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Development 103 Supplement, 233-239 (1988)
Printed in Great Britain © The Company of Biologists Limited 1988
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
2
Department of Biochemistry and Molecular Genetics, St Mary's Hospital Medical School, University of London, London W2 IPG, UK,
Department of Plastic Surgery and ^Genetics Division of the Blood Bank, National University Hospital, Reykjavik, Iceland
Summary
Congenital malformations such as secondary cleft
palate can be exclusively monogenic or polygenic, but
most cases have a multifactorial origin involving both
environmental and genetic factors, making genetic
analysis difficult. The new techniques of molecular
genetics have allowed the successful chromosomal
localization of mutant genes in disorders that show a
simple Mendelian segregation, whether autosomal
dominant (e.g. Huntington's disease), autosomal recessive (cystic h'brosis) or X-linked (Duchenne muscular dystrophy). Recently, a large Icelandic family
(over 280 members) with X-linked secondary cleft
palate and ankyloglossia (tongue-tied) has been used
as a model to localize the mutant gene associated with
this craniofacial clefting. The gene has been subchromosomally localized to Xql3-q21.1, using anonymous probe DXYS1; a LOD score of 3 0 7 was obtained.
We are preparing cosmid libraries from DNA from
mouse cell lines containing only the relevant part of
the human X chromosome, introduced by chromosome-mediated gene transfer. Cosmids that contain
human X-chromosome sequences will be isolated and
analysed for overlapping sequences and RFLPs (restriction fragment length polymorphisms) and the
regions further defined by pulsed-field gel electrophoresis and the identification of coding sequences. This
should give data on the location and structure of a gene
involved in the craniofacial development of the human
palatine shelves. This gene, and its protein product,
will identify one component of the pathway that causes
nonfusion of the palate. In the long term, the understanding of the expression of this sex-linked gene for
secondary cleft palate and ankyloglossia will provide a
model for the molecular identification of other genes
regulating processes in craniofacial development
whose expression is hidden in phenotypic, polygenic
complexity.
Introduction
weeks 5-7 and occurs at an incidence of approximately 1 in 1000 births (Thompson & Thompson,
1986). Although CL is frequently associated with cleft
palate (CP), CL and CP are different both temporally
and with respect to developmental lineage.
CP alone results from failure of the mesenchymal
masses of the palatine processes to fuse during weeks
7-12. 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-
The majority of congenital craniofacial malformations occur during the 5-12 weeks of development.
The earlier part of this period approximates to the
time in which teratogens can harm the developing
fetus, spanning the embryonic period from 3 to 9
weeks postconception (Moore, 1982). The most common congenital abnormality of the face is cleft lip
(CL), a condition which involves nonfusion of the
upper lip and the anterior part of the maxilla during
Key words: molecular genetics, cleft palate, DNA, Xchromosome, Y-linked disorders, ankyloglossia,
craniofacial malformation.
234
G. Moore and others
Molecular genetics of craniofacial abnormality
235
Table 1. Recurrence risks (%) for cleft lip ± cleft palate
Number sibs affected
0
Neither parent affected
One parent affected
Both parents affected
01
3
34
3
11
40
9
19
45
One sib and
one seconddegree relative
affected
One sib and
one thirddegree relative
affected
6
16
43
4
14
44
Data from Bonaiti-Pellie & Smith, 1974.
Andersen, 1942). Due to both the genetic and developmental evidence it is deemed justifiable to treat
cleft lip with or without cleft palate (CL±CP) as a
separate 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 being
less important than ethnic differences. However, CP
alone has an average incidence of 0-7/1000 and shows
little variation in different racial groups. This may
mean that CP alone will not fit the purely multifactorial model. Such a model includes both polygenic
origin and undefined 'environmental' factors that will
increase the variation in incidence both geographically and to some extent racially. Previous studies
have shown that CP may include both polygenic and
monogenic types (Bixler, Fogh-Anderson & Conneally, 1971; Bear, 1976). To date, there have been
only three pedigrees reported in which CP is clearly
inherited as a single-gene X-linked disorder (Lowry,
1970; Rushton, 1979; Bjornsson & Arnason, 1986).
Fig. 1. The pedigree of 293 individuals in the family
studied is shown in Fig. 1; three of us (O.J., A. A. and
A.B.) have personally investigated 182 individuals in
generations IV-VII. Information on other family
members was gained either from written or family
records. Blood was collected from 82 individuals for
RFLP genotyping. These include nine males with
secondary cleft palate and ankyloglossia (CP+A) and ten
females with ankyloglossia alone (A). One female had
CP alone and one male had a patent but high-vaulted
palate characteristic of those affected. For purposes of
genetic analysis, each family member was designated
affected if they had any of the features of CP or A. The
gene is almost certainly sex-linked, since of the 14
matings where a CP+A father has the opportunity to
pass a mutant gene to his son, in no case is a son
affected. This shows that this gene is almost certainly sexlinked as opposed to an autosomal gene with a sexdifference in expression. Phenotypically unaffected
females who may be assumed to carry the mutant allele
can have affected sons and daughters, verifying that this
gene is an X-linked dominant with reduced penetrance.
Penetrance was calculated from within the family and was
found to be 82 % in the female carriers.
Multifactorial traits are defined as those that are
determined by a combination of factors, genetic and
nongenetic, each with a minor but additive effect
(such as blood pressure) (Thompson & Thompson,
1986). It is important to distinguish between multifactorial and polygenic inheritance; the latter term
should be used in a more restricted sense with
reference to conditions determined exclusively by a
large number of genes, each with a small effect,
acting additively, such as hair colour (Fraser, 1976).
Multifactorial inheritance is more difficult to analyse
than other types of inheritance, but is thought to
account for much of the normal variation in families,
as well as for many common disorders, including
congenital malformations. The genetic contribution
to multifactorial inheritance has traditionally been
analysed by comparing the ratio of the concordance
rate of the condition in monozygotic and dizygotic
twin pairs with the incidence of the trait in the general
population.
It appears that many congenital malformations
result from a failure in a specific developmental
process so that subsequent normal stages cannot be
reached. The normal rate of development can be
thought of as a continuous distribution, resulting
from many environmental and genetic factors. If the
normal rate of development is perturbed, a serious
malformation may result, dividing the continuous
variable distribution into a normal and abnormal class
separated by a threshold. Several human congenital
malformations show family patterns that fit this
multifactorial-threshold model, CL ± CP and CP being two of them. Analysis is made by collection of
information on the frequency of the malformation in
the general population and in different categories of
relatives. This allows the empirical risk of the malformation to be estimated in subsequent pregnancies.
This risk is based solely on past occurrences and does
not rely on knowledge of the genetic and environmental factors that give rise to the malformation
(Table 1 gives recurrence risks).
Research on both the genetics and environmental
factors involved in cleft palate has been carried out
236
G. Moore and others
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Fig. 3. To^I-digested DNA samples from the pedigree
are shown hybridized to the anonymous DXYSlmarker.
Section of the Icelandic family showing the X-linked
inheritance of CP and A.
Methods
DNA extraction
Genomic DNA was prepared from 10 ml of whole
blood collected in EDTA (Kunkel et al. 1977).
Restriction enzyme analysis
The analysis of inheritance of RFLPs was carried out
using standard techniques. 4/zg DNA was digested with
the restriction enzyme revealing the polymorphism for
each X-chromosome probe. The digested DNA was
fractionated by electrophoresis on 1 % agarose gels and
transferred to Hybond-N™ membranes (Amersham
International) (Southern, 1975). The probes used were
labelled to a specific activity of lxl0 9 disintsmin~' /ig"1
by synthesis using random oligonucleotide primers
(Feinberg & Vogelstein, 1983). The filters were washed
down to a final salt concentration of OlxSSC (SSC is
015M-NaCl, 015M-sodium citrate) in the presence of
0-1 % SDS at 65°C. Autoradiography was for 24 h at
-70°C with an intensifying screen. For the linked probe
pDP34 in this figure, three bands are seen. The 13 kb
band is Y-chromosome specific and can be seen in all
males. The polymorphism for this probe gives two bands
at 12 and 11 kb. The 12 kb band segregates with the
CP+A mutation, i.e. all affected males and female
carriers must show the 12 kb band.
using rats and different mouse strains, and epidemiological studies followed in man. The nucleotide analogue FUDR has been found to inhibit acid mucopolysaccharide synthesis and to decrease palatine
shelf force inducing CP in rat fetuses (Ferguson,
1978). Mutant genes in mice have been associated
with CP cho, which inhibits jaw growth (Seegmiller &
Fraser, 1977) and ur, which reduces palatine shelf
width (Fitch, 1957).
In man, associations of CP and CL ± CP with HLA
typings have been excluded (Van Dyke, Goldman,
Spielman & Zmijewski, 1983; Van Dyke, Goldman,
Spielman, Zmijewski & Oka, 1980) despite evidence
of associations of the mouse MHC regions H-2 and
H-3 with glucocorticoid- and phenytoin-induced CP
(Gasser, Mele, Lees & Goldman, 1981a; Gasser,
Mele & Goldman, 19816). It is still not clear exactly
how the genes involved in the failure of the palate to
fuse, and potential thresholds to teratogenic agents,
combine; large parts of the complex biochemical
mechanisms are still only postulated at the theoretical
level. There have been few definitive findings which
hold up in more than a single model system, and any
genetic or environmental factor that appears critical
in one case can be excluded in another.
The analysis of single gene mutations using RFLPs
for linkage studies has had considerable success in
determining the chromosomal location of several
common inherited disorders. RFLPs are specific
enzyme sites that are polymorphic and segregate in
Mendelian fashion. These sites can segregate in a
linked or unlinked manner depending on their proximity to the disease locus. Both in cases where the
affected chromosome is known, as for Duchenne
muscular dystrophy (Davies et al. 1983) and chronic
granulomatous disease (Royer-Pokura et al. 1986),
and when neither the autosome nor the biochemical
defect is identified, as for Huntington's disease
(Gusella et al. 1983) and cystic fibrosis (Knowlton et
al. 1985; Wainwright et al. 1985; White et al. 1985),
linkage to within five centimorgans has been
achieved. However, the analysis of polygenic disorders by linkage studies with RFLP markers is at
present more complex both practically and theoretically.
One approach to dissecting the aetiology of disorders with complex combinations of genetic and
environmental factors is to use as a model a family in
which the phenotype is due to a single gene defect,
but displays the same features as more common
multifactorial and sporadic cases. Such a 'model' for
midline congenital defects has been found in a large
Icelandic family (over 280 individuals) (Fig. 1) showing Mendelian inheritance of X-linked secondary
cleft palate and ankyloglossia ('tongue-tied')
(CP+A) (Fig. 2). Both the large size of this pedigree
and the availability of many well-localized X-chromosome probes has made it possible to localize this
defect subchromosomally. The CP+A locus has been
found 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 and
ankyloglossia. (A) Partial clefting of the secondary palate; (B) complete clefting of the secondary palate; (C)
ankyloglossia or tongue-tied.
Molecular genetics of craniofacial abnormality
KY6
237
D1C 56
22-3
pD2
22-2
22-1
C7
21-3
21-2
pERT
211
11-4
11-3
11-23
11-22
11-21
111
11
121
12-2
pOTC
I
Ll-28
CpX203
p581
I
G3-1
pDP34
7b
13
X65H7
211
21-2
21-3
S21/S9
221
22-2
22-3
S21/S9
23
24
25
4315
43-15
A2-7
26
A2-7
27
DX13
DX13
28
-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 their
maximum 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 +3
and —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 0-5
for 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 multipoint
linkage was feasible between these probes due to their low information content within this family. However from these
data large sections of Xq and Xp have been excluded.
238
G. Moore and others
probe DXYS1 (probe name pDP34), which maps to
Xql3-21.1 (Moore et al. 1987) (Fig. 3).
Once localization on the X chromosome has been
more accurately achieved with finer mapping, cosmid
libraries can be used to jump or walk from specific
restriction enzyme sites to the physical location of the
gene. Subsequent cloning and sequencing of the
normal gene and its mutation and analysis of its
functional expression using cDNA libraries and
Northern blots will allow one part of the pathway that
leads to cleft palate to be clarified. Autosomal sequences may either be homologous, or specify other
parts of the same pathway, or at least give clues to
potential candidate genes involved in neural crest cell
and craniofacial development.
Discussion
Localization of the mutation causing cleft palate in
this family to Xql3-21.1 is a first step in understanding the genetic component of congenital neural crest
defects. This region of the X chromosome contains an
XY homologous region (Page, Harper, Love &
Botstein, 1982). As the limits of genetic mapping in
this family are approached, the techniques of cosmid
walking and jumping and pulsed-field gel electrophoresis (Schwartz & Cantor, 1984; Poustka & Lehrach, 1986) will allow the gap between the genetic and
physical map to be bridged.
The isolation of the gene and studies of its spatial
and temporal expression during development of the
secondary palate should lead us to a greater understanding of mechanisms which may lead to the failure
of closure of the palate. This mechanism can be
compared with those which may cause sporadic cases
in humans, or in animal models. There are also
families known in which an X-linked form of spina
bifida and anencephaly occur (Burn & Gibbens, 1979;
Jensson, Arnason, Gunnarsdottir, Petursdottir, Fossdal & Hreidarsson, 1987); any homology in linkage
between these families and the CP+A Icelandic
family would be of interest.
The cloning of this mutation can now be used as a
starting point for the analysis of other developmental
abnormalities. For example, this gene may be a
member of a multigene family involved in the embryonic development of other tissues. Bodmer (1983) has
suggested that genes with related functions whose
coordinated expression gives rise to complex phenotypes may be clustered on one chromosomal region as
supergenes. In mouse, five related homoeotic genes
are known to be clustered in a supergene family at a
single genetic locus. Such structure-function relationships may allow direct entry into complex
genetic systems involved in pattern formation and
ontogeny. Analysis of CP+A as a sex-linked single
gene provides a model for other genes regulating
processes in embyronic development.
We thank Action Research for the Crippled Child and
the Medical Research Council for generous financial support, Kay Davies, Ken Kidd, Sue Chamberlain and Keith
Johnson for many helpful discussions, all those who generously provided probes, and the family for their full cooperation.
References
BEAR, J. C. (1976). A genetic study of facial clefting in
Northern England. Clin. Gen. 9, 277-284.
BIXLER, D., FOGH-ANDERSEN, P. & CONNEALLY, P. M.
(1971). Incidence of cleft lip and palate in the offspring
of cleft parents. Clin. Gen. 2, 155-159.
BJORNSSON, A. & ARNASON, A. (1986). X-linked midline
defect in an Icelandic family. Abstract. 19th Congress of
Scandinavian Association of Plastic Surgeons,
Gothenburg.
BODMER, W. F. (1983). In Evolution from Molecules to
Man (ed. D. S. Bendall), pp. 197-208. Cambridge
University Press.
BONAITI-PELLIE, C. & SMITH, C. (1974). Risk tables for
genetic counselling in some common congenital
malformation. J. med. Gen. I I , 374-377.
BURN, J. & GIBBENS, D. (1979). May spina bifida result
from an X-linked defect in a selective abortion
mechanism? 'Hypothesis'. J. med. Gen. 16, 210-214.
DAVIES, K. E., PEARSON, P. L., HARPER, P. S., MURRAY,
J. H., O'BRIEN, T., SARFARAZI, M. & WILLIAMSON, R.
(1983). Linkage analysis of two cloned DNA sequences
flanking the Duchenne muscular dystrophy locus on the
short arm of the human X chromosome. Nucl. Acids
Res. 11,2303-2312.
FARRALL, M., SCAMBLER, P., NORTH, P. & WILLIAMSON, R.
(1986). The analysis of multiple polymorphic loci on a
single human chromosome to exclude linkage to
inherited disease: cystic fibrosis and chromosome 4.
Am. J. hum. Gen. 38, 75-83.
FEINBERG, A. P. & VOGELSTEIN, B. (1983). A technique
for radiolabelling DNA restriction endonuclease
fragments to high specific activity. Analyt. Biochem.
132, 6-13.
FERGUSON, M. W. J. (1978). The teratogenic effects of 5fluoro-2-desoxyuridine (F.U.D.R.) on the Wistar rat
fetus, with particular reference to cleft palate. J. Anat.
126, 37-49.
FITCH, N. (1957). An embryological analysis of two
mutants in the house mouse both producing cleft
palate. /. exp. Zool. 136, 329-361.
FOGH-ANDERSEN, P. (1942). In Inheritance of Harelip and
Cleft Palate. Copenhagen: A. Busck.
FRASER, F. C. (1976). The multifactorial/threshold
concept - uses and misuses. Teratology 14, 267-280.
GASSER, D. L., MELE, L. & GOLDMAN, A. S. (1981a).
Immune response genes and drug-induced
developmental abnormalities. In MHC and Diseases,
pp. 320-328. Basel: Karger.
Molecular genetics of craniofacial abnormality
GASSER, D. L., MELE, L., LEES, D. D. & GOLDMAN,
239
PAGE, D. C , HARPER, M. E., LOVE, J. & BOTSTEIN, D.
WATKINS, P. C., OTTINA, K., WALLACE, M. R.,
(1982). Occurrence of a transposition from the Xchromosome long arm to the Y-chromosome short arm
during human evolution. Proc. natn. Acad. Sci. U.S.A.
79, 5352-5326.
POUSTKA, A. & LEHRACH, H. (1986). Jumping libraries
and linking libraries: the next generation of molecular
tools in mammalian genetics. TIG 2, 174-179.
SAKAGUCHI, A. Y., YOUNG, A. B., SHOULSON, I.,
ROYER-POKURA, B . , KUNKEL, L. M . , MONACO, A . P . ,
A. S. (19816). Genes in mice that affect susceptibility
to cortisone - induced cleft palate are closely linked to
Ir genes on chromosomes 2 and 17. Proc. natn. Acad.
Sci. U.S.A. 78, 3147-3150.
GUSELLA, J. F., WEXLER, N. S., CONNEALLY, P. M.,
NAYLOR, S. L., ANDERSON, M. A., TANZI, R. E.,
BONILLA, E. & MARTIN, J. B. (1983). A polymorphic
DNA marker genetically linked to Huntington's
disease. Nature, Load. 306, 234-238.
JENSSON, O., AKNASON, A., GUNNARSDOTTIR, H.,
PETURSDOTTIR, I., FOSSDAL, R. & HREIDARSSON, S.
(1988). A family showing apparent X-linked inheritance
of both anencephaly and spina bifida. J. med. Gen. (in
press).
KERNAHAN, D. A. & STARK, R. B. (1958). A new
classification for cleft lip and cleft palate. Plastic
Reconst. Surg. 22, 435-441.
KNOWLTON, R. G., COHEN-HAGNENAUER, O., VAN CONG,
N., FREZAL, J., BROWN, V. A., BARKER, D., BRAMAN,
J. C , SCHUMM, J. W., Tsui, L - C , BUCHWALD, M. &
DONIS-KELLER, H. (1985). A polymorphic DNA marker
linked to cystic fibrosis is located on chromosome 7.
Nature, bond. 318, 360-382.
KUNKEL, L. M., SMITH, K. D., BOYER, S. H.,
BORGAONKER, D. S., WACHTEL, S. S., MILLER, O. J.,
BREG, W. R., JONES, H. W. & RARY, M. D. (1977).
Analysis of human Y-chromosome specific reiterated
DNA in chromosome variants. Proc. natn. Acad. Sci.
U.S.A. 74, 1245-1249.
LATHROP, G. M., LALONEL, J. M., JULIER, C. & OTT, J.
(1984). Strategies for multilocus linkage analysis in
humans. Proc. natn. Acad. Sci. U.S.A. 81, 3443-3446.
LECK, I. (1984). The geographical distribution of neural
tube defects and oral clefts. Br. med. Bull. 40.
390-395.
LOWRY, R. B. (1970). Sex-linked cleft palate in a British
Columbian Indian family. Pediatrics 46, 123-128.
MOORE, G. E., IVENS, A., CHAMBERS, J., FARRALL, M.,
WILLIAMSON, R., PAGE, D. C , BJORSSON, A.,
ARNASON, A. & JENSSON, O. (1987). Linkage of an X-
chromosome cleft palate gene. Nature, Lond. 326,
91-92.
MOORE, K. L. (1982). In The Developing Human, 3rd
edn, pp. 206-212. Philadelphia: W. B. Saunders.
GOFF, S. C , NEWBURGER, P. E., BAEHNER, R. L.,
COLE, F. S., CURNUTTE, J. T. & ORKIN, S. H. (1986).
Cloning the gene for an inherited human disorder chronic granulomatous disease - on the basis of its
chromosomal location. Nature, Lond. 322, 32-38.
RUSHTON, A. R. (1979). Sex-linked inheritance of cleft
palate. Hum. Gen. 48, 179-181.
SCHWARTZ, D. C. & CANTOR, C. R. (1984). Separation of
Yeast-Chromosome sized DNAs by pulsed field
Gradient Electrophoresis. Cell 37, 67-75.
SEEGMILLER, R. E. & FRASER, F. C. (1977). Mandibular
growth retardation as a cause of cleft palate in mice
homozygous for the chondrodysplasia gene.
J. Embryol. exp. Morph. 38, 227-238.
SOUTHERN, E. M. (1975). Detection of specific sequences
among DNA fragments separated by gel
electrophoresis. / . molec. Biol. 98, 503-517.
THOMPSON, J. S. & THOMPSON, M. W. (1986).
Multifactorial Inheritance. In Genetics in Medicine, 4th
edn, pp. 210-225. Philadelphia: W. B. Saunders.
VAN DYKE, D. C , GOLDMAN, A. S., SPIELMAN, R. S. &
ZMIJEWSKI, C. M. (1983). Segregation of HLA in
families with oral clefts: Evidence against linkage
between isolated cleft palate and HLA. Am. J. med.
Gen. 15, 85-88.
VAN DYKE, D. C , GOLDMAN, A. S., SPIELMAN, R. S.,
ZMUEWSKI, C. M. & OKA, S. W. (1980). Segregation of
HLA in sibs with cleft lip or cleft lip and palate:
Evidence against genetic linkage. Cleft Palate J. 17,
189-193.
WAINWRIGHT, B. J., SCAMBLER, P. J., SCHMIDTKE, J.,
WATSON, E. A., LAW, H-Y., FARRALL, M., COOKE, H.
J., EIBERG, H. & WILLIAMSON, R. (1985). Localisation
of cystic fibrosis locus to human chromosome 7cen-q22.
Nature, Lond. 318, 384-385.
WHITE, R., WOODWARD, S., LEPPERT, M., O'CONNEL, P.,
HOFF, M., HERBST, J., LALOUEL, J-M., DEAN, M. &
VANDE WOUDE, G. (1985). A closely linked genetic
marker for cystic fibrosis. Nature, Lond. 318, 382-384.