Single allele mutations at the of congenital disease

Nadia Rosenthal, Richard P. Harvey

J Clin Invest. 1999;104(11):1483-1484. https://doi.org/10.1172/JCI8825.

Commentary

A prevailing dream among today’s developmental biologists is that the genes they discover controlling basic embryonic processes in animal models will eventually surface in mutant form as the cause of human genetic disorders. This hope has now been realized for the NKX2.5 gene, which encodes a member of the family that is expressed in cardiac muscle during embryonic, fetal, and adult life (1). In the 7 years since it was first cloned from the mouse, Nkx2.5 has emerged as a key regulator of cardiac development in numerous vertebrate model systems, even those with the most primitive (2, 3). In genetically engineered mice lacking both copies of the Nkx2.5 gene, heart development is arrested, and the heart remains a primitive linear tube, which fails to loop or to develop definitive chambers (1). The early lethality caused by mutation of Nkx2.5 in mice is not surprising, given that its homologue tinman is absolutely essential for formation of the muscular heartlike structure in the fruit fly Drosophila (1). The number of downstream cardiac regulatory genes shown to be dependent upon Nkx2.5 has placed this gene near the top of a genetic hierarchy responsible for heart development in vertebrates. The disruption of a gene in laboratory mice involves first producing heterozygous offspring that carry a single null allele. The apparently […]

Find the latest version: https://jci.me/8825/pdf Single allele mutations at the heart Commentary of congenital disease See related article, pages 1567–1573. Nadia Rosenthal1 and Richard P. Harvey2,3

1Cardiovascular Research Center, Massachusetts General Hospital-East, Charlestown, Massachusetts 02129, USA 2Victor Chang Cardiac Research Institute, Darlinghurst, Sydney 2010, Australia 3Faculties of Medicine and Life Sciences, University of New South Wales, Kensington, Sydney 2052, Australia Address correspondence to: Nadia Rosenthal, Cardiovascular Research Center, Massachusetts General Hospital, 149 13th Street, Charlestown, Massachusetts 02129, USA. Phone: (617) 724-9560; Fax: (617) 724-9561; E-mail: [email protected].

A prevailing dream among today’s Diverse cardiac defects in families with gotes, looking for clues to the pheno- developmental biologists is that the NKX2.5 mutations. Several NKX2.5 mu- typic variability seen in families with genes they discover controlling basic tations have been discovered in fami- NKX2.5 mutations. In one mouse embryonic processes in animal models lies affected with cardiac defects. strain, preliminary studies have re- will eventually surface in mutant form Although the functional impact of vealed atrial septal defects in 20% of as the cause of human genetic disor- some of these mutations is uncertain, Nkx2.5 heterozygotes, along with con- ders. This hope has now been realized others, affecting amino acids within duction abnormalities (S. Izumo, per- for the NKX2.5 gene, which encodes a the DNA-binding homeodomain, sonal communication). In the original member of the homeobox transcrip- would certainly be disruptive to pro- knockout line, only milder atrial sep- tion factor family that is expressed in tein function. Unlike well-character- tal defects were found (R.P. Harvey, cardiac muscle during embryonic, ized dominant conditions such as unpublished data). Whether these dif- fetal, and adult life (1). In the 7 years Holt-Oram syndrome (6), in which car- ferences are due to genetic back- since it was first cloned from the diac and forelimb abnormalities are ground or to the nature of the target- mouse, Nkx2.5 has emerged as a key prominent features, specific NKX2.5 ed mutation is not yet clear. However, regulator of cardiac development in alleles do not appear to cosegregate it has become evident that back- numerous vertebrate model systems, with particular clinical phenotypes. A ground genetic differences be-tween even those with the most primitive wide variety of cardiac malformations individuals can profoundly modify hearts (2, 3). In genetically engineered appear in these families, predominant- the effect of a particular mutation. In mice lacking both copies of the Nkx2.5 ly atrial septal defect and atrioventric- Nkx2.5 knockout experiments, such gene, heart development is arrested, ular conduction block, but also ven- modifier genes that would be identical and the heart remains a primitive lin- tricular septal defects, tricuspid valve between individual laboratory mice ear tube, which fails to loop or to abnormalities, double-outlet right ven- kept on genetically homogeneous in- develop definitive chambers (1). tricle, and tetralogy of Fallot, a com- bred backgrounds might well vary The early lethality caused by muta- mon cause of la maladie bleue (blue between mice with different inbred tion of Nkx2.5 in mice is not surpris- babies). Ebstein’s malformation, a dis- backgrounds. The genetic homogene- ing, given that its homologue tinman is placement of the hinge-points of tri- ity of laboratory mice contrasts with absolutely essential for formation of cuspid valve leaflets, is now for the first the genetic heterogeneity of human the muscular heartlike structure in the time associated with a genetic locus (4). populations, but represents a power- fruit fly Drosophila (1). The number of Together, these studies represent a ful jumping-off point for tracking downstream cardiac regulatory genes significant breakthrough in our under- down the identity of genetic modifiers shown to be dependent upon Nkx2.5 standing of human heart defects. Car- that may alter the nature or severity of has placed this gene near the top of a diac abnormalities have been ascribed defects in humans. genetic hierarchy responsible for heart more often to complex interactions Rethinking haploinsufficiency. Although development in vertebrates. between genes and environment than modifier genes no doubt underlie some The disruption of a gene in laborato- to mendelian inheritance of single gene of the variability in NKX2.5-related ry mice involves first producing het- mutations. This perspective may now defects, the mouse studies hint at other erozygous offspring that carry a single need to be reevaluated. Single-allele causes. Abnormalities resulting from null allele. The apparently normal car- mutations underlying congenital ab- single null mutations are attributed to diac development in mice heterozy- normalities may be masked by factors haploinsufficiency, a term that suggests gous for an Nkx2.5 targeted mutation such as the less-than-perfect cosegre- that 2-fold reduction of expression initially led researchers to believe that gation of mutation and malformation underlies the observed defect. For genes the effects of its disruption were reces- (partial penetrance), variability in sever- such as NKX2.5 that encode transcrip- sive, but this view is now challenged by ity and nature of the abnormality (par- tion factors, haploinsufficiency is usu- two recent clinical studies (one in this tial expressivity), and asymptomatic ally defined as a below-threshold level issue of the JCI) reporting that multi- individuals seen in affected families (7). of protein produced from the single ple congenital heart defects can be In the wake of human studies, devel- normal allele, leading to pathologically ascribed to inherited mutations in one opmental biologists were quick to low levels of expression of downstream allele of the human NKX2.5 gene (4, 5). reexamine mouse Nkx2.5 heterozy- target genes. The molecular conse-

The Journal of Clinical Investigation | December 1999 | Volume 104 | Number 11 1483 quences of haploinsufficiency may could have profound will require further analysis. Neverthe- need to be reinterpreted in light of implications for the morphological less, the issues raised by these studies accumulating evidence that gene outcome of single allele mutations. are likely to generate new principles rel- expression is controlled in a probabilis- The range of defects in affected fam- evant to a wide spectrum of dominant tic manner. Thus, transcriptional ilies also suggests that the NKX2.5 human genetic diseases. enhancers, which bind transcription transcription factor participates in a factors, are thought to increase only the number of distinct morphogenetic Acknowledgments probability of gene activation within a processes in heart development that The authors wish to thank Seigo Izumo given cell, rather than the level of were previously obscured in homozy- for sharing unpublished results. expression (8). The implications of this gous mice due to early lethality. 1. Harvey, R.P. 1996. NK-2 homeobox genes and slight change in emphasis are pro- Although this notion is intriguing, the heart development. Dev. Biol. 178:203–216. found: a low level of NKX2.5 protein observed defects could also reflect 2. Fu, Y., Yan, W., Mohun, T.J., and Evans, S.M. expressed in heterozygotes may mean milder manifestations of the arrest of 1998. Vertebrate tinman homologues XNkx2-3 and XNkx2-5 are required for heart formation in that some cells, on a probabilistic basis, looping seen in mouse Nkx2.5 homo- a functionally redundant manner. Development. will not activate target genes at all, or zygotes, with individual outcomes 125:4439–4449. will activate them only sporadically. determined by modifier genes and 3. Grow, M.W., and Krieg, P.A. 1998. Tinman func- tion is essential for vertebrate heart development: Developmental events that operate in a probabilistic mechanisms. Looping elimination of cardiac differentiation by dominant small number of cells, or that occur sets the stage for many downstream inhibitory mutants of the tinman-related genes, during a narrow temporal window may remodeling events in heart develop- XNkx2-3 and XNkx2-5. Dev. Biol. 204:187–196. 4. Benson, D.W., et al. 1999. Mutations in the car- be especially prone to the effects of dis- ment, and failure to complete looping diac transcription factor NKX2.5 affect diverse rupting single alleles of regulatory has been proposed to explain the cardiac developmental pathways. J. Clin. Invest. genes. To complicate matters, an impressive variety of cardiac defects 104:1567–1573 5. Schott, J.-J., et al. 1998. Congential heart disease increasing number of transcription-fac- seen in trisomy 16 mice, which repre- caused by mutations in the transcription factor tor genes are being found that show sent a partial model of Down’s syn- NKX2-5. Science. 281:108–111. random monoallelic expression, i.e., drome (10). For the moment, there- 6. Basson, C.T., et al. 1999. Different TBX5 interac- tions in heart and limb defined by Holt-Oram only one allele is ever active in any one fore, we should not assume that all syndrome mutations. Proc. Natl. Acad. Sci. USA. cell (9). In individuals who are het- abnormal structures arising from 96:2919–2924. erozygous for a mutation, some cells NKX2.5 mutation reflect a direct role 7. Benson, D.W., et al. 1998. Reduced penetrance, vari- able expressivity, and genetic heterogeneity of may express the functional allele, but for the gene in their formation. familial atrial septal defects. Circulation. others express only the mutant allele, What are the clinical implications of 97:2043–2048. and therefore no functional protein at these studies? Genetic screening for 8. Fiering, S., Whitelaw, E., and Martin, D.I.K. 1999. To be or not to be active: the stochastic nature of all. If these mechanisms are operative in NKX2.5 mutations within families with enhancer action. Bioessays. In press. NKX2.5 heterozygotes, cells that acti- congenital abnormalities would be of 9. Nutt, S.L., et al. 1999. Independent regulation of vate NKX2.5 target genes during heart clear prognostic value in prenatal coun- the two Pax5 alleles during B-cell development. Nat. Genet. 21:390–395. development may be randomly deter- seling. Whether familial NKX2.5 muta- 10. Mjaatvedt, C.H., et al. 1998. Mechanisms of seg- mined, and may therefore vary enor- tions turn out to be more frequent than mentation, septation, and remodelling of the mously in number and spatial distribu- expected in the general population, and tubular heart: endocardial cushion fate and car- diac looping. In Heart development. R.P. Harvey and tion between affected individuals. In whether NKX2.5 mutations are signifi- N. Rosenthal, editors. Academic Press. San Diego, this way, probabilistic mechanisms of cant genetic factors in idiopathic cases CA. 159–177.

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