Transcript DYNC2H1 Clipson Family Variants 27.11.09 1.I2526S/N c.7577T>G

Genome wide SNP analysis leads to the
identification of compound heterozygous
mutations in a pedigree with multiple
pregnancies affected with short rib
polydactyly type III
Sian Ellard1, Ann-Marie Patch1, Elizabeth Young1, Anna L. Gloyn3 and Peter D.
Turnpenny2
Departments of Molecular Genetics1 and Clinical Genetics2, Royal Devon & Exeter NHS
Foundation Trust and Oxford Centre for Diabetes3, Endocrinology and Metabolism,
Churchill Hospital
Family referred to Peninsula Clinical
Genetics service in 2000
Born 1996
TOP 20/40 1999
Short rib polydactyly
 A group of skeletal dysplasias characterized by short
ribs, short limbs, polydactyly and visceral abnormalities
 Lethal in the newborn period
 Four types (I-IV), genes not known
 Presumed AR inheritance
 Diagnosed on ultrasound scan
Family referred to Peninsula Clinical
Genetics service in 2000
Born 1996
TOP15-20 weeks’ gestation between 1999 and 2003
Aim
 To identify the causative gene mutation(s) in
the affected foetuses
Methods
 DNA extracted from paraffin-embedded fixed tissue
stored from the 5 affected foetuses, their unaffected sibling
and both parents
 Genome wide linkage analysis (Illumina Golden Gate
n=6008 SNPs)
 Fine mapping using microsatellite markers
 Sequence analysis of candidate gene
Results:Genome wide SNP scan
 Genotype call rate 98.9% for FFPE DNA vs 99.7% for
lymphocyte DNA
 No homozygous regions exceeding 3cM
 Perl script written to identify informative markers where
affected offspring share a genotype but the unaffected child
does not
Results:Genome wide SNP scan
Results:Genome wide SNP scan
Regions where genotypes were shared by the affected offspring but not the unaffected child.
Chromosome
Physical Start
Physical End
Size (Mb)
5
166.1
172.3
6.2
6
2.2
3.7
1.5
11
97.6
107.3
9.8
17
24.9
28.1
3.2
Results:Microsatellite analysis
 16 microsatellites tested
Chromosome
Physical Start
Physical End
Size (Mb)
5
166.1
172.3
6.2
6
2.2
3.7
1.5
11
97.6
103.6
6.0
17
24.9
28.1
3.2
Results:Candidate genes
6Mb containing 21 genes
Chromosome 11
Results:DYNC2H1 candidate gene
 DYNC2H1 encodes a dynein motor protein required for
generation and maintenance of cilia
 Jeune asphyxiating thoracic dystrophy is a skeletal
dysplasia caused by mutations in the IFT80 gene that
encodes a protein involved in intraflagellar transport of
primary cilia (Beales et al 2007 Nature Genetics)
Where next?
 Sequence best functional candidate
(DYNC2H1, 90 exons) or
 Array capture of 6Mb region for next
generation sequencing
Results:Sequence analysis
Results:Sequence analysis
Results:Splicing prediction
p.Ala940ValfsX8
Results of genetic testing given to
family in 2010
c.2819-14A>G/N
N/N
p.Ile2526Ser/N
c.2819-14A>G/p.Ile2526Ser
Conclusions
 DYNC2H1 mutations confirmed in a 9th family with short rib
polydactyly type III
 First example of linkage mapping to identify compound
heterozygous mutations in an outbred family affected with a
recessive disorder
 Ten years from referral to report; how long using next
generation sequencing technology?
Acknowledgements
 The family
 Helen Butler and Cecilia Lindgren, Wellcome Trust Centre, Oxford
 Nathalie Dagoneau and Valerie Cormier-Daire, Paris
 Karen Stals, Exeter
 Northcott Devon Medical Foundation