 stabilizes the myelin sheath and plays a role in the generation of oligodendrocyte precursor cells[8]. PLP1 gene deficiency results in PLP1 protein overexpression (PLP1 gene duplication mutation) or reduction (PLP1 gene point mutation), which induces abnormal myelination and/or oligodendrocyte death, further leading to myelin sheath deficiency or reduced white matter. The PLP1 gene is associated with SPG2 and encodes PLP protein, which is an endogenous myelin protein. The PLP1 gene promotes myelin sheath maturation and helps to maintain membrane structure. Following a PLP gene mutation, PLP expression is abnormal or absent, which affects neurite myelin sheath maturation and leads to neurite swelling and degeneration[910]. In the present study, single-nucleotide polymorphisms were detected in Genecards (http://www.genecards.org/), and known single-nucleotide polymorphisms were distributed in exon 1, 3, 4, and 5, which were subsequently amplified.

PCR amplification of PLP1 gene exon 1, exon 3, exon 4, and exon 5 revealed a T-to-C mutation in position 844 in proband exon 4 of sample number 10; the 282 amino acid codon resulted in a UUC-to-CUC mutation, i.e., phenylalanine to leucine. Amino acid changes have been shown to result in axonal degeneration and demyelination of the corticospinal tract[11]. Five pathogenic point mutations were detected, including His139Tyr, Trp144Term, Ser169Phe, Ile186Thr, and Phe236Ser. The mutation locus in the present study has not been previously reported or published in a database. Gene detections for three generations of one HSP family, comprising 10 family members, revealed a novel mutation locus, which provides significant information for screening and diagnosis of HSP genes.

SUBJECTS AND METHODS

Design

A hereditary gene mutation study.

Time and setting

The study was performed at the Laboratory of Molecular Biology, Heping Campus Jilin University, China from July 2008 to January 2009.

Subjects

There were three HSP patients in the enrolled family. The patients from the third generation were aged 25–36 years. The proband male, aged 25 years, was admitted to China-Japan Union Hospital of Jilin University in March 12, 2008 due to lower limb weakness and stiffness, which had been present for over 18 years, but had become aggravated over the past 4 years. The patient exhibited an abnormal gait since three years of age. The patient also exhibited difficulties in walking and climbing, and was not willing to participate in sports activities. The condition gradually worsened and calcium deficiency was considered following three separate diagnoses from Yanbian Hospital, Jilin Province, China. The patient was subsequently treated with calcium supplementation. However, symptoms were not ameliorated. Diagnosis was performed according to HPS diagnosis standards proposed by Harding in 1983[12]. Informed consent was obtained from all participants according to Administrative Regulations on Medical Institution, issued by the State Council of the People's Republic of China[13].

Nervous system examination

The patient exhibited an abnormal gait, clear consciousness, and had clear language. Cranial nerve examination was normal, but muscular tension in both lower extremities was high. Muscular strength was normal, with normal, deep, and superficial sensations. In addition, the patient performed well in the finger-nose test and heel-knee- tibia test, which was accompanied by active bilateral biceps brachii reflex, bilateral patella tendon hyperreflexia, patellar clonus, ankle clonus, and Babinski signs.

Family history

The grandfather and male cousin of the proband also exhibited similar symptoms, so the proband's condition was preliminarily considered to be X-linked recessive inheritance.

Auxiliary examination

Head and cervical MRI were normal, as was the electromyogram of the lower limbs.

Family survey

In Figure 1, subject 5 suffered from disease onset at age 10, which was manifested by difficult walking, stiff lower limbs, and an abnormal gait. He was diagnosed with spastic paraplegia by several hospitals. The grandfather of subject 10 suffered from disease onset at > 20 years of age and presented with walking difficulties and an abnormal gait; the patient was bed-ridden for 4–5 years, and died at 40 years of age. Subjects 1, 2, 3, 4, 6, 7, 8, and 9 did not present with any abnormalities.

Ten blood samples were collected from the family (two patients and eight normal subjects). Blood samples from subjects 1–10 were collected and anti-coagulated.

Methods

Whole genomic DNA extraction from subjects 1–10

Whole genomic DNA was extracted using the TIANamp Genomic DNA Mini-prep Kit (Tiangen, Beijing, China). Extracted DNA samples were analyzed using 0.8% agarose gel electrophoresis[1415].

Location and acquisition of the SPG2 exon sequence

According to clinical symptoms, mode of inheritance, and data from Online Mendelian Inheritance in Man (http://www.ncbi.nlm.nih.gov), the chromosome was localized at Xq21-22 and the pathogenic gene was determined to be SPG2, i.e., PLP1. The human PLP1 gene sequence was searched for in GenBank (http://www.ncbi.nlm.nih.gov/).

Primer design

SNP detection in Genecards (http://www.genecards.org/) showed that published SNPs were distributed in exon 1, 3, 4, and 5. Therefore, these four fragments were subsequently amplified. The clone primer sequence was designed using Premier 5.0 program (www.PremierBioft.com).

Real-time PCR amplification of PLP1 exon 1, exon 3, exon 4, and exon 5 target genes

Exon 1, 3, 4, and 5 upstream and downstream primers were used for PCR amplification to obtain target genes. The thermocycler was purchased from Shanghai Scientific Instrument, Shanghai, China (process described in supplementary information online).

Reaction system was 25 μL in total under the following cycle conditions: 94°C denaturation for 5 minutes; 94°C for 30 seconds; 52°C annealing for 30 seconds except PLP1 exon 3 target gene primers at 57°C; 72°C for 30 seconds, 30 cycles in total, followed by extension at 72°C for 8 minutes. The PCR products were identified by 0.8% agarose gel electrophoresis, were observed by an ultraviolet lamp, and were stored at 4°C[16].

Reaction system of PCR amplification for the PLP1 target gene (25 μL total): 2 μL human genomic DNA, 1 μL sense primer, 1 μL anti-sense primer, 2 μL dNTP mixture, 2.5 μL 10× Ex Tabuffer, 16 μL ddH2O, and 0.5 μL Ex Taq DNA polymerase (a heat-resistant enzyme).

Vector and plasmid linkage system: 2 μL retrieval product, 5 μL solution I, 0.5 μL pMD18-T Simple Vector, 2.5 μL ddH2O; the reaction was mixed and incubated in a water bath at 16°C for 4 hours.

PCR ligation product transformation

The ligation product was mixed with Escherichia coli (DH5α) competent cells (prepared by our laboratory)[171819], placed in an ice bath for 30 minutes, followed by a water bath at 42°C for 90 seconds, and cooled immediately in an ice-bath for 2 minutes. Then, 600 μL Luria-Bertani medium was added to the mixture, which was shaken at 200 r/min at 37°C for 45 minutes. The product was evenly spread on LB-Amp (50 mg/L) agar plates and dried. The flat plates were inversed and incubated at 37°C for 12–16 hours. Single colony growth was observed.

T vector plasmid extraction

Under sterile conditions, 40 µL colony containing T vector plasmid was seeded in 4 mL LB liquid medium containing 50 μg/mL kanamycin/ampicillin and was shaken overnight at 37°C[20], followed by centrifugation in a microcentrifuge to precipitate bacteria and disperse the solution in viscous bacterium lysate. The product was mixed with equal amount of phenol/chloroform, and the supernatant was transferred to a new centrifuge tube and mixed with absolute alcohol at room temperature to precipitate DNA. The sample was shaken, rinsed, mixed with pancreatic RNase free of DNase (20 μg/mL), shaken, incubated, and stored at −20°C[21].

Sequencing of PLP1 exon 1, exon 3, exon 4, exon 5

The extracted plasmids were sequenced by Shanghai Sangon Biotech, China. Sequencing result alignment was performed using DNAMAN Version 5.2 software (www.Lynnonbiosoft.com) for nucleic acid and protein sequence alignments.