Jan Marshall Friedman

PLPHP deficiency is a recently discovered form of vitamin B6-dependent epilepsies (B6Es) that is caused by recessive mutations in PLPBP. PLPHP is involved in pyridoxal 5’-phosphate (PLP) homeostatic regulation. However, the mechanism by which PLPHP dysfunction disrupts PLP homeostasis and leads to the observed encephalopathy in patients was still elusive. We characterized the clinical, genomic and biochemical abnormalities in a new series of 12 PLPHP deficiency patients. Our results identified previously undescribed clinical features of PLPHP deficiency, including non-epileptic movement disorder, fatal mitochondrial encephalopathy and folinic acid-responsive seizures. We characterized the pathogenicity of patients’ PLPBP variants using in silico tools and 3D modelling of PLPHP and developed a system of clinical severity score. We generated and characterized PLPBP knockout models in HEK293 cells, yeast and zebrafish. Our plpbp-KO zebrafish model replicated the clinical phenotype of PLPHP-deficient patients by showing vitamin B6-dependent seizures and death in untreated KO larvae. Consistent with the biochemical picture in patients, Plphp-deficient fish displayed decreased systemic levels of PLP. In the future this model can be utilized as a tool for investigating the disease pathophysiology, drug screening and identifying diagnostic biomarkers. Pyridoxine-dependent epilepsy (PDE-ALDH7A1) is another form of B6Es that is caused by mutations in ALDH7A1, a gene which encodes an enzyme within the lysine catabolism pathway. We have successfully generated and characterized transgenic mouse strain with constitutive genetic ablation of Aldh7a1. Results showed that KO mice accumulated high concentrations of upstream lysine metabolites including ∆¹-piperideine-6-carboxylic acid (P6C), α-aminoadipic semialdehyde (α-AASA) and pipecolic acid (PIP), similar to the biochemical picture in ALDH7A1-defiecint patients. KO mice fed the regular diet (0.9% lysine) did not exhibit seizures based on EEG analysis. When KO mice are switched to a diet containing higher amount of lysine (4.7%), they developed severe recurrent seizures which led to their quick death. In analogy to the patients’ picture also, treating KO mice under high lysine diet with pyridoxine injections prevented seizures and prolonged their survival. This study provides a proof-of-concept for the utility of the model to study PDE-ALDH7A1 biochemistry and to test new therapeutics.

View record

Neurofibromatosis 1 (NF1) is an autosomal dominant disorder with an estimated prevalence of 1/3000. NF1 is characterized by multiple café-au-lait spots, iris hamartomas, and multiple nerve sheath tumors. Patients may also present with heart disease, cerebrovascular disease, ischemia, or aneurysm. Though well documented, vascular disease in NF1 patients remains poorly understood. Previous in vitro studies suggest that endothelial and vascular smooth muscle function is altered in Nf1+/- mice; however, it is unknown how these alterations affect vascular function in vivo. Haploinsufficiency for neurofibromin, the protein affected in patients with NF1, results in prolonged Ras hyperactivation. We hypothesized that this may result in vascular endothelial dysfunction and impaired cardiac function. To study this hypothesis we examined vascular function in Nf1+/- and control mice using wire myography. Isometric force measurements in thoracic and abdominal aorta at 6-months of age were similar, with Nf1+/- mice demonstrating altered smooth muscle function, enhanced relaxation, and upregulation of the PI3K/Akt/eNOS pathway. To determine if the alterations observed at 6 months of age remain stable or progress to a more dysfunctional state, we examined the abdominal aorta in older mice. Interestingly, we observed increased contraction and reduced relaxation in 9-to-12 month old Nf1+/-mice compared to control littermates, indicative of endothelial dysfunction and progression to a more dysfunctional state. Vascular dysfunction is likely to impact cardiac performance, and Ras hyperactivation has also been linked to cardiac dysfunction. We, therefore, used 2-dimensional echocardiography with color Doppler to measure cardiac function in Nf1+/- and control littermates. We found that Nf1+/- mice have increased left ventricular wall thickness and reduced cardiac contractility. We also observed alterations in cardiomyocyte organization in Nf1+/- animals. The results presented in this thesis support the hypothesis that neurofibromin haploinsufficiency in Nf1+/- mice results in vascular endothelial and cardiac dysfunction. Whether these findings extend to humans with NF1, who have the same genetic defect, is unknown, but if so, our observations may have important clinical implications. The role of neurofibromin in other kinds of vascular disease needs to be studied, and the possibility that neurofibromin may provide a novel therapeutic target should be explored. 

View record

Intellectual disability affects 1-3% of individuals globally, and, for half the cases, the cause is unknown. Recent studies using whole genome microarray genomic hybridization have shown that submicroscopic genomic imbalance causes intellectual disability in at least 10% of idiopathic cases with normal conventional cytogenetic analysis. I established genotype-phenotype correlations for de novo copy number variants detected by previous whole genome array genome hybridization studies performed by our group in children with intellectual disability. These genotype-phenotype correlations show that genomic imbalance of genes belonging to the epigenetic regulatory category, among others, are causative of intellectual disability. I hypothesized that dosage changes in the broad functional category of genes encoding epigenetic regulatory proteins are more likely to be pathogenic for intellectual disability than dosage changes in other kinds of genes. Epigenetic regulatory proteins include those with DNA methylation, histone modification or chromatin remodeling activity. I have selected all known genes encoding epigenetic regulatory proteins and defined probes to interrogate these candidate genes for copy number alteration as part of a custom targeted microarray design that selectively investigates all candidate genes associated with intellectual disability. We have conducted comparative genome hybridization on 177 patients with idiopathic intellectual disability using this array and on both normal parents of each affected child. We identified and independently validated 16 cases with de novo CNVs involving the epigenetic regulatory candidates. 7 of the 16 CNVs involve the same exon of the JARID2 gene, while the other 9 CNVs affect different genes. I discuss genotype-phenotype correlations for these cases and show that epigenetic perturbation by way of disruption of genes that encode epigenetic regulators is an important cause for intellectual disability.

View record

Major birth defects occur in 2% to 3% of liveborn infants and are the leading cause of infant mortality. The cause of most birth defects is unknown. The objectives of this thesis are to (1) assess the risk of having a birth defect in human pregnancy following maternal use of common antidepressant medications and to (2) evaluate the risk of a birth defect or subsequent adverse outcome in relation to restricted fetal growth in early pregnancy. I used data from the National Birth Defects Prevention study, a population-based case-control study of birth defects risk factors in the US to study the rates and patterns of antidepressant medication use around pregnancy and compare the prevalence of common antidepressant medication use among mothers of cases and mothers of controls. I found that selective serotonin reuptake inhibitors (SSRIs) were the most commonly used antidepressants among pregnant women, followed by bupropion, and that the rate of maternal antidepressant use significantly increased over the period between 1998 to 2005. I found that maternal use of SSRIs in early pregnancy was associated with the occurrence of anencephaly, craniosynostosis and omphalocele in the infant, whereas early pregnancy exposure to bupropion was associated with an increased risk for left outflow tract heart defects. I also conducted a retrospective cohort study using ultrasound examination data on singleton pregnancies in women with regular menstrual cycles who had crown-rump length (CRL) measurements at the Ultrasound Unit of British Columbia Women’s Hospital. I found that a first trimester CRL in the 10th centile or less was strongly associated with subsequent spontaneous abortion, delivering through a cesarean section or having an infant with low birth weight or length. The results presented in this thesis indicate that maternal treatment with common antidepressant medications may increase the risk for certain birth defects and that restricted growth of the embryo may adversely affect subsequent pregnancy outcomes.

View record