Human Genome Research Project

Genetic Metabolic Diseases Research Group

• Head of Unit
• Research Aspect One - The Biology of Rett Syndrome: MECP2 Mutations and Beyond
• Research Aspect Two - Development and Evaluation of New Treatments for Phenylketonuria (PKU)

See also: Genetic Metabolic Diseases in the hospital's Directory of Services

Head of Unit

Research Aspect One - The Biology of Rett Syndrome: MECP2 Mutations and Beyond

Background Information

Rett syndrome (RTT) is a severe neurodevelopmental disorder, with a cumulative incidence of 1 per 10,000 females by the age of twelve in Australia Most cases of RTT are due to mutations in the X-linked methyl CpG-binding protein 2 (MECP2), however even with the most comprehensive mutation screening strategies 5 – 10% of RTT patients do not appear to have a disease-causing mutation in the MECP2 gene.  We have previously shown that some, but not all, of the phenotypic variability may be explained on genetic epidemiological grounds by the site and type of mutation, and by the level of skewing of X-inactivation.

In our ongoing investigation of the downstream biological consequences of MECP2 mutations, we have studied the expression profile in the brains of RTT patients by microarray analysis (in collaboration with Dr Barry Slobedman, Westmead Millennium Institute) to identify genes that consistently show altered expression.  We have identified a subset of genes that may serve important functions in vesicular dynamics, mitochondrial bioenergetics or apoptosis.  These findings were verified by real-time (quantitative) PCR, and in RNAi studies using a neuronal cell line, SH-SY5Y.  Our findings raise the possibilities that the pathogenesis of RTT may in part be a consequence of dysregulation of the cellular functions regulated by these genes.

Our group has also discovered a second gene (cyclin dependent kinase-like 5; CDKL5), mutations in which cause a phenotype that has a strong overlap with RTT, particularly those individuals with early onset infantile spasms, and may also be associated with autism.

Research Directions

Our group currently has two major directions of research relating to RTT:

* CDKL5 Related Research:

• Evaluation of the role of mutations in CDKL5 in RTT and other disorders (including X-linked mental retardation, X-linked Infantile Spasm syndrome [ISSX], and autism).
• Study of the intracellular location and function of CDKL5 and its interaction with MeCP2, and examination of the effect of CDKL5 mutations on the stability and function of its gene product.
• Development of a knock-out mouse model for Cdkl5 (in collaboration with Dr Patrick Tam’s group, Children’s Medical Research Institute).

* MeCP2 Related Research:

• Identification of novel proteins which interact with MeCP2 using pull-down assays, using wild-type and mutant GST-MeCP2 fusion proteins.  The novel protein partners will be characterised by peptide mass fingerprint analyses or direct amino acid sequencing (in collaboration with Dr Philip Robinson’s group, Children’s Medical Research Institute).
• Examination of whether the genes identified through microarray analysis of RTT brains and cell lines are under the direct transcriptional control of MeCP2 using a combination of chromatin immunoprecipitation (ChIP) and gene promoter analyses (in collaboration with Dr Assam El-Osta’s group, Baker Medical Research Institute).

Evaluation of the significance of these downstream MeCP2 targets on the pathogenesis of the neurological dysfunction in RTT (using a number of structural and functional studies of a neuronal cell (SH-SY5Y) RNAi knockdown model and various animal models at our disposal.

The Research Team

Dr Patrick Tam - Mouse model development and study
Dr Phil Robinson - GST-pull down research

Dr Barry Slobedman - Microarray research

Dr Helen Leonard - Phenotype-genotype studies

Professor David Ravine - Molecular and functional studies
Dr Alka Saxena

Associate Professor Jozef Gécz - CDKL5 mutation screening

Professor Huda Zoghbi - Mouse model studies

Professor Angus Clarke - CDKL5 mutation screening and phenotype-genotype studies
Dr Hayley Archer
Dr Julie Evans

Research Aspect Two - Development and Evaluation of New Treatments for Phenylketonuria (PKU)

Background Information

PKU is a rare disorder caused by an inherited enzyme deficiency. A person with PKU is unable to break down the amino acid phenylalanine, found in all protein containing foods, and a build-up of this amino acid occurs.  Without treatment from soon after birth, the persistent elevation of phenylalanine is toxic to the brain and leads to profound intellectual handicap. Fortunately, nowadays all babies are tested for this disorder soon after birth, and treatment leads to normal development.  The only way of treating PKU is with a very strict diet, extremely low in protein, and supplemented with specifically designed medical foods. The affected children can’t eat meat, chicken, fish, or dairy products, and even must have special low-protein bread and pasta. The diet is very difficult, and the protein substitutes taste bad to most children.

Research Directions

* Tetrahydrobiopterin-Responsive PKU:
We are currently investigating different ways in which the diet can be made more acceptable, or for some children not necessary at all. One research project is to further investigate the use of a “co-factor” tetrahydrobiopterin (BH4), which increases the activity of the defective enzyme. We need to find out which children can attain much lower levels of phenylalanine just by taking this medicine, and importantly, we need to find out the lowest doses we can use, as the medicine is extremely expensive. Research currently under way, which involves testing DNA of the affected children, will also be a clue as to which children will respond.

* Effect of Large Neutral Amino acids on Brain Phenylalanine in PKU:

There is the occasional individual with PKU, who despite poor control of the diet, and chronic marked elevations of blood phenylalanine, seems to escape any significant neurological impairment.  It is believed that this protective effect is a consequence of variability in the transport kinetics of the blood-brain barrier phenylalanine transporter.  Large neutral amino acids (LNAA) compete with phenylalanine for this transporter, and we are currently evaluating the potential usefulness of supplemental LNAA in blocking the uptake of excess amounts of phenylalanine into the brain.  This work, which includes a combination of biochemical, brain MRS and neuropsychological studies, is being done in collaboration with Dr Pamela Joy and Ms Antonella Rocca of the Child Development Unit, Mr Allan Kemp, previously of the Medical Imaging Department, and Professor Lindy Rae of the University of NSW.