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GHR gene transcript heterogeneity may explain phenotypic variability in GHR pseudoexon (6?) patients.

ABSTRACT: OBJECTIVES:The homozygous GH receptor (GHR) pseudoexon (6?) mutation leads to growth hormone insensitivity (GHI) with clinical and biochemical heterogeneity. We investigated whether transcript heterogeneity (6?-GHR to WT-GHR transcript ratio) and/or concurrent defects in other short stature (SS) genes contribute to this. METHODS:6?-GHR and WT-GHR mRNA transcripts of 4 6? patient (height SDS -4.2 to -3.1) and 1 control fibroblasts were investigated by RT-PCR. Transcripts were quantified by qRT-PCR and delta delta CT analysis and compared using ANOVA with Bonferroni correction. In eleven 6? patients, 40 genes known to cause GHI/SS were analysed by targeted next generation sequencing. RESULTS:RT-PCR confirmed 6?-GHR transcript in the 6? patients but not control. 6?-GHR transcript levels were comparable in patients 1 and 3 but significantly different among all other patients. The mean 6?:WT transcript ratios ranged from 29-71:1 for patients 1-4 and correlated negatively with height SDS (R=-0.85; p<0.001). Eight deleterious variants in 6 genes were detected but the number of gene hits did not correlate with the degree of SS in individual 6? patients. CONCLUSION:Variable amounts of 6?- and WT-GHR transcripts were identified in 6? patients but no 6? transcript was present in the control. Higher 6?:WT GHR transcript ratio correlated with SS severity and may explain the phenotypic variability. Analysis of known SS genes suggested that phenotypic variation is independent of the genetic background. This is the first report of transcript heterogeneity producing a spectrum of clinical phenotypes in different individuals harbouring an identical homozygous genetic mutation.

SUBMITTER: Chatterjee S

PROVIDER: S-EPMC7077524 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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GHR gene transcript heterogeneity may explain phenotypic variability in GHR pseudoexon (6Ψ) patients.

Chatterjee Sumana S Cottrell Emily E Rose Stephen J SJ Mushtaq Talat T Maharaj Avinash Vickram AV Williams Jack J Savage Martin O MO Metherell Louise A LA Storr Hl H

Endocrine connections 20200201

<h4>Objectives</h4>The homozygous GH receptor (GHR) pseudoexon (6Ψ) mutation leads to growth hormone insensitivity (GHI) with clinical and biochemical heterogeneity. We investigated whether transcript heterogeneity (6Ψ-GHR to WT-GHR transcript ratio) and/or concurrent defects in other short stature (SS) genes contribute to this.<h4>Methods</h4>6Ψ-GHR and WT-GHR mRNA transcripts of 4 6Ψ patient (height SDS -4.2 to -3.1) and 1 control fibroblasts were investigated by RT-PCR. Transcripts were quant ...[more]

PMID: 32061156

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Project description:The scale and capability of single-cell and single-nucleus RNA-sequencing technologies are rapidly growing, enabling key discoveries and large-scale cell mapping operations. However, studies directly comparing technical differences between single-cell and single-nucleus RNA sequencing are still lacking. Here, we compared three paired single-cell and single-nucleus transcriptomes from three different organs (Heart, Lung and Kidney). Differently from previous studies that focused on cell classification, we explored disparities in the transcriptome output of whole cells relative to the nucleus. We found that the major cell clusters could be recovered by either technique from matched samples, but at different proportions. In 2/3 datasets (kidney and lung) we detected clusters exclusively present with single-nucleus RNA sequencing. In all three organ groups, we found that genomic and gene structural characteristics such as gene length and exon content significantly differed between the two techniques. Genes recovered with the single-nucleus RNA sequencing technique had longer sequence lengths and larger exon counts, whereas single-cell RNA sequencing captured short genes at higher rates. Furthermore, we found that when compared to the whole host genome (mouse for kidney and lung datasets and human for the heart dataset), single transcriptomes obtained with either technique skewed from the expected proportions in several points: a) coding sequence length, b) transcript length and c) genomic span; and d) distribution of genes based on exons counts. Interestingly, the top-100 DEG between the two techniques returned distinctive GO terms. Hence, the type of single transcriptome technique used affected the outcome of downstream analysis. In summary, our data revealed both techniques present disparities in RNA capture. Moreover, the biased RNA capture affected the calculations of basic cellular parameters, raising pivotal points about the limitations and advantages of either single transcriptome techniques.

Dataset Information

GHR gene transcript heterogeneity may explain phenotypic variability in GHR pseudoexon (6?) patients.

Publications

GHR gene transcript heterogeneity may explain phenotypic variability in GHR pseudoexon (6Ψ) patients.

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