Frances Bond West Midlands Regional Genetics Laboratory 12/04/10 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

Aim of project & methods Overview Micro syndrome Aim of project & methods Results Conclusion

Micro Syndrome Rare autosomal recessive disorder Eyes Brain Genital Small eyes Congenital cataracts Loss of optic nerve fibres Brain Severe mental retardation Severe cortical visual impairment Genital Microgenitalia The most reliable diagnostic signs that are present from birth are eye features Several children have died by the age of 12 years (respiratory complications). The oldest patient known with this condition is 23 (patient 1). Patient 1 at 6 and 21. Patient 2 (brother of patient 1) at 5 and 20.

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 2005, Inactivation of the Rab3 GTPase Activating Protein Dr Aligianis’ research group from the Section of Medical and Molecular Genetics, University of Birmingham These variants segregated with disease status and were not present in 400 control chromosomes

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 clinically diagnosed with Micro syndrome included 1 patient from the each of the families used for autozygosity mapping for confirmation purposes Clarify the contribution of MICRO3 mutations in the aetiology of Micro syndrome performed with a view to offering screening of MICRO3 as a diagnostic service in the future. all fragments amplified using the same conditions and M13 tag Covered exons and the intron-exon boundaries. Dosage analysis was not performed for exons 4_1, 4_2, 5 and 7 because probes that met MRC Holland’s guidelines could not be designed. Unfortunately, no reference control probes were used so patients that appeared normal and had no heterozygous snps by sequence analysis may have a heterozygous whole gene deletion. 5

Results Variants were detected in 13 unrelated patients (21%) 10 different novel variants 6 patients had variants that are likely to be pathogenic (10%) All variants previously identified were confirmed but not detected in any other patients in the cohort The variants are located throughout the gene, Green boxes = exons in major transcript 4 different variants likely to be pathogenic, 2 of unknown significance, 3 likely to be benign polymorphisms, 1 likely to be a technical MLPA artefact but needs to be confirmed using another dosage method Five of the patients with pathogenic or likely pathogenic variants have variants that affect exon 3 suggesting a possible mutation hotspot in this location. 6

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 Homozygous in all four families: Parents and siblings, 24 individuals Previous work by the Section of Medical and Molecular Genetics, University of Birmingham had suggested there was evidence of a founder effect because these patients and their extended families shared the same haplotype for a 5.5Mb region of chromosome 10. Identified during an unrelated study investing the protein’s role in the secretory pathway performed by Dr Aligianis’ research group Confirms is a pathogenic mutation which results in loss of protein function 7

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? Heterozygous Parents and 1 affected and 1 unaffected sibling. 8

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 not possible to perform in silico analysis using PolyPhen or Sift for other 2 variants of c.619T>C (p.X207GlnextX21) and c.577G>A (p.Gly193Ser) have not been possible within the time constraints of this project performed by Dr Aligianis’ research group (Section of Medical and Molecular Genetics, University of Birmingham) Confirms is a pathogenic mutation which results in loss of protein function This suggests, functional studies needed to confirm 9

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 Expected because PCR amplification had repeatedly failed during attempts to sequence this exon Needs to be confirmed with functional studies Apparent single exon deletion should be treated with caution unfortunately this was not possible within the time constraints of this project. However, exon 3 failing to amplify using PCR with primers in a different location to the probe binding site and the patient’s parents both having an apparent heterozygous deletion of exon 3 is also evidence to support the case for the deletion being real. Exon 3 10

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 Mutations in MICRO3 can cause Micro syndrome 4 had the same one Current testing strategies are suited to first screening for mutations in genes with the highest mutation detection rates and, if no mutations are found, screening other genes with lower detection rates can be beneficial if the clinical diagnosis is certain. – Both offered at WMRGL evidence of a founder effect of a pathogenic mutation in exon 3 in this population MLPA for MICRO3 should not be put into diagnostic service without further validation and the introduction of reference control probes in order to allow the exclusion of heterozygous whole gene deletions in all patients. only 20 patients in the project cohort have been screened for mutations in RAB3GAP1 in order to accurately calculate the MICRO3 variant detection rate in this cohort 11

Acknowledgments Dr Aligianis’ Research Group WMRGL 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 12