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The trend in distribution of Q223R mutation of leptin receptor gene in amoebic liver abscess patients from North India: a prospective study.

Verma AK, Ahuja V, Paul J - Biomed Res Int (2014)

Bottom Line: Host genetic susceptibility is an important risk factor in infectious diseases.The frequency of allele "G" (coding for arginine) was in general high in Indian population irrespective of the disease.Our results of Fisher exact test shows that heterozygous mutant (QQ versus QR, P=0.049) and homozygous mutant (QQ versus RR, P=0.004) were significantly associated with amoebic liver abscess when compared with homozygous wild (QQ).

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.

ABSTRACT
Host genetic susceptibility is an important risk factor in infectious diseases. We explored the distribution of Q223R mutation in leptin receptor gene of amoebic liver abscess (ALA) patients of North India. A total of 55 ALA samples along with 102 controls were subjected to PCR-RFLP analysis. The frequency of allele "G" (coding for arginine) was in general high in Indian population irrespective of the disease. Our results of Fisher exact test shows that heterozygous mutant (QQ versus QR, P=0.049) and homozygous mutant (QQ versus RR, P=0.004) were significantly associated with amoebic liver abscess when compared with homozygous wild (QQ).

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(a) PCR-RFLP analysis of Q223R SNP (rs1137101) from leptin receptor gene. Lane M = 100 bp Marker, lane AG, lane GG, and lane AA represent restriction enzyme digested PCR product of ALA pus DNA of different genotypes. After digestion with BseNI restriction enzyme, wild type AA is visible as two bands 221 bp, 146 bp and the third band of 19 bp is invisible due to smaller size. Heterozygous AG is visible as three bands 367 bp, 221 bp, 146 bp and 19 bp (invisible) whereas homozygous mutant GG is visible as bands of 221 bp, 146 bp and 19 bp (invisible). (b) Sequencing results of wild “A” and mutant “G” are detected by PCR-RFLP confirming the presence of SNP in DNA of ALA patients.
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fig2: (a) PCR-RFLP analysis of Q223R SNP (rs1137101) from leptin receptor gene. Lane M = 100 bp Marker, lane AG, lane GG, and lane AA represent restriction enzyme digested PCR product of ALA pus DNA of different genotypes. After digestion with BseNI restriction enzyme, wild type AA is visible as two bands 221 bp, 146 bp and the third band of 19 bp is invisible due to smaller size. Heterozygous AG is visible as three bands 367 bp, 221 bp, 146 bp and 19 bp (invisible) whereas homozygous mutant GG is visible as bands of 221 bp, 146 bp and 19 bp (invisible). (b) Sequencing results of wild “A” and mutant “G” are detected by PCR-RFLP confirming the presence of SNP in DNA of ALA patients.

Mentions: A total of 55 ALA samples along with 102 controls were subjected to PCR-RFLP analysis. DNA was amplified using leptin receptor specific primers. PCR was performed in a touch gene (Nugen Scientific, USA) machine. Thin walled 0.2 mL tubes were used for amplification. A typical PCR reaction (20 μL) included 7.8 μL of autoclaved milliQ water, 2 μL of 10X PCR buffer with MgCl2 (containing 750 mM Tris-HCl (pH 8.8 at 25°C), 200 mM (NH4)2SO4, 0.1% Tween-20, 1.5 mM MgCl2), 2 μL of dNTP mix (containing 2 mM of each dNTP), 2 μL (20 pmol) of each primer forward as well as reverse, and 0.2 μL of TaqDNA polymerase (5 U/μL, MBI Fermentas, USA) and 2.0 μL of template DNA. The amplification conditions were one cycle of 94°C for 5 min followed by 30 cycles of 94°C for 30 s, annealing 55°C for 1 min, extension at 72°C for 30 sec, and finally one cycle of 72°C for 10 min and finally held at to 4°C. Volume of template DNA used (2.0 μL; ~50 ng) worked fine for PCR amplification. The sample containing all reagents except the template DNA was treated as the negative control. The size and integrity of the products were checked by electrophoresis. 10 μL of the PCR product was run on a 0.8–1.2% agarose gel at 5 V/cm for an appropriate time period. Restriction enzyme BseNI was used to digest the PCR amplified product of 386 bp and the fragments generated upon digestion are represented in Figure 1. Restriction enzyme BseNI digests only when the sequence reads nucleotide A at the locus. Thus digestion of 386 bp PCR product yielded three bands of 221 + 146 + 19 bp in case of homozygous (AA, assuming A as wild allele) wild and two bands of 367 + 19 bp in case of homozygous mutant (GG) (Figure 1). As expected, the digestion of heterozygous mutant yielded four bands of 367 + 221 + 146 + 19 bp as shown in a representative gel. All bands except 19 bp were visible on 1.5% agarose gel. Sequencing of mutated fragment confirmed the presence of mutation in Indian population (Figures 2(a) and 2(b)).


The trend in distribution of Q223R mutation of leptin receptor gene in amoebic liver abscess patients from North India: a prospective study.

Verma AK, Ahuja V, Paul J - Biomed Res Int (2014)

(a) PCR-RFLP analysis of Q223R SNP (rs1137101) from leptin receptor gene. Lane M = 100 bp Marker, lane AG, lane GG, and lane AA represent restriction enzyme digested PCR product of ALA pus DNA of different genotypes. After digestion with BseNI restriction enzyme, wild type AA is visible as two bands 221 bp, 146 bp and the third band of 19 bp is invisible due to smaller size. Heterozygous AG is visible as three bands 367 bp, 221 bp, 146 bp and 19 bp (invisible) whereas homozygous mutant GG is visible as bands of 221 bp, 146 bp and 19 bp (invisible). (b) Sequencing results of wild “A” and mutant “G” are detected by PCR-RFLP confirming the presence of SNP in DNA of ALA patients.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4121093&req=5

fig2: (a) PCR-RFLP analysis of Q223R SNP (rs1137101) from leptin receptor gene. Lane M = 100 bp Marker, lane AG, lane GG, and lane AA represent restriction enzyme digested PCR product of ALA pus DNA of different genotypes. After digestion with BseNI restriction enzyme, wild type AA is visible as two bands 221 bp, 146 bp and the third band of 19 bp is invisible due to smaller size. Heterozygous AG is visible as three bands 367 bp, 221 bp, 146 bp and 19 bp (invisible) whereas homozygous mutant GG is visible as bands of 221 bp, 146 bp and 19 bp (invisible). (b) Sequencing results of wild “A” and mutant “G” are detected by PCR-RFLP confirming the presence of SNP in DNA of ALA patients.
Mentions: A total of 55 ALA samples along with 102 controls were subjected to PCR-RFLP analysis. DNA was amplified using leptin receptor specific primers. PCR was performed in a touch gene (Nugen Scientific, USA) machine. Thin walled 0.2 mL tubes were used for amplification. A typical PCR reaction (20 μL) included 7.8 μL of autoclaved milliQ water, 2 μL of 10X PCR buffer with MgCl2 (containing 750 mM Tris-HCl (pH 8.8 at 25°C), 200 mM (NH4)2SO4, 0.1% Tween-20, 1.5 mM MgCl2), 2 μL of dNTP mix (containing 2 mM of each dNTP), 2 μL (20 pmol) of each primer forward as well as reverse, and 0.2 μL of TaqDNA polymerase (5 U/μL, MBI Fermentas, USA) and 2.0 μL of template DNA. The amplification conditions were one cycle of 94°C for 5 min followed by 30 cycles of 94°C for 30 s, annealing 55°C for 1 min, extension at 72°C for 30 sec, and finally one cycle of 72°C for 10 min and finally held at to 4°C. Volume of template DNA used (2.0 μL; ~50 ng) worked fine for PCR amplification. The sample containing all reagents except the template DNA was treated as the negative control. The size and integrity of the products were checked by electrophoresis. 10 μL of the PCR product was run on a 0.8–1.2% agarose gel at 5 V/cm for an appropriate time period. Restriction enzyme BseNI was used to digest the PCR amplified product of 386 bp and the fragments generated upon digestion are represented in Figure 1. Restriction enzyme BseNI digests only when the sequence reads nucleotide A at the locus. Thus digestion of 386 bp PCR product yielded three bands of 221 + 146 + 19 bp in case of homozygous (AA, assuming A as wild allele) wild and two bands of 367 + 19 bp in case of homozygous mutant (GG) (Figure 1). As expected, the digestion of heterozygous mutant yielded four bands of 367 + 221 + 146 + 19 bp as shown in a representative gel. All bands except 19 bp were visible on 1.5% agarose gel. Sequencing of mutated fragment confirmed the presence of mutation in Indian population (Figures 2(a) and 2(b)).

Bottom Line: Host genetic susceptibility is an important risk factor in infectious diseases.The frequency of allele "G" (coding for arginine) was in general high in Indian population irrespective of the disease.Our results of Fisher exact test shows that heterozygous mutant (QQ versus QR, P=0.049) and homozygous mutant (QQ versus RR, P=0.004) were significantly associated with amoebic liver abscess when compared with homozygous wild (QQ).

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.

ABSTRACT
Host genetic susceptibility is an important risk factor in infectious diseases. We explored the distribution of Q223R mutation in leptin receptor gene of amoebic liver abscess (ALA) patients of North India. A total of 55 ALA samples along with 102 controls were subjected to PCR-RFLP analysis. The frequency of allele "G" (coding for arginine) was in general high in Indian population irrespective of the disease. Our results of Fisher exact test shows that heterozygous mutant (QQ versus QR, P=0.049) and homozygous mutant (QQ versus RR, P=0.004) were significantly associated with amoebic liver abscess when compared with homozygous wild (QQ).

Show MeSH
Related in: MedlinePlus