‘The hunt for the clubfoot gene’

Mr S L Barker BSc(Hons) MD FRCSEd(Tr&Orth)

The hunt for the clubfoot gene…

•

Why bother ?

•

The toolbox

•

Stumbling blocks

•

Where are we up to ?

•

ACRG

•

What next ?

Why bother?•

Scientific curiosity

•

Preventative potential•

Therapeutic potential

•

Parental ‘need to know’–

Is it my fault?

–

It’s his/her fault–

It’s not my side of the family

–

Will it happen again?

What are the tools?

•

Genetic epidemiology–

Census, Birth registers, Morbidity/Mortality databases, Case note review, Questionnaires, Pedigree studies, Direct interviews.

•

Molecular genetics"the analysis of human DNA, RNA, chromosomes, proteins, and certain metabolites in order to detect heritable disease-related genotypes, mutations, phenotypes, or karyotypes for clinical purposes"

(Holtzman & Watson 1997)

•

Genetic epidemiology–

Census, Birth registers, Morbidity/Mortality databases, Case note review, Questionnaires, Pedigree studies, Direct interviews.

•

Molecular genetics"the analysis of human DNA, RNA, chromosomes, proteins, and certain metabolites in order to detect heritable disease-related genotypes, mutations, phenotypes, or karyotypes for clinical purposes"

(Holtzman & Watson 1997)

What are the tools?

Macro-level, ‘Birds eye’ study

Micro-level, lab study

•

Genetic epidemiology–

Census, Birth registers, Morbidity/Mortality databases, Case note review, Questionnaires, Pedigree studies, Direct interviews.

•

Molecular genetics"the analysis of human DNA, RNA, chromosomes, proteins, and certain metabolites in order to detect heritable disease-related genotypes, mutations, phenotypes, or karyotypes for clinical purposes"

(Holtzman & Watson 1997)

What are the tools?

Macro-level, ‘Birds eye’ study

The case for heritability•

Twins (Idelberger, 1939)–

32% Monozygotic (Identical)

–

2.9% Dizygotic

(Non-identical)•

Relatives also affected –

Maori 1978 (Beals) 12%

–

Texans 1998 (Lochmiller) 24%–

Auckland, Polynesians 54%

–

Glasgow, 1984 (Cartlidge) 30%

The case for heritability

•

Risk to siblings (Wynne Davies, 1965)

–

Population risk of 1 in 800 live births–

Risk to a second child 1 in 80.

•

2 males: 1 female•

Major gene…Polygenic (Palmer, 1964, Wang, 1968)

•

Single gene, 2 alleles (Rebbeck

1993)

The case for heritability•

Dominant gene with 33% penetrance

(Chapman, 2000)

•

ECCE Pedigree Study (Cardy, 2007)

–

785 families–

26% had affected relative, 7.8% 1st

degree

–

Absolute risk to 1st

or 2nd

degree relative

•

1.2% ♂

proband•

0.8% ♀

proband

–

Greatest risk to male relatives of male proband

–

1.5%

Models of heritability

•

Dominance•

Dominance (incomplete penetrance)

•

Recessive•

Sex linkage

•

Chromosomal•

Polygenic

Models of heritability

•

Dominance•

Dominance (incomplete penetrance)

•

Recessive•

Sex linkage

•

Chromosomal•

Polygenic

Where are we up to?•

Pressure theories–

Oligohydramnios

–

Abnormal fetal

position–

Unstretched

uterus

•

Placental insufficiency•

Constriction bands

•

Toxin•

Temperature

•

Infective pathogen (enterovirus)

•

Drugs•

EM radiation

•

Chromosomal abnormality•

Sex-linked

•

Single dominant•

Single recessive

•

Polygenic

Where are we up to?•

Pressure theories–

Oligohydramnios

–

Abnormal fetal

position–

Unstretched

uterus

•

Placental insufficiency•

Constriction bands

•

Toxin•

Temperature

•

Infective pathogen (enterovirus)

•

Drugs•

EM radiation

•

Chromosomal abnormality•

Sex-linked

•

Single dominant•

Single recessive

•

Polygenic

Multifactorial inheritance

Genotype A+

Environmental factors B

Phenotype

•

Genetic epidemiology–

Census, Birth registers, Morbidity/Mortality databases, Case note review, Questionnaires, Pedigree studies, Direct interviews.

•

Molecular genetics"the analysis of human DNA, RNA, chromosomes, proteins, and certain metabolites in order to detect heritable disease-related genotypes, mutations, phenotypes, or karyotypes for clinical purposes"

(Holtzman & Watson 1997)

What are the tools?

Micro-level, lab study

Finding Clubfoot Genes

•

Candidate genes•

Gene-gene interaction studies–

Biochemical pathways

•

Gene-environment interaction

•

Identification of associated genes

The Human Genome Mapping Project

•

Mapping of all chromosomes

•

Identification of gene sequence & order

•

Nothing about function

•

Enables identification of disease associated genes

Congenital Acquired

TalipesEquinovarus

Talipes Calcaneovalgus

Metatarsus Adductus

Postural/Mild

Structural/Severe

Idiopathic Syndromic

Club footClub foot

Aetiological heterogeneity

Congenital Acquired

TalipesEquinovarus

Talipes Calcaneovalgus

Metatarsus Adductus

Postural/Mild

Structural/Severe

Idiopathic Syndromic

Club footClub foot

Aetiological heterogeneityCVA,Guillan-Barré, Intraspinal

tumours,

Poliomyelitis

Cerebral Palsy, Diastematomyelia, Downs, Arthrogryposis

Intra-uterine environment, Genotype

…Clinical homogeneity

Homology

‘Ontogeny recapitulates phylogeny’

Homology

‘Ontogeny recapitulates phylogeny’

Homology

Human ICTEV

Mouse/Chick clubfoot

Mousegenome

Humangenome

Homology

?

Human ICTEV

Mouse/Chick clubfoot

Mousegenome

Humangenome

?structural

genetic

•

DTDST –

sulphate transporter

gene (Paris, 2001)

•

MTHFR gene –

C677T allele protective (Aberdeen 2007)

& Impaired homocysteine

metabolism (Turkey 2003)

•

CASP10 -

apoptosis regulation, 2q31-33 (Texas 2005)

•

Wnt7 –

limb dorsoventral

axis& LM07 (Iowa 2005)

•

Limb development control genes –HOX D10

Limb bud development control

•

Apical ectodermal

ridge controls proximal-distal axis -

FGF4 gene

•

Zone of proliferating activity controls antero-posterior axis -

SHH gene

•

Dorsal ectoderm controls dorso-ventral axis -

Wnt

genes

•

Homeobox

genes -

Hox

A & D via BMPs

Limb bud development control

•

Apical ectodermal

ridge controls proximal-distal axis -

FGF4 gene

•

Zone of proliferating activity controls antero-posterior axis -

SHH gene

•

Dorsal ectoderm controls dorso-ventral axis –

Wnt7a genes via mesodermal

LMX1B•

Homeobox

genes -

Hox A & D via

BMPs

Aberdeen Clubfoot Research Group

• Orthopaedics: S Barker• Genetics: Z Miedzybrodzka, G Liu, D Madrigal• Epidemiology: L Sharp, A Cardy• MRI: C Tickle, S Chudek, S Duce

ACRG strategies

•

Questionnaire data, UK and Netherlands•

Candidate genes -

folate

pathway,

HoxD10, Wnt-7a•

Gene-gene and gene-environment interaction studies

•

Homozygosity

mapping•

Genetic linkage in multi-case families

•

Mouse clubfoot model development

pma E18.5

CD1 E17.5

What Makes a Foot Rotate?

•

Not a lot in the literature!•

Differential growth in 3 axes, apoptosis.

•

16mm (6wiu) -

90°

inward rotation•

22mm (7wiu) –

feet sagittal

•

27mm (8wiu) –

elongation of talus•

Fetus

–

differential calcaneal

growth, foot

d/flexes & pronates

to adult position.•

Which genes control the process?–

HOX C10 & 11 ?

Where to next ?•

Mouse model –

Validation –

MRI, Histology

–

Limb rotation control•

Pedigree studies

•

Define the condition!•

Males > Females–

550 males (70%)

–

235 female (30%) ECCE•

Racial variability

Acknowledgements

Thank you