Next-generation Sequencing Better for Genetic Testing of AS, Study Finds

Ana Pena, PhD avatar

by Ana Pena, PhD |

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Next-generation sequencing

Next-generation sequencing (NGS) offers more advantages over conventional Sanger sequencing for the genetic testing of people who might have Alport syndrome (AS), a study has found.

Both have a high diagnostic ability, but NGS can identify harmful mutations missed by Sanger sequencing and detect other inherited kidney diseases that might be misdiagnosed as AS.

The study, “Comparison between conventional and comprehensive sequencing approaches for genetic diagnosis of Alport syndrome,” was published in the journal Molecular Genetics & Genomic Medicine.

Alport syndrome is an inherited disease caused by mutations in the COL4A3, COL4A4, and COL4A5 genes, which provide instructions for making protein components of type IV collagen alpha chains. This protein is vital in supporting cells in many tissues, particularly the kidneys, eyes, and inner ear, which is why AS primarily affects the functioning of these organs.

When doctors suspect the disease, based on symptoms or family history, genetic testing can be requested to get a definitive diagnosis. Such tests are widely available and have a high rate of accuracy.

Sanger sequencing was the method typically used for genetic diagnosis of AS, but screening of all three genes with this technology is time-consuming and expensive. Sanger sequencing can only sequence short pieces of DNA, so it takes a long time to run through long genes and is impractical for sequencing all of a person’s genes (genome).

More recently, next-generation sequencing (NGS) has sped up the process (taking only days to weeks to sequence a human genome) and reduced costs.

NGS enables comprehensive screening of many genes and offers a potential advantage over Sanger sequencing — the ability to detect mutations in genes other than COL4A3-5 that cause other inherited diseases, such as kidney disorders, that can be mistaken for AS.

However, few studies have employed NGS for comprehensive gene testing in AS and no study has looked at how well NGS compares with conventional Sanger sequencing in identifying AS-causing mutations.

So in this study, researchers at Japan’s Kobe University Graduate School of Medicine sought to compare the screening efficacy of the two methods for AS.

The team recruited 441 patients clinically suspected of AS, and did genetic screenings on DNA collected from patients’ blood samples.

Participants were divided into two groups — in one, patients had genetic tests using targeted exome sequencing using NGS (147 patients); in the other, patients were screened for COL4A3-5 mutations by Sanger sequencing (294 patients).

Exome sequencing means that only the protein-coding region of genes will be sequenced. Here, a targeted approach was used — rather than sequencing the entire genome, the analysis was restricted to a custom panel of genes, pre-selected by researchers.

Results showed that in the NGS group, 126 patients (86%) tested positive for AS, while two subjects had harmful mutations in other genes — NPHS1 and EYA1.

Alterations of these genes are known to cause other inherited disorders affecting the kidneys and the ears, namely congenital nephrotic syndrome and brachio‐otorenal syndrome. So NGS detection of those mutations helped doctors dismiss AS and re-diagnose the patients.

In the group analyzed by Sanger sequencing, 239 patients (81%) were diagnosed with AS. But within this group, 13 patients tested negative for COL4A3-5 mutations and were subsequently sent for NGS testing.

That revealed that three patients indeed carried AS mutations, and two had mutations in CLCN5 or LAMB2  — two genes linked to other inherited kidney diseases.

Overall, the results show that both Sanger sequencing and targeted exome sequencing with NGS have high diagnostic ability for patients clinically suspected of having AS, with a final variant detection rate of 90%.

However, “various benefits of a comprehensive diagnostic approach using NGS for the diagnosis of AS were revealed,” researchers said.

NGS has a higher diagnostic ability, identifying harmful mutations missed by Sanger sequencing. It also has the advantage of detecting other inherited kidney diseases.


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