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Development of low-density oligonucleotide microarrays for detecting mutations causing Wilson's disease.

Mathur M, Singh E, Poduval TB, Rao AV - Indian J. Med. Res. (2015)

Bottom Line: The hybridization reaction were found to be consistent across the surface of a given microarray.Our results have shown that 52 °C post-hybridization wash yields better reproducibility across experiments compared to 42 °C.The present results demonstrate the design and evaluation of a low-density microarray for the detection of 62 mutations in ATP7B gene, and show that a microarray based approach can be cost-effective for detecting a large number of mutations simultaneously.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai, India.

ABSTRACT

Background & objectives: Wilson's disease (WD) is an autosomal recessive disorder caused by mutations in ATP7B, a copper transporter gene, leading to hepatic and neuropsychiatric manifestations due to copper accumulation. If diagnosed early, WD patients can be managed by medicines reducing morbidity and mortality. Diagnosis of this disease requires a combination of tests and at times is inconclusive due to overlap of the symptoms with other disorders. Genetic testing is the preferred alternative in such cases particularly for individuals with a family history. Use of DNA microarray for detecting mutations in ATP7B gene is gaining popularity because of the advantages it offers in terms of throughput and sensitivity. This study attempts to establish the quality analysis procedures for microarray based diagnosis of Wilson's disease.

Methods: A home-made microarrayer was used to print oligonucleotide based low-density microarrays for addressing 62 mutations causing Wilson's disease reported from Indian population. Inter- and intra- array comparisons were used to study quality of the arrays. The arrays were validated by using mutant samples generated by site directed mutagenesis.

Results: The hybridization reaction were found to be consistent across the surface of a given microarray. Our results have shown that 52 °C post-hybridization wash yields better reproducibility across experiments compared to 42 °C. Our arrays have shown > 80 per cent sensitivity in detecting these 62 mutations.

Interpretation & conclusions: The present results demonstrate the design and evaluation of a low-density microarray for the detection of 62 mutations in ATP7B gene, and show that a microarray based approach can be cost-effective for detecting a large number of mutations simultaneously. This study also provides information on some of the important parameters required for microarray based diagnosis of genetic disorders.

No MeSH data available.


Related in: MedlinePlus

Normalized spot intensities obtained for 61 perfect match probes from six independent hybridizations. The error bars indicate one sigma (Standard Deviation) from the mean.
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Figure 4: Normalized spot intensities obtained for 61 perfect match probes from six independent hybridizations. The error bars indicate one sigma (Standard Deviation) from the mean.

Mentions: Inter-experiment comparison of spot intensities: Reproducibility of intensity data (of either PM or MM probes) across experiments is necessary for applying any further processing to the data. The reproducibility was assessed by comparing the intensity data from 61 perfect match probes from six independent experiments each carried out at 52 and at 42°C. The dispersion of intensities (standard deviation computed from inter-array experiments) were found to be lower for the hybridizations done at 52°C (Fig. 4) as compared to those at 42°C (data not shown). Inspite of the lower mean intensities obtained from post-hybridization washes at 52°C, better data reproducibility was observed at higher temperature. Hence the 52°C data were used for all further analysis.


Development of low-density oligonucleotide microarrays for detecting mutations causing Wilson's disease.

Mathur M, Singh E, Poduval TB, Rao AV - Indian J. Med. Res. (2015)

Normalized spot intensities obtained for 61 perfect match probes from six independent hybridizations. The error bars indicate one sigma (Standard Deviation) from the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4418154&req=5

Figure 4: Normalized spot intensities obtained for 61 perfect match probes from six independent hybridizations. The error bars indicate one sigma (Standard Deviation) from the mean.
Mentions: Inter-experiment comparison of spot intensities: Reproducibility of intensity data (of either PM or MM probes) across experiments is necessary for applying any further processing to the data. The reproducibility was assessed by comparing the intensity data from 61 perfect match probes from six independent experiments each carried out at 52 and at 42°C. The dispersion of intensities (standard deviation computed from inter-array experiments) were found to be lower for the hybridizations done at 52°C (Fig. 4) as compared to those at 42°C (data not shown). Inspite of the lower mean intensities obtained from post-hybridization washes at 52°C, better data reproducibility was observed at higher temperature. Hence the 52°C data were used for all further analysis.

Bottom Line: The hybridization reaction were found to be consistent across the surface of a given microarray.Our results have shown that 52 °C post-hybridization wash yields better reproducibility across experiments compared to 42 °C.The present results demonstrate the design and evaluation of a low-density microarray for the detection of 62 mutations in ATP7B gene, and show that a microarray based approach can be cost-effective for detecting a large number of mutations simultaneously.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai, India.

ABSTRACT

Background & objectives: Wilson's disease (WD) is an autosomal recessive disorder caused by mutations in ATP7B, a copper transporter gene, leading to hepatic and neuropsychiatric manifestations due to copper accumulation. If diagnosed early, WD patients can be managed by medicines reducing morbidity and mortality. Diagnosis of this disease requires a combination of tests and at times is inconclusive due to overlap of the symptoms with other disorders. Genetic testing is the preferred alternative in such cases particularly for individuals with a family history. Use of DNA microarray for detecting mutations in ATP7B gene is gaining popularity because of the advantages it offers in terms of throughput and sensitivity. This study attempts to establish the quality analysis procedures for microarray based diagnosis of Wilson's disease.

Methods: A home-made microarrayer was used to print oligonucleotide based low-density microarrays for addressing 62 mutations causing Wilson's disease reported from Indian population. Inter- and intra- array comparisons were used to study quality of the arrays. The arrays were validated by using mutant samples generated by site directed mutagenesis.

Results: The hybridization reaction were found to be consistent across the surface of a given microarray. Our results have shown that 52 °C post-hybridization wash yields better reproducibility across experiments compared to 42 °C. Our arrays have shown > 80 per cent sensitivity in detecting these 62 mutations.

Interpretation & conclusions: The present results demonstrate the design and evaluation of a low-density microarray for the detection of 62 mutations in ATP7B gene, and show that a microarray based approach can be cost-effective for detecting a large number of mutations simultaneously. This study also provides information on some of the important parameters required for microarray based diagnosis of genetic disorders.

No MeSH data available.


Related in: MedlinePlus