Establishing Assays for Detecting SMNT Gene Mutation in Single Cell Using Nested-PCR Method

The worldwide incidence of SMA is 1/10,000 live births and the incidence of disease carriers ranges from 1/40 to 1/60 [1-3]. According to the current international classification, SMA is divided into three categories based on age of disease onset and severity of disease: Severe, intermediate and juvenile form of SMA type I, II, and III. SMN gene including exon 9 encodes the SMN protein molecules 294 amino acids in length. The SMN gene has two copies; SMNt (SMN 1) and SMNc (SMN 2) differ only in 5 base pairs: one in intron 6, one in exon 7, two in exon 7 and one in exon 8. Differences in exon 7 and exon 8 were used to distinguish between SMNt and SMNc in SMA diagnosis (Figure 1). Differ in one nucleotide of exon 7 between SMNt and SMNc, making SMNt synthesize molecules enough length SMN protein and functional, while protein synthesis by SMNc have very limited functionality.


Introduction
Spinal muscular atrophy (SMA) is a severe neurodegenerative autosomal recessive mutation. SMA is characterized by the progressive degeneration of spinal anterior horn cells lead to muscle weakness symmetrical stem limbs, muscle tone and tendon reflex is lost or reduced, chest deformity and stiffness. The worldwide incidence of SMA is 1 / 10,000 live births and the incidence of disease carriers ranges from 1/40-1/60 [1][2][3]. According to the current international classification, SMA is divided into three categories based on age of disease onset and severity of disease: Severe, intermediate and juvenile form of SMA type I, II, and III [8]3. SMN gene including exon 9 encodes the SMN protein molecules 294 amino acids in length. The SMN gene has two copies, SMNT (SMN1) and SMNc (SMN2) (Figure 1) differ only in 5 base pairs: one in intron 6, one in exon 7, two in exon 7 and one in exon 8. Differences in exon 7 and exon 8 were used to distinguish between SMNT and SMNc in SMA diagnosis [4]. Differ in one nucleotide of exon 7 between SMNT and SMNc, making SMNT synthesize molecules enough length SMN protein and functional, while protein synthesis by SMNc have very limited functionality. There are three types of SMN gene mutations that cause SMA disease:

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Treating for these patients creates a burden to both economically and morally for the family and society. Therefore, genetic diagnosis before embryo transfer (preimplantation genetic diagnosis-PGD) SMA families who have a profile with SMA to select healthy embryos transferred to the uterus for implantation mother, from which was born Healthy babies are important and important [10,11]. As the result, we started researching the project: "Establishing assays for detecting SMNt gene mutation in single cell using nested-PCR method". With target: Developed a procedure to identify SMNt gene mutations that cause muscular atrophy from a white blood cell, separated from the peripheral blood of the patient's family.

Research Objects
Pick out 4 families with children aged 0 to 18 years to be examined and treated at Vietnam National Children's Hospital, was diagnosed with spinal muscular atrophy due to loss of exon 7 homozygous gene SMNT.

Separating Leukocytes from Peripheral Blood Samples:
Leukocyte extraction from whole blood according to the Ficollpaque protocol. Dilute the white blood cell with 1x PBS solution, pick up a white blood cell on a microscope and place it on a 0.2 ml PCR tube. After that, the cell lysis was 5μL KOH 0.2M, annealed 65°C for 10 minutes, neutralize KOH with 5μl tricine 0.2 M.

Results and Discussion
In Figure 2, exon 7 PCR products were not incubated with DraI (Ko DraI) enzyme, and exon 8 was not incubated with DdeI (Ko DdeI) enzyme from Patient C3's father (B3), patient C3's mother (M3), C3 patients are the same, with 188bp -product of exon 7 and 190bp -product exon 8. But when incubated with the enzyme DraI PCR products that: a) B3 and M3 both have 188bp and 164bp respectively, meaning both B3 and C3 have exon 7 SMNT (uncutted-188bp), exon 7 SMNc is cut into two pieces (164bp and 24bp). b) Meanwhile the only one C3 has 164bp -loss homozygous exon 7 SMNt, only exon 7 SMNc was cutted by restriction enzymes into two band (164bp, 24bp). This result is consistent with PCR conclusions from the whole sample of Vietnam National Children's Hospital.
Thus, patients with C3 loss have co-exon 7 and 8 SMNT genes. This illustrate, we were successful in amplified SMN gene from a human leukemia cells and incubated in step restriction enzymes. However, because of the 2% agarose gel electrophoresis results, the two bands of exon 7 SMNc cutted by the enzyme DraI in sample B3, C3 were not clearly separated. In Figure 3, both father (B7) and mother (M7) of the C7 patient have a SMNt exon 7 gene. Therefore, when incubating the PCR products with restriction enzyme DraI have two-stranded (180bp in exon 7 SMNt and 164bp in exon 7 SMNc), similar with exon 8 of the SMN gene. Thus, patients also lose C7 exon 7 and exon homozygous gene 8 SMNT. Conducting research on other patient families, we obtained the following results: All 4 patients with spinal muscular atrophy research are lost exon 7 SMNt. This result is consistent with the genetic diagnosis SMNt from whole blood samples of Vietnam National Children's Hospital as well as the research of other authors [7][8][9]. Also, all parents of patients have the exon 7.8 SMNt there and in fact, the parents are non-SMA expression (Table 1). That is, just one chromosome in the pair of homologous 5 gene SMNT normal (heterozygous) can translate enough protein SMN (survival motor neuron) for nerve cells mobilize normal operation.

Conclusion and Recommendations
From the results obtained in the study, we can confirm that the first step we have succeeded in building processes identified gene mutation that causes SMA SMNT from a cell.