American Journal of Medical Genetics 101:356±358 (2001)

Genetics of Human Situs Abnormalities

Brett Casey*

Department of Pathology, Baylor College of Medicine and Texas Children’s Hospital, Houston, Texas

Human left-right malformations are usuallysporadic, but many familial cases have beendescribed. Recognition of these families aswell as sporadic cases with underlyingcytogenetic abnormalities suggest geneticpredisposition for many instances of situsmalformations. Studies in nonhuman verte-brates have led to the discovery of severalgenes conserved in normal left-right devel-opment, and mutations in some of thesehave been identi®ed in humans. In addition,positional cloning efforts have yielded somesuccess in enlarging our understanding ofthe molecular genetics of human left-rightanomalies. ß 2001 Wiley-Liss, Inc.

KEY WORDS: left-right axis; situs inver-sus; situs ambiguus; hetero-taxia; ZIC3

NOMENCLATURE AND CLINICALCONSIDERATIONS

The normal left-right anatomic arrangement is calledsitus solitus. Mirror-image reversal of all asymmetricstructures has been given a variety of labels, mostcommonly situs inversus, complete or total situsinversus, and situs inversus totalis. The resultinganatomy is more of a curiosity than a hazard. Whenthe entire anatomic left±right axis is neither normalnor mirror-image reversed, the resulting phenotypehas been called situs ambiguus, partial situs inversus,heterotaxy or heterotaxia (sometimes accompanied bythe adjective visceral), laterality or isomerismsequence, and Ivemark, asplenia, or polyaspleniasyndrome. All of these terms describe the same generalphenotype: discordant left±right anatomy within andamong the lateralized structures of the chest andabdomen. Complex, often fatal heart malformationsare common (although not invariable), as are intestinalmalrotation and abnormalities of spleen position and/ornumber.

Rough estimates place the incidence of left±rightmalformations at 1/5000 births, with cases dividedequally between situs inversus and situs ambiguus[Ferencz et al., 1993; Afzelius and Mossberg, 1995].This ®gure may underestimate the actual incidence ofeach. Complete left±right reversal (situs inversus) mayescape detection because it poses no detriment to theindividual, and cases of situs ambiguus with normalhearts or those with clinically silent cardiac malforma-tions also may not come to medical attention. Forexample, in a retrospective review of 18 patients withintestinal malrotation, seven were found also to havepolysplenia, and six of these also had either interruptedor double inferior vena cava [Zissin et al., 1999]. None ofthese patients had been determined previously toharbor left±right axis malformations.

Many familial cases of situs abnormalities have beendescribed in which also one or more family membershave only cardiac malformations that would not bedescribed by most cardiologists as being the conse-quence of abnormal left±right development [Alonsoet al., 1995; Casey, 1998]. However, given the familybackground and the fact that these isolated heartmalformations also occur among individuals withobvious situs ambiguus, one might entertain thehypothesis that these and perhaps other cases ofisolated heart malformations may be the result ofabnormal laterality. Recently, Bouvagnet and collea-gues [MeÂgarbane et al., 2000] have provided experi-mental evidence in support of this hypothesis (seebelow).

GENES AND ENVIRONMENT

Clearly, both genes and environment can contributeto the development of left±right axis malformations.Retinoic acid exposure, for example, can inducelaterality defects in a variety of vertebrates, includingHomo sapiens. Much more important from a publichealth standpoint, however, is the increased risk ofleft±right malformations in the offspring of motherswith (nongestational) diabetes mellitus [Splitt et al.,1999]. Whether or not left±right malformationsdevelop in the offspring may depend on his or hergenetic background. Morishima and colleagues havedeveloped lines of nonobese diabetic (NOD) mice with ahigh incidence of situs abnormalities among offspring ifthe dam is hyperglycemic early in gestation [Morishimaet al., 1991]. The incidence is highest (65%) if the sire is

*Correspondence to: Brett Casey, M.D., Department of Pathol-ogy, Baylor College of Medicine, One Baylor Plaza, Houston, TX77030. E-mail: bcasey@bcm.tmc.edu

Received 8 November 2000; Accepted 1 December 2000

ß 2001 Wiley-Liss, Inc.

from the NOD strain, decreases to 24% when the sire isfrom strain ICR (from which NOD was derived), anddecreases to background levels with C57Bl sires[Morishima et al., 1996]. These results suggest thatthe genetic background of the embryo lowers thethreshold for malformations induced by environmentalagents.

CYTOGENETICS

Recognition of familial situs abnormalities during thepast several decades has led to the obvious conclusionthat a strong genetic predisposition underlies theseanomalies, at least in some cases. Cytogenetic abnorm-alities in association with situs malformations supportthis conclusion. Trisomies and monosomies (completeand partial), translocations (balanced and unbalanced),inversions, deletions, and even isodisomy have all beendescribed [Kosaki and Casey, 1998]. Presumably thesecytogenetic alterations provide clues to the location ofgenes involved in left±right axis development.

Unfortunately, no such gene has been discovered byexploiting a cytogenetic rearrangement. Iida andcolleagues recently reported that UVRAG, a genepreviously implicated in the pathogenesis of xerodermapigmentosum, was disrupted in the 11q inversion foundin a case of situs ambiguus [Iida et al., 2000]. This genehas not been implicated in left±right development ofnonhuman vertebrates, and no mutation has beenfound in an unrelated case, so it remains unclearwhether or not disruption of this gene is causallyrelated to the accompanying phenotype.

A more convincing connection between left±rightgenes and cytogenetic abnormalities is found in thecase of a de novo interstitial deletion in chromosome10q22 [Carmi et al., 1992]. Situs ambiguus and midlinemalformations are the accompanying phenotype. Themurine gene nodal and its counterpart in othervertebrates has been shown to be required for normalleft±right axis development. Human NODAL maps to10q22 and, based on polymorphic microsatelllite ana-lysis, is deleted in the 10q22 deletion chromosome [B.Casey, unpublished results].

LINKAGE MAPPING

Cytogenetic abnormalities associated with left±rightmalformations are quite uncommon, and when identi-®ed have not been helpful to date in identifying newgenes associated with left±right axis development.Linkage studies have for the most part been equallyunhelpful. Several reasons account for this. Largefamilies with many affecteds and obligate carriers arerare, in part because the malformations are often lethalbefore the individuals reach reproductive age. Giventhe number of genes involved in left±right development(and therefore potentially involved in left±right mal-formations), the underlying genetic defect is unlikely tobe the same in any two or more large families.

Morelli and colleagues have detected linkage tochromosome 3p in a single family with LR malforma-tions, and Vitale and colleagues have provided sugges-

tive (but not convincing) evidence for linkage of a left±right gene to chromosome 6p [Vitale et al., in press;Morelli et al., 2001]. The results point to very largeregions within their respective chromosomes, suggest-ing that it will be very dif®cult to identify the causativegenes by traditional positional cloning without addi-tional data to narrow the critical regions.

CANDIDATE GENES

Despite the dif®culties, some data have accumulatedconnecting phenotype with underlying genotype inhuman left±right malformations. Spectacular successin the study of left±right development among verte-brate model organisms has yielded a large number ofcandidate genes for study in human left±right mal-formations. Mutations in human homologues of themouse genes nodal, ActRIIb, and lefty have beendescribed [Bassi et al., 1997; Kosaki K. et al., 1999;Kosaki R. et al., 1999] Most recently, Muenke andcolleagues have reported loss-of-function mutations inthe human homologue of cryptic, a gene essential fornormal left±right development in mouse [Muenke,personal communication].

ZIC3: MUTATION, MOUSE, AND MIDLINE

Mutations in ZIC3, an X-linked gene encoding atranscription factor, have been identi®ed among bothsporadic and familial cases of situs abnormalities[Gebbia et al., 1997]. Males hemizygous for loss-of-function mutations manifest situs ambiguus and,occasionally, midline abnormalities as well. In fact,situs ambiguus and midline anomalies (particularly ofthe posterior neural tube or of the hindgut) suggest thepresence of an underlying ZIC3 mutation. Usuallycarrier females are free of malformations, but in onefamily, some females heterozygous for a ZIC3 mutationare situs inversus, whereas the affected males are situsambiguus.

Two recent studies suggest that ZIC3 may beinvolved in malformations not necessarily thought tobe directly associated with normal left±right develop-ment. Purandare and colleagues (personal communica-tion) have reported discovery of an interstitial deletionin Xq26 that includes ZIC3. Several family membershave inherited the deletion, including an affected malewith features of VACTERL-H but without character-istic situs abnormalities [B. Casey, unpublishedresults]. Bouvagnet and colleagues have described afamily harboring a ZIC3 mutation in which affectedmales have transposition of the great vessels andmidline anomalies but no obvious left±right malforma-tions.[Megarbane et al., 2000]. Furthermore, there is amale in this family who harbors the mutation but isanatomically normal.

Midline malformations associated with ZIC3 muta-tions suggest that this gene may be essential for normalmidline development rather than directly involved inleft±right axis development per se. Targeted disruptionof murine Zic3 supports this hypothesis. All Zic3-nullmice that survive intrauterine development have a

Left±Right Malformations and Mutations 357

kinked tail, and approximately 10% manifest othermalformations. Among these are heart anomalies,altered lung lobation, and gut malrotation, all char-acteristic of abnormal left±right speci®cation. Malfor-mations of the neural tube and axial skeleton appearindependently of left±right-axis anomalies and withequal frequency. Skeletal abnormalities include asym-metric, homeotic transformations.

Symmetric nodal expression at the node in Zic3-de®cient mice begins appropriately but disappearsrather than becoming asymmetric as in the wild type.Subsequent expression of nodal and the downstreamgene Pitx2 in the lateral plate mesoderm is randomizedrather than consistently left-sided. These resultssuggest that Zic3 functions in murine left±rightdevelopment by helping to maintain nodal expressionat the node.

CONSIDERATIONS FOR THE FUTURE

Most of what we know about vertebrate left±rightasymmetry has been discovered only in the last fewyears. Chick, mouse, frog, and zebra®sh are proving tobe powerful, complementary systems in which to sortout the biology of this developmental process. However,the application of this new knowledge to understandingthe etiology of human left±right malformations isproving to be complicated. Already, more than 20 geneshave been implicated in vertebrate left±right asym-metry, and surely there are many more to be uncov-ered. All are reasonable candidates for human disease,but none is likely to be associated with more than just asmall percentage of cases, given the genetic complexityof the system. Furthermore, work in mouse has shownus that normal left±right development is sensitive toappropriate gene dosage, such that mice doubly-heterozygous for some gene knock-outs manifest left±right malformations, whereas the single heterozygotesdo not. This observation, along with the NOD mouseexperiments, suggests the somewhat daunting hypoth-esis that the underlying genetics of many human casesmay be quite complex, just as it is for adult-onsetdiseases such as hypertension and diabetes. Theunusually rich supply of mouse models for humanleft±right malformations will prove to be invaluable insorting out this hypothesis.

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