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


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          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.


          6Ψ- GHR and WT -GHR mRNA transcripts of four 6Ψ patients (height SDS −4.2 to −3.1) and one control fibroblast 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 6Ψ patients, 40 genes known to cause GHI/SS were analysed by targeted next generation sequencing.


          RT-PCR confirmed 6Ψ- GHR transcript in the 6Ψ patients but not in the control. 6Ψ- GHR transcript levels were comparable in patients 1 and 3 but significantly different among all other patients. The mean 6Ψ: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 six genes were detected, but the number of gene hits did not correlate with the degree of SS in individual 6Ψ patients.


          Variable amounts of 6Ψ- and WT- GHR transcripts were identified in 6Ψ patients but no 6Ψ transcript was present in the control. Higher 6Ψ: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.

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          Intrauterine growth retardation and postnatal growth failure associated with deletion of the insulin-like growth factor I gene.

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            Computational definition of sequence motifs governing constitutive exon splicing.

            We have searched for sequence motifs that contribute to the recognition of human pre-mRNA splice sites by comparing the frequency of 8-mers in internal noncoding exons versus unspliced pseudo exons and 5' untranslated regions (5' untranslated regions [UTRs]) of transcripts of intronless genes. This type of comparison avoids the isolation of sequences that are distinguished by their protein-coding information. We classified sequence families comprising 2069 putative exonic enhancers and 974 putative exonic silencers. Representatives of each class functioned as enhancers or silencers when inserted into a test exon and assayed in transfected mammalian cells. As a class, the enhancer sequencers were more prevalent and the silencer elements less prevalent in all exons compared with introns. A survey of 58 reported exonic splicing mutations showed good agreement between the splicing phenotype and the effect of the mutation on the motifs defined here. The large number of effective sequences implied by these results suggests that sequences that influence splicing may be very abundant in pre-mRNA.
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              The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: causes and consequences.

              A total of 101 different examples of point mutations, which lie in the vicinity of mRNA splice junctions, and which have been held to be responsible for a human genetic disease by altering the accuracy of efficiency of mRNA splicing, have been collated. These data comprise 62 mutations at 5' splice sites, 26 at 3' splice sites and 13 that result in the creation of novel splice sites. It is estimated that up to 15% of all point mutations causing human genetic disease result in an mRNA splicing defect. Of the 5' splice site mutations, 60% involved the invariant GT dinucleotide; mutations were found to be non-randomly distributed with an excess over expectation at positions +1 and +2, and apparent deficiencies at positions -1 and -2. Of the 3' splice site mutations, 87% involved the invariant AG dinucleotide; an excess of mutations over expectation was noted at position -2. This non-randomness of mutation reflects the evolutionary conservation apparent in splice site consensus sequences drawn up previously from primate genes, and is most probably attributable to detection bias resulting from the differing phenotypic severity of specific lesions. The spectrum of point mutations was also drastically skewed: purines were significantly over-represented as substituting nucleotides, perhaps because of steric hindrance (e.g. in U1 snRNA binding at 5' splice sites). Furthermore, splice sites affected by point mutations resulting in human genetic disease were markedly different from the splice site consensus sequences. When similarity was quantified by a 'consensus value', both extremely low and extremely high values were notably absent from the wild-type sequences of the mutated splice sites. Splice sites of intermediate similarity to the consensus sequence may thus be more prone to the deleterious effects of mutation. Regarding the phenotypic effects of mutations on mRNA splicing, exon skipping occurred more frequently than cryptic splice site usage. Evidence is presented that indicates that, at least for 5' splice site mutations, cryptic splice site usage is favoured under conditions where (1) a number of such sites are present in the immediate vicinity and (2) these sites exhibit sufficient homology to the splice site consensus sequence for them to be able to compete successfully with the mutated splice site. The novel concept of a "potential for cryptic splice site usage" value was introduced in order to quantify these characteristics, and to predict the relative proportion of exon skipping vs cryptic splice site utilization consequent to the introduction of a mutation at a normal splice site.

                Author and article information

                Endocr Connect
                Endocr Connect
                Endocrine Connections
                Bioscientifica Ltd (Bristol )
                March 2020
                12 February 2020
                : 9
                : 3
                : 211-222
                [1 ]Centre for Endocrinology , William Harvey Research Institute, Barts and the London School of Medicine & Dentistry, Queen Mary University of London, London, UK
                [2 ]Birmingham Heartlands Hospital , University Hospitals Birmingham NHS Foundation Trust, Birmingham, UK
                [3 ]The Leeds Teaching Hospital NHS Trust , Leeds, UK
                Author notes
                Correspondence should be addressed to H L Storr: h.l.storr@ 123456qmul.ac.uk
                © 2020 The authors

                This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

                : 23 January 2020
                : 12 February 2020

                short stature,growth hormone insensitivity,ghr pseudoexon,splicing,gene sequencing


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