Comparison of NGS panel and Sanger sequencing for genotyping CAG repeats in the AR gene

Maria Santa Rocca; Margherita Ferrarini; Aichi Msaki; Cinzia Vinanzi; Marco Ghezzi; Maurizio De Rocco Ponce; Carlo Foresta; Alberto Ferlin

doi:10.1002/mgg3.1207

Research Methods · Preclinical Research

Comparison of NGS panel and Sanger sequencing for genotyping CAG repeats in the AR gene

Rocca MS, Ferrarini M, Msaki A, Vinanzi C, Ghezzi M, De Rocco Ponce M, Foresta C, Ferlin A

Published June 2020 Molecular genetics & genomic medicine

DOI 10.1002/mgg3.1207 PMID 32216057 PMC PMC7284049

RRM Academy Synopsis

HipSTR accurately sizes androgen receptor CAG repeats in 46,XY men

HipSTR software called every CAG repeat genotype correctly in 46,XY men, 100 out of 100 against 99 out of 100 for Sanger sequencing. In men with Klinefelter syndrome, HipSTR was correct for 64 out of 100 and Sanger for 88 out of 100. The retrospective comparison covered 228 men evaluated for fertility at one center.

Key Findings

Of 114 men with 46,XY chromosomes, 103 samples passed quality filters. The two methods matched in 102; HipSTR called the one discordant genotype correctly. Sanger accuracy was 102/103 and HipSTR accuracy 103/103.
Among 109 Klinefelter samples that passed quality filters, Sanger and HipSTR agreed in 57 and disagreed in 52. Microsatellite analysis showed Sanger correct in 39 and HipSTR in 13.
Accuracy in the Klinefelter samples was 88% (96/109) for Sanger and 64% (70/109) for HipSTR.
Capillary electrophoresis found 67 heterozygous and 42 homozygous Klinefelter samples. HipSTR identified all heterozygous genotypes and 7% of homozygous genotypes; Sanger identified 94% of heterozygous genotypes.
In four Klinefelter samples HipSTR detected heterozygous genotypes that Sanger missed. One of the four came from a man with 50% 47,XXY/46,XY mosaicism.

Interpretation

The study is a retrospective method comparison in 228 men referred to one center. All were of Caucasian ethnicity and Italian origin by self-report. Microsatellite analysis served as an independent check of CAG repeat length. The authors suggest the NGS panel for infertile men with 46,XY chromosomes and for men with suspected low-level mosaicism. For men with a 47,XXY karyotype, the authors advise caution, because HipSTR frequently called homozygous genotypes heterozygous. The authors add that short reads may miss longer CAG expansions. The panel covered GGC repeats insufficiently. The study reports genotyping accuracy and no clinical outcomes.

RRM Context

Restorative reproductive medicine seeks the cause of infertility in both partners. Men contribute to infertility in many couples. The paper says Klinefelter syndrome is the most common genetic cause of male infertility. It adds that longer CAG repeats have been linked to infertility in men and women. Accurate repeat counts help in checking the male partner.

Our editorial summary of this paper, not the article's abstract.

Abstract

Background

The androgen receptor (AR) is a nuclear receptor, encoded by the AR gene on the X chromosome. Within the first exon of the AR gene, two short tandem repeats (STR), CAG and GGC, are a source of polymorphism in the population. Therefore, high-throughput methods for screening AR, such as next-generation sequencing (NGS), are sought after; however, data generated by NGS are limited by the availability of bioinformatics tools. Here, we evaluated the accuracy of the bioinformatics tool HipSTR in detecting and quantify CAG repeats within the AR gene.

Method

The AR gene of 228 infertile men was sequenced using NGSgene panel. Data generated were analyzed with HipSTR to detect CAG repeats. The accuracy was compared with the results obtained with Sanger.

Results

We found that HipSTR was more accurate than Sanger in genotyping normal karyotype men (46,XY), however, it was more likely to misidentify homozygote genotypes in men with Klinefelter syndrome (47,XXY).

Conclusion

Our findings show that the bioinformatics tool HipSTR is 100% accurate in detecting and assessing AR CAG repeats in infertile men (46,XY) as well as in men with low-level mosaicism.

Topics

Research Methods › Preclinical Research › Laboratory Methods · Genetics and Immunology › Reproductive Genetics › Single Gene Conditions

Maria Santa Rocca, Margherita Ferrarini, Aichi Msaki, Cinzia Vinanzi, Marco Ghezzi, Maurizio De Rocco Ponce, Carlo Foresta, Alberto Ferlin

M Rocca, M Ferrarini, A Msaki, C Vinanzi, M Ghezzi, M Ponce, C Foresta, A Ferlin

PMID 32216057 32216057 DOI 10.1002/mgg3.1207 10.1002/mgg3.1207 Rocca et al. 2020, Rocca 2020

Comparison of NGS panel and Sanger sequencing for genotyping CAG repeats in the AR gene

HipSTR accurately sizes androgen receptor CAG repeats in 46,XY men

Key Findings

Interpretation

RRM Context

Abstract

Topics

By this author

Related research