Development 103 Supplement, 233-239 (1988)Printed in Great Britain © The Company of Biologists Limited 1988

233

The application of molecular genetics to detection of craniofacial

abnormality

GUDRUN MOORE1, ALASDAJR JVENS1, JOANNA CHAMBERS', ARNI BJORNSSON2,

ALFRED ARNASON3, OLAFUR JENSSON3 and ROBERT WILLIAMSON1

1 Department of Biochemistry and Molecular Genetics, St Mary's Hospital Medical School, University of London, London W2 IPG, UK,2Department of Plastic Surgery and ^Genetics Division of the Blood Bank, National University Hospital, Reykjavik, Iceland

Summary

Congenital malformations such as secondary cleftpalate can be exclusively monogenic or polygenic, butmost cases have a multifactorial origin involving bothenvironmental and genetic factors, making geneticanalysis difficult. The new techniques of moleculargenetics have allowed the successful chromosomallocalization of mutant genes in disorders that show asimple Mendelian segregation, whether autosomaldominant (e.g. Huntington's disease), autosomal re-cessive (cystic h'brosis) or X-linked (Duchenne muscu-lar dystrophy). Recently, a large Icelandic family(over 280 members) with X-linked secondary cleftpalate and ankyloglossia (tongue-tied) has been usedas a model to localize the mutant gene associated withthis craniofacial clef ting. The gene has been sub-chromosomally localized to Xql3-q21.1, using anony-mous probe DXYS1; a LOD score of 307 was ob-tained.

We are preparing cosmid libraries from DNA frommouse cell lines containing only the relevant part ofthe human X chromosome, introduced by chromo-some-mediated gene transfer. Cosmids that contain

human X-chromosome sequences will be isolated andanalysed for overlapping sequences and RFLPs (re-striction fragment length polymorphisms) and theregions further defined by pulsed-field gel electrophor-esis and the identification of coding sequences. Thisshould give data on the location and structure of a geneinvolved in the craniofacial development of the humanpalatine shelves. This gene, and its protein product,will identify one component of the pathway that causesnonfusion of the palate. In the long term, the under-standing of the expression of this sex-linked gene forsecondary cleft palate and ankyloglossia will provide amodel for the molecular identification of other genesregulating processes in craniofacial developmentwhose expression is hidden in phenotypic, polygeniccomplexity.

Key words: molecular genetics, cleft palate, DNA, X-chromosome, Y-linked disorders, ankyloglossia,craniofacial malformation.

Introduction

The majority of congenital craniofacial malforma-tions occur during the 5-12 weeks of development.The earlier part of this period approximates to thetime in which teratogens can harm the developingfetus, spanning the embryonic period from 3 to 9weeks postconception (Moore, 1982). The most com-mon congenital abnormality of the face is cleft lip(CL), a condition which involves nonfusion of theupper lip and the anterior part of the maxilla during

weeks 5-7 and occurs at an incidence of approxi-mately 1 in 1000 births (Thompson & Thompson,1986). Although CL is frequently associated with cleftpalate (CP), CL and CP are different both temporallyand with respect to developmental lineage.

CP alone results from failure of the mesenchymalmasses of the palatine processes to fuse during weeks7-12. Generally, combined cleft lip and palate(CL+P) and CL alone are more frequent in males,whereas for isolated CP the reverse is true (Fogh-

234 G. Moore and others

Molecular genetics of craniofacial abnormality 235

Table 1. Recurrence risks (%) for cleft lip ± cleft palate

Number sibs affected

0

One sib andone second-

degree relativeaffected

Neither parent affected 01 3 9One parent affected 3 11 19Both parents affected 34 40 45

Data from Bonaiti-Pellie & Smith, 1974.

One sib andone third-

degree relativeaffected

61643

41444

Andersen, 1942). Due to both the genetic and devel-opmental evidence it is deemed justifiable to treatcleft lip with or without cleft palate (CL±CP) as aseparate entity from CP alone (Kernahan & Stark,1958).

The incidence of CL ± CP is higher than that of CP,varying from 2-1/1000 in Japan to 0-4/1000 in Nigeria(Leek, 1984), with the geographical variation beingless important than ethnic differences. However, CPalone has an average incidence of 0-7/1000 and showslittle variation in different racial groups. This maymean that CP alone will not fit the purely multifac-torial model. Such a model includes both polygenicorigin and undefined 'environmental' factors that willincrease the variation in incidence both geographi-cally and to some extent racially. Previous studieshave shown that CP may include both polygenic andmonogenic types (Bixler, Fogh-Anderson & Con-neally, 1971; Bear, 1976). To date, there have beenonly three pedigrees reported in which CP is clearlyinherited as a single-gene X-linked disorder (Lowry,1970; Rushton, 1979; Bjornsson & Arnason, 1986).

Fig. 1. The pedigree of 293 individuals in the familystudied is shown in Fig. 1; three of us (O.J., A. A. andA.B.) have personally investigated 182 individuals ingenerations IV-VII. Information on other familymembers was gained either from written or familyrecords. Blood was collected from 82 individuals forRFLP genotyping. These include nine males withsecondary cleft palate and ankyloglossia (CP+A) and tenfemales with ankyloglossia alone (A). One female hadCP alone and one male had a patent but high-vaultedpalate characteristic of those affected. For purposes ofgenetic analysis, each family member was designatedaffected if they had any of the features of CP or A. Thegene is almost certainly sex-linked, since of the 14matings where a CP+A father has the opportunity topass a mutant gene to his son, in no case is a sonaffected. This shows that this gene is almost certainly sex-linked as opposed to an autosomal gene with a sex-difference in expression. Phenotypically unaffectedfemales who may be assumed to carry the mutant allelecan have affected sons and daughters, verifying that thisgene is an X-linked dominant with reduced penetrance.Penetrance was calculated from within the family and wasfound to be 82 % in the female carriers.

Multifactorial traits are defined as those that aredetermined by a combination of factors, genetic andnongenetic, each with a minor but additive effect(such as blood pressure) (Thompson & Thompson,1986). It is important to distinguish between multifac-torial and polygenic inheritance; the latter termshould be used in a more restricted sense withreference to conditions determined exclusively by alarge number of genes, each with a small effect,acting additively, such as hair colour (Fraser, 1976).Multifactorial inheritance is more difficult to analysethan other types of inheritance, but is thought toaccount for much of the normal variation in families,as well as for many common disorders, includingcongenital malformations. The genetic contributionto multifactorial inheritance has traditionally beenanalysed by comparing the ratio of the concordancerate of the condition in monozygotic and dizygotictwin pairs with the incidence of the trait in the generalpopulation.

It appears that many congenital malformationsresult from a failure in a specific developmentalprocess so that subsequent normal stages cannot bereached. The normal rate of development can bethought of as a continuous distribution, resultingfrom many environmental and genetic factors. If thenormal rate of development is perturbed, a seriousmalformation may result, dividing the continuousvariable distribution into a normal and abnormal classseparated by a threshold. Several human congenitalmalformations show family patterns that fit thismultifactorial-threshold model, CL ± CP and CP be-ing two of them. Analysis is made by collection ofinformation on the frequency of the malformation inthe general population and in different categories ofrelatives. This allows the empirical risk of the malfor-mation to be estimated in subsequent pregnancies.This risk is based solely on past occurrences and doesnot rely on knowledge of the genetic and environ-mental factors that give rise to the malformation(Table 1 gives recurrence risks).

Research on both the genetics and environmentalfactors involved in cleft palate has been carried out

236 G. Moore and others

\ZTTQ

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no •

samP

n n0 0

s sa am mP P1 Ie e

1211Kb

Ankyloglossia

Cleft 2° palate

Obligate carrier

and Unaffected

* Y-specific band

Fig. 3. To^I-digested DNA samples from the pedigreeare shown hybridized to the anonymous DXYSlmarker.Section of the Icelandic family showing the X-linkedinheritance of CP and A.Methods

DNA extractionGenomic DNA was prepared from 10 ml of whole

blood collected in EDTA (Kunkel et al. 1977).Restriction enzyme analysisThe analysis of inheritance of RFLPs was carried out

using standard techniques. 4/zg DNA was digested withthe restriction enzyme revealing the polymorphism foreach X-chromosome probe. The digested DNA wasfractionated by electrophoresis on 1 % agarose gels andtransferred to Hybond-N™ membranes (AmershamInternational) (Southern, 1975). The probes used werelabelled to a specific activity of lxl09disintsmin~' /ig"1

by synthesis using random oligonucleotide primers(Feinberg & Vogelstein, 1983). The filters were washeddown to a final salt concentration of OlxSSC (SSC is015M-NaCl, 015M-sodium citrate) in the presence of0-1 % SDS at 65°C. Autoradiography was for 24 h at-70°C with an intensifying screen. For the linked probepDP34 in this figure, three bands are seen. The 13 kbband is Y-chromosome specific and can be seen in allmales. The polymorphism for this probe gives two bandsat 12 and 11 kb. The 12 kb band segregates with theCP+A mutation, i.e. all affected males and femalecarriers must show the 12 kb band.

using rats and different mouse strains, and epidemio-logical studies followed in man. The nucleotide ana-logue FUDR has been found to inhibit acid muco-polysaccharide synthesis and to decrease palatine

shelf force inducing CP in rat fetuses (Ferguson,1978). Mutant genes in mice have been associatedwith CP cho, which inhibits jaw growth (Seegmiller &Fraser, 1977) and ur, which reduces palatine shelfwidth (Fitch, 1957).

In man, associations of CP and CL ± CP with HLAtypings have been excluded (Van Dyke, Goldman,Spielman & Zmijewski, 1983; Van Dyke, Goldman,Spielman, Zmijewski & Oka, 1980) despite evidenceof associations of the mouse MHC regions H-2 andH-3 with glucocorticoid- and phenytoin-induced CP(Gasser, Mele, Lees & Goldman, 1981a; Gasser,Mele & Goldman, 19816). It is still not clear exactlyhow the genes involved in the failure of the palate tofuse, and potential thresholds to teratogenic agents,combine; large parts of the complex biochemicalmechanisms are still only postulated at the theoreticallevel. There have been few definitive findings whichhold up in more than a single model system, and anygenetic or environmental factor that appears criticalin one case can be excluded in another.

The analysis of single gene mutations using RFLPsfor linkage studies has had considerable success indetermining the chromosomal location of severalcommon inherited disorders. RFLPs are specificenzyme sites that are polymorphic and segregate inMendelian fashion. These sites can segregate in alinked or unlinked manner depending on their prox-imity to the disease locus. Both in cases where theaffected chromosome is known, as for Duchennemuscular dystrophy (Davies et al. 1983) and chronicgranulomatous disease (Royer-Pokura et al. 1986),and when neither the autosome nor the biochemicaldefect is identified, as for Huntington's disease(Gusella et al. 1983) and cystic fibrosis (Knowlton etal. 1985; Wainwright et al. 1985; White et al. 1985),linkage to within five centimorgans has beenachieved. However, the analysis of polygenic dis-orders by linkage studies with RFLP markers is atpresent more complex both practically and theoreti-cally.

One approach to dissecting the aetiology of dis-orders with complex combinations of genetic andenvironmental factors is to use as a model a family inwhich the phenotype is due to a single gene defect,but displays the same features as more commonmultifactorial and sporadic cases. Such a 'model' formidline congenital defects has been found in a largeIcelandic family (over 280 individuals) (Fig. 1) show-ing Mendelian inheritance of X-linked secondarycleft palate and ankyloglossia ('tongue-tied')(CP+A) (Fig. 2). Both the large size of this pedigreeand the availability of many well-localized X-chromo-some probes has made it possible to localize thisdefect subchromosomally. The CP+A locus has beenfound to be closely linked to one anonymous DNA

2A

B

Fig. 2. Photographs of the disorder in affected male children showing the variation in the severity of the cleft palate andankyloglossia. (A) Partial clefting of the secondary palate; (B) complete clefting of the secondary palate; (C)ankyloglossia or tongue-tied.

Molecular genetics of craniofacial abnormality 237

22-3

22-2

22-1

21-3

21-2

211

11-4

11-3

11-23

11-2211-211111112112-2

13

211

21-2

21-3

22122-222-3

23

24

25

26

27

28

KY6 D1C 56

pD2

C7

pERT

pOTC

ILl-28

p581

IpDP34

CpX203

G3-1

7b

X65H7

S21/S9

A2-7

DX13

4315

S21/S9

43-15

A2-7

DX13

-3 - 2 - 1 +1 +2 +3

LOD score

Fig. 4. Diagramatic description of the X chromosome showing both the in situ localization of the X probes and theirmaximum LOD scores in the linkage analysis of the CP+A family.Linkage and segregation analysis

Combined LOD scores were calculated using the computer program package LINKAGE as described by Lathrop(1984). LOD = decimal logarithm of odds ratio, likelihood of observed recombination frequency to likelihood at 50 %recombination (completely unlinked). The standard significant cut off points for positive linkage and exclusion are +3and —2 respectively. Multipoint linkage analysis between informative markers was carried out as described in Farrall,Scambler, North & Williamson (1986). Fig. 4 gives the maximum LOD scores at recombination fractions from 0 to 0-5for all the informative probes. These data locate CP+A to Xql3-Xq21-1.

The exclusion map on Fig. 4 is composed of 14 probes that did not show linkage to CP+A. Minimal multipointlinkage was feasible between these probes due to their low information content within this family. However from thesedata large sections of Xq and Xp have been excluded.

238 G. Moore and others

probe DXYS1 (probe name pDP34), which maps toXql3-21.1 (Moore et al. 1987) (Fig. 3).

Once localization on the X chromosome has beenmore accurately achieved with finer mapping, cosmidlibraries can be used to jump or walk from specificrestriction enzyme sites to the physical location of thegene. Subsequent cloning and sequencing of thenormal gene and its mutation and analysis of itsfunctional expression using cDNA libraries andNorthern blots will allow one part of the pathway thatleads to cleft palate to be clarified. Autosomal se-quences may either be homologous, or specify otherparts of the same pathway, or at least give clues topotential candidate genes involved in neural crest celland craniofacial development.

Discussion

Localization of the mutation causing cleft palate inthis family to Xql3-21.1 is a first step in understand-ing the genetic component of congenital neural crestdefects. This region of the X chromosome contains anXY homologous region (Page, Harper, Love &Botstein, 1982). As the limits of genetic mapping inthis family are approached, the techniques of cosmidwalking and jumping and pulsed-field gel electro-phoresis (Schwartz & Cantor, 1984; Poustka & Lehr-ach, 1986) will allow the gap between the genetic andphysical map to be bridged.

The isolation of the gene and studies of its spatialand temporal expression during development of thesecondary palate should lead us to a greater under-standing of mechanisms which may lead to the failureof closure of the palate. This mechanism can becompared with those which may cause sporadic casesin humans, or in animal models. There are alsofamilies known in which an X-linked form of spinabifida and anencephaly occur (Burn & Gibbens, 1979;Jensson, Arnason, Gunnarsdottir, Petursdottir, Foss-dal & Hreidarsson, 1987); any homology in linkagebetween these families and the CP+A Icelandicfamily would be of interest.

The cloning of this mutation can now be used as astarting point for the analysis of other developmentalabnormalities. For example, this gene may be amember of a multigene family involved in the embry-onic development of other tissues. Bodmer (1983) hassuggested that genes with related functions whosecoordinated expression gives rise to complex pheno-types may be clustered on one chromosomal region assupergenes. In mouse, five related homoeotic genesare known to be clustered in a supergene family at asingle genetic locus. Such structure-function re-lationships may allow direct entry into complexgenetic systems involved in pattern formation andontogeny. Analysis of CP+A as a sex-linked single

gene provides a model for other genes regulatingprocesses in embyronic development.

We thank Action Research for the Crippled Child andthe Medical Research Council for generous financial sup-port, Kay Davies, Ken Kidd, Sue Chamberlain and KeithJohnson for many helpful discussions, all those who gener-ously provided probes, and the family for their full coop-eration.

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