Download Micro Syndrome

Identification of mutations
in a novel gene for
Micro syndrome
in a cohort of
international patients
Frances Bond
West Midlands Regional Genetics Laboratory
12/04/10
Overview
• Micro syndrome
• Aim of project & methods
• Results
• Conclusion
Micro Syndrome
• Rare autosomal recessive disorder
Eyes
Brain
Small eyes
Congenital cataracts
Loss of optic nerve fibres
Genital
Microgenitalia
Severe mental retardation
Severe cortical visual impairment
Novel Gene Discovery
~60% of cases
• RAB3GAP is a key regulator of the Rab3 protein
– Converts active Rab3-GTP to inactive Rab3-GDP
– Involved in exocytosis of neurotransmitters and hormones
• Autozygosity mapping studies
– 5.5Mb region of homozygosity on chromosome 10
– Sequence analysis of candidate genes within this region
identified variants in the ‘MICRO3’ gene
Aim of Project
• To screen 63 unrelated patients to identify mutations and
copy number abnormalities in MICRO3
• Bi-directional sequencing
– Plate-based high-throughput strategy
– All the exons in the 4 alternative transcripts of MICRO3
• MLPA
– Synthetic probes for exons 1_1, 1_2, 2, 3, 6, 8 and 9
Results
• Variants were detected in 13 unrelated patients (21%)
– 10 different novel variants
– 6 patients had variants that are likely to be pathogenic (10%)
c.88C>A
1_1
1_2
Deletion of exon
1_1 and 1_2
c.187-55T>A
c.71T>A
2
3
Deletion
of exon 3
4-1a
4_1b
c.186+57G>A
4_1c
4_2
5
6
c.277_279delAGA
7
c.260-7dupA
8
c.619T>C
9
c.577G>A
p.Leu24Gln
• Identified in 4 patients
– Consanguineous Pakistani families
– Segregated with Micro syndrome
• 2 codons downstream of a published variant
– Results in an inactive GDP-bound protein
• In silico analysis
– Little effect on splicing
– Predicted to affect protein function
• Functional studies
– Disrupted protein does not bind to
GDP or GTP
p.Arg93del, p.X207GlnextX21 &
p.Gly193Ser
• Identified in 1 patient
– Non-consanguineous
American Caucasian family
– Segregated with Micro syndrome
– p.X207GlnextX21 and p.Gly193Ser in cis
• Suggests one is not pathogenic
• p.X207GlnextX21: Extends protein
– ?Abolish C-terminal post-translational modification and
subsequent membrane targeting?
p.Arg93del, p.X207GlnextX21 &
p.Gly193Ser
• In silico analysis
– All: Little effect on splicing
– P.Gly193Ser: Predicted to be tolerated
• Functional studies – p.Arg93del
– Disrupted protein does not bind to GDP or GTP
– Pathogenic: p.Arg93del & p.X207GlnextX21
– Non-pathogenic: p.Gly193Ser
Homozygous deletion of exon 3
• Identified in 1 patient
– Consanguineous family
– Heterozygous deletion of exon 3 identified in parents
• Predicted to result in a truncated protein
• Needs to be confirmed using another method
Exon 3
Exon 3
Conclusion
• 6 patients had likely pathogenic variants in MICRO3 (10%)
– 5 of these had variants that affect exon 3
• Analysis of RAB3GAP should be the first-line test
followed by MICRO3 testing
– Screen exon 3 first in consanguineous Pakistani families
• Dr Aligianis’ Research Group, MRC Human Genetics
Unit, Edinburgh
– RAB3GAP testing for any patient with a negative result
– Functional studies to confirm pathogenicity of variants
– Research into MICRO3 role in development
Acknowledgments
I would like to thank everyone who has contributed to this project:
Dr Aligianis’ Research Group
– Irene Aligianis
– Danai Bem
WMRGL
– Fiona Macdonald
– Carol Hardy
– Sequencing and fragment analysis technical staff