Cis‐Regulatory Mutations in Human Disease


Enhancers are cis‐regulatory elements that accurately control the spatiotemporal pattern of gene expression and these can function over large genomic distances to activate the target gene. Genomewide methodologies have revealed general chromatin signatures for enhancers and the human and mouse noncoding genomes have undergone an extensive functional annotation. An increasing number of enhancer mutations are showing association with human disease, and disease mechanisms are being described that result from altered gene expression. Owing to the modular nature of enhancers, mutations can generate disease phenotypes that represent either a subset of the symptoms of coding mutations or due to a change in specificity of enhancer activity, a novel phenotype. Topologically associated domains, also known as TADs, have emerged as a fundamental structural unit to limit enhancer regulatory activity and disruptions of these boundaries result in gene misexpression and disease.

Key Concepts

  • Enhancers function over large genomic distances to activate the target gene.
  • Methodologies have been developed to annotate the functional elements in the noncoding genome.
  • Mutations in enhancers that regulate developmental gene expression can cause congenital abnormalities.
  • Owing to the modular nature of enhancers the disease presentation may be different from mutations in the coding region of the target gene.
  • Disruption of TAD boundaries can result in gene misexpression and disease.

Keywords: cis‐regulatory mutation; enhancer; congenital abnormalities; gene expression; topological associated domains

Figure 1. The roles of enhancers in regulating gene expression in the PAX6, SOX9 and SHH loci. (a) In the PAX6 locus, PAX6 protein binds to the SIMO enhancer leading to its interaction with the PAX6 gene. This maintains an auto regulatory feedback loop resulting in the production of additional PAX6 (i). A G>T point mutation alters the PAX6 binding site within the SIMO enhancer leading to reduced PAX6 binding and loss of the auto regulatory loop (ii). (b) In the SOX9 locus a collection of highly conserved noncoding elements (HCNEs) between the SOX9 and KCNJ2 genes are responsible for regulating optimal SOX9 levels (i). Increased binding of MSX1 (which acts as an inhibitor) at one of these HCNEs due to a T>C point mutation weakens its influence on the SOX9 gene expression leading to a disease phenotype (ii). Removal of this HCNE completely due to a 75 kb deletion also effects SOX9 levels (iii). In contrast, a duplication event may lead to overproduction of SOX9 due to the action of additional HCNEs on the SOX9 gene (iv). (c) In the limb bud the SHH gene is regulated by the ZRS, while in the forebrain it is regulated by SBE2. Within the limb, SHH is maintained in the posterior regions due to the correct ratio of ETS1/GABPα to ETV4/ETV5 proteins bound at the ZRS (i). Point mutations within this enhancer can lead to additional ETS1/GABPα sites at the detriment of ETV4/ETV5 sites, leading to the production of SHH in both anterior and posterior regions and the associated limb phenotypes (ii). Likewise, a 2 kb deletion may remove a silencer acting on the SHH gene leading to ectopic SHH expression (iii). In the forebrain the SHH gene is regulated by SBE2, which is situated roughly equidistant between the ZRS and SHH (iv). An inversion event removes the influence of SBE2, instead allowing an enhancer (HCNE2) within the EMID2 gene to regulate SHH expression, a process referred to as enhancer adoption (v).
Figure 2. Point mutations can effect transcription factor binding sites (TFBSs) within an enhancer leading to alterations in gene expression. (a) The precise quantity and type of transcription factor binding to enhancers are responsible for regulating the normal expression levels of a gene. (b) Point mutations within an enhancer may remove some of these TFBSs leading to reduced binding and reduced expression of the target gene. (c) Sometimes mutations may create sites for repressor proteins, which again leads to a reduction in target gene expression. (d) Alternatively, point mutations may produce additional TFBSs for activator proteins, which may result in ectopic gene expression.
Figure 3. Topological associated domains (TADs) are separated by boundary regions, which, when removed, allow enhancers from previously inaccessible regions to influence the expression of genes within the TAD. (a) Within a TAD, boundary regions such as those provided by CTCF sites restrict the influence of enhancers to genes within a defined area. (b) However, when a TAD boundary is removed, perhaps due to a deletion, enhancers from the adjacent TAD may act on genes within this deconstructed TAD thereby leading to abnormal gene expression levels.


Adey A, Morrison HG, Asan, et al. (2010) Rapid, low‐input, low‐bias construction of shotgun fragment libraries by high‐density in vitro transposition. Genome Biology 11: R119.

Anderson E, Peluso S, Lettice LA and Hill RE (2012a) Human limb abnormalities caused by disruption of hedgehog signaling. Trends in Genetics 28: 364–373.

Anderson E and Hill RE (2014) Long range regulation of the sonic hedgehog gene. Current Opinion in Genetics and Development 27: 54–59.

Barutcu AR, Fritz AJ, Zaidi SK, et al. (2016) C‐ing the genome: a compendium of chromosome conformation capture methods to study higher‐order chromatin organization. Journal of Cellular Physiology 231: 31–35.

Benko S, Fantes JA, Amiel J, et al. (2009) Highly conserved non‐coding elements on either side of SOX9 associated with Pierre Robin sequence. Nature Genetics 41: 359–364.

Bhatia S, Bengani H, Fish M, et al. (2013) Disruption of autoregulatory feedback by a mutation in a remote, ultraconserved PAX6 enhancer causes aniridia. American Journal of Human Genetics 93: 1126–1134.

Buenrostro JD, Giresi PG, Zaba LC, Chang HY and Greenleaf WJ (2013) Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA‐binding proteins and nucleosome position. Nature Methods 10: 1213–1218.

Buenrostro JD, Wu B, Litzenburger UM, et al. (2015) Single‐cell chromatin accessibility reveals principles of regulatory variation. Nature 523: 486–490.

Chandrasegaran S and Carroll D (2016) Origins of programmable nucleases for genome engineering. Journal of Molecular Biology 428: 963–989.

Chen X, Xu H, Yuan P, et al. (2008) Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 133: 1106–1117.

Cotney J, Leng J, Oh S, et al. (2012) Chromatin state signatures associated with tissue‐specific gene expression and enhancer activity in the embryonic limb. Genome Research 22: 1069–1080.

Dekker J, Rippe K, Dekker M and Kleckner N (2002) Capturing chromosome conformation. Science 295: 1306–1311.

Dorschner MO, Hawrylycz M, Humbert R, et al. (2004) High‐throughput localization of functional elements by quantitative chromatin profiling. Nature Methods 1: 219–225.

Douglas AT and Hill RD (2014) Variation in vertebrate cis‐regulatory elements in evolution and disease. Transcription 5: e28848.

ENCODE Project Consortium (2012) An integrated encyclopedia of DNA elements in the human genome. Nature 489: 57–74.

Erokhin M, Vassetzky Y, Georgiev P and Chetverina D (2015) Eukaryotic enhancers: common features, regulation, and participation in diseases. Cellular and Molecular Life Sciences 72: 2361–2375.

Fakhouri WD, Rahimov F, Attanasio C, et al. (2014) An etiologic regulatory mutation in IRF6 with loss‐ and gain‐of‐function effects. Human Molecular Genetics 23: 2711–2720.

Giorgio E, Robyr D, Spielmann M, et al. (2015) A large genomic deletion leads to enhancer adoption by the lamin B1 gene: a second path to autosomal dominant adult‐onset demyelinating leukodystrophy (ADLD). Human Molecular Genetics 24: 3143–3154.

Giresi PG, Kim J, McDaniell RM, Iyer VR and Lieb JD (2007) FAIRE (Formaldehyde‐Assisted Isolation of Regulatory Elements) isolates active regulatory elements from human chromatin. Genome Research 17: 877–885.

Girisha KM, Bidchol AM, Kamath PS, et al. (2014) A novel mutation (g.106737G>T) in zone of polarizing activity regulatory sequence (ZRS) causes variable limb phenotypes in Werner mesomelia. American Journal of Medical Genetics Part A 164A: 898–906.

Goryshin IY and Reznikoff WS (1998) Tn5 in vitro transposition. Journal of Biological Chemistry 273: 7367–7374.

Heintzman ND, Stuart RK, Hon G, et al. (2007) Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nature Genetics 39: 311–318.

Hyon C, Chantot‐Bastaraud S, Harbuz R, et al. (2015) Refining the regulatory region upstream of SOX9 associated with 46,XX testicular disorders of Sex Development (DSD). American Journal of Medical Genetics Part A 167A: 1851–1858.

Jeong Y, Leskow FC, El‐Jaick K, et al. (2008) Regulation of a remote Shh forebrain enhancer by the Six3 homeoprotein. Nature Genetics 40: 1348–1353.

Kleinjan DJ and van Heyningen V (1998) Position effect in human genetic disease. Human Molecular Genetics 7: 1611–1618.

Kurth I, Klopocki E, Stricker S, et al. (2009) Duplications of noncoding elements 5′ of SOX9 are associated with brachydactyly‐anonychia. Nature Genetics 41: 862–863.

Lettice LA, Daniels S, Sweeney E, et al. (2011) Enhancer‐adoption as a mechanism of human developmental disease. Human Mutation 32: 1492–1499.

Lettice LA, Williamson I, Wiltshire JH, et al. (2012) Opposing functions of the ETS factor family define Shh spatial expression in limb buds and underlie polydactyly. Developmental Cell 22: 459–467.

Levine M (2010) Transcriptional enhancers in animal development and evolution. Current Biology 20: R754–R763.

Li W, Notani D, Ma Q, et al. (2013) Functional roles of enhancer RNAs for oestrogen‐dependent transcriptional activation. Nature 498: 516–520.

Logan M, Simon HG and Tabin C (1998) Differential regulation of T‐box and homeobox transcription factors suggests roles in controlling chick limb‐type identity. Development 125: 2825–2835.

Lohan S, Spielmann M, Doelken SC, et al. (2014) Microduplications encompassing the Sonic hedgehog limb enhancer ZRS are associated with Haas‐type polysyndactyly and Laurin‐Sandrow syndrome. Clinical Genetics 86: 318–325.

Lupiáñez DG, Kraft K, Heinrich V, et al. (2015) Disruptions of topological chromatin domains cause pathogenic rewiring of gene‐enhancer interactions. Cell 161: 1012–1025.

Lupiáñez DG, Spielmann M and Mundlos S (2016) Breaking TADs: how alterations of chromatin domains result in disease. Trends in Genetics 32: 225–237.

Marinić M, Aktas T, Ruf S and Spitz F (2013) An integrated holo‐enhancer unit defines tissue and gene specificity of the Fgf8 regulatory landscape. Developmental Cell 24: 530–542.

Montavon T, Soshnikova N, Mascrez B, et al. (2011) A regulatory archipelago controls Hox genes transcription in digits. Cell 147: 1132–1145.

Murakawa Y, Yoshihara M, Kawaji H, et al. (2016) Enhanced identification of transcriptional enhancers provides mechanistic insights into diseases. Trends in Genetics 32: 76–88.

Niederriter AR, Varshney A, Parker SC and Martin DM (2015) Super enhancers in cancers, complex disease, and developmental disorders. Genes (Basel) 6: 1183–1200.

Noonan JP and McCallion AS (2010) Genomics of long‐range regulatory elements. Annual Review of Genomics and Human Genetics 11: 1–23.

Norbnop P, Srichomthong C, Suphapeetiporn K and Shotelersuk V (2014) ZRS 406A>G mutation in patients with tibial hypoplasia, polydactyly and triphalangeal first fingers. Journal of Human Genetics 59: 467–470.

Ovcharenko I, Nobrega MA, Loots GG and Stubbs L (2004) ECR Browser: a tool for visualizing and accessing data from comparisons of multiple vertebrate genomes. Nucleic Acids Research 32: W280–W286.

Palstra RJ, Tolhuis B, Splinter E, et al. (2003) The beta‐globin nuclear compartment in development and erythroid differentiation. Nature Genetics 35: 190–194.

Patrinos GP, de Krom M, de Boer E, et al. (2004) Multiple interactions between regulatory regions are required to stabilize an active chromatin hub. Genes and Development 18: 1495–1509.

Petit F, Jourdain AS, Holder‐Espinasse M, et al. (2016) The disruption of a novel limb cis‐regulatory element of SHH is associated with autosomal dominant preaxial polydactyly‐hypertrichosis. European Journal of Human Genetics 24: 37–43.

Rada‐Iglesias A, Bajpai R, Prescott S, et al. (2012) Epigenomic annotation of enhancers predicts transcriptional regulators of human neural crest. Cell Stem Cell 11: 633–648.

Smemo S, Campos LC, Moskowitz IP, et al. (2012) Regulatory variation in a TBX5 enhancer leads to isolated congenital heart disease. Human Molecular Genetics 21: 3255–3263.

Spielmann M, Brancati F, Krawitz PM, et al. (2012) Homeotic arm‐to‐leg transformation associated with genomic rearrangements at the PITX1 locus. American Journal of Human Genetics 91: 629–635.

Spitz F and Furlong EE (2012) Transcription factors: from enhancer binding to developmental control. Nature Reviews. Genetics 13: 613–626.

Tolhuis B, Palstra RJ, Splinter E, Grosveld F and de Laat W (2002) Looping and interaction between hypersensitive sites in the active beta‐globin locus. Molecular Cell 10: 1453–1465.

VanderMeer JE, Lozano R, Sun M, et al. (2014) A novel ZRS mutation leads to preaxial polydactyly type 2 in a heterozygous form and Werner mesomelic syndrome in a homozygous form. Human Mutation 35: 945–948.

Visel A, Blow MJ, Li Z, et al. (2009) ChIP‐seq accurately predicts tissue‐specific activity of enhancers. Nature 457: 854–858.

Weedon MN, Cebola I, Patch AM, et al. (2014) Recessive mutations in a distal PTF1A enhancer cause isolated pancreatic agenesis. Nature Genetics 46: 61–64.

Wieczorek D, Pawlik B, Li Y, et al. (2010) A specific mutation in the distant sonic hedgehog (SHH) cis‐regulator (ZRS) causes Werner mesomelic syndrome (WMS) while complete ZRS duplications underlie Haas type polysyndactyly and preaxial polydactyly (PPD) with or without triphalangeal thumb. Human Mutation 31: 81–89.

Xiao B, Ji X, Xing Y, Chen YW and Tao J (2013) A rare case of 46, XX SRY‐negative male with approximately 74‐kb duplication in a region upstream of SOX9. European Journal of Medical Genetics 56: 695–698.

Yao B, Wang Q, Liu CF, et al. (2015) The SOX9 upstream region prone to chromosomal aberrations causing campomelic dysplasia contains multiple cartilage enhancers. Nucleic Acids Research 43: 5394–5408.

Further Reading

Anderson E, Peluso S, Lettice LA and Hill RE (2012b) Human limb abnormalities caused by disruption of hedgehog signaling. Trends in Genetics 28: 364–373.

Dekker J and de Laat W (eds) (2012) 3D chromatin architecture. Methods 58: 189–314.

Diehl AG and Boyle AP (2016) Deciphering ENCODE. Trends in Genetics 32: 238–249.

Lam MT, Li W, Rosenfeld MG and Glass CK (2014) Enhancer RNAs and regulated transcriptional programs. Trends in Biochemical Sciences 39: 170–182.

Smith E and Shilatifard A (2014) Enhancer biology and enhanceropathies. Nature Structural and Molecular Biology 21: 210–219.

Zhang F and Lupski JR (2015) Non‐coding genetic variants in human disease. Human Molecular Genetics 24: R102–R110.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
Douglas, Adam, Peluso, Silvia, Lettice, Laura A, and Hill, Robert E(Aug 2016) Cis‐Regulatory Mutations in Human Disease. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0024920]