Variants in the 5'UTR reduce SHOX expression and contribute to SHOX haploinsufficiency.
Journal
European journal of human genetics : EJHG
ISSN: 1476-5438
Titre abrégé: Eur J Hum Genet
Pays: England
ID NLM: 9302235
Informations de publication
Date de publication:
01 2021
01 2021
Historique:
received:
14
01
2020
accepted:
23
06
2020
revised:
08
06
2020
pubmed:
11
7
2020
medline:
17
8
2021
entrez:
11
7
2020
Statut:
ppublish
Résumé
SHOX haploinsufficiency causes 70-90% of Léri-Weill dyschondrosteosis (LWD) and 2-10% of idiopathic short stature (ISS). Deletions removing the entire gene or enhancers and point mutations in the coding region represent a well-established cause of haploinsufficiency. During diagnostic genetic testing on ISS/LWD patients, in addition to classic SHOX defects, five 5'UTR variants (c.-58G > T, c.-55C > T, c.-51G > A, c.-19G > A, and c.-9del), were detected whose pathogenetic role was unclear and were thus classified as VUS (Variants of Uncertain Significance). The purpose of the present study was to investigate the role of these noncoding variations in SHOX haploinsufficiency. The variants were tested for their ability to interfere with correct gene expression of a regulated reporter gene (luciferase assay). The negative effect on the mRNA splicing predicted in silico for c.-19G > A was assayed in vitro through a minigene splicing assay. The luciferase assay showed that c.-51G > A, c.-19G > A, and c.-9del significantly reduce luciferase activity by 60, 35, and 40% at the homozygous state. Quantification of the luciferase mRNA showed that c.-51G > A and c.-9del might interfere with the correct SHOX expression mainly at the post-transcriptional level. The exon trapping assay demonstrated that c.-19G > A determines the creation of a new branch site causing an aberrant mRNA splicing. In conclusion, this study allowed us to reclassify two of the 5'UTR variants identified during SHOX diagnostic screening as likely pathogenic, one remains as a VUS, and two as likely benign variants. This analysis for the first time expands the spectrum of the genetic causes of SHOX haploinsufficiency to noncoding variations in the 5'UTR.
Identifiants
pubmed: 32647378
doi: 10.1038/s41431-020-0676-y
pii: 10.1038/s41431-020-0676-y
pmc: PMC7852508
doi:
Substances chimiques
5' Untranslated Regions
0
SHOX protein, human
0
Short Stature Homeobox Protein
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
110-121Références
Rao E, Weiss B, Fukami M, Rump A, Niesler B, Mertz A, et al. Pseudoautosomal deletions encompassing a novel homeobox gene cause growth failure in idiopathic short stature and Turner syndrome. Nat Genet. 1997;16:54–63.
pubmed: 9140395
doi: 10.1038/ng0597-54
Sabherwal N, Bangs F, Röth R, Weiss B, Jantz K, Tiecke E, et al. Long-range conserved non-coding SHOX sequences regulate expression in developing chicken limb and are associated with short stature phenotypes in human patients. Hum Mol Genet. 2007;16:210–20.
pubmed: 17200153
doi: 10.1093/hmg/ddl470
Chen J, Wildhardt G, Zhong Z, Röth R, Weiss B, Steinberger D, et al. Enhancer deletions of the SHOX gene as a frequent cause of short stature: the essential role of a 250 kb downstream regulatory domain. J Med Genet. 2009;46:834–9.
pubmed: 19578035
pmcid: 2778764
doi: 10.1136/jmg.2009.067785
Leka SK, Kitsiou-Tzeli S, Kalpini-Mavrou A, Kanavakis E. Short stature and dysmorphology associated with defects in the SHOX gene. Hormones. 2006;5:107–18.
pubmed: 16807223
doi: 10.14310/horm.2002.11174
Jorge AA, Souza SC, Nishi MY, Billerbeck AE, Libório DC, Kim CA, et al. SHOX mutations in idiopathic short stature and Leri-Weill dyschondrosteosis: frequency and phenotypic variability. Clin Endocrinol (Oxf). 2007;66:130–5.
Rappold GA, Fukami M, Niesler B, Schiller S, Zumkeller W, Bettendorf M, et al. Deletions of the homeobox gene SHOX (short stature homeobox) are an important cause of growth failure in children with short stature. J Clin Endocrinol Metab. 2002;87:1402–6.
pubmed: 11889216
doi: 10.1210/jcem.87.3.8328
Rappold G, Blum WF, Shavrikova EP, Crowe BJ, Roeth R, Quigley CA, et al. Genotypes and phenotypes in children with short stature: clinical indicators of SHOX haploinsufficiency. J Med Genet. 2007;44:306–13.
pubmed: 17182655
doi: 10.1136/jmg.2006.046581
pmcid: 17182655
Stuppia L, Calabrese G, Gatta V, Pintor S, Morizio E, Fantasia D, et al. SHOX mutations detected by FISH and direct sequencing in patients with short stature. J Med Genet. 2003;40:E11.
pubmed: 12566529
pmcid: 1735371
doi: 10.1136/jmg.40.2.e11
Hirschfeldova K, Solc R, Baxova A, Zapletalova J, Kebrdlova V, Gaillyova R, et al. SHOX gene defects and selected dysmorphic signs in patients of idiopathic short stature and Léri-Weill dyschondrosteosis. Gene. 2012;491:123–7.
pubmed: 22020182
doi: 10.1016/j.gene.2011.10.011
pmcid: 22020182
Genoni G, Monzani A, Castagno M, Ricotti R, Rapa A, Petri A, et al. Improving clinical diagnosis in SHOX deficiency: the importance of growth velocity. Pediatr Res. 2018;83:438–44.
pubmed: 29211059
doi: 10.1038/pr.2017.247
Marchini A, Ogata T, Rappold GA. A track record on SHOX: from basic research to complex models and therapy. Endocr Rev. 2016;37:417–48.
pubmed: 27355317
pmcid: 4971310
doi: 10.1210/er.2016-1036
Bertorelli R, Capone L, Ambrosetti F, Garavelli L, Varriale L, Mazza V, et al. The homozygous deletion of the 3’ enhancer of the SHOX gene causes Langer mesomelic dysplasia. Clin Genet. 2007;72:490–1.
pubmed: 17935511
doi: 10.1111/j.1399-0004.2007.00875.x
Bunyan DJ, Baker KR, Harvey JF, Thomas NS. Diagnostic screening identifies a wide range of mutations involving the SHOX gene, including a common 47.5 kb deletion 160 kb downstream with a variable phenotypic effect. Am J Med Genet A. 2013;161:1329–38.
doi: 10.1002/ajmg.a.35919
Gatta V, Antonucci I, Morizio E, Palka C, Fischetto R, Mokini V, et al. Identification and characterization of different SHOX gene deletions in patients with Leri-Weill dyschondrosteosys by MLPA assay. J Hum Genet. 2007;52:21–7.
pubmed: 17091221
doi: 10.1007/s10038-006-0074-5
Benito-Sanz S, Barroso E, Heine-Suñer D, Hisado-Oliva A, Romanelli V, Rosell J, et al. Clinical and molecular evaluation of SHOX/PAR1 duplications in Leri-Weill dyschondrosteosis (LWD) and idiopathic short stature (ISS). J Clin Endocrinol Metab. 2011;96:E404–12.
pubmed: 21147883
doi: 10.1210/jc.2010-1689
Fukami M, Naiki Y, Muroya K, Hamajima T, Soneda S, Horikawa R, et al. Rare pseudoautosomal copy-number variations involving SHOX and/or its flanking regions in individuals with and without short stature. J Hum Genet. 2015;60:553–6.
pubmed: 26040210
doi: 10.1038/jhg.2015.53
Hirschfeldova K, Solc R. Comparison of SHOX and associated elements duplications distribution between patients (Lėri-Weill dyschondrosteosis/idiopathic short stature) and population sample. Gene. 2017;627:164–8.
pubmed: 28629824
doi: 10.1016/j.gene.2017.06.034
Monzani A, Babu D, Mellone S, Genoni G, Fanelli A, Prodam F, et al. Co-occurrence of genomic imbalances on Xp22.1 in the SHOX region and 15q25.2 in a girl with short stature, precocious puberty, urogenital malformations and bone anomalies. BMC Med Genomics. 2019;12:5.
pubmed: 30626445
pmcid: 6327496
doi: 10.1186/s12920-018-0445-8
Benito-Sanz S, Aza-Carmona M, Rodríguez-Estevez A, Rica-Etxebarria I, Gracia R, Campos-Barros A, et al. Identification of the first PAR1 deletion encompassing upstream SHOX enhancers in a family with idiopathic short stature. Eur J Hum Genet. 2012;20:125–7.
pubmed: 22071895
doi: 10.1038/ejhg.2011.210
Kant SG, Broekman SJ, de Wit CC, Bos M, Scheltinga SA, Bakker E, et al. Phenotypic characterization of patients with deletions in the 3’-flanking SHOX region. PeerJ. 2013;1:e35.
pubmed: 23638371
pmcid: 3629036
doi: 10.7717/peerj.35
Blaschke RJ, Töpfer C, Marchini A, Steinbeisser H, Janssen JW, Rappold GA. Transcriptional and translational regulation of the Leri-Weill and Turner syndrome homeobox gene SHOX. J Biol Chem. 2003;278:47820–6.
pubmed: 12960152
doi: 10.1074/jbc.M306685200
pmcid: 12960152
Cacciari E, Milani S, Balsamo A, Spada E, Bona G, Cavallo L, et al. Italian cross-sectional growth charts for height, weight and BMI (2 to 20 yr). J Endocrinol Invest. 2006;29:581–93.
pubmed: 16957405
doi: 10.1007/BF03344156
pmcid: 16957405
Bogin B, Varela-Silva MI. Leg length, body proportion, and health: a review with a note on beauty. Int J Environ Res Public Health. 2010;7:1047–75.
pubmed: 20617018
pmcid: 2872302
doi: 10.3390/ijerph7031047
Tanner JM, Whitehouse RH, Cameron N, Marshall WA, Healy WA. H G. Assessment of skeletal maturity and prediction of adult height (TW2 method). San Diego, CA: Academic Press; 1988.
Desmet FO, Hamroun D, Lalande M, Collod-Béroud G, Claustres M, Béroud C. Human splicing finder: an online bioinformatics tool to predict splicing signals. Nucleic Acids Res. 2009;37:e67.
pubmed: 2685110
pmcid: 2685110
doi: 10.1093/nar/gkp215
Sticht C, De La Torre C, Parveen A, Gretz N. miRWalk: an online resource for prediction of microRNA binding sites. PLoS One. 2018;13:e0206239.
pubmed: 30335862
pmcid: 6193719
doi: 10.1371/journal.pone.0206239
Kopanos C, Tsiolkas V, Kouris A, Chapple CE, Albarca Aguilera M, Meyer R, et al. VarSome: the human genomic variant search engine. Bioinformatics. 2019;35:1978–80.
doi: 10.1093/bioinformatics/bty897
Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17:405–24.
pubmed: 4544753
pmcid: 4544753
doi: 10.1038/gim.2015.30
Giordano M, Godi M, Giacopelli F, Lessi M, Mellone S, Paracchini R, et al. A variation in a Pit-1 site in the growth hormone gene (GH1) promoter induces a differential transcriptional activity. Mol Cell Endocrinol. 2006;249:51–7.
pubmed: 16517055
doi: 10.1016/j.mce.2006.01.006
pmcid: 16517055
Dauber A, Rosenfeld RG, Hirschhorn JN. Genetic evaluation of short stature. J Clin Endocrinol Metab. 2014;99:3080–92.
pubmed: 24915122
pmcid: 4154097
doi: 10.1210/jc.2014-1506
Araujo PR, Yoon K, Ko D, Smith AD, Qiao M, Suresh U, et al. Before it gets started: regulating translation at the 5’ UTR. Comp Funct Genomics. 2012;2012:475731.
pubmed: 22693426
pmcid: 3368165
doi: 10.1155/2012/475731
Jackson RJ, Hellen CU, Pestova TV. The mechanism of eukaryotic translation initiation and principles of its regulation. Nat Rev Mol Cell Biol. 2010;11:113–27.
pubmed: 20094052
pmcid: 4461372
doi: 10.1038/nrm2838
Semler O, Garbes L, Keupp K, Swan D, Zimmermann K, Becker J, et al. A mutation in the 5’-UTR of IFITM5 creates an in-frame start codon and causes autosomal-dominant osteogenesis imperfecta type V with hyperplastic callus. Am J Hum Genet. 2012;91:349–57.
pubmed: 22863195
pmcid: 3415541
doi: 10.1016/j.ajhg.2012.06.011
Kramer M, Sponholz C, Slaba M, Wissuwa B, Claus RA, Menzel U, et al. Alternative 5’ untranslated regions are involved in expression regulation of human heme oxygenase-1. PLoS One. 2013;8:e77224.
pubmed: 24098580
pmcid: 3788786
doi: 10.1371/journal.pone.0077224
Wang G, Guo X, Floros J. Differences in the translation efficiency and mRNA stability mediated by 5’-UTR splice variants of human SP-A1 and SP-A2 genes. Am J Physiol Lung Cell Mol Physiol. 2005;289:L497–508.
pubmed: 15894557
doi: 10.1152/ajplung.00100.2005
Cannons AC, Cannon J. The stability of the Chlorella nitrate reductase mRNA is determined by the secondary structure of the 5’-UTR: implications for posttranscriptional regulation of nitrate reductase. Planta. 2002;214:488–91.
pubmed: 11855653
doi: 10.1007/s00425-001-0679-z
Hua XJ, Van de Cotte B, Van Montagu M, Verbruggen N. The 5’ untranslated region of the At-P5R gene is involved in both transcriptional and post-transcriptional regulation. Plant J. 2001;26:157–69.
pubmed: 11389757
doi: 10.1046/j.1365-313x.2001.01020.x
Khamis A, Palmen J, Lench N, Taylor A, Badmus E, Leigh S, et al. Functional analysis of four LDLR 5’UTR and promoter variants in patients with familial hypercholesterolaemia. Eur J Hum Genet. 2015;23:790–5.
pubmed: 25248394
doi: 10.1038/ejhg.2014.199
Bisio A, Nasti S, Jordan JJ, Gargiulo S, Pastorino L, Provenzani A, et al. Functional analysis of CDKN2A/p16INK4a 5’-UTR variants predisposing to melanoma. Hum Mol Genet. 2010;19:1479–91.
pubmed: 20093296
doi: 10.1093/hmg/ddq022
Andreotti V, Bisio A, Bressac-de Paillerets B, Harland M, Cabaret O, Newton-Bishop J, et al. The CDKN2A/p16(INK) (4a) 5’UTR sequence and translational regulation: impact of novel variants predisposing to melanoma. Pigment Cell Melanoma Res. 2016;29:210–21.
pubmed: 26581427
doi: 10.1111/pcmr.12444
pmcid: 26581427
de Boer M, van Leeuwen K, Hauri-Hohl M, Roos D. Activation of cryptic splice sites in three patients with chronic granulomatous disease. Mol Genet Genom Med. 2019;7:e854.
Vivenza D, Guazzarotti L, Godi M, Frasca D, di Natale B, Momigliano-Richiardi P, et al. A novel deletion in the GH1 gene including the IVS3 branch site responsible for autosomal dominant isolated growth hormone deficiency. J Clin Endocrinol Metab. 2006;91:980–6.
pubmed: 16368751
doi: 10.1210/jc.2005-1703
pmcid: 16368751