Overcoming the pitfalls of NGS-based molecular diagnosis of Shwachman-Diamond syndrome

Peng, Xiaomin; Dong, Xinran; Wang, Yaqiong; Wu, Bingbing; Wang, Huijun; Lü, Wei; Xiao, Fei; Yang, Lin; Li, Gang; Zhou, Wenhao; Liu, Bo; Lu, Yulan

doi:10.1101/2021.08.22.21262444

Cited by 2 publications

(2 citation statements)

References 31 publications

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“…Since the common SBDS pathogenic variants c.185_184TA>GT and c.258+2T>C are at the functional PSV loci, it has been suggested that recombination and gene conversion might occur between SBDS and SBDSP1 [ 11 ]. Based on the above factors, researchers have recognized the complexity and challenges of NGS analysis of the SBDS gene, and therefore suggest using PCR, RT-PCR or specific bioinformatics tools to increase the sensitivity and specificity of detecting pathogenic variants in SBDS [ 12 – 14 ].…”

Section: Introductionmentioning

confidence: 99%

Improved detection of SBDS gene mutation by a new method of next-generation sequencing analysis based on the Chinese mutation spectrum

Wu¹,

Zhang²,

Qiang³

et al. 2022

PLoS ONE

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Next-generation sequencing (NGS) is a useful molecular diagnostic tool for genetic diseases. However, due to the presence of highly homologous pseudogenes, it is challenging to use short-read NGS for analyzing mutations of the Shwachman-Bodian-Diamond syndrome (SBDS) gene. The SBDS mutation spectrum was analyzed in the Chinese population, which revealed that SBDS variants were primarily from sequence exchange between SBDS and its pseudogene at the base-pair level, predominantly in the coding region and splice junction of exon two. The c.258+2T>C and c.185_184TA>GT variants were the two most common pathogenic SBDS variants in the Chinese population, resulting in a total carrier frequency of 1.19%. When analyzing pathogenic variants in the SBDS gene from the NGS data, the misalignment was identified as a common issue, and there were different probabilities of misalignment for different pathogenic variants. Here, we present a novel mathematical method for identifying pathogenic variants in the SBDS gene from the NGS data, which utilizes read-depth of the paralogous sequence variant (PSV) loci of SBDS and its pseudogene. Combined with PCR and STR orthogonal experiments, SBDS gene mutation analysis results were improved in 40% of clinical samples, and various types of mutations such as homozygous, compound heterozygous, and uniparental diploid were explored. The findings effectively reduce the impact of misalignment in NGS-based SBDS mutation analysis and are helpful for the clinical diagnosis of SBDS-related diseases, the research into population variation, and the carrier screening.

show abstract

Section: Introductionmentioning

confidence: 99%

Improved detection of SBDS gene mutation by a new method of next-generation sequencing analysis based on the Chinese mutation spectrum

Wu¹,

Zhang²,

Qiang³

et al. 2022

PLoS ONE

View full text Add to dashboard Cite

show abstract

“…Since the common SBDS pathogenic variants c.185_184TA>GT and c.258+2T>C are at the functional PSV loci, it has been suggested that recombination and gene conversion might occur between SBDS and SBDSP1 [11] . Based on the above factors, researchers have recognized the complexity and challenges of NGS analysis of the SBDS gene, and therefore suggest using PCR, RT-PCR or specific bioinformatics tools to increase the sensitivity and specificity of detecting pathogenic variants in SBDS [12][13][14] .…”

Section: Introductionmentioning

confidence: 99%

Improved detection of SBDS gene mutation by a new method of next-generation sequencing analysis based on the Chinese mutation spectrum

Wu¹,

Zhang²,

Qiang³

et al. 2021

Preprint

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Next-generation sequencing (NGS) is a useful molecular diagnostic tool for genetic diseases. However, due to the presence of highly homologous pseudogenes, it is challenging to use short-read NGS for analyzing mutations of the Shwachman-Bodian-Diamond syndrome (SBDS) gene. The SBDS mutation spectrum was analyzed in the Chinese population, which revealed that SBDS variants were primarily from sequence exchange between SBDS and its pseudogene at the base-pair level, predominantly in the coding region and splice junction of exon two. The c.258+2T>C and c.185_184TA>GT variants were the two most common pathogenic SBDS variants in the Chinese population, resulting in a total carrier frequency of 1.19%. When analyzing pathogenic variants in the SBDS gene from the NGS data, the misalignment was identified as a common issue, and there were different probabilities of misalignment for different pathogenic variants. Here, we present a novel mathematical method for identifying pathogenic variants in the SBDS gene from the NGS data, which utilizes read-depth of the paralogous sequence variant (PSV) loci of SBDS and its pseudogene. Combined with PCR and STR orthogonal experiments, the results of SBDS gene mutation analysis were improved in 40% of clinical samples, and various types of mutations such as homozygous, compound heterozygous, and uniparental diploid were explored. The findings effectively reduce the impact of misalignment in NGS-based SBDS mutation analysis and are helpful for the clinical diagnosis of SBDS-related diseases, the research into population variation, and the carrier screening.

show abstract

Overcoming the pitfalls of NGS-based molecular diagnosis of Shwachman-Diamond syndrome

Cited by 2 publications

References 31 publications

Improved detection of SBDS gene mutation by a new method of next-generation sequencing analysis based on the Chinese mutation spectrum

Improved detection of SBDS gene mutation by a new method of next-generation sequencing analysis based on the Chinese mutation spectrum

Improved detection of SBDS gene mutation by a new method of next-generation sequencing analysis based on the Chinese mutation spectrum

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