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Human Genome Research Program

Research arm of the Western Sydney Genetics Program

Publications 2006 - 2008

NSW Centre for Rett Syndrome Research

Genetic Metabolic Diseases Research Group (including the NSW Centre for Rett Syndrome Research)

Marfan Research Group

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

Head: Prof. John Christodoulou
Telephone: (02) 9845 3452
Fax: (02) 9845 1864
Location: The Kerry Packer Institute for Child Health Research, Level 3

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 8,500 females by the age of fifteen years 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 three 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.

Identification of phosphorylation targets of CDKL5 as possible contributors to the pathogenesis of RTT.

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 that interact with MeCP2 using a proteomics approach, namely 2D gel electrophoresis. The novel interacting protein partners will be characterised by peptide mass fingerprint analyses or direct amino acid sequencing (in collaboration with Patrick Tam's and Dr Philip Robinson's groups, 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 primary neuronal cultures various animal models at our disposal.

* Other RTT Related Research:

Gene discovery approaches to identify new RTT genes.

Evaluation of potential new therapies based on our enhanced understanding of the pathogenesis of RTT.

The Research Team

  • Professor John Christodoulou - Head of Unit
  • Dr Carolyn Ellaway - Clinical Researcher
  • Ms Gladys Ho - RettBASE Coordinator
  • Dr Sarah Williamson - Research Officer
  • Dr Gregory Pelka (based at CMRI) - Postdoctoral Fellow
  • Mr Abidali Mohamedali (based at CMRI) - PhD student
  • Ms Vidya Vasudevan - PhD student
  • Ms Roksana Armani - PhD student
  • Ms Katrina Slater - MPhil student

Collaborating Researchers

  • Children's Medical Research Institute (Sydney) Collaborators
    • Dr Patrick Tam - Mouse model development and study
    • Dr Phil Robinson - MeCP2 protein partner proteomics studies
  • Westmead Millennium Institute (Sydney) Collaborator
    • Dr Barry Slobedman - Microarray research
  • TVW Telethon Research Institute (Perth, WA) Collaborators
    • Dr Helen Leonard - Phenotype-genotype studies
  • West Australian Institute for Medical Research (Perth, WA) Collaborators
    • Professor David Ravine - Molecular and functional studies
    • Dr Alka Saxena
  • Dept of Genetic Medicine, Women's & Children's Hospital (Adelaide, SA) Collaborator
    • Associate Professor Jozef Gécz - CDKL5 mutation screening
  • University of Wales College of Medicine (UK) Collaborator
    • Professor Angus Clarke - CDKL5 mutation screening and phenotype-genotype studies
    • Dr Hayley Archer

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.

* Development of Novel Treatment Strategies for Phenylketonuria:

This study proposes to genetically engineer Lactobacillus organisms to enable strains that colonise the human small bowel to produce an enzyme that has the potential to ameliorate a specific inborn error of metabolism. Eventual animal experiments will provide biological proof of principle, and will demonstrate therapeutic safety. Based on current data, it is feasible to deliver enzymatically active products within the lumen of the small intestine designed to metabolise toxic products that are pathological in some inherited disorders. PKU is an excellent model disease to evaluate this novel treatment modality. The ability to reduce the levels of phenylalanine in the small intestine would allow a relaxation of the diet, better compliance, better growth, and improve the quality of life of individuals with PKU.

* Interallelic Complementation in Phenylketonuria:

Phenylalanine hydroxylase (PAH) is the enzyme responsible for catalysing the conversion of phenylalanine into tyrosine in the presence of the co-factors (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) and O2. The enzyme is a homotetramer, consisting of four identical subunits 452 residues in length. Each subunit has three domains, the regulatory domain, the catalytic domain and a tetramerisation domain. Interallelic complementation is the phenomenon in which the heteroallelic (carrying different mutations) protein regains partial or complete function compared to either of the homoallelic proteins.

We hypothesise that positive interallelic complementation exists in PAH and that some structural mutants, not only kinetic mutants, are BH4-responsive alleles, and that BH4 supplementation would increase the degree of complementation involving these alleles.

The aims of this study are:

  1. to study single mutations of PAH and classify their effect on the expression and function of the protein
  2. to investigate (or verify) the effects of BH4 on expression and function of certain PAH mutants
  3. to study combinations of PAH mutations for evidence of interallelic complementation, both with and without augmented levels of BH4

The Research Team

  • Professor John Christodoulou - Head of Unit
  • Dr Xinzhang Tong - Research Officer
  • Ms Gladys Ho - PhD student

Collaborating Researchers

  • Children's Hospital at Westmead Collaborator
    • Dr Ian Alexander - development of genetically modified probiotic for the treatment of PKU

Research Aspect Three - Molecular and Functional Studies of Disorders of the Mitochondrial Respiratory Chain

Background Information

Mitochondrial respiratory chain (RC) disorders are a highly variable group of disorders, often with their onset in infancy, with a minimum birth prevalence of around 1 in 5,000. Disease severity can be markedly different between individuals, with multiple organs being affected in some individuals, potentially resulting in severe disability. Although mutations in genes that form the respiratory chain system have been identified, a distinct correlation between these mutations and disease severity remains unclear.

Diagnosis of a mitochondrial RC defect is primarily based on enzymatic measures, which are often tissue specific and not demonstrable in cultured skin fibroblasts in about 50% of cases with a demonstrated muscle RC enzyme defect. For most patients with mitochondrial disorders, the underlying genetic defect is unknown, and prenatal diagnosis rests with enzyme analyses in cultured chorionic villus cells or amniocytes, which best reflect results observed in cultured fibroblasts. Therefore, prenatal testing is only available for families where the defect is clearly demonstrable in cultured skin fibroblasts. However, tissue specificity (ie a lack of expression of the functional defect in cultured skin cells) severely limits the availability of reliable prenatal diagnosis.

Research Directions

Our group currently has two major active research projects relating to the mitochondrial respiratory chain disorders:

* Unmasking Mitochondrial Respiratory Chain Disorders by Forced Myogenesis of Cultured Cells:

The transcription factor MyoD plays a key regulatory role in the differentiation of the skeletal-muscle cell lineage, and overexpression of MyoD in skin fibroblasts, chorionic villus and amniocytes produces fused multi-nucleated myotubes. We hypothesise that forced myogenesis of patient skin fibroblasts will unmask mitochondrial RC defects in patients who demonstrate abnormal enzymology in skeletal muscle, but exhibit normal or equivocal results in fibroblasts.

We aim to study the usefulness of MyoD-forced myogenesis for unmasking of mitochondrial RC disorders in cultured fibroblasts derived from patients with:

  1. A mitochondrial disorder resulting from a definite or presumed nuclear defect, who show enzyme abnormalities in skeletal muscle, but normal results in cultured fibroblasts.
  2. A family history of mitochondrial RC dysfunction, to investigate whether MyoD-forced myogenesis can be applied to the prenatal diagnosis of nuclear-encoded RC defects.
  3. A mitochondrial disorder resulting from a known mtDNA defect, who show enzyme abnormalities in skeletal muscle, but normal results in cultured fibroblasts.

* Discovery of Novel Genes Responsible for Mitochondrial Respiratory Chain Disorders:

Mitochondrial respiratory chain disorders are seven times more common in the NSW Lebanese population than the non-Lebanese population, and much of this can be attributed to a high level of consanguinity in this ethnic group. This has given us an opportunity to use autozygosity mapping to identify candidate genes for further study. We have recently identified a novel gene responsible for MLASA (Myopathy, Lactic Acidosis and Sideroblastic Anaemia) in two Lebanese families. Autozygosity analysis is currently under way in several other consanguineous families.

The Research Team

  • Professor John Christodoulou - Head of Unit
  • Dr Lisa Riley - Research Officer
  • Children's Hospital at Westmead Collaborator
    • Dr Sandra Cooper - MyoD forced myogenesis and mitochondrial disorders gene discovery projects

Marfan Research Group

Head: Dr Lesley Adès
Telephone: 9845 3219
Fax: 9845 3204
Location: Diagnostic Services building, level 1

The Marfan Research Group provides a research and diagnostic facility for the screening of the FBN1, TGFBR1 and TGFBR2 genes in patients referred with a possible diagnosis of Marfan syndrome, or Marfan-related conditions. Seven per cent of aortic dissections occur in young subjects, aged less than 40 years. Aortic dissection in this subgroup is most often related to an underlying connective tissue disorder, such as Marfan syndrome, "incomplete" Marfan syndrome (usually without ectopia lentis), the vascular subtype of Ehlers Danlos syndrome, non-syndromic familial thoracic aortic aneurysm and dissection (TAAD), or the rare, more recently described and probably clinically under/mis-diagnosed Loeys Dietz syndromes, types I, Ia and II. Loeys Dietz syndrome type II is an important differential diagnosis in patients thought to have the vascular subtype of Ehlers Danlos syndrome, but in whom collagen studies prove normal. We have studied more than 1000 individuals from approximately 460 families. A total of 135 FBN1, 7 TGFBR1 and 10 TGFBR2 gene mutations have been characterised. We are transitioning from a sole DHPLC approach with or without MLPA in selected circumstances, to direct DNA sequencing of FBN1. Currently, 30 of the 65 FBN1 exons are now sequenced directly, whilst the rest are screened by DHPLC. All exons of both TGFBR1 and TGFBR2 are directly DNA sequenced. We have investigated the usefulness of MLPA in FBN1 mutation-negative patients who may have a large FBN1 insertion or deletion, and have noted a low yield of additional mutations (deletions or duplications). The group's success in FBN1 gene mutation detection rivals those of international groups. The methodology allows for detection of single base substitutions, deletions and insertions. Mutation detection is 75-80% for those who meet the Ghent diagnostic criteria for Marfan syndrome, and 25% in those who do not. We have developed a Marfan database that incorporates clinical and molecular data, links to the international databases and allows for powerful statistical analyses and genotype-phenotype correlations. Data entry into the database has been updated recently. We have strong collaborative links with international groups involved in the study of aortic aneurysms and dissections, arterial tortuosity syndromes, bicuspid aortic valve disease and the TGFB signalling pathway. We regularly contribute data to the International Marfan Database. We have participated in a "hybrid" documentary on Marfan syndrome, "Jabe Babe", short-listed at the 2005 Cannes Film Festival, and nominated for three AFI and one IF award.

Our goals are to:

  • Educate physicians and the community about Marfan syndrome and related conditions
  • Maintain a high FBN1 mutation detection capability
  • Develop a high TGFBR1 and TGFBR2 mutation detection capability
  • Establish genotype-phenotype correlations in Marfan syndrome and in related disorders
  • Discover the disease pathogenesis in individuals who are FBN1, TGFBR1 and TGFBR2 mutation negative
  • Establish mutation screening for the ACTA2 gene
  • Continue to publish our research findings in peer-reviewed international journals
  • Become the National Reference Laboratory in Australasia for Marfan syndrome and related disorders
This document was updated on Monday, 9 February 2009

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