Allelic Expression of MMACHC and Evidence for Genotype-Phenotype Correlations in Cblc Disease

Allelic Expression of MMACHC and Evidence for Genotype-Phenotype Correlations in cblC Disease

Thesis by

Natascia Anastasio

Department of Human Genetics McGill University Montreal, Quebec, Canada

May 2010

A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Masters of Science

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Abstract

Mutations in the MMACHC gene cause cblC, the most common inborn error of cobalamin metabolism, with approximately 400 known cases. It results in the inability to convert vitamin B12 (cobalamin) into its two active

coenzyme forms, methylcobalamin and adenosylcobalamin, required by methionine synthase and methylmalonyl-CoA mutase respectively. It can be

characterized according to age of onset, with early onset patients presenting

within the first year of life with a number of pathologies and later onset

patients presenting after the age of four with predominantly neurological

symptoms. Individuals with the c.394C>T (p.R132X) as well as a number of

missense mutations generally have later onset of disease whereas patients

with the c.331C>T (p.R111X) and c.271dupA (p.R91KfsX14) mutations

usually present in infancy. Expression experiments measuring allele-specific

transcripts and quantitative real-time RT-PCR measuring overall MMACHC

transcript amount revealed increased transcription from alleles bearing late

onset related mutations when compared to early onset mutation-bearing

alleles. Understanding the mechanisms underlying early and late onset of

disease may improve treatment and prognosis for cblC patients.

2 Résume:

cblC, causé par des mutations dans le gène MMACHC, est la maladie génétique

la plus commune du métabolisme de la vitamine B12 (cobalamine), avec plus de

400 cas dans le monde. La maladie se traduit par une inhabilité à convertir la

cobalamine en deux formes actives de coenzymes nécessaires au bon

fonctionnement des cellules chez les mammifères. La première, la

méthylcobalamine est nécessaire pour la conversion de l’homocysteine en

méthionine par l’enzyme méthionine synthase. La seconde,

l’adénosylcobalamine est nécessaire pour la conversion de la méthylmalonyl-

CoA en succinyl-CoA par l’enzyme méthylmalonyl-CoA synthase. cblC peut

être caractérisé par l’âge d’apparition des symptômes. Les patients qui

présentent des symptômes pendant leur première année de vie ont de

nombreuses pathologies, alors que les patients qui se présentent les symptômes

plus tardivement, après l’âge de quatre ans, ont principalement des affections

neurologiques. Les individus avec la mutation c.394C>T (p.R132X) ainsi que

ceux avec une mutation faux-sens sont symptomatiques en général plus tard dans leur vie, alors que les individus avec les mutations c.331C>T (p.R111X) et c.271dupA (p.R91KfsX14) le sont habituellement dans leur petite enfance. Dans cette étude, nous avons analysé l’expression spécifique des allèles de MMACHC

et mesuré la quantité totale d’ARN messager pour MMACHC. Les résultats

3 montrent que la quantité d’ARN messager est plus élevée pour les allèles reliés à

une apparition tardive des symptômes, et que la quantité d’ARN messager total

pour MMACHC dans les cellules des patients porteurs des allèles reliés à une apparition tardive des symptômes est-elle aussi plus élevée. Comprendre les mécanismes sous-jacents à l’apparition précoce ou tardive des symptômes de la maladie cblC pourrait nous aider dans le traitement et le pronostic des patients.

4 TABLE OF CONTENTS

ABSTRACT………………………………………………….……………….....2 RESUME………………………………………………………………………..3 TABLE OF CONTENTS…………………………………………………...... 5 LIST OF ABBREVIATIONS………………………………………………….9 LIST OF FIGURES…………………………………………………………...10 LIST OF TABLES…………………………………………………………….11 ACKNOWLEDGEMENTS…………………………………………………..12 RATIONALE AND OBJECTIVES OF STUDY……………………………13

CHAPTER 1 Introduction to Cobalamin Metabolism and MMACHC…………….……...15

1.1 Cobalamin Structure and Dietary Requirements…………………………...15 1.1.1 Cobalamin Structure and Derivatives………………………………..15 1.1.2 Dietary Sources and Requirements of Cobalamin…………………...16

1.1.3 Dietary Insufficiency of Vitamin B12………………………………..16 1.2 Cobalamin Uptake and Metabolism………………………………………..18 1.2.1 Cobalamin Uptake in the Gastrointestinal Tract……………………..18 1.2.2 Cellular Uptake and Metabolism of Cobalamin……………………..18 1.3 Inherited Defects of Cobalamin Metabolism……………………………....23 1.3.1 Defects in Cobalamin Absorption, Transport and Uptake…………...23 1.3.1.1 Haptocorrin Deficiency……………………………………23 1.3.1.2 Intrinsic-factor Deficiency………………………………...23 1.3.1.3 Imerslund-Gräsbeck Syndrome…………………………...24 1.3.1.4 TC Deficiency……………………………………………..25 1.3.1.5 TC Receptor Defects……………………………………....25

5 1.3.2 Intracellular Defects…………………………………………...…….26 1.3.2.1 Complementation Class Assignment……………………...26 1.3.2.2 Inborn errors that result in combined methylmalonic aciduria and homocystinuria………………………………27 1.3.2.3 Inborn Errors that result in isolated homocystinuria….…...29 1.3.2.4 Inborn Errors that result in isolated methylmalonic aciduria………………………………………………....…30 1.4 Combined methylmalonic aciduria and homocystinuria, cblC type……….33 1.4.1 Subdivisions: Age of Onset………………………………………….33 1.4.2 Pathology of cblC disease………………………….………………...34 1.4.2.1 Hematological Abnormities……………….………………34 1.4.2.2 Neurological Symptoms…………………….…………….35 1.4.2.3 Dysmorphism……………………………………………...37 1.4.2.4 Dermatological Symptoms………………………………...39 1.4.2.5 Ocular Phenotype………………………………………….40 1.4.2.6 Structural Heart Defects…………………………………...41 1.4.2.7 Vascular Symptoms……………………………………….42 1.4.2.8 Renal Dysfunction………………………………………...43 1.4.3 Treatment…………………………………………………………….43 1.4.4 MMACHC……………………………………………………………45 1.4.4.1 MMACHC gene structure and mutations………………….45 1.4.4.2 Proposed Protein Function………………………………...47 1.4.4.2.1 MMACHC and Cancer………………………….50 1.4.5 Genotype-Phenotype Correlations in cblC Disease………………….52 1.5 Differential Allelic Expression……………………………………………..53

6 CHAPTER 2 Materials and Methods………………………………………………………..56

2.1 Cell Culture and Cell Line Selection………………………………….……56 2.2 DNA and RNA Extraction and cDNA Synthesis…………………………..57 2.2.1 DNA Extraction…………………………………………………...57 2.2.2 RNA Extraction…………………………………………………...57 2.2.3 DNA and RNA Quality Control………………………….……….60 2.2.4 cDNA synthesis…………………………………………………...60 2.3 Allelic Expression Analysis………………………………………………...61 2.3.1 Primer Design……………………………………………………..61 2.3.2 PCR………………………………………………………….…….62 2.3.3 Sequencing and Allelic Expression Analysis……………………..62 2.3.4 Allelic Expression Data and Statistical Analysis…………….……62 2.4 Quantitative Real Time RT-PCR…………………………………………...66 2.4.1 Cell Line Selection………………………………………………...66 2.4.2 RNA extraction, RNA Quality Control and cDNA Synthesis……66 2.4.3 Primer Design……………………………………………………..66 2.4.4 Quantitative Real-Time RT-PCR Analysis……………………..…66 2.4.5 Statistical Analysis………………………………………………...67 CHAPTER 3 Results………………………………………………………………………….69

3.1 Allelic Imbalance Analysis…………………………………….…………...69 3.1.1 Haplotype Analysis in Control Cell Lines………………………...69 3.1.2 Allelic Expression Analysis in Patient Cell Lines………………...69 3.1.3 Allelic expression of the c.666C>A mutation…………………….74 3.2 Quantitative Real-Time RT-PCR Analysis………………………………..76

7 CHAPTER 4 Discussion……………………………………………………………………...78

SUMMARY AND CONCLUSIONS…………………………………………86

ORIGINAL CONTRIBUTIONS TO SCIENCE……………………………87

BIBLIOGRAPHY……………………………………………………………..88

APPENDIX A: List of Publications and Presentations……………………103

APPENDIX B: Published Abstracts………………………………………..106

APPENDIX C: Published Article…….……………………………………..108

8 LIST OF ABBREVIATIONS

AdoCbl: 5’deoxyadenosylcobalamin AMN: Gene name for amnionless ATP: Adenosine triphosphate Cbl: Cobalamin cDNA: Complementary DNA CN-Cbl: Cyanocobalamin Co: Cobalt CUBN: Gene name for cubilin DNA: Deoxyribose nucleic acid HC: Haptocorrin HLH: Hemophagocytic lymphohistiocytosis HUS: Hemolytic uremic syndrome IF: Intrinsic factor LMBRD1: Gene name for methylmalonic aciduria and homocystinuria CblF type MeCbl: Methylcobalamin MMA: Methylmalonic acid MMAA: Gene name for methylmalonic aciduria cblA type MMAB: Gene name for methylmalonic aciduria cblB type MMACHC: Gene name for methylmalonic aciduria and homocystinuria cblC type MMADHC: Gene name for methylmalonic aciduria and homocystinuria cblD type MS: Methionine synthase enzyme MTRR: Gene name for methionine synthase reductase MTR: Gene name for methionine synthase MTHFR: methylenetetrahydrofolate reductase enzyme NMD: Nonsense-mediated decay OHCbl: Hydroxocobalamin PTC: Premature stop codon RNA: Ribonucleotide tri-phosphate RT-PCR: Reverse transcription-polymerase chain reaction TC : Transcobalamin TCblR: Transcobalamin receptor

9 LIST OF FIGURES

Page

1- Molecular Structure of Vitamin B12 17 2- Gastrointestinal Uptake of Cobalamin 20 3- Summary of Intracellular Cobalamin Trafficking 22 4- Mechanism of R-Group Removal by MMACHC Protein 51 5- PeakPicker Program Output 71 6- Results of allelic expression analysis in patient fibroblast cell lines carrying the c.271dupA, c.331C>T, c.394C>T and c.482G>A mutations 73 7- Results of allelic expression analysis in patient fibroblast 75 cell lines carrying the c.666C>A mutation 8- Quantitative real-time RT-PCR results 77

10 LIST OF TABLES

Page 1- Cell Line Selection for Allelic Expression Analysis 58 2- Primers for Allelic Expression and Quantitative Real-Time RT-PCR Analysis 64 3- PCR Master Mix and Cycling Protocol 65 4- Cell Line Selection for Quantitative Real-Time RT-PCR 68

11 ACKNOWLEDGEMENTS

First and foremost, I would like to thank my supervisor, Dr. David Rosenblatt, for all of the support and encouragement that any graduate student could ever ask for. The time spent in his lab is and shall remain one of the most formative experiences in my life and everything that I learnt over the course of my three years there, both in terms of scientific knowledge and general life lessons will never be forgotten.

I would also like to thank Dr. David Watkins, who has been both a scientific mentor as well as a friend throughout my time here. Thank you for the science chats, friendly visits and musical trivia.

I would like to thank my supervisory committee members: Drs. Nancy Braverman and Tomi Pastinen for all of their help over the past few years. Their time, insight, feedback and encouragement have and always will be greatly appreciated.

I would like to thank Jocelyne Lavallée, Maria Galvez and Angeline Boulay for their company and help around the lab. I would especially like to thank Laura Benner for being not only a great help, but also a great friend during my time at the lab.

I would like to thank Gail Dunbar for teaching me all I ever wanted to know about tissue culture and for providing me with countless cell lines.

I would like to thank all of the lab-mates that I have worked with over the past three years: Lama Yamani, Isabelle Racine-Miousse, Junhui Liu, Amanda Loewy, Fei Li, Michael Quinn, Karen Gambaro, Luca Cavallone and Nancy Hamel. I would like to thank all of them for their support, encouragement, advice, help and their company in general; you helped make my time here something truly amazing. I want to also thank all of the students that have passed through: Mireille Sayegh, Lara Reichman, Simon Zhu, Laura Dempsey-Nunez and Lydia Vezina.

Last but absolutely not least, I want to thank my family: my Mom, Dad, my sister Sandra and my Nonna for putting up with me and encouraging me like my own personal cheerleaders. There is no way I could do anything without your love and support and I appreciate it every day.

12 RATIONALE AND OBJECTIVES OF STUDY

Vitamin B12 (cobalamin, Cbl) is required by all mammalian cells as a

cofactor for two intracellular reactions. In the mitochondria, 5’-

deoxyadenosylcobalamin is required by the enzyme methylmalonyl-CoA mutase

for the conversion of L-methylmalonyl-CoA to succinyl-CoA. In the cytoplasm,

methylcobalamin is required by the enzyme methionine synthase for the

conversion of homocysteine to methionine. Patients with defects in this pathway

present with either isolated methylmalonic aciduria, isolated homocystinuria or

combined methylmalonic aciduria and homocystinuria. cblC is the most common inborn error of Cbl metabolism and is caused by mutations in the gene

MMACHC, located on chromosome 1p34.1.

There is evidence for genotype-phenotype correlations in cblC disease, with the c.394C>T (p.R132X) as well as a number of missense mutations generally associated with later onset of disease and the c.331C>T (p.R111X) and c.271dupA (p.R91KfsX14) mutations correlating to earlier onset of disease. The objective of this study was to examine the underlying cause of these genotype- phenotype correlations. Allele specific expression analysis was performed in order to determine whether transcripts bearing later onset-related mutations showed increased levels of MMACHC transcripts when compared to early onset

13 mutation-bearing transcripts. Overall MMACHC transcript levels were also assessed using quantitative real-time RT-PCR.

14 Chapter 1

Introduction to Cobalamin Metabolism and MMACHC

1.1 Cobalamin Structure and Dietary Requirements

1.1.1 Cobalamin Structure and Derivatives

Vitamin B12 (cobalamin, Cbl) is a water-soluble vitamin essential to many microorganisms and animals. Its structure consists of a corrin ring formed by four linked pyrrol rings (Figure 1). This corrin structure makes up the core of the

molecule. At the center of this ring resides a cobalt atom, which can exist in its

three reduction states. Below the corrin ring, a 1-α-D-ribofuranosyl-5,6-

dimethyl benzimidazoyl-3-phosphate (DMB) is linked via phosphodiester and

central cobalt-coordinated linkages. Cbl can be referred to as either “base-on” or

“base-off” depending on whether the DMB is linked to the central cobalt atom,

in the case of “base-on”, or not, as is the case in “base-off”. A variety of upper

axial ligands can be coordinated to the central cobalt atom. The different Cbl

forms are distinguished by their differing upper-axial ligands, which can be a

hydroxy (OH) molecule in the case of hydroxycobalamin, or a cyanide molecule

in the case of cyanocobalamin, among others (Seetharam & Alpers, 1982). The

two Cbl enzymatic cofactors required by all mammalian cells are 5’-

deoxyadenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl).

15 1.1.2 Dietary Sources and Requirements of Cobalamin

In nature, Cbl can only be synthesized by microorganisms, particularly soil and colon-dwelling microorganisms. As humans are unable to absorb the Cbl produced by their colonic bacteria, they are required to acquire the vitamin from dietary sources, particularly meat and dairy products. The recommended daily intake of Cbl for a healthy adult is 2.4μg (Stabler and Allen, 2004); however,

Bor et al (2010) suggest that in order to maintain optimal biomarker status

(healthy methylmalonic acid (MMA), total and holo-transcobalamin, plasma Cbl levels, etc) in individuals with normal vitamin B12 absorption, a higher daily dietary intake of 4.2–7.0μg may be required.

1.1.3 Dietary Insufficiency of Vitamin B12

The liver serves as a storage site for Cbl and this is able to compensate for a lack of Cbl in the diet, therefore, the symptoms of a dietary lack of the vitamin may not be apparent for a number of years (Rothenberg et al, 1999). Insufficient Cbl intake can result in a multitude of symptoms, most commonly neurological and hematological symptoms. Initial symptoms can include weakness and fatigue, paraesthesias and sore tongue. Macrocytic anemia is also common, specifically megaloblastic anemia. Progressive demyelination leading to neuropathy may also occur (Seetharam & Alpers, 1982).

Upper Axial Ligand

Corrin Ring

DMB

Figure 1: Molecular Structure of Vitamin B12. The corrin ring with central cobalt

atom is depicted centrally and R denotes the variable upper axial ligand bound to

the central cobalt atom. The 1-α-D-ribofuranosyl-5,6-dimethyl benzimidazoyl-3-

phosphate (DMB) is positioned at the lower axial position. The molecule in this

figure is depicted as “base-on” as the DMB is linked to the central cobalt atom.

Adapted from Banerjee and Ragsdale, 2003.

17 1.2 Cobalamin Uptake and Metabolism

1.2.1 Cobalamin Uptake in the Gastrointestinal Tract

Dietary cobalamin initially binds salivary haptocorrin (HC, TCI, TCIII), a

glycoprotein secreted by the salivary glands in the mouth (Burger and Allen,

1974). In the acidic environment of the stomach, the HC-Cbl complex is tightly

bound, but once it enters the alkaline environment of the small intestine, the

complex is hydrolyzed by pancreatic proteases, freeing Cbl (Allen et al, 1978)

and allowing it to bind to intrinsic factor (IF) in the proximal ileum (Gräsbeck,

1984). IF is released by the acid-secreting parietal cells of the gastric fundic mucosa and facilitates the transport and uptake of Cbl (Gräsbeck and Kouvonen,

1983). In the distal ileum, the IF-Cbl complex binds cubam, a specific apical brush-border receptor formed by the products of the cubilin (CUBN) and amnionless (AMN) genes (Aminoff et al, 1999; Tanner et al, 2003). The interaction between the two cubam components is essential for the proper functioning of the receptor. Once the enterocyte absorbs the complex, it is believed that endogenous transcobalamin (TC) binds the Cbl and the complex is then delivered into the portal circulation by means of transcytosis and Cbl is delivered to the body’s tissues (Figure 2) (Seetharam et al, 1999).

1.2.2 Cellular Uptake and Metabolism of Cobalamin

The cell-surface receptor that recognizes the TC-Cbl complex is the

transcobalamin receptor (TCblR), a 58-kDa protein encoded by the CD320 gene

18 (Quadros et al, 2009). When the complex binds the receptor, the TCblR-TC-Cbl

complex in endocytosed and the complex is degraded in the lysosome. The

TCblR is then recycled and the free Cbl is shuttled out of the lysosome and into

the cytoplasm through interaction with LMBD1, a lysosomal membrane protein

with homology to lipocalin membrane receptor LIMR (Rutsch et al, 2009). The

exact mechanisms through which Cbl is shuttled throughout the cell remain

elusive, yet it is thought that an intra-lysosomal protein is responsible for

presenting free Cbl to LMBD1 and that LMBD1 then transfers the Cbl to the

putative next member of the pathway, MMACHC (methylmalonic aciduria and homocystinuria cblC type). MMACHC is thought to directly interact with

MMADHC (methylmalonic aciduria and homocystinuria CblD type), the next

protein in the pathway, which is hypothesized to shuttle the Cbl to one of two

final destinations: the mitochondria or the cytoplasm. Cbl meanwhile, is also

undergoing a series of reduction reactions from cob(III)alamin, which has the

cobalt atom (Co) in the +3 oxidation state, to cob(I)alamin, with the Co in the +1

oxidation state. The exact mechanism by which the Cbl is reduced is not

completely understood. It is thought that MMACHC and MMADHC play a part.

Figure 2: Gastrointestinal uptake of Cbl. Dietary Cbl enters the stomach bound to salivary haptocorrin (HC). Cbl is then freed by pancreatic proteases and is then bound by intrinsic factor (IF). This complex is then recognized by the cubam receptor found on the apical brush border of enterocytes. The receptor is made up of two proteins encoded by the Amnionless and Cubilin genes. Once absorbed, the Cbl is freed from the complex and can then be bound by endogenous transcobalamin protein (TC). This complex then enters portal circulation. From Li and Watkins, 2009.

20 Once in its fully reduced form, it can then be converted into the two Cbl cofactors required by methionine synthase and methylmalonyl-CoA mutase,

MeCbl and AdoCbl respectively (Figure 3).

In the mitochondria, the MMAB (methylmalonic aciduria, B type) protein transfers an adenosyl moiety from ATP (adenosine triphosphate) to the reduced

Cbl, converting it into the required AdoCbl form. The AdoCbl can then be used by the methylmalonyl-CoA mutase enzyme in the conversion of methylmalonyl-

CoA into succinyl-CoA. The MMAA (methylmalonic aciduria, A type) gene product MMAA is postulated to play a role in the protection of radical intermediates, which result from methylmalonyl-CoA mutase turnover (Banerjee et al, 2009).

In the cytoplasm, the Cbl is converted into MeCbl by methionine synthase reductase (MSR) and methionine synthase (MS), and the MeCbl can then be utilized by MS in the conversion of homocysteine to methionine (Quadros,

2009).

21 Figure 3: Summary of Intracellular Cobalamin Trafficking. Cbl enters the cell

bound to transcobalamin (TC) via the TC-Receptor. In the lysosome the TC-Cbl

complex is hydrolyzed and free Cbl can then enter the cytoplasm through

interaction with LMBD1, a lysosomal membrane protein. Cbl then interacts with

MMACHC and MMADHC proteins that aid in the reduction of cob(III)alamin

to cob(I)alamin and shuttle the Cbl to either the mitochondrion or the

cytoplasm. In the mitochondrion, Cbl interacts with MMAB to be converted to

AdoCbl, which is then used by methylmalonyl-CoA mutase to convert

methylmalonyl-CoA into succinyl-CoA. MMAA, a protein thought to play a role in the protection of radical intermediates, which result from methylmalonyl-CoA

mutase turnover. Cbl is also kept in the cytoplasm, where it is converted into

MeCbl by methionine synthase reductase to be used in the conversion of

homocysteine to methionine by methionine synthase. From Rutsch et al, 2009

22 1.3 Inherited Defects of Cobalamin Metabolism

1.3.1 Defects in Cobalamin Absorption, Transport and Uptake

1.3.1.1 Haptocorrin Deficiency

Haptocorrin (HC) is the least understood of the human Cbl-binding

proteins and its function remains unclear. Other than low serum Cbl levels,

patients are typically asymptomatic and because transcobalamin is the protein

required for normal cellular uptake of Cbl, outside of the liver, HC deficiency does not affect Cbl metabolism or clinical status. The gene that encodes HC is

TCN1 and it is located on chromosome 11q11-q12.3 (Carmel et al, 2009). It has

been shown that haptocorrin binds cobalamin analogues as well. These are Cbl

analogues that are unable to be used in cellular metabolism. It has been

suggested that HC acts a scavenger protein, binding these analogs in circulation

and then clearing them into the bile via hepatic asialoglycoprotein receptors

(Hardlei and Nexo, 2009).

1.3.1.2 Intrinsic-factor Deficiency

Patients with intrinsic factor (IF) deficiency usually present with

megaloblastic anemia and developmental delay between the ages of one and five

years of age, although some patients have been known to present well into their

third decade of life (Cooper and Rosenblatt, 1987, Carmel 1983). IF deficiency

is the inability to produce IF or the production of IF with decreased or absent

23 function (Spurling et al, 1964; McIntyre et al, 1965; Katz et al, 1972). Some patients have also presented due to the production of IF with reduced affinity for either the receptor or Cbl (Katz et al, 1974, Yang et al, 1985). The gene, which encodes IF is GIF (gastric intrinsic factor) and is located on chromosome 11q13.

It encodes a protein 45 kDa in size (Orkin et al, 2009).

1.3.1.3 Imerslund-Gräsbeck Syndrome

Imerslund-Gräsbeck Syndrome (IGS) was first described in 1960

(Gräsbeck, 1960; Imerslund, 1960) and is also referred to as enterocyte cobalamin malabsorption and megaloblastic anemia 1. It is a rare autosomal recessive disorder and patients can present with proteinuria, neurological symptoms, failure to thrive, megaloblastic anemia, genitourinary tract abnormalities and recurrent respiratory and gastrointestinal infections between the ages of one and five years of age. IGS is also sometimes accompanied by diabetes mellitus (Madhavan et al, 2009; Gräsbeck, 2006). The defect in IGS is a lack of functional cubam. As the cubam receptor is made up of two separate proteins, mutations in the genes encoding either of these two proteins result in

IGS. The cubilin gene (CUBN) was mapped to 10p12.1 (Aminoff et al, 1999) and the amnionless gene (AMN) was mapped to 14q32 (Tanner et al, 2003).

However, a study conducted by Tanner et al. (2004) indicates further genetic heterogeneity as a number of families have presented with IGS yet have no

24 mutations in either the AMN or CUBN gene and no evidence of linkage to either of the two genes has been found.

1.3.1.4 TC Deficiency

TC deficiency can be caused in three ways. Patients may have undetectable levels of TC, may have TC that is unable to bind Cbl, or TC that is able to complex with Cbl but unable to complex with the receptor on the cell surface

(Haurani et al, 1979). It is an autosomal recessive disorder and patients can present within the first few months of life with failure to thrive, lethargy, mucosal irritation, severe hematological problems such as macrocytic anemia, megaloblastic anemia, pancytopenia, neutropenia, reticulocytopenia, neurological problems, diarrhea, vomiting, immunological deficiency and

thrombocytopenia. TC deficiency is the result of mutations in the TCN2 gene

located on chromosome 22 (Prasad et al, 2008; Ratschmann et al, 2009).

1.3.1.5 TC Receptor Defects

The gene for the TC receptor, CD320, encodes a protein that consists of a

282 amino acid sequence producing a 58-kDa protein. This includes a signal

peptide, an extracellular, a transmembrane and a cytoplasmic domain. The

receptor is highly glycosylated and the extracellular domain contains two low-

density lipoprotein receptor type domains separated by a cysteine-rich CUB

(complement C1r/C1s, Uegf, Bmp1) domain. The receptor’s high affinity for

25 holo-TC and lack of affinity for other ligands shows its extreme specificity

(Quadros et al, 2009). Mutations in the TC receptor have been associated with elevated levels of MMA in the blood of infants undergoing routine newborn screens. Two common mutations have been identified through sequencing of genomic DNA from individuals showing decreased uptake of Cbl: c.262_264delGAG (p.E88del) and c.297delA (p.Q99HfsX33) (Anastasio et al,

2009). As patients have only been identified via elevations in MMA on newborn screens, it is still unclear as to whether mutations in the TC receptor result in a true disease phenotype or whether it is simply an anomalous biochemical finding.

1.3.2 Intracellular Defects

1.3.2.1 Complementation Class Assignment

Complementation of cultured fibroblasts can be used to determine which intracellular defect a cell line has (Gravel et al, 1975; Mellman et al, 1978;

Rosenberg, 1979). Before complementation is performed, a number of tests are first done in order to assess the biochemical features of a patient cell line. These tests include: 1) Measurement of total uptake of labelled [57Co]CN-Cbl into cells and its conversion into the two intracellular cofactors MeCbl and AdoCbl

(Rosenblatt and Cooper, 1990); 2) measurement of methionine synthase function through the incorporation of label from 5-[14C]methyltetrahydrofolate into acid-

26 precipitable material (Willard et al, 1978) and 3) measurement of methylmalonyl-CoA mutase function via incorporation of label from

[14C]propionate into cellular macromolecules (Rosenblatt et al, 1984).

Complementation class is assessed through the fusion of patient cell lines to cells from known complementation classes. This is done using polyethylene glycol. Propionate or methyltetrahydrofolate incorporation is then measured in these heterokaryons (Watkins and Rosenblatt, 1986). When cells from different complementation classes, i.e. with mutations in different genes, are fused together, the metabolic defect is corrected. When cells with the same genetic defect are fused, no complementation occurs and the biochemical defect remains. This is particularly useful when biochemical or clinical presentations are unable to distinguish which cellular defect the patient has.

1.3.2.2 Inborn errors that result in combined methylmalonic

aciduria and homocystinuria

Defects in the genes encoding proteins involved in the early steps of the

Cbl pathway result in the cell’s inability to produce either Cbl cofactor, resulting in the failure to convert methylmalonyl-CoA to succinyl-CoA and homocysteine to methionine. The cblF disorder results in defects in the lysosomal export protein, LMBD1 encoded by LMBRD1 on chromosome 2q23.2. Cbl is trapped within the lysosome and unable to undergo further transport and processing

27 within the cytoplasm. Patients present with a number of symptoms that can include poor feeding, failure to thrive, macrocytic anemia, thrombocytopenia, pancytopenia and some individuals also present with congenital heart defects and facial abnormalities (Rutsch et al., 2009).

A defect in the MMACHC gene results in cblC disease, which will be further discussed in section 1.4. A defect in the MMADHC gene, located on chromosome 2q23.2, can result in isolated methylmalonic aciduria, isolated homocystinuria or combined methylmalonic aciduria and homocystinuria, depending upon the location of the mutation. N-terminal mutations result in isolated methylmalonic aciduria, whereas C-terminal mutations result in isolated homocytinuria. The MMADHC protein contains a sequence homologous to a putative ATPase component of a bacterial ATP-binding cassette transporter and also contains a putative mitochondrial targeting sequence and a Cbl binding site.

It is hypothesized that the MMADHC protein is involved in the further processing and localization of the Cbl to either the mitochondria or the cytoplasm (Coehlo et al, 2008) (Figure 3). In patients with classic cblD

(combined homocystinuria and methylmalonic aciduria) developmental delay, megaloblastic anemia and neurological abnormalities characteristic of cblC, cblE and cblG (which are further discussed in section 1.3.2.3) are seen. cblD patients with isolated homocystinuria (variant 1) present with neurological abnormalities similar to those seen in cblC, cblE and cblG as well as megaloblastic anemia.

28 Patients with cblD with isolated methylmalonic aciduria (variant 2) present with

symptoms similar to those in cblA and cblB (further discussed in section 1.3.2.3), specifically metabolic crises (Suormala et al, 2004, Miousse, 2009).

1.3.2.3 Inborn Errors that result in isolated Homocystinuria

Mutations in the enzymes involved in converting homocysteine to methionine will result in patients presenting with isolated homocystinuria, as is the case in cblE and cblG which result from mutations in the genes encoding methionine synthase reductase (MTRR) and methionine synthase (MTR) respectively. Methionine synthase reductase is responsible for keeping methionine synthase-bound cobalamin in its active form (Watkins et al, 2008).

Patients with cblE disease usually present in childhood with megaloblastic anemia, failure to thrive, developmental delay and cerebral atrophy with white matter abnormalities (Zavadáková et al, 2002).

Methionine synthase (MS) is the enzyme that converts homocysteine to methionine (Watkins et al, 2008) by catalyzing the transfer of a methyl group from methyltetrahydrofolate to homocysteine to form methionine and tetrahydrofolate (Koutmos et al, 2009). It is a 136-kDa polypeptide that consists of four modules, 2 of which are the homocysteine-binding acceptor domain and the methyltetrahydrofolate-donating domain, which catalyze the methyl transfer reaction in conjunction with the cobalamin-binding domain. A third domain

29 binds Cbl and a fourth reactivates the enzyme after MeCbl has been oxidized to form cob(II)alamin (Matthews et al, 2008). Patients with defects in MS present with cblG disease. This disease can manifest as early as the newborn period of age or well into adulthood (Watkins et al, 2002). Patients present with symptoms identical to those in cblE disease and can include ataxia, megaloblastic anemia, neonatal seizures severe developmental delay, cerebral atrophy, and blindness

(Whitehead, 2006). As previously mentioned, isolated homocystinuria is also found in cases of variant 1 cblD disease.

1.3.2.4 Inborn Errors that result in isolated Methylmalonic

Aciduria

Elevated methylmalonic acid in blood and urine may be a result of the cell’s inability to convert methylmalonyl-CoA into succinyl-CoA in the mitochondria. In general, patients with methylmalonic acidurias present early in life with severe disease that can be fatal if left untreated. Patients can experience acute metabolic acidosis. Neuronal damage, caused by ‘metabolic strokes’, which are the result of the accumulation of organic acids, may also occur

(Morath, 2008). During metabolic crises, normal cell functions may be altered and bone marrow may be suppressed resulting in leukopenia, anemia and thrombocytopenia. Renal involvement has also been reported and can include tubulointerstitial nephritis, renal tubular acidosis and progressive renal

30 insufficiency resulting in end-stage renal disease (Srinivas et al, 2001).

Mitochondrial dysfunction is also a characteristic result of methylmalonic acidemia. It is thought that acryloyl-CoA species derived from propionyl-CoA, which are thought to form in methylmalonic acidemia, are excreted into the mitochondrial matrix leading to respiratory chain failure. This would lead to an increase in reactive oxygen species resulting in oxidative stress. Oxidative stress also can cause a loss of mitochondrial membrane potential and can cause severe structural damage to the mitochondria (Chandler et al, 2009).

cblA, cblB and mutase (mut) are all inborn errors of Cbl metabolism that result in isolated MMA. These defects are the result of mutations in the genes for

MMAA, MMAB (ATP:cob(I)alamin adenosyltransferase) and methylmalonyl-

CoA mutase respectively. The MMAB gene product is responsible for the transfer of an adenosyl moiety from ATP to form adenosylcobalamin. Patients with defects in this gene present very early in life with cblB disease. Symptoms include severe biochemical and clinical manifestations such as hypotonia, pancytopenia, neurological problems and hyperammonaemia (Merinero et al,

2008). Interestingly, MMAB has been linked to high-density lipoprotein cholesterol (HDL-C) levels by genome-wide association studies (Junyent et al,

2009). Transcript level studies have found that higher levels of MMAB protein correlate to lower levels of HDL-C (Fogarty et al, 2010). As much of the cholesterol regulation happens in the liver, and MMAB is highly expressed in

31 this tissue, it is thought that this may be the site of action, though the biology behind the mechanism remains unclear. It is hypothesized that due to the protein’s involvement in the catabolism of certain amino acids, short chain fatty acids and the side chain of cholesterol to the citric acid cycle intermediate succinyl-CoA, increased MMAB protein levels may lower levels of cholesterol as a whole, including HDL-C levels.

During the degradation of propionate towards the Krebs cycle, the mitochondrial matrix protein methylmalonyl-CoA mutase catalyzes the isomerization of L-methylmalonyl-CoA into succinyl-CoA. This enzyme is a homodimer in its mature form and utilizes AdoCbl as a cofactor (Ledley and

Rosenblatt, 1997). A disruption of this enzyme results in disease, which is characterized according to enzyme activity. Patients can have a complete absence (mut0) of the enzyme or residual activity of the enzyme (mut-). Patients with no enzyme function present within the first few days of life and many patients die. Symptoms can include hypertonia, vomiting, hyperammonaemia, acidosis, renal problems, and polypnea. Patients with residual mutase function present early in life with a number of symptoms including vomiting, metabolic acidosis and hypotonia (Merinero et al, 2008).

The function of the MMAA gene remains elusive, but its bacterial ortholog

MeaB has been characterized quite extensively. MeaB is thought to play a part in gating the transfer of AdoCbl from adenosyltransferase to the mutase enzyme

32 and may also serve in screening the Cbl docking to the mutase enzyme, ensuring that only the active AdoCbl is bound and not the inactive cob(II)alamin form

(Banerjee et al, 2009). Patients with cblA tend to present in infancy with vomiting, metabolic acidosis, respiratory and neurological problems and cortical atrophy (Merinero et al, 2008). As previously mentioned, isolated methylmalonic aciduria can also be found in cases of variant-2 cblD disease.

1.4 Combined methylmalonic aciduria and homocystinuria, cblC type

1.4.1 Subdivisions: Age of onset

The cblC disorder was first described in 1969 (Mudd et al) and is the most common inborn error of cobalamin metabolism. Over 400 cases have been reported worldwide. The disorder can be subdivided according to age of onset, with the more common early onset patients presenting within the first year of life and later onset patients presenting after the age of four. Early onset patients may present with any number of phenotypes (Lerner-Ellis et al, 2009), described in the following section, whereas later onset patients tend to present with acute onset of neurological symptoms (Ben-Omran et al, 2007) and ocular phenotypes are rarely seen (Gerth et al, 2008). Early onset disease is far more severe and approximately one quarter of patients do not survive. Patients that do survive tend to have permanent neurological impairment (Rosenblatt et al, 1997). Later onset patients tend to present with a less severe form of the disease and respond

33 better to treatment with pharmacological doses of OHCbl (further discussed in

section 1.4.3), some showing almost complete reversal of neurological

symptoms (Augoustides-Savvopoulou et al, 1999).

1.4.2 Pathology of cblC disease

1.4.2.1 Hematological Abnormalities

Patients with cblC disease may present with a number of defects in red

blood cell production including macrocytic anemia, megaloblastic anemia and

myeloid dysplasia (Mamlok et al, 1985; Linnell et al, 1983). This is thought to

be due to the inability to recycle folate beyond the methyltetrahydrofolate point

due to an inability to use methyltetrahydrofolate for transmethylation reactions.

Also, N5, N10-methylene tetrahydrofolate is unable to act as a cofactor for thymidylate synthase, impairing the conversion of dUMP to dTMP. This subsequently impairs DNA synthesis, which may result in abnormal turnover of

red blood cells (Mamlok et al, 1985).

Other haematological abnormalities such as decreased platelets in the blood

(thrombocytopenia) and low number of neutrophils in the blood (neutropenia)

have also been reported (Enns et al, 1999). Neutrophil hypersegmentation, in

which one or more six-lobed or five or more five-lobed neutrophils are found

among 100 segmented neutrophils, is also characteristic of Cbl-deficient

patients, including patients with cblC (Watkins et al, 2009).

34 One case of hemophagocytic lymphohistiocytosis (HLH) in conjunction with cblC disease has been reported (Wu et al, 2005). HLH is characterized by immune dysregulation and proliferation of macrophages and lymphocytes and manifests as fever or splenomegaly, cytopenia, hemophagocytosis in bone marrow, lymph nodes and spleen, hypertriglyceridemia and/or hypofibrinogenemia. It is unclear as to whether cblC caused the HLH in this patient, although altered DNA synthesis may result in dysregulation of natural killer cell and immune system function.

1.4.2.2 Neurological Symptoms

Neurological problems in cblC disease are common in both the early and late-onset form of the disease. Early onset disease is characterized by a severe neurological phenotype that may or may not improve with treatment, whereas later onset neurological symptoms appear suddenly and are often less severe and respond better to treatment (Enns et al, 1999; Thauvin-Robinet et al, 2008).

Behavioural symptoms and changes such as social withdrawal and attention problems have been documented (Enns et al, 1999; Powers et al, 2001;

Beauchamp et al, 2009). Psychological problems such as dementia and psychiatric disturbances may appear suddenly in the case of late onset cblC disease (Shinnar and Singer, 1984; Augoustides-Savvopoulou et al, 1999;

Powers et al, 2001; Roze et al, 2003; Tsai et al, 2007; Thauvin-Robinet et al,

35 2008). Developmental delay, mild to severe mental retardation, cognitive impairment, primarily in frontal/executive function and progressively decreasing intellect has been seen in a number of patients (Dillon et al, 1974; Anthony and

McLeay, 1976; Boxer et al, 2005; Yuen et al, 2007; Beauchamp et al, 2009).

Seizures, ataxia and loss of bladder and bowel control have also been noted

(Biancheri et al, 2002; Dillon et al, 1974; Tsai et al, 2007; Bodamer et al, 2001).

Many of the above-mentioned neurological symptoms are due, at least in part, to the progressive neuropathy and myelopathy observed in cblC (Roze et al,

2003; Shinnar and Singer, 1984; Tsai et al, 2007; Enns et al, 1999; Powers et al,

2001; Thauvin-Robinet et al, 2008). Post-mortem and MRI analysis has also revealed white matter loss, cerebral and cortical atrophy, interhemispheric fissures, ventriculomegaly and lesions of the basal ganglia (Sharma et al, 2007;

Enns et al, 1999; Longo et al, 2005; Geraghty et al, 1992).

The relationship between Cbl and subacute combined degeneration of the spinal cord has been known for decades and it is a well-documented manifestation of cblC disease (Dillon et al, 1974; Roze et al, 2003).

It is thought that the myelopathy seen in cblC is the result of a deficiency of S-adenosylmethionine due to decreased function of methionine synthase and a subsequent decrease in available methionine. This leads to the interference with the methylation required by myelin basic protein or the lipids that maintain the integrity of the myelin sheath (Enns et al, 1999).

36 The causes of the lesions are a topic of debate. Some believe their cause to

be a result of metabolic strokes caused by mitochondrial dysfunction due to toxic build-up of methylmalonic acid, an inhibitor of the respiratory chain. It is also thought that homocysteine plays a role and that the lesions have a vascular origin and the toxic effects of homocysteine are the cause (Longo et al, 2005).

Homocysteine is also thought to be responsible for the intellectual decline as well as the seizures some patients manifest as it has long been associated with cognitive decline and has been proven to induce seizures in animal models

(Beauchamp et al, 2009; Biancheri et al, 2002).

1.4.2.3 Dysmorphism

Patients with cblC have sometimes presented with minor facial abnormalities. Cerone et al (1999) reviewed seven patients diagnosed with cblC disease and noted a number of minor facial anomalies common to all. Patients ranged in age from 3 to 10 years and were characterized as having a high forehead, long face, large, low-set ears and a flat infranasal depression. It was also observed that the characteristics became even more prominent as the patients aged. Ben-Omran et al (2007) described a patient diagnosed with the late-onset form of cblC disease who also showed some facial dysmorphism.

These features included a triangular face, large, prominent nose, micrognathia and down-slanting eyes. However, as this patient resembled her healthy mother

37 and sister and the dysmorphism described was quite different from that which

was previously reported by Cerone et al, the authors were uncertain as to how

related the dysmorphism was to the disease. They concluded that while it may be

present, facial dysmorphism is not a consistent feature of cblC disease,

particularly in the late onset form.

Patients have also been reported with physical features similar to those

seen with Marfan syndrome, such as increased arm span, scoliosis,

arachnodactyly and joint hyperlaxity (Heil et al, 2007). Folic acid, vitamin B6 and B12 and carnitine treatment improved the Marfanoid features in one patient.

It is thought that the dysmorphism in cblC patients may be due to an as-of-

yet unidentified role of MMACHC in early development. The importance of

vitamin B12 during development is a current topic of discussion and there is

evidence that the vitamin may play an important role in the prevention of neural

tube defects as well as other birth defects (Li et al, 2009). In the case of the

patient with Marfanoid features, due to improvement of the dysmorphism with

treatment, the authors postulated that the Marfan-like features might be due to

low cysteine levels, as this morphology is also seen in individuals with classic

homocystinuria (Heil et al, 2007).

38 1.4.2.4 Dermatological Symptoms

Patients with cblC disease have presented with a number of dermatological features as well. Howard et al (1997) described two patients who were diagnosed with early onset cblC disease who manifested skin lesions in the neonatal period. Patient 1 presented with disproportionately large lips and skin peeling. The condition worsened, and skin reddening (erythema), shedding of the outer skin layers (desquamation), a thickening of the stratum corneum

(hyperkeratosis), lip inflammation (cheilitis) and lesions around the mouth appeared. Histological tests showed changes at the epidermal-dermal border, necrotic keratinocytes, infiltration of lymphocytes into the dermis, pale keratinocytes and parakeratosis, which is the abnormal formation of the horn cells of the epidermis caused by inflammation of the cells, incomplete formation of keratin and the persistence of nuclei. Dietary restrictions and treatment with methionine and hydroxycobalamin resolved the problems.

Patient 2 presented with diffuse dermatitis after a previous presentation of reddening of the oral mucosa and splitting of the lips at the corners of the mouth.

Desquamation and erosions were seen, particularly in the diaper area. The patient also presented with hair loss (Enns et al, 1999). Dermatological symptoms were resolved after carnitine supplementation, dietary restrictions and intramuscular hydroxycobalamin injections.

39 Dermatological symptoms in a number of other metabolic diseases appear

only once treatment, which may include dietary restrictions, has begun. In the

case of cblC disease, and other diseases characterized by MMA, it is thought that

these symptoms are a direct result of the defect and skin lesions improve once

treatment is started. It is unclear what the cause of these skin eruptions is,

although it has been postulated that perhaps, since number of metabolic disorders manifest similar dermatological symptoms, a common biochemical disturbance, i.e. prostaglandin metabolism, may be responsible (Howard et al,

1997).

1.4.2.5 Ocular Phenotype

Common opthamological findings in cblC disease include involuntary eye movements, such as nystgamus, strabismus and epileptiform ocular and eyelid movements. Poor photopic and scotopic vision, myopia and hyperopia have also been observed (Cogan et al, 1980; Robb et al, 1984; Schimel and Mets, 2006;

Gerth et al, 2008; Grant et al, 2009).

In terms of ocular morphology, retinal nerve fiber layer loss and macular and optic atrophy with “bone spicule formation”, lamellar lens opacities and fundus changes have also been noted (Gerth et al, 2008).

A common characteristic of cblC disease is progressive retinal degeneration, specifically a “salt-and-pepper” retinopathy (Tsina et al, 2005;

40 Schimel and Mets, 2006; Gerth et al, 2008). Patients have presented with hypo- and hyper-pigmented changes to the retinal pigment epithelia, often in a bull’s eye formation. Retinal damage was progressive in some cases, as demonstrated by electroretinogram testing and examination of the fundus at 7, 25 and 37 months of age (Shimel and Mets, 2006). The progressive nature of the degeneration suggests that it is not due to the lack of MMACHC protein at some key retinal developmental time point, but that the degeneration is a result of disease progression.

1.4.2.6 Structural Heart Defects

In one study, ten patients with early onset cblC diseased were assessed for the presence of structural defects. The authors found that 50% of these patients had structurally and clinically significant heart defects, a rate much higher than that found in the population in general (3-13/1000 live births) (Profitlich et al,

2009). Another study, which studied the long-term outcome in treated cblC patients found them to have a higher incidence of congenital heart disease, with

2/8 patients presenting with heart issues (Andersson et al, 1999). Other studies have also identified cardiomyopathies in several cblC patients (Heinemann et al,

2001; Tomaske et al, 2001). Defects included atrial septal defects, focal left- ventricular non-compaction, muscular ventricular septal defect and mitral regurgitation.

41 There are a number of hypotheses as to why cblC patients have an increased incidence of structural heart defects. As previously mentioned,

MMACHC may play a pivotal role in embryonic development. Another possibility, put forward by Gerth et al (2009), lies in DNA and histone methylation. The authors postulate that due to decreased activity of methionine synthase, lower methionine levels alter S-adenosylmethionine and S- adenosylhomocysteine levels. As S-adenosylmethionine is an important methyl- group donor, decreased levels may alter DNA and histone methylation that could alter gene expression during development, affecting organogenesis.

1.4.2.7 Vascular Symptoms

cblC disease has also manifested through a number of cardiovascular events. An infant presented with retinal and subdural and intraventricular cerebral haemorrhages (Francis et al, 2004). In many cases, subsequent issues such as cor pulmonale, are a result of thromboemboli (Brandstetter et al, 1990).

Cases of vascular damage have also been reported, with the occurrence of lesions in multiple arteries of multiple organs (Smith et al, 2006). Cardiovascular involvement in cblC disease is attributed to direct damage to the vascular endothelium due to elevated homocysteine levels in patient blood (Francis et al,

2004).

42 1.4.2.8 Renal Dysfunction

Renal dysfunction as a result of haemolytic uremic syndrome (HUS) has been described in many cblC patients (Baumgartner et al, 1979; Geraghty et al,

1992; Van Hove et al, 2002; Guigonis et al, 2005; Sharma et al, 2007). HUS is characterized by the presence of azotemia, thrombocytopenia and microangiopathic haemolytic anemia, with or without bloody diarrhea. It is thought that the renal damage is a result of injury to the endothelium of the kidney’s glomeruli and blood vessels, due to the elevation of homocysteine and its derivatives in the blood of cblC patients. But, as HUS has not been described in patients with cystathionine-β-synthase deficiency that also have elevated homocysteine in blood, a specific characteristic of cblC disease may also play an important role in the development of HUS (Geraghty et al, 1992).

Methylmalonic acid is also a putative nephrotoxin, although the exact mechanism through which it would induce kidney damage is unclear (Morath et al, 2008).

1.4.3 Treatment

It has been known for decades that patients with cblC disease do not fully respond to treatment with cyanocobalamin (CN-Cbl). A study comparing the responsiveness of cblC and cblD patient fibroblasts to treatment with hydroxycobalamin (OHCbl) and CN-Cbl showed that cblC fibroblasts had a

43 diminished response to treatment with CN-Cbl when compared to cblD fibroblasts as demonstrated by the function of methylmalonyl-CoA mutase and methionine synthase (Mellman et al, 1979). cblC cells retained less CN-Cbl vs.

OHCbl and were found to be unable to remove the cyano group from the CN-

Cbl molecule. When one compared CN-Cbl to OHCbl treatment in patients with cblC, the results were similar. Treatment of two patients with CN-Cbl (three times per week in patient 1 and daily for patient 2) reduced plasma homocysteine (Hcy) to near normal levels, but urine MMA remained above normal levels in both patients. Treatment with 1 mg OHCbl either daily or three times per week reduced urine MMA to levels below detection (Andersson et al,

1998). Level of responsiveness to OHCbl treatment is also variable and may be dependent on the mutations present (Froese et al, 2009).

Treatment with betaine has also been shown to be effective in reducing homocysteine levels and increasing methionine levels. Betaine acts as a substrate for betaine-homocysteine methyltransferase and becomes an excellent methyl donor for the methylation of homocysteine. It is therefore able to substitute for defective methionine synthase activity, caused by a lack of MeCbl (Lawson-

Yuen et al, 2005).

44 1.4.4 MMACHC

1.4.4.1 MMACHC gene structure and mutations

The gene responsible for cblC disease was discovered in 2006 and is

located on chromosome 1p34.1. It was denoted MMACHC for methylmalonic

aciduria and homocystinuria cblC type (Lerner-Ellis et al, 2006). The gene is made up if 5 exons and has an 846 base pair open reading frame which encodes a protein 282 amino acids in length and with a molecular weight of 31.7 kDa.

Residues 118-138 of the gene share a 52% amino acid similarity to residues in methylmalonyl-CoA mutase of Streptomyces avermitilis, which are part of its cobalamin-binding region. MMACHC retains the canonical 122-HXXGX126-

154GG-156 cobalamin-binding motif. A study calls to question the importance of

the histidine residue at position 122, as MMACHC protein bearing the mutation

H122A was still able to strongly bind OHCbl (Froese et al, 2009).

The last 100 C-terminal residues of the protein showed similarity to the

C-terminal domain of the TonB protein-family found in Gram-negative bacteria.

In E.coli, TonB is a 25 kDa protein that complexes to a number of other proteins, harnessing the energy from a proton-motive force and transferring this energy to

the BtuB membrane receptor in order to transport cobalamin across the outer bacterial membrane (James et al, 2009). Three-dimensional modelling and comparison showed similarity between MMACHC and TonB proteins. Residues

185-282 of MMACHC were 3-D modelled and then aligned to residues 152-239

45 of E.coli TonB. Although the amino acid sequence identity was only 14% across these regions, the protein models superimposed quite closely, with the exception of MMACHC residues 198-203. This region of TonB is thought to interact with the Ton box of outer membrane TonB-dependent receptors and in MMACHC it is though that this region may be required for the direct interaction with another protein. (Lerner-Ellis et al, 2006).

Over sixty disease-causing MMACHC mutations have been identified in approximately 400 individuals. The most common is c.271dupA

(p.Arg91LysfsX13), which accounts for the majority of all pathogenic alleles, specifically in European and North American populations, and is found in individuals from diverse ethnic backgrounds including people from Hispanic,

Australian, Pakistani, Syrian, Turkish, Jewish and European ancestry. This mutation is found on 4 haplotypes constructed from 13 polymorphisms surrounding or within the gene. These 4 haplotypes differ from each other at one polymorphism and it is thought that this mutation is the result of a common ancestral mutational event (Lerner-Ellis et al, 2006; Morel et al, 2006). A number of other mutations cluster by ethnicity. The c.394C>T (p.Arg132X) mutation is found predominantly in Asiatic-Indian/Pakistani/Middle Eastern populations (Morel et al, 2006) but has also been found to account for up to 16% of pathogenic alleles in Italian and Portuguese patients as well (Nogueira et al,

2008). Another common mutation, c.331C>T (p.Arg111X) is frequently found

46 in French-Canadian and Cajun populations, but has also been found in people of

Italian, central European and Palestinian ancestry (Lerner-Ellis et al, 2009). The c.482G>A (p.Arg161Gln) mutation is common to the Hispanic population. The c.440G>A (p.Gly147Asp) mutation has only been found in the homozygous state in two individuals of Native American origin (Morel et al, 2006). The c.3G>A mutation has been reported in Italians, Australians and Europeans

(Lerner-Ellis et al, 2009). The 609G>A (W203X) mutation has been found to account for a large majority of the mutations in the Chinese population (Wang et al, 2009). The c.328_331delAACC (p.Asn110AspfsX13) mutation has been described in people of Hispanic origin. A number of mutations are found primarily, but not exclusively, in Italians: c.457C>T (p.Arg153X), c.468_469delCT (p.Trp157ValfsX24), c.666C>A (p.Tyr222X) (Lerner-Ellis et al, 2009).

1.4.4.2 Proposed protein function

The exact function of MMACHC protein remains elusive, but it has long been hypothesized that the protein is required for the Cbl processing that precedes the biosynthesis of the two Cbl forms required by the cell, AdoCbl and

MeCbl. A 2008 study (Kim et al) demonstrated the ability of the MMACHC gene product to decyanate CN-Cbl. The protein utilizes electrons that are transferred via cytosolic flavoprotein oxidoreductases to NADPH in order to cleave the

47 carbon-cobalt bond and reductively eliminate the cyano ligand. This is consistent with the inability of cblC patients to respond to treatment with CN-Cbl, a non- physiologic form of Cbl.

There is still disagreement as to whether MMACHC binds CN-Cbl in the

“base-on” or “base-off” form. Studies have found contradicting results with regards to how MMACHC binds CN-Cbl. A 2008 study by Kim et al utilized spectrophotometry to visualize changes in absorption brought about by

MMACHC binding CN-Cbl. They were unable to detect major absorption changes characteristic of the “base-on” to “base-off” shift, suggesting that

MMACHC binds CN-Cbl in the “base-on” conformation and is unable to catalyze the change to the “base-off” form. However, a 2009 study, (Froese et al) done using higher concentrations of MMACHC, observed binding of CN-Cbl in the “base-off” form.

Mutations in the MMACHC protein have also been a useful tool in the clarification of the protein’s function. As previously mentioned, patients have differential responses to OHCbl treatment as well and a study examined the reasons for this (Froese et al, 2009). Mutant MMACHC proteins were created bearing either a mutation that is associated to later onset of cblC disease and high responsiveness to OHCbl treatment, c.482G>A (p.R161Q), or an early onset-related mutation, c.440G>A (p.G147D), which is not responsive to therapy. The early onset mutation was unable to bind either CN-Cbl or OHCbl.

48 The late onset mutation bound OHCbl with wild-type affinity, but CN-Cbl was more weakly bound and decyanation was impaired.

MMACHC is also able to de-alkylate alkylcobalamins such as MeCbl and

AdoCbl. A study found that cblC patient fibroblast cell lines with defective

MMACHC protein were unable to perform this dealkylation whereas aortic

endothelial cells with functional protein were. MMACHC protein also showed a

wide range of substrate specificity, being able to dealkylate a number of xenobiotic straight-chain alkyl-Cbls ranging from ethylcobalamin to hexylcobalamin. The study proposed a number of mechanisms as to precisely how this dealkylation may occur: Homolysis of the C-Co bond resulting in cob(II)alamin and an alkyl radical; nucleophilic displacement resulting in cob(I)alamin and the transfer of the alkyl group to an acceptor molecule; or lastly, the reductive dealkylation of the alkylcobalamin resulting in either cob(I) or cob(II)alamin and a carbanion or alkylradical (Hannibal et al, 2009).

A study has provided evidence for the nucleophilic displacement

hypothesis proposed by Hannibal et al (2009) and gluthathione was shown to be

required for the dealkylation activity of MMACHC (Kim et al, 2009).

MMACHC uses the thiolate of the glutathione molecule for a nucleophilic

displacement that results in cob(I)alamin and a glutathione thioether (Figure 4).

Other biologically relevant thiols such as cysteine and homocysteine were

unable to substitute for the glutathione.

49 1.4.4.2.1 MMACHC and Cancer

A study investigated MMACHC expression in a methionine dependent tumorigenic human melanoma cell line MeWo-LC1 that was derived from the methionine independent non-tumorigenic line, MeWo. MeWo-LC1 cells show decreased synthesis of the Cbl cofactors as well as decreased activity of the Cbl- dependent enzymes. This is a cellular phenotype identical to that of cblC cell lines, as further evidenced by the fact that cblC cell lines were unable to complement MeWo-LC1 cells and that infection of MeWo-LC1 cells with wild- type MMACHC corrected the phenotype. Sequencing revealed no mutations in the MMACHC gene in MeWo-LC1 cell lines and gene expression was not detected by non-quantitative and quantitative PCR. Bisulphite sequencing revealed nearly complete methylation of the CpG island at the 5’ end of

MMACHC. Epigenetic modification of MMACHC is responsible for the methionine-dependence of the MeWo-LC1 cell line (Loewy et al, 2009).

Figure 4: Mechanism of R-group removal by MMACHC protein. CN-Cbl is reductively decyanated to cob(II) alamin and cyanide and alkylcobalamins are reduced via nucleophilic displacement to give cob(I)alamin, which is oxidized to cob(II)alamin and then can subsequently undergo conversion to the active cofactor forms (AdoCbl and MeCbl). From Kim et al, 2009.

51 1.4.5 Genotype-Phenotype Correlations in cblC Disease

A number of genotype-phenotype correlations have been found which relate MMACHC mutations with ages of onset. Two common mutations, c.271dupA and c.331C>T have been found primarily in patients with early onset disease. All reported patients homozygous for the c.271dupA mutation, 96 to date, have presented within the first year of life, with the exception of two who presented between the ages of one and four (Lerner-Ellis et al, 2009). Most patients bearing the c.331C>T mutation have presented within the first six months of life (Morel et al, 2006). The c.394C>T mutation has been consistently linked to later onset of cblC disease. To date, at least twelve patients homozygous for the c.394C>T mutation have been reported to present with late onset disease. Patients compound heterozygous for the c.394C>T mutation as well as an early onset-related mutation, such as c.271dupA, have had more variable ages of onset, presenting as early as the neonatal period to as late as 14 years of age (Lerner-Ellis et al, 2009). All of these mutations result in the creation of premature stop codons and result in the abolition of the putative Cbl- binding domain as well as the TonB homology domain. A number of less common missense mutations, such as c.482G>A (p.R161Q), have also been correlated to later onset of disease, with individuals presenting as late as 10 to 20 years of age. Most of these individuals are compound heterozygous for the

52 c.271dupA mutation and a missense mutation (Lerner-Ellis et al, 2006; Morel et

al, 2006, Tsai et al, 2007, Nogueira et al, 2008, Thauvin-Robinet et al, 2008).

It is hypothesized that mutation-bearing transcripts have differential

stabilities, with later-onset mutation transcripts able to avoid degradation and be

translated into a protein with residual function. Other factors such as background

genetic variation, and environmental and dietary exposures may contribute to

cblC’s clinical heterogeneity.

1.5 Differential Allelic Expression

It has become apparent that genetic variation that affects gene expression

may result in phenotypic and disease variability. It has been established that unequal transcript expression is a common phenomenon, with 5%-20% of

individuals showing differential allelic expression in 15%-50% of genes (Ge et

al, 2005). This variation may be the result of a polymorphism or mutation within

the gene, which affects the stability of a specific allele’s transcript. For example,

compound heterozygous mutations resulting in premature termination codons

(PTC) in the first few exons of the muscle chloride channel gene, CLCN1, can

result in the autosomal recessive form of congenital myotonia, as the transcripts

are degraded by nonsense-mediated decay (NMD). However, PTC mutations in

the last exon on one allele of a gene allow the transcript to escape nonsense-

mediated decay. The transcript is translated into a protein that has a dominant-

53 negative effect resulting in a dominant form of congenital myotonia (Khajavi et al, 2006).

Regulatory polymorphisms can also affect the amount of transcript being produced. A G to A change in the promoter region of the tumor necrosis factor alpha (TNF) gene has been shown to correlate with susceptibility to severe malaria in a number of countries. The A allele is associated with higher constitutive and inducible levels of TNF-α transcription compared with the G allele. TNF-α has been shown to directly inhibit iron absorption and increased levels of this protein would increase iron concentration in the blood (Atkinson et al, 2008). It is thought that increased levels of iron in the blood lead to susceptibility to severe malarial infection, by an unknown mechanism, in patients for which access to malaria diagnosis and treatment resources are not readily available (Prentice et al, 2007).

Translational efficiency may also be affected by regulatory polymorphisms, such as the C>T polymorphism 4 base pairs upstream of the translation initiation codon (ATG) of the serine protease factor XII gene (F12), the first coagulation factor in the coagulation cascade. The T allele has been associated with increased risk of coronary artery disease. The SNP creates a novel ATG site, reducing the translational efficiency of the transcript (Knight,

2005).

54 Allele-specific expression may also be the result of polymorphisms or

mutations that affect methylation status in an allele-specific manner. Allele-

specific methylation has been reported in VNN1, whose transcript levels have

been linked to HDL cholesterol levels, as well as for CYP2A7, which is thought

to play some role in the control of nicotine levels in blood and subsequently affect smoking behavior. In both cases, this allele-specific methylation has been linked to genotype at specific SNPs (Kerkel et al, 2008).

Differences in the allelic expression of the MMACHC gene, as well as increased or decreased overall transcript amounts in patients, may provide clues as to why cblC patients present with such clinical heterogeneity. As early and late onset cblC disease present with markedly different phenotypes and have differential responses to treatment, screening that enables early detection of the disease is critical and mutation analysis may allow for better assessment and management for patients.

The objective of this study is to examine the underlying relationship between MAMCHC transcript levels and cblC’s genotype-phenotype correlations through allele specific expression analysis and quantitative real-time RT-PCR.

55 CHAPTER 2

Materials and Methods

2.1 Cell Culture and Cell Line Selection

Cell lines were obtained from the Repository for Mutant Human Cell Strains

of the Montreal Children’s Hospital (http://www.cellbank.mcgill.ca) and were

determined to be negative for mycoplasma contamination by the modified

protocol of Schneider et al. (1974). Cells were routinely cultured in Eagle’s

minimum essential medium (MEM) containing Earle’s salts and non-essential

amino acids (Invitrogen Canada, Burlington, Ontario), supplemented with

2.2 g/L sodium bicarbonate, 0.11 g/L sodium pyruvate, 10 mg/L ferric nitrate,

1.5 g/L glucose, and 10% fetal bovine serum (Cansera International, Etobicoke,

Ontario). Tissue cultures were maintained in 75cm2 flasks, incubated at 37 °C in

5% CO2, and fed twice weekly.

A total of 33 cblC patient (compound heterozygotes and carriers) and control

cell lines were chosen for allelic expression analysis. Each patient cell line was

previously found to belong to the cblC complementation class through complementation analysis. Mutation analysis confirmed cell lines to be compound heterozygous for MMACHC mutations previously linked to genotype- phenotype correlations within the disease (Morel et al, 2007) (Table 1).

56 Mutations studied were c.271dupA (p.Arg91LysfsX13), c.394C>T (p.Arg132X),

c.331C>T (p.Arg111X) and c.482G>A (p.Arg161Gln). The c.666C>A

(p.Tyr222X) was chosen as well, as it is also a mutation that results in a

premature stop codon, but is downstream of the c.394C>T mutation.

Comparison between their expression levels may give some indication of levels of nonsense-mediated decay.

2.2 DNA and RNA Extraction and cDNA Synthesis

2.2.1 DNA Extraction

Confluent cells in T75 tissue culture flasks were trypsinized and genomic

DNA was extracted using the Qiagen gDNA Extraction Kit for Cultured cells

according to manufacturer’s instructions (Qiagen, Mississauga, Ontario).

Extracted gDNA was then stored at -20ºC.

2.2.2 RNA Extraction

RNA was extracted from confluent cultured patient fibroblasts in T75

tissue culture flasks using Trizol reagent according to the manufacturer’s

instructions (Molecular Research Center Inc, Cincinnati, OH). Extracted RNA

was then stored at -80ºC.

57 Cell Line Status Mutations in MMACHC Age of Onset

MCH39* Control - - N/A MCH55* Control - - N/A MCH58* Control - - N/A MCH64* Control - - N/A C024 Affected c.271dupA c.394C>T 20 months C072 Affected c.271dupA c.394C>T ? C075 Affected c.271dupA c.394C>T ? C101 Affected c.271dupA c.394C>T 3.5 months WG1070 Affected c.271dupA c.394C>T 6 months WG1127 Affected c.271dupA c.331C>T 4 weeks WG1129 Affected c.271dupA c.394C>T 4 years WG1135 Carrier c.331C>T - N/A WG1391 Affected c.271dupA c.482G>A 14 years WG1404 Affected c.394C>T c.666C>A < 14 years WG1481 Affected c.271dupA c.394C>T ? WG1496 Affected c.271dupA c.331C>T ? WG1513 Carrier c.271dupA - N/A WG1514 Carrier c.271dupA - N/A WG1692 Affected c.271dupA c.394C>T ? WG1958* Control - - N/A WG2113* Control - - N/A WG2328* Control - - N/A WG2339 Affected c.271dupA c.482G>A ~18years WG2460 Affected c.271dupA c.666C>A Birth WG2599 Affected c.271dupA c.394C>T ? WG3014 Affected c.271dupA c.331C>T Birth WG3072 Affected c.271dupA c.331C>T 2 months WG3130 Affected c.271dupA c.482G>A 36 years WG3281 Affected c.331C>T c.666C>A 1 month WG3480 Affected c.271dupA c.482G>A 3.5 years WG3514 Affected c.271dupA c.666C>A ? WG3665 Affected c.271dupA c.331C>T Early WG3686 Affected c.271dupA c.482G>A Early

Table 1: Cell lines selected for Allelic Expression Analysis. In total, 23 cblC

patient cell lines, 7 control cells lines and 3 carrier cell lines were tested. Age of

onset of cblC disease and MMACHC mutations present are also shown. In the

age of onset column, “Early” implies onset before 1 year of age, “N/A” denotes

individuals who do not have cblC disease, and “?” denotes individual for whom age of onset is unknown.

59 2.2.3 DNA and RNA quality control

RNA and DNA concentration was assessed using the Nanodrop spectrophotometer (ND 1000). RNA quality was evaluated using the Agilent

2100 Bioanalyzer RNA NANO 6000 chips according to manufacturer’s protocol

(Agilent, Mississauga, Ontario). The chip utilizes traditional gel electrophoresis methods but in a chip format.

2.2.4 cDNA synthesis

In order to ensure purity of RNA used for cDNA synthesis, 10μg of extracted RNA was incubated in 2µl of Dnase I (Ambion) in water, 10μl of buffer and water up to a volume of 100μl for 30 minutes at 37ºC.

RNA was the precipitated by adding 100μl of phenol-chloroform

(Invitrogen Canada, Burlington, Ontario). This RNA and phenol-chloroform was mixed and then spun at 14 000 G for 5 minutes. The aqueous layer was removed then incubated at -80ºC in 10μl 3M sodium acetate and 300μl of 100% ethanol and then centrifuged at 14 000 G at 4ºC for 15 minutes. Samples were then washed twice with 100μl of 75% ethanol and centrifuged at 14 000 G at 4ºC for

10 minutes. The pellet was subsequently resuspended in 500ng of random primers in 10 μl of water (Invitrogen Canada, Burlington, Ontario) and then incubated in a master mix containing 1 μl 10mM dNTP, 2μl 100mM DTT, 4μl

5X First Strand Buffer, 1μl Rnase Out Rnase Inhibitor and 2μl of SuperScriptII

60 enzyme (Invitrogen Canada, Burlington, Ontario) per tube for 1 hour at 42ºC. cDNA was then stored at -80ºC.

2.3 Allelic Expression Analysis

2.3.1 Primer Design

Primers for allelic expression analysis were designed using the default settings of Primer 3.0 (http://frodo.wi.mit.edu/primer3/). Single nucleotide polymorphisms were masked prior to primer design. Primers targeted the same sequence in genomic DNA and mRNA sequences, when possible, but if a targeted variant localized close to exon-intron junction separate genomic DNA and mRNA specific primer pairs were designed to share one common primer and to yield similar fragment size. The primer used for sequencing was located at least 70 base pairs upstream of the mutation/polymorphism of interest to ensure that ABI sequence traces would be of sufficient quality for quantitative analysis of allele ratios. Primers specific to the rs7903, rs11580609 and rs2275276 SNPs were designed in order to allow individual haplotypes to be assessed. These

SNPs were found to be highly polymorphic in patient and control cell lines.

Primers were also designed to capture c.271dupA, c.331C>T, c.394C>T, c.482G>A and c.666C>A mutations (Table 2).

61 2.3.2 PCR

PCR was performed according to Table 3 specifications. For each PCR reaction, 5ng cDNA or 2ng of gDNA was used. PCR product formation was

verified by electrophoresis in 1.1% agarose gels. For each patient cell line

sample, 1 gDNA replicate and 3 cDNA replicates were amplified in parallel.

2.3.3 Sequencing and Allelic Expression Analysis

Primers were removed before sequencing by incubating PCR products with exonuclease 1 and shrimp alkaline phosphatase for 1 hour at 37ºC (USB

(Affymetrix), Cleveland, Ohio, USA). Sequencing was carried out at the McGill

University and Genome Quebec Innovation Centre using the BigDye Terminator v.3.1 cycle sequencing kit (Applied Biosystems).

2.3.4 Allelic Expression Data and Statistical Analysis

In order to establish allele-specific expression, the sequencing products were

run on an Applied Biosystems 3730XL DNA analyzer. ‘‘PeakPicker’’ software

was then used to assess allelic ratios. gDNA and cDNA samples were run in

parallel and heterozygous gDNA was used to confirm as well as to establish the

cut-offs for the expected 1:1 allelic ratio. Specific allele expression was

examined using two tests. For the sample-specific test, a 95% confidence

interval was established for equal allelic expression based on the peak height in

62 the heterozygous gDNA sample. Allelic expression differences were established only when expression in the cDNA triplicates deviated from this interval. The locus-specific test is a two-tailed t-test that compares the average peak ratios from the gDNA and cDNA replicates. This test uses all of the replicate values and increases the sensitivity of result evaluation. (Ge et al, 2005).

In order to establish statistical significance, ratio values at each mutation and polymorphism were averaged for each replicate and then the three values averaged as well. Values were compared to control sample values and a T-Test used to establish significance.

63 a)

Table 2: a) Primers used for allelic expression analysis b) Primers used for quantitative real time RT-PCR analysis

Table 3: PCR Master Mix and cycling protocol

65 2.4 Quantitative Real Time RT-PCR

2.4.1 Cell Line Selection

Cell lines homozygous for the c.271dupA, c.331C>T and c.394C>T

mutations, as well as a number of control cell lines were selected (Table 4).

2.4.2 RNA extraction, RNA quality control and cDNA synthesis

RNA extraction, quality control and cDNA synthesis were performed as

previously described in section 2.2.2, 2.2.3 and 2.2.4.

2.4.3 Primer Design

Primers for MMACHC were designed using Primer 3.0 software to capture

a 200 base pair region (Table 2b). Primers were verified in dbSNP

(http://www.ncbi.nlm.nih.gov/projects/SNP/) ensure no polymorphisms were

present. Primers for GAPDH were based on primers used by Lesouhaitier et al

(2001).

2.4.4 Quantitative Real-Time RT-PCR

Quantitative real-time RT-PCR was performed on the RotorGene 6000 (Corbett

Technologies, San Francisco, CA) using SYBR Green (Invitrogen) according to manufacturer’s instructions. Differences in amount of cDNA generated from

RNA transcripts were detected via fluorescence. SYBR Green fluoresces when

66 bound to double-stranded DNA. As PCR reaction proceeds, each cycle produces increased amounts of double-stranded to which SYBR Green can bind. The excitation and emission maxima of SYBR Green are at 494 nm and 521 nm respectively. The RotorGene 6000 is able to detect this fluorescence. Increased fluorescence indicates increased transcript amount. GAPDH transcript levels were used a control to assess and account for any variations in baseline transcription between cell lines.

2.4.5 Statistical Analysis

Statistical analysis of the data was performed using the Kruskal-Wallis one-way analysis of variance test as well as analysis of variance (ANOVA).

Mutation Cell Lines Age of Onset WG1176 ? c.394C>T WG2722 1 year WG3183 ~10 years WG2279 <4 weeks c.271dupA WG2452 6 weeks WG3366 18 days WG1539 Birth c.331C>T WG1422 <2 months WG2076 <3 months MCH58 N/A Controls MCH64 N/A MCH65 N/A

Table 4: Cell Lines selected for quantitative real-time RT-PCR. 3 cell lines, each homozygous for either the c.271dupA, c.331C>T or c.394C>T mutation were chosen. Age of onset of cblC disease is also included.

68 CHAPTER 3

Results

3.1 Allelic Expression Analysis

3.1.1 Haplotype analysis in control cell lines

In order to assess the effect background haplotype has on gene expression, control cell lines heterozygous for three SNPs within the MMACHC gene region

(rs2275276, rs11580609 and rs7903) were sequenced and the allele ratio established. Analysis revealed the expected 1:1 ratio (control ratio = 0.99) in control cell lines, indicating that transcription of MMACHC is not affected by the underlying haplotype as typed by these SNPs (Figure 6, Control group). The two haplotypes observed were G/C/G or A/G/C at rs2275276/ rs11580609/rs7903.

3.1.2 Allelic expression of patient cell lines

The PeakPicker software output is shown in Figure 5. The program places the gDNA ratio (red bars) and mRNA ratio in all three replicates (blue bars) for all tested cell line samples together in order to allow for easier comparison. The gDNA ratio for all samples are within the 95% confidence interval (designated by the red horizontal lines) of the 1:1 expected allele ratio (blue horizontal line),

69 indicating that the alleles are present in a 1:1 ratio in the gDNA and that any deviation from the ratio in mRNA is a result of transcription rate differences.

There was increased transcription from alleles bearing late onset mutations when compared to early onset mutations. The c.394C>T mutation was expressed

an average of 3.67 times that of the c.271dupA allele (P value = 3.6x10-6) in cell

lines from patients heterozygous for these two mutations and 3.08 times that of

the c.331C>T allele (P value = 9.56x10-6). The c.482G>A missense mutation was also expressed at an increased rate of 1.88 times that of the c.271dupA mutation (p=1.1x10-11) (Figure 6).

Early onset mutations were expressed at decreased rates when compared to

the wild-type allele as well. The c.271dupA mutation was expressed at 0.51

times that of the wild-type allele (P value = 3.4x10-6) in carriers and the

c.331C>T allele was expressed at a ratio of 0.58 times that of the wild-type allele

(P value = 3.0x10-10). When the two early onset-related mutations were in

compound heterozygous form, the c.331C>T allele was expressed at 0.89 times

that of the c.271dupA allele (P value = 0.04) (Figure 6).

Figure 5: PeakPicker program output for allelic expression analysis of the c.271dupA allele in patient fibroblast cell lines. The ratio represents that of the c.271dupA allele to the other allele. The Y-axis indicates the allele ratio. The red bars indicate the ratio of the alleles in gDNA from a given cell line, and the adjacent blue bars represent the allele ratios of mRNA from the same cell line

(triplicate samples). All the ratios for gDNA are within the 95% confidence interval (red horizontal lines) indicating the expected 1:1 ratio (blue horizontal line). Cell lines (mutations) in figure are:

WG1070 (c.271dupA/c.394C>T), WG1129 (c.271dupA/c.394C>T),

WG1481 (c.271dupA/c.394C>T), WG 1513 (c.271dupA carrier),

WG1514 (c.271dupA carrier), WG1692 (c.271dupA/c.394C>T),

WG2460 (271dupA/c.666C>A), WG3130 (c.271dupA/c.482G>A),

WG3480 (c.271dupA/c.482G>A), WG3514 (c.271dupA/c.666C>A),

WG3665 (c.271dupA/c.331C>T), WG3686 ((c.271dupA/c.482G>A)

Figure 6: Results of allelic expression analysis in patient fibroblast cell lines carrying the c.271dupA, c.331C>T, c.394C>T and c.482G>A mutation. Control cell line allele ratios cluster at the expected allele ratio of 1. Deviation from the

1:1 ratio indicates a difference in relative amounts of allelic mRNA transcripts attributable to the MMACHC mutation present. n=number of patient cell lines sampled.

73 3.1.3 Allelic expression of the c.666C>A mutation

The c.666C>A mutation, another mutation that also results in the

creation of a premature stop codon but has not been seen to directly correlate

with age of onset of cblC disease, was also assessed. Expression analysis

revealed that the c.271dupA allele was expressed at 0.4 times that of the

c.666C>A mutation (P value = 2.33x10-13) and the c.331C>T mutation was

expressed at 0.38 times that of the mutation (P value = 6.07x10-13). Interestingly, the c.666C>A mutation was expressed at decreased levels when compared to the late onset c.394C>T allele, with the c.394C>T allele being expressed at 1.36 times that of the c.666C>A mutation (P value = 2.7x10-4) (Figure 7).

Figure 7: Results of allelic expression analysis in patient fibroblast cell lines carrying the c.666C>A mutation. Control cell line allele ratios cluster at the expected allele ratio of 1. Deviation from the 1:1 ratio indicates a difference in relative amounts of allelic mRNA transcripts attributable to the MMACHC mutation present. n=number of patient cell lines sampled.

75 3.2 Quantitative Real-Time RT-PCR Analysis

Overall MMACHC mRNA transcript levels were assessed by quantitative real-time RT-PCR in cell lines homozygous for mutations of interest. Patient cell lines homozygous for the late onset-related c.394C>T mutation showed significantly increased amounts of MMACHC mRNA transcript when compared to cell lines homozygous for either the c.271dupA mutation (P value = 0.00018) or the c.331C>T mutation (P value = 0.00024). Transcript levels form all three mutation groups did not differ significantly from control levels (Figure 8).

Expression levels were normalized using a housekeeping gene, GAPDH.

Expression of this gene was used to control for baseline expression levels in individual cell lines.

Figure 8: Quantitative real-time RT-PCR results. Relative concentration of

MMACHC mRNA transcript in fibroblast cell lines from controls and patients homozygous for one of the early or late onset-related mutations (c.271dupA, c.331C>T or c.394C>T) is shown. Expression is normalized to that of the control gene GAPDH. ** indicates statistically significant (p<0.01) difference in transcript levels between c.271dupA and c.394C>T homozygotes and c.331C>T and c.394C>T homozygotes.

77 CHAPTER 4

Discussion

Genotype-phenotype correlations have been noted in cblC disease, with certain mutations generally relating to early onset of the disease; c.271dupA and c.331C>T, and other mutations relating to later onset of the disease; c.394C>T and a number of missense mutations including c.482G>A. In order to understand the relationship between expression of different alleles and clinical phenotype of cblC disease, allele-specific and overall transcript levels of MMACHC were examined in cblC patient fibroblasts bearing age of onset–related mutations.

Analysis of allele-specific expression in compound heterozygotes and carriers of common mutations associated to either late or early disease onset revealed markedly decreased expression of the early onset c.271dupA and c.331C>T when compared to both the wild-type transcript and the later onset-associated c.394C>T allele. The c.271dupA mutation was also under-expressed when compared to the late onset-related missense mutation c.482G>A. These results compliment the quantitative real-time RT-PCR results, which showed that cell lines homozygous for the late onset-related c.394C>T allele had significantly increased transcript levels when compared to both early onset-related mutations: c.271dupA and c.331C>T.

78 Increased expression of the c.394C>T allele was also seen when it was in a compound heterozygous state with another nonsense mutation, c.666C>A. This is of interest when one begins to consider the mechanisms which may cause the c.394C>T mutation-bearing allele to be over-expressed, which will be further discussed below.

Analysis of control cell lines heterozygous for the two haplotypes observed in patient cell lines revealed that the underlying haplotype had no effect on transcript expression and therefore any differences can be attributed to the mutations present.

Increased expression from the c.394C>T allele was seen regardless of patient’s age of onset. cblC’s clinical heterogeneity, particularly in relation to this mutation, has been well documented (Morel et al, 2006; Lerner-Ellis et al,

2009) and may be the result of a number of different causes. cblC disease can present at varying ages or manifest itself differently within the same family or even between siblings (Augoustides-Savvopoulou et al, 1999; Heil et al, 2007).

For example, two brothers both presented with late onset cblC, but had very different neurological phenotypes. Both presented after thirty years of age, but the first brother exhibited myelopathy and the other with neuropsychiatric problems and dementia as a result of severe cerebral perivascular demyelination

(Powers et al, 2001). For this reason, it is possible that other factors play a role in the disease’s presentation and act in conjunction with the mutations in

79 MMACHC. Even with increased levels of expression from the c.394C>T mutation-bearing allele, other factors may influence the final phenotype.

Causes of the differences in clinical manifestations of cblC disease may lie in other genotypic differences among patients with the same MMACHC mutations, specifically the c.394C>T mutation. A phenomenon such as this has been observed in Bardet-Biedl syndrome (McCandless and Cassidy, 2006). It was observed that in several families, a number of family members were homozygous for mutations in a specific gene related to the disease, but not all homozygous individuals were affected by the disease. Further investigation revealed that the affected individuals were also heterozygous for a mutation in another gene related to Bardet-Biedl syndrome. In the case of cblC, heterozygous mutations or polymorphisms in different genes encoding proteins that interact with MMACHC or belong to the Cbl or interacting pathways could act in conjunction with the c.394C>T mutation in MMACHC to affect the onset of symptoms and perhaps even the severity of disease. Therefore, even with increased expression of the c.394C>T allele and its possible translation into a protein with residual function, SNPs in genes encoding interacting proteins may prevent proper interaction or hamper the residual function of mutated

MMACHC protein, modulating age and severity of onset. In order to investigate whether this is the case for cblC disease, haplotype analysis as well as mapping of SNPs in related genes could prove helpful.

80 It is also possible that environmental factors play a role in differential onset

of cblC symptoms among individuals with the same mutation. Studies have

shown that environmental factors can trigger the onset and affect the severity of

a disease, even when the same mutations are present. For example, smoking and

alcohol consumption have been shown to precipitate the onset of Leber

hereditary optic neuropathy, a genetic disorder caused by mutations in

mitochondrial DNA. Incomplete penetrance of mutations is observed and

investigation of a number of affected and unaffected carriers found that

penetrance reached as high as 93% in smokers and was also much higher in

individuals with high alcohol consumption (Kirkman et al, 2009). Diet may be

particularly important for cblC disease as diets with increased or decreased levels of vitamin B12, folate and vitamin B6 may help prevent or precipitate onset

of symptoms (Tanaka et al, 2009). It is possible that a c.394C>T mutant

MMACHC protein with residual function would be more or less able to function

depending on the absence or presence of environmental stressors such as over-

abundance or severe lack of B vitamins in the diet. This hypothesis could be

tested using animal models with an MMACHC protein carrying the c.394C>T

mutation. The environments and diets of these animals can be easily altered in

order to investigate these effects. The influence of factors such as diet could then

be systematically assessed.

81 Although age of onset has been used to sub-categorize patients with cblC

disease, it is difficult to assess the accuracy of patient reports. Patients may

suffer from less severe disease-related symptoms that go undiagnosed until later

in life and differential interpretation of symptoms by doctors as well as non-

uniform reporting of patients’ histories may help explain the clinical variation

seen in patients with the same MMACHC genotype.

The three most common age of onset-related mutations, c.271dupA

(p.Arg91LysfsX13), c.331C>T (p.Arg111X) and c.394C>T (p.Arg132X), all result in the creation of premature stop codons within the third exon of

MMACHC. Increased transcription from the late onset related c.394C>T allele could indicate the c.394C>T mutation may be protective as increased levels of transcript could allow for some protein with residual function to be produced. If this is the case, the protein produced by the c.394C>T mutation-bearing transcript would be substantially truncated, missing part of the putative B12- binding motif as well as the TonB-homology domain.

The increase in transcript amount may be the result of resistance to the mechanism of nonsense-mediated decay (NMD). Eukaryotic cells maintain this system as a method of quality control leading to the degradation of transcripts that terminate prematurely during translation (Matsuda et al, 2008). Differences in NMD have previously been noted to result in differential neurological phenotypes, for example the gene SOX10 (Inoue et al, 2004). Mutations that

82 resulted in transcripts that were more stable resulted in the translation of proteins

that created a dominant negative effect, which in turn resulted in a far more

severe phenotype. The milder phenotype was the result of transcripts that were

degraded by the NMD machinery. While it seems that the opposite effect is

observed in cblC disease, with the more stable transcript resulting in a milder

phenotype, it is evident that the stability of mRNA transcript can play a role in

the manifestation and severity of disease.

However, if the c.394C>T transcript is immune to NMD, this contradicts what is so far known about the NMD machinery and the rules for transcript degradation. In general, all transcripts that result in a premature termination codon (PTC) more than 55 base-pairs before the final exon-exon junction are degraded (Matsuda et al, 2008). The c.394C>T mutation creates a PTC well before the last exon-exon junction complex. Further complicating matters, the allele bearing the c.394C>T mutation is also more highly expressed than the allele bearing another mutation which creates a PTC further downstream than the one created by the c.394C>T mutation, c.666C>A (p.Tyr222X).

Previous studies have also observed this phenomenon; in which expected

NMD substrates have managed to avoid degradation. A study examining MMAA mRNA levels found normal or close to normal levels of transcript, even though they bore mutations which resulted in PTCs (Merinero et al, 2008). A number of mechanisms through which transcripts evade NMD have been proposed and

83 include RNA editing and concealment of the PTC by a protein complex

(Holbrook et al, 2004). Another study investigated the susceptibility to NMD of six truncating mutations in HNF-1β, with two of the mutations (p.H69fsdelAC and p.P159fsdelT) producing transcripts that were unexpectedly immune to

NMD. It is thought that translation was reinitiated at a site downstream of the

PTC.

It is also possible that a process other than NMD carries out the regulation of MMACHC transcript levels. Proteins or miRNAs may bind the transcript and guide its expression or degradation. A study suggests the possibility that overlapping genes may produce natural anti-sense transcripts of one another, and that these anti-sense transcripts may have regulatory roles (Makalowska, 2008).

MMACHC overlaps 2 genes in the opposite orientation, PRDX1 and

LOC126661. If antisense transcripts were to bind at the mRNA position +394, then a mutation at this site would result in a mismatch, rendering the antisense transcript ineffective, thereby increasing transcript levels.

Many patients compound heterozygous for the c.271dupA, as well as a missense mutation, such as c.347C>T (p.L116P), c.440G>C(p.G147A), c.482G>A (p.R161Q) or c.565C>A (p.R189S) have been reported to present with late onset cblC disease (Thauvin-Robinet et al, 2008; Lerner-Ellis et al,

2006; Morel et al, 2006). Increased transcription was seen from the c.482G>A allele when compared to the c.271dupA allele, most likely a result of the

84 degradation of the PTC-bearing c.271dupA transcript. It is possible that transcripts bearing missense mutations are translated into proteins with residual function, resulting in less severe disease. As previously mentioned, mutant

MMACHC proteins bearing the c.482G>A (p.R161Q) bound OHCbl with wild- type affinity (Froese et al, 2009).

cblC disease is clinically heterogeneous and late onset disease is particularly interesting as patients who were previously healthy manifest neurological symptoms quite suddenly. cblC patients also respond differentially to treatment with OHCbl, some showing a complete reversal of symptoms and other showing no response, indicating that an alternative treatment method is required. Expanded newborn screening may help to detect patients pre- symptomatically. Understanding the disease mechanisms and underlying causes of genotype-phenotype correlations will be necessary to ensure optimal patient treatment and ensure positive patient outcomes.

85 SUMMARY AND CONCLUSIONS

In this study, the relationship between the clinical phenotype of cblC disease and the expression levels of mRNA transcripts from alleles related to age of onset was examined. It was observed that there were increased transcript amounts from alleles bearing late onset- related mutations (c.394C>T and c.482G>A) when compared to early onset-related mutations (c.271dupA and c.331C>T). Cell lines from patients homozygous for either late or early onset- related mutations revealed that cell lines homozygous for the late onset-related c.394C>T had increased levels of MMACHC mRNA overall.

86 ORIGINAL CONTRIBUTIONS TO SCIENCE

Further delineation of genotype-phenotype correlations in cblC disease

Correlating genotype to the amount of mRNA transcript in fibroblast cell lines from patients with cblC disease

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Thauvin-Robinet C, Roze E, Couvreur G, Horellou MH, Sedel F, Grabli D, Bruneteau G, Tonneti C, Masurel-Paulet A, Perennou D, Moreau T, Giroud M, de Baulny HO, Giraudier S, Faivre L. 2008. The adolescent and adult form of cobalamin C disease: clinical and molecular spectrum. J Neurol Neurosurg Psychiatry. 79(6): 725-8.

Tomaske M, Bosk A, Heinemann MK, Sieverding L, Baumgartner ER, Fowler B, Trefz FK. 2001. CblC/D defect combined with haemodynamically highly relevant VSD. J Inherit Metab Dis. 24(4):511-2.

Tsai AC, Morel CF, Scharer G, Yang M, Lerner-Ellis JP, Rosenblatt DS, Thomas JA. 2007. Late-onset combined homocystinuria and methylmalonic aciduria (cblC) and neuropsychiatric disturbance. Am J Med Genet A. 143A(20): 2430-4.

Tsina EK, Marsden DL, Hansen RM, Fulton AB. 2005. Maculopathy and retinal degeneration in cobalamin C methylmalonic aciduria and homocystinuria. Arch Ophthalmol. 123(8): 1143-6.

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102 APPENDIX A List of Publications and Presentations

Publications:

1. Lerner-Ellis JP*, Anastasio N*, Liu J, Coelho D, Suormala T, Stucki M, Loewy A, Gurd S, Grundberg E, Morel CF, Watkins D, Baumgartner MR, Pastinen T, Rosenblatt DS, Fowler B. 2009. Spectrum of mutations in MMACHC, allelic expression and evidence for genotype-phenotype correlations. Human Mutation, 2009 30(7): 1072-1081. *These authors contributed equally to this work

2. Loewy AD, Niles KM, Anastasio N, Watkins D, Lavoie J, Lerner-Ellis JP, Pastinen T, Trasler JM, Rosenblatt DS. 2009. Methionine dependence in a human melanoma cell line due to epigenetic modification of MMACHC expression. Molecular Genetics and Metabolism. 96(4): 261- 7.

Presentations (oral and posters):

1. Allelic imbalance and genotype-phenotype correlations of cblC disease. Poster presentation for the Canadian Human Genetics Conference. Mont- Saint-Sauveur. April 2008.

2. Allelic imbalance and genotype-phenotype correlations in cblC disease. Oral project presentation for the McGill University Human Genetics Graduate Research Day. June 2008.

103 3. Allelic expression of the MMACHC gene and genotype-phenotype correlations in cblC disease. Poster Presentation for FASEB: Folic acid,

Vitamin B12 and One-Carbon Metabolism, August 2008, Lucca, Italy.

4. Allelic expression of the MMACHC gene and genotype-phenotype correlations in cblC disease [1450]. Poster presentation for The American Society of Human Genetics, November 12, 2008, Philadelphia, Pennsylvania. http://www.ashg.org/2008meeting/abstracts/fulltext/*

5. Allelic Expression of the MMACHC Gene and Genotype Phenotype Correlations in cblC Disease. Oral project presentation CIHR Trainee Get-Together. Montreal. QC. December 2008.

6. Allelic Expression of the MMACHC Gene and Genotype Phenotype Correlations in cblC Disease. Oral project presentation for Montreal Children’s Hospital Neurosciences Day. December 2008.

7. Spectrum of Mutations in MMACHC, Allelic Expression and Evidence for Genotype-Phenotype Correlations. Oral project presentation for the Garrod Symposium. Montreal, QC. May 2009

8. Mutations in TCblR, the gene for the transcobalamin receptor, result in

decreased cellular uptake of vitamin B12 and methylmalonic aciduria. Poster presentation for the 11th International Congress of Inborn Errors of Metabolism; 2009 Aug 29-Sept 3; San Diego, California. p.122.

104 9. Mutations in TCblR, the gene for the transcobalamin receptor, result in

decreased cellular uptake of vitamin B12 and methylmalonic aciduria. Poster presentation for the Canadian Human Genetics Conference. Mont- Saint-Sauveur. April 2010.

105 APPENDIX B Published Abstracts American Society of Human Genetics

Allelic expression of the MMACHC gene and genotype-phenotype correlations in cblC disease

Natascia Anastasio1, Jordan P. Lerner-Ellis1, Tomi Pastinen1,2, Junhui Liu1, David Coelho3,4, Terttu Suormala3,4, Martin Stucki, 3,4, Amanda Loewy1, Scott Gurd1,2, Elin Grundberg1,2, Chantal F. Morel5, Mathias R. Baumgartner3,4, David Watkins1, Brian Fowler3,4, David S. Rosenblatt1 1McGill University, Montreal, Canada, 2McGill University and Genome Quebec Innovation Centre, 3Metabolic Unit, University Children’s Hospital, Basel, Switzerland, 4Division of Metabolism and Molecular Paediatrics, University Children’s Hospital, Zurich, Switzerland, 5Department of Medicine, University of Toronto, Canada

Combined methylmalonic aciduria and homocystinuria, cblC type, caused by mutations in MMACHC located in chromosome region 1p34.1, results from the inability to convert intracellular vitamin B12 (cobalamin) into its two active coenzyme forms. Methylcobalamin is required by methionine synthase in the conversion of homocysteine to methionine, and adenosylcobalamin is required by methylmalonyl-CoA mutase in the conversion of methylmalonyl CoA to succinyl CoA. Individuals with cblC disease may present early in life or at a much later age. With regard to genotype-phenotype correlation, individuals with the c.394C>T (p.R132X) mutation generally have late onset of disease whereas patients with the c.331C>T (p.R111X) and c.271dupA (p.R91KfsX14) mutations usually present in infancy. Previous quantitative real-time RT-PCR studies showed increased MMACHC mRNA transcript levels in patients homozygous for the late-onset c.394C>T mutation when compared to both controls and patients homozygous for the early onset c.271dupA and c.331C>T mutations. Allele-specific expression analysis was carried out on human cblC fibroblasts with compound heterozygous mutations. Increased transcript levels were consistently observed from the c.394C>T allele when compared to the c.271dupA and c.331C>T alleles. Understanding the mechanisms underlying differential MMACHC transcript levels may provide a clue as to why individuals carrying the c.394C>T mutation generally present later in life.

106 11th International Congress of Inborn Errors of Metabolism

Mutations in TCblR, the gene for the transcobalamin receptor, result in

decreased cellular uptake of vitamin B12 and methylmalonic aciduria.

Natascia Anastasio1,2, David Watkins1,2, Lydia Vezina1,2, Lara Reichman1,2,

Laura Dempsey-Nunez1,2, Edward V. Quadros3 and David S. Rosenblatt1,2 1Department of Medical Genetics, McGill University Health Centre, Montreal, Quebec, 2Department of Human Genetics, McGill University, Montreal, Quebec, 3Department of Medicine and Cell Biology, State University of New York Downstate Medical Centre, Brooklyn, New York

Vitamin B12 (cobalamin) is necessary for the conversion of homocysteine to methionine and of methylmalonyl CoA to succinyl CoA. Cells take up circulating cobalamin bound to the transport protein transcobalamin (TC) by endocytosis, mediated by the transcobalamin-cobalamin receptor (TCblR). This 282 amino acid highly glycosylated receptor has a single 12 amino acid transmembrane region, a cytoplasmic tail of 22 amino acids and an extracellular domain of 200 amino acids, which contains two LDLR type A domains separated by a cysteine rich CUB domain.

The recently discovered gene encoding TCblR (CD320), located on chromosome 19p13.2, spans a region of 6.224kb and contains five exons. Decreased uptake of the TC-bound cobalamin was identified in cultured skin fibroblasts from six unrelated patients referred to our laboratory because of a positive newborn screening test for methylmalonic aciduria. No other defects in cellular cobalamin metabolism were detected. A 3-base pair deletion resulting in loss of a glutamic acid residue (c.262_264delGAG, p.E88del) was identified in homozygous form in four patients and in heterozygous form in two others. A second mutation (c.297delA, p.Q99HfsX33) was identified in one of the heterozygotes. The c.262_264delGAG mutation appeared on a single haplotype, suggesting an ancestral mutational event. Thus, patients with a positive newborn screen for methylmalonic aciduria and low cobalamin uptake by cultured fibroblasts should be tested for mutations in the TCblR gene. The clinical significance of these mutations in long-term follow up remains to be determined, as all patients were picked up by newborn screening.

107 APPENDIX C Published Article

Human Mutation, 30(7): 1072-1081 July 2009

Spectrum of Mutations in MMACHC, Allelic Expression, and Evidence for Genotype–Phenotype Correlations

Jordan P. Lerner-Ellis1,4,*, Natascia Anastasio1,2,* , Junhui Liu1,2 , David Coelho4, Terttu Suormala4 , Martin Stucki5,6 , Amanda D. Loewy1,2 , Scott Gurd2,3, Elin Grundberg2,3, Chantal F. Morel7 , David Watkins1,2 Matthias R. Baumgartner1,3, Tomi Pastinen1–3 , David S. Rosenblatt1,2 and Brian Fowler4

1Department of Medical Genetics, McGill University Health Centre, Montreal, Quebec, Canada; 2Department of Human Genetics, McGill University, Montreal, Quebec, Canada; 3McGill University and Genome Quebec Innovation Centre, Montreal, Quebec, Canada; 4Metabolic Unit, University Children’s Hospital, Basel, Switzerland; 5Division of Metabolism and Molecular Pediatrics, University Children’s Hospital, Zurich, Switzerland; 6Zurich Center for Integrative Human Physiology, University of Zurich, Zurich, Switzerland; 7Department of Medicine, University Health Network, University of Toronto, Toronto, Ontario, Canada * These authors contributed equally to this work

Thesis Author’s Contribution: Natascia Anastasio performed all experiments in article sections “Allelic Expression Studies” and “Quantitative Real-Time RT-PCR”. N. Anastasio also contributed to writing and editing of article.

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