Human malformations caused by mutations in the 5' Hox genes

(1)

HUMAN MALFORMATIONS CAUSED BY MUTATIONS

IN THE 5' H O X GENES

FRANCES REBECCA GOODMAN

A dissertation submitted to the

University of London

in candidature for the degree of

Doctor of Philosophy

Molecular Medicine Unit

Institute of Child Health, London

(2)

P ro Q u e s t N um ber: U 1 2 2 0 8 8

All rights re s e rv e d

INFORMATION TO ALL U S E R S

T h e quality of this rep ro d u ctio n is d e p e n d e n t u pon th e quality of th e co p y su b m itte d . In th e unlikely e v e n t th a t th e a u th o r did not s e n d a c o m p le te m a n u sc rip t a n d th e re a re m issing p a g e s , th e s e will be no ted . Also, if m aterial had to be rem oved,

a note will indicate th e d eletion.

uest.

P ro Q u e s t U 1 2 2 0 8 8

P u b lish e d by P ro Q u e s t LLC(2016). C opyright of th e D issertatio n is held by th e Author. All rights re s e rv e d .

This w ork is p ro te c te d a g a in s t u n a u th o riz e d copying u n d e r Title 17, United S ta te s C o d e . M icroform Edition © P ro Q u e s t LLC.

P ro Q u e s t LLC

789 E a s t E ise n h o w e r P arkw ay P.O. Box 1346

(3)

ABSTRACT

The Hox genes are fundamental regulators of embryonic development in virtually all multicellular organisms. Here I describe a number of human malformations due to mutations in two of these genes, H0XD13 and H0XA13.

The dominantly-inherited limb malformation synpolydactyly (SPD) is caused by expansions of an N-terminal polyalanine tract in H0XD13. Analysis of 20 affected families shows a highly significant increase in penetrance and phenotypic severity with increasing expansion size. In a family with a 14-residue expansion, the longest so far reported, affected individuals have an unusually severe limb phenotype; affected males also have hypospadias, not previously described in SPD. The expansions appear to confer a progressive gain of function on the mutant protein, perhaps by perturbing interactions with another protein.

A novel phenotype, similar to but distinct from classical SPD, co-segregates with deletions in H0XD13 in two further families. These deletions truncate the first exon and the homeobox respectively, probably resulting in non-fimctional proteins. A virtually identical phenotype occurs in a father and daughter hemizygous for a microdeletion encompassing H0XD8 to H0XD13 and EVX2. These patients’ phenotype nevertheless differs strikingly from that in Hoxdl3 knock-out mice.

In another family, a novel polydactyly/brachydactyly syndrome including distal ulnar abnormalities co-segregates with an amino acid substitution in the homeodomain of H0XD13. Here, altered DNA-binding capacity appears to affect protein function in a way different to either the gain-of-function or the loss-of-function mutations described above.

(4)

PREFACE

This dissertation describes work carried out in the Molecular Medicine and Clinical Genetics Units at the Institute of Child Health, University College, London, between January 1996 and June 1999. The dissertation, and all the work reported in it, are my own, except where otherwise stated in the text. The dissertation is not substantially the same as any that I have submitted for a degree or diploma or other qualification at any other university, and no part of it already has been or is being concurrently submitted for any such degree, diploma or other qualification.

First and foremost, I should like to thank my supervisor. Prof. Peter Scambler, for his enthusiastic encouragement and support throughout the course of this project. I should also like to thank Prof. Robin Winter for his advice and assistance with the clinical aspects of the study; Prof. Bjom Olsen for generously sharing data prior to publication and providing helpful advice fi*om across the Atlantic; and Prof. Richard Mann for many stimulating discussions during my recent visit to his laboratory.

I am also most grateful to the many clinical colleagues who referred and helped assess the patients described in this study, including Dr. Frits Beemer, Dr Louise Brueton, Dr Amanda Collins, Prof. Dian Donnai, Dr Alan Donnenfeld, Dr Murray Feingold, Prof. Jean-Pierre Fryns, Dr Christine Garrett, Prof. Maria-Luisa Giovannucci-Uzielli, Dr Raoul Hennekam, Dr Susan Huson, Dr Elisabeth Lapi, Prof. Frank Majewski, Dr Julie McGaughran, Dr Carole McKeown, Dr Ruth Newbury-Ecob, Dr Anne Slavotinek, Dr William Reardon, Dr Karen Temple, Dr Joseph Upton and Dr Andrew Wilkie. I am especially grateful to Dr Christine Hall for her help with the interpretation of radiographs and to Dr Angela Wade for her help with the statistical analysis. I also greatly appreciate the interest and support I have received fi'om the many patients who participated in this study.

(5)

Finally, my most particular thanks go to my friend and partner, Nicholas Short, who during the course of this project, as in all things, was the sine qua non of my existence.

The work described in this thesis was supported by the award of a Clinical Training Fellowship from the Medical Research Council, which is also generously supporting a new program of research I am about to undertake, aimed at continuing and building upon this work. The next four years thus promise to be even more exciting (and busier!) than the last.

Molecular Medicine Unit,

Institute of Child Health, London

(6)

SELECT QUOTATIONS

Each hand hath naturally five fingers onely; whatever is more or less is against nature, and if there bee fewer it is a fault not to bee helped by art. But if there bee more, that fo r the most part may bee helped by art...If by chance they shall grow together by a little and thin skin and flesh, they shall forthwith bee divided with a sharp razor; but i f they bee joined by the interposition o f a more gross and dens substance, to wit the nerves, tendons and vessels, being knit together on each side, it will be best not to meddle at all with the dividing them.

‘The Works of that famous Chimgion, Ambrose Parey’, translated by T. Johnson, 1649

I am able to show you tonight skiagrams o f three cases o f syndactylism, and also to detail to you the family history o f this affection as present in five generations...Holt describes it as one o f the ‘stigmata o f degeneration and states that it is more common in persons o f weak intellect...Osier states that the deformity is commoner in the children o f syphilitic parents. We have no information on this point in regard to these cases. The family puts forward a tradition to explain their affliction, and which I give without comment. “The father was a bad man, while the mother was a good woman, and prayed that her children

should be marked, so that she should know them. ”

J. G. Edwards, Medical Journal o f Australia 2, 319,1916

There is no record o f intermarriage or illegitimacy; mentality is average or above... The family belongs to a sect which shuns the use o f alcoholic and other harmful beverages, tobacco, etc. The above mentioned facts fa il to support suggestions that syndactyly is associated with such characteristics as low mentality, alcoholism and inbreeding.

R. M. Alvord, Journal o f Heredity 38,49-53,1947

(7)

ago, with no further interest being shown. This village and the affected inhabitants have come to public attention through a television programme...

B. S. Sayli et a l. Journal o f Medical Genetics 32,421-434, 1995

Fellas with big feet have the largest manhoods, scientists claimed yesterday. Many women believe a man’s shoe size and willy size are connected. Now researchers have discovered a gene that controls the growth o f fingers, toes andpenises. Mice bred without the hox gene (sic) had no digits or male organs.

Report in a popular British newspaper, 1997

How is HOX regulation integrated with other pattern regulators?... What genes are regulated by HOX genes and how do they affect growth and patterning? Why are the A13 and D13 genes but not, as yet, 37 others, implicated in human developmental diseases? When the remaining mysteries are mustered, it seems that we understand very little about how HOX genes work. The future holds great excitement—at the moment, w e’re just limbering up.

(8)

LIST OF FIGURES

Figure 1.1 The bones of the human hand...23

Figure 1.2 The bones of the human foot...24

Figure 1.3 Organisation of the Hox genes in Drosophila and mouse...38

Figure 3.1 Primers used to amplify if0AD75 from genomic D N A ...78

Figure 3.2 Sequence analysis of exon 1 of HOXDl 3... 82

Figure 3.3 Secondary structures in exon 1 of HOXDl 3 and HOXAl 3...83

Figure 3.4 Genomic sequence of HOXDl 3...86

Figure 3.5 Amino acid sequence o f HOXDl 3... 88

Figure 3.6 Corrections to nucleotide sequence of HOXDl 3... 89

Figure 3.7 Primers used to amplify HOXAl3 from genomic DNA...91

Figure 3.8 Sequence analysis of exon 1 o f HOXAl 3... 94

Figure 3.9 Genomic sequence of HOXAl 3...96

Figure 3.10 Amino acid sequence of HOXAl 3...97

Figure 3.11 Corrections to nucleotide sequence of HOXAl 3...99

Figure 3.12 Single nucleotide polymorphism in HOXDl3... 101

Figure 4.1 Pedigree A ... 106

Figure 4.2 Limb phenotype in Pedigree A ... 108

Figure 4.3 Pedigrees B, C, D and E ... 110

Figure 4.4 Limb phenotype in Pedigree B ... I l l Figure 4.5 Pedigrees F, G and 2... 114

Figure 4.6 Pedigrees H, I and 1... 116

Figures 4.7 Limb phenotype in Pedigree H ... 117

Figure 4.8 Pedigrees J, K, L and M ... 119

Figure 4.9 Limb phenotype in Pedigrees J and M ... 120

Figure 4.10 Pedigrees N, O and P ... 122

Figure 4.11 Pedigrees 3 and Q ... 124

Figure 4.12 Limb phenotype in Pedigree Q ... 126

Figure 4.13 Sequence encoding normal and expanded polyalanine tracts in H0XD13 128 Figure 4.14 Segregation of the polyalanine tract expansion in Pedigree M ... 130

(15)

Figure 4.17 Correlation between expansion size and number of limbs affected in SPD 136 Figure 4.18 Correlation between expansion size and level of branching in SPD hands 137

Figure 5.1 Pedigree R ...141

Figure 5.2 Limb phenotype in proband from Pedigree R ... 142

Figure 5.3 Foot phenotype in further individuals from Pedigree R ... 144

Figure 5.4 Pedigree S ...146

Figure 5.5 Hand phenotype in individuals from Pedigree S ... 147

Figure 5.6 Foot phenotype in individuals from Pedigree S ... 149

Figure 5.7 14-bp deletion in exon 1 of HOXDl 3 in Pedigree R ... 151

Figure 5.8 Sites of the HOXDl3 deletions in Pedigrees R and S ... 152

Figure 5.9 Segregation of the deletion in Pedigree R ... 153

Figure 5.10 Effect of the deletion in Pedigree R ... 154

Figure 5.11 1-bp deletion in exon 2 of HOXDl 3 in Pedigree S ... 156

Figure 5.12 Segregation of the deletion in Pedigree S... 157

Figure 5.13 Effect of the deletion in Pedigree S ... 158

Figure 6.1 Pedigree T...163

Figure 6.2 Limb phenotype in Pedigree T ... 164

Figure 6.3 Limb phenotype in Case 2 ... 167

Figure 6.4 Haplotype analysis in Pedigree T ... 170

Figure 6.5 Segregation of polymorphism in H0XD8 in Pedigree T... 171

Figure 6.6 Map of the HOXD gene cluster... 173

Figure 6.7 FISH studies in Pedigree T... 174

Figure 6.8 Example of detection of cosmids from the LL02NC02 hbrary... 176

Figure 6.9 EcoKL restriction map of part of the HOXD cluster... 180

Figure 6.10 Control Southern blots probed with HOXD3 A, H0XD3B and H0XD4P 181 Figure 6.11 EcoKL restriction digest of cosmids from the HOXD cluster... 183

Figure 6.12 Cosmid contig from the HOXD cluster... 184

Figure 6.13 Southern blots of DNA from II. 1 in Pedigree T probed with 100KT7... 187

Figure 6.14 Southern blot of DNA from 5 members of Pedigree T probed with 100KT7.... 188

Figure 6.15 Southern blots of fragment El probed with 100KT7 and H0XD8U... 190

Figure 6.16 Restriction map of fragment E l ... 191

Figure 6.17 Extent of deletions in Cases 1-4... 194

Figure 7.1 Pedigree U ... 205

(16)

Figure 7.3 Limb phenotype in further individuals from Pedigree U ... 208

Figure 7.4 Hand phenotype in Case 6 ... 212

Figure 7.5 Base substitution in exon 2 of HOXDl 3 in Pedigree U ...214

Figure 7.6 Sites of the HOXD 13 base substitutions in Pedigree U and Case 6 ... 215

Figure 7.7 Segregation of the base substitution in Pedigree U ...216

Figure 7.8 Base substitution in exon 1 o îHOXDl3 in Case 6 ...218

Figure 7.9 Segregation of the base substitution in Case 6 and his parents...219

Figure 7.10 Structure of the homeodomain complexed with target DNA... 222

Figure 8.1 Pedigree V ... 227

Figure 8.2 Limb phenotype in Pedigree V ... 228

Figure 8.3 Pedigree W ... 230

Figure 8.4 Limb and genital tract phenotype in Pedigree W ...232

Figure 8.5 Pedigree X ... 234

Figure 8.6 Limb phenotype in Case 7 ... 236

Figure 8.7 Base substitution in exon 1 o ïHOXAl3 in Pedigree V ...238

Figure 8.8 Sites of the HOXAl3 mutations in Pedigrees V and W and Case 7 ... 239

Figure 8.9 Segregation of the base substitution in Pedigree V ...240

Figure 8.10 Effect of the base substitution in Pedigree V ...241

Figure 8.11 HOXAl3 polyalanine tract expansion in Pedigree W... 243

Figure 8.12 Base substitution in exon 2 of HOXAl 3 in Case 7 ... 245

(17)

LIST OF TABLES

Table 2.1 Primers used for PCR amplification o ï HOXDl3 and HOXAl3... 56

Table 2.2 Primers used for PCR amplification of HOXD cluster probes...56

Table 2.3 Primers used for cycle sequencing of cloned DNA...56

Table 2.4 Chromosome 2q31 microsatellite repeats...57

Table 3.1 Corrections to amino acid sequence of HOXD 1 3 ...87

Table 3.2 Corrections to nucleotide sequence of HOXAl 3...87

Table 4.1 Clinical findings in SPD pedigrees...103

Table 5.1 Distal limb phenotype in mutation carriers fi'om Pedigrees R and S...160

Table 6.1 Cosmids identified by screening library LL02NC02...177

Table 6.2 Cosmids identified with probes H0XD3P, H0XD4P and H 0X D9P...178

Table 6.3 Restriction mapping of fi-agment E l ...191

Table 6.4 Microsatellite marker analysis in Cases 1-4...195

(18)

LIST OF ABBREVIATIONS

A adenine

BSA bovine serum albumin

bp base pair

C cytosine

°C degree Celsius

CCD cleidocranial dysplasia

cM centimorgan

cm centimetre

CNS central nervous system

dATP deoxyadenosine triphosphate

ddNTP dideoxynucleoside triphosphate

dGTP deoxyguanosine triphosphate

DMSO dimethyl sulphoxide

DNA deoxyribonucleic acid

dNTP deoxynucleoside triphosphate

dpc day post-coitum

dTTP deoxythymidine triphosphate

dUTP deoxyuridine triphosphate

E embryonic day

B. coli Escherichia coli

EDTA ethylenediamine tetra-acetic acid

FBS fetal bovine serum

FISH fluorescence in situ hybridisation

FITC fluoroscein isothiocyanate

g gram

G guanine

H d hypodactyly

HFGS hand-foot-genital syndrome

HPE holoprosencephaly

XU international unit

(19)

kPa kilopascal

lod logarithm of the odds

M molar

mM millimoles per litre

mg milligram

ml millilitre

Hi microlitre

mm millimetre

ng

nanogram

nm nanometre

OPMD oculopharyngeal muscular dystrophy

P short arm of chromosome

P probability

PAC PI-derived artificial chromosome

PCR polymerase chain reaction

q

long arm of chromosome

RNA ribonucleic acid

rpm revolutions per minute

SDS sodium dodecyl sulphate

SPD synpolydactyly

spdh synpolydactyly homolog

ssc

saline sodium citrate

T thymine

UTR untranslated region

uv

ultraviolet

V Volt

v/v volume per volume

w/v weight per volume

(20)

GLOSSARY OF CLINICAL TERMS

Autopod

Blepharophimosis

Brachydactyly

Camptodactyly

Chordee

Clinodactyly

Craniosynostosis

Cryptorchidism

Ectrodactyly

Hypertelorism

Hypospadias

Meso-axial

Mesomelic

Distal segment (along the proximal-distal axis) of the limb: carpals, metacarpals and phalanges in the upper limb; tarsals, metatarsals and phalanges in the lower limb

Reduction (in both height and width) of the space between the eyelids

Shortening of the digits

Bending of the digits in a plane at right angles to the plane of the palm/sole, due to flexion contractures of the interphalangeal joints

Downwards (ventral) curvature of the shaft of the penis often accompanying severe hypospadias

Curving of the fingers (most commonly the 5^^ fingers) in the plane of the palm, usually towards the midline, due to hypoplasia of the middle phalanges

Premature fusion of the bones of the skull, causing an abnormal skull shape. Can result in neurological complications.

Undescended testes

Longitudinal deficit affecting the central digital rays (also called split hand / split foot and lobster-claw deformity)

Wide-set eyes (distance between the pupils more than 3 standard deviations above the mean)

Displacement of the opening of the urethra from its normal position at the tip of the glans of the penis to a site on the under (ventral) surface of the penis

Involving the middle (central) axis of the limb, which runs down the centre of its length to the end of the middle digit

(21)

Microcephaly Micrognathia Ocular coloboma Palpebral fissure Pelvi-ureteric junction obstruction Polydactyly Post-axial Pre-auricular pit Pre-axial Proptosis Ptosis Oligodactyly Stylopod Symphalangism Syndactyly Synostosis

Small skull size, as determined by measurement of the occipto- frontal circumference of the head

Small lower jaw, caused by undergrowth of the mandible

Cleft (usually inferior) in the iris and/or the retina and optic nerve, due to failure of normal closure of the choroid fissure Space between the upper and lower eyelids

Obstruction of normal outflow of urine firom pelvicalyceal system of kidney into ureter. Commonly caused by narrowing/functional abnormality of upper end of ureter. Can result in dilation of pelvicalyceal system (hydronephrosis) and progressive kidney damage.

Increased number of digits

Involving the posterior (ulnar/fibular) half of the limb, which runs down its length to the end of the 5^^ finger/toe

Blind skin pit in fi"ont of ear, probably remnant of a branchial cleft

Involving the anterior (radial/tibial) half of the limb, which runs down its length to the end of the thumb/hallux

Protrusion of the eyes

Drooping of the upper eyelids Reduced number of digits

Proximal segment (along the proximal-distal axis) of the limb: humerus in the upper limb, femur in the lower limb

Bony fusion of the phalanges at the interphalangeal joints, preventing joint movement

(22)

Talipes equinovarus Type of club foot deformity, in which the foot is sharply plantar flexed, with the sole turned towards the midline

Thenar eminence Elliptical muscle mass overlying the 1®^ metacarpal at the base of the thumb

Vesico-ureteric reflux

Zeugopod

Reflux of urine during bladder emptying back up ureters into pelvicalyceal system of kidneys. Most commonly caused by abnormal insertion of lower end of ureters into bladder wall. Can result in progressive kidney damage (reflux nephropathy).

(23)

CHAPTER 1. INTRODUCTION; SYNPOLYDACTYLY,

HAND-FOOT-GENITAL SYNDROME AND THE ITOY GENES

Synpolydactyly (SPD) is the first human malformation syndrome shown to be caused by mutations in a HOX gene, HOXDl3 (Muragaki et al., 1996). In the year following this discovery, another human malformation syndrome, hand-foot-genital syndrome (HFGS) was shown to be caused by a mutation in a closely related HOX gene,

HOXAl3 (Mortlock and Innis, 1997). To date, these are the only two HOX genes known to be mutated in human disorders.

This Chapter gives an account of the clinical features and molecular basis of SPD and HFGS, followed by a short review of the recent literature on the Hox genes, including their roles in development and the regulation of transcription, with particular emphasis on

HOXD 13 and HOXAl3. The Chapter ends with an overview of the work to be described in this thesis.

For ease of reference, schematic drawings of the bones of the human hand are shown in Figs. 1.1 and 1.2.

Synpolydactyly

Clinical features

SPD (OMIM No. 186000) is a rare congenital malformation of the distal limbs, in which there is a distinctive combination of syndactyly (webbed digits) and polydactyly (supernumerary digits). The malformation occurs in a characteristic distribution, which differs in the hands and in the feet. Thus, there is syndactyly between the and 4^^ fingers and between the 4^^ and 5^^ toes, with partial or complete digit duplication in the syndactylous web.

(24)

Head of phalanx

Base of phalanx Head of fourth

metacarpal

Hamate Pisiform

Triquetral

Distal phalanx Middle phalanx

•Proximal phalanx

Second metacarpal Sesamoid bone Trapezoid Trapezium Scaphoid Lunate

Capitate

B

Second metacarpal

First metacarpal Trapezoid

Trapezium Scaphoid

Distal phalanx Middle phalanx

Proximal phalanx Head of phalanx

Head of fourth metacarpal Base of phalanx

Capitate Pisiform Hamate riquetral unate

Figure 1.1 The carpal, metacarpal and phalangeal bones o f the human hand. A , V iew o f the palmar surface. B, View o f the dorsal surface.

(25)

Proximal phalanx

First

metatarsal _j _Intermediate

"l cuneiform bone

\ Lateral

J cuneiform bone

M ed ial L /

cuneiform bone

Navicular Cuboid

Talus

Calcaneum

B Distal phalanx

Groove for ; y

peroneus longus Cuboid

Calcaneum

Middle phalanx

Proximal phalanx

Sesam oid bones

Medial cuneiform bone Intermediate cuneiform bone

Navicular

Talus

Lateral cuneiform bone

surface.

(26)

syndactyly comprised webbed 3^^^ and 4* fingers, often with an additional finger in the web but normal toes, while ‘Bell Type BT syndactyly comprised webbed 4^^ and 5*^ toes, often with an additional toe in the web but normal fingers. In her review of the literature. Bell noted 17 pedigrees with Type A2 syndactyly and 3 pedigrees with Type A2 plus Type B1 syndactyly, most of which were probably instances of what is now termed SPD. This classification was superseded in 1978 by that of Temtamy and McKusick, who identified five clinically distinct types of syndactyly after reviewing previously published cases and studying additional families (Temtamy and McKusick, 1978b). In this system SPD was classed as a syndactyly (‘Syndactyly Type 11’) rather than a polydactyly, because syndactyly can occur in the condition in the absence of polydactyly, but polydactyly does not occur in the absence of syndactyly. Temtamy and McKusick were also the first to use the term ‘synpolydactyly’, to emphasise the importance of the syndactyly component of the condition. This contrasted with their findings in one of the Polydactylies (Pre-axial Polydactyly Type IV), in which polydactyly can occur in the absence of syndactyly, but syndactyly does not occur in the absence of polydactyly, and which they therefore termed ‘polysyndactyly’. Not surprisingly, the similarity between these two names has sometimes led to confusion as to which of the two conditions is under discussion. More recently, an alternative classification of syndactylies and Polydactylies has been suggested by Winter and Tickle (1993), based on whether patterning of the digits is normal or disturbed, and whether secondary modelling of the limb bud is normal or defective. In this system SPD is regarded as an example of normal pattern formation, but increased digit number in the meso-axial line, with abnormal digit separation. This classification emphasises the importance of the polydactyly component of the condition.

(27)

disease (Anderson, 1898). “The fingers of both hands are contracted at the middle and distal joints, and the middle and distal phalanges of the fourth finger on each hand are duplicated, the two digits being enclosed in one cutaneous investment...His mother and sister, and three out of four of his own children, had congenital deformities like his own.” In 1916, the first radiographs (‘skiagrams’) of patients with ‘syndactylism’ were published, clearly showing bilateral 3^*^ finger duplication and bifid metacarpals in a 5- generation Australian family with 12 affected members (Edwards, 1916).

Since then, numerous other large affected families have been reported world-wide. In 1927, a 7-generation Danish family with 42 affected members was reported by Thomsen (1927), who named the syndrome ‘Vordingborgtyp’ syndactyly after the small city in Denmark where the family lived. In 1947, a 6-generation American family fi-om Utah with 21 affected members was reported by Alvord (1947), who again noted incomplete penetrance (“There are three instances of ‘skipped’ generations.”), as well as variable expressivity (“The appearance of the affected digits is not constant. Zygodactyly may appear in both hands or in the right hand only; it may occur in the feet but not in the hands.”). In 1967, a 5-generation American family fi-om Ohio with 27 affected members was reported by Cross et al. (1967), while in 1986, a 6-generation Israeli family with 16 affected members was reported by Merlob and Grunebaum (1986). In 1990, a 5- generation Italian family with 11 affected members was reported by Chessa Ricotti et al.

(1990). The clinical and molecular findings in this family, which is designated Pedigree P in this study, are described in detail in Chapter 4. In 1992, a 4-generation Belgian family with 14 affected members was reported by De Smet et al. (1992), while in 1995 a 4- generation Italian family with 8 affected members was reported by Camera et al. (1995). The largest family identified to date is the remarkable pedigree studied by Sayli et al.

(1995), fiom an isolated village in rural Turkey, in which the malformation was present in 182 individuals and could be traced back over 7 generations, spanning at least 140 years.

A review of these reports reveals that affected individuals share a characteristic set of distal limb abnormalities, as illustrated in Chapter 4. In the hands, the most striking feature is partial or complete soft tissue syndactyly between the 3"* and 4^*^ fingers, usually with an extra finger lying between the 3^^ and 4^^ fingers in the syndactylous web, due to partial or complete duplication of the 3^^ or 4* finger. The webbed fingers may be held in fixed flexion, due to contractures at the interphalangeal joints, and there is often also clinodactyly and/or camptodactyly of the 5^^ fingers (Camera et al., 1995; Cross et al.,

(28)

is partial or complete soft tissue syndactyly between the 4^^ and 5^ toes, usually with an extra toe lying between the 4^^ and 5^^ toes in the syndactylous web, due to partial or complete duplication of the 5^^ toe. Without surgical intervention, patients may have difficulty finding sufficiently broad footwear to accommodate the extra toe. The soft tissue syndactyly may extend from the 2"^ to the 5* toes, sometimes with associated brachydactyly, and the nails of the syndactylous toes may be dystrophic. Radiological examination is necessary to determine the proximal extent of the digit duplication, which can range from a bifid distal phalanx to duplication of all three phalanges, with involvement of the metacarpals and metatarsals. Thus, in the hands the 3*^*^ metacarpal may be enlarged, bifid or Y-shaped, or there may be an additional rudimentary metacarpal lying between the 3"^^ and 4* metacarpals, while in the feet there may be an additional metatarsal, either rudimentary or well-formed, lying between the 4* and 5^^ metatarsals (Camera et al., 1995; Merlob and Grunebaum, 1986; Sayli et ah, 1995). Radiological examination also usually reveals hypoplasia of the middle phalanges of the 5‘^ fingers and hypoplasia or aplasia of the middle phalanges of the 2"*^ to 5^^ toes (Camera

et al., 1995; Sayli et al., 1995). Involvement does not extend as far proximally as the carpal bones in the wrists or the tarsal bones in the feet, however, and there are no other skeletal or extra-skeletal manifestations. Nowadays the majority of patients undergo surgical division of the syndactyly and/or removal of the extra digits, partly to improve hand and foot function, and partly for cosmetic reasons.

(29)

had bilateral hand involvement, and there was digit duplication in 164/179 affected hands, with syndactyly alone in only 15/179 affected hands (Sayli et ah, 1995). Even more striking, there is often variable expressivity within a single affected individual. Thus, there may be upper limb involvement without lower limb involvement, or lower limb involvement without upper limb involvement, although the upper limbs are more commonly affected than the lower limbs. Moreover, both hand and foot involvement may be unilateral or asymmetrical, so that one hand or foot may be severely affected, while the opposite hand or foot is normal or only mildly affected.

Recent reports of rare patients apparently homozygous for SPD, who have much more severe deformities than those seen in heterozygotes, indicate that the condition is semi-dominant. One such patient (individual 8 in Pedigree 2, shown in Fig. 4.5) was the offspring of a first cousin marriage. Her parents were deceased, but her mother reportedly had typical SPD, and although her father was reportedly unaffected (and thus a non penetrant mutation carrier), her paternal grandfather was reportedly typically affected, and both her sons were typically affected on clinical examination (Muragaki et a l, 1996). This patient’s hands and feet were abnormally small. In the hands, she had very short fingers, with syndactyly between fingers 3 to 5, which shared a single knuckle. A radiograph showed duplication of the distal phalanges of the thumbs; hypoplastic middle phalanges in fingers 2 to 5; shortened, rounded metacarpals; and loss of the trapezium bone in the wrist, with two accessory carpal bones. The proximal carpals, radius and ulna were normal. In the feet, she had just three very short toes. A radiograph showed a very short 2"^ metatarsal and a single short bone replacing metatarsals 3 to 5. The tarsals, tibia and fibula were normal.

(30)

radius and ulna were normal. Radiographs of the feet revealed loss of the normal phalangeal structures, with fusion of some phalanges and severe middle phalanx hypoplasia or aplasia, but no polydactyly. In addition, the metatarsals, cuboid and all 3 cuneiforms were replaced by large bony islands representing fusions between the metatarsals and the cuboid or cuneiforms. The talus, calcaneus, navicular, tibia and fibula were normal. Radiographs of the entire spine of two of these severely affected patients were also obtained, and one was found to have total coccygeal agenesis (Akarsu et al.,

1996).

Molecular basis

The clue to the molecular basis of SPD came from linkage studies carried out in the large pedigree documented by Sayli et al (1995). Individuals from five different branches of the family, yielding a total of 62 informative meioses, were analysed with polymorphic markers mapping to chromosomal regions where genes involved in limb development were known to be located (Sarfarazi et al., 1995). These included markers mapping to chromosome 2q23-36, the site of the HOXD gene cluster. Evidence of significant linkage was first obtained with one of these markers, D2S111, which gave a lod score of z = 4.71 at 0 = 0.15. Typing with 14 other markers mapping to the region gave a maximum lod score of z = 8.73 at 0 = 0.05 for H0XD8 (H0X4E), a polymorphic TA repeat in the 3’UTR of the HOXD8 gene (Rosen and Brown, 1993). Multipoint linkage and haplotype analysis indicated that the SPD locus was likely to lie in a 1.7cM interval centromeric to the H0XD8 marker (lod score of z = 12.96 at 0 = 0.17). This interval contains the five most 5’ members of the HOXD gene cluster, H0XD9 to H0XD13, as well as the EVX2, DLXÎ and DLX2 genes, all of which are involved in limb development. Sarfarazi et al.

suggested that H0XD13 was the most likely candidate, partly because this gene’s expression pattern in the developing mouse limb corresponds most closely to the distribution of abnormalities in SPD, and partly because the limb deformities in patients thought to be homozygous for SPD have some features in common with those reported in homozygous Hoxdl3 knock-out mice, including delayed ossification and abnormalities in the length, segmentation and branching of distal bony elements. Significant linkage to marker D2S138 on chromosome 2q31 was subsequently reported in a Mexican SPD family with 12 affected members (Polymeropoulos et al., 1995).

(31)

patient, the child of clinically normal parents, who was therefore likely to carry a de novo

mutation, while Pedigrees 2 and 3 (Figs. 4.5 and 4.11) were large multi-generation families with 8 and 24 living affected members respectively. In Pedigrees 2 and 3, linkage was first established to markers D2S111 and D2S1391, which flank a 2cM interval on chromosome 2q31, as well as to the H0XD8 marker. HOXD 12, HOXD 13 and EVX2 were then selected as the most plausible candidate genes in the region, since they are all expressed in the most distal part of the developing mouse limb. Sequence analysis of the homeoboxes of these genes in affected patients revealed no abnormalities. The 5’ coding regions of H0XD12 and HOXD13 were therefore cloned and sequenced, and exon 1 of

HOXD 13 was found to contain an imperfect trinucleotide repeat encoding a 15-residue polyalanine tract.

(32)

in a HOX gene. The possible functional consequences of these mutations are discussed in Chapter 10.

Hand-foot-genital syndrome

Clinical features

HFGS (OMIM No. 140000) is a rare congenital malformation syndrome in which there is a distinctive combination of distal limb and genital tract abnormalities. It was first described in 1970 by Stem et al. (1970), who reported a 4-generation American family with 17 affected individuals. Details of the radiological findings in this family were published separately in the same year (Poznanski et al., 1970). All 13 affected individuals available for examination had characteristic abnormalities of the hands and feet. In addition, 4/7 affected females had a bifid or double uterus. The condition was initially therefore termed hand-foot-uterus syndrome (HFUS), and is still sometimes called by this name. In three Swiss brothers with the same characteristic hand and foot abnormalities, however, hypospadias was subsequently noted (Giedion and Prader, 1976). This prompted a review of the original American family, in which 2/10 affected males were also found to have hypospadias. As there was now evidence of male as well as female genital tract involvement in the condition, the name hand-foot-genital syndrome (HFGS) was adopted (Giedion and Prader, 1976; Poznanski et al., 1975).

(33)

a phenotype similar to but not typical of HFGS was reported by Hennekam (1989). The clinical and molecular findings in this family, which is designated Pedigree X in this study, are also described in detail in Chapter 8. In 1990, another American affected father and daughter were reported by Cleveland and Holmes (1990). Most recently, in 1993, an affected Belgian male with three affected sons was reported by Fryns et aL (1993). The clinical and molecular findings in this family, which is designated Pedigree V in this study, are again described in detail in Chapter 8.

Review of these reported cases reveals that affected individuals share a characteristic set of distal limb abnormalities, illustrated in Figs. 8.2 and 8.4 A. The most striking features in the hands are short, stubby thumbs, often proximally-placed and radially-deviated, with hypoplastic thenar eminences. The hands are also usually small overall, with clinodactyly and/or brachydactyly of the 5*^ fingers, and sometimes ulnar deviation of the 2"^ fingers. Radiological findings in the hands include short, pointed 1®* distal phalanges and/or short 1®^ metacarpals, short 5* middle phalanges, and sometimes short, malformed 2"^ middle phalanges. In addition, there are usually abnormalities of the carpal bones in the wrists, including malformation of the scaphoid in children, and fusion of the scaphoid and trapezium in adults, often with an os centrale and long ulnar styloid (Poznanski et aL, 1970). The most striking features in the feet are short, stubby halluces, often medially deviated (Cleveland and Holmes, 1990). The feet are also small overall (the original report describes them as ‘doll-like’), so that patients generally require an abnormally small shoe size for their age. Radiological findings in the feet include short, pointed 1®* distal phalanges and/or short 1®* metatarsals, as well as short or fused middle and distal phalanges of toes 2-5. In addition, there are usually abnormalities of the tarsal bones, including malformation of the navicular, shortening of the calcaneus and fusion of the cuneiforms to the navicular or the metatarsals (Poznanski et aL, 1970). Although ossification of the phalanges, metacarpals and metatarsals appears to occur normally, ossification of many of the carpal and tarsal bones is delayed, a phenomenon known as dysharmonie maturation. There are no skeletal abnormalities outside the hands and feet.

(34)

(1975) and subsequently confirmed by Giedion and Prader (1976) and Cleveland and Holmes (1990).

In marked contrast to the limb phenotype in SPD, the pattern of abnormalities in the hands in HFGS is closely analogous to that in the feet. Moreover, all individuals with hand and foot abnormalities have bilateral symmetrical involvement of both hands and both feet, and little variation in the severity of these abnormalities has been noted either within or between families. The hand and foot abnormalities cause few practical problems to patients, however, apart from an occasional inability to oppose the thumb and 5^^

finger, and difficulty in balancing while standing still, due to the small size of the feet. Surgery to the hands and feet is hardly ever required.

The characteristic abnormality in the female genital tract is a Müllerian duct fusion defect, as illustrated in Fig. 8.4 B. The caudal ends of the two Müllerian (paramesonephric) ducts normally join together in early embryonic development, giving rise to a single uterus, cervix and upper vagina. If this fusion fails to occur, varying degrees of genital tract ‘duplication’ result. The uterus may consist of two separate cavities (uterus duplex), or a single cavity which is bifid (uterus bicomis) or partially divided by a longitudinal septum (septate uterus). The cervix may be double (bicollis) instead of single (unicollis), and the vagina may also be divided by a longitudinal septum. Defects ranging from isolated longitudinal vaginal septum to double uterus with double cervix (uterus didelphys) have been reported in 8/16 females with HFGS. (These figures do not include the family described by Hennekam (1989), which is not entirely typical of HFGS.) In addition, a tight hymeneal constriction ring may be present. Although fertility is normal, these malformations may cause serious problems during pregnancy and labour (midtrimester miscarriages, premature labour, fetal malpresentation, still birth), and have led to fetal loss or neonatal death in at least three affected families (Donnenfeld et al.,

1992; Halal, 1988; Stem et a l, 1970).

(35)

An unusual additional feature, malformation of the urinary tract, has also been reported in five females with HFGS. The young girl described by Poznanski et al. (1975) presented with urinary incontinence and recurrent urinary tract infections. She was found to have a patulous intravaginal urethra, bilateral ectopic ureteric orifices and vesico- ureteric reflux on the left. Two of the five affected females in Pedigree W had similar urinary tract malformations (Elias et aL, 1978; Verp, 1989; Verp et at., 1983), described in detail in Chapter 8. The young girl reported by Halal also had a patulous urethra, while her mother had a patulous intravaginal urethra and an ectopic accessory ureteric orifice, which led to recurrent urinary tract infections, chronic pyelonephritis, renal insufficiency, and renal transplant at the age of 47 (Halal, 1988). Only two affected males, however, have been noted to have urinary tract malformations, and they were both from Pedigree W, as described in Chapter 8. These findings led Donnenfeld et al. to propose that the name of the condition be changed again to hand-foot-genito-urinary syndrome (HFGUS) (Donnenfeld et al., 1992), but this suggestion has not been generally adopted.

HFGS is inherited in an autosomal dominant manner (8 instances of male-to-male transmission were observed in the family first described, definitively excluding sex linkage (Stem et al., 1970)). Pedigree analysis suggests that the penetrance of the limb abnormalities is complete, since there are no clear instances of obligate carriers with normal hands and feet. The only possible exception is one of the clinically unaffected parents of the three affected brothers reported by Giedion and Prader (1976), but this finding could reflect gonadal mosaicism or even a rare autosomal recessive form of the disorder rather than non-penetrance. Of the three affected brothers reported by Fryns et al., however, one had hypospadias with normal hands and feet, suggesting that the penetrance of the limb abnormalities may in fact be incomplete (Fryns et al., 1993). By contrast, the genital and urinary tract malformations are clearly only partially penetrant in both males and females. As stated previously, levels of penetrance in a syndrome can only be assessed exactly when the molecular basis in each affected family has been identified.

Molecular basis

(36)

(Hd/Hd) die in utero. Those that are bom usually survive to adulthood but are infertile and have only a single digit on each paw.

The distal limb abnormalities in these mice have been studied in detail by Mortlock

et al. (1996) Skeletal staining of the forelimbs of 3-day-old Hd!+ mice reveals short or absent distal phalanges of digit 1 (14/15), delayed ossification of the 5^^ middle phalanx (4/15), premature ossification of carpal d4 (4/15) and fusion of carpals dl and carpal d2-c (6/15). Similar hindlimb preparations reveal shortening or absence of one or both phalanges of digit 1 (12/12), delayed ossification of the 2nd and/or 5* middle phalanges (7/12 and 1/12 respectively), and fusion of tarsals cunéiforme 3 and naviculare (12/12). Similar preparations of 3-day-old HdlHd mice show a single incompletely formed digit, most probably the 4*^ digit, in the forelimbs and hindlimbs, with reduction or absence of the carpal and tarsal bones. Neither heterozygotes nor homozygotes exhibit any skeletal abnormalities outside the distal limb. The pattern of skeletal abnormalities in heterozygotes is thus virtually identical to that seen in the hands and feet of patients with HFGS, as described above.

In female mice, there are normally two separate uterine horns, which join together at the level of the cervix, so Müllerian duct fusion defects would be expected to manifest only as a double cervix and/or a septate vagina. No genital or urinary tract abnormalities have been observed in Hd!-^ mice, however, although abnormalities of the female genital tract (hypoplasia of the clitoris and vagina, vesico-uterine fistula) and the urinary tract (cystic enlargement of the bladder and ureters) have occasionally been seen in HdlHd

mice. The high rate of embryonic lethality and the infertility in homozygotes remain unexplained.

(37)

In the light of the extreme similarity between the limb abnormalities in Hdl+ mice and those in patients with HFGS, Mortlock and Innis (1997) re-visited the family in which HFGS was first described and were able to show that in this family one allele at a highly polymorphic locus mapping close to the HOXA gene cluster on chromosome 7pl4 segregated with the disease phenotype. Sequence analysis in an affected individual revealed a single base substitution (A to G) in exon 2 of H0XA13 which creates a premature stop. This mutation destroys a NlaYV restriction site, allowing its presence to be demonstrated in all 16 family members with hand and foot abnormalities available for testing, but not in any family member with normal hands and feet. The distal limb abnormalities in this family were thus shown to be fully penetrant, although only 1/10 male and 3/6 female mutation carriers had genital tract abnormalities. The mutation is predicted to result in a truncated protein lacking the final 20 C-terminal amino acids of the wild-type protein, including the last 13 amino acids of the homeodomain. A very similar premature stop mutation in exon 2 has recently been identified in the father and daughter reported by Cleveland and Holmes (1990; Innis, 1997). HFGS is thus the second human malformation shown to be caused by mutations in a HOX gene. The possible functional consequences of these mutations are discussed in Chapter 9.

These studies illustrate both the value and the limitations of mouse models of human malformation syndromes. The extreme similarity of the distal limb abnormalities in Hd!-^ mice and HFGS patients meant that identification of the molecular basis of Hd

led directly to the identification of the molecular basis of HFGS. Because the normal anatomy of the female genital tract is different in mice and in humans, however, Hd mice cannot be used to study the Müllerian duct fusion defects that occur in HFGS, even though it is this feature of the syndrome that causes the most serious clinical problems.

The

Hox

genes

(38)

The genomic organisation and evolution of Hox genes

In all species in which they have been studied to date, the Hox genes are organised into clusters (Krumlauf, 1994). Thus, Drosophila has 8 Hox genes organised into a single cluster, the Homeotic complex (HOM-Q, which is divided into two parts, the Antennapedia complex (ANTP-Q and the Bithorax complex {BX-C), as shown in Fig 1.3 A. Vertebrates have 39 Hox genes organised into four clusters, named HoxA though

HoxD, as shown in Fig 1.3 B. Each vertebrate cluster is approximately 120 kb long, and contains 9 to 11 genes, all orientated in same 5' to 3’ direction of transcription. These clusters are located on separate chromosomes. Thus, in humans, the HOXA cluster lies on chromosome 7pl4, the HOXB cluster on chromosome 17q21, the HOXC cluster on chromosome 12ql3, and the HOXD cluster on chromosome 2q31.

Both the Drosophila and the vertebrate Hox gene clusters are believed to have arisen from a single ancestral cluster, which must thus have predated the divergence of arthropods and chordates about 600 million years ago. This ancestral cluster is itself presumed to have arisen from the tandem duplication and diversification of a single Hox

gene (Kenyon, 1994; McGinnis and Krumlauf, 1992). Over the course of evolution, the ancestral cluster has become split into two parts in Drosophila (in some other arthropods, it is intact), while in vertebrates it has undergone at least two successive duplication events, followed by divergence (Carroll, 1995).

The vertebrate Hox genes can be divided into 13 different subsets or ‘paralogous groups’, on the basis of sequence similarity and relative position within the clusters, as shown in Fig. 1.1 B. Members of a given paralogous group show greater sequence similarity to each other than they do to other members of their respective clusters, since they share a common evolutionary ancestor. No one cluster contains a representative from all 13 paralogous groups, since different sets of genes were lost from the different clusters early in their separate evolution, but analysis in many species has shown that in any given cluster the same paralogous subsets have been retained in all vertebrates, indicating a strong selective pressure very early in vertebrate evolution to maintain the particular combination currently observed (Krumlauf, 1994). Specific vertebrate paralogous groups show homology to specific Drosophila Hox genes, as indicated in Fig 1.3 (Mann and Chan, 1996). Thus, paralogous groups 1, 2 and 4 are most similar to labial {lab\ proboscipedia (pb) and Deformed (Dfd) respectively. Group 3 may be specific to

(39)

A Drosophila BX-C ANTP-C

Abd-B Abd-A Ubx Antp Scr Dfd pb lab

B M ouse

HoxA

(H o x I) 5'

aI3 1.10 IJ

a l l 1.9 II alO 1.8 IH a9 1.7 IG a7 1.1 lA a6 1.2 IB aS 1.3 1C a4 1.4 ID a3 1.5 y a2 1 . 1 1

a l 1.6

HoxB

bl3

y

b9 b8 b7 b6 bS b4 b3 b2 b l

2.5 2.4 2.3 2.2 2.1 2.6 2.7 2.8 2.9 2E 2 0 2 C 2 8 2A 2F 2 0 2H 21

y

H oxC

(H o x 3 )

c l3 c l2 c l l 3.7 3 0 clO 3.6 3F c9 3.2 3 8 c8 3.1 3A c6 3.3 3 0 c5 3.4 3 0 c4 3.5 3F

y

HoxD

(H o x 4 )

dl3 d l2 4.7

d l l 4.6 4F

dlO d9

4.5 4.4 4 0 4 0

d8 4.3 4F

y

d4 4.2 4 8 d3 4.1 4A d l 4.9

Figure 1.3 Organisation o f the Hox genes A , in Drosophila and B, in mouse. A , In

Drosophila, a single HOM-C cluster containing 8 genes is split into two complexes, BX-C

and ANTP-C. In the mouse, there are four separate clusters, HoxA through HoxD, each

(40)

Groups 5 to 8 are equally similar to Sex combs reduced {Scr\ Antennapedia {Antp\ Ultrabithorax {Ubx) and Abdominal-A (Abd-A), while groups 9 to 13 are most similar to

Abdominal-B (Abd-B). It is the most 5’ member of the ancestral cluster, therefore, that has undergone the greatest diversification during evolution, giving rise to 16 different genes in vertebrates, including HOXD 13 and HOXAl3.

The role of the Hox proteins in development

In almost all metazoans, Hox proteins play a fundamental role in organising the body plan during embryonic development. Indeed, their evolution during the pre- Cambrian era may have made an important contribution to the explosion of diverse body plans that subsequently occurred in the early Cambrian period (McGinnis and Krumlauf, 1992). Rather than specifying the development of particular structures, Hox proteins act by assigning distinct positional identities to cells in different regions along the developing body axes. In Drosophila, Hox proteins determine segment identity along the main anterior-posterior (head-to-tail) axis. Mutations in Drosophila Hox genes result in the loss of specific body segments or the transformation of one segment into another, as reviewed by Gehring (1994). Thus, loss-of-function mutations in the Ubx gene transform the 3^** thoracic segment, which normally carries small balancing organs (halteres), into a 2"^ thoracic segment, carrying wings, resulting in a four-winged fly. Similarly, if the Antp

gene, which is normally expressed in the 2"^ thoracic segment, is ectopically expressed in the developing antennae, the result is a fly whose antennae are replaced by legs. It is this ability of the Drosophila Hox genes, when mutated, to transform one part of the insect body into the ‘likeness’ of another that originally earned them the name ‘homeotic’ genes, from the Greek word ‘opoioç (homoios) meaning ‘alike’. Vertebrate Hox genes also control the organisation of the embryo along the primary anterior-posterior body axis, and therefore play an important role in the development of the central nervous system, axial skeleton, urogenital tract and gastrointestinal tract (McGinnis and Krumlauf, 1992). In addition, they help regulate patterning along the secondary axes that arise during the outgrowth of the vertebrate limb and genital buds, and are therefore crucial for the development of the limbs and external genitalia (Krumlauf, 1994).

(41)

expressed most posteriorly, whereas the more 3’ genes are expressed in progressively more anterior regions. Since the 3’ genes are generally also expressed in the posterior regions, sets of overlapping ‘nested’ or ‘Russian doll’ expression domains often result. In the mouse, for example, ordered arrays of spatially-restricted Hox gene expression domains with sharply demarcated anterior boundaries have been observed in the hindbrain, branchial arches, neural crest, neural tube, paraxial mesoderm, digestive tract, and Wolffian and Müllerian ducts (reviewed in (Krumlauf, 1994). In addition, in vertebrates (although not in Drosophila) the time in embryonic development at which the different Hox genes are expressed corresponds to their physical position on the chromosomes. This phenomenon is termed ‘temporal colinearity’. Thus, expression of genes in a more 3’ position in the clusters is activated before that of the more 5’ genes. This sequential activation of vertebrate Hox genes correlates with the sequential determination of identity along the body axes. Both forms of colinearity imply the existence of complex-wide regulatory mechanisms for co-ordinating gene expression. The timing of expression of individual mouse Hoxd genes has already been shown to depend on their relative position within the cluster (van der Hoeven et a/., 1996), and a regulatory element has recently been identified which appears to direct timing of Hoxd gene activation (Kondo and Duboule, 1999).

The specific fimction of a given vertebrate Hox gene cannot readily be deduced from its expression pattern alone, since members of the same paralogous group often have very similar expression domains and the expression domains of different paralogous groups overlap extensively. Moreover, individual Hox genes often have complex expression patterns which involve many cell types and change rapidly over short periods of time. In the absence o f any obvious naturally occurring mutations in the vertebrate Hox

genes (the HoxalS mutation in the hypodactyly mouse and the H0XD13 mutations in SPD patients were only discovered in 1996), studies of vertebrate Hox gene function have therefore relied largely on the introduction of loss-of-function (and a few gain-of- function) Hox gene germline mutations into mice using gene targeting, as well as on Hox

gene misexpression experiments in mice and chicks using retroviral vectors (reviewed by McGinnis and Krumlauf (1992), Krumlauf (1994), and Favier and Dollé (1997).

Targeted disruptions of several mouse Hox genes have been shown to result in homeotic transformations (to more anterior or posterior structures), just as in Drosophila^

(42)

than a change of identity, absence or malformation of structures has been observed. In particular, in mice with targeted disruptions of Hox genes that are normally expressed in the developing limbs (Davis and Capecchi, 1994; Dollé et al., 1993; Fromental-Ramain et a l, 1996), individual skeletal elements often show reductions in size, changes in shape and a delay in their rate of maturation, indicating an important role for vertebrate Hox

genes in the control of cell growth. Mutation of paralogous and non-paralogous Hox

genes in combination has also shown that there are likely to be complex interactions between different Hox proteins, as suggested by their overlapping expression domains. Analysis of compound mouse mutants has provided evidence for functional compensation, suggestive of redundancy, both within and between different paralogous groups, as well as synergistic interactions (reviewed by Rijli and Chambon (1997). Different Hox proteins may thus have overlapping as well as unique functions, and different members of a paralogous group may share regulation of the same target genes. There is also evidence for a hierarchy amongst Hox proteins, whereby the more 5’ members of a cluster predominate over the more 3’ members, not by downregulating their transcription, but by overriding their functional effects. This phenomenon, which is termed ‘phenotypic suppression’ in Drosophila, and ‘posterior prevalence’ in the mouse (Duboule, 1991), may be particularly important in the case of HOXD 13, since evidence from both targeted mutations in mouse and misexpression studies in mouse and chick suggests that Hoxd 13 can exert functional dominance over both group 12 and group 11 paralogs (Davis and Capecchi, 1996) (Kondo et al., 1996) (van der Hoeven et a l, 1996) (Goff and Tabin, 1997).

The role of HOXD13 and HOXAl3 in development

As might be expected fi-om their position at the 5’ end of their respective clusters, both Hoxd 13 and HoxalS are expressed in the most posterior region of the primary anterior-posterior (head-to-tail) body axis, as well as in the most posterior and distal regions of the two secondary axes (the limb and genital buds). Accordingly, both genes play a major role in the development of the distal portion of the limbs, as well as the lower portion of the urogenital tract.

(43)

segment, the autopod, consists of the carpals, metacarpals and phalanges in forelimb, and the tarsals, metatarsals and phalanges in the hindlimb. The development of these structures in embryonic life involves a series of condensation, branching, and segmentation events in chondrogenic cells (Shubin and Alberch, 1986). Mesenchymal cells in the limb bud first aggregate to form a central condensation, which grows by the addition of cells at its distal end. This primary condensation then undergoes sequential dichotomous branching, which generates first the two elements of the zeugopod, and then the carpal/tarsal and digital elements of the autopod. Finally, in the developing autopod, segmentation of the primary digital condensations occurs, generating the metacarpals/metatarsals and phalanges. This overall process is highly conserved across species. The different morphology in different tetrapod limbs thus depends on the absence of certain branching events, the absence of chondrification or ossification of certain elements, and differential growth or secondary fusions between condensations.

Numerous signalling molecules have now been identified which provide growth and/or patterning information along the proximal-distal, anterior-posterior and dorsal- ventral axes of the developing limb, as reviewed by Wolpert (1999). Hoxd and Hoxa

genes of the paralog groups 9 to 13 (the 5’ Hoxd and Hoxa genes) are likely to act as downstream effectors of many of these signals, since their activation is spatially and temporally coupled to limb bud outgrowth. Moreover, their expression domains have been noted to centre around the zone of polarising activity (the main pattern-promoting region in the posterior and distal part of the limb bud).