Hedgehog Signaling Modulates Cholesterol Homeostasis in Chondrocytes and in Osteoarthritis

HEDGEHOG SIGNALING MODULATES CHOLESTEROL HOMEOSTASIS IN CHONDROCYTES AND IN OSTEOARTHRITIS

Shabana Amanda Ali

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Institute of Medical Science University of Toronto © Copyright by Shabana Amanda Ali 2014

HEDGEHOG SIGNALING MODULATES CHOLESTEROL HOMEOSTASIS IN CHONDROCYTES AND IN OSTEOARTHRITIS Shabana Amanda Ali Doctor of Philosophy Institute of Medical Science University of Toronto 2014

Abstract

Osteoarthritis (OA) is a common degenerative disease of the joint that is characterized by degradation and calcification of articular cartilage, and subchondral bone changes. Hedgehog (Hh) signaling is known to be activated in human and murine

OA. Since Hh signaling regulates Gli‐mediated gene expression, we identified Hh target genes that are expressed in chondrocytes. Microarray analyses were performed to detect changes in gene expression when the Hh pathway was modulated in human OA cartilage samples. Results from the Affymetrix Human Gene 1.0 ST microarray were analyzed for differentially expressed genes from three patient samples. Using

Ingenuity® Pathway analysis, several genes known to be involved in sterol homeostasis were found to be modulated with Hh inhibition. We hypothesized that Hh signaling regulates cholesterol biosynthesis in chondrocytes, and that modulating cholesterol homeostasis impacts the severity of OA.

To investigate the function of cholesterol in the cartilage, mice with chondrocyte‐specific cholesterol accumulation were generated. This was achieved by excising Insig1 and Insig2, major negative regulators of cholesterol homeostasis. Over time, mice with chondrocyte‐specific cholesterol accumulation exhibited impaired growth of the long bones. With aging or surgically induced joint instability, these mice

developed more severe OA than control littermates. They expressed typical OA

markers, such as type X collagen, indicating chondrocyte hypertrophy in the articular

cartilage. Genetic manipulation of Hh signaling in these mice suggests that Hh signaling

is modulating the phenotype by regulating sterol homeostasis. Hh reduction and statin

treatment both lowered cholesterol production and rescued the phenotype, reducing

the severity of OA. Here we identified novel Hh target genes in chondrocytes which

regulate intracellular cholesterol levels, and found that cholesterol dysregulation in the

chondrocytes predisposes to OA. These data suggest that pharmacologic correction of

intra‐articular sterol imbalance can be used as a treatment for osteoarthritis.

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Acknowledgements

For taking a chance on me, I thank my supervisor Dr. Benjamin Alman. His vision saw beyond the limited scientific experience I brought to the lab, and his support afforded me the training environment to flourish as a scientist. To my past and present lab mates, I am forever indebted for the patience, assistance, and inspiration provided to me. For their scientific guidance and/or experimental assistance I thank Dr. Louisa

Ho, Dr. Claire Hsu, Dr. ZhuJuan (Sue) Li, Dr. Jason Rockel, Dr. Saeid Amini‐Nik, Dr.

Farasat Zaman, Dr. Alvin Lin, Mushriq Al‐Jazrawe, Heather Whetstone, Raymond Poon,

Henry Ma, and Puviindran Nadesan. For his contributions, both significant (help with mathematical calculations) and trivial (changing my outlook on life), I thank my desk‐ mate Dr. Gurpreet Baht.

I am thankful for the scientific expertise offered by my supervisory committee,

Dr. Christopher McCulloch, Dr. Khosrow Adeli, and Dr. Jane Aubin. These individuals were critical in shaping the direction of this project. The participation of my esteemed thesis examination committee, Dr. Frank Beier, Dr. Mohit Kapoor, and Dr. Dominic Ng, is greatly appreciated. For his participation in both my reclassification exam and my final committee meeting, I thank Dr. Chi‐Chung Hui. His advice, both scientific and personal, was instrumental to my graduate experience. He is a dedicated mentor and I am grateful for his investment in me.

Without collaboration this work would not be possible. I am thankful to Dr.

Khosrow Adeli, Dr. Philip Connelly, Dr. Carolyn Cummins and their respective laboratories for providing experimental guidance, and to Dr. David Backstein at Mount

Sinai Hospital (Toronto, Canada) for granting access to human cartilage specimens.

For making me the person I am today, I thank my grandparents, my parents, my brother, and all of my good friends along the way. My grandmother who raised me during the early years taught me my first, and arguably most important, life lessons. Her struggle with osteoarthritis motivated me to pursue this field of research. If my small contribution makes a difference, it is for her.

My parents worked tirelessly to provide me with support of all kinds. My mother showed me the meaning of strength, of hard work, of unconditional love. My father encouraged my curiosity, gave me a sense of worth, and instilled in me the importance of education. Together, my parents created a life for me that they themselves could not have. Despite the room for improvement that remains (of which you’re always reminding me), I hope I have made you proud.

My big little brother Jordan is constantly pushing me to be better, leading me with his own incredible example. I know that he will always be there for me, because he has always been there for me. I did the thing which I thought I could not do; time to set new goals. My best friend Natalie is a perpetual source of inspiration, leading me with her own incredible example. She sees the best in me, and firmly reminds me when I forget. These two give me the perfect balance of tough and unconditional love, adjusted accordingly on a daily basis, at their wise and trusted discretion. To them I owe my sanity (a state that is constantly challenged in graduate school).

These words only begin to express my gratitude.

I share this accomplishment with each of you.

Contributions

Under the supervision of Dr. Benjamin Alman, I designed experiments,

interpreted results, and wrote the manuscripts resulting from the work described here.

The results described in Chapter Two, as partly published in Analytical Biochemistry

(Ali and Alman 2012), were acquired independently. The results described in Chapters

Three and Four, the manuscript for which is being prepared for publication, were

acquired with the assistance of the following individuals. Mushriq Al‐Jazrawe assisted

with histological grading, protein blots, and statistical analyses. Heather Whetstone

assisted with histological sectioning and staining. Raymond Poon and Henry Ma assisted with primary human chondrocyte culture and reporter construct experiments.

Sarah Farr and Mark Naples from the laboratory of Dr. Khosrow Adeli performed

radiotracer experiments to assess cholesterol biosynthesis. The contributions made by

these individuals are also described in‐text.

Table of Contents

Abstract ii

Acknowledgements iv

Contributions vi

List of Figures x

List of Tables xii

Chapter One: Introduction 1

Background

Osteoarthritis: Prevalence, Pathophysiology, Etiology, Burden of disease 2

Experimental models of osteoarthritis 7

Articular joints: Articular cartilage, Subchondral bone 9

Articular chondrocytes and growth plate chondrocytes 12

Hedgehog signaling and osteoarthritis 14

Gene expression analyses in osteoarthritis 17

Systemic influences in osteoarthritis 19

Cholesterol homeostasis 21

Cholesterol homeostasis and Hedgehog signaling 25

Cholesterol homeostasis and osteoarthritis 27

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Current treatment strategies in osteoarthritis: Pharmacologics, Lifestyle changes, Surgery, Cartilage restoration 30

Rationale 35

Hypothesis 36

Objectives 36

Chapter Two: Hh signaling regulates expression of cholesterol biosynthetic genes in chondrocytes 37

Summary 38

Introduction 39

Results

Optimized RNA extraction from human osteoarthritic cartilage 41

Gene expression profiling in osteoarthritic cartilage: microarray analyses 47

Identifying Hh targets: cholesterol biosynthetic genes 52

Discussion 58

Materials and Methods 62

Chapter Three: Hh signaling modulates cholesterol accumulation in chondrocytes 67

Summary 68

Introduction 69

Results

Cholesterol accumulation in chondrocytes: InsigDKO mice 72

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Hedgehog signaling regulates cholesterol biosynthesis in chondrocytes 78

Cholesterol accumulation in chondrocytes impairs growth of the long bones 81

Discussion 88

Materials and Methods 92

Chapter Four: Cholesterol modulation can alter the severity of osteoarthritis 95

Summary 96

Introduction 97

Results

Cholesterol accumulation in chondrocytes predisposes to osteoarthritis 99

Cholesterol inhibition protects against osteoarthritis in mice 105

Statin treatment reduces OA markers in human cartilage 115

Discussion 118

Materials and Methods 122

Chapter Five: Conclusions and future research 127

Future Research 130

Relevance 139

References 140

Appendix: Microarray gene list 155

List of Figures

Figure 1. Representative total knee arthroplasty sample showing features of osteoarthritis.

Figure 2. Schematic representation of articular chondrocytes and growth plate chondrocytes.

Figure 3. Schematic representation of the Hedgehog (Hh) signaling pathway.

Figure 4. Schematic representation of critical regulators of cholesterol homeostasis.

Figure 5. Representative electropherograms comparing RNA quality.

Figure 6. Hh inhibition in human articular cartilage.

Figure 7. Microarray analysis: gene filtering.

Figure 8. Heatmap of cholesterol homeostatic genes.

Figure 9. Real‐time PCR validation of cholesterol homeostatic genes.

Figure 10. Western blot of INSIG1 in human OA.

Figure 11. In silico analyses of cholesterol homeostatic genes.

Figure 12. SREBF2 chromatin immunoprecipitation.

Figure 13. Schematic of the cholesterol biosynthetic pathway.

Figure 14. Western blot of INSIG1 in InsigDKO mice.

Figure 15. Real‐time PCR of Insig1 in murine cartilage.

Figure 16. Visualizing total lipid and sterol accumulation in chondrocytes.

Figure 17. Quantifying total lipid and sterol accumulation in chondrocytes.

Figure 18. Real‐time PCR for Hh target genes in mice.

Figure 19. Western blot of INSIG1 in mice with Hh modulation.

Figure 20. Quantifying cholesterol biosynthesis in chondrocytes.

Figure 21. Gross size of InsigDKO mice.

Figure 22. Bone length in InsigDKO mice.

Figure 23. Growth plate analyses in InsigDKO mice.

Figure 24. Secondary ossification centre development in InsigDKO mice.

Figure 25. Skeletal analyses in InsigDKO mice.

Figure 26. Histologic analyses of InsigDKO cartilage.

Figure 27. Type X collagen staining of InsigDKO cartilage.

Figure 28. Radiographs of InsigDKO knees.

Figure 29. Real‐time PCR of OA markers in InsigDKO cartilage.

Figure 30. Verification of statin treatment effectiveness in the cartilage.

Figure 31. Serum cholesterol level following statin treatment.

Figure 32. Statin treatment in Control, Col2a1‐Gli2, InsigDKO, and Col2a1‐Gli2;InsigDKO mice.

Figure 33. Statin treatment in mice with surgically‐induced OA.

Figure 34. Type X collagen staining of Col2a1‐Gli2, InsigDKO, and Col2a1‐Gli2;InsigDKO cartilage.

Figure 35. Type X collagen staining of cartilage after surgically‐induced OA.

Figure 36. Real‐time PCR of OA markers after statin treatment.

Figure 37. ADAMTS5 reporter construct assay.

Figure 38. ADAMTS5 promoter analysis.

Figure 39. Working model: cholesterol homeostasis mediates Hedgehog signaling in osteoarthritis.

List of Tables

Table 1. Comparing RNA obtained from different extraction methods.

Table 2. Comparing gene expression results using RNA obtained from different extraction methods.

Table 3. Microarray analysis: top 20 gene changes.

Table 4. Ingenuity Pathway Analysis of microarray results.

Table 5. ICRS Scoring of Gli2+/‐, InsigDKO, and Gli2+/‐;InsigDKO cartilage.

Table 6. ICRS Scoring of Col2a1‐Gli2, InsigDKO, and Col2a1‐Gli2;InsigDKO cartilage.

Table 7. ICRS Scoring of cartilage after surgically‐induced OA.

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Chapter One:

Introduction

Background

Osteoarthritis (OA)

Prevalence

Currently, 10% of men and 18% of women over 60 years of age develop

osteoarthritis. Musculoskeletal disorders, of which OA is the third most prevalent, are

the second largest contributors to years lived with disability (Vos et al. 2012). In

Canada, as many as 1 in 8 experience symptoms of this disease (Bombardier et al.

2011), and the prevalence will rise as the population ages (Felson et al. 1987). By 2040,

it is expected that almost 30% of the Canadian labour force will have symptomatic

osteoarthritis (Bombardier et al. 2011). The cost associated with treatment

interventions and lost productivity creates an economic burden (Woolf and Pfleger

2003).

Risk factors for this disease can be classified into systemic factors and

biomechanical factors (Felson et al. 2000). Systemic factors include age, sex, ethnicity,

bone density, genetics, weight, menstrual history, and nutrition. Local biomechanical

factors include joint loading, injury or trauma, joint deformity, physical activity, and

muscle weakness. Despite being the most common degenerative disease of the joints,

there is currently no cure and only limited treatment options for osteoarthritis (Felson

et al. 2000; Neogi and Zhang 2013) (see Current treatment strategies in

osteoarthritis).

Pathophysiology

Osteoarthritis primarily affects the articular cartilage lining the joints and the

subchondral bone (Poole 1999). The main feature of OA is degradation and erosion of the articular cartilage. Chondrocytes are the cells responsible for building and maintaining the cartilage matrix. During OA, the articular chondrocytes undergo changes which contribute to disease pathogenesis (Sandell and Aigner 2001). The structure of the matrix changes with alterations to chondrocyte distribution and matrix components (Mainil‐Varlet et al. 2003). The cartilage becomes calcified and fibrillated,

with degradation commonly beginning at a focal lesion and progressing outward

(Figure 1). Inflammation of the synovial joint can also occur, but is usually local to the

joint (Poole 1999).

As OA severity increases, the cartilage becomes completely eroded, exposing

subchondral bone. Changes to the subchondral bone include irregularity of the contour,

increases in bone remodeling, and sclerosis (increased density/hardening of the bone)

(Sanchez et al. 2005). Osteophytes, or bone spurs, also develop and are thought to be an

attempt to restore stability to the joint (Kellgren and Lawrence 1957) (Figure 1). The

resulting discomfort, pain, stiffness, and swelling restrict physical activity for those

afflicted (Felson et al. 2000).

Any articulating joint can be affected by osteoarthritis, especially following an

injury. The most commonly affected joints include the knees, hips, hands, and spine, but

other joints such as the shoulders can also be affected (Hassett et al. 2006; Hunter and

Eckstein 2009). Osteoarthritis of the knee accounts for 83% of the total osteoarthritis

burden, affecting 250 million people globally, and almost twice as many women as men

(Vos et al. 2012).

Figure 1. Representative total knee arthroplasty sample showing features of osteoarthritis. Typical features of osteoarthritis include cartilage fibrillation, cartilage erosion, and osteophyte (bone spur) formation. Progression of this disease typically correlates with degeneration of the menisci. The medial side tends to be more severely affected.

Etiology

Because weight‐bearing joints are most susceptible, osteoarthritis has been

framed as a mechanical disease which results from load, wear‐and‐tear (with aging),

and/or instability (from trauma). Features such as flattening of the meniscus in knee

joints (Figure 1) support this notion, but mechanical factors alone cannot explain the origins of this disease since non‐weight‐bearing joints are also affected (Hassett et al.

2006). OA can be classified as idiopathic, or primary OA, when the causes are unknown,

or secondary OA, when the causes are known (Felson et al. 2000).

In addition to mechanical factors, other known contributors to OA include

mutations which compromise joint architecture or cartilage integrity (Y. Li et al. 2007).

Regarding joint architecture, mutations which result in chondrodysplasia

(compromised joint and bone development) have been shown to cause premature OA

(Patterson and Dealy 2014). Regarding cartilage integrity, mutations to type II collagen, the major structural component of the extracellular matrix of cartilage, can lead to osteoarthritis (Ala‐Kokko et al. 1990; Sahlman et al. 2004). Aside from these structural contributors, an emerging body of evidence suggests that perturbations to systemic factors may also contribute to OA pathogenesis. Regardless of the cause of osteoarthritis, the body’s inability to regenerate cartilage leads to progression in severity with age (Sudo et al. 2008). The multi‐factorial etiology of OA necessitates careful experimental modeling for scientific study of the disease.

Burden of disease

The disease burden created by osteoarthritis can be categorized into three

groups: direct costs, indirect costs, and intangible costs (Hunter et al. 2014). The direct

costs are the obvious costs associated with treatment interventions such as surgery,

pharmacologic treatment, and non‐pharmacologic treatment. Because OA is a chronic

disease, there are direct costs associated with health care provision, adverse effects of

treatment (such as revisions required following total joint replacement), and long‐term

care (see Current treatment strategies in osteoarthritis).

Easily overlooked are the indirect costs associated with OA. These typically

relate to lost or reduced productivity in the workforce or at home. Regarding the

workforce, sufferers of OA can experience reduced employment due to their limited

physical capabilities, increased absenteeism, and early retirement (Hunter et al. 2014).

Regarding household responsibilities, sufferers of OA may require assistance with daily tasks from a caregiver. Premature death becomes a risk due to a variety of contributing factors, including the ramifications of the sedentary lifestyle that is frequently adopted

(see Current treatment strategies in osteoarthritis).

Reduction in social participation begins to cross over from the indirect costs to the intangible costs associated with OA. The pain and fatigue that is commonly experienced limits activity, including social outings, and decreases quality of life

(Hunter et al. 2014). The psychosocial toll of chronic pain and limited physical ability is difficult to capture. Mobility can be equated with freedom and independence, so sufferers of OA (as well as their social network) are faced with the added mental and emotional burden which results from limited mobility and inevitable dependence.

Experimental models of osteoarthritis

To study OA, many experimental models are available, each offering unique advantages in capturing the disease. Mouse models are perhaps the most widely used, with genetic, mechanical, age‐related, and systemically‐induced OA all being reported.

Harnessing type II collagen as a tissue‐specific driver for alterations to chondrocyte gene expression has proven to be a powerful tool for investigating gene function in the cartilage (Grant et al. 2000; Grover and Roughley 2006). Mice with mutations in type II collagen itself, such as the Col2a1‐sedc mouse, develop several cartilage defects, including OA (Donahue et al. 2003).

More than one surgical model of OA has been described, with studies comparing the resulting severity from different surgical procedures (Kamekura et al. 2005).

Whether the meniscus or various ligaments are transected, the result is mechanical instability in the joint which provides a model of trauma‐ or injury‐induced OA (Janusz et al. 2002). To capture spontaneous age‐related OA, researchers have allowed mice to reach 12 months of age, and even 18 months in some cases (Miyaki et al. 2010). A 12‐ month‐old mouse is middle‐aged, estimated to be equivalent to a 42‐year‐old human

(Flurkey et al. 2007). This facilitates questions related to disease development; capturing both progressive changes (beginning in middle age, representative of early‐ stage disease) and cumulative changes (occurring in older age, representative of late‐ stage disease). Using 6‐month‐old mice can prove advantageous for investigating preventative strategies to inhibit joint changes and cartilage degradation in earlier stages of OA.

Mouse models of OA continue to evolve, with systemic models describing

cytokine‐ and diet‐induced OA (Griffin et al. 2010; Seeuws et al. 2010). In general, mice

are advantageous because they are relatively inexpensive, they are amenable to genetic

manipulation (Grover and Roughley 2006), and their knee anatomy resembles that of

other mammals (Glasson et al. 2010). However, given the differences, such as the fact

that mice are approximately 2500‐fold lighter in weight than humans (Glasson et al.

2010), alternative models have been developed to study OA. Rats, guinea pigs, rabbits, horses, and other larger animals have been used (van den Berg 2008).

The most clinically relevant model is offered by human osteoarthritic cartilage itself, which is available from total joint replacement surgeries (see Current treatment strategies in osteoarthritis). Primary human cartilage can be treated as explants to maintain the extracellular environment of the chondrocyte. This offers a major advantage over cell culture, since chondrocytes have been shown to lose their phenotype when cultured for extended periods of time (von der Mark et al. 1977).

Explants are a system for investigating whether experimental findings from animal studies are sustained in human samples. Administering pharmacologics to human samples in vitro facilitates translation of findings. Dose responses, toxicity, and efficacy of pharmacologics can all be queried safely and inexpensively prior to clinical trials.

Used together, mouse models of OA and human OA samples are complimentary for robust testing of hypotheses.

Articular joints

Articular cartilage

Articular chondrocytes are responsible for producing and maintaining the

extracellular matrix that comprises the articular cartilage. The function of the articular

cartilage is to line the ends of the bones which make up the joints, such that the joints

can articulate in a frictionless manner and absorb impact. Articular cartilage consists of

three distinct zones that are identifiable by shape of the resident chondrocytes and by

orientation of the type II collagen fibres (Figure 2). The superficial zone (Zone I)

contains flattened chondrocytes with dense, horizontal collagen fibrils and minimal

proteoglycans. The middle zone (Zone II) contains rounded chondrocytes with crossed,

intersecting collagen fibrils and more proteoglycans. The deep radial zone (Zone III)

contains spherical chondrocytes with radial collagen fibrils and abundant

proteoglycans. A tidemark can be observed microscopically and delineates the

transition from the lower radial zone that is un‐calcified (Zone IV) to the underlying

zone of calcified cartilage (Zone V) (Lyons et al. 2006; Mainil‐Varlet et al. 2003).

Figure 2. Schematic representation of articular chondrocytes and growth plate chondrocytes. The articular cartilage is divided into several zones which can be identified by articular chondrocyte shape and type II collagen fibre orientation. The growth plate chondrocytes undergo a controlled process of differentiation from resting, to proliferating, to pre‐hypertrophic, to hypertrophic. This enables elongation of the long bones.

Subchondral bone

Beneath Zone V lies the subchondral bone which has been shown to undergo OA‐

associated changes. These include irregularity of the contour, increased remodeling,

sclerosis, necrosis, and fracture (Mainil‐Varlet et al. 2003). Hypotheses have been put

forth to suggest that subchondral bone alterations can impact articular cartilage (R.

Zhang et al. 2012). For example, changes to mechanical properties such as the stiffness

of subchondral bone may reduce shock absorption and promote cartilage degeneration

(Muraoka et al. 2007). In vitro experiments show that co‐culture of subchondral osteoblasts can decrease gene expression of chondrogenic markers such as type II collagen in chondrocytes. The simultaneous shift towards hypertrophy (as seen in OA) suggests that the subchondral bone may play a role in that shift (Sanchez et al. 2005). A recent study reported that key OA‐associated genes show similar changes in the subchondral bone as they do in the articular cartilage (Chou et al. 2013). It remains to be determined whether changes to the subchondral bone precede or result from changes to the articular cartilage; nevertheless, these reports suggest that OA should be considered a total joint disease which affects multiple tissues.

Articular chondrocytes and growth plate chondrocytes

In osteoarthritis, the chondrocytes in the articular cartilage undergo characteristic changes which recapitulate the differentiation process of the chondrocytes in the growth plate cartilage (Tchetina et al. 2005). Normal bone development begins with condensation of mesenchymal cells. Endochondral

ossification proceeds under the control of signaling pathways which direct the cells to

become chondrocytes. The chondrocytes proliferate and produce a matrix rich in type II

collagen and aggrecan (Grant et al. 2000). A subset of chondrocytes then becomes

hypertrophic, producing a matrix rich in type X collagen. These hypertrophic

chondrocytes, which are also found in the articular cartilage during osteoarthritis,

regulate matrix mineralization. Blood vessels enter the developing structure, bringing

with them bone‐forming cells. Hypertrophic chondrocytes then undergo cell death,

leaving behind a matrix on which bone can be formed (Kronenberg 2003).

Through the controlled process of endochondral ossification, the growth plate architecture is established for the primary and secondary ossification centres (Figure

2). Resting chondrocytes transition into proliferating chondrocytes, which change from

a round shape to a flat shape, and organize into columns. Pre‐hypertrophic

chondrocytes modify their expression to become hypertrophic and are replaced by bone as described above (Figure 2). Developmental signaling pathways, such as the

Hedgehog pathway, tightly regulate this process and determine the rate of growth of the long bones (Goldring et al. 2006; Provot and Schipani 2005).

Several of the phenotypic changes which occur in the growth plate chondrocytes are observed in articular chondrocytes during osteoarthritis disease progression.

Recapitulating the behavior of growth plate chondrocytes, articular chondrocytes in OA

proliferate (become clonal), become hypertrophic, and form bone (osteophytes). The proliferation that replenishes the chondrocyte pool in the growth plate is hypothesized to be mimicked by the chondrocyte clonality that is seen in OA articular chondrocytes

(Sandell and Adler 1999). Articular chondrocytes have been shown to adapt an anabolic profile during OA, secreting matrix in an attempt to rebuild and compensate for the degradation that occurs. This is a shift away from the normal resting phenotype of

articular chondrocytes (Kruegel et al. 2008). Chondrocyte hypertrophy is a hallmark of

osteoarthritis, where chondrocytes become enlarged and begin expressing type X

collagen, exactly as they do in the growth plate (Girkontaite et al. 1996). Finally, the

entire process of endochondral ossification has been hypothesized to be recapitulated

in osteophyte formation (Sandell and Aigner 2001).

The similarities that can be drawn between growth plate chondrocytes and OA

articular chondrocytes suggest that there may be a common biology behind the

controlled changes that occur in both populations of cells. In support of this, markers

that are typically found in differentiating growth plate chondrocytes become expressed

in the articular cartilage during OA progression (Pfander et al. 2001). This suggests that

the genetic profile of articular chondrocytes shifts towards that of growth plate

chondrocytes (Tchetina et al. 2005). Signaling pathways, such as the Hedgehog

pathway, which govern growth plate development, also become activated in OA. As

such, these two populations of chondrocytes are inextricably linked, and information on

the biology of the growth plate can be used to inform hypotheses on the biology of OA.

Hedgehog signaling and osteoarthritis

Previous data have shown that modulation of the Hedgehog (Hh) signaling

pathway impacts the severity of osteoarthritis (Lin et al. 2009; J. Zhou et al. 2014). Of

the three extracellular Hh ligands, Indian hedgehog (Ihh) has been shown to regulate chondrocyte proliferation and hypertrophy in the growth plate of the long bones (Lai

and Mitchell 2005; Vortkamp et al. 1996). The Hh signaling cascade begins with

extracellular Hh ligand binding to the transmembrane receptor Ptch1 (Figure 3). This relieves inhibition of Smo, a second transmembrane protein. Through a mechanism that is incompletely elucidated, Smo governs processing of the Gli transcription factors

(Rockel and Alman 2011). In vertebrates, Hh signaling is mediated by three Gli transcription factors: while Gli1 is not essential for development (H. L. Park et al. 2000),

Gli2 is thought to be the main transcriptional activator and Gli3 the main transcriptional repressor (Jiang and Hui 2008; Ruiz i Altaba 1999). These transcription factors regulate the expression of Hh target genes, including Gli1, Ptch1, and Hhip

(McMahon 2000; Wang et al. 2007) (Figure 3).

Aberrant activation of Hedgehog signaling in articular chondrocytes causes them to undergo phenotypic changes that resemble growth plate chondrocyte differentiation

(Aigner et al. 2007; Mak et al. 2008). For example, changes such as chondrocyte hypertrophy are observed in OA (see Articular chondrocytes and growth plate chondrocytes). Based on the similarities in chondrocyte phenotype in the growth plate and articular cartilage during OA, Lin et al. hypothesized and ultimately demonstrated that Hh signaling was involved in mediating OA pathogenesis (Lin et al. 2009).

Figure 3. Schematic representation of the Hedgehog (Hh) signaling pathway. In the absence of Hh ligand, the transmembrane receptor Ptch represses a second transmembrane receptor Smo, resulting in pathway inhibition. In the presence of Hh ligand, repression of Smo by Ptch is relieved, and the Gli transcription factors become active, resulting in pathway activation and upregulation of target genes. PM = plasma membrane.

When OA is surgically induced in mice by medial meniscectomy, the Hh pathway becomes activated in the articular cartilage. An increase in the expression of OA‐ associated genes Adamts5, Col10a1, and Mmp13, is accompanied by Hh activation. In this model, Hh pathway blockade reduces the severity of OA, suggesting that this pathway is mediating the phenotype (J. Zhou et al. 2014). In genetically modified mice with chondrocyte‐specific Hh activation, in which Gli2 is expressed under type II collagen (Col2a1) regulatory elements (Col2a1‐Gli2), an OA phenotype is observed by 6 months (Hopyan et al. 2002). This phenotype includes typical radiographic and histologic features of OA in the knees of mutants compared to wildtype littermates. In human articular cartilage, OA severity correlates with Hh pathway activation. Severity is evidenced by upregulated expression of typical OA markers, ADAMTS5, COL10A1, and

MMP13, and accompanying Hh activation is demonstrated by an increase in expression of the Hh targets PTCH1, GLI1, and HHIP (Lin et al. 2009).

Taken together, the evidence from mouse and human studies indicate that activated Hh signaling predisposes to OA. The applications of this are evidenced in existing patents which describe the use of Ihh as a marker to predict OA and the use of

Hh inhibitors to prevent OA (Bumcrot 2005; L. Wei 2012). Despite this, the mechanisms by which Hh signaling results in OA remain unknown.

Gene expression analyses in osteoarthritis

Gene expression analyses can be used to improve our understanding of specific pathways and systems within multi‐factorial pathologies such as OA. With technologies such as real‐time polymerase chain reaction (PCR), microarray, and sequencing, our ability to assess and validate gene expression changes in a high‐throughput manner is improving (Loven et al. 2012). The utility of gene expression data is far‐reaching, as it can be used to identify mutations, dysregulated biological networks or signaling pathways, and other genetic signatures in normal and diseased tissues.

Identifying perturbations to gene expression patterns in the context of disease can elucidate underlying causes and illuminate targets for treatment. Microarray analyses have been conducted to compare normal and osteoarthritic samples such as the cartilage (Aigner et al. 2006; Karlsson et al. 2010), subchondral bone (R. Zhang et al.

2012), synovium (Kato et al. 2007), and blood (Ramos et al. 2013), but not without limitations (Loven et al. 2012). The input and output of gene expression experiments must be carefully considered to maximize the utility of the results, the input being high quality RNA, and the output being final outcomes of downstream analysis.

Gene expression profiles of the articular cartilage are commonly probed, but articular cartilage presents specific challenges to the isolation of high‐quality RNA that is sufficient for experimentation. Articular cartilage consists primarily of type II collagen and the proteoglycan aggrecan (Carney and Muir 1988; Roughley and Lee

1994). It is a largely acellular tissue, containing only 1% to 2% articular chondrocytes.

Because the proteoglycans carry a negative charge, they tend to co‐purify with RNA, and because the number of cells is limited, there is a small amount of RNA per gram of

tissue. Without proper RNA isolation techniques, there is a risk of low‐quality RNA

being used to conduct gene expression experiments, the results of which may be

inaccurate and misleading (Perez‐Novo et al. 2005).

The results that are obtained from high‐throughput gene expression screening

can be overwhelming and difficult to interpret. The value of global gene assessment can

be lost if analyses are too broad or too focused, if data are not normalized correctly, if

results are not verifiable in independent samples, and so on. Results from expression

experiments are typically filtered down to a list of genes which show changes based on

criteria set by the investigator. Because this list can contain hundreds of genes, it is

common to choose one gene (or a small subset of genes) as the focus of subsequent

analyses (Ijiri et al. 2008; H. Zhang et al. 2002). Unfortunately this strategy fails to

capture the global overview that is the advantage of high‐throughput experiments.

With evolving technologies for assessing gene expression come new platforms

for analyzing and interpreting results (Downey 2006). If gene lists are categorized

functionally into biological networks or signaling pathways, then the high‐throughput

value is maintained (Loven et al. 2012), investigators have a functional network or

pathway with which to conduct subsequent analyses (Olex et al. 2014), and targeted

treatment for pathologies can be pursued.

Systemic influences in osteoarthritis

OA has been described as a metabolic disease whereby systemic factors (such as lipids, inflammatory molecules, neuroendocrine factors) impact skeletal tissue development and integrity. Disruption to cartilage integrity leaves it susceptible to secondary factors which can induce cartilage degradation and other typical features of

OA (Aspden et al. 2001). Framing OA as a total joint disease with systemic contributors offers an explanation as to why OA affects non‐weight‐bearing joints such as the hands.

Jungmann et al. examine four metabolic risk factors, including high abdominal circumference, hypertension, high fat consumption, and self‐reported diabetes mellitus, in the context of OA. At baseline, the authors report each of these risk factors to be independently associated with cartilage degeneration, but this association was lost after the two‐year follow‐up period (Jungmann et al. 2013). Other groups have described an association between obesity and OA, not only for the increase in mechanical load to the joints, but also for the increase in factors that induce inflammation (Karvonen‐Gutierrez et al. 2013). It is thought that excessive adipose tissue serves as a source of inflammatory factors which can induce cartilage degradation (Griffin et al. 2010; Issa and Griffin 2012). These results point to a relationship between systemic metabolism and OA, but the mechanism and causality remain unclear. This problem is common with epidemiological studies (Al‐Arfaj 2003;

Sturmer et al. 1998).

Studies which examine metabolic factors tend to assay serum levels of markers

such as cytokines or sterols, while neglecting to examine intra‐articular levels (Al‐Arfaj

2003; Griffin et al. 2010). Gierman et al. report that a high cholesterol diet in mice

predisposes to OA development, but they report only on serum cholesterol and offer no

mechanistic explanation as to why some drugs which lower serum cholesterol are

effective at preventing OA while others are not (Gierman et al. 2014). Considering OA a

total joint disease, disruptions to cell and tissue homeostasis (in the cartilage, bone,

muscle, ligaments, joint capsule, and surrounding fat) must be considered.

Because chondrocytes are the only cell type in the cartilage and are responsible

for maintaining cartilage integrity, their homeostatic balance is of critical importance to

OA pathogenesis. OA cartilage does show altered lipid metabolism, with cholesterol and

lipid localization in the superficial zone (Cillero‐Pastor et al. 2012; Villalvilla et al.

2013). OA chondrocytes show intracellular lipid deposits and altered expression of

regulators of lipid and sterol homeostasis (Kostopoulou et al. 2012; Tsezou et al. 2010).

Furthermore, articular chondrocytes have been shown to differ from growth plate

chondrocytes in terms of lipid content and free cholesterol (Le Lous et al. 1981).

Taken together, existing literature support a role for both local and systemic factors in OA pathogenesis due to their potential impact on chondrocyte metabolism and homeostasis. Because serum levels may not always be representative of intra‐ articular levels of various factors (Prete et al. 1993), future studies should focus more closely on the intra‐articular environment (see Cholesterol homeostasis and osteoarthritis). In order to elucidate the pathophysiology of OA (the biology behind cartilage degradation) and understand the etiology of OA (why non‐weight‐bearing joints are affected), a more careful examination of contributors to chondrocyte homeostasis is required.

Cholesterol homeostasis

Cholesterol is essential to cell and tissue homeostasis. It is a major component of

mammalian cell membranes, a constituent of myelin in the nervous system, and a

precursor to hormones and other signaling molecules (Jira 2013). In the lipid bilayer of

the cell membrane, cholesterol contributes to membrane fluidity, and exists in

unesterified (“free”) form. Within the cell or tissue, cholesterol typically exists in

esterified form, bound to phospholipids (Tabas 2002).

Because dietary intake of cholesterol can vary, and insufficient or excessive

cholesterol can have deleterious effects, cholesterol biosynthesis and trafficking is

tightly controlled to maintain homeostasis. The LDL receptor (LDLR) is responsible for

cellular cholesterol uptake while the ATP‐binding cassette transporter A1 (ABCA1) is

responsible for cholesterol removal (Goedeke and Fernandez‐Hernando 2012) (Figure

4). These transporters, and other genes involved in the cholesterol biosynthetic

pathway, are regulated by mechanisms which detect intracellular cholesterol levels and

maintain them within a physiologic range.

Figure 4. Schematic representation of critical regulators of cholesterol homeostasis. Intracellular cholesterol levels are governed via cholesterol efflux (ATP‐ binding cassette transporter A1; ABCA1), cholesterol import (low‐density lipoprotein receptor; LDLR), and cholesterol biosynthesis which is regulated by INSIGs (insulin‐ induced genes), SCAP (SREBP cleavage‐activating protein), SREBFs (sterol regulatory element‐binding factors), and HMGCR (3‐hydroxy‐3methylglutaryl coenzyme A reductase). Once processed, the SREBFs translocate to the nucleus to regulate expression of cholesterol homeostatic genes via the SRE binding site. PM = plasma membrane. ER = endoplasmic reticulum.

Low intracellular cholesterol levels are detected by a protein complex consisting

of Insig, Srebf, Scap, and Hmgcr, all of which are tethered to the endoplasmic reticulum

(ER) membrane (Figure 4). Cholesterol depletion causes Scap to undock from the

complex and take Srebf to the Golgi for processing to activator forms (Brown and

Goldstein 1997; Engelking et al. 2005). Srebf proteins are transcription factors which

regulate expression of all lipid and sterol regulatory genes. Srebf1a and Srebf1c are

thought to regulate fatty acid synthesis genes and Srebf2 is thought to regulate cholesterol biosynthesis genes (Horton et al. 2003). When Srebf2 target genes are upregulated, cholesterol production in the cell ultimately increases.

When intracellular cholesterol levels rise, cholesterol binds to the sterol‐sensing domain of Scap and prevents its dissociation from the protein complex in the ER membrane. This causes nuclear Srebf2 to decrease along with transcription of its target genes, and cholesterol biosynthesis decreases (Brown and Goldstein 1997; Engelking et al. 2005). The two mammalian Insig genes, Insig1 and Insig2, function redundantly to

regulate cholesterol biosynthesis and in their absence, mice accumulate cholesterol and

triglycerides (Engelking et al. 2005; Engelking et al. 2006). In addition to tethering the

protein complex to the ER membrane, Insig has been shown to regulate the proteolytic

degradation of HMG‐CoA reductase (Hmgcr) (Sever et al. 2003). This establishes the

Insig proteins as major negative regulators of cholesterol biosynthesis.

Insig1 is regulated at both transcript and protein levels. When intracellular

levels of sterols are low and the Scap/Srebf complex dissociates from Insig, residue‐

specific ubiquitination of Insig1 targets it for proteasomal degradation (Gong et al.

2006). Processed Srebf2 translocates to the nucleus to activate transcription of

cholesterol‐related genes, including Insig1. Newly synthesized Insig1 binds the

Scap/Srebf complex and prevents further activation of the cholesterol pathway (Gong et

al. 2006). This is an important feature of the feedback‐inhibition which maintains

homeostatic levels of sterols in the cell.

The nuclear receptors LXRs (liver X receptors) act as cholesterol sensors which

also regulate levels of intracellular cholesterol. The LXRs are activated by endogenous

oxysterols which accumulate with increasing intracellular cholesterol. To prevent over‐

accumulation of cholesterol, LXR activation induces transcription of genes which are

involved in cholesterol transport and regulation, among other genes (Zhao and

Dahlman‐Wright 2010). This represents another mechanism through which cholesterol

homeostasis is maintained in the cell.

Cholesterol homeostasis and Hedgehog signaling

Multiple lines of evidence point to a relationship between cholesterol

homeostasis and Hedgehog signaling. Hh ligands have cholesterol moieties, Hh pathway proteins have sterol‐sensing domains, and defects in either Hh signaling or cholesterol homeostasis produce similar central nervous system abnormalities, facial dysmorphisms, and skeletal defects (Eaton 2008). Smith–Lemli–Opitz syndrome (SLOS) includes multiple malformation and mental retardation which is known to result from defects in cholesterol synthesis (Jira 2013). Because some of the developmental abnormalities observed in SLOS are comparable to those which result from compromised Hh signaling, it has been proposed that the abnormal cholesterol metabolism of SLOS impairs Hh signaling (Koide et al. 2006). In support of this, Cooper et al. show that insufficient levels of intracellular sterol can diminish response to Hh signal in embryonic chick tissues (Cooper et al. 2003).

Whereas depletion of intracellular sterol has been hypothesized to diminish Hh signaling, increased intracellular sterol has been hypothesized to augment Hh signaling.

Oxysterols have been shown to regulate Hh signaling, with 25‐hydroxycholesterol activating Hh signaling in medulloblastoma cells (Corcoran and Scott 2006). Lipid rafts in the plasma membrane have been shown to concentrate Smoothened (Figure 3) and potentiate transduction of Hh signal (Shi et al. 2013). These and other studies indicate that Hh signal transduction depends on cholesterol metabolism (Stottmann et al. 2011).

Cholesterol has been hypothesized to play a role in Hh ligand trafficking, including secretion from the cell and distribution across the tissue (Eaton 2008). In the growth plate, Hh ligand is released by pre‐hypertrophic chondrocytes but signals

distally to regulate the rate of chondrocyte differentiation (Vortkamp et al. 1996).

Modulation of sterol level has been shown to cause altered growth plate phenotypes,

which could potentially arise from impaired Hh ligand trafficking. Impaired growth of

the long bones, as described to result from cholesterol depletion (Gofflot et al. 2003; S.

Wu and De Luca 2004), has also been shown to result from decreasing Hh signaling

(Kobayashi et al. 2005). While these overlapping phenotypes in SLOS and the growth

plate highlight the interdependency between Hh signaling and sterol homeostasis, the

exact regulatory relationship remains unclear.

Cholesterol homeostasis and osteoarthritis

The effects of disrupted cholesterol homeostasis are commonly considered in the context of cardiovascular diseases (Goedeke and Fernandez‐Hernando 2012; Ng and Hegele 1993), but the relative importance of cholesterol and its functions to cell homeostasis makes perturbations to this molecule deleterious in other systems as well.

Deficiencies in cholesterol metabolism during development have been reported to result in a variety of human disorders, including skeletal defects, behavioural deficits, and Smith‐Lemli‐Opitz syndrome (SLOS; described in Hedgehog signaling and cholesterol homeostasis), among others (Jira 2013; Porter 2003).

Regarding skeletal development, disruption to cholesterol equilibrium in the cartilage may have direct or indirect effects on joint architecture, chondrocyte metabolism, matrix synthesis or degradation, bone remodeling, and signaling pathways

(Girkontaite et al. 1996; Ijiri et al. 2008; Kanbe et al. 2006; Woods et al. 2009; H. Zhang et al. 2002). In rats treated with pharmacologic cholesterol inhibitors, impaired development (Gofflot et al. 2003) and growth of the long bones (S. Wu and De Luca

2004) have been reported. Aberrant limb patterning (Gofflot et al. 2003) and reductions in growth plate chondrocyte proliferation and hypertrophy (S. Wu and De Luca 2004) are hypothesized to be caused by impaired Hedgehog signaling resulting from cholesterol deficiency. Such perturbations to normal chondrocyte behaviour may contribute to OA development.

To date, studies examining the relationship between sterol profiles and the occurrence of OA in humans have been largely epidemiological, with confounding variables making results difficult to interpret (Al‐Arfaj 2003; Sturmer et al. 1998). Al‐

Arfaj shows an association between increased serum cholesterol levels and

osteoarthritis which persists when adjusted for potential confounding variables including age, sex, body mass index, serum uric acid, and serum triglycerides (Al‐Arfaj

2003). This study is limited by the relatively small sample size (246 patients), the short duration (7 months) for a chronic disease, and the oversight of other potential confounding variables (activity level, diet, ethnicity, and so on). Other epidemiological studies show a similar positive relationship between systemic sterol levels and OA but are subject to the same confounds (Sturmer et al. 1998).

A recent study profiled phospholipids in human synovial fluid (a viscous fluid

filling the articular joint, thought primarily to provide lubrication). Kosinska et al. used

mass spectrometry to identify lipid species in the synovial fluid and showed significant

differences between controls, early OA, and late OA. The authors speculate that

alterations to phospholipid profiles may affect joint lubrication, scavenging of reactive

oxygen species, or inflammatory processes, all of which can contribute to OA

pathogenesis (Kosinska et al. 2013). With respect to the articular cartilage, reports of

cholesterol localization to the superficial zone have been made (Cillero‐Pastor et al.

2012), and comparisons of candidate lipids between growth plate and articular

chondrocytes have been made (Le Lous et al. 1981), but no complete profiles of sterol

or lipid species in osteoarthritic chondrocytes have been made.

Statins are widely used drugs which inhibit HMG‐CoA reductase and lower

cholesterol production. Based on epidemiological studies associating elevated

cholesterol and osteoarthritis, statins have been suggested as a potential therapeutic

intervention for OA (Baker et al. 2011). The major limitation of these studies is the

assumption that systemic sterol levels are representative of intra‐articular sterol levels.

Prete et al. compiled data showing that total cholesterol levels of control patients

ranged from 148‐269 mg/dL in the plasma to 7‐8 mg/dL in the synovial fluid, while that

of OA patients ranged from 127‐252 mg/dL in the plasma to 4‐169 mg/dL in the

synovial fluid (Prete et al. 1993). This suggests that any pharmacologic cholesterol

inhibitor should target the synovial joint specifically, to improve OA prognosis.

Mouse studies offer further support for targeting cholesterol intracellularly in

OA. Gierman et al. report a rescue of the OA phenotype when mice were treated with a

statin, which reduces intracellular production of cholesterol, but not with ezetimibe,

which reduces absorption of dietary cholesterol. While serum cholesterol was lowered

by both drugs, only statin (presumably by intracellular reduction of cholesterol) was

effective in attenuating OA. Unfortunately the authors do not explicitly report on the

efficacy of statin treatment in the chondrocytes (Gierman et al. 2014). In other reports,

statins have been shown to prevent cartilage degradation by reducing enzymes such as

MMP13 (Barter et al. 2010), and promote anabolism by inducing expression of collagen

type II and aggrecan (Simopoulou et al. 2010). These findings point to a role for

cholesterol homeostasis in OA pathogenesis.

Current treatment strategies in osteoarthritis

Pharmacologics

There is no cure for osteoarthritis and treatment options are limited. The three

most common interventions are pharmacologic, lifestyle changes, and surgical

(Bombardier et al. 2011). Emerging interventions include cell therapies and cartilage

replacement strategies, but these are still largely experimental. Analgesics and anti‐

inflammatory drugs are commonly prescribed to patients suffering from the chronic

pain associated with OA, but the side‐effects caused by long‐term use of these drugs can

prove to be detrimental.

Formulations of glucosamine have been investigated for their putative benefits

in cartilage maintenance, but inconsistent results from different studies make

conclusions difficult to draw (Altman et al. 2006; Kapoor et al. 2012; Uitterlinden et al.

2007). Illustrating this point, Vlad et al. found the heterogeneity among glucosamine

trials to be larger than could be expected by chance alone. The authors speculate that

the differences in effect size across trials could be due to differences in glucosamine

preparations or industry bias/involvement, among other factors (Vlad et al. 2007).

Another challenge is whether oral pharmacologics can reach the synovial joint and be

effective, or whether local administration via intra‐articular injection directly into the

synovial joint is required. These are important considerations for any pharmacological

treatment of OA.

Lifestyle changes

Obesity is among the top modifiable risk factors for OA development. Although

commonly thought to increase load and exacerbate wear‐and‐tear on the joints, obesity

has also been shown to correlate with OA symptoms in non‐weight‐bearing joints

(Griffin et al. 2010) (see Articular joints). Weight management can help reduce symptoms of OA by reducing inflammation and alleviating load on the joints

(Bombardier et al. 2011; Messier et al. 2013b). Weight reduction has been shown to

have a dose‐response effect in alleviating symptoms of knee OA. Messier et al. followed

patients over an 18‐month period with combination diet and exercise interventions

(Messier et al. 2013b). “High” weight loss consisted of 32.5% to 10.1%, “medium” 9.9%

to 5.0%, and “low” 4.9% to 9.9% body weight. For outcomes including knee

compressive force, inflammation, pain, and function, participants in the “high” weight

loss category had significantly less joint load, reduced systemic inflammation and pain,

and improved function (Messier et al. 2013b).

Lifestyle changes which promote weight reduction such as dietary restriction

and regular exercise have been shown to improve OA outcomes (Hunter and Eckstein

2009; Messier et al. 2013b). Recommending exercise to a population of patients with

restricted physical mobility may seem counterintuitive and even detrimental, but

studies indicate that moderate exercise can improve OA outcomes, while excessive

exercise can exacerbate outcomes (Galois et al. 2004). A major consideration regarding

exercise is the frequency, duration, and type (aerobic vs. strength) recommended.

Different regulatory bodies make different recommendations. For example, the

World Health Organization (WHO) recommends 30 minutes of moderate physical

activity 5 days per week, while the Osteoarthritis Research Society International

(OARSI) guidelines recommend referral to a physical therapist and subsequent “regular

exercise” including aerobic, strengthening, and range of motion exercises (Iversen

2010). These guidelines are ambiguous at best, since “strengthening” exercises can

refer to a variety of exercises. Current clinical trials seek to assess the effects of

localized strength training to the thigh muscle specifically, in an attempt to reduce pain

and improve mobility (Messier et al. 2013a). Studies of this nature are required to

elucidate a specific dose of exercise that physicians can prescribe to OA patients.

Despite the known benefits of exercise, patients suffering from late‐stage OA

may find physical activity prohibitive due to pain, stiffness, and swelling in the joints. As

such, weight gain may be both a cause and result of OA, since sufferers become

sedentary. Although people do not die from OA directly, they can experience premature

mortality from OA indirectly, due to the sedentary lifestyle they adopt. Rahman et al.

show that patients with OA had a higher risk of cardiovascular disease (Rahman et al.

2013). While metabolic perturbations may be at play, physical inactivity may also

contribute. For these reasons, those who are able should pursue weight loss through

diet and exercise to reduce OA symptoms.

Surgery

Despite some arthroscopic interventions showing minimal results in alleviating

pain (Moseley et al. 2002), surgery is still commonly prescribed to patients who suffer

from disabling OA (Bombardier et al. 2011). Joint replacement surgeries are most

frequently performed on the knee and hip, but can also be done for the shoulder and

ankle (Proffen et al. 2013). In the United States between 1991 and 2010, the volume of

annual total knee replacement surgeries increased 162%. Patients are typically white

(~90%) and female (~65%), with a mean age of 74 (Cram et al. 2012). This option is

drastic and invasive, coming with all the complications of surgery in addition to such risks as joint infection, thrombosis and embolism, and mechanical failure of implants resulting in dislocation or even bone fracture (Proffen et al. 2013).

Although implant survival rates can reach 85‐95% even after 10‐15 years

(Proffen et al. 2013), a significant proportion of patients are unsatisfied with post‐

operative results and many require hospital readmission or surgical revision (Cram et

al. 2012; Liddle et al. 2013). Total joint replacements are restricted to patients with

advanced disease (Liddle et al. 2013), making this expensive procedure one that can

only offer relief after years of suffering (Bombardier et al. 2011). With our aging

population, the demand for joint replacement surgeries will continue to rise, especially

if improved preventative measures are not sought (Carr et al. 2012).

Cartilage restoration

Still in experimentation are more controversial treatment strategies for OA

which include mechanical therapies, cartilage restoration, and various pharmacologics.

Brighton et al. describe delivery of a specific electrical signal to the articular cartilage to

stimulate anabolic activity and reduce protease expression (Brighton et al. 2008).

Protease inhibitors have been met with challenges, but are still in development, especially against MMP13 and ADAMTS5, the two major proteases known to mediate

cartilage degradation in OA. For a full review of potential disease‐modifying

osteoarthritis drugs, see Abramson et al., 2006 (Abramson et al. 2006).

Cartilage grafts and cell‐based therapies are becoming more feasible as our

understanding of cartilage biology improves (Craft et al. 2013). This improved

understanding also facilitates development of pharmacologics which target specific

biological pathways. The discovery that Hh modulation can alter the severity of OA (Lin

et al. 2009; J. Zhou et al. 2014) has led to patents describing Hh inhibitors for the

treatment of OA (Bumcrot 2005; L. Wei 2012) (see Hedgehog signaling and osteoarthritis). The observation that lipid and sterol imbalance correlates with OA severity (Al‐Arfaj 2003; Aspden et al. 2001) has led to patents describing statins for the

treatment of OA (M. L. Ho et al. 2013) (see Cholesterol homeostasis). Current

treatment options are clearly inadequate, but advances are being made as our

understanding of the pathophysiology of OA improves.

Rationale

Hedgehog signaling has been shown impact OA severity but the mechanisms by

which this occurs remain unknown (Lin et al. 2009; J. Zhou et al. 2014). Given the

technologies available for studying gene expression in a high‐throughput manner

(Loven et al. 2012), identifying Hh target genes in OA cartilage is a viable strategy for

elucidating those unknown mechanisms. Using human cartilage samples that are

available from total knee replacement surgeries, I will identify Hh signaling target genes

in osteoarthritic chondrocytes. By grouping those target genes functionally, I will

elucidate biological networks that are regulated by Hh signaling. Using genetically

modified mice, I will investigate the importance of a chosen biological network in

chondrocyte homeostasis, and assess the effects in the growth plate cartilage and the

articular cartilage. Finally, I will target the biological network pharmacologically in both

mouse and human samples, to determine whether modulation can alter the severity of

osteoarthritis.

Hypothesis

Hh signaling regulates cholesterol biosynthesis in chondrocytes, and modulating

cholesterol homeostasis can impact the severity of OA.

Objectives

1. Identify Hh signaling target genes in chondrocytes.

2. Determine whether modulation of Hh signaling target genes can impact chondrocyte

homeostasis.

3. Assess the impact of Hh signaling target gene modulation on the severity of

osteoarthritis.

Chapter Two:

Hh signaling regulates expression of cholesterol biosynthetic genes in chondrocytes

RNA extraction from human articular cartilage by chondrocyte isolation

(Ali and Alman 2012)

Published in Analytical Biochemistry

Summary

Gene expression analyses can be used to elucidate the role of signaling

pathways, such as the Hh signaling pathway, in osteoarthritis. These studies require

sufficient quantities of high‐quality RNA, and functional analysis of results to uncover

the biological relevance. Here we describe an optimized method for RNA extraction

from human articular cartilage. Chondrocytes are isolated from the extracellular matrix and modifications are made to the traditional TRIzol® protocol, including two RNA–

DNA–protein phase separations. With the optimized method described, RNA recovery increased by approximately 1 µg per 100 mg of cartilage, and RNA integrity number

(RIN) improved from 2.0 to 7.5. Using this method, RNA was isolated from three human

OA cartilage samples which were treated with a pharmacological Hh inhibitor to

modulate the Hh signaling pathway. After being tested for effective Hh inhibition, these

samples were subjected to the Affymetrix Human Gene 1.0 ST microarray. Using

Ingenuity® Pathway analysis to capture global changes in gene networks, several genes

known to be involved in sterol homeostasis were found to be dysregulated with Hh

inhibition. Independent human cartilage samples were used to verify Hh‐mediated

changes in expression to cholesterol biosynthetic genes. A potential mechanism behind

this relationship is identified, whereby Hh signaling regulates expression of SREBF2,

the major transcriptional activator of all cholesterol biosynthetic genes. Taken together,

we show that genes involved in cholesterol homeostasis are subject to regulation by Hh

signaling.

Introduction

Gene expression studies of human articular cartilage can improve our

understanding of OA, but transcriptomics require sufficient quantities of high‐quality

RNA (Geyer et al. 2009). Samples from patients undergoing total joint replacement

surgeries serve as physiologically relevant models for experimentation, but the low

cellularity and high proteoglycan content of cartilage make efficient and effective

isolation of high‐quality RNA difficult (Carney and Muir 1988; Roughley and Lee 1994).

Because total cartilage consists of only 1% to 2% chondrocytes, RNA content per gram

of tissue is limited; and because the major proteoglycan, aggrecan, is negatively

charged, RNA purity is compromised by co‐purification of protein. Existing methods for

RNA extraction from human cartilage do not succeed in addressing these specific

challenges. Some methods require specialized equipment such as micro‐

dismembranators and freezer mills, which may not be readily available in all

laboratories. These methods describe the use of columns for RNA extraction or

purification (e.g., Qiagen RNeasy columns) which improve quality but compromise yield

(Gehrsitz et al. 2001; McKenna et al. 2000). Given the value of studying gene expression

in chondrocytes, the inherent challenges in isolating RNA from cartilage, and the

limitations of current methodologies, we optimized a method for RNA extraction from

human articular cartilage (Ali and Alman 2012).

Our optimized method of RNA extraction was used to conduct a series of

microarray experiments in order to identify Hh signaling target genes in human

osteoarthritic cartilage. Because Hh signaling has been shown to be dysregulated in

human and mouse OA (Lin et al. 2009; J. Zhou et al. 2014), we hypothesize that aberrant

expression of Hh target genes in chondrocytes promotes OA pathogenesis by disrupting

normal articular chondrocyte behaviour. To elucidate the biological networks that

could be responsible for this disruption, functional pathway analysis of microarray data was conducted (Downey 2006). Regulation of target genes by Hh signaling was verified using independent human samples that were treated with a Hh antagonist or a Hh agonist. Additionally, in silico analyses were conducted to determine whether target genes contained Gli consensus binding sites and whether target genes were dysregulated in osteoarthritic cartilage when compared to normal cartilage. The results of these experiments identify Hh target genes that are expressed in osteoarthritic cartilage.

Results

Optimized RNA extraction from human osteoarthritic cartilage

To maximize RNA yield while minimizing degradation and contamination, an optimized method for RNA extraction from human osteoarthritic cartilage was developed (Ali and Alman 2012). Samples were collected from 20 patients undergoing total knee replacement surgery for the treatment of clinically diagnosed OA. Informed consent was obtained from each patient. Articular cartilage explants were harvested and divided into groups, then incubated overnight at 37°C and 5% CO2 in culture media.

For a full description of this protocol, see Methods.

For a comparison group, some cartilage samples were subjected to the traditional TRIzol® protocol (Invitrogen, CA). These samples were frozen with liquid nitrogen, pulverized using mortar and pestle, suspended in 1 mL of TRIzol® reagent per 50 mg of cartilage, and homogenized by Polytron sonication. RNA extraction was conducted as per the TRIzol® manufacturer’s protocol. A subset of these samples was subjected to Qiagen RNeasy column purification (Qiagen, MD), according to the manufacturer’s protocol.

In Part 1 of the modified method (see Methods: Isolation of Primary

Chondrocytes), chondrocytes were isolated from the surrounding proteoglycan‐rich matrix (Goldring 2005). Briefly, cartilage explants were incubated in trypsin while in rotation with glass beads to provide physical homogenization. After being washed, collagenase A1 was applied to the explants for a 6‐hour or overnight digestion.

Following chondrocyte isolation, an average of 1.1 x 106 (±0.3 x 106, n = 5) viable cells

were obtained per gram of cartilage, as determined by the trypan blue exclusion assay.

Isolated chondrocytes were lysed with TRIzol®, transferred to cryotubes containing

smaller glass beads, and stored until further use.

In Part 2 (see Methods: RNA Extraction by Repeated Phase Separations),

RNA was extracted from the chondrocytes by modifying the TRIzol® manufacturer’s protocol (Dell'accio et al. 2008). The key modification was to the RNA–DNA–protein phase separation step. The aqueous phase containing RNA was transferred to a fresh tube, but before precipitating the RNA, an equal volume of TRIzol® was added.

Incubation and phase separation were repeated to remove residual protein and DNA without compromising RNA yield. RNA in the aqueous phase was then precipitated and resuspended in nuclease‐free water.

RNA extracted by traditional and optimized methods was compared by spectrophotometry, Bioanalyzer, and real‐time PCR. RNA quality was defined by RNA

integrity (lack of degradation) and RNA purity (absence of contaminants). The Agilent

2100 Bioanalyzer (RNA 6000 Nano LabChip, Agilent Technologies, CA) was used to

determine the RNA integrity number (RIN), a measure of RNA degradation (Schroeder

et al. 2006) where 0 represents the poorest quality or degraded RNA and 10 represents

the best quality or intact RNA. Integrity is visualized by electropherogram based on the

identification of 18S and 28S ribosomal RNA (rRNA) peaks/bands. The NanoDrop 1000

spectrophotometer (NanoDrop Technologies, DE) was used to determine RNA purity

and concentration. Low A260:A280 and A260:A230 ratios (<1.5) reflect RNA that is

contaminated with protein, phenol, and other organic compounds, while ratios of

approximately 2.0 reflect relatively pure RNA. In particular, low A260:A280 ratios reveal

protein contamination given that nucleic acids absorb maximally at 260 nm and

proteins at 280 nm.

Using the optimized RNA extraction protocol, 1.1 µg of RNA per 100 mg of

cartilage was recovered, whereas only 0.2 µg of RNA was recovered from the same

amount of cartilage using the traditional TRIzol® protocol with column purification

(Table 1). RNA from both methods showed reasonable A260:A280 ratios and low

A260:A230 ratios, but the optimized protocol significantly improved A260:A230 from 0.42 to

1.33 (Table 1). Additionally, RNA integrity was improved when the modified protocol

was used, with an increase in RIN from 2.0 to 7.5 (Table 1). RNA from the traditional

TRIzol® protocol was degraded, with low‐molecular‐weight contamination and

background noise (Figure 5a). RNA from the optimized protocol exhibited clearly defined peaks and bands for 18S and 28S rRNA on the electropherogram,

demonstrating its high quality (Figure 5b).

Table 1. Comparing RNA obtained from different extraction methods. In comparison with the traditional TRIzol® protocol, the modified protocol improves the quality and quantity of RNA extracted from human articular cartilage. RIN (RNA integrity number) and RNA parameters are presented with mean values in bold and the range of values in parentheses (*P < 0.05).

Figure 5. Representative electropherograms comparing RNA quality. RNA extracted using the traditional TRIzol® method coupled with Qiagen column purification (a, RIN 2.3) and RNA extracted using the modified protocol (b, RIN 8.2). Peaks and bands labeled as 18S and 28S represent rRNA. RIN = RNA integrity number.

To determine the effects of RNA quality on gene expression assays, cartilage from a representative sample was divided and RNA was extracted using the traditional

TRIzol® method and the optimized method. RNA was reverse transcribed to complementary DNA (cDNA) using Superscript II (Invitrogen) as per the manufacturer’s protocol. To show the impact of RNA quality on gene expression assays, real‐time PCR was performed for COL2A1, COL10A1, MMP13, and β‐ACTIN using

TaqMan assays from Applied Biosystems (Carlsbad, CA). Results are presented according to the comparative CT method (∆∆CT) of analysis (Livak and Schmittgen

2001). Although the RNA yields from the two methods appear to be comparable, the

value for the traditional TRIzol® method likely reflects protein contamination, as

shown by the low A260:A280 ratio (Table 2a). Results from a bicinchoninic acid (BCA)

assay reveal 0.36 ng of protein per nanogram of RNA extracted from the traditional

method, whereas no protein was detected in RNA extracted from the optimized method.

Real‐time PCR results show that the RNA isolated using the traditional method

consistently requires more amplification cycles (higher CT values) for detection of

COL2A1, COL10A1, MMP13, and β‐ACTIN transcripts, genes that are typically assayed in cartilage experiments (Table 2b). The resulting differences in ∆CT values (Table 2b)

ultimately alter ∆∆CT calculations in the determination of gene expression given that a

∆CT of 1 equals a 2‐fold change in expression (Livak and Schmittgen 2001).

Furthermore, since standard deviation shows an increase in error with higher CT values, gene expression results that are based on higher CT values are considered less

accurate (Karlen et al. 2007). This indicates that poor RNA quality limits accurate

detection of gene expression changes. Taken together, these results demonstrate that

our reported optimized method for RNA extraction from human articular cartilage

improves the yield of high‐quality RNA that is suitable for gene expression analyses.

Table 2. Comparing gene expression results using RNA obtained from different extraction methods. (a) Comparing quality and quantity of RNA isolated from the same cartilage sample using two different extraction methods. (b) CT values for genes typically assayed in cartilage experiments, COL2A1, COL10A1, and MMP13. In this representative sample, the quality of RNA [shown in (a)] impacts gene expression results by altering ∆CT values.

Gene expression profiling in osteoarthritic cartilage: microarray analyses

To elucidate the mechanisms by which Hh activation predisposes to OA (Lin et al. 2009; J. Zhou et al. 2014), we sought to identify Hh target genes in human OA cartilage using microarray analysis. With the consent of each patient, human OA samples were obtained from total knee arthroplasty surgeries. Articular cartilage

explants were incubated overnight with the Hh antagonist N‐[(3S,5S)‐1‐(2H‐benzo[3,4‐

d]1,3‐dioxolan‐5‐ylmethyl)‐5‐(piperazinylcarbonyl)pyrrolidin‐3‐yl]‐N‐[(3‐

methoxyphenyl)methyl]‐3,3‐dimethylbutanamide (C31H42N4O5) or carrier (Chen et al.

2002; Williams et al. 2003). Our optimized protocol for RNA extraction was used to maximize the quality and quantity of RNA isolated from the cartilage, which is known to have low cell and high proteoglycan content (Ali and Alman 2012; Carney and Muir

1988; Mankin et al. 1971) (see Optimized RNA extraction from human osteoarthritic cartilage). To verify modulation, real‐time PCR was used to confirm downregulation of the known Hh target gene GLI1 in the treated group (Figure 6).

Figure 6. Hh inhibition in human articular cartilage. Inhibition of Hh signaling following treatment with a Hh antagonist is verified by downregulation of the known Hh target gene GLI1. Error bar is 95% confidence interval (n = 3; *P < 0.05).

Microarray analyses were performed to assess differential gene expression using

the Affymetrix Human Gene 1.0 ST platform (Affymetrix, CA). Unlike arrays with a 3’

bias, this array offers the advantage of assaying the whole gene transcript with probes

designed against well‐annotated exons. In addition, the target labeling protocol that is

used generates biotinylated sense DNA versus cRNA. This allows target‐probe

hybridization to occur as DNA‐DNA duplexes which have been shown to be more specific than cRNA‐DNA duplexes (Pradervand et al. 2008). Following hybridization, microarray chips were scanned using the Affymetrix GeneChip Scanner 3000

(generating CEL files), by The Centre for Applied Genomics at The Hospital for Sick

Children. Probe set data were generated (CHP files) for each of the chips using the

Affymetrix Expression Console. These data and a description of the microarray experiment have been entered into the GEO database under GSE54749.

For each of three biological replicates, data from treated samples were

normalized to data from control samples using Partek® Genomics Suite. This generated

three lists of genes (one for each of the three biological replicates) which were

differentially expressed in the treatment group over the control group. Due to the

genetic variability that is inherent to human samples, gene changes were not averaged

across datasets but rather differentially expressed genes were filtered for those which

changed in the same direction in three of three datasets (Figure 7). No additional

criteria, such as arbitrary fold‐change cut off, were used for gene filtering. A complete

list of the 511 downregulated and 1189 upregulated transcripts can be found in

Appendix: Microarray gene list.

Figure 7. Microarray analysis: gene filtering. Each colour represents a different microarray dataset after identifying gene changes in the treated (Media+Hh Antagonist) group over the control group (Media). Each spike represents a different gene, where inward spikes represent decreases in gene expression and outward spikes represent increases in gene expression. Differentially expressed genes were filtered for those which changed in the same direction across datasets.

A list of the top 10 upregulated genes and the top 10 downregulated genes was

generated (Table 3). The top three genes to show an increase in expression include SCD

[stearoyl‐CoA desaturase (delta‐9‐desaturase)], LDLR (low density lipoprotein

receptor), and INSIG1 (insulin induced gene 1). The top three genes to show a decrease

in expression include TGFBI (transforming growth factor, beta‐induced), MXRA5

(matrix‐remodelling associated 5), and FAM29A (family with sequence similarity 29,

member A) (Table 3).

To capture the global changes to gene expression that occurred as a result of Hh

modulation, pathway analysis was used to identify signaling pathways, molecular

networks, and biological processes that were represented in the microarray data

(Table 4). Results from Ingenuity® Pathway analysis identified the top dysregulated

networks to be lipid metabolism, and endocrine system development and function. The

top dysregulated biological functions included the inflammatory response, lipid

metabolism, and tissue morphology. Among the top dysregulated canonical pathways

were the biosynthesis of steroids, and LXR/RXR activation. The top toxicity list was

cholesterol biosynthesis (Table 4). From these analyses emerged a common theme,

genes and networks related to lipid metabolism and cholesterol biosynthesis were

dysregulated with Hh modulation.

Table 3. Microarray analysis: top 20 gene changes. The top 10 upregulated and top 10 downregulated genes in human OA chondrocytes with Hh inhibition, after filtering for genes which changed in the same direction across three datasets.

Table 4. Ingenuity Pathway Analysis of microarray results. Microarray results were analyzed in an unbiased manner for biological networks, signaling pathways, and other functional gene groups using Ingenuity Pathway Analysis. The top results for each category are shown.

Identifying Hh targets: cholesterol biosynthetic genes

Microarray analyses revealed genes involved in the cholesterol homeostatic

pathway to be among the top dysregulated with Hh pathway inhibition (Figure 8).

Figure 8. Heatmap of cholesterol homeostatic genes. Results from Affymetrix Human Gene 1.0 ST microarray of human osteoarthritic cartilage treated with Hh antagonist. Each of three pairs of samples (Control vs. Treated) were analyzed independently and filtered for genes which were either upregulated or downregulated across all three samples. Greater intensity of red represents increased gene expression with Hh inhibition (Treated).

Because cholesterol is vital for cellular processes and because its dysregulation has

been implicated in OA, we sought to assess the role of Hh signaling in regulating the

expression of cholesterol biosynthetic genes (Kostopoulou et al. 2012; Sturmer et al.

1998). Human cartilage samples were subjected to Hh modulation and assayed for

expression of HMGCR, HMGCS1, DHCR7, LDLR, ABCA1, and INSIG1. These genes, which are known to be involved in sterol homeostasis (Gill et al. 2008; Horton et al. 2003), show increased expression with Hh inhibition (evidenced by downregulation of GLI1,

Figure 9a) and decreased expression with Hh activation (evidenced by upregulation of

GLI1, Figure 9b). In addition to validating microarray results by confirming changes to

top dysregulated genes such as LDLR, INSIG1, and HMGCS1 (Table 3), these data confirm Hh‐mediated regulation of cholesterol homeostatic genes.

Figure 9. Real‐time PCR validation of cholesterol homeostatic genes. Validation of Hh regulation of cholesterol homeostatic genes identified by microarray. Independent human osteoarthritic cartilage samples were treated identically to those used in microarray analyses. Expression in the control group was arbitrarily defined as ‘1’ (dashed line) and data from the groups treated with (a) Hh antagonist and (b) Hh agonist given as the mean. Error bars are 95% confidence intervals (n = 3; *P < 0.05).

To determine whether the observed changes in gene expression translate to

changes in protein expression, Western blot analysis was performed using human OA

cartilage with Hh modulation. Results demonstrate that changes to INSIG1 translate to

the protein level, since treatment with Hh antagonist increased INSIG1 protein and

treatment with Hh agonist decreased INSIG1 protein (Figure 10). In silico analyses

were performed to determine whether cholesterol homeostatic genes contain Gli

binding sites. Mulan analyses (http://mulan.dcode.org) (Ovcharenko et al. 2005)

revealed Gli consensus binding sites that are conserved between humans and mice in

10 of the 19 genes identified by microarray (Figure 11). Analysis of publicly available

microarray data comparing normal cartilage to osteoarthritic cartilage identified 8 of

the 19 genes to be dysregulated in osteoarthritis (Karlsson et al. 2010; Kostopoulou et

al. 2012) (Figure 11).

Figure 10. Western blot of INSIG1 in human OA. Representative Western blot showing INSIG1 protein from human OA cartilage explants treated with Hh antagonist or Hh agonist. ACTIN is shown as a loading control.

Figure 11. In silico analyses of cholesterol homeostatic genes. Mulan inquiries (http://mulan.dcode.org) were conducted to examine the 5 kb promoter region of cholesterol homeostatic genes identified by microarray analyses (3 of 3 arrays) for Gli consensus sequence‐binding sites that are conserved between humans and mice (Gli site). Publicly available expression data comparing osteoarthritic cartilage to normal cartilage were filtered for changes to cholesterol biosynthetic genes (OA vs. normal). Genes with a Gli site or with dysregulated expression in OA are designated by black boxes in the respective columns.

From in silico analyses, SREBF2 was found to have conserved Gli consensus binding sites and to be dysregulated in OA (Kostopoulou et al. 2012). SREBF2 is the

major transcriptional regulator of cholesterol homeostatic genes such as INSIG1, which

negatively regulates cholesterol biosynthesis (Horton et al. 2003). When intracellular

cholesterol is high, the Insig proteins tether the transcriptional activators of cholesterol

biosynthetic genes (Srebfs) to the ER membrane, effectively preventing expression of

target genes and subsequent cholesterol synthesis (Figure 4). This end‐product feedback inhibition stipulates that when intracellular cholesterol is low, the Srebfs translocate to the nucleus to activate transcription and restore homeostasis (Engelking et al. 2005) (see Chapter One: Cholesterol homeostasis).

To confirm whether SREBF2 is targeted by Hh signaling, we performed a chromatin immunoprecipitation assay with antibodies directed against Gli1 and relevant controls. PCR results using primers designed specifically for human SREBF2 confirmed that Gli transcription factors bind to this gene (Figure 12). This suggests that

Hh signaling may be regulating expression of cholesterol homeostatic genes via Gli‐ mediated regulation of SREBF2. As a known target of SREBF2 and a critical regulator of cholesterol biosynthesis, the gene and protein changes observed to INSIG1 provides support for the role of Hh signaling in regulating cholesterol homeostatic genes.

Figure 12. SREBF2 chromatin immunoprecipitation. Chromatin from primary human OA chondrocytes was probed with anti‐Gli1, IgG (Neg), and anti‐H3 (Pos) antibodies. PCR amplification of the SREBF2 gene shows enrichment in the Gli1 fraction, with primers designed to flank the putative Gli binding site. Additional controls included primers designed adjacent to the Gli‐binding site and primers designed to amplify randomly selected DNA on the same chromosome.

Discussion

To obtain sufficient quantities of high‐quality RNA for gene expression analyses,

we have optimized a method for RNA extraction from human articular cartilage.

Because of the low cell density and high proteoglycan content of cartilage (McKenna et

al. 2000; Roughley and Lee 1994), there are many advantages to isolating chondrocytes from the extracellular matrix prior to RNA extraction: protein contamination is

minimized, homogenization is simplified, exposure to nucleases is limited, and RNA

yield is maximized. Removing the proteinaceous matrix prevents copurification of

protein with RNA, effectively improving RNA purity. Homogenization is made easy with

cell lysis in TRIzol®, which immediately protects RNA from nucleases and prevents

degradation, effectively improving RNA integrity. Finally, chondrocyte isolation allows

concentration of RNA such that the yield per sample preparation is maximized. These

improvements make chondrocyte isolation a practical strategy for ameliorating RNA

quality and yield from human cartilage.

When chondrocytes are isolated from the cartilaginous matrix, gene expression

patterns may be altered (Hayman et al. 2006). Whether chondrocyte isolation changes

gene expression could not be objectively determined here because two variables are

altered dependently: chondrocyte microenvironment and RNA quality (Ruettger et al.

2010). Hayman et al. reported that chondrocytes which underwent a 6‐hour digestion

had the fewest gene expression changes relative to native cartilage, so this supports the

use of a 6‐hour digestion (Hayman et al. 2006). Whether the changes in gene expression

they observed are a result of chondrocyte isolation or RNA quality cannot be

ascertained because the authors did not report the RNA quality of the samples

compared. This is relevant since RNA quality has been demonstrated to have an

independent effect on gene expression assays such as real‐time PCR (Perez‐Novo et al.

2005).

The explant model described here can be considered more physiologically

relevant to OA pathology than in vitro experiments with extended cell culture periods;

the extracellular matrix is preserved during experimental manipulations and is digested

just prior to RNA extraction (von der Mark et al. 1977). To preserve the effects on gene expression during chondrocyte isolation, pharmacological modulators or other effectors of gene expression can be spiked into the digestion media. Changes to gene expression that are due to the chondrocyte isolation process are equalized in all groups, removing it as an independent effector on gene expression (Hayman et al. 2006). When comparing experimental to control groups, such as Hh antagonist‐treated versus untreated groups as done here, changes in gene expression that are due to the experimental manipulation (such as Hh pathway inhibition) are identified.

For groups that prefer extracting RNA from total cartilage, traditional homogenization methods can be used, followed by multiple rounds of RNA–DNA–

protein phase separation to trap protein and DNA in the organic phase without

diminishing RNA in the aqueous phase. Dell’Accio et al. described a method that involves three phase separations using a sequential phenol–chlorophorm–isoamyl alcohol procedure with incubation and centrifugation times extending over 4 hours

(Dell'accio et al. 2008). The optimized protocol described here prescribes only two rounds of phase separation with TRIzol® reagent that can be completed in approximately 2 hours. Furthermore, Dell’Accio et al. reported a recovery rate of 5 ng

RNA per milligram of adult human articular cartilage (Dell'accio et al. 2008), but using

the same tissue with our optimized method, we were able to recover an average of 10 ng RNA per milligram of cartilage.

The optimized method described here for chondrocyte isolation and RNA extraction confers the ability to efficiently extract high‐quality RNA from human articular cartilage. This method was applied to the study of Hh signaling target genes in osteoarthritic cartilage. Following microarray analysis, global changes to gene expression were assessed, and several biological networks were identified. From these data, changes to the cholesterol biosynthetic pathway were evident. Gene changes to master regulators such as INSIG1 and SREBF2, as well as their targets (see Chapter

One: Cholesterol homeostasis), suggest that Hh signaling is involved in regulating this network.

This is the first study to identify Hh signaling as a regulator of cholesterol homeostatic genes. Several lines of evidence point to a relationship between Hedgehog signaling and cholesterol homeostasis. Hh ligands carry cholesterol moieties, Hh pathway proteins contain sterol‐sensing domains, and deficits in either Hh signaling or cholesterol homeostasis produce similar central nervous system abnormalities, facial dysmorphisms, and skeletal defects (Eaton 2008). Sterol levels have been shown to regulate Hh signaling (Corcoran and Scott 2006), and Hh signal transduction has been shown to require cholesterol metabolism (Stottmann et al. 2011). Cholesterol has been hypothesized to play a role in Hh ligand trafficking, including secretion from the cell and distribution across the tissue (Eaton 2008). The present study identifies cholesterol homeostatic genes as Hh signaling targets, supporting the possibility of a mutual

regulatory relationship between Hh signaling and cholesterol biosynthesis. While

others have investigated the role of sterols in modulating Hh signaling (Corcoran and

Scott 2006; Stottmann et al. 2011), ours is the first evidence to demonstrate the role of

Hh signaling in regulating sterol homeostasis.

As described in Chapter One, cholesterol is vital for cellular function and

perturbations in its regulation can disrupt tissue homeostasis; cholesterol and lipid

dysregulation has been implicated in OA pathogenesis; and finally, an interplay exists

between cholesterol and Hh signaling, suggesting a mutual regulatory relationship. For

these reasons, we pursued the hypothesis that cholesterol homeostatic genes are true

targets of Hh signaling. Results from real‐time PCR validation experiments (Figure 9),

Western blot experiments (Figure 10), in silico analyses (Figure 11), and chromatin

immunoprecipitation (Figure 12) all confirmed a role for Hh signaling in regulating

expression of cholesterol homeostatic genes.

We put forth regulation of SREBF2 as the mechanism through which Hh

signaling regulates cholesterol homeostasis, but other possibilities exist. For example,

LXR/RXR activation was identified by microarray analyses. Modulation of the LXR

pathway is another mechanism through which Hh signaling may impact cholesterol

homeostasis. These data suggest that Hh signaling regulates cholesterol homeostasis in

the cartilage, and that dysregulation may contribute to chondrocyte‐related

pathologies.

Materials and Methods

Isolation of Primary Chondrocytes

Human cartilage samples were obtained from patients undergoing total knee replacement surgery for clinically diagnosed osteoarthritis (mean age 64.7 years). All samples were obtained with informed consent under the approval of the Mount Sinai

Hospital Research Ethnics Board (Toronto, Canada). Articular cartilage was dissected away from subchondral bone and connective tissue. Cartilage was weighed then

washed with PBS 2‐3 times in a 10‐cm dish. A No.10 scalpel was used to finely mince

the cartilage into pieces of less than 1 mm3. For Hh modulation, Hh antagonist or Hh

agonist was added to the culture media, and cartilage was incubated at 37°C and 5%

CO2 overnight. Culture media consisted of modified DMEM (Wisent, Cat No. 319‐005‐

CL) supplemented with 50 ug/mL ascorbic acid, 0.1% BSA, 10 mM glycerol‐2‐

phosphate, and 1X penicillin‐streptomycin solution (Wisent, Cat No. 450‐201‐EL),

passed through a 0.20‐micron filter.

Cartilage pieces were transferred to 50‐mL polypropylene tubes containing 5 mL

of 5.0‐mm glass beads and 10 mL of 0.25% trypsin per gram of cartilage. With end‐to‐

end rotation, tubes were incubated for 45 min at 37°C. After washing away trypsin with

PBS, 10 mL of 1 mg/mL collagenase A1 per gram of cartilage was added to the tubes.

Digestion media consisted of modified DMEM (Wisent, Cat No. 319‐005‐CL)

supplemented with 1X antibiotic‐antimycotic solution (Wisent, Cat No. 450‐115‐EL)

and collagenase A1, passed through a 0.20‐micron filter. Hh antagonist or Hh agonist

was also added to the media, to maintain modulation of Hh signaling during digestion.

Once cartilage was 95% digested, the cell suspension was filtered through a 70‐

micron cell strainer into a fresh 50‐mL tube and centrifuged at 1000 g for 5 minutes at

room temperature. Cells were washed twice with PBS, and lysed with TRIzol® reagent

(approximately 1 mL per gram of digested cartilage). TRIzol® (with cells) was

transferred to 2‐mL polypropylene cryotubes containing 0.25 mL of 1.0‐mm glass beads

and tubes were stored at ‐80°C until RNA extraction.

RNA Extraction by Repeated Phase Separations

Isolated chondrocytes stored in TRIzol® were thawed from ‐80°C. Samples were homogenized thrice using a Mini‐BeadbeaterTM (BioSpec Products, OK) for 30 seconds

at 4200 rpm, alternating between ice. Following a 20‐minute incubation on ice, tubes

were centrifuged at 10000 g for 10 minutes at 4°C to remove cellular debris. The

supernatant was transferred to fresh 1.5‐mL polypropylene microcentrifuge tubes, and

0.2 mL chloroform per 1 mL TRIzol® was added. After shaking tubes by hand for 30

seconds and a 2‐minute incubation on ice, tubes were centrifuged at 10000 g for 12‐15

minutes at 4°C. The aqueous phase was transferred (~50% of total volume) to fresh

tubes and mixed with an equal volume of TRIzol®. The organic phases were stored at

‐80°C for future protein extraction. Following a 30‐minute incubation on ice, 0.2 mL

chloroform per 1 mL TRIzol® was added again. After shaking tubes by hand for 30 seconds and a 2‐minute incubation on ice, tubes were centrifuged at 10000 g for 12‐15 minutes at 4°C. Again, the aqueous phase was transferred (~50% of total volume) to fresh tubes, but this time mixed with an equal volume of isopropanol (0.5 mL per 1 mL

TRIzol®). Samples were incubated on ice for 5 minutes and centrifuged at 12000 g for

25‐30 minutes at 4°C. The resulting RNA pellet was washed with 1 mL of 70% or 75% ethanol per 1 mL TRIzol® and centrifuged at 7000 g for 5 minutes at 4°C. The

supernatant was discarded and the RNA pellet was dried for 5 minutes at room

temperature. To dissolve the RNA pellet, nuclease‐free water (approximately 25‐40 uL

per gram of digested cartilage) was added and samples were incubated for 5 minutes at

room temperature (vs. 55‐60°C as recommended by the TRIzol® protocol).

Microarray

The experimental design included three human osteoarthritic articular cartilage

samples treated in vitro with a Hh antagonist or control, as described above in Isolation

of Primary Chondrocytes. The Affymetrix Human Gene 1.0 ST platform was used, which is spotted with synthesized oligonucleotides designed against exons. RNA was

extracted from human chondrocytes treated with 10 μM C31H42N4O5 (Hh antagonist) or

control, as described above in RNA Extraction by Repeated Phase Separations (Ali and Alman 2012). Labeling and hybridizations were conducted by The Centre for

Applied Genomics at the Hospital for Sick Children (Toronto, Canada). Results were analyzed independently for paired samples from each of the three patients (Control1 vs.

Hh antag1, Control2 vs. Hh antag2, Control3 vs. Hh antag3) using Affymetrix Expression

Console and Partek® Genomics Suite. Differentially expressed genes were filtered for

those which were either upregulated or downregulated across all three samples.

Ingenuity® Pathway analysis was used to identify functional gene networks

represented in the microarray data. Multiple‐sequence alignment analysis

(http://mulan.dcode.org/) was used to detect conserved transcription factor binding

sites in genes of interest (Ovcharenko et al. 2005). Data have been reported in

compliance with MIAME (minimum information about a microarray experiment)

(Brazma et al. 2001), and can be accessed through the GEO database (GSE54749).

Real‐time PCR

Real‐time PCR experiments were conducted using TaqMan assays from Applied

Biosystems. Results were normalized to an endogenous control (ASNS

Hs00155888_m1, ACTB Hs99999903_m1, or GAPDH Hs99999905_m1), and analyzed according to the comparative CT method (∆∆CT). RNA quality was assessed using

COL2A1 (Hs00264051_m1), COL10A1 (Hs00166657_m1), and MMP13

(Hs00233992_m1). To determine the effectiveness of Hh antagonist [10 μM N‐[(3S,5S)‐

1‐(2H‐benzo[3,4‐d]1,3‐dioxolan‐5‐ylmethyl)‐5‐(piperazinylcarbonyl)pyrrolidin‐3‐yl]‐

N‐[(3‐methoxyphenyl)methyl]‐3,3‐dimethylbutanamide (C31H42N4O5)] and Hh agonist

[(10 μM purmorphamine, Cayman Chemical Company or Hh ligand (5 μg/ml Shh‐N,

R&D Systems)] in modulating pathway activity, levels of the Hh target gene GLI1

(Hs00171790_m1) were assayed. Microarray validation was performed using HMGCR

Hs00168352_m1, HMGCS1 Hs00940429_m1, DHCR7 Hs01023087_m1, LDLR

Hs00181192_m1, ABCA1 Hs01059118_m1, and INSIG1 Hs00175767_m1.

Statistical analyses

Real‐time PCR data analysis was conducted such that expression in the control group was arbitrarily set to 1. Data are reported as the mean with 95% confidence

intervals. Statistical analyses were conducted using the Student’s t test for two samples assuming unequal variances with alpha set to 0.05.

Western blot analysis

Human cartilage explants were treated identically as described in Isolation of

Primary Chondrocytes. Whole‐cell protein lysates from isolated chondrocytes were harvested using Reporter Lysis Buffer (Promega), according to the manufacturer’s instructions. Antibodies against INSIG1 (1:100, sc‐25124‐R, Santa Cruz) were used, with ACTIN (1:5000, A5441, Sigma) as a loading control. The signals were detected and quantified using the ChemiDoc MP Imaging System (Bio‐Rad, Hercules, CA). These experiments were conducted with the assistance of Mushriq Al‐Jazrawe.

Chromatin Immunoprecipitation (ChIP)

ChIP was performed using the ChIP‐IT kit (Active Motif) according to the manufacturer’s protocol. Briefly, primary human chondrocytes (freshly isolated from cartilage explants as described above in Isolation of Primary Chondrocytes) were fixed with 1% formaldehyde to maintain protein‐DNA binding. DNA was sheared with nine pulses at power level two using a Sonic Dismembrator (Fisher Scientific), with 15 seconds of sonication followed by 30 seconds incubation on ice. A Gli1 antibody

(AF3324, R&D Systems), negative control IgG (Active Motif) and positive control histone H3 (Active Motif) were used to immunoprecipitate DNA‐protein complexes which were purified and analyzed by PCR.

Chapter Three:

Hh signaling modulates cholesterol accumulation in chondrocytes

Cholesterol homeostasis mediates Hedgehog signaling in osteoarthritis

Shabana Amanda Ali, Mushriq Al‐Jazrawe, Heather Whetstone, Raymond Poon, Henry Ma, Sarah Farr, Mark Naples, Khosrow Adeli, Benjamin Alman

Manuscript under preparation

Summary

Results from gene expression analyses in Chapter Two suggest that Hh

signaling regulates expression of cholesterol homeostatic genes in chondrocytes.

Because several important regulators of cholesterol homeostasis were found to be

regulated by Hh signaling, the net effect on total cholesterol levels was unclear. Here we

generate mice with robust chondrocyte‐specific cholesterol accumulation. Using

genetically modified mice, we excise Insig1, the major negative regulator of cholesterol

biosynthesis, from Col2a1‐expressing cells, in a background Insig2 knockout. To

interrogate the role of Hh signaling in regulating cholesterol accumulation, we cross the

resulting progeny to mice with Hh activation (Col2a1‐Gli2) and mice with Hh reduction

(Gli2+/‐). Using assays to measure lipid accumulation and cholesterol biosynthesis, we

show that Hh activation increases cholesterol production, and Hh reduction decreases

cholesterol production. Cholesterol homeostasis is shown to be important for normal

growth of the long bones, as cholesterol accumulation results in delayed differentiation

of the growth plate chondrocytes and delayed development of the secondary

ossification centre. This is the first evidence to demonstrate a role for Hh signaling in

regulating cholesterol biosynthesis in chondrocytes.

Introduction

With evidence from gene expression analyses to support a role for Hh signaling

in regulating cholesterol homeostatic genes, we sought to determine the effect of Hh

signaling on total cholesterol levels. Because there were gene expression changes to

many regulators of cholesterol biosynthesis and homeostasis (Figure 13), the net effect of Hh modulation on total cholesterol levels was unclear. As a major negative regulator

of cholesterol biosynthesis, the inverse relationship we observed between INSIG1

expression and Hh signaling suggested that Hh reduction decreased cholesterol levels

and conversely, that Hh activation increased cholesterol levels. This is consistent with

the increase in transcription of cholesterol biosynthetic genes we observed with Hh

inhibition (Figure 8), an increase which typically occurs when intracellular cholesterol

is low in an attempt to restore homeostasis (see Cholesterol homeostasis) (Engelking

et al. 2005).

To assess the role of Hh signaling in modulating cholesterol levels and the

subsequent role of cholesterol in chondrocyte biology, a robust model with cholesterol

perturbation in chondrocytes was needed. Cholesterol homeostasis has been the

subject of many studies and as a result, there are several transgenic mouse lines which

target cholesterol homeostasis (Horton et al. 2002). Because cholesterol is so vital to

cell homeostasis, genetic manipulation of some genes has minimal effect, likely due to

functional redundancy by other genes and/or regulatory mechanisms which maintain

cholesterol levels within homeostatic range. For some cholesterol‐related genes,

ubiquitous transgenic mice exhibit lethality (Engelking et al. 2005), which again reflects

the importance of cholesterol homeostasis. Brown and Goldstein, who won the

Figure 13. Schematic of the cholesterol biosynthetic pathway. Coloured boxes signify altered gene expression with Hh inhibition, as determined by microarray experiments. Red indicates upregulation and green represents downregulation. Figure adapted from GenMAPP 2.0.

Nobel Prize in Medicine in 1985 for their work on cholesterol homeostasis, describe a

mouse with a floxed Insig1 gene and germline disruption of the Insig2 gene (Brown and

Goldstein 1996; Engelking et al. 2005; Engelking et al. 2006). When subjected to Cre‐

mediated excision of Insig1 (Nagy 2000), the resulting Insig double‐knock‐out

(InsigDKO) mice show robust accumulation of cholesterol and triglycerides in the target

tissue (Engelking et al. 2005; Engelking et al. 2006).

To determine whether sterol dysregulation plays a role in cartilage homeostasis,

we generated a chondrocyte‐specific knockout of the Insig1 gene, using the mouse line

described by Brown and Goldstein (Engelking et al. 2005; Engelking et al. 2006). To

determine the role of Hh signaling in modulating cholesterol, we used transgenic mice

which have higher (Hopyan et al. 2002) or lower (Mo et al. 1997) levels of Gli2, the

major transcriptional activator of Hh target genes, in order to activate or inhibit Hh

signaling, respectively. By crossing these mice, transgenics were generated in which Hh

signaling and cholesterol biosynthesis were modulated separately and together. This

allowed us to investigate the role of Hh signaling in cholesterol homeostasis, and to

characterize the role of cholesterol homeostasis in chondrocytes.

Results

Cholesterol accumulation in chondrocytes: InsigDKO mice

To modulate cholesterol biosynthesis in chondrocytes, we removed the major negative regulator, Insig1. Double‐knock‐out experiments have shown that the two mammalian Insig genes, Insig1 and Insig2, function redundantly to regulate sterol

biosynthesis and in their absence, mice accumulate cholesterol and triglycerides

(Engelking et al. 2005; Engelking et al. 2006). We crossed the Insig1(fl/fl);Insig2(‐/‐)

mouse line with the Col2a1‐Cre mouse line to excise Insig1 in chondrocytes (Engelking

et al. 2005; Ovchinnikov et al. 2000). To confirm Cre‐mediated excision in chondrocytes,

articular cartilage was harvested from knee joints of Insig1(‐/‐);Insig2(‐/‐) mice,

Insig1(‐/fl);Insig2(‐/‐) mice, and Insig1(fl/fl);Insig2(‐/‐) mice. Western blot analysis

revealed Insig1 protein to be reduced in Insig1(‐/‐);Insig2(‐/‐) cartilage but not Insig1(‐

/fl);Insig2(‐/‐) cartilage (Figure 14), so all subsequent analyses were performed with

double‐knock‐out Insig1(‐/‐);Insig2(‐/‐) mice (InsigDKO) and “control” littermates

[Insig1(fl/fl);Insig2(‐/‐)]. Reduction of Insig1 expression in InsigDKO cartilage was also

confirmed by real‐time PCR (Figure 15).

Figure 14. Western blot of INSIG1 in InsigDKO mice. Protein analysis for INSIG1 in murine cartilage resulting from the cross between Insig1(fl/fl);Insig2(‐/‐) and Col2a1‐ Cre. Reduction of INSIG1 was observed in Insig1(‐/‐) cartilage [Insig1(‐/‐);Insig2(‐/‐), subsequently designated InsigDKO] but not Insig1(‐/fl) cartilage [Insig1(‐/fl);Insig2(‐/‐ )], so all analyses focused on Insig1(‐/‐) and Insig1(fl/fl) cartilage [Insig1(fl/fl);Insig2(‐ /‐), subsequently designated Control]. ACTIN is shown as a loading control.

Figure 15. Real‐time PCR of Insig1 in murine cartilage. Mice with Hh reduction (Gli2+/‐), Hh activation (Col2a1‐Gli2), and/or Insig1 excision (InsigDKO) were assayed for expression of Insig1. Expression in the Control group was arbitrarily defined as ‘1’ and data from other groups given as the mean. Error bars are 95% confidence intervals (n = 3; *P < 0.05).

The role of Insig is to tether the multi‐protein complex containing the Srebf transcription factors to the ER membrane (Figure 4). In the absence of Insig, Srebf activity can be expected to increase, since it is free for processing and translocation to the nucleus (Engelking et al. 2005). The result of this is increased expression of cholesterol biosynthetic genes, and subsequent increased production of cholesterol. As a functional assay to measure total sterol and lipid accumulation in InsigDKO mice, Oil‐

Red‐O staining (Tsezou et al. 2010) was performed on primary cultured chondrocytes

(Figure 16, compare InsigDKO to Control) (Gosset et al. 2008). An increase in red staining was apparent upon visual inspection, and was also confirmed by spectrophotometric readings of alcohol‐extracted stain (Figure 17, compare InsigDKO to Control). These results verify accumulation of sterol and lipids in the chondrocytes as a result of Insig1 excision.

Figure 16. Visualizing total lipid and sterol accumulation in chondrocytes. Representative images of cultured primary chondrocytes stained with Oil‐Red‐O (red colour) to show total sterol and lipid accumulation according to genotype. Scale bar, 100 μm.

Figure 17. Quantifying total lipid and sterol accumulation in chondrocytes. Spectrophotometric quantification of alcohol‐extracted Oil‐Red‐O stain from cultured murine chondrocytes shown in Figure 16, normalized to crystal violet stain. Error bars are 95% confidence intervals (n = 3; *P < 0.05).

To address the possibility of a mutual regulatory relationship between Hh signaling and cholesterol homeostasis, InsigDKO cartilage was probed for expression of

Hh target genes. If cholesterol levels impact Hh pathway activity, then InsigDKO

chondrocytes with sterol and lipid accumulation are expected to show altered

expression of Hh target genes. Results from real‐time PCR showed no change to the Hh

target genes Gli1, Ptch1, and Hhip (Figure 18, InsigDKO). This demonstrates that Hh

pathway activity is not altered by cholesterol accumulation in the chondrocytes.

Figure 18. Real‐time PCR for Hh target genes in mice. Gene expression for Hh targets Gli1, Ptch1, and Hhip in the articular cartilage of mice with cholesterol accumulation (InsigDKO), reduction of Hh signaling (Gli2+/‐;InsigDKO), or activation of Hh signaling (Col2a1‐Gli2;InsigDKO). Expression in Control mice was arbitrarily defined as ‘1’ (dashed line) and data for each genotype given as the mean. Error bars are 95% confidence intervals (n = 3; *P < 0.05).

Hedgehog signaling regulates cholesterol biosynthesis in chondrocytes

To establish whether Hh signaling regulates expression of Insig1 in murine cartilage, we performed Western blot and real‐time PCR experiments using our transgenic mice with Hh modulation. We first confirmed effective modulation of Hh signaling in our mice by real‐time PCR for Hh target genes (Figure 18, Gli2+/‐;InsigDKO and Col2a1‐Gli2;InsigDKO). Consistent with Hh modulation in human cartilage, an inverse relationship between Hh signaling and Insig1 expression was found in murine

cartilage. Reduction of Hh signaling in Gli2+/‐ cartilage (Mo et al. 1997) increased gene

and protein expression of Insig1, while activation of Hh signaling in Col2a1‐Gli2

cartilage (Hopyan et al. 2002) decreased gene and protein expression of Insig1 (Figure

15 and Figure 19). Real‐time PCR was also used to confirm excision of Insig1 in the

compound mutants Col2a1‐Gli2;InsigDKO, and Gli2+/‐;InsigDKO (Figure 15).

Figure 19. Western blot of INSIG1 in mice with Hh modulation. Protein analysis for INSIG1 in murine cartilage with Hh reduction (Gli2+/‐) or Hh activation (Col2a1‐Gli2). ACTIN is shown as a loading control.

We investigated the effect of Hh modulation on overall sterol and lipid level by performing Oil‐Red‐O staining of chondrocytes from each mouse line. We hypothesized that Hh reduction lowers cholesterol levels, based on the observed increase in the negative regulator Insig1, and the increase in transcription of cholesterol biosynthetic genes, which is the typical response to low intracellular cholesterol. Primary chondrocyte cultures were established as previously described from each of the genetically modified mice with modulated Hh signaling and/or cholesterol biosynthesis

(Gosset et al. 2008). As predicted, Oil‐Red‐O staining for total sterol and lipid accumulation showed lower levels with Hh reduction (Gli2+/‐) and higher levels with Hh activation (Col2a1‐Gli2; Figure 16). Consistent with removal of the negative regulator

Insig1, InsigDKO chondrocytes showed lipid and sterol accumulation, and despite the absence of this regulator, Hh signaling still modulated sterol and lipid levels (Figure

16). With Hh activation (Col2a1‐Gli2;InsigDKO), accumulation increased, and with Hh reduction (Gli2+/‐;InsigDKO), accumulation decreased. These findings were apparent

upon visual inspection and were confirmed by spectrophotometric quantification

(Figure 17).

To determine whether the sterol accumulation could be attributed to cholesterol

biosynthesis, radiotracer experiments were conducted with the help of Sarah Farr and

Mark Naples in the laboratory of Dr. Khosrow Adeli. Primary chondrocyte cultures from

each of the six mouse lines were incubated with 50 µCi/mL 3H‐acetic acid sodium salt

overnight to measure cholesterogenesis from acetate. Lipid was extracted from the

chondrocytes and subjected to thin layer chromatography to separate cholesterol.

Results from this experiment were similar to those of Oil‐Red‐O, showing decreased

cholesterol synthesis with Hh reduction (Gli2+/‐), and increased cholesterol synthesis with Hh activation (Col2a1‐Gli2) and Insig1 removal (InsigDKO). Again Hh signaling had

effects independent of Insig1 (Col2a1‐Gli2;InsigDKO and Gli2+/‐;InsigDKO),

demonstrating a novel role for Hh signaling in the regulation of intracellular cholesterol

biosynthesis in chondrocytes (Figure 20).

Figure 20. Quantifying cholesterol biosynthesis in chondrocytes. Cholesterol biosynthesis as measured by 3H‐ acetic acid sodium salt incorporation in pooled primary chondrocytes from each genotype shown in Figure 16. Measured in triplicate and reported as the mean relative to Control in counts per minute (cpm). Error bars are SEM.

Cholesterol accumulation in chondrocytes impairs growth of the long bones

To begin assessing the effect of cholesterol accumulation in chondrocyte biology, gross phenotypic observations were made. Visually apparent was the smaller size of

InsigDKO mice (Figure 21). Weight measurements taken at 24 weeks and 52 weeks confirmed that InsigDKO mice were smaller in size than their control littermates

(Figure 21). These findings were consistent with those reported by Engelking et al. in

2006, who found that ubiquitous InsigDKO mice were smaller than their control littermates at 18.5 days post coitum (dpc) (Engelking et al. 2006). To determine whether this size deficiency also applied to the skeleton, measurements of the long bones were taken at 16 weeks, 24 weeks, and 52 weeks. Both the femur and tibia of

InsigDKO mice were shorter than those of control mice, at all time points (Figure 22).

These data indicate that cholesterol accumulation in the chondrocytes impairs growth of the long bones.

Figure 21. Gross size of InsigDKO mice. (a) InsigDKO mice are visibly smaller than their Control littermates at 52 weeks. (b) InsigDKO mice (grey bars) weigh significantly less than their Control littermates (black bars) at 24 weeks and 52 weeks. Error bars are 95% confidence intervals (n = 5‐9; *P < 0.05).

Figure 22. Bone length in InsigDKO mice. (a) The femur and (b) the tibia of InsigDKO mice (grey bars) are significantly shorter than those of Control mice (black bars) at 16 weeks, 24 weeks, and 52 weeks. Error bars are 95% confidence intervals (n = 10‐32; *P < 0.05).

To elucidate the cause of the growth impairment in InsigDKO mice, growth plate

analyses were conducted since the growth plate is responsible for elongation of the

long bones. Analysis of the growth plate in 17.5 dpc embryos suggests that the

proliferative zone is expanded and the hypertrophic zone is condensed in InsigDKO

mice (Figure 23). Used to mark hypertrophic chondrocytes, type X collagen staining

confirmed reduction in the hypertrophic zone of the growth plate (Figure 23). This

deficiency in chondrocyte differentiation likely contributed to the impaired growth of

the long bones in InsigDKO mice. In addition, InsigDKO mice exhibited delayed

development of the secondary ossification centre (SOC). By 1 week of age, the SOC is

apparent by radiography in control mice, but not InsigDKO mice (Figure 24). Despite this, total skeleton preparations did not show overt differences in bone (Alizaren red staining) nor cartilage (Alcian blue staining) distribution in InsigDKO mice as compared to control mice (Figure 25). These observations suggest that cholesterol accumulation in chondrocytes delays growth of the long bones by delaying growth plate chondrocyte differentiation.

Figure 23. Growth plate analyses in InsigDKO mice. The tibiae of 17.5 dpc mice were stained with an antibody against type X collagen (Col10) to identify the hypertrophic zone in the growth plate. InsigDKO mice had a shorter hypertrophic zone than Control littermates. Error bar is 95% confidence interval (n = 3; *P < 0.05).

Figure 24. Secondary ossification centre development in InsigDKO mice. InsigDKO mice showed a delay in development of the secondary ossification centre, visible by radiography at 7 days in Control mice (red arrow).

Figure 25. Skeletal analyses in InsigDKO mice. Total skeletons were stained with Alcian blue to identify cartilage and Alizaren red to identify bone. There were no overt differences between InsigDKO and Control mice at 2 days.

Discussion

There is mounting evidence to support an important role for lipid and sterol

homeostasis in chondrocyte biology and pathology. Disrupted sterol equilibrium in the

cartilage may directly or indirectly impact developmental signaling pathways, joint

architecture, chondrocyte metabolism, matrix synthesis or degradation, bone

remodeling, and other biological processes (Eaton 2008; Girkontaite et al. 1996; Ijiri et

al. 2008; Kanbe et al. 2006; Woods et al. 2009; S. Wu and De Luca 2004; H. Zhang et al.

2002). Here we generated mice with chondrocyte‐specific cholesterol accumulation in

order to study these possibilities. As the first reported mouse with cholesterol

accumulation specific to the cartilage, this is a useful tool for furthering our

understanding of cholesterol homeostasis in chondrocyte biology. Using this model, we

report two important findings pertaining to chondrocytes; first, that cholesterol

accumulation is regulated by Hh signaling, and second, that cholesterol accumulation

results in growth impairment in the long bones.

Gene expression analyses in human osteoarthritic cartilage demonstrate that Hh

signaling regulates expression of cholesterol homeostatic genes. We show that this

relationship also exists in murine cartilage, as Hh signaling altered expression of Insig1

and effectively modulated lipid accumulation and cholesterol biosynthesis. Combined

with the in silico data presented in Chapter Two showing conservation of Gli consensus

binding sites between human and mouse (Figure 11), these results suggest that Hh

signaling may have a role in regulating cholesterol biosynthesis across mammals.

Because this is the first report of Hh signaling regulating cholesterol homeostasis, the

universality of this relationship across species remains to be explored.

Our findings that Hh signaling was able to modulate cholesterol accumulation

with or without Insig1 support our initial results that Hh signaling regulates multiple

mediators of cholesterol homeostasis. Our data suggest that there are Insig1‐dependent

and Insig1‐independent mechanisms through which Hh signaling regulates cholesterol

homeostasis. We show that Hh modulation inversely altered gene and protein

expression of Insig1, and there were subsequent effects on total lipid accumulation and

cholesterol production. Because Insig is a major driver of cholesterol homeostasis, its

modulation by Hh signaling was likely mediating this effect. However, we show that Hh

modulation continued to have effects on total lipid accumulation and cholesterol

production in the absence of Insig1. This highlights the role of Hh signaling in regulating

cholesterol homeostatic genes other than Insig1, such that alterations to overall

cholesterol homeostasis can still be achieved without Insig1. In Chapter Two, we point

to SREBF2 as a likely mediator of Hh signaling on cholesterol homeostasis.

The mice examined here carry germline disruption of Insig2, whose function is

redundant to that of Insig1 in the liver. Engelking et al. have shown that loss of either

Insig1 alone or Insig2 alone in the liver results in normal levels of cholesterol and

triglycerides, and that loss of either gene does not cause an increase in expression of the

other gene (Engelking et al. 2005). Despite this, Insig2 has been shown to have unique

functions in different tissues, for example, polymorphisms in adipose tissues is

associated with obesity (Krapivner et al. 2008). Potential cartilage‐specific functions of

Insig2 remains subject for future investigation, but the germline disruption examined

here is one explanation for the weight discrepancy between Control and InsigDKO mice.

Although the role of Hh signaling in regulating cholesterol homeostasis has not

been documented, the reverse interaction has been examined by several groups (Eaton

2008; Gill et al. 2008; Riobo 2012). Hh ligands are modified with cholesterol moieties,

the role of which is unclear, but thought to be involved in Hh ligand secretion and

trafficking. In support of this, the transmembrane proteins which secrete Hh ligand

(Dispatched) and detect Hh ligand (Patched) contain sterol‐sensing domains, much like

the domains found in the cholesterol biosynthetic genes HMGCoA reductase (HMGCR)

and sterol‐regulatory‐element‐binding protein cleavage‐activating protein (SCAP)

(Eaton 2008). Taken with the current findings which identify cholesterol biosynthetic

genes as Hh signaling targets, a mutual regulatory relationship between Hh signaling

and cholesterol biosynthesis is plausible.

Our findings do not support the hypothesis that Hh signaling regulates cholesterol levels in order to regulate activity of its own pathway, as InsigDKO mice did not show alterations to Hh signaling. These findings are consistent with those of

Engelking et al., who reported no differences in gene expression levels of Shh, Smo,

Ptch1, or Gli1 in the palate tissues at 13.5 dpc in total InsigDKO embryos (Engelking et al. 2006). Because the exact relationship between sterol levels and Hh activity remains

unresolved, this interaction merits further investigation. One possibility is that the

accumulation of cholesterol and other sterol intermediates generated in InsigDKO mice are different from those which are responsible for altering Hh activity. Other groups

have described a role for specific oxysterols in regulating Hh signaling (Corcoran and

Scott 2006; Dwyer et al. 2007; Gill et al. 2008). In the current study, we do not analyze

the complete sterol profile that is generated in InsigDKO mice, rather we take a

candidate approach and quantify changes to cholesterol specifically. As such, we

conclude that cholesterol accumulation in chondrocytes does not alter Hh activity.

Because Hh target gene expression remained unaltered in our mice with

cholesterol accumulation, it is unlikely that the phenotypic changes are attributable to

perturbations in Hh signaling in the cartilage. We observed a delay in growth of the long

bones that can be attributed to impaired growth plate chondrocyte differentiation, yet

overall skeletons appeared normal. This suggests that the cholesterol accumulation in

InsigDKO mice impacted the controlled process of chondrocyte differentiation but not skeletal patterning. Impaired growth of the long bones has also been reported to result from reduced levels of cholesterol in the bones (S. Wu and De Luca 2004), so it may be that a fine balance of cholesterol is required for normal chondrocyte differentiation.

When cholesterol is lowered with statin treatment, an increase in bone formation is

observed, but results depend on the method of administration, dosage, and carrier used

(Mundy et al. 1999; J. B. Park 2009; Woods et al. 2009).

Whether the effects of cholesterol dysregulation on the growth plate are

mediated by Hh signaling, which is known to regulate growth plate chondrocyte

differentiation (Kobayashi et al. 2002), is unclear. One possibility is that cholesterol

levels alter Hh signal trafficking, ultimately impacting signal transduction and

chondrocyte differentiation. In support of this, mutants with deficits in either Hh

signaling or cholesterol homeostasis show similar skeletal defects (Eaton 2008), but the

precise interplay, if any, is subject for future study.

Materials and Methods

Genetically modified mice

We crossed Col2a1‐Cre mice (Ovchinnikov et al. 2000) with Insig1(fl/fl);Insig2(‐

/‐) mice (Engelking et al. 2005) to excise Insig1 in Col2a1‐expressing cells and generate

mice with chondrocyte‐specific (Grant et al. 2000) cholesterol accumulation. Mice

expressing Cre are referred to as InsigDKO [Insig1(‐/‐);Insig2(‐/‐);Cre] and are

compared to their Cre‐negative [Insig1(fl/fl);Insig2(‐/‐); “control”] littermates. Cre‐

mediated recombination was confirmed by real‐time PCR and Western blot analysis. To

activate Hh signalling, InsigDKO mice were crossed with Col2a1‐Gli2 mice (Hopyan et al.

2002), the progeny of which were designated Col2a1‐Gli2;InsigDKO. To reduce Hh

signalling, InsigDKO mice were crossed with Gli2zfd mice (Gli2+/‐) (Mo et al. 1997), the

progeny for which were designated Gli2+/‐;InsigDKO. All animal studies were approved

by the Toronto Centre for Phenogenomics.

Gene and protein analysis

RNA extraction was conducted using TRIzol® Reagent (Invitrogen, CA)

according to the manufacturer’s protocol. Real‐time PCR experiments were conducted using TaqMan assays from Applied Biosystems, as described in the Materials and

Methods section of Chapter Two. Results were normalized to an endogenous control

(Gapdh 4352932E or ACTB 4352933E), and analyzed according to the comparative CT method (∆∆CT). Excision or modulation of Insig1 was confirmed using

Mm00463389_m1. Hh pathway modulation was verified using Gli1 (Mm00494645_m1),

Ptch1 (Mm00436026_m1), and Hhip (Mm00469580_m1). Protein extraction was

conducted using Reporter Lysis Buffer (Promega), according to the manufacturer’s

instructions. Western blot analyses were performed as described in the Materials and

Methods section of Chapter Two, with the assistance of Mushriq Al‐Jazrawe. Whole‐cell

protein lysates from primary mouse chondrocytes were assayed using antibodies

against INSIG1 (1:100, sc‐25124‐R, Santa Cruz) and ACTIN (1:5000, A5441, Sigma).

Lipid and cholesterol measurement

Primary murine chondrocyte cultures were established using articular cartilage

from the knees of 5 day old mice (Gosset et al. 2008). Briefly, dissected cartilage was

digested overnight in collagenase to liberate chondrocytes. Cells were filtered and

cultured for 2‐3 days, until used for experimentation. For total sterol and lipid levels,

primary chondrocytes were fixed with 10% phosphate‐buffered formalin for 10

minutes. Cells were stained with Oil‐Red‐O and extracted stain was quantified by

spectrophotometry (OD 500), with readings normalized to crystal violet stain (OD 540).

For cholesterol synthesis, primary mouse chondrocytes were incubated with 50 µCi/mL

3H‐ acetic acid sodium salt overnight. Lipid extracted from the cells underwent thin

layer chromatography for separation of components, including cholesterol.

Incorporated 3H was measured in triplicate and reported as the relative change in

counts per minute (cpm).

Skeletal analyses

After sacrifice, mouse joints were harvested and fixed in 10% phosphate‐

buffered formalin for 4‐7 days, depending on age. Radiographs of joints were taken

using the Faxitron MX20 X‐ray system. Total skeletons were harvested and fixed in 95%

ethanol until stained for cartilage with Alcian blue and bone with Alizaren red. Bone

samples were decalcified in 20% EDTA (pH 8.0), dehydrated, and embedded into

paraffin for sectioning as previously described (Lin et al. 2009). For growth plate

analyses, immunohistochemistry was performed using antibodies against type X

collagen (X53, Quartett, Germany). These experiments were conducted with the

assistance of Heather Whetstone. Briefly, 5 µM serial sections were deparaffinized with

xylene, and rehydrated through an alcohol gradient (100%, 95%, 80%, 70%), ending

with water. Following digestion, endogenous peroxide activity was blocked by 3%

peroxide in methanol for 15 minutes at room temperature. Non‐specific binding was

blocked with 2% normal horse serum (Vectorlabs) in PBS for 30 minutes. Primary

antibody was incubated overnight, then biotinylated secondary antibody and

avidin‐linked peroxidase (Vectastain Universal Elite ABC kit, Vectorlabs, Burlingame,

CA) were used to detect primary antibody binding.

Statistical analyses

Values are reported as the mean and error bars represent 95% confidence

intervals. Student’s t test was used to determine statistical significance with alpha set to

0.05.

Chapter Four:

Cholesterol modulation can alter the severity of osteoarthritis

Cholesterol homeostasis mediates Hedgehog signaling in osteoarthritis

Shabana Amanda Ali, Mushriq Al‐Jazrawe, Heather Whetstone, Raymond Poon, Henry Ma, Sarah Farr, Mark Naples, Khosrow Adeli, Benjamin Alman

Manuscript under preparation

Summary

In Chapter Two and Chapter Three, we demonstrate that Hh signaling regulates cholesterol biosynthesis in chondrocytes and that dysregulation can impact growth of the long bones. Here we assess the effect of cholesterol accumulation in the articular cartilage and the resulting implications for osteoarthritis pathogenesis. Using histology, radiography, and gene expression analyses, we show that InsigDKO mice develop typical features of OA. This phenotype can be modulated by genetic reduction of Hh signaling via Gli2+/‐, which normalizes cholesterol accumulation. Markers of OA can also be reduced by pharmacologic normalization of cholesterol levels, with statin

treatment in vivo in murine cartilage, and in vitro in human cartilage. We suggest a potential mechanism of action in which statin treatment reduces expression of

ADAMTS5, the major protease responsible for cartilage degradation in OA.

Introduction

Activation of Hedgehog signaling has been shown to predispose to the

development of osteoarthritis, yet the mechanisms through which this occurs remain

unknown (Lin et al. 2009). In Chapter Two, we identify cholesterol homeostatic genes

to be targets of Hh signaling. In Chapter Three, we show that Hh signaling modulates

cholesterol accumulation in chondrocytes, with implications for growth plate

chondrocyte differentiation. Here we seek to determine whether cholesterol

accumulation in the chondrocytes predisposes to osteoarthritis development, and to

verify whether cholesterol accumulation mediates the effects of Hh signaling on

osteoarthritis development.

As described in Chapter One: Experimental models of osteoarthritis, there

are many established models of osteoarthritis which allow researchers to capture the

heterogeneity of the disease as it is observed in patient populations. Genetically‐

induced osteoarthritis has been demonstrated through modification of various genes

and pathways (Y. Li et al. 2007), including the Hh signaling pathway, in which activation

predisposes to osteoarthritis (Lin et al. 2009). Age‐related osteoarthritis is captured

when animals are allowed to reach advanced age and develop spontaneous

osteoarthritis (Miyaki et al. 2010; Poulet et al. 2014). Mechanically‐induced

osteoarthritis is captured by surgical procedures which create instability in the joint

(Kamekura et al. 2005); this models osteoarthritis that results from trauma in patients.

In the current study we use genetic modification by Insig1 excision and/or Gli2

overexpression in Col2a1‐expressing cells, aging of up to 24 weeks, and mechanical

instability by surgical excision of the medial meniscus to determine whether cholesterol

modulation in the chondrocytes can alter the severity of osteoarthritis.

To assess development of the osteoarthritic phenotype, there are established

radiographic features, histologic changes, and gene expression markers. Radiographs

are most commonly used to diagnose OA in the patient population. Indicators of OA

include subchondral bone sclerosis (thickening) and irregularity of the bone contour

(Kellgren and Lawrence 1957). Histologic analyses are typically conducted following

total joint replacement surgeries, to confirm OA diagnosis. Grading scales are used to

identify the stage and severity of OA based on predefined criteria (Glasson et al. 2010;

Mainil‐Varlet et al. 2003). Indicators of OA include cartilage fibrillation and erosion, and

chondrocyte hypertrophy. Gene changes typically include upregulated expression of the

proteases which mediate cartilage degradation, such as MMP13 and ADAMTS5 (Glasson

et al. 2005). We use these radiographic, histologic, and gene expression markers to

assess the severity of osteoarthritis in our mice with modulated cholesterol levels.

Modulation of cholesterol is achieved by cholesterol accumulation in InsigDKO mice

and Col2a1‐Gli2 mice, or by cholesterol reduction in Gli2+/‐ mice or with statin

treatment. We also assess whether pharmacologic cholesterol inhibition with statin

treatment can attenuate the expression of OA markers in human cartilage.

Results

Cholesterol accumulation in chondrocytes predisposes to osteoarthritis

To determine whether cholesterol accumulation in the cartilage impacts OA development, knees of 24‐week‐old InsigDKO mice and relevant controls were examined for typical markers of OA (Poole 1999). Methods used include histology, radiography, and gene expression analysis. Examination of the articular cartilage in

InsigDKO mice revealed alterations to subchondral bone, cartilage mineralization, irregular cell distribution, and cartilage erosion (Figure 26). This histologic evaluation was based on criteria used for scoring features of osteoarthritis, as set out by the

International Cartilage Repair Society (ICRS) (Mainil‐Varlet et al. 2003). Overall ICRS scores were reduced from 17.3 in control mice to 11.3 in InsigDKO mice (P < 0.05),

where lower scores represent more severe osteoarthritis (Mainil‐Varlet et al. 2003)

(Table 5). InsigDKO mice exhibited increased chondrocyte hypertrophy in the

superficial zone, as marked by type X collagen staining (Figure 27). Radiographic results showed cartilage mineralization as mentioned above, but also irregularity in the bone contour with changes to the subchondral bone, including sclerosis (Figure 28).

InsigDKO mice had increased expression of Mmp13 and Adamts5, proteases known to

mediate cartilage degradation (Glasson et al. 2005) (Figure 29). Although the

phenotype is not severe, it is robust, consistent with osteoarthritis development in

InsigDKO mice.

Figure 26. Histologic analyses of InsigDKO cartilage. Representative histological sections showing haematoxylin and eosin staining of the knee joints in 24‐week‐old mice. Boxes delineate magnified regions shown below to highlight changes in chondrocyte morphology and distribution. InsigDKO mice show characteristic OA changes, including cartilage erosion. Scale bar, 100 μm.

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Gli2+/-; ICRS Cartilage Scoring Scale Control Gli2+/- InsigDKO InsigDKO 1) Surface 3.0 ± 0.0 3.0 ± 0.0 2.3 ± 0.9 3.0 ± 0.0 2) Matrix 3.0 ± 0.0 2.7 ± 0.7 2.5 ± 1.0 3.0 ± 0.0 3) Cell Distribution 2.5 ± 0.6 2.0 ± 0.0 2.0 ± 0.8 2.3 ± 0.5 4) Cell Population Viability 3.0 ± 0.0 2.7 ± 0.7 2.8 ± 0.5 2.8 ± 0.5 5) Subchondral Bone 2.8 ± 0.5 3.0 ± 0.0 0.5 ± 0.6 2.5 ± 0.6 6) Cartilage Mineralization 3.0 ± 0.0 3.0 ± 0.0 1.3 ± 0.5 2.5 ± 0.6 ICRS Summary Score 17.3 16.3 11.3 16.0 P-value (U-test) P=0.190 P=0.020 P=0.178

Table 5. ICRS Scoring of Gli2+/‐, InsigDKO, and Gli2+/‐;InsigDKO cartilage. The International Cartilage Repair Society (ICRS) score was used to grade histologic sections in a blinded manner. The mean and 95% confidence interval are given for each criterion, and a summary score is provided with Mann‐Whitney U statistical analysis to determine significant differences. Knee joints from 24‐week‐old mice were graded for each genotype shown in Figure 26. Statistical analyses compared each of Gli2+/‐, InsigDKO, and Gli2+/‐;InsigDKO to Control. Only the score between InsigDKO and Control was significant (P < 0.05).

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Figure 27. Type X collagen staining of InsigDKO cartilage. Representative histological sections showing type X collagen immunohistochemistry in the articular cartilage of the femur at 24 weeks for each genotype shown in Figure 26. InsigDKO mice show increased chondrocyte hypertrophy in the superficial zone. Scale bar, 100 μm.

Figure 28. Radiographs of InsigDKO knees. Representative radiographic images of mouse knees corresponding to the genotypes shown in Figure 26, showing a lateral view at 16 weeks of age. InsigDKO mice show irregularity in the bone contour and increased subchondral sclerosis.

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Figure 29. Real‐time PCR of OA markers in InsigDKO cartilage. Gene expression of proteases known to mediate cartilage degradation in osteoarthritis, Mmp13 and Adamts5, from cartilage microdissected from the knees of 24‐week‐old mice. Expression of Control was arbitrarily defined as ‘1’, and data for each genotype given as the mean. Error bars are 95% confidence intervals (n = 4, *P < 0.05).

The characteristic OA changes observed by histology, radiography, and gene expression in InsigDKO mice were all rescued when cholesterol was lowered through genetic reduction of Hh signaling (Gli2+/‐;InsigDKO). Results in Chapter Three show

that Gli2+/‐;InsigDKO mice have cholesterol levels that are comparable to control mice, a rescue of the cholesterol accumulation seen in InsigDKO mice. Demonstrated by previous studies and confirmed here with the Gli2+/‐ mice, Hh reduction protects against

osteoarthritis development (Lin et al. 2009; J. Zhou et al. 2014), a potential mechanism

through which is by lowering cholesterol. In Gli2+/‐;InsigDKO mice, cartilage

mineralization and cartilage erosion were reduced, and the subchondral bone and cell

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distribution was comparable to that of control mice (Figure 26). Overall ICRS scores

were improved from 11.3 in InsigDKO mice to 16.0 in Gli2+/‐;InsigDKO mice, which was

not significantly different from the score of 17.3 in control mice (P < 0.05; Table 5).

Hypertrophy in the superficial zone of Gli2+/‐;InsigDKO cartilage was also reduced, as

marked by type X collagen staining (Figure 27). Radiographs were comparable to those

of control mice (Figure 28) and expression of Mmp13 and Adamts5 was reduced

(Figure 29). Taken together, these results suggest that reduction of Hh signaling in

Gli2+/‐;InsigDKO mice normalized cholesterol accumulation and attenuated the

osteoarthritis phenotype.

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Cholesterol inhibition protects against osteoarthritis in mice

To assess whether pharmacologic sterol normalization could mitigate osteoarthritis progression, mice were treated with 3mg/kg/day lovastatin, by surgical implantation of a slow‐release drug pellet (Innovative Research of America, FL). The dosage is physiologically relevant with no toxicity effects being reported (MacDonald et al. 1988; Reagan‐Shaw et al. 2008). Pellets were placed adjacent to the synovial membrane of the knee to inhibit cholesterol production for 8 weeks. We examined mice which reliably develop osteoarthritis, including mice with cholesterol accumulation

(InsigDKO), Hh activation [Col2a1‐Gli2 (Lin et al. 2009) and Col2a1‐Gli2;InsigDKO], and mechanical joint instability [medial meniscectomy surgery (Kamekura et al. 2005; Lin et al. 2009) performed on control and InsigDKO mice].

Effective modulation of the cholesterol pathway was confirmed by an increase in

HMGCR expression in the articular chondrocytes, an increase which occurs when intracellular cholesterol is low, as expected with statin treatment (Figure 30). There were no detectable alterations to systemic levels of cholesterol; serum levels remained unchanged in statin‐treated mice as compared to placebo‐treated mice after the 8‐week period (Figure 31). This suggests that the statin treatment was locally effective in the articular cartilage of the synovial joint without having a significant systemic effect.

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Figure 30. Verification of statin treatment effectiveness in the cartilage. Increased HMGCR expression is used to indicate decreased intracellular cholesterol which is the anticipated effect of statin treatment. (a) Representative histological sections showing HMGCR immunohistochemistry (brown) in the articular cartilage at 16 weeks for InsigDKO mice treated with placebo or statin. Scale bar, 50 μm. (b) Real‐time PCR of HMGCR in human osteoarthritic articular cartilage explants treated with statin. Expression in the control group was arbitrarily defined as ‘1’ and data from the statin‐ treated group given as the mean. Error bar is 95% confidence interval (n = 4; *P < 0.05).

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Figure 31. Serum cholesterol level following statin treatment. Systemic levels of cholesterol in the serum of mice that were implanted with a slow‐release Placebo or Statin pellet were measured using the Amplex Red assay. No statistically significant differences were detected after the 8‐week treatment period. Error bars are 95% confidence intervals (n = 3).

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In each mouse with osteoarthritis, statin treatment rescued the OA phenotype.

Examination of the histology revealed reduced cartilage fibrillation and erosion, with

normalized cell distribution (Figure 32 and Figure 33). There were no obvious differences in Control (Figure 32) or Sham‐operated (Figure 33) mice that were

treated with statin. In mice with an OA phenotype, statin treatment significantly improved overall ICRS scores (Table 6 and Table 7). For example, InsigDKO + Surgery mice scored 11.3 with placebo and 16.7 with statin treatment (P < 0.05), where higher scores represent less severe osteoarthritis (Mainil‐Varlet et al. 2003) (Table 7).

Furthermore, statin treatment attenuated chondrocyte hypertrophy in the superficial zone, as indicated by reduced type X collagen staining (Figure 34 and Figure 35).

These data suggest that statin treatment can protect against osteoarthritis development in mice.

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Figure 32. Statin treatment in Control, Col2a1‐Gli2, InsigDKO, and Col2a1‐ Gli2;InsigDKO mice. Representative histological sections showing haematoxylin and eosin staining of the knee joints in 16-week-old mice that were implanted with a slow-release Placebo or Statin pellet at 8 weeks of age. Statin treatment reduces features of OA. Scale bar, 100 μm.

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Figure 33. Statin treatment in mice with surgically‐induced OA. Representative histological sections showing haematoxylin and eosin staining of the knee joints in 16‐ week‐old mice that were implanted with a slow‐release Placebo or Statin pellet. Mice were subject to excision of the medial meniscus (Surgery) or sham operations (Sham) at 8 weeks of age. Statin treatment reduces features of OA. Scale bar, 100 μm.

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Col2a1-Gli2; ICRS Cartilage Scoring Col2a1-Gli2 InsigDKO InsigDKO Scale Placebo Statin Placebo Statin Placebo Statin 1) Surface 2.7 ± 0.7 3.0 ± 0.0 2.8 ± 0.5 3.0 ± 0.0 2.3 ± 0.7 3.0 ± 0.0 2) Matrix 2.7 ± 0.7 2.7 ± 0.7 2.8 ± 0.5 3.0 ± 0.0 2.0 ± 0.0 3.0 ± 0.0 3) Cell Distribution 2.0 ± 0.0 2.7 ± 0.7 2.3 ± 0.9 2.7 ± 0.7 2.0 ± 0.0 2.7 ± 0.7 4) Cell Population Viability 2.7 ± 0.7 3.0 ± 0.0 2.8 ± 0.5 3.0 ± 0.0 2.7 ± 0.7 3.0 ± 0.0 5) Subchondral Bone 2.7 ± 0.7 3.0 ± 0.0 1.5 ± 0.6 3.0 ± 0.0 2.0 ± 0.0 2.7 ± 0.7 6) Cartilage Mineralization 2.0 ± 1.1 3.0 ± 0.0 1.8 ± 0.5 3.0 ± 0.0 2.7 ± 0.7 3.0 ± 0.0 ICRS Summary Score 14.7 17.3 13.8 17.7 13.7 17.3 P-value (U-test) P=0.043 P=0.026 P=0.046

Table 6. ICRS Scoring of Col2a1‐Gli2, InsigDKO, and Col2a1‐Gli2;InsigDKO cartilage. The International Cartilage Repair Society (ICRS) score was used to grade histologic sections in a blinded manner. The mean and 95% confidence interval are given for each criterion, and a summary score is provided with Mann‐Whitney U statistical analysis to determine significant differences. Knee joints from 16‐week‐old mice were graded for genotypes shown in Figure 32. Statistical analyses compared placebo to statin treatment for each of Col2a1‐Gli2, InsigDKO, and Col2a1‐Gli2;InsigDKO. Statin treatment was found to have a significant difference in all groups (P < 0.05).

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Surgery Control InsigDKO ICRS Cartilage Scoring Scale Placebo Statin Placebo Statin 1) Surface 2.0 ± 0.0 2.7 ± 0.7 1.3 ± 0.7 2.7 ± 0.7 2) Matrix 2.7 ± 0.7 3.0 ± 0.0 2.0 ± 1.1 3.0 ± 0.0 3) Cell Distribution 1.7 ± 0.7 2.7 ± 0.7 1.3 ± 0.7 2.3 ± 0.7 4) Cell Population Viability 2.0 ± 0.0 2.7 ± 0.7 2.3 ± 0.7 2.7 ± 0.7 5) Subchondral Bone 1.7 ± 0.7 2.3 ± 0.7 1.7 ± 0.7 3.0 ± 0.0 6) Cartilage Mineralization 2.7 ± 0.7 3.0 ± 0.0 2.7 ± 0.7 3.0 ± 0.0 ICRS Summary Score 12.7 16.3 11.3 16.7 P-value (U-test) P=0.046 P=0.046

Table 7. ICRS Scoring of cartilage after surgically‐induced OA. The International Cartilage Repair Society (ICRS) score was used to grade histologic sections in a blinded manner. The mean and 95% confidence interval are given for each criterion, and a summary score is provided with Mann‐Whitney U statistical analysis to determine significant differences. Knee joints from 16‐week‐old mice were graded for genotypes shown in Figure 33. Statistical analyses compared placebo to statin treatment in Control and InsigDKO mice that underwent excision of the medial meniscus (Surgery). Statin treatment was found to have a significant difference in both groups (P < 0.05).

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Figure 34. Type X collagen staining of Col2a1‐Gli2, InsigDKO, and Col2a1‐ Gli2;InsigDKO cartilage. Representative histological sections showing type X collagen immunohistochemistry in the articular cartilage of the femur at 16 weeks for each genotype/treatment group shown in Figure 32. Statin treatment reduces type X collagen staining in the superficial zone. Scale bar, 100 μm.

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Figure 35. Type X collagen staining of cartilage after surgically‐induced OA. Representative histological sections showing type X collagen immunohistochemistry in the articular cartilage of the femur at 16 weeks for each genotype/treatment group shown in Figure 33. Statin treatment reduces type X collagen staining in the superficial zone. Scale bar, 100 μm.

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Statin treatment reduces OA markers in human cartilage

To investigate the utility of cholesterol inhibition as a potential therapeutic intervention in human osteoarthritis, articular cartilage explants were treated with 10

µM lovastatin. This concentration was chosen because it is widely used in experiments,

showing an effect on the cholesterol pathway without toxicity effects (Jakobisiak et al.

1991; MacDonald et al. 1988). We confirmed effective modulation of the cholesterol

pathway with an increase in HMGCR expression in the cartilage, an increase which

occurs when intracellular cholesterol is low, as expected with statin treatment (Figure

30).

Treatment of human osteoarthritic cartilage with statin resulted in

downregulated expression of the proteases MMP13 and ADAMTS5 (Figure 36). These

proteases are known to mediate cartilage degradation in OA (Glasson et al. 2005). To

explore the relationship between statin treatment and ADAMTS5 expression, human osteoarthritic chondrocytes were transfected with an ADAMTS5 luciferase reporter construct and treated with lovastatin. ADAMTS5 promoter activity was reduced following lovastatin treatment (Figure 37). These experiments were conducted with the assistance of Raymond Poon and Henry Ma. Because cholesterol levels are known to regulate Srebfs (Brown and Goldstein 1997), we examined the ADAMTS5 promoter for potential Srebf binding sites. In silico analyses (using the Mulan tool ‐ http://mulan.dcode.org) (Ovcharenko et al. 2005) revealed a SRE binding site that is conserved between human and mouse, suggesting that this gene is a potential target of the Srebf transcription factors (Figure 38). These data indicate that the cholesterol

pathway can impact osteoarthritis by modulating expression of ADAMTS5.

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Figure 36. Real‐time PCR of OA markers after statin treatment. Gene expression showing a reduction in OA markers MMP13 and ADAMTS5 in human osteoarthritic cartilage explants treated with statin (grey bars). Expression in the control group (black bars) was arbitrarily defined as ‘1’ and data from the statin‐treated group given as the mean. Error bars are 95% confidence intervals (n = 4; *P < 0.05).

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Figure 37. ADAMTS5 reporter construct assay. Luciferase activity from the ADAMTS5 promoter construct transfected into primary human OA chondrocytes, treated with control or statin. Measured in triplicate and reported as relative light units (RLU). Error bars are SEM.

Figure 38. ADAMTS5 promoter analysis. Schematic of the ADAMTS5 promoter showing a SREBF consensus binding site (red tick) that is conserved between mouse and human (from MULAN analysis, http://mulan.dcode.org).

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Discussion

The importance of cholesterol homeostasis in articular chondrocyte function and

articular cartilage integrity is demonstrated here, as mice with cholesterol

accumulation develop characteristic features of OA. The role of cholesterol

dysregulation in osteoarthritic cartilage has not been thoroughly explored.

Epidemiological studies describe systemic cholesterol levels correlating with

radiographic OA (Al‐Arfaj 2003; Sturmer et al. 1998), but these findings are

complicated by the fact that serum levels of cholesterol do not necessarily correlate with intra‐articular levels of cholesterol (Prete et al. 1993). We examine known models of osteoarthritis (Col2a1‐Gli2 and medial meniscectomy) as well as our mice with cartilage‐specific cholesterol accumulation, and find that statin treatment can rescue the OA phenotype in each. There are several implications of these findings.

Finding that statin treatment rescued the OA phenotype in Col2a1‐Gli2 mice suggests that the Hh activation in these mice causes an accumulation of cholesterol

which is normalized by statin treatment. In support of this, we show that Hh activation

results in cholesterol accumulation in chondrocytes in Chapter Three. Finding that

statin treatment rescued the OA phenotype induced by medial meniscectomy in control

mice suggests that there is an abnormal level of cholesterol accumulation in these mice as well, a level that is subsequently normalized by statin treatment. One possibility is that cholesterol accumulation in these control mice results from activation of Hh signaling, which we have previously shown to become active in mechanical models of

OA (Lin et al. 2009). Hh is known to be a mechanoresponsive gene, where mechanical stress (such as that which results following medial meniscectomy) induces the

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expression of Ihh in chondrocytes (Q. Wu et al. 2001). Once Hh becomes active in the

chondrocytes, it may induce expression of cholesterol biosynthetic genes, cause cholesterol accumulation, and induce OA pathogenesis.

Finding that statin treatment rescued the OA phenotype in InsigDKO mice

suggests that the OA changes are attributable to cholesterol accumulation, and not Hh

modulation. Since real‐time PCR results showed no change to Hh target genes in

InsigDKO cartilage, the OA phenotype can be attributed to cholesterol accumulation and

not perturbations to Hh signaling (see Chapter Three for further discussion). Despite

hypotheses surrounding a mutual regulatory relationship between Hh signaling and the

cholesterol pathway, our data support a role for Hh signaling in upstream regulation of

cholesterol homeostasis only. We show that Hh reduction in Gli2+/‐;InsigDKO mice

reduces the cholesterol accumulation seen in InsigDKO mice, and effectively rescues the

OA phenotype. As described in Chapter Three, this supports an Insig1‐independent

mechanism through which Hh signaling regulates cholesterol homeostasis, a

mechanism that is likely dependent on Hh regulation of Srebf2.

Finding that statin treatment rescued the OA phenotype in InsigDKO mice also

suggests that the OA changes are attributable to cholesterol accumulation and not

deformity of the joint during development. OA is known to result from skeletal

dysplasia (Sahlman et al. 2004) so the possibility exists that cholesterol accumulation in

InsigDKO mice compromises normal skeletal and joint development, which

subsequently predisposes to OA. This possibility is partially supported by our findings

in Chapter Three, which show impaired growth of the long bones in InsigDKO mice;

but partially negated by our findings in Chapter Three, which show normal total

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skeletons in InsigDKO mice. Our best evidence to negate the possibility that the OA

phenotype in InsigDKO mice results from skeletal dysplasia is provided by the statin

rescue. Pharmacologic cholesterol inhibition reduced the OA features in InsigDKO mice

after 8 weeks. This is supported by findings from Wu et al., who show that cholesterol inhibition in rat long bones reduced chondrocyte hypertrophy in vitro (S. Wu and De

Luca 2004). If the phenotype were being caused solely by joint deformity, reduction in

cholesterol levels would not have an impact. Rescue by statin treatment suggests that

the OA phenotype was caused at least in part by cholesterol accumulation in the adult

cartilage.

The implications of the statin effect in human osteoarthritic cartilage are also

far‐reaching. Reducing expression of the proteases that are responsible for cartilage

degradation is a viable strategy for preventing the cartilage erosion that is

characteristic of osteoarthritis. This is of particular importance since there are

currently no effective therapeutics for preventing cartilage degradation. We have

previously demonstrated that Hh signaling is activated in human osteoarthritic

cartilage (Lin et al. 2009), so the mechanism of action in human cartilage may be the

same as that in mouse cartilage, whereby activation of Hh signaling causes cholesterol

accumulation and exacerbates OA progression. Instances of OA resulting from skeletal

deformity or trauma in patients (Hunter and Eckstein 2009) may be comparable to our

medial meniscectomy, where compromised joint architecture and abnormal joint

loading causes mechanotransduction of Hh signaling and subsequent accumulation of

cholesterol. Our data suggest that patients who suffer these deformities or traumas may

benefit from local statin treatment to prevent cartilage loss.

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Results from our statin experiments highlight a potential mechanism through

which cholesterol accumulation may be mediating OA progression: regulation of

ADAMTS5, the major protease responsible for cartilage degradation (Glasson et al.

2005). In silico analyses revealed a SRE binding site in the proximal promoter region of the ADAMTS5 gene that is conserved between humans and mice. The Srebf transcription factors are known to bind to the SRE site and mediate transcription of target genes. The Srebf transcription factors are responsive to intracellular cholesterol levels. When cholesterol is high, these factors are tethered to the ER membrane, and transcription of target genes is prevented (Goedeke and Fernandez‐Hernando 2012).

Here we show cholesterol accumulation positively correlates with OA progression, so if

Srebf2 is tethered to the ER membrane, it can no longer prevent repression of ADAMTS5 transcription, and levels of this protease increase. In the alternative scenario when statin treatment is applied and intracellular cholesterol is lowered, processed Srebf2 translocates to the nucleus to repress expression of ADAMTS5 and prevent cartilage degradation. The Srebf transcription factors have been shown to function with co‐ regulators to negatively regulate target gene expression in this manner (Bennett et al.

1999; Edwards et al. 2000).

Although the exact mechanism of Srebf‐mediated regulation of ADAMTS5 remains to be elucidated, we hypothesize that the level of intracellular cholesterol is in part responsible for regulating Srebf2 and the resulting OA phenotype. Whether cholesterol accumulation has additional effects in contributing to OA pathogenesis remains to be determined, and is discussed further in Chapter Five.

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Materials and Methods

OA assessment

Radiography

Mouse knee joints were assessed for the development of OA by radiography,

histology, and real‐time PCR. For radiographs, mice were sacrificed, their joints

harvested, fixed in 10% phosphate‐buffered formalin for 4‐7 days (depending on age)

and imaged using the Faxitron MX20 X‐ray system. Lateral and anterior‐posterior

radiographs were taken and assessed for subchondral bone sclerosis (localized

whitening), irregularity in bone contour, and mineralization of the cartilage. These

features were used to evaluate samples for ICRS scoring (see below).

Histology

For histology, knee joints were decalcified in 20% ethylenediaminetetraacetic

acid (EDTA) at pH 7.4, dehydrated in serial concentrations of ethanol, and embedded

into paraffin wax. For OA assessment, 5 μm serial sections were made, and

representative sections stained with hematoxylin and eosin (H&E). For

immunohistochemistry, sections were deparaffinized, rehydrated, enzyme‐digested,

and stained with type X collagen (X53, Quartett, Germany) or HMG CoA‐reductase

(ab174830, abcam; used to verify effectiveness of statin treatment in murine

chondrocytes, see Lovastatin treatment). A description of this is given in the Materials and Methods section of Chapter Three. These experiments were conducted with the assistance of Heather Whetstone.

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Grading for OA severity was performed in a blinded manner using the ICRS scoring system (Mainil‐Varlet et al. 2003), as described by Lin et al. previously (Lin et al.

2009). This scoring system includes six categories which can be assigned values of 0 for most severe phenotype to 3 for least severe phenotype. The categories include surface continuity, matrix composition, cell distribution, cell viability, subchondral bone integrity, and cartilage mineralization. The authors recommend that each of these categories be considered separately, but here we provide an overall summary score to ease interpretation of phenotype severity. As such, for overall scores, the most severe possible score is 0 while the least severe possible score is 18 (Mainil‐Varlet et al. 2003).

Statistical significance was determined using a Mann‐Whitney U test, with the assistance of Mushriq Al‐Jazrawe.

The ICRS scoring system was chosen over other scoring systems, such as the

OARSI scale (Glasson et al. 2010), for three reasons. First, to compare scores to previously published ICRS scores from our lab group (Lin et al. 2009), facilitating direct comparison of the severity of the OA phenotype. Second, to assess the repair that was anticipated to result from Hh reduction in Gli2+/‐;InsigDKO mice and from statin treatment in mice. The ICRS scale is uniquely focused on capturing cartilage repair.

Third, to circumvent the lack of Safranin‐O‐stained histology, which is part of the OARSI assessment. Despite trouble‐shooting efforts, Safranin‐O, Alcian blue, and Toluidine blue staining of cartilage proteoglycan was not achieved in InsigDKO nor control sections.

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Gene expression

For real‐time PCR, experiments were conducted using TaqMan assays from

Applied Biosystems, as described in the Materials and Methods section of Chapter Two.

For mouse cartilage, RNA extraction was conducted using TRIzol® Reagent (Invitrogen,

CA) according to the manufacturer’s instructions. For human cartilage, RNA extraction

was conducted using the modified protocol described in Chapter Two. Real‐time PCR

results were normalized to an endogenous control (Gapdh 4352932E or ACTB

4352933E for mouse samples; ACTB Hs99999903_m1 or GAPDH Hs99999905_m1 for human samples), and analyzed according to the comparative CT method (∆∆CT).

Markers of OA included Mmp13 Mm01168713_m1 and Adamts5 Mm00478620_m1 for mouse; MMP13 Hs00233992_m1 and ADAMTS5 Hs00199841_m1 for human. To verify cholesterol inhibition by statin treatment in human cartilage, HMGCR

(Hs00168352_m1) was used (see Lovastatin treatment).

Medial meniscectomy surgery

To destabilize the joint and recapitulate mechanically‐induced OA, we performed medial meniscectomy, or surgical excision of the medial meniscus as previously described (Kamekura et al. 2005; Lin et al. 2009). Briefly, anesthetized mice had their knees shaved and surgically prepared under sterile conditions. Using a No.15 scalpel, a medial incision was made along the patella tendon and then through the joint capsule, revealing the medial meniscus to be excised. The incision was closed using two sutures, and covered with antibiotic cream. Analgesic was administered subcutaneously prior to surgery and for 72 hours post‐operatively, twice daily. Sham controls received

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an identical procedure except the medial meniscus was left intact. No mice perished

during surgery nor post‐operatively. In sum, the medial meniscus was removed from

the left knee of 8‐week‐old mice, and OA developed predictably 8 weeks post‐

operatively. All animal studies were approved by the Toronto Centre for

Phenogenomics.

Lovastatin treatment

Mouse

To reduce sterol levels in vivo, mice were treated with 3mg/kg/day lovastatin by surgical implantation of slow‐release drug pellets (Innovative Research of America, FL).

At 8 weeks of age, pellets were placed adjacent to the synovial membrane of the knee.

Pellets were 3mm in diameter, appropriate for implantation in 25 gram mice. Pellets were implanted in Control, Col2a1‐Gli2, InsigDKO, and Col2a1‐Gli2;InsigDKO mice, and for mechanically‐induced OA, in Control and InsigDKO mice. Effective statin treatment in the cartilage was confirmed by immunohistochemistry for HMG CoA‐reductase (see

OA assessment) in the chondrocytes.

Serum levels of cholesterol were assayed using the Amplex Red cholesterol assay kit (Molecular Probes). Approximately 1 mL of blood was collected from each mouse by cardiac puncture at the time of sacrifice. Samples were allowed to coagulate and serum was collected. The assay was conducted according to the manufacturer’s protocol.

Briefly, this is a fluorometric assay which detects products of cholesterol, using H2O2

detection (by the Amplex Red reagent) as a readout (M. Zhou et al. 1997).

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Human

To reduce sterol levels in vitro, human cartilage explants were established and

treatment applied as described in Chapter Two. Explants were treated overnight with

10 µM lovastatin hydroxy acid (Cayman Chemical Company, MI). Effective statin

treatment was confirmed by real‐time PCR for HMGCR (see OA assessment) in the

cartilage.

ADAMTS5 reporter construct

Primary cultures were established from human osteoarthritic cartilage using a method previously described (Otero et al. 2012), with the assistance of Henry Ma.

Chondrocytes were transfected with an ADAMTS5 reporter construct or negative control vector (GeneCopoeia, MD) using the Neon® Transfection System (Life

Technologies, ON). The construct contained a luciferase reporter under the control of human ADAMTS5 regulatory elements, and a β‐gal reporter for normalization of the transfection efficiency. Promoter activity was assayed following overnight treatment with 10 µm lovastatin and is reported as relative light units (RLU). These experiments were conducted with the assistance of Raymond Poon.

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Chapter Five:

Conclusions and future research

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Conclusions

Examining the expression of genes regulated by Hh signaling in human osteoarthritic cartilage led us to identify a mechanism through which Hh signaling impacts osteoarthritis severity (Figure 39). Our data support the hypothesis that Hh signaling regulates cholesterol biosynthesis in chondrocytes, and that modulating cholesterol homeostasis can impact the severity of OA. Higher levels of Hh signaling are associated with accumulation of intracellular cholesterol in chondrocytes. In genetically modified mice, we show that cholesterol accumulation causes impaired growth of the long bones; it also causes an osteoarthritis phenotype which can be modulated by Hh signaling. Reducing cholesterol accumulation attenuates the expression of osteoarthritis markers in vivo in murine cartilage and in vitro in human

cartilage. The protection against cartilage degradation is mediated in part by reduction

in ADAMTS5 expression. These findings demonstrate that Hh signaling regulates

cholesterol biosynthesis in chondrocytes, and provide support for the use of

cholesterol‐lowering agents to slow articular cartilage degeneration in OA.

Our key findings show that Hh signaling regulates expression of cholesterol

biosynthetic genes in chondrocytes; that cholesterol accumulation in chondrocytes

impairs growth of the long bones and predisposes to osteoarthritis; and that cholesterol

inhibition in the cartilage attenuates the severity of osteoarthritis. The broader

relevance of these findings and their contribution to the advancement of scientific

knowledge can be facilitated with additional experiments. Furthermore, to realize the

potential of this biomedical study, additional studies are required to translate these

basic scientific findings to clinical practice.

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Figure 39. Working model: cholesterol homeostasis mediates Hedgehog signaling in osteoarthritis. Intracellular cholesterol concentration is governed in part by endogenous cholesterol biosynthesis which is regulated by INSIGs (insulin‐induced genes) and SREBFs (sterol regulatory element‐binding factors), in a protein complex found in the ER (Figure 4). Once processed, the SREBFs translocate to the nucleus to regulate expression of target genes via the Sre binding site. The Hedgehog signaling pathway is mediated by processing of the GLI transcription factors which also translocate to the nucleus to regulate expression of target genes via the conserved Gli binding site (Figure 3). Putative target genes of both SREBF and GLI include cholesterol homeostatic genes (choles. genes) such as INSIG1, and genes involved in osteoarthritis (OA genes) pathogenesis such as ADAMTS5. PM = plasma membrane. ER = endoplasmic reticulum. + = homeostasis, ‐ = decrease, ++ = increase.

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Future Research

Hh signaling regulates expression of cholesterol homeostatic genes in chondrocytes.

Does Hh signaling regulate cholesterol homeostatic genes via Srebf2?

We performed microarray analyses in human osteoarthritic cartilage to identify

Hh signaling target genes. Among the many genes and pathways that were identified, we chose to pursue cholesterol biosynthesis due to its essential role in cellular homeostasis. We observed an Insig1‐independent mechanism through which Hh signaling regulates cholesterol homeostasis, and show that Hh signaling may regulate cholesterol homeostatic genes via Gli‐mediated targeting of Srebf2. To confirm that

Srebf2 is responsible for mediating the effect of Hh signaling on cholesterol homeostasis, chondrocyte‐specific Srebf2‐knockout experiments are required. If Srebf2 is responsible, then cholesterol levels in Srebf2‐knockout chondrocytes will be unchanged with Hh modulation. If cholesterol levels can still be altered in the absence of Srebf2, additional pathways which govern cholesterol homeostasis, such as LXR activation (Gentili et al. 2005), can be explored.

What other pathways are regulated by Hh signaling in chondrocytes?

Other pathways which were identified by our microarray analyses, such as lipid metabolism and steroid biosynthesis, may also contribute to OA pathogenesis under the

regulation of Hh signaling. This can be investigated using the same experimental design

as the current study. Transgenic mice which lack critical regulators in pathways of

interest can be used to determine the contribution of those pathways to chondrocyte biology and osteoarthritis development. The inter‐dependency of pathways also merits

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further investigation since for example, sterol intermediates produced in the cholesterol biosynthetic pathway feed into the steroid biosynthetic pathway (Jefcoate et al. 1992).

What other sterol intermediates are regulated by Hh signaling?

While we confirm a positive relationship between Hh signaling and cholesterol levels, we do not identify other sterol intermediates that were regulated by Hh signaling. Previous studies have shown there to be differences in lipid composition between growth plate and articular chondrocytes (Le Lous et al. 1981), so it is reasonable to expect differences in osteoarthritic chondrocytes, potentially governed by

Hh signaling. Specifying the exact sterol intermediates will allow refinement of hypotheses regarding pathway cross‐talk, as well as hypotheses regarding the specific role of those sterol intermediates in chondrocyte biology.

Establishing a sterol profile is relevant in continuing to explore the potential mutual regulatory relationship between Hh signaling and cholesterol biosynthesis.

Oxysterols have been shown to regulate Hh signaling in some tissues (Corcoran and

Scott 2006; Dwyer et al. 2007). If levels of these are regulated by Hh signaling via the cholesterol biosynthetic pathway in chondrocytes, a mutual regulatory relationship may emerge. Mass spectrometry experiments will prove useful in testing this hypothesis by determining whether specific oxysterols are regulated by Hh signaling in chondrocytes.

Does Hh signaling regulate cholesterol homeostasis in other systems?

We show for the first time a role for Hh signaling in regulating cholesterol biosynthesis. We investigate this relationship in cartilage and osteoarthritis, but it may

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also translate to other tissue types and other cartilage pathologies. Regarding other

tissues, simple stains such as Oil‐Red‐O can be used to crudely assess whether Hh

modulation alters lipid content. Regarding other cartilage pathologies, the contribution

of cholesterol dysregulation to chondrosarcoma (L. Ho et al. 2009), which also results from activation of Hh signaling, merits further investigation. This can be explored using the mice with cartilage‐specific cholesterol accumulation and Hh modulation described

in Chapter Three.

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Cholesterol accumulation in chondrocytes impairs growth of the long bones and predisposes to osteoarthritis.

Does Insig2 have chondrocyte‐specific roles?

To elucidate the role of cholesterol accumulation in chondrocyte biology, we excised Insig1 specifically in Col2a1‐expressing cells, in a background Insig2 knockout.

These mice showed delayed growth of the long bones and a predisposition to osteoarthritis. The mice examined in this study [including “control” Insig1(fl/fl);Insig2(‐

/‐) mice] carry germline disruption of Insig2, whose function is redundant to that of

Insig1 in the liver (Engelking et al. 2005). Loss of either Insig1 alone or Insig2 alone in the liver results in normal levels of cholesterol and triglycerides, and loss of either gene does not cause a compensating increase in expression of the other gene (Engelking et al.

2005). Despite this, Insig2 can serve unique roles in different tissues, for example, it is a colon cancer biomarker (C. G. Li et al. 2008), and polymorphisms in Insig2 in adipose tissues is associated with obesity (Krapivner et al. 2008). To assess potential chondrocyte‐specific functions, reintroduction of the Insig2 gene in InsigDKO cartilage is required. Differences, if any, between Insig1(fl/fl);Insig2(‐/‐) cartilage and

Insig1(fl/fl);Insig2(+/+) cartilage would highlight chondrocyte‐specific functions of

Insig2.

Is the OA phenotype in InsigDKO mice due to developmental perturbations?

Because InsigDKO mice exhibited cholesterol accumulation in chondrocytes throughout development, we were unable to definitively ascertain whether the phenotypes observed were due to developmental perturbations in chondrocyte biology and subsequent bone formation, or to the cholesterol accumulation itself. Future work

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to address this includes use of an inducible Col2a1 promoter (Grover and Roughley

2006) to drive Cre expression and excise Insig1 in adult cartilage. If cholesterol

accumulation that is induced in adult cartilage also creates an osteoarthritic phenotype

as we observe here, then the contribution of developmental abnormalities can be

excluded. Phenotypic analyses of these mice can be conducted in a similar manner as

described in Chapter Four, using radiography, histology, and gene expression to assess

typical features of osteoarthritis.

Where does cholesterol accumulate in InsigDKO chondrocytes?

Removal of the major negative regulator of cholesterol biosynthesis caused robust cholesterol accumulation in the chondrocytes. This allowed us to examine the effects of tissue‐specific cholesterol dysregulation on osteoarthritis outcomes, offering specificity over studies which examine systemic cholesterol dysregulation. To achieve greater specificity, the precise sub‐cellular localization of the excess cholesterol in our

InsigDKO mice is required. Techniques as crude as sub‐cellular fractionation or as

sophisticated as electron microscopy could be used to determine localization. This

would inform hypotheses surrounding the mechanisms through which cholesterol

accumulation results in osteoarthritic changes. For example, accumulation in the

plasma membrane may facilitate lipid raft formation and potentiate signal transduction

of pathways which mediate OA pathogenesis.

How does cholesterol accumulation exacerbate OA pathogenesis?

Additional studies are required to elucidate the mechanism through which

cholesterol accumulation contributes to OA pathogenesis. Because cholesterol is a

component of the lipid bilayer, alterations in free (unesterified) cholesterol may

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compromise membrane fluidity and disrupt membrane domains, ultimately perturbing

cell homeostasis by disrupting function of integral membrane proteins and/or signaling pathways (Tabas 2002). For example, lipid rafts have been shown to facilitate high activation levels of Hedgehog signaling by concentrating Smoothened, the pathway

transducer (Shi et al. 2013). Activation of Hedgehog signaling may increase cholesterol

accumulation which potentiates transduction of the Hh signal past a homeostatic

threshold, pushing the chondrocyte into an OA‐profile.

ER stress has been explored as a contributor to OA (Patterson and Dealy 2014).

Existing data suggest that chondrocytes can recover from ER stress that is induced by

accumulation of misfolded proteins, but those chondrocytes exhibit altered phenotypes

and behavior which ultimately compromises tissue structure (Tsang et al. 2007). Patra

et al. show that lack of S1P (site‐1 protease, which regulates SREBF processing and the

unfolded protein response) in the cartilage compromises type II collagen formation due

to retention of subunits in the ER, ultimately affecting endochondral bone development

(Patra et al. 2014). Cholesterol accumulation in our InsigDKO mice may induce the ER

stress response which compromises chondrocyte function and promotes OA (Patterson

and Dealy 2014). ER stress can be investigated by probing validated gene markers such

as PERK (EIF2AK3; eukaryotic translation initiation factor 2‐alpha kinase 3) and IRE1

(ERN1; endoplasmic reticulum to nucleus signaling 1), among others (Ron and Walter

2007), in our InsigDKO chondrocytes.

Determining whether lipid raft formation or ER stress are altered by Hh

signaling‐induced cholesterol modulation will clarify our understanding of the

contribution of cholesterol to OA pathogenesis. One mechanism explored in the current

135 study is the role for cholesterol‐mediated regulation of ADAMTS5 expression. Alternate mechanisms are equally possible, such as induction of chondrocyte hypertrophy by cholesterol‐mediated activation of the lipid regulator Ror‐alpha (Woods et al. 2009).

Barter et al. highlight the role of protein geranylgeranylation, showing that statin treatment (and subsequent reduction of the isoprenoid intermediate geranylgeranyl pyrophosphate) can reduce proteases, which are known contributors to cartilage degradation and OA pathogenesis (Barter et al. 2010). As described above, additional information regarding the exact sterol intermediates and their subcellular localization will be useful in refining hypotheses and directing future studies to elucidate the contribution of cholesterol to OA pathogenesis.

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Cholesterol inhibition in the cartilage attenuates the severity of osteoarthritis.

In mouse cartilage in vivo, lowering cholesterol by Hh reduction or statin treatment attenuates the severity of osteoarthritis. In human cartilage in vitro, lowering cholesterol by statin treatment reduces expression of osteoarthritis markers.

Cholesterol inhibition, therefore, is a viable strategy for mitigating the cartilage degradation that is characteristic of osteoarthritis. Current therapeutics for osteoarthritis are largely limited to analgesics, with no pharmacologic available for slowing cartilage degradation. Perhaps the most important implication of our results pertaining to statin treatment is the potential translation to the clinic for treating osteoarthritis. Because statins are widely used to lower cholesterol in cardiovascular diseases, there is existing data on the efficacy and safety of these drugs, which removes a barrier to translation (Lv et al. 2014).

Can cholesterol inhibition attenuate the severity of other diseases which result from Hh activation?

Our finding that statin treatment rescued the OA phenotype in mice with Hh activation (Col2a1‐Gli2) points to the possibility that statin treatment may be useful in attenuating other diseases which result from Hh activation. Medulloblastoma and basal cell carcinoma, for example, can result from Hh activation (Goodrich et al. 1997;

Grachtchouk et al. 2000). Because the medulloblastoma patient population is pediatric, and Hh signaling is crucial to the normal development of several tissues,

pharmacologics which inhibit Hh signaling are not an ideal therapy (Jiang and Hui

2008). As described above, it is important to explore whether the relationship between

Hh signaling and cholesterol regulation translates to other tissues, such as the brain and

137

skin. If this is indeed the case, statin treatment may be efficacious and relatively safe in

treating medulloblastoma, basal cell carcinoma, and other diseases which are known to

result from Hh activation.

Is local administration of statin required to improve OA outcomes?

Phase 2 clinical trials are currently being conducted by Stanford University to assess the benefits of statin treatment on OA outcomes (Hydroxychloroquine/

Atorvastatin in the Treatment of Osteoarthritis of the Knee; http://clinicaltrials.gov/show/NCT01645176). An anticipated limitation of this clinical

trial is the method of drug delivery, for which they chose oral administration. Studies

which examine the bioavailability and efficacy of statins in the synovial joint tissues of

patients following oral administration have yet to be conducted. Existing studies

examining the effect of statin treatment on OA outcomes show inconsistent results

(Kadam et al. 2013; Valdes et al. 2014; W. Wei et al. 2014) which may be explained in

part by method of drug delivery and resulting bioavailability of the drug in the joint.

Since our treatments were applied locally, our findings support the use of intra‐

articular statin injection to reduce cartilage degradation in OA patients. Further

research into statin pharmacokinetics in cartilage are required, in addition to more carefully designed clinical trials to translate these basic findings into the clinical setting.

138

Relevance

Identifying Hh target genes and networks improves our understanding of

cartilage biology and pathology, and proposes potential mechanisms by which Hh activation contributes to OA pathogenesis. With human and animal samples, we show that Hh signaling regulates cholesterol homeostasis and that cholesterol modulation can alter OA pathogenesis. These results highlight the need to further explore the complex interactions among Hh signaling, cholesterol homeostasis, and chondrocyte biology. The ability to pharmacologically correct sterol imbalances in the cartilage to prevent OA pathology represents a promising therapeutic intervention.

139

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Appendix: Microarray gene list

Fold change Fold change Fold change AVERAGE Transcripts ([Treatment1] ([Treatment2] ([Treatment3] Fold Cluster Id vs [Control1]) vs [Control2]) vs [Control3]) change Gene symbol 7896754 ‐1.239296 ‐1.028587 ‐1.2576101 ‐1.18 7896859 ‐1.1681848 ‐1.1480374 ‐1.0222958 ‐1.11 7897520 ‐1.1661352 ‐1.0919778 ‐1.179166 ‐1.15 7898371 ‐1.1814516 ‐1.1686509 ‐1.0819559 ‐1.14 LOC644634 7898736 ‐1.0208108 ‐1.2438325 ‐1.1888715 ‐1.15 HSPC157 7899534 ‐1.0573555 ‐1.115906 ‐1.2213967 ‐1.13 EPB41 7900635 ‐1.0297656 ‐1.1724788 ‐1.176768 ‐1.13 hCG_23177 7901565 ‐1.1557424 ‐1.2017087 ‐1.0000958 ‐1.12 DIO1 7901951 ‐1.1134347 ‐1.0275791 ‐1.1232364 ‐1.09 PGM1 7902425 ‐1.1350677 ‐1.1362531 ‐1.0528945 ‐1.11 ST6GALNAC3 7902861 ‐1.1970338 ‐1.0311054 ‐1.2001237 ‐1.14 LRRC8B 7903203 ‐1.1277745 ‐1.0156893 ‐1.1988994 ‐1.11 SNX7 7903391 ‐1.1768523 ‐1.2460194 ‐1.0103074 ‐1.14 7905033 ‐1.124537 ‐1.1265637 ‐1.1901158 ‐1.15 7905035 ‐1.2440093 ‐1.1450274 ‐1.0724686 ‐1.15 7905308 ‐1.1481949 ‐1.0208989 ‐1.1683198 ‐1.11 BNIPL 7905481 ‐1.0264015 ‐1.1864425 ‐1.214625 ‐1.14 7906330 ‐1.0612032 ‐1.1251994 ‐1.1199013 ‐1.10 CD1D 7906954 ‐1.331442 ‐1.1345092 ‐1.1225402 ‐1.20 PBX1 7908861 ‐1.2182635 ‐1.136407 ‐1.091596 ‐1.15 OCR1 7909422 ‐1.3482366 ‐1.2777729 ‐1.0128561 ‐1.21 LOC642587 7909990 ‐1.2309741 ‐1.0400394 ‐1.2586567 ‐1.18 7910010 ‐1.0530959 ‐1.3169386 ‐1.2339808 ‐1.20 7910186 ‐1.2155724 ‐1.1121147 ‐1.1317921 ‐1.15 7910257 ‐1.1233639 ‐1.1575162 ‐1.0553399 ‐1.11 GJC2 7910377 ‐1.6061957 ‐1.4717146 ‐1.040646 ‐1.37 7910727 ‐1.1677496 ‐1.0868012 ‐1.1296215 ‐1.13 ACTN2 7911049 ‐1.2393565 ‐1.1588407 ‐1.0629451 ‐1.15 C1orf100 7911349 ‐1.0628706 ‐1.1624547 ‐1.1226256 ‐1.12 7911413 ‐1.2520198 ‐1.1395662 ‐1.054782 ‐1.15 TNFRSF4 7911578 ‐1.217956 ‐1.1199672 ‐1.0720618 ‐1.14 CDC2L2|CDC2L1 7912861 ‐1.1867154 ‐1.1722811 ‐1.0707278 ‐1.14 LOC644634 7915184 ‐1.1162472 ‐1.1842861 ‐1.018505 ‐1.11 RHBDL2 7915229 ‐1.1994841 ‐1.1562371 ‐1.0787473 ‐1.14 HEYL 7915252 ‐1.0718507 ‐1.2693925 ‐1.1473501 ‐1.16 BMP8B 7915331 ‐1.0315639 ‐1.2819028 ‐1.2351078 ‐1.18 7915896 ‐1.2836509 ‐1.155666 ‐1.3532797 ‐1.26 CYP4Z2P 7916112 ‐1.3307526 ‐1.0329095 ‐1.2239927 ‐1.20 RAB3B 7916743 ‐1.133749 ‐1.173384 ‐1.0798877 ‐1.13 7917634 ‐1.0058423 ‐1.12566 ‐1.1890664 ‐1.11 HFM1 7917695 ‐1.13969 ‐1.0306325 ‐1.1260939 ‐1.10 7918048 ‐1.1583831 ‐1.1604854 ‐1.0550547 ‐1.12 7919155 ‐1.1688854 ‐1.8558902 ‐1.1456141 ‐1.39 7919299 ‐1.221169 ‐1.0376744 ‐1.1911544 ‐1.15 LOC100130236 7919380 ‐1.2176822 ‐1.0196455 ‐1.1871936 ‐1.14 LOC100130236

155

7919390 ‐1.1749278 ‐1.1701393 ‐1.1066655 ‐1.15 7919405 ‐1.174467 ‐1.8295774 ‐1.1409572 ‐1.38 7920193 ‐1.1835285 ‐1.2680614 ‐1.1082168 ‐1.19 LCE1C 7920285 ‐1.2409769 ‐1.3551095 ‐1.3021888 ‐1.30 S100A2 7920567 ‐1.0126534 ‐1.1684762 ‐1.1436824 ‐1.11 PMVK 7921031 ‐1.2109495 ‐1.1401324 ‐1.0691005 ‐1.14 7922389 ‐1.0073905 ‐1.127707 ‐1.1371604 ‐1.09 7922404 ‐1.0381347 ‐1.2563999 ‐1.3448906 ‐1.21 SNORD80|GAS5 7923037 ‐1.1190828 ‐1.1477737 ‐1.1112567 ‐1.13 7923974 ‐1.1193688 ‐1.0059763 ‐1.1330968 ‐1.09 7924445 ‐1.1240531 ‐1.0341871 ‐1.202964 ‐1.12 7924819 ‐1.2243763 ‐1.1744118 ‐1.1711066 ‐1.19 7925062 ‐1.1920491 ‐1.1615798 ‐1.0512623 ‐1.13 SIPA1L2 7925182 ‐1.0364329 ‐1.202894 ‐1.2015712 ‐1.15 SNORA14B 7925242 ‐1.0110915 ‐1.1201626 ‐1.1419328 ‐1.09 7925318 ‐1.5950552 ‐1.3424733 ‐1.4629917 ‐1.47 7925718 ‐1.1497971 ‐1.0367104 ‐1.1552188 ‐1.11 OR2B11 7925846 ‐1.0420406 ‐1.1636237 ‐1.1852438 ‐1.13 C10orf110 7926297 ‐1.3219967 ‐1.2300143 ‐1.1535416 ‐1.24 7927186 ‐1.116926 ‐1.2489287 ‐1.0269914 ‐1.13 RASSF4 7927681 ‐1.117609 ‐1.1308718 ‐1.0092411 ‐1.09 BICC1 7928514 ‐1.2361337 ‐1.1492356 ‐1.0223653 ‐1.14 7929032 ‐1.0980208 ‐1.1755128 ‐1.1332781 ‐1.14 FAS 7929533 ‐1.2300106 ‐1.0713491 ‐1.208209 ‐1.17 CC2D2B|LOC100131720 7929653 ‐1.1190768 ‐1.321037 ‐1.1438507 ‐1.19 ANKRD2 7929779 ‐1.1462467 ‐1.0364449 ‐1.1603695 ‐1.11 ABCC2 7931108 ‐1.1349638 ‐1.1980145 ‐1.0000949 ‐1.11 DMBT1 7931204 ‐1.124299 ‐1.1153541 ‐1.1310067 ‐1.12 LHPP 7931838 ‐1.1907548 ‐1.0391468 ‐1.2228552 ‐1.15 7933008 ‐1.0160373 ‐1.1437327 ‐1.2838147 ‐1.15 7933209 ‐1.1713217 ‐1.1251779 ‐1.0760181 ‐1.12 C10orf25 7933665 ‐1.1675456 ‐1.1430777 ‐1.0292453 ‐1.11 MBL2 7935679 ‐1.097733 ‐1.1846622 ‐1.1227124 ‐1.14 CPN1 7937950 ‐1.1202292 ‐1.299221 ‐1.1406789 ‐1.19 OR51D1 7938758 ‐1.0261204 ‐1.29826 ‐1.239581 ‐1.19 SAA1|SAA2 7939052 ‐1.1756849 ‐1.0749184 ‐1.1778132 ‐1.14 FIBIN 7939365 ‐1.1141043 ‐1.2389454 ‐1.0409287 ‐1.13 FJX1 7940822 ‐1.0201366 ‐1.1302205 ‐1.2145262 ‐1.12 7941749 ‐1.1619815 ‐1.1304215 ‐1.0315908 ‐1.11 SYT12 7944049 ‐1.2701621 ‐1.1039438 ‐1.1240085 ‐1.17 SIDT2 7944867 ‐1.2389716 ‐1.1459262 ‐1.337283 ‐1.24 7944970 ‐1.2426654 ‐1.0866597 ‐1.1966279 ‐1.18 7945663 ‐1.1985242 ‐1.048137 ‐1.2108531 ‐1.15 CTSD|LOC402778 7946569 ‐1.146311 ‐1.2217132 ‐1.1331683 ‐1.17 RNF141 7946977 ‐1.1775659 ‐1.2733252 ‐1.0118728 ‐1.15 SAA4 7946983 ‐1.5070485 ‐1.1571165 ‐1.1382565 ‐1.27 SAA2|SAA1 7947230 ‐1.072386 ‐1.1168576 ‐1.2412677 ‐1.14 BDNF 7947462 ‐1.1457043 ‐1.2049994 ‐1.0657629 ‐1.14 ABTB2 7947551 ‐1.1180062 ‐1.2851707 ‐1.0894003 ‐1.16 7947742 ‐1.1297166 ‐1.0042957 ‐1.115263 ‐1.08

156

7948133 ‐1.1340811 ‐1.2189684 ‐1.0596061 ‐1.14 OR5M3 7948344 ‐1.1773208 ‐1.1347039 ‐1.0178528 ‐1.11 GLYAT 7948369 ‐1.2485758 ‐1.0573007 ‐1.1535907 ‐1.15 7948834 ‐1.3164091 ‐1.1715544 ‐1.0344025 ‐1.17 7948987 ‐1.0112492 ‐1.1268858 ‐1.1415362 ‐1.09 HRASLS3 7950036 ‐1.1263306 ‐1.1254836 ‐1.0950731 ‐1.12 FGF3 7950553 ‐1.09396 ‐1.2258033 ‐1.1205388 ‐1.15 7951004 ‐1.1444405 ‐1.0152844 ‐1.1616305 ‐1.11 C11orf75 7951429 ‐1.2179612 ‐1.1664883 ‐1.164392 ‐1.18 KBTBD3 7951668 ‐1.1966258 ‐2.26246 ‐1.0374669 ‐1.50 7952339 ‐1.2256712 ‐1.2279723 ‐1.0599637 ‐1.17 LOC85389 7952631 ‐1.1697 ‐1.1174647 ‐1.0379763 ‐1.11 P53AIP1 7953032 ‐1.1515633 ‐1.2214835 ‐1.0870336 ‐1.15 LRTM2 7953665 ‐1.1767784 ‐1.1308128 ‐1.1538184 ‐1.15 DPPA3 7954100 ‐1.2158893 ‐1.1309227 ‐1.0168245 ‐1.12 7955043 ‐1.029899 ‐1.1148893 ‐1.2694701 ‐1.14 7955694 ‐1.1697878 ‐1.0139592 ‐1.1990726 ‐1.13 IGFBP6 7955729 ‐1.2305291 ‐1.0022773 ‐1.2645892 ‐1.17 MFSD5 7957023 ‐1.1383976 ‐1.0393449 ‐1.1166416 ‐1.10 LYZ 7957161 ‐1.1278166 ‐1.1962087 ‐1.1203734 ‐1.15 THAP2 7958207 ‐1.2753422 ‐1.2590711 ‐1.1286881 ‐1.22 7958453 ‐1.0169996 ‐1.2286311 ‐1.2156032 ‐1.15 7960338 ‐1.2524924 ‐1.1274483 ‐1.1650988 ‐1.18 7960436 ‐1.3187519 ‐1.0697742 ‐1.2069689 ‐1.20 FLJ44874 7961891 ‐1.1398716 ‐1.116494 ‐1.0586067 ‐1.10 BHLHB3 7962139 ‐1.2944684 ‐1.6259124 ‐1.2575294 ‐1.39 OVOS2|LOC100132881 7962236 ‐1.1196722 ‐1.2289126 ‐1.1078498 ‐1.15 7963502 ‐1.1831151 ‐1.3382368 ‐1.0556839 ‐1.19 KRT77 7963689 ‐1.1279773 ‐1.1819038 ‐1.0707328 ‐1.13 NPFF 7963946 ‐1.1534932 ‐1.0060945 ‐1.1139452 ‐1.09 MMP19 7964246 ‐1.1678979 ‐1.0677938 ‐1.2752719 ‐1.17 SNORD59B 7964602 ‐1.1717365 ‐1.1507206 ‐1.1221786 ‐1.15 LRIG3 7965767 ‐1.0049801 ‐1.1923171 ‐1.1170946 ‐1.10 7966127 ‐1.1933887 ‐1.1227053 ‐1.0343099 ‐1.12 SELPLG 7966225 ‐1.1991614 ‐1.3619182 ‐1.114256 ‐1.23 7966293 ‐1.1580918 ‐1.0965353 ‐1.1602176 ‐1.14 FLJ40142 7967107 ‐1.1083332 ‐1.3466547 ‐1.1840038 ‐1.21 C12orf27 7967193 ‐1.1167853 ‐1.2344662 ‐1.0399429 ‐1.13 MORN3 7967698 ‐1.122709 ‐1.2789763 ‐1.6820436 ‐1.36 7967872 ‐1.1310834 ‐1.0743521 ‐1.1848722 ‐1.13 7970388 ‐1.0185413 ‐1.1809233 ‐1.1150835 ‐1.10 7971526 ‐1.0544024 ‐1.1821325 ‐1.2638721 ‐1.17 HTR2A 7972546 ‐1.1597128 ‐1.1144748 ‐1.021918 ‐1.10 7973652 ‐1.145908 ‐1.0977293 ‐1.1228678 ‐1.12 TSSK4 7974214 ‐1.0512942 ‐1.1815953 ‐1.2344646 ‐1.16 KLHDC1 7974335 ‐1.0273261 ‐1.1712015 ‐1.1275516 ‐1.11 7974687 ‐1.1308441 ‐1.1638987 ‐1.075547 ‐1.12 7975309 ‐1.1326947 ‐1.1706564 ‐1.2940979 ‐1.20 7976239 ‐1.1648555 ‐1.0687666 ‐1.2078676 ‐1.15 PTMAP7 7976810 ‐1.1919054 ‐1.1559659 ‐1.1252129 ‐1.16 SNORD113‐3

157

7977761 ‐1.2426963 ‐1.1237935 ‐1.0531527 ‐1.14 SALL2 7978653 ‐1.1632799 ‐1.2946237 ‐1.0140741 ‐1.16 GARNL1 7978690 ‐1.1967659 ‐1.0102891 ‐1.2214823 ‐1.14 7980603 ‐1.2631735 ‐1.0843284 ‐1.1148542 ‐1.15 7980605 ‐1.0962623 ‐1.1209263 ‐1.3158514 ‐1.18 KCNK10 7980859 ‐1.0359589 ‐1.1238269 ‐1.1359125 ‐1.10 PP8961 7980889 ‐1.1272774 ‐1.162957 ‐1.0199138 ‐1.10 7981742 ‐1.1492268 ‐1.1603285 ‐1.0562541 ‐1.12 LOC90925 7981947 ‐1.1578717 ‐1.0548266 ‐1.2465389 ‐1.15 SNRPN 7981972 ‐1.1312745 ‐1.6127473 ‐1.0674124 ‐1.27 SNRPN 7982098 ‐1.160935 ‐1.0550139 ‐1.2484871 ‐1.15 SNRPN 7982230 ‐1.0053731 ‐1.1952736 ‐1.1650352 ‐1.12 FAM7A1|GOLGA9P|FAM7A3 7982284 ‐1.1217887 ‐1.170737 ‐1.1689181 ‐1.15 GOLGA9P 7982535 ‐1.054629 ‐1.1901615 ‐1.1304559 ‐1.13 C15orf41 7982876 ‐1.1565158 ‐1.0874544 ‐1.122222 ‐1.12 FAM92A1|FAM92A2 7983500 ‐1.1225733 ‐1.394044 ‐1.0550944 ‐1.19 7983630 ‐1.108441 ‐1.1242946 ‐1.1825603 ‐1.14 FGF7 7983771 ‐1.003591 ‐1.2217277 ‐1.2300467 ‐1.15 7984436 ‐1.2748382 ‐1.2105106 ‐1.0067405 ‐1.16 LBXCOR1 7985037 ‐1.226634 ‐1.3714616 ‐1.2055959 ‐1.27 7985221 ‐1.0213271 ‐1.1221585 ‐1.1226256 ‐1.09 LOC646934 7985587 ‐1.1192448 ‐1.151928 ‐1.0999775 ‐1.12 SCAND2 7986561 ‐1.3620731 ‐1.1061167 ‐1.1974267 ‐1.22 7987066 ‐1.0151405 ‐1.2709823 ‐1.145822 ‐1.14 7987310 ‐1.0802622 ‐1.2658659 ‐1.1149721 ‐1.15 GJD2|GJA9 7988077 ‐1.1254036 ‐1.0084178 ‐1.169678 ‐1.10 LCMT2 7988424 ‐1.0237318 ‐1.4947435 ‐1.4157429 ‐1.31 7988440 ‐1.1148093 ‐1.0219171 ‐1.349166 ‐1.16 7989968 ‐1.2974492 ‐1.0074338 ‐1.1229858 ‐1.14 CALML4 7990452 ‐1.0519546 ‐1.2617414 ‐1.2692586 ‐1.19 LOC554175|LOC729809 7991047 ‐1.1691381 ‐1.1899309 ‐1.1677655 ‐1.18 LOC100131860 7991542 ‐1.2985655 ‐1.0675676 ‐1.1495607 ‐1.17 FLJ42289 7991815 ‐1.1650152 ‐1.1390272 ‐1.0242103 ‐1.11 PDIA2 7992863 ‐1.1628617 ‐1.1943543 ‐1.2081535 ‐1.19 OR1F1 7992877 ‐1.1656126 ‐1.1188745 ‐1.0911243 ‐1.13 ZNF75A|LOC100128510 7993754 ‐1.1725152 ‐1.1423855 ‐1.0191922 ‐1.11 LOC100129488 7995332 ‐1.1419132 ‐1.2413559 ‐1.0103017 ‐1.13 7995350 ‐1.0441247 ‐1.1841248 ‐1.2310412 ‐1.15 7996498 ‐1.1525491 ‐1.1862032 ‐1.0500996 ‐1.13 SLC9A5 7997372 ‐1.1426249 ‐1.1882234 ‐1.004234 ‐1.11 7997875 ‐1.053784 ‐1.1225687 ‐1.185921 ‐1.12 C16orf81 7998485 ‐1.107941 ‐1.225454 ‐1.1583207 ‐1.16 LOC283951 7998927 ‐1.1506369 ‐1.1043873 ‐1.2603927 ‐1.17 7999886 ‐1.1707772 ‐1.1728144 ‐1.0082905 ‐1.12 7999981 ‐1.2192432 ‐1.0627617 ‐1.1467855 ‐1.14 ACSM1 8000738 ‐1.0783455 ‐1.1448008 ‐1.1565655 ‐1.13 KCTD13 8001102 ‐1.1731738 ‐1.2441498 ‐1.1090258 ‐1.18 8001371 ‐1.0241971 ‐1.1183219 ‐1.1471221 ‐1.10 8004184 ‐1.079198 ‐1.1509663 ‐1.1279597 ‐1.12 XAF1 8004309 ‐1.1344624 ‐1.1494528 ‐1.1473053 ‐1.14 SLC2A4

158

8004408 ‐1.2454047 ‐1.1325206 ‐1.0803822 ‐1.15 FGF11 8005225 ‐1.1170769 ‐1.2349257 ‐1.1674564 ‐1.17 LOC162632 8005685 ‐1.1663718 ‐1.0901207 ‐1.1965191 ‐1.15 8005753 ‐1.003376 ‐1.1873051 ‐1.4082209 ‐1.20 8005785 ‐1.1949079 ‐1.1610156 ‐1.0469292 ‐1.13 KSR1 8005809 ‐1.178125 ‐1.2208102 ‐1.2303269 ‐1.21 LGALS9 8005953 ‐1.0350748 ‐1.2790414 ‐1.2803051 ‐1.20 SNORD4A 8006081 ‐1.1821449 ‐1.1199597 ‐1.0864234 ‐1.13 8006433 ‐1.1256804 ‐1.0086231 ‐1.1455703 ‐1.09 CCL2 8006865 ‐1.2422552 ‐1.194151 ‐1.0407062 ‐1.16 PPP1R1B 8007263 ‐1.125596 ‐1.1608901 ‐1.1517155 ‐1.15 HSD17B1 8007603 ‐1.1137339 ‐1.1158684 ‐1 ‐1.08 8008350 ‐1.1626219 ‐1.1449513 ‐1.0000218 ‐1.10 MYCBPAP 8008588 ‐1.0730299 ‐1.1852114 ‐1.1709827 ‐1.14 HLF 8008965 ‐1.1299677 ‐1.0466642 ‐1.2586576 ‐1.15 8009844 ‐1.1973641 ‐1.1401241 ‐1.1038592 ‐1.15 LLGL2 8010078 ‐1.1731684 ‐1.248726 ‐1.1627487 ‐1.19 SNORD1C 8010897 ‐1.1377529 ‐1.1930258 ‐1.0499513 ‐1.13 METRNL 8011671 ‐1.2097199 ‐1.1669418 ‐1 ‐1.13 GGT6 8011732 ‐1.2225828 ‐1.0491917 ‐1.1758633 ‐1.15 LOC100132978 8012309 ‐1.1198332 ‐1.1229507 ‐1.0949949 ‐1.11 ALOX12B 8012951 ‐1.4136238 ‐1.0232565 ‐1.1511729 ‐1.20 CDRT1|TRIM16 8013356 ‐1.0691361 ‐1.2525641 ‐1.1390085 ‐1.15 8013527 ‐1.183047 ‐1.062078 ‐1.1270676 ‐1.12 8013529 ‐1.3972099 ‐1.0783712 ‐1.2473458 ‐1.24 8013987 ‐1.1142015 ‐1.4133202 ‐1.5968835 ‐1.37 8014704 ‐1.1859324 ‐1.2775048 ‐1.2140096 ‐1.23 8015681 ‐1.1925843 ‐1.2735271 ‐1.0437707 ‐1.17 CCR10 8015906 ‐1.1705462 ‐1.1195908 ‐1.1021883 ‐1.13 8016487 ‐1.1224438 ‐1.1857282 ‐1.0014987 ‐1.10 HOXB13 8016532 ‐1.1204137 ‐1.1956154 ‐1.0427014 ‐1.12 GNGT2 8016739 ‐1.1387917 ‐1.1250964 ‐1.120523 ‐1.13 TOB1 8017378 ‐1.1446226 ‐1.0477349 ‐1.1547782 ‐1.12 CYB561 8018652 ‐1.361631 ‐1.1638983 ‐1.0695252 ‐1.20 RNF157 8019643 ‐1.1769046 ‐1.1450365 ‐1.120059 ‐1.15 DOC2B 8020123 ‐1.2161952 ‐1.1715344 ‐1.2168515 ‐1.20 TXNDC2 8020760 ‐1.2144672 ‐1.1808996 ‐1.1884449 ‐1.19 8021245 ‐1.1288146 ‐1.3623563 ‐1.0556556 ‐1.18 DCC 8023528 ‐1.0272005 ‐1.3503045 ‐1.1576416 ‐1.18 ALPK2 8023926 ‐1.3542663 ‐1.1436003 ‐1.0449892 ‐1.18 PARD6G 8025826 ‐1.1852858 ‐1.1862342 ‐1.0969282 ‐1.16 8026182 ‐1.2166154 ‐1.1255115 ‐1.0662421 ‐1.14 MGC3207 8026787 ‐1.1088161 ‐1.2473087 ‐1.2091366 ‐1.19 FAM129C 8026926 ‐1.138226 ‐1.155456 ‐1.0498385 ‐1.11 MAST3 8027233 ‐1.1181829 ‐1.0932386 ‐1.1441342 ‐1.12 ZNF101 8027377 ‐1.1775936 ‐1.120066 ‐1.0015619 ‐1.10 8027429 ‐1.2911018 ‐1.3320327 ‐1.0229461 ‐1.22 8028206 ‐1.2205637 ‐1.0949863 ‐1.3631274 ‐1.23 ZNF345 8028309 ‐1.1849595 ‐1.2031429 ‐1.1301652 ‐1.17 8029375 ‐1.165125 ‐1.2562487 ‐1.0103853 ‐1.14

159

8029914 ‐1.0083652 ‐1.1516474 ‐1.2063702 ‐1.12 GPR77 8030831 ‐1.056192 ‐1.2895998 ‐1.1884161 ‐1.18 ZNF175 8032491 ‐1.1193259 ‐1.1811864 ‐1.0043415 ‐1.10 LMNB2 8034320 ‐1.1282841 ‐1.1623744 ‐1.1057303 ‐1.13 ZNF433 8034326 ‐1.2614471 ‐1.0487839 ‐1.1463664 ‐1.15 8034349 ‐1.2122726 ‐1.027877 ‐1.2724067 ‐1.17 ZNF44 8034390 ‐1.1742053 ‐1.0433823 ‐1.3176584 ‐1.18 ZNF799 8034694 ‐1.1095688 ‐1.1832575 ‐1.2473186 ‐1.18 8035847 ‐1.1244503 ‐1.0256978 ‐1.4222808 ‐1.19 ZNF675 8036351 ‐1.1939071 ‐1.2281318 ‐1.0104808 ‐1.14 LOC342892 8037231 ‐1.463514 ‐1.2127107 ‐1.0521618 ‐1.24 PSG3 8037259 ‐1.1891646 ‐1.1960526 ‐1.2747034 ‐1.22 PSG11 8037283 ‐1.1774278 ‐1.1716328 ‐1.2232862 ‐1.19 PSG4 8037621 ‐1.1437429 ‐1.0150472 ‐1.1588085 ‐1.11 EML2 8038314 ‐1.0293359 ‐1.1624051 ‐1.1162008 ‐1.10 FLJ10490 8038874 ‐1.2146318 ‐1.169089 ‐1.1225892 ‐1.17 FLJ30403 8038989 ‐1.1474153 ‐1.1355653 ‐1.1895802 ‐1.16 ZNF600 8040365 ‐1.237787 ‐1.1225224 ‐1.1014553 ‐1.15 TRIB2 8040712 ‐1.1338233 ‐1.1207978 ‐1.016668 ‐1.09 CENPA 8042038 ‐1.1501498 ‐1.0612081 ‐1.213359 ‐1.14 RPL23AP13 8042308 ‐1.1846063 ‐1.219864 ‐1.178802 ‐1.19 8042324 ‐1.0209745 ‐1.2211428 ‐1.2123812 ‐1.15 8042464 ‐1.016172 ‐1.2357031 ‐1.1922982 ‐1.15 8042532 ‐1.0403354 ‐1.2517078 ‐1.1173948 ‐1.14 VAX2 8043043 ‐1.1452181 ‐1.2406706 ‐1.0604295 ‐1.15 DNHL1|LOC200383 8043071 ‐1.1253717 ‐1.0869683 ‐1.2295554 ‐1.15 LOC200383 8043502 ‐1.041727 ‐1.1712826 ‐1.4214735 ‐1.21 8043512 ‐1.0799599 ‐1.1247827 ‐1.233348 ‐1.15 ZNF2 8044035 ‐1.1625024 ‐1.0278059 ‐1.2220144 ‐1.14 IL18R1 8044764 ‐1.0152888 ‐1.1281484 ‐1.1196922 ‐1.09 8045279 ‐1.0548402 ‐1.2509687 ‐1.2248049 ‐1.18 8046922 ‐1.1524653 ‐1.0906504 ‐1.1461868 ‐1.13 COL3A1 8047337 ‐1.2136518 ‐1.1245916 ‐1.0514058 ‐1.13 8047557 ‐1.1737839 ‐1.1763889 ‐1.1030033 ‐1.15 8049487 ‐1.2017003 ‐1.1330436 ‐1.1553528 ‐1.16 MLPH 8050060 ‐1.0316539 ‐1.1242983 ‐1.2154266 ‐1.12 TSSC1 8050071 ‐1.0232592 ‐1.1345288 ‐1.2759494 ‐1.14 ADI1 8050687 ‐1.2850941 ‐1.1354996 ‐1.1409444 ‐1.19 8052784 ‐1.0668502 ‐1.2068669 ‐1.2600424 ‐1.18 NFU1 8052866 ‐1.0840428 ‐1.1528002 ‐1.1390525 ‐1.13 FAM136A 8053231 ‐1.1174583 ‐1.2038665 ‐1.2263949 ‐1.18 LOXL3 8053379 ‐1.3621267 ‐1.140152 ‐1.0118667 ‐1.17 LOC129293 8054295 ‐1.1482404 ‐1.0459356 ‐1.2824177 ‐1.16 8055643 ‐1.1755197 ‐1.1234406 ‐1.319307 ‐1.21 8056877 ‐1.1515645 ‐1.209793 ‐1.0940906 ‐1.15 CHRNA1 8056995 ‐1.1414709 ‐1.0475291 ‐1.6364006 ‐1.28 TTC30B 8057599 ‐1.0779386 ‐1.1524447 ‐1.2133337 ‐1.15 TFPI 8057817 ‐1.0712577 ‐1.1770087 ‐1.144253 ‐1.13 8058106 ‐1.0137728 ‐1.2836937 ‐1.1311256 ‐1.14 8059578 ‐1.3709984 ‐1.2950534 ‐1.0034155 ‐1.22

160

8059864 ‐1.1359365 ‐1.1819448 ‐1.0493227 ‐1.12 GBX2 8059996 ‐1.1858007 ‐1.0751188 ‐1.1151752 ‐1.13 PER2 8060765 ‐1.1278725 ‐1.2077934 ‐1.0838586 ‐1.14 PRND 8061404 ‐1.1667578 ‐1.0267144 ‐1.2232623 ‐1.14 8062864 ‐1.1299961 ‐1.1280899 ‐1.0402436 ‐1.10 WISP2 8063074 ‐1.161579 ‐1.3275356 ‐1.030984 ‐1.17 ZSWIM1 8063410 ‐1.0729451 ‐1.1434417 ‐1.1414875 ‐1.12 PARD6B 8063531 ‐1.167091 ‐1.0631039 ‐1.1878158 ‐1.14 C20orf107 8064868 ‐1.2455668 ‐1.2226378 ‐1.2192042 ‐1.23 RP5‐1022P6.2 8065194 ‐1.0284455 ‐1.4689733 ‐1.2417064 ‐1.25 8066745 ‐1.1293821 ‐1.0006018 ‐1.1949793 ‐1.11 ZNF334 8067585 ‐1.1276138 ‐1.0314044 ‐1.1306652 ‐1.10 BHLHB4 8068383 ‐1.1109526 ‐1.2716205 ‐1.1471775 ‐1.18 CLIC6 8069804 ‐1.2963073 ‐1.1182044 ‐1.0640442 ‐1.16 KRTAP26‐1 8069811 ‐1.1415263 ‐1.1699111 ‐1.0471985 ‐1.12 KRTAP23‐1 8070097 ‐1.2714323 ‐1.1978985 ‐1.0551244 ‐1.17 C21orf55 8070681 ‐1.2725102 ‐1.0779703 ‐1.1427877 ‐1.16 C21orf84 8071168 ‐1.1572989 ‐1.0671101 ‐1.1214296 ‐1.12 DKFZP434P211|LOC646074 8071272 ‐1.159128 ‐1.2243245 ‐1.1595939 ‐1.18 GP1BB 8073015 ‐1.1491311 ‐1.0548085 ‐1.1289803 ‐1.11 KDELR3 8073088 ‐1.166444 ‐1.2652293 ‐1.041814 ‐1.16 APOBEC3G|APOBEC3F 8074057 ‐1.1644158 ‐1.2275028 ‐1.1066564 ‐1.17 8074714 ‐1.1829728 ‐1.1847116 ‐1.0770009 ‐1.15 DKFZP434P211|LOC646074 8075785 ‐1.140406 ‐1.116487 ‐1.0849069 ‐1.11 FOXRED2 8075924 ‐1.1899663 ‐1.1209122 ‐1.2011963 ‐1.17 MFNG 8076113 ‐1.1582625 ‐1.1279755 ‐1.0208787 ‐1.10 RP1‐199H16.1 8077323 ‐1.0886214 ‐1.4730018 ‐1.2605501 ‐1.27 CNTN4 8077595 ‐1.1630408 ‐1.0093642 ‐1.128436 ‐1.10 BRPF1 8077635 ‐1.121752 ‐1.0662351 ‐1.1674826 ‐1.12 OGG1 8078155 ‐1.1356629 ‐1.1786535 ‐1.0015367 ‐1.11 GALNTL2 8079613 ‐1.1212101 ‐1.0444125 ‐1.12985 ‐1.10 8079988 ‐1.3122028 ‐1.1141529 ‐1.00804 ‐1.14 C3orf45 8081233 ‐1.2021098 ‐1.1782302 ‐1.2279524 ‐1.20 8081348 ‐1.0009781 ‐1.1224022 ‐1.3112178 ‐1.14 PCNP 8081503 ‐1.046727 ‐1.1794485 ‐1.1418022 ‐1.12 DZIP3 8083061 ‐1.182843 ‐1.1338469 ‐1.2538469 ‐1.19 8083214 ‐1.176936 ‐1.204946 ‐1.0205301 ‐1.13 CHST2 8084217 ‐1.1498879 ‐1.2449409 ‐1.1839526 ‐1.19 8084766 ‐1.2596422 ‐1.0055674 ‐1.1228255 ‐1.13 TP63 8085732 ‐1.2460425 ‐1.1491847 ‐1.0675083 ‐1.15 EFHB 8087825 ‐1.1354399 ‐1.1155609 ‐1.0376215 ‐1.10 ABHD14B 8088285 ‐1.15899 ‐1.1783205 ‐1.0223178 ‐1.12 HESX1 8088952 ‐1.0175797 ‐1.1159927 ‐1.2305539 ‐1.12 8089993 ‐1.0024654 ‐1.1609001 ‐1.1301271 ‐1.10 WDR5B 8090291 ‐1.2615534 ‐1.0849414 ‐1.2922174 ‐1.21 LOC200810 8090559 ‐1.0148567 ‐1.1266266 ‐1.1708612 ‐1.10 RPL32P3 8090674 ‐1.0479083 ‐1.2538457 ‐1.169852 ‐1.16 8091537 ‐1.1301827 ‐1.2261946 ‐1.0756636 ‐1.14 IGSF10 8091922 ‐1.1779809 ‐1.2007847 ‐1.1210434 ‐1.17 WDR49 8092169 ‐1.5019922 ‐1.336261 ‐1.0817556 ‐1.31 TNFSF10

161

8092541 ‐1.1631415 ‐1.1345767 ‐1.1707602 ‐1.16 LIPH 8092596 ‐1.1804836 ‐1.1404588 ‐1.0028856 ‐1.11 DGKG 8095251 ‐1.0698718 ‐1.1722296 ‐1.2220927 ‐1.15 8096411 ‐1.0418754 ‐1.1272963 ‐1.1589227 ‐1.11 TIGD2 8096955 ‐1.2390587 ‐1.2746465 ‐1.3130344 ‐1.28 8097447 ‐1.021497 ‐1.1401143 ‐1.1438525 ‐1.10 8098506 ‐1.1622338 ‐1.0544153 ‐1.1623598 ‐1.13 8098671 ‐1.1506845 ‐1.182949 ‐1.2312691 ‐1.19 F11 8099025 ‐1.1469216 ‐1.2548261 ‐1.0398202 ‐1.15 8099255 ‐1.1978335 ‐1.1434828 ‐1.0145589 ‐1.12 PSAPL1 8099570 ‐1.0125599 ‐1.1833833 ‐1.1953404 ‐1.13 C4orf30 8099922 ‐1.3966119 ‐1.489955 ‐1.2042067 ‐1.36 8100229 ‐1.1427068 ‐1.3490162 ‐1.1700697 ‐1.22 8100362 ‐1.0851167 ‐1.1568668 ‐1.1268337 ‐1.12 LNX1 8100519 ‐1.1381354 ‐1.0797079 ‐1.1146693 ‐1.11 TXNDC9 8100541 ‐1.0728738 ‐1.173856 ‐1.1154237 ‐1.12 IGFBP7 8100555 ‐1.0007436 ‐1.2045817 ‐1.1667786 ‐1.12 LOC391656 8100699 ‐1.1165882 ‐1.1587764 ‐1.0265064 ‐1.10 SYT14L 8101061 ‐1.0544167 ‐1.1955289 ‐1.1307522 ‐1.13 8101780 ‐1.1413743 ‐1.0857375 ‐1.153593 ‐1.13 PGDS 8102781 ‐1.2576001 ‐1.1657729 ‐1.2119558 ‐1.21 LOC646187 8103023 ‐1.114999 ‐1.089999 ‐1.1910535 ‐1.13 8103166 ‐1.1877081 ‐1.0424626 ‐1.1459823 ‐1.13 SH3D19|FLJ46481 8103516 ‐1.0159528 ‐1.1446517 ‐1.1705551 ‐1.11 8103853 ‐1.2490022 ‐1.1504657 ‐1.3950586 ‐1.26 MGC45800 8104348 ‐1.0803001 ‐1.1395029 ‐1.3162934 ‐1.18 8104617 ‐1.1307904 ‐1.1290511 ‐1.0110162 ‐1.09 LOC391764 8104654 ‐1.2701516 ‐1.002062 ‐1.1434624 ‐1.14 8105989 ‐1.1451144 ‐1.4596436 ‐1.0017636 ‐1.20 FLJ40092|LOC441081 8106743 ‐1.0679519 ‐1.1786653 ‐1.2343683 ‐1.16 VCAN 8106776 ‐1.0397291 ‐1.1978922 ‐1.3132606 ‐1.18 COX7C 8107162 ‐1.169216 ‐1.0817614 ‐1.1604179 ‐1.14 LOC134505 8107857 ‐1.08141 ‐1.2999642 ‐1.1465683 ‐1.18 8108217 ‐1.8351796 ‐1.3044615 ‐1.069777 ‐1.40 TGFBI 8108832 ‐1.0880775 ‐1.1365558 ‐1.1258345 ‐1.12 KIAA0141 8109486 ‐1.0083958 ‐1.408165 ‐1.2317425 ‐1.22 8109612 ‐1.1516181 ‐1.0644593 ‐1.1284639 ‐1.11 ADRA1B 8109693 ‐1.1391666 ‐1.137372 ‐1.0107262 ‐1.10 8110668 ‐1.019779 ‐1.1163096 ‐1.1654474 ‐1.10 8112258 ‐1.2392946 ‐1.1847371 ‐1.1845342 ‐1.20 8112476 ‐1.0388913 ‐1.390674 ‐1.2577717 ‐1.23 FLJ40092|LOC441081 8112615 ‐1.1377119 ‐1.1698558 ‐1.1605732 ‐1.16 ENC1 8113658 ‐1.1629851 ‐1.3860811 ‐1.1062201 ‐1.22 LOC100132014 8113724 ‐1.0659521 ‐1.2787704 ‐1.3962063 ‐1.25 8114111 ‐1.7492881 ‐1.2535564 ‐1.0454283 ‐1.35 8114300 ‐1.2430623 ‐1.1569777 ‐1.0910265 ‐1.16 KLHL3 8114780 ‐1.246393 ‐1.0990406 ‐1.149239 ‐1.16 PCDH12 8117583 ‐1.0010923 ‐1.261282 ‐1.1729195 ‐1.15 HIST1H2AI|HIST1H3H 8117760 ‐1.0341878 ‐1.2558444 ‐1.1446778 ‐1.14 HLA‐F 8117869 ‐1.0342736 ‐1.1565961 ‐1.1581645 ‐1.12 TRIM39|TRIM39R

162

8118322 ‐1.2387516 ‐1.054911 ‐1.1256804 ‐1.14 SNORD52|C6orf48 8119423 ‐1.1338044 ‐1.161085 ‐1.2753941 ‐1.19 LOC221442 8119896 ‐1.1155329 ‐1.1786734 ‐1.011395 ‐1.10 8120057 ‐1.0891224 ‐1.2881671 ‐1.1179605 ‐1.17 8120247 ‐1.1962273 ‐1.1585102 ‐1.0011622 ‐1.12 8120271 ‐1.2747245 ‐1.0986568 ‐1.1650711 ‐1.18 FBXO9 8120460 ‐1.0332854 ‐1.204305 ‐1.211022 ‐1.15 LOC442229 8121130 ‐1.1169311 ‐1.0591099 ‐1.1551149 ‐1.11 8121741 ‐1.0830096 ‐1.1472548 ‐1.1256161 ‐1.12 8123644 ‐1.069684 ‐1.1721658 ‐1.1153345 ‐1.12 TUBB2A 8124553 ‐1.1262603 ‐1.0392927 ‐1.2153076 ‐1.13 ZKSCAN4 8125234 ‐1.0007002 ‐1.1907489 ‐1.1743116 ‐1.12 TNXB|TNXA 8125289 ‐1.0670452 ‐1.2549875 ‐1.1605295 ‐1.16 TNXA|TNXB 8125859 ‐1.0436913 ‐1.113749 ‐1.1736687 ‐1.11 TAF11 8126184 ‐1.3638836 ‐1.0601834 ‐1.1557374 ‐1.19 KIF6 8126442 ‐1.1755081 ‐1.1922375 ‐1.1053839 ‐1.16 TBCC 8126484 ‐1.3382208 ‐1.1082193 ‐1.1804925 ‐1.21 8127660 ‐1.1673032 ‐1.1541712 ‐1.0719168 ‐1.13 8127932 ‐1.1354865 ‐1.0172201 ‐1.1865585 ‐1.11 TBX18 8129392 ‐1.1739697 ‐1.1553757 ‐1.0564686 ‐1.13 KIAA0408|C6orf174 8129876 ‐1.1449543 ‐1.0052804 ‐1.2982544 ‐1.15 PBOV1 8130181 ‐1.121079 ‐1.1840845 ‐1.0383615 ‐1.11 8130660 ‐1.3080856 ‐1.2649312 ‐1.5105453 ‐1.36 8131919 ‐1.1996411 ‐1.1020441 ‐1.1206433 ‐1.14 NPY 8132245 ‐1.1235428 ‐1.010256 ‐1.2548987 ‐1.13 FLJ20712 8132250 ‐1.172944 ‐1.0751791 ‐1.1522795 ‐1.13 BMPER 8132830 ‐1.2432225 ‐1.6542674 ‐1.3796985 ‐1.43 8132843 ‐1.0427817 ‐1.7100819 ‐1.2685616 ‐1.34 FAM29A (HAUS6) 8135224 ‐1.0270871 ‐1.2254953 ‐1.1621358 ‐1.14 NF‐E4 8135915 ‐1.1398461 ‐1.0279154 ‐1.1221076 ‐1.10 HIG2 8136063 ‐1.0105916 ‐1.1974611 ‐1.1231867 ‐1.11 8136078 ‐1.3011712 ‐1.3270377 ‐1.4341699 ‐1.35 8136658 ‐1.1611602 ‐1.2796263 ‐1.1266106 ‐1.19 8136954 ‐1.1303827 ‐1.0141361 ‐1.1445726 ‐1.10 FAM139B|LOC652795 8137232 ‐1.1385602 ‐1.123844 ‐1.1237752 ‐1.13 GIMAP8 8137464 ‐1.2233617 ‐1.3429384 ‐1.1178629 ‐1.23 8137927 ‐1.3463244 ‐1.2116418 ‐1.2346703 ‐1.26 PAPOLB 8137931 ‐1.1515138 ‐1.2659065 ‐1.193679 ‐1.20 MMD2 8141560 ‐1.2044296 ‐1.0075576 ‐1.1465406 ‐1.12 TFR2 8142763 ‐1.1865283 ‐1.2030901 ‐1.4484302 ‐1.28 8143154 ‐1.2802789 ‐1.3185332 ‐1.0211422 ‐1.21 DGKI 8143463 ‐1.1763363 ‐1.1017003 ‐1.155456 ‐1.14 LOC136242 8144230 ‐1.0584028 ‐1.1357356 ‐1.2626258 ‐1.15 ZNF596 8144621 ‐1.1177125 ‐1.3881612 ‐1.1920708 ‐1.23 C8orf12 8144717 ‐1.0508242 ‐1.1644509 ‐1.1943152 ‐1.14 8144878 ‐1.1645075 ‐1.0226784 ‐1.2938935 ‐1.16 8146330 ‐1.1271794 ‐1.0504675 ‐1.1689004 ‐1.12 8146517 ‐1.039835 ‐1.2308658 ‐1.3265978 ‐1.20 CHCHD7 8146859 ‐1.0421897 ‐1.5340792 ‐1.2190603 ‐1.27 8148158 ‐1.1408248 ‐1.0781295 ‐1.3091106 ‐1.18 WDR67

163

8148474 ‐1.0087148 ‐1.1766478 ‐1.1217109 ‐1.10 8149275 ‐1.3396361 ‐1.2699971 ‐1.535426 ‐1.38 8149809 ‐1.1729199 ‐1.2145904 ‐1.0561767 ‐1.15 8149865 ‐1.1649675 ‐1.193713 ‐1.0087445 ‐1.12 EBF2 8150687 ‐1.2286807 ‐1.22496 ‐1.0197637 ‐1.16 8151215 ‐1.125452 ‐1.032116 ‐1.1211839 ‐1.09 LOC728774 8151223 ‐1.2021682 ‐1.161791 ‐1.0005025 ‐1.12 SLCO5A1 8151310 ‐1.1394893 ‐1.1520045 ‐1.029043 ‐1.11 EYA1 8152597 ‐1.1296767 ‐1.0231435 ‐1.2725624 ‐1.14 MRPL13 8153304 ‐1.1329118 ‐1.1144217 ‐1.0485513 ‐1.10 TSNARE1 8153320 ‐1.0592437 ‐1.2180276 ‐1.1353679 ‐1.14 8153363 ‐1.1578265 ‐1.1863923 ‐1.0210525 ‐1.12 CYP11B1 8154447 ‐1.0598923 ‐1.1965804 ‐1.1690933 ‐1.14 8154491 ‐1.1434791 ‐1.3207484 ‐1.3826722 ‐1.28 ADAMTSL1 8154512 ‐1.178788 ‐1.0925812 ‐1.1565659 ‐1.14 ADAMTSL1 8155508 ‐1.1404332 ‐1.0695829 ‐1.4007633 ‐1.20 8156523 ‐1.0563921 ‐1.1138076 ‐1.1858414 ‐1.12 8157027 ‐1.0185847 ‐1.258886 ‐1.1265827 ‐1.13 NIPSNAP3B 8157731 ‐1.1228418 ‐1.1581258 ‐1.0029973 ‐1.09 CRB2 8158081 ‐1.074475 ‐1.2064265 ‐1.1591502 ‐1.15 C9orf117 8158542 ‐1.1450524 ‐1.2212651 ‐1.0259508 ‐1.13 LOC100128501 8159004 ‐1.1418691 ‐1.2781873 ‐1.1137958 ‐1.18 RPL7A|SNORD24 8160439 ‐1.258943 ‐1.2016276 ‐1.0595245 ‐1.17 8160459 ‐1.1193366 ‐1.1142873 ‐1.0563816 ‐1.10 ELAVL2 8160663 ‐1.1149685 ‐1.2610207 ‐1.1951705 ‐1.19 AQP7 8160900 ‐1.1776699 ‐1.1401237 ‐1.0365562 ‐1.12 C9orf144|RP11‐251H13.2 8161513 ‐1.1618793 ‐1.0299528 ‐1.1498747 ‐1.11 PGM5P2 8161520 ‐1.215008 ‐1.0410358 ‐1.1574442 ‐1.14 PGM5P2 8161943 ‐1.096678 ‐1.5495085 ‐1.179431 ‐1.28 LOC644714 8162214 ‐1.1449199 ‐1.1378958 ‐1.0113769 ‐1.10 LOC645961 8162929 ‐1.1644909 ‐1.1324682 ‐1.1883187 ‐1.16 OR13C4 8163533 ‐1.2028567 ‐1.194494 ‐1.0311501 ‐1.14 FLJ31713 8163618 ‐1.1829689 ‐1.3177207 ‐1.0297363 ‐1.18 TNFSF15 8163637 ‐1.2208376 ‐1.1397737 ‐1.0292698 ‐1.13 TNC 8164438 ‐1.2453928 ‐1.1182964 ‐1.0166935 ‐1.13 8166565 ‐1.1291627 ‐1.183872 ‐1.0187716 ‐1.11 MAGEB18 8166619 ‐1.087334 ‐1.1201719 ‐1.1349844 ‐1.11 MAGEB1|MAGEB4 8167027 ‐1.1201622 ‐1.2636591 ‐1.018737 ‐1.13 RGN 8167854 ‐1.2211274 ‐1.1944569 ‐1.1137935 ‐1.18 APEX2 8168079 ‐1.8974931 ‐2.0799263 ‐1.214796 ‐1.73 8168855 ‐1.1354032 ‐1.1902515 ‐1.0709162 ‐1.13 ARMCX4 8169002 ‐1.0021577 ‐1.1306237 ‐1.2562608 ‐1.13 8169634 ‐1.1175219 ‐1.1776197 ‐1.2227252 ‐1.17 8170015 ‐1.0306959 ‐1.1264628 ‐1.193451 ‐1.12 ZNF449 8170247 ‐1.1763655 ‐1.2534027 ‐1.0686966 ‐1.17 CXorf18 8170282 ‐1.1169677 ‐1.1205648 ‐1.0308256 ‐1.09 SPANXN4 8171172 ‐1.465611 ‐1.4290162 ‐1.1539816 ‐1.35 MXRA5 8171449 ‐1.1684867 ‐1.1505685 ‐1.0259542 ‐1.12 ACE2 8172028 ‐1.0661342 ‐1.138698 ‐1.4076415 ‐1.20 8172191 ‐1.0958965 ‐1.2049588 ‐1.1820956 ‐1.16

164

8172453 ‐1.1303902 ‐1.0334401 ‐1.251748 ‐1.14 TIMM17B 8173208 ‐1.1529659 ‐1.1574439 ‐1.0678133 ‐1.13 SPIN4 8173366 ‐1.2473128 ‐1.2616489 ‐1.1043935 ‐1.20 P2RY4 8174119 ‐1.0489181 ‐1.238366 ‐1.1494266 ‐1.15 ZMAT1 8174513 ‐1.1753209 ‐1.1280079 ‐1.062383 ‐1.12 CHRDL1 8175537 ‐1.1147419 ‐1.2108519 ‐1.0806557 ‐1.14 CXorf18 8175900 ‐1.1410948 ‐1.1629108 ‐1.0659852 ‐1.12 ARHGAP4 8176427 ‐1.1547782 ‐1.1477331 ‐1.0614046 ‐1.12 8177114 ‐1.1616321 ‐1.1432308 ‐1.0622799 ‐1.12 8177269 ‐1.1526574 ‐1.1473463 ‐1.1023531 ‐1.13 8177717 ‐1.0475904 ‐1.1508293 ‐1.1399816 ‐1.11 HLA‐F 8177770 ‐1.0348558 ‐1.1373097 ‐1.1595732 ‐1.11 TRIM39|TRIM39R 8178193 ‐1.00785 ‐1.1741405 ‐1.228931 ‐1.14 HLA‐DRA 8178712 ‐1.0386152 ‐1.2842276 ‐1.1668032 ‐1.16 TNXB|TNXA 8179088 ‐1.0336882 ‐1.1408814 ‐1.1610003 ‐1.11 TRIM39|TRIM39R 8179935 ‐1.0352781 ‐1.2866459 ‐1.1692953 ‐1.16 TNXB|TNXA 7897076 1.1510614 1.0922247 1.1336693 1.13 7897295 1.1296413 1.1846787 1.1459758 1.15 TAS1R1 7897620 1.0159584 1.1886483 1.0875065 1.10 PGD 7897648 1.0742676 1.0919988 1.0921133 1.09 PEX14 7897774 1.081394 1.2034408 1.1758256 1.15 CLCN6 7897955 1.0289028 1.2011805 1.171541 1.13 AADACL4 7897964 1.1254687 1.0040089 1.131356 1.09 7898355 1.2891978 1.2042652 1.2153044 1.24 7898516 1.009878 1.3230559 1.2712504 1.20 ACTL8 7898537 1.2447776 1.1138451 1.2788566 1.21 PAX7 7898549 1.2348049 1.3195752 1.1745745 1.24 MRTO4 7898663 1.0843025 1.100151 1.0889727 1.09 PINK1 7898739 1.2353425 1.0166035 1.1003829 1.12 CDC42 7898793 1.1355668 1.0965538 1.139344 1.12 C1QA 7898856 1.2672035 1.2139896 1.2527413 1.24 7899075 1.085975 1.0914208 1.0482724 1.08 EXTL1 7899462 1.1110444 1.093446 1.0471518 1.08 RCC1|SNHG3‐RCC1 7900167 1.0893992 1.1468533 1.0830995 1.11 CDCA8 7900201 1.1359103 1.3685161 1.0178107 1.17 UTP11L 7900228 1.1390303 1.1057076 1.0194008 1.09 NDUFS5 7901477 1.2765534 1.1397523 1.0855181 1.17 7901744 1.045477 1.1447905 1.1884142 1.13 7902205 1.2056524 1.1910024 1.2658346 1.22 IL12RB2 7902223 1.2519118 1.3212445 1.0250919 1.20 7902623 1.0964338 1.0446494 1.0905982 1.08 DNASE2B 7902891 1.1400611 1.2392255 1.0652698 1.15 ZNF326 7903113 1.0922941 1.1563128 1.1760311 1.14 7903117 1.1081648 1.1664362 1.0551777 1.11 7903294 1.0723974 1.1966654 1.1622926 1.14 HIAT1 7903945 1.0037584 1.1432108 1.1668503 1.10 CHIA 7903972 1.1381075 1.0802768 1.106901 1.11 ATP5F1 7904166 1.1696506 1.1603684 1.1469288 1.16 7904244 1.0837214 1.0459411 1.0839715 1.07 C1orf161 7904923 1.2396585 1.3253466 1.1765984 1.25

165

7905145 1.0588506 1.1196725 1.1170189 1.10 7905500 1.0511688 1.2970569 1.1326501 1.16 LCE2D 7905510 1.0440439 1.0986836 1.0889133 1.08 LCE4A 7905831 1.0230776 1.1363654 1.1631527 1.11 FLAD1 7905848 1.1692652 1.0885009 1.0860368 1.11 LENEP 7905862 1.1145992 1.1555128 1.2117912 1.16 DCST1 7905986 1.3869632 1.7196069 1.1069486 1.40 FDPS 7906056 1.1189053 1.1951638 1.0007069 1.10 MSTO1 7906374 1.0072769 1.1109996 1.1015762 1.07 OR6K6 7906602 1.0904256 1.0040228 1.1228875 1.07 VANGL2 7906904 1.4218113 1.2711087 1.1200882 1.27 HSD17B7|HSD17B7P2 7906948 1.1174898 1.4249172 1.1140597 1.22 7907156 1.0840542 1.0169584 1.2204956 1.11 XCL1 7907213 1.1200727 1.1662153 1.1467499 1.14 SCYL1BP1 7907396 1.0090443 1.1240501 1.2126802 1.12 C1orf105 7907655 1.3123606 1.1973704 1.2120551 1.24 7907972 1.1202178 1.142938 1.029045 1.10 C1orf120 7908347 1.0988703 1.062123 1.1159657 1.09 OCLM 7908931 1.0695376 1.3283019 1.1153204 1.17 OPTC 7909146 1.0852911 1.2265384 1.0553892 1.12 FAM72A|FAM72B|GCUD2 7909400 1.1327516 1.2043886 1.0362749 1.12 CD46 7909839 1.1213129 1.3361613 1.2022796 1.22 7909866 1.1986332 1.101886 1.0296522 1.11 MOSC2 7909896 1.1353232 1.0421346 1.141205 1.11 7910054 1.0979434 1.0001305 1.1058396 1.07 DNAH14|C1orf67 7910140 1.0114939 1.0947012 1.1365994 1.08 7910217 1.1021385 1.1127702 1.0617039 1.09 WNT3A 7910379 1.148149 1.3181341 1.0679494 1.18 DUSP5P 7910383 1.1402104 1.0086511 1.0853363 1.08 7910694 1.0433437 1.1133544 1.1981535 1.12 EDARADD|ENO1P 7911108 1.2828081 1.1870481 1.1030408 1.19 7911199 1.0091224 1.1293362 1.1507008 1.10 C1orf150 7911213 1.1964147 1.1321957 1.0847105 1.14 7911258 1.1114 1.3891696 1.0549266 1.19 OR2L3 7911285 1.0558071 1.140611 1.1309208 1.11 OR2T5|OR2T29 7911333 1.1230642 1.1506171 1.0293548 1.10 MGC70863|FLJ45055 7911351 1.1160182 1.1789832 1.1202208 1.14 FLJ22639 7911444 1.1064351 1.1174395 1.0151646 1.08 UBE2J2 7911600 1.0068133 1.279063 1.0944381 1.13 NADK 7912527 1.0142835 1.1646087 1.1273626 1.10 7912622 1.0601285 1.1916683 1.1488372 1.13 LRRC38 7912627 1.1789157 1.1858736 1.0238465 1.13 7912968 1.0082502 1.1276168 1.1568687 1.10 TAS1R2 7914015 1.1174606 1.14287 1.1878577 1.15 FAM46B 7914326 1.079862 1.1228778 1.1078744 1.10 NKAIN1 7914334 1.1119776 1.2920896 1.1981182 1.20 WDR57 7914342 1.1617012 1.3512344 1.5707223 1.36 FABP3 7914550 1.1483771 1.0828764 1.0230229 1.08 ZBTB8OS 7914555 1.0936936 1.0962142 1.0451782 1.08 7914630 1.050928 1.1656728 1.1586297 1.13 TRIM62

166

7914748 1.0315499 1.1066 1.1204625 1.09 7914758 1.0936173 1.1922749 1.014112 1.10 DLGAP3 7914898 1.0482907 1.1133273 1.1456944 1.10 C1orf78 7914992 1.0759733 1.2026142 1.1429445 1.14 7915238 1.0408008 1.1267428 1.2073572 1.12 NT5C1A 7915261 1.0372485 1.085276 1.1448836 1.09 TRIT1 7915275 1.3849834 1.3089769 1.042553 1.25 7915347 1.0418375 1.1573647 1.211245 1.14 CITED4 7916432 1.2098842 1.64858 1.2048577 1.35 DHCR24 7916541 1.0414288 1.1376148 1.3182639 1.17 DAB1 7917741 1.1115043 1.0984734 1.0592563 1.09 TMED5 7917902 1.0128384 1.1041617 1.1562387 1.09 7917942 1.6705587 1.1041701 1.1271787 1.30 FLJ35409 7918235 1.0988994 1.2054938 1.027409 1.11 C1orf62 7918550 1.0542278 1.159891 1.1082348 1.11 7918558 1.2091035 1.2154393 1.2108947 1.21 KCND3 7918755 1.2315357 1.0314361 1.1361754 1.13 DENND2C 7919038 1.0523465 1.1045549 1.1197166 1.09 WARS2 7919578 1.054149 1.255575 1.2388761 1.18 ATG9B 7919591 1.3053297 1.6244799 1.0333732 1.32 GCUD2|FAM72D|FAM72A 7919598 1.003229 1.1634614 1.1458443 1.10 LOC440570 7920971 1.2899604 1.2021253 1.0843492 1.19 C1orf85 7921014 1.1146063 1.1519368 1.0498892 1.11 MEF2D 7921319 1.0344708 1.1278032 1.0983605 1.09 FCRL1 7922297 1.1045629 1.2463247 1.0308124 1.13 7922610 1.1290392 1.0493231 1.0866148 1.09 ABL2 7922994 1.0058644 1.1389682 1.093652 1.08 FAM5C 7923183 1.0312009 1.1985829 1.2430952 1.16 ZNF281 7923440 1.256046 1.0613726 1.1499282 1.16 7923705 1.0341591 1.0831965 1.2549328 1.12 7924150 1.0795887 1.1059997 1.1198283 1.10 TMEM206 7924603 1.0399779 1.296999 1.2516326 1.20 LBR 7924823 1.0687306 1.1632595 1.0935035 1.11 JMJD4 7924956 1.0293341 1.0849783 1.0909822 1.07 ABCB10 7925043 1.1420517 1.030057 1.1220131 1.10 EXOC8 7925229 1.092141 1.1935173 1.0363517 1.11 B3GALNT2 7925500 1.0417713 1.1028578 1.1574033 1.10 CHML 7925525 1.08177 1.1719398 1.1723881 1.14 CEP170 7925747 1.0585822 1.0971439 1.1531976 1.10 OR2T29|OR2T5 7925761 1.0779314 1.1119624 1.230066 1.14 OR14I1 7925763 1.1406046 1.0677868 1.1155417 1.11 SH3BP5L 7926117 1.2071596 1.0595473 1.3675073 1.21 LOC389936 7926817 1.10835 1.0796828 1.1206197 1.10 7927513 1.1023061 1.2055846 1.0079533 1.11 FAM21C|FAM21D|FAM21A 7927560 1.0337442 1.1759382 1.0979863 1.10 FAM21A|FAM21B|FAM21C 7927784 1.0175204 1.1400449 1.0830547 1.08 MGC14425 7927872 1.1065938 1.1735663 1.0344774 1.10 7928489 1.1326423 1.1186335 1.028298 1.09 7928524 1.0620534 1.1777104 1.0836878 1.11 VDAC2 7928695 1.1559491 1.0789545 1.2232057 1.15 C10orf58

167

7928705 1.0606804 1.1212865 1.1762773 1.12 TSPAN14 7928838 1.0029606 1.3414345 1.2627882 1.20 LDB3 7929282 1.0360326 1.1672171 1.1315142 1.11 HHEX 7929550 1.0913357 1.1902641 1.0615526 1.11 CCNJ 7929677 1.1078944 1.2551439 1.3218403 1.23 PI4K2A 7929744 1.1790128 1.0968956 1.1068331 1.13 NKX2‐3 7929816 1.5850128 1.9766299 1.3361237 1.63 SCD 7929945 1.119577 1.126624 1.0864425 1.11 7929952 1.1187308 1.3101586 1 1.14 7930378 1.027271 1.0892463 1.2632704 1.13 7931159 1.1748859 1.0861951 1.0713869 1.11 PSTK 7931346 1.196999 1.1290709 1.1652806 1.16 7931469 1.0455859 1.0909204 1.1101393 1.08 PWWP2B 7931561 1.1201508 1.0559185 1.083648 1.09 ZNF511 7931681 1.3009871 1.3304496 1.2147334 1.28 7931754 1.231239 1.4713528 1.2925997 1.33 IDI1 7932014 1.2965382 1.1418325 1.0369838 1.16 FLJ45983 7933129 1.1843921 1.1452696 1.0921273 1.14 LOC100129482|ZNF37B 7933147 1.1312752 1.0122029 1.2116097 1.12 7933192 1.0854497 1.3804832 1.1720936 1.21 HNRNPA3P1 7933296 1.1154513 1.292157 1.1341951 1.18 7933421 1.1193206 1.2825233 1.1166298 1.17 7933638 1.0474097 1.1015114 1.1408814 1.10 FLJ31958 7933821 1.1985829 1.1087329 1.0333914 1.11 RHOBTB1 7933933 1.2579958 1.2026166 1.1425769 1.20 DNAJC12 7934074 1.0786033 1.1573533 1.144447 1.13 TACR2 7934244 1.1751884 1.1735554 1.0545021 1.13 DNAJB12 7934271 1.136074 1.0507158 1.087107 1.09 PLA2G12B 7934544 1.0885196 1.0293219 1.0935862 1.07 COMTD1 7934613 1.1336018 1.1092608 1.0699803 1.10 7934698 1.0384477 1.0872359 1.1559892 1.09 SFTPA2B|SFTPA1B 7934708 1.0388824 1.0823795 1.1559365 1.09 SFTPA2B|SFTPA1B 7934812 1.1261479 1.0725825 1.0846485 1.09 WAPAL 7934868 1.1136621 1.2912312 1.1214911 1.18 7935027 1.0178965 1.1333126 1.1398641 1.10 IDE 7935139 1.3728848 1.0030696 1.1046494 1.16 PIPSL|LOC100101438 7935421 1.2131793 1.091393 1.033727 1.11 FRAT2 7935588 1.0740135 1.1189808 1.1826713 1.13 HPS1 7935707 1.1037136 1.0849199 1.0068731 1.07 CHUK 7935855 1.0938482 1.0066755 1.1499692 1.08 LBX1 7936050 1.0600826 1.4191047 1.1628467 1.21 CYP17A1 7936683 1.0954373 1.1046684 1.0176779 1.07 TIAL1 7937287 1.0887424 1.0952773 1.0305613 1.07 PSMD13 7937698 1.0787804 1.1278588 1.200434 1.14 LOC399851 7938059 1.1703485 1.4165095 1.1346682 1.24 OR52N2 7938111 1.1257846 1.2397081 1.013732 1.13 FXC1 7938299 1.2610881 1.2292888 1.2756087 1.26 7938592 1.0952924 1.1346829 1.0691513 1.10 MLSTD2 7938683 1.1110739 1.04629 1.1285176 1.10 LOC441453 7938702 1.3140308 1.1609738 1.0365065 1.17

168

7938730 1.177552 1.0083178 1.0988424 1.09 MYOD1 7938746 1.1688286 1.0548737 1.1260433 1.12 MRGPRX4 7938762 1.1071415 1.0946639 1.1324207 1.11 GTF2H1 7938812 1.0423496 1.1353784 1.2142669 1.13 TMEM86A 7939102 1.1423005 1.0973692 1.039132 1.09 ELP4 7939184 1.0911779 1.0858637 1.0518315 1.08 TCP11L1 7939368 1.0951831 1.0864446 1.0221207 1.07 TRIM44 7939418 1.0984491 1.0007416 1.1906186 1.10 7939465 1.111458 1.2245847 1.0204897 1.12 HSD17B12 7939640 1.2565863 1.0807068 1.1328729 1.16 7939657 1.216549 1.0448631 1.2185317 1.16 DGKZ 7939751 1.1183647 1.0890802 1.0074081 1.07 NR1H3 7939922 1.0266309 1.2180333 1.0940413 1.11 7940187 1.1143329 1.0950109 1.1184886 1.11 OR4D11 7940565 1.3872017 1.7684774 1.2019056 1.45 FADS2 7940643 1.1741626 1.181732 1.0231256 1.13 ASRGL1 7941401 1.0975063 1.0172814 1.2586476 1.12 OVOL1 7941879 1.1098359 1.1003135 1.1989672 1.14 TBC1D10C 7942261 1.0690874 1.0934864 1.2273028 1.13 KRTAP5‐9 7942379 1.0375736 1.1976575 1.0957266 1.11 7942812 1.3635063 1.2716907 1.0301949 1.22 7942964 1.2704325 1.4564365 1.0640963 1.26 TMEM135 7943345 1.1077659 1.0215929 1.1010072 1.08 7943777 1.0491816 1.13531 1.153611 1.11 7944656 1.2045447 1.2950277 1.2545313 1.25 SC5DL 7944763 1.1613625 1.253128 1.0034267 1.14 7944765 1.0282325 1.0983852 1.2368402 1.12 7944795 1.1469857 1.1459273 1.1776855 1.16 OR4D5 7944843 1.0414501 1.1149102 1.2490575 1.14 OR8A1 7945031 1.0249689 1.0884944 1.1658881 1.09 7945069 1.1631116 1.0412408 1.2805629 1.16 7945241 1.1454351 1.1015193 1.0055007 1.08 C11orf44 7945657 1.3039912 1.4724731 1.2182237 1.33 KRTAP5‐4 7945660 1.5319107 1.4647156 1.1134888 1.37 FAM99A|FAM99B 7945781 1.0217073 1.1350085 1.1727331 1.11 PHLDA2 7945892 1.1008136 1.1695052 1.1381199 1.14 7946008 1.00011 1.1394441 1.1971904 1.11 7946071 1.129915 1.1219008 1.0397937 1.10 OR51B5 7946082 1.111807 1.0947167 1.0247531 1.08 UBQLNL 7946278 1.1363707 1.0970964 1.0222907 1.09 OR2AG2 7946504 1.0945919 1.0172179 1.144433 1.09 TMEM41B 7946849 1.060365 1.1535525 1.2143649 1.14 7947332 1.0968391 1.1908724 1.0286076 1.11 IMMP1L 7947423 1.1941888 1.3714901 1.6846529 1.42 7947490 1.0571951 1.2150092 1.2498909 1.17 APIP 7947563 1.129081 1.0794399 1.1245191 1.11 ALX4 7947969 1.0690955 1.1012129 1.0934912 1.09 FNBP4 7948148 1.3908156 1.4505529 1.096228 1.31 OR5M10 7948176 1.0941694 1.0229589 1.0897971 1.07 TNKS1BP1 7948314 1.194864 1.0590371 1.2500573 1.17 OR10Q1

169

7948324 1.0332417 1.2115849 1.1202226 1.12 OR5B3 7948612 1.389518 1.7409058 1.272718 1.47 FADS1|FADS3 7950067 1.3876474 1.832188 1.1571846 1.46 DHCR7 7950136 1.1195134 1.0001894 1.2956498 1.14 PHOX2A 7950162 1.0858828 1.1071364 1.1212779 1.10 PDE2A 7950501 1.1048265 1.0198611 1.1152036 1.08 GDPD5 7950726 1.1184409 1.3252301 1.1168975 1.19 MGC33846 7951040 1.0746651 1.1269238 1.1651138 1.12 GPR83 7951157 1.1260823 1.1829653 1.083151 1.13 CCDC82 7951246 1.1862001 1.0646971 1.1608717 1.14 MMP8 7952022 1.0447702 1.0909963 1.1357466 1.09 AMICA1 7953590 1.1206003 1.0188628 1.2319261 1.12 7953765 1.0099763 1.2900819 1.2698985 1.19 FAM80B 7953967 1.1011623 1.0506123 1.1270609 1.09 PRH2|PRB4|PRH1 7954938 1.1795764 1.0999851 1.1194687 1.13 7954985 1.076839 1.1501229 1.095421 1.11 TMEM117 7954995 1.0341314 1.2999603 1.1168126 1.15 7955535 1.0911982 1.0106016 1.1407554 1.08 ACVR1B 7955817 1.1311092 1.0987116 1.0532029 1.09 PCBP2 7955983 1.1368139 1.1433543 1.363487 1.21 OR10A7 7956005 1.0583957 1.1199627 1.106764 1.10 OR2AP1 7956522 1.0336608 1.1303023 1.405738 1.19 KIF5A 7956741 1.0825801 1.1973653 1.1008457 1.13 7956987 1.0862237 1.091143 1.046039 1.07 PRO2268 7957052 1.0643302 1.1715544 1.0828124 1.11 CCT2 7957417 1.2373077 1.014112 1.0966556 1.12 TMTC2 7957499 1.1622119 1.1353277 1.1585225 1.15 7957514 1.0783886 1.1922607 1.0920544 1.12 PLEKHG7 7957570 1.1432176 1.0110654 1.1663827 1.11 PLXNC1 7957631 1.088002 1.3721077 1.1352185 1.20 7957649 1.1610371 1.224155 1.080859 1.16 SNRPF 7957850 1.1165051 1.0808126 1.2338009 1.14 GAS2L3 7957960 1.1097082 1.0739101 1.0983471 1.09 7958051 1.0225999 1.1275226 1.1127305 1.09 ASCL1 7958147 1.1144184 1.0850046 1.0941907 1.10 TDG 7958724 1.02681 1.0909945 1.1304996 1.08 LOC100131138 7958884 1.1535778 1.2142336 1.2014108 1.19 OAS1 7959012 1.370048 1.1330421 1.3531141 1.29 7959251 1.0851576 1.2516458 1.0322863 1.12 P2RX7 7959322 1.1088755 1.1379831 1.0196953 1.09 PSMD9 7959386 1.0339738 1.1046001 1.1514525 1.10 LRRC43 7960730 1.2675259 1.4684078 1.0755075 1.27 MBOAT5 7960863 1.081137 1.1636052 1.1822531 1.14 7962137 1.2396445 1.2175374 1.3502492 1.27 7962146 1.032202 1.2679836 1.1003829 1.13 FAM60A 7962246 1.0311723 1.0905982 1.1257217 1.08 7962479 1.4602299 1.4280101 1.5332865 1.47 7963203 1.0689492 1.1160101 1.0937091 1.09 LOC121006 7963289 1.083213 1.0129496 1.1010953 1.07 BIN2 7963333 1.0512803 1.0989691 1.1194476 1.09 KRT80

170

7963406 1.1009116 1.2505653 1.0876651 1.15 KRT6B 7963664 1.0119687 1.1286112 1.1865722 1.11 SP7 7963817 1.144295 1.4497429 1.0557984 1.22 GTSF1 7964303 1.0955354 1.3246254 1.0217543 1.15 TAC3 7964870 1.1928493 1.1166136 1.0773939 1.13 7964907 1.1051848 1.1004604 1.0611215 1.09 PTPRR 7965384 1.220489 1.0686723 1.0885041 1.13 C12orf12 7965436 1.1297354 1.098101 1.0283854 1.09 EEA1 7965469 1.1091039 1.4083502 1.1535625 1.22 7965573 1.1028498 1.036066 1.0910089 1.08 NTN4 7966072 1.025804 1.1294109 1.1071484 1.09 PRDM4 7966223 1.1547279 1.3182181 1.1657082 1.21 7966441 1.2536273 1.1330818 1.0028051 1.13 C12orf47 7967021 1.1387017 1.0625207 1.1101751 1.10 PXN 7967028 1.2020141 1.3786241 1.090111 1.22 RNU4‐2 7967034 1.0327302 1.151292 1.2710814 1.15 PLA2G1B 7967109 1.111036 1.0445648 1.2158339 1.12 C12orf43 7967544 1.0834718 1.333641 1.1164919 1.18 SCARB1 7968052 1.2335387 1.102786 1.0110296 1.12 C1QTNF9 7968197 1.2333361 1.1292721 1.0094873 1.12 7968746 1.160036 1.1529062 1.0059245 1.11 WBP4 7969241 1.3022971 1.095593 1.0411228 1.15 7969256 1.0910633 1.3244904 1.1587361 1.19 LOC220115|LOC100134095 7969286 1.1325064 1.0711763 1.1349784 1.11 RP11‐431O22.2 7969569 1.0429975 1.1060424 1.2404983 1.13 SLITRK5 7969576 1.0354006 1.1117365 1.1242694 1.09 MIRHG1 7969881 1.0288134 1.2028539 1.1012675 1.11 TPP2 7969933 1.3986505 1.580853 1.2018008 1.39 7970542 1.3067088 1.1880211 1.3679639 1.29 7970949 1.0546083 1.196276 1.2209293 1.16 MAB21L1 7971013 1.0821723 1.0899265 1.1500072 1.11 7971124 1.006146 1.3127251 1.178963 1.17 UFM1 7971222 1.0251362 1.2545222 1.1603802 1.15 MTRF1 7971539 1.023081 1.1282327 1.2724336 1.14 7971573 1.0836498 1.1699401 1.0020424 1.09 RCBTB2 7971602 1.027613 1.1974037 1.1060293 1.11 RCBTB1 7972601 1.0371648 1.2099782 1.0865897 1.11 NALCN 7972650 1.1956738 1.0518117 1.1586274 1.14 FGF14 7972663 1.1712626 1.1278383 1.1559732 1.15 7972674 1.170441 1.2206416 1.1669599 1.19 C13orf27 7972682 1.1372461 1.1010579 1.0048937 1.08 KDELC1 7973014 1.1571643 1.1704097 1.0060112 1.11 OR4K5 7973032 1.0701144 1.1795222 1.4005175 1.22 OR11H7P 7973371 1.1175157 1.0799937 1.1743419 1.12 C14orf119 7973618 1.0128793 1.117256 1.1019509 1.08 IRF9 7973894 1.084861 1.103896 1.1849309 1.12 LOC644384 7973900 1.277512 1.2312207 1.0148386 1.17 C14orf19 7973943 1.112018 1.0763191 1.084629 1.09 INSM2 7974257 1.0565803 1.1360556 1.1161568 1.10 ATP5S 7974341 1.194222 1.0767535 1.1049204 1.13 GNG2

171

7974814 1.1724974 1.0842123 1.0359843 1.10 7975613 1.0894377 1.0432116 1.1125485 1.08 ACOT6 7976059 1.1047761 1.0872284 1.0662763 1.09 7976156 1.1090075 1.2369903 1.08833 1.14 7976241 1.106283 1.0497328 1.1598787 1.11 7976571 1.0948288 1.2413256 1.161264 1.17 C14orf129 7976783 1.0099055 1.124259 1.1557459 1.10 DLK1 7976826 1.1132116 1.1682184 1.094083 1.13 SNORD114‐26 7977340 1.1935812 1.0985421 1.1226556 1.14 BTBD6 7977592 1.067013 1.109497 1.0887517 1.09 GAFA1 7977801 1.0938928 1.3290436 1.0973006 1.17 7977906 1.031997 1.1426003 1.1025349 1.09 ACIN1 7977933 1.12468 1.1275591 1.1175241 1.12 SLC7A8 7978628 1.0935526 1.1938313 1.0122821 1.10 PPP2R3C 7978776 1.1141639 1.22823 1.0455937 1.13 C14orf106 7979085 1.0848105 1.1967698 1.0032496 1.09 PYGL 7979129 1.0666901 1.1493925 1.1010994 1.11 7979721 1.300675 1.3126569 1.01888 1.21 C14orf83 7979743 1.1067533 1.3534411 1.2476686 1.24 RDH11 7980003 1.1816949 1.2180598 1.1220624 1.17 7980304 1.5645561 1.2236837 1.2202382 1.34 7980309 1.0865674 1.3893831 1.0114367 1.16 C14orf1 7980485 1.0197796 1.1656303 1.416423 1.20 DIO2 7980496 1.0140839 1.2891518 1.1298579 1.14 C14orf145 7980718 1.0863254 1.0807147 1.1453567 1.10 7980828 1.3185114 1.1558684 1.0060302 1.16 CCDC88C 7981290 1.094767 1.0923302 1.0449519 1.08 WARS 7981309 1.033831 1.1132084 1.1284893 1.09 BEGAIN 7981320 1.1120496 1.109826 1.1998976 1.14 MEG3 7981346 1.1711774 1.2496294 1.0841571 1.17 RAGE 7981951 1.3169165 1.2044766 1.2997162 1.27 SNRPN|SNORD116‐2 7982341 1.113481 1.1026537 1.0791156 1.10 CHRNA7 7982574 1.1251559 1.1889567 1.0456314 1.12 FAM98B 7982663 1.1635729 1.0039536 1.1874813 1.12 BUB1B 7982792 1.0273577 1.1636568 1.1217232 1.10 RAD51 7983051 1.020741 1.0987304 1.1223521 1.08 7983405 1.1588959 1.081942 1.2900145 1.18 DUOXA2 7984016 1.1804863 1.163158 1.1531154 1.17 FAM148A 7984152 1.062806 1.2502553 1.1483809 1.15 FBXL22 7984952 1.0143697 1.0855261 1.1534745 1.08 C15orf39 7985025 1.180257 1.093124 1.0016623 1.09 ODF3L1 7985259 1.048015 1.1042227 1.168297 1.11 ZFAND6 7985266 1.2341272 1.2666059 1.195359 1.23 7985577 1.127577 1.0449512 1.0859976 1.09 ZSCAN2 7986092 1.1753209 1.2619061 1.0093756 1.15 FURIN 7986442 1.0110474 1.2182997 1.6815044 1.30 7987439 1.0421199 1.1289489 1.137474 1.10 GPR176 7987454 1.1417826 1.0999888 1.1277804 1.12 BMF 7987466 1.0325743 1.1813614 1.1756399 1.13 7987554 1.1673186 1.2118264 1.0806401 1.15 DNAJC17

172

7987772 1.131289 1.086165 1.0134532 1.08 EHD4 7988344 1.4107089 1.1495728 1 1.19 7988605 1.2267666 1.0405142 1.1007311 1.12 COPS2 7988767 1.2261326 1.1154406 1.1879791 1.18 CYP19A1 7988970 1.1497496 1.1412175 1.1024679 1.13 KIAA1370 7989132 1.1146766 1.1231774 1.0022594 1.08 RFXDC2 7989243 1.1848475 1.3885108 1.0430361 1.21 7989387 1.0235163 1.1058377 1.0896668 1.07 VPS13C 7989628 1.0783031 1.1652937 1.1153659 1.12 CSNK1G1 7990080 1.0855659 1.1390898 1.007214 1.08 LARP6 7990391 1.1897768 1.1844732 1.1232929 1.17 CYP1A1 7990736 1.1062205 1.0785295 1.0869116 1.09 ADAMTS7 7991332 1.0101154 1.1088485 1.1400498 1.09 MESP1 7991512 1.2363735 1.0002819 1.1832633 1.14 C15orf51 7991540 1.085924 1.2476517 1.4647379 1.27 7991598 1.3920518 1.0886657 1.0132167 1.16 SNRPA1 7992010 1.0860816 1.2000749 1.4015839 1.23 RHBDL1 7992269 1.0117226 1.1604125 1.0945789 1.09 7992439 1.1792384 1.2640398 1.0726395 1.17 GFER 7992795 1.1520286 1.3738303 1.0463477 1.19 THOC6 7992861 1.112365 1.0961595 1.174602 1.13 7992987 1.1692795 1.1235157 1.0373291 1.11 HMOX2 7993114 1.0809804 1.0895361 1.1123701 1.09 C16orf68 7993310 1.0996104 1.1430331 1.0326347 1.09 MKL2 7993737 1.0832559 1.1292201 1.2053181 1.14 ACSM2A|ACSM2B 7993798 1.1459872 1.0068792 1.1207037 1.09 7993821 1.1516284 1.012576 1.1332433 1.10 FLJ41766 7994582 1.02038 1.1979686 1.1432679 1.12 SULT1A3|GIYD1|GIYD2 7994781 1.0222586 1.1966896 1.1289283 1.12 SULT1A3|GIYD1|GIYD2 7994804 1.1244626 1.0482734 1.1149449 1.10 MYLPF 7994824 1.2689855 1.266793 1.0655808 1.20 7994926 1.2178738 1.1528318 1.0649657 1.15 SNORA30 7995258 1.0618587 1.2270639 1.119456 1.14 ZNF267 7995310 1.0873858 1.0350366 1.1259755 1.08 MGC34800 7995477 1.3889308 1.2058476 1.095362 1.23 7995712 1.0662756 1.2237407 1.0878661 1.13 CAPNS2 7995895 1.1074691 1.0690786 1.1036417 1.09 HERPUD1 7996185 1.0644273 1.1119875 1.108667 1.10 MMP15 7996593 1.0860633 1.1640873 1.009804 1.09 CTCF 7996608 1.0863581 1.1592932 1.0789808 1.11 RLTPR 7996759 1.3815032 1.1655991 1.1755847 1.24 7996837 1.092714 1.0253667 1.1531295 1.09 CDH1 7996947 1.0351788 1.1888369 1.1153455 1.11 CYB5B 7997336 1.0015212 1.142313 1.1092359 1.08 KIAA1576 7997396 1.1956921 1.0283272 1.1252043 1.12 ATMIN 7997626 1.0137795 1.1514218 1.1088341 1.09 C16orf44 7997700 1.1996664 1.178701 1.0882423 1.16 7997746 1.048049 1.1584375 1.1705872 1.13 JPH3 7997962 1.1052253 1.134366 1.0507773 1.10 DPEP1 7998759 1.1218729 1.1241769 1.2996548 1.18 LOC283871

173

7999253 1.1995828 1.1639338 1.1589242 1.17 PPL 7999387 1.1006489 1.2138785 1.0006566 1.11 EMP2 7999427 1.4258915 1.0068566 1.2340657 1.22 TNP2 7999435 1.2868649 1.0909429 1.0098978 1.13 PRM2 7999752 1.002207 1.0943358 1.1343687 1.08 7999965 1.0174118 1.1235164 1.1832852 1.11 ACSM2B|ACSM2A 8000407 1.0893899 1.1842877 1.1664674 1.15 8000411 1.163313 1.0741888 1.1427313 1.13 8000465 1.2093744 1.282371 1.0333343 1.18 8000467 1.1141713 1.0798538 1.0841471 1.09 GSG1L 8000746 1.1087912 1.0956571 1.0253481 1.08 8000957 1.0944146 1.124809 1.0086572 1.08 LOC283932 8000974 1.0978557 1.0160508 1.1209348 1.08 ZNF668 8001030 1.070668 1.3272351 1.2469769 1.21 PYCARD 8001552 1.1898173 1.1506544 1.2058214 1.18 CIAPIN1 8001800 1.3694799 1.2314364 1.0838622 1.23 CDH11 8002181 1.1280044 1.1114668 1.1424183 1.13 DPEP3 8002209 1.0488133 1.0910089 1.1467783 1.10 8002262 1.095937 1.0961037 1.079372 1.09 8002266 1.0887408 1.1591797 1.0508189 1.10 CTF8 8002312 1.1312715 1.093 1.053097 1.09 NOB1 8002344 1.0808529 1.0929419 1.1625277 1.11 EXOSC6 8002992 1.0847833 1.1017444 1.114893 1.10 C16orf46 8003332 1.3093789 1.5064822 1.1704128 1.33 MVD 8003444 1.0311412 1.2269135 1.0863422 1.11 LOC146429 8003605 1.1772624 1.5924035 1.1016129 1.29 8003656 1.041855 1.2235769 1.3967426 1.22 SERPINF2 8003812 1.1337674 1.1440598 1.4369242 1.24 OR3A3 8003824 1.1893297 1.1343563 1.1907091 1.17 CTNS 8004167 1.0845137 1.0399116 1.0970066 1.07 FAM64A 8004219 1.0243803 1.1837707 1.1942095 1.13 8005089 1.1931292 1.1968703 1.0642089 1.15 COX10 8005743 1.0322092 1.1908324 1.1875083 1.14 FLJ36000 8005839 1.1996229 1.8263687 1.1805004 1.40 TMEM97 8006323 1.1130961 1.0405288 1.1000903 1.08 8006367 1.1356021 1.0156969 1.1704766 1.11 RHBDL3 8006423 1.006006 1.1000518 1.2387348 1.11 SPACA3 8006590 1.1136106 1.0010692 1.2378942 1.12 8006779 1.181573 1.053165 1.1849493 1.14 ARHGAP23 8006786 1.0556784 1.2208792 1.124136 1.13 8006836 1.0827494 1.036712 1.1177361 1.08 FLJ43826 8007071 1.2016506 1.1071901 1.0730101 1.13 CDC6 8007148 1.1042011 1.1127062 1.3389119 1.19 GAST 8007388 1.0688294 1.0922204 1.2640142 1.14 CNTD1 8007548 1.0356264 1.1140163 1.3520757 1.17 C17orf88 8007828 1.1870351 1.035157 1.2893823 1.17 MAPT 8007902 1.100092 1.0929043 1.0254744 1.07 8008711 1.0514582 1.1205696 1.1228697 1.10 OR4D1 8009727 1.1611476 1.1709166 1.1948096 1.18 ICT1 8009796 1.047944 1.1041256 1.0857812 1.08 KIAA0195

174

8009951 1.0943238 1.1527853 1.0367186 1.09 ITGB4 8010512 1.0197479 1.1625643 1.0972586 1.09 KIAA1303 8010550 1.1127979 1.1419733 1.0158436 1.09 CHMP6 8010780 1.0210466 1.1943929 1.1506366 1.12 FLJ35767 8010963 1.0291283 1.0850339 1.3442773 1.15 8011320 1.241717 1.0955245 1.1100123 1.15 OR1D5|OR1D4 8011375 1.0474495 1.1840708 1.0983831 1.11 TRPV1 8012079 1.124576 1.1046549 1.038746 1.09 DVL2 8012528 1.0289361 1.1140468 1.1560159 1.10 LOC388333 8013035 1.0829638 1.0050365 1.2267103 1.10 ZNF624 8013135 1.1092952 1.3578025 1.0151284 1.16 SREBF1 8013319 1.2802929 1.3376375 1.3635235 1.33 GRAP 8013473 1.043633 1.2505599 1.0908455 1.13 LOC339240 8014035 1.006125 1.2857502 1.1005998 1.13 8014487 1.2498661 1.4064792 1.1593804 1.27 ACACA 8014787 1.1199657 1.0600078 1.1281208 1.10 LOC100131933 8015511 1.1029085 1.199477 1.0060179 1.10 DHX58 8015642 1.2235154 1.1619585 1.0923183 1.16 PSMC3IP 8015735 1.1893553 1.0870671 1.0214889 1.10 8015737 1.1043023 1.1071491 1.2865762 1.17 8015806 1.1039271 1.0006282 1.1479646 1.08 ETV4 8016094 1.0247376 1.198096 1.2717092 1.16 GJC1 8016239 1.1103065 1.1368725 1.0243989 1.09 PLEKHM1 8016463 1.0607456 1.2082378 1.0924212 1.12 HOXB6 8016473 1.0922648 1.1810955 1.1847837 1.15 HOXB8 8016519 1.1186882 1.0624428 1.1135101 1.10 8016615 1.0436248 1.1360942 1.1520369 1.11 SAMD14 8017096 1.1301181 1.1502517 1.2292368 1.17 8017253 1.0226841 1.0836982 1.4480606 1.18 LOC729621 8017476 1.1296633 1.2560225 1.0370692 1.14 CSH2|CSH1|GH1 8019280 1.0399817 1.2170421 1.1059258 1.12 PCYT2 8019347 1.0839396 1.095706 1.0279282 1.07 8019392 1.5260879 1.4667207 1.0959201 1.36 FASN 8019711 1.0231706 1.1080948 1.1088847 1.08 SCGB1C1|hCG_1741344 8019754 1.0455555 1.1280317 1.2422535 1.14 LOC348262 8019778 1.1103601 1.2853881 1.1253959 1.17 PCYT2 8020226 1.1863377 1.050289 1.3032066 1.18 SLMO1 8020795 1.094254 1.0483708 1.294714 1.15 TTR 8020847 1.0267242 1.120407 1.1183462 1.09 DTNA 8020878 1.1741852 1.0350024 1.1097856 1.11 MAPRE2 8021129 1.2592072 1.0747237 1.1220658 1.15 C18orf23|RNF165 8021243 1.0818232 1.2520355 1.1809367 1.17 8021286 1.1411254 1.141305 1.1350343 1.14 C18orf54 8021482 1.0956249 1.0971795 1.0196384 1.07 8021914 1.0636635 1.0911989 1.146274 1.10 ADNP2 8022412 1.1131451 1.1215338 1.1953112 1.14 MC2R 8022488 1.0961356 1.0162147 1.1937114 1.10 ABHD3 8022531 1.0441654 1.1991677 1.2249871 1.16 NPC1 8022801 1.150295 1.2398093 1.131391 1.17 8023646 1.0969764 1.0155355 1.1099946 1.07 BCL2

175

8023672 1.1556892 1.1132587 1.0901805 1.12 VPS4B 8023710 1.088721 1.0689185 1.112376 1.09 CDH19 8024358 1.1561661 1.1478014 1.0544721 1.12 CSNK1G2 8024637 1.0345742 1.0935128 1.0924107 1.07 FZR1 8025183 1.0135192 1.198838 1.0943351 1.10 MCOLN1 8025285 1.1476917 1.0255924 1.1771255 1.12 C19orf59 8025828 1.342854 1.9312289 1.4032624 1.56 LDLR 8026024 1.0718602 1.171986 1.1050807 1.12 ASNA1 8026294 1.2343777 1.0090717 1.2273779 1.16 LOC113230 8026383 1.1044307 1.0832781 1.0194547 1.07 8026533 1.0324129 1.1247005 1.1223755 1.09 HSH2D 8027006 1.1277384 1.087998 1.0673132 1.09 SSBP4 8027018 1.0507933 1.089239 1.1005405 1.08 C19orf50 8027268 1.1017265 1.0956006 1.067694 1.09 ZNF66 8027297 1.2429205 1.1064311 1.050386 1.13 ZNF738 8027368 1.0881437 1.0816481 1.1237062 1.10 ZNF254 8028613 1.0221069 1.2882127 1.1100236 1.14 IL29 8029289 1.0601476 1.0913533 1.2017281 1.12 TEX101 8029726 1.0596019 1.1251943 1.1883341 1.12 8029978 1.0973939 1.1716045 1.155868 1.14 CRX 8030092 1.1213454 1.1434053 1.0661032 1.11 SEC1 8030360 1.0969989 1.2803886 1.020122 1.13 SNORD32A|RPL13A 8030416 1.1490396 1.1047487 1.0032324 1.09 SCAF1 8030641 1.0926497 1.2245147 1.0509089 1.12 POLD1 8030753 1.1023731 1.0852035 1.0846374 1.09 KLK3 8031122 1.1010189 1.2503743 1.0276161 1.13 CNOT3 8031475 1.2750847 1.1506517 1.0362085 1.15 TMEM190 8031550 1.113002 1.0083191 1.1990746 1.11 EPN1 8031686 1.1528356 1.1079568 1.1188302 1.13 USP29 8032212 1.0423386 1.1248897 1.5171665 1.23 8032249 1.0215588 1.2098322 1.1780297 1.14 ADAMTSL5 8032265 1.0865175 1.1059024 1.0423824 1.08 MEX3D 8032530 1.027805 1.1515329 1.0959002 1.09 SGTA 8032996 1.2373424 1.1093712 1.0713977 1.14 P117 8033043 1.0367426 1.1414834 1.1871414 1.12 FUT6 8034565 1.1639488 1.1730564 1.0133333 1.12 DNASE2 8034722 1.1947049 1.12514 1.1961514 1.17 RFX1 8034974 1.0388033 1.2565236 1.1935713 1.16 ABHD9 8035040 1.1099932 1.2761459 1.023432 1.14 WIZ 8035737 1.0305477 1.116048 1.099045 1.08 ATP13A1 8035808 1.1343454 1.2239212 1.0771935 1.15 ZNF100 8035884 1.0671002 1.091376 1.2106774 1.12 8036025 1.1145389 1.1227331 1.0890173 1.11 LOC388532 8036055 1.0108515 1.108182 1.1061075 1.08 LGI4 8036318 1.1038665 1.1310426 1.2296996 1.15 ZNF566 8036389 1.1304739 1.1949619 1.1592311 1.16 ZNF585B 8036707 1.1006631 1.1334991 1.1710659 1.14 LRFN1 8037032 1.1217613 1.0707406 1.1420189 1.11 B3GNT8 8037309 1.1122673 1.2900051 1.0348873 1.15 PHLDB3 8037363 1.1151527 1.0059854 1.213049 1.11 CADM4

176

8037444 1.1404817 1.1333302 1.0846306 1.12 ZNF235 8037513 1.0016457 1.1998651 1.1394354 1.11 EXOC3L2 8037537 1.132253 1.2893683 1.0663266 1.16 ERCC2 8037594 1.1484252 1.1225996 1.0559969 1.11 RTN2 8037872 1.0363613 1.091552 1.1110451 1.08 BBC3 8037959 1.1015583 1.0117561 1.1456702 1.09 BSPH1 8038139 1.0949053 1.1953843 1.1232353 1.14 LOC126147 8038213 1.0035601 1.1302202 1.104783 1.08 HSD17B14 8038309 1.1308845 1.2382035 1.0388597 1.14 KCNA7 8038861 1.0080763 1.2064408 1.1206759 1.11 SIGLEC6 8038949 1.1100028 1.0883803 1.0168601 1.07 LOC284371 8039054 1.112999 1.1247158 1.0682883 1.10 ZNF347 8039062 1.0534581 1.2284671 1.1171508 1.13 ZNF665 8039070 1.142879 1.0263453 1.0875217 1.09 ZNF677 8039484 1 1.1864887 1.0863243 1.09 IL11 8039771 1.0088001 1.1140265 1.1560957 1.09 ZNF132 8039937 1.1295047 1.5711663 1.05858 1.25 GLTPD1 8040018 1.0855099 1.1221952 1.0110089 1.07 TTC15 8040077 1.1542975 1.2321513 1.157515 1.18 FLJ41046 8040247 1.1002251 1.2290207 1.1047484 1.14 8040334 1.0220647 1.1960003 1.4564226 1.22 8040340 1.3005749 1.2337475 1.2187976 1.25 LPIN1 8040490 1.0156469 1.1791928 1.1015697 1.10 KLHL29 8040690 1.2535655 1.3191674 1.1322863 1.24 LOC339778 8041553 1.0976213 1.31082 1.0003332 1.14 GEMIN6 8041568 1.1762931 1.0331163 1.1027021 1.10 8041570 1.0248408 1.1375709 1.1730393 1.11 TMEM178 8041913 1.0997006 1.0136445 1.1250601 1.08 CCDC128 8042109 1.1179589 1.484534 1.0827075 1.23 8042283 1.1357613 1.2061709 1.0283625 1.12 HSPC159 8042291 1.0960356 1.119539 1.0009179 1.07 AFTPH 8042859 1.1315758 1.1109452 1.0331717 1.09 ZNHIT4|WBP1 8042925 1.0081285 1.1003175 1.0906909 1.07 SEMA4F 8042993 1.0168675 1.126982 1.0827183 1.08 CTNNA2 8043155 1.2174774 1.0572513 1.1033552 1.13 8043381 1.0889463 1.1176873 1.1514727 1.12 SMYD1 8043666 1.1672145 1.1239223 1.063958 1.12 CNNM3 8043861 1.0895234 1.1593146 1.0300586 1.09 EIF5B 8043902 1.1886759 1.0432957 1.0948567 1.11 PDCL3 8044698 1.0093678 1.0879905 1.1151546 1.07 8044745 1.1210052 1.1475796 1.0170625 1.10 DDX18 8045291 1.118862 1.0385957 1.0863498 1.08 TUBA3D|TUBA3E|TUBA3C 8045309 1.2214955 1.0734627 1.1152003 1.14 CCDC74A|CCDC74B 8045339 1.1033952 1.0515451 1.1184154 1.09 8045398 1.0073398 1.0879704 1.0879517 1.06 RAB3GAP1 8045664 1.2966574 1.0488575 1.140409 1.16 LOC130576 8045846 1.1076678 1.211239 1.0775093 1.13 8046588 1.112647 1.2116097 1.1271175 1.15 8046746 1.0297823 1.1811045 1.125388 1.11 PPP1R1C 8046848 1.1410908 1.1064249 1.0727328 1.11 ZC3H15

177

8047125 1.1709634 1.2413892 1.0409181 1.15 8047702 1.073869 1.1250708 1.1406028 1.11 ICOS 8047709 1.1260886 1.1332166 1.0359287 1.10 PARD3B 8047865 1.0234149 1.2048035 1.1312759 1.12 PIP5K3 8047965 1.1697171 1.1214617 1.1816348 1.16 KIAA1843 8048249 1.0850217 1.264646 1.1255829 1.16 GPBAR1 8048707 1.0918534 1.1125114 1.030358 1.08 CCDC140 8049297 1.067084 1.1029832 1.1025652 1.09 SCARNA5 8049574 1.0171082 1.1155137 1.1413985 1.09 UBE2F 8050190 1.0062757 1.1033624 1.092167 1.07 ADAM17 8050238 1.0096729 1.3419073 1.1066549 1.15 8050423 1.1664112 1.3680679 1.2281935 1.25 MYCNOS 8050869 1.0368019 1.1180809 1.1286008 1.09 8051028 1.1733855 1.1356539 1.6870941 1.33 8051119 1.1335284 1.1746734 1.016246 1.11 PPM1G 8051275 1.1168337 1.2598221 1.0694344 1.15 CAPN13 8051573 1.135777 1.0954293 1.031939 1.09 CDC42EP3 8051622 1.0942366 1.0432395 1.1174754 1.08 SFRS7 8052233 1.1260399 1.1629074 1.0693333 1.12 C2orf63 8052581 1.1199516 1.1583039 1.1684129 1.15 8052902 1.2715251 1.0886272 1.0388349 1.13 FIGLA 8052932 1.1997668 1.014188 1.1005005 1.10 8052940 1.1032085 1.1099741 1.1336812 1.12 PAIP2B 8052947 1.1271831 1.0678142 1.094717 1.10 CYP26B1 8053288 1.2063495 1.1299982 1.0958154 1.14 C2orf3 8053349 1.0942814 1.2931936 1.1237864 1.17 LRRTM1 8054329 1.010041 1.1065339 1.1565193 1.09 RNF149 8054344 1.0864116 1.0311092 1.0844786 1.07 FLJ42986 8054467 1.1713191 1.1276996 1.0094216 1.10 SEPT10 8054866 1.0865089 1.0417964 1.094415 1.07 FLJ14816 8055173 1.1143352 1.1167365 1.112515 1.11 CCDC74A|CCDC74B 8055261 1.139507 1.0840005 1.0080233 1.08 RAB6C|RAB6A|LOC150786 8055314 1.0853919 1.1059898 1.2114271 1.13 LYPD1 8056968 1.2236205 1.0058295 1.172049 1.13 LOC375295 8057394 1.1193746 1.034479 1.0885063 1.08 SESTD1 8057480 1.0305 1.1719116 1.1271939 1.11 NEUROD1 8057959 1.1910133 1.0351857 1.0883445 1.10 PGAP1 8058203 1.0884304 1.0544676 1.1163502 1.09 ALS2CR12 8058295 1.0471013 1.113925 1.0889208 1.08 ALS2 8058348 1.1621474 1.2068162 1.0971066 1.16 8058458 1.0146344 1.1046023 1.3692735 1.16 LOC645978 8059275 1.0914459 1.0932302 1.0192562 1.07 8059277 1.1198806 1.0619369 1.1881032 1.12 8059350 1.0939044 1.1000866 1.0825136 1.09 AP1S3 8059475 1.0328757 1.1228226 1.1184309 1.09 8059712 1.3682551 1.2878159 1.0861412 1.25 SNORD82 8060020 1.2291523 1.12031 1.0969434 1.15 FLJ43879 8060344 1.015644 1.114084 1.2066394 1.11 TRIB3 8060501 1.0879031 1.0058022 1.1641188 1.09 SNORA51 8060940 1.0840851 1.0896542 1.1466302 1.11 C20orf103

178

8060997 1.1012875 1.0691595 1.0847528 1.09 SPTLC3 8061073 1.1521202 1.033899 1.1530662 1.11 8061082 1.0414917 1.0997874 1.0952533 1.08 OTOR 8061562 1.1270437 1.371754 1.0547127 1.18 8061772 1.1140752 1.1123098 1.0031422 1.08 MAPRE1 8062034 1.0123056 1.1108696 1.0953751 1.07 TP53INP2 8062251 1.2576936 1.1678226 1.0021715 1.14 EPB41L1 8062395 1.2002261 1.2184396 1.037624 1.15 NNAT 8062404 1.135014 1.1217039 1.1953064 1.15 8062539 1.0212471 1.2342712 1.1742077 1.14 SLC32A1 8062576 1.1551306 1.3200786 1.1851801 1.22 DHX35 8063380 1.1745375 1.4031214 1.1093928 1.23 8064098 1.6033744 2.116185 1.076793 1.60 8064203 1.372524 1.2163411 1.0273006 1.21 C20orf135 8064208 1.0832462 1.1532807 1.101936 1.11 DNAJC5 8064904 1.0382837 1.1172911 1.1067946 1.09 FERMT1 8065084 1.1607689 1.094352 1.2964474 1.18 LOC613266 8065120 1.1970372 1.2106566 1.0758492 1.16 LOC100131642 8065416 1.0996456 1.0684042 1.2246082 1.13 CST2 8065421 1.105321 1.0262113 1.1512756 1.09 CST5 8065668 1.3222821 1.0968618 1.0237637 1.15 CDK5RAP1 8065752 1.1070796 1.0944349 1.0386262 1.08 8065756 1.1156029 1.4172776 1.1648909 1.23 8065868 1.0857489 1.1283491 1.0045909 1.07 EIF6 8066482 1.0643868 1.113456 1.2204403 1.13 WFDC5 8067087 1.0878898 1.0982342 1.3012619 1.16 SALL4 8067546 1.0952411 1.1349968 1.2036597 1.14 LOC100135777 8067798 1.0356162 1.0900875 1.1397492 1.09 SOX18 8067818 1.1533815 1.250562 1.1654543 1.19 NPBWR2 8068200 1.083691 1.0558423 1.1111436 1.08 C21orf119 8068952 1.1247782 1.134591 1.0444733 1.10 AGPAT3 8069085 1.1040555 1.1546607 1.0059222 1.09 TRPM2 8069470 1.1211905 1.1510926 1.0290399 1.10 FLJ45055 8069505 1.0241995 1.2080194 1.200622 1.14 C21orf15 8069764 1.0947967 1.0162036 1.1071374 1.07 8069868 1.2053615 1.1780374 1.2567815 1.21 KRTAP7‐1 8070295 1.2947075 1.0891966 1.0799944 1.15 8070689 1.1884036 1.3389566 1.021784 1.18 HSF2BP 8070716 1.1459842 1.1244931 1.1530684 1.14 C21orf32 8070777 1.05599 1.0951904 1.096032 1.08 KRTAP10‐2 8070961 1.1622511 1.3456504 1.0130118 1.17 LSS 8071196 1.0285329 1.1128931 1.0904436 1.08 TSSK2 8071532 1.1072937 1.1031947 1.0157355 1.08 RIMBP3|RIMBP3B 8072304 1.0353328 1.1104218 1.2180107 1.12 HORMAD2 8072454 1.0355922 1.0928086 1.0835334 1.07 RNF185 8072482 1.1206452 1.3031067 1.3505179 1.26 8072494 1.1465476 1.088265 1.067767 1.10 SFI1 8072678 1.060807 1.1958834 1.1087348 1.12 HMOX1 8072817 1.0296036 1.3502027 1.1260772 1.17 CDC42EP1 8072894 1.0502821 1.1948193 1.1617411 1.14 TRIOBP

179

8072924 1.1786925 1.1181917 1.0637169 1.12 8073522 1.3821472 1.2623159 1.1185522 1.25 SREBF2 8073605 1.067412 1.0979903 1.2558036 1.14 BIK 8073631 1.011309 1.1952214 1.126076 1.11 8073633 1.3042239 1.3415449 1.07715 1.24 PNPLA3 8074335 1.161173 1.0233363 1.2035892 1.13 PRODH 8074769 1.0485368 1.1116483 1.1457751 1.10 RIMBP3B 8074897 1.0035793 1.1316619 1.1104409 1.08 LOC388882 8075164 1.1207159 1.2166504 1.0036118 1.11 CHEK2 8075217 1.2406746 1.1246005 1.0622226 1.14 AP1B1 8075695 1.1291765 1.108224 1.0531102 1.10 APOL3 8076300 1.0409745 1.1611803 1.2756956 1.16 8077779 1.0604779 1.2618691 1.1101829 1.14 8077879 1.0093129 1.1707269 1.0968992 1.09 SYN2 8077942 1.1739038 1.0586137 1.1473857 1.13 8078448 1.1046932 1.1408131 1.085699 1.11 8078688 1.0041285 1.4246714 1.1846654 1.20 8078918 1.139042 1.0443114 1.2673261 1.15 SNORA62|RPSA 8079392 1.1149122 1.0621967 1.093917 1.09 CCR2|FLJ78302 8079401 1.1760712 1.0099475 1.2082905 1.13 CCR5|LOC727797 8079407 1.1910795 1.1463323 1.127524 1.15 CCRL2|LOC727811 8079415 1.0463772 1.1503224 1.1702793 1.12 RTP3 8080676 1.0623767 1.1652055 1.0970656 1.11 PDE12 8080683 1.0524477 1.2931359 1.3070289 1.22 8080855 1.10761 1.2061267 1.0740469 1.13 SYNPR 8081676 1.0431571 1.2572551 1.1175374 1.14 GTPBP8 8081758 1.0426064 1.1470599 1.1918478 1.13 GRAMD1C 8081820 1.0916423 1.349632 1.2146478 1.22 8082254 1.1193899 1.0157654 1.387496 1.17 LOC100125556 8082368 1.0587201 1.1356084 1.1381934 1.11 PODXL2 8082431 1.0926493 1.0371193 1.1514267 1.09 RAB7A 8082583 1.3166375 1.1310732 1.0166894 1.15 UNQ6125 8082767 1.0137396 1.2310498 1.1265763 1.12 TMEM108 8082965 1.1457464 1.0566317 1.1443876 1.12 MRAS 8083599 1.062492 1.0926054 1.1026024 1.09 C3orf55 8083656 1.0814718 1.11551 1.0839983 1.09 MFSD1 8084219 1.1930149 1.0136374 1.0891447 1.10 KLHL24 8084323 1.0866141 1.1163129 1.0222502 1.08 DVL3 8084648 1.1018784 1.0638505 1.1023946 1.09 AHSG 8084929 1.1677003 1.0857611 1.0269703 1.09 OSTalpha 8084955 1.1056045 1.3706174 1.0796925 1.19 PIGX 8085114 1.173778 1.790764 1.3402411 1.43 8085195 1.0221235 1.0875745 1.1205 1.08 LHFPL4 8086352 1.1327134 1.1000757 1.0693913 1.10 ULK4 8086462 1.1142468 1.0197085 1.1575338 1.10 C3orf39 8086698 1.1149176 1.1143717 1.0651959 1.10 CCDC12 8086876 1.1161406 1.0864607 1.2975092 1.17 8087611 1.0802108 1.1485919 1.1062574 1.11 HYAL1 8087624 1.1683686 1.0012949 1.0874135 1.09 HYAL2 8087640 1.0071754 1.2355593 1.1221403 1.12 RASSF1

180

8087746 1.3380895 1.0796232 1.1351863 1.18 8087830 1.1304798 1.0322174 1.2474369 1.14 RPL29|RPL29P4 8088106 1.1322964 1.2320483 1.0179752 1.13 TKT 8088339 1.0807765 1.1483634 1.0853542 1.10 ARF4 8088384 1.1198887 1.134029 1.1049134 1.12 PDHB 8088548 1.0891746 1.0561428 1.1148387 1.09 LOC401070 8088560 1.0756171 1.0907398 1.0868533 1.08 ADAMTS9 8088739 1.1240164 1.08984 1.0071647 1.07 LMOD3 8088915 1.244291 1.1418823 1.3153348 1.23 8089185 1.0518382 1.1373851 1.1892256 1.13 IMPG2 8089261 1.1746129 1.0142635 1.1008526 1.10 CBLB 8089372 1.0270263 1.1540025 1.1148137 1.10 KIAA1524 8090030 1.086557 1.1068846 1.0102016 1.07 HSPBAP1 8090349 1.0709288 1.1256685 1.1785047 1.13 8090639 1.1267011 1.1218644 1.0621141 1.10 PIK3R4 8090772 1.1096172 1.1863471 1.0246999 1.11 TOPBP1 8091032 1.2216873 1.0976093 1.0455153 1.12 FOXL2 8091118 1.0624642 1.1157618 1.0915153 1.09 8091511 1.0380384 1.1226084 1.0856854 1.08 P2RY14 8091546 1.0925367 1.2684324 1.0154489 1.13 LOC645843 8091696 1.07076 1.2137485 1.0859147 1.12 8091941 1.163363 1.035801 1.1197432 1.11 PDCD10 8092187 1.0052066 1.0869352 1.108785 1.07 SPATA16 8092358 1.0216057 1.0890774 1.3083009 1.14 MCF2L2 8092552 1.1022503 1.1281611 1.120264 1.12 IGF2BP2 8092578 1.0024505 1.162364 1.2097371 1.12 ETV5 8092627 1.096805 1.1555724 1.0299456 1.09 TBCCD1 8093166 1.098469 1.0498685 1.2169946 1.12 PIGZ 8093298 1.2313472 1.0112141 1.208703 1.15 CCR5|LOC727797 8093304 1.2640184 1.1644728 1.0646574 1.16 CCRL2|LOC727811 8093332 1.4434346 1.1100413 1.0831928 1.21 MGC26356 8094028 1.2191719 1.0076215 1.1901772 1.14 MGC10981 8094271 1.112554 1.1025108 1.0232978 1.08 MED28 8095043 1.1092124 1.0972053 1.1730796 1.13 RASL11B 8095535 1.0796311 1.2166375 1.6135966 1.30 ENAM 8095562 1.1259034 1.4725097 1.01102 1.20 8095806 1.111116 1.0660285 1.1184926 1.10 ART3 8096744 1.2097026 1.2136682 1.055665 1.16 CYP2U1 8096905 1.0509305 1.1191146 1.2114524 1.13 C4orf16 8097080 1.0627059 1.1816308 1.3656329 1.20 SYNPO2 8097305 1.3179964 1.0714883 1.085143 1.16 8097521 1.1564451 1.3110752 1.0437499 1.17 SCOC 8097813 1.0393463 1.17111 1.1088755 1.11 ARFIP1 8097938 1.0057414 1.1278822 1.1116755 1.08 NPY2R 8098103 1.2633426 1.117578 1.1821023 1.19 FNIP2 8098163 1.1315991 1.0858716 1.1595863 1.13 8098195 1.2796769 1.4979651 1.3891957 1.39 SC4MOL 8098246 1.0937886 1.0596247 1.1885862 1.11 ANXA10 8098307 1.1009861 1.1176859 1.0522985 1.09 GALNT17 8098439 1.0369273 1.1587243 1.1990623 1.13

181

8098697 1.0422035 1.159538 1.1514883 1.12 TRIML1 8098902 1.2467214 1.0078471 1.3266749 1.19 8098904 1.0364634 1.1885725 1.114094 1.11 SLBP 8099093 1.0000705 1.1748334 1.2199346 1.13 OTOP1 8099130 1.0985798 1.1651673 1.1660734 1.14 8099713 1.0274769 1.1424938 1.2204685 1.13 8099897 1.1094203 1.1026253 1.025056 1.08 UGDH 8100360 1.087425 1.1283834 1.0169301 1.08 8100476 1.0193365 1.1923935 1.115482 1.11 8100941 1.1273556 1.1903751 1.4589944 1.26 8100990 1.0672321 1.2177129 1.211691 1.17 PPBPL2 8101065 1.0861491 1.2985483 1.1014524 1.16 PPEF2 8101945 1.0859692 1.1368676 1.1283267 1.12 H2AFZ 8101957 1.0923392 1.1965808 1.0426807 1.11 EMCN 8102127 1.1270326 1.0923514 1.006276 1.08 TACR3 8102350 1.1348528 1.0902828 1.0047051 1.08 8102695 1.0326033 1.1007409 1.3635288 1.17 8102800 1.1098788 1.0252416 1.1082915 1.08 SLC7A11 8102988 1.2251945 1.2743807 1.0597644 1.19 GYPE 8103289 1.1344129 1.0945115 1.0350981 1.09 PLRG1 8103431 1.0082549 1.1053396 1.1180351 1.08 LOC201725 8103542 1.0352918 1.0856032 1.1446664 1.09 8103847 1.1530807 1.0351614 1.2246268 1.14 8103951 1.0803565 1.445028 1.1406714 1.22 ACSL1 8105061 1.358969 1.1496751 1.0837351 1.20 FYB 8105681 1.1498834 1.0063429 1.098101 1.08 ERBB2IP 8106098 1.1452423 1.0476351 1.1141897 1.10 MAP1B 8106280 1.2202277 1.5617967 1.3801378 1.39 HMGCR 8106352 1.062583 1.2256234 1.093025 1.13 NBLA11895|LOC100129566 8106512 1.1874758 1.0509329 1.1594855 1.13 8106602 1.0681391 1.1958035 1.1203319 1.13 ZFYVE16 8106727 1.0116898 1.1098315 1.1117427 1.08 FLJ41309|LOC92270 8107202 1.090789 1.1154649 1.0394652 1.08 8107208 1.0993909 1.0778117 1.1067924 1.09 FER 8107353 1.0800687 1.2695737 1.0975372 1.15 ZRSR1 8107563 1.1601192 1.1742254 1.1195197 1.15 PRR16 8107769 1.1997886 1.1773881 1.025725 1.13 SLC12A2 8107850 1.0545132 1.2956613 1.1010921 1.15 CHSY3 8108180 1.2168953 1.161417 1.1113706 1.16 8108593 1.1227034 1.1118246 1.0625745 1.10 WDR55 8108713 1.6116 1.0085168 1.3863844 1.34 PCDHB8 8108979 1.1151649 1.0594062 1.2000531 1.12 8109350 1.1005517 1.2102486 1.0461987 1.12 SLC36A1 8109752 1.2340273 1.3939427 1.0959743 1.24 ODZ2 8109908 1.0152781 1.0830003 1.1265793 1.07 LOC257358 8109990 1.1623844 1.199245 1.095869 1.15 LOC644994 8110327 1.0882304 1.1181097 1.0494618 1.09 RGS14 8110562 1.0387175 1.1065565 1.117091 1.09 LTC4S|MAML1 8110872 1.098481 1.0046712 1.1118968 1.07 IRX2 8110980 1.0905156 1.0389407 1.0924009 1.07

182

8111668 1.1349878 1.1425977 1.0859965 1.12 8111922 1.0538038 1.1525743 1.1088209 1.11 LOC648987 8111941 1.4250731 1.7165734 1.262038 1.47 HMGCS1 8112014 1.1258032 1.0370429 1.1068466 1.09 8112337 1.0531186 1.2481421 1.1109082 1.14 SFRS12IP1 8112666 1.2467922 1.2033628 1.3176323 1.26 8112886 1.2623788 1.1329335 1.3279916 1.24 8112890 1.1336977 1.129739 1.01999 1.09 MED27|LOC441089 8112894 1.1183105 1.1006234 1.0622013 1.09 8112994 1.1253554 1.2606223 1.0932415 1.16 8112996 1.0903474 1.0537603 1.0966951 1.08 8113120 1.0996453 1.0146316 1.1621674 1.09 8113344 1.0998285 1.1377695 1.0084978 1.08 8113491 1.3779963 1.3541964 1.3548331 1.36 STARD4 8113691 1.086562 1.1103528 1.0602566 1.09 DTWD2 8113914 1.0611106 1.1483223 1.1667798 1.13 FNIP1|RAPGEF6 8114207 1.0997052 1.1278207 1.0101378 1.08 8114625 1.0487309 1.2473701 1.6051635 1.30 DND1 8114647 1.0981504 1.098264 1.1449388 1.11 SLC25A2 8114658 1.132524 1.0943061 1.0678016 1.10 DIAPH1 8114787 1.1468227 1.113735 1.0373682 1.10 GNPDA1 8114918 1.1911771 1.2217528 1.2318499 1.21 8115144 1.0824567 1.1476694 1.3145565 1.18 ARSI 8115166 1.040689 1.2585461 1.11497 1.14 8115455 1.2573137 1.104576 1.0736351 1.15 HAVCR1 8115679 1.3310843 1.2092618 1.1996447 1.25 8115681 1.1716567 1.1275505 1.0761662 1.13 PANK3 8115849 1.0153888 1.187256 1.185961 1.13 8115927 1.0281916 1.1903431 1.0939788 1.10 RNF44 8116502 1.1097827 1.1479262 1.0350091 1.10 8116571 1.1174418 1.0007075 1.3770894 1.17 8117079 1.4278411 1.1212153 1.0609981 1.20 LOC644714 8117118 1.0966759 1.1001091 1.0727743 1.09 8117301 1.0517519 1.1838669 1.0905877 1.11 HIST1H2BA 8117389 1.2761223 1.0722673 1.1558055 1.17 HIST1H2BE|HIST1H2BG 8117531 1.0982335 1.2382228 1.1764798 1.17 8117653 1.0600208 1.1165568 1.2274873 1.13 8117900 1.0145338 1.2092681 1.0839106 1.10 DDR1 8118061 1.1019957 1.0777109 1.1147577 1.10 DPCR1 8118142 1.1294072 1.0544529 1.2123348 1.13 TNF 8118235 1.1177952 1.1244166 1.0588737 1.10 C6orf21|LY6G6D 8118655 1.0902076 1.1091813 1.0495194 1.08 B3GALT4 8118945 1.0081528 1.1200165 1.0910082 1.07 PPARD 8119338 1.1160557 1.1959937 1.063687 1.13 GLP1R 8119466 1.1144029 1.1686428 1.0180607 1.10 MDFI 8119712 1.0626606 1.308009 1.1943839 1.19 SRF 8119842 1.0459995 1.1777766 1.0956429 1.11 POLR1C 8119951 1.0823082 1.0530636 1.100164 1.08 CAPN11 8120208 1.1089935 1.089553 1.1232083 1.11 8120269 1.1018332 1.0085509 1.2281367 1.11 FBXO9

183

8120717 1.2461965 1.1134405 1.1070837 1.16 8121159 1.1346071 1.0684094 1.1058168 1.10 8121429 1.1429939 1.2051648 1.0138301 1.12 FIG4 8121936 1.0853385 1.0917534 1.1721994 1.12 C6orf58 8122240 1.0931258 1.1349849 1.0266112 1.08 8122554 1.1558781 1.0947547 1.0226614 1.09 RAB32 8122703 1.1244128 1.8360835 1.2831087 1.41 8123060 1.2967709 1.0687546 1.1604788 1.18 8123463 1.0863329 1.1762267 1.0999709 1.12 C6orf120 8123760 1.0669852 1.2443653 1.2079962 1.17 RP3‐398D13.1 8123910 1.0067434 1.0937933 1.2388757 1.11 GCM2 8123949 1.1122205 1.0973488 1.0792947 1.10 8124055 1.0881318 1.0048698 1.2527914 1.12 8124430 1.0724534 1.3050238 1.1286482 1.17 HIST1H1D 8124467 1.0915481 1.2284136 1.1877557 1.17 8125123 1.1193928 1.0120867 1.2569854 1.13 8125321 1.0766495 1.1435021 1.1522696 1.12 PRRT1 8125415 1.105344 1.1179084 1.0882872 1.10 C6orf10 8125426 1.1444012 1.0987494 1.0235386 1.09 BTNL2 8125752 1.1063268 1.1001848 1.0114374 1.07 CUTA 8126760 1.1200553 1.0241125 1.2982603 1.15 RCAN2 8126770 1.2401724 1.1886314 1.0504571 1.16 CYP39A1 8126855 1.0534406 1.0997034 1.1302661 1.09 C6orf138 8127145 1.0878912 1.2473116 1.1333523 1.16 ELOVL5 8127824 1.1008563 1.1100299 1.1133629 1.11 UBE2CBP 8127854 1.1345043 1.2509531 1.0559585 1.15 ME1 8128034 1.0537056 1.1417592 1.0854051 1.09 C6orf166 8128133 1.0916628 1.2349828 1.0931118 1.14 LYRM2|ZC3H11A 8128447 1.0992764 1.2497706 1.0947865 1.15 MCHR2 8128737 1.1578338 1.2576853 1.0104086 1.14 MICAL1 8128888 1.1014436 1.085251 1.0590638 1.08 8129039 1.0239207 1.258759 1.1480939 1.14 LOC442249 8129099 1.0938382 1.0643182 1.0943335 1.08 TSPYL1 8129309 1.1805983 1.0473307 1.133782 1.12 8129773 1.0912577 1.0901452 1.1183296 1.10 BCLAF1 8129783 1.2213676 1.0321215 1.1300102 1.13 MAP7 8129963 1.1100801 1.1043829 1.046939 1.09 ADAT2 8130013 1.1299796 1.0474256 1.1172918 1.10 EPM2A 8130071 1.1348224 1.3036649 1.017729 1.15 C15orf29|LOC729176 8131263 1.1120886 1.1187074 1.0206047 1.08 RNU13P2 8131519 1.1575855 1.115584 1.0321887 1.10 PHF14 8131583 1.1750431 1.2364291 1.0243549 1.15 BZW2 8131844 1.216325 1.1027594 1.2336179 1.18 GPNMB 8131867 1.0847112 1.1424341 1.1795359 1.14 8131917 1.0924782 1.1092836 1.1579192 1.12 8131975 1.1089488 1.0925981 1.0933639 1.10 TAX1BP1 8132118 1.1180189 1.1455827 1.1367306 1.13 AQP1 8132840 1.1391896 1.1352553 1.1271812 1.13 DKFZp564N2472 8132851 1.1390684 1.0205723 1.2631643 1.14 VSTM2A 8132897 1.1741191 1.2215388 1.083567 1.16 LANCL2

184

8133018 1.0998638 1.1385399 1.0315083 1.09 ZNF716 8133106 1.0961124 1.1659393 1.0406518 1.10 SNORA22 8133504 1.1306741 1.0362352 1.0846815 1.08 GTF2I 8133860 1.0571015 1.2628796 1.123619 1.15 GNAI1 8134211 1.1107507 1.2066586 1.0527855 1.12 DKFZP564O0523 8134420 1.2928214 1.3182987 1.0322874 1.21 TAC1 8134550 1.2847217 1.0577432 1.2523807 1.20 8135514 1.0406098 1.1427038 1.1652428 1.12 IFRD1 8135544 1.0810394 1.1690712 1.1870645 1.15 FOXP2 8135734 1.0278134 1.1014451 1.1489328 1.09 C7orf58 8135922 1.1247734 1.1646588 1.051844 1.11 METTL2B 8135969 1.0996795 1.2272773 1.2276428 1.18 CCDC136 8136177 1.0057164 1.1160557 1.1873981 1.10 KIAA0265 8136181 1.1465778 1.112018 1.0075703 1.09 C7orf45 8136471 1.1087356 1.0379505 1.0832505 1.08 8136709 1.1194812 1.0214102 1.2165209 1.12 LOC93432 8136849 1.1088686 1.1205922 1.0638769 1.10 GSTK1 8137240 1.1169957 1.0674512 1.1516277 1.11 GIMAP7 8137517 1.2553642 1.3094624 1.232021 1.27 HTR5A 8137526 1.4404664 1.6578734 1.4730247 1.52 INSIG1 8137833 1.1655428 1.1064253 1.1164203 1.13 SNX8 8138189 1.1341456 1.2267083 1.1122195 1.16 RPA3 8138527 1.190082 1.1214306 1.1195275 1.14 MGC87042 8138757 1.091138 1.0596775 1.1024911 1.08 HOXA10 8138922 1.1590073 1.0231063 1.0837289 1.09 KBTBD2 8138941 1.261633 1.2117302 1.133226 1.20 NT5C3 8138988 1.1472188 1.0710167 1.241623 1.15 DPY19L2P1 8139125 1.0752083 1.1607711 1.1070405 1.11 8139201 1.0141542 1.0841954 1.0879797 1.06 8139242 1.2266543 1.0536234 1.2966862 1.19 8139314 1.0477213 1.2403716 1.1955312 1.16 GCK 8139458 1.1357976 1.0963519 1.0003008 1.08 LOC100128364 8139697 1.1830213 1.1421118 1.0582925 1.13 8139780 1.2792262 1.184631 1.0897791 1.18 8140420 1.2636291 1.1881655 1.1385956 1.20 FDPSL2A 8140443 1.2641546 1.2175925 1.1312666 1.20 FDPSL2A 8140864 1.1610378 1.5999483 1.0626613 1.27 CYP51A1 8141076 1.0629809 1.1230657 1.1098076 1.10 PON2 8141140 1.0823333 1.1239539 1.0400367 1.08 DLX5 8141169 1.0813296 1.1053166 1.1755702 1.12 MGC72080 8141173 1.1597561 1.1373818 1.1190207 1.14 LOC4951|OCM 8141317 1.1454536 1.1653842 1.0191869 1.11 CYP3A4 8141526 1.0811384 1.3324625 1.1056919 1.17 TSC22D4 8142538 1.087222 1.0115049 1.1107397 1.07 8142880 1.1199117 1.1034762 1.0433085 1.09 8142882 1.2430623 1.121002 1.0093288 1.12 8143385 1.0690697 1.2041085 1.0866547 1.12 8143482 1.1329223 1.0776333 1.2899702 1.17 TAS2R38 8143605 1.1130792 1.3609872 1.3286572 1.27 FAM115A 8143714 1.1734108 1.2201434 1.0077955 1.13 ZNF777

185

8143790 1.0657513 1.1108384 1.0942352 1.09 TMEM176B 8144044 1.0942674 1.2373546 1.5165994 1.28 FLJ42291 8144405 1.0462593 1.1942734 1.1430331 1.13 DEFB107A|DEFB107B 8144669 1.200071 1.432527 1.2145431 1.28 FDFT1 8144703 1.1446048 1.0871285 1.056406 1.10 8144810 1.116982 1.2208017 1.1979531 1.18 8144931 1.0566058 1.2122667 1.1473868 1.14 ATP6V1B2 8144995 1.1096909 1.0097656 1.2555625 1.13 FGF17 8145272 1.2131665 1.1013486 1.0646366 1.13 R3HCC1 8145660 1.1903507 1.095768 1.0505766 1.11 DCTN6 8145766 1.038845 1.1204685 1.3566672 1.17 MST131 8145795 1.035105 1.2928765 1.1616582 1.16 8146649 1.1009473 1.0917014 1.0511045 1.08 MTFR1 8146906 1.1519033 1.324599 1.2853665 1.25 8147112 1.0694556 1.1934143 1.1056988 1.12 CA13 8147503 1.0373627 1.1714802 1.1794018 1.13 LAPTM4B 8147724 1.1003222 1.0182929 1.1793674 1.10 ATP6V1C1 8148280 1.1962905 1.4785942 1.1612251 1.28 SQLE 8148293 1.1620045 1.1548103 1.0442897 1.12 NSMCE2 8148325 1.1278055 1.1723021 1.0545069 1.12 8148783 1.1246827 1.0939466 1.160998 1.13 KIAA1833|LOC377711 8148824 1.131059 1.1234322 1.0237825 1.09 HSF1|LOC642255 8148982 1.1262897 1.370911 1.0435061 1.18 LOC389607 8149104 1.0007354 1.2497393 1.1223966 1.12 DEFA6 8149230 1.0436689 1.1653006 1.1399734 1.12 DEFB107A|DEFB107B 8149438 1.0737433 1.1049719 1.0954887 1.09 SGCZ 8149918 1.0094689 1.2678624 1.2215368 1.17 CHRNA2 8150034 1.495197 1.4631358 2.0955126 1.68 8150197 1.0871303 1.0276362 1.0869039 1.07 DUSP26 8150217 1.1834346 1.0967858 1.2147366 1.16 hCG_1640171 8150757 1.1027207 1.0755843 1.0892757 1.09 RB1CC1 8150868 1.0098327 1.1944404 1.1704402 1.12 8151123 1.1908764 1.0870786 1.0427063 1.11 PTTG3 8151369 1.4045244 1.1299374 1.2180384 1.25 RPESP 8151492 1.0846169 1.0358583 1.0831907 1.07 8151795 1.025104 1.094058 1.1386987 1.09 CDH17 8151917 1.0828693 1.2314999 1.0549304 1.12 MTERFD1 8152340 1.1488284 1.0392759 1.0903848 1.09 NUDCD1 8152617 1.1743839 1.1277674 1.1468053 1.15 HAS2 8152648 1.0036291 1.1065599 1.1796013 1.10 C8orf76 8153336 1.1064296 1.0030155 1.0925183 1.07 SLURP1 8153727 1.1019484 1.1238774 1.0066296 1.08 CPSF1 8153828 1.0900439 1.0115197 1.2051448 1.10 FOXH1 8154357 1.1772017 1.0942771 1.0517457 1.11 LOC392285 8154365 1.0978593 1.2612057 1.0520822 1.14 8154476 1.11959 1.0183512 1.1087927 1.08 SH3GL2 8154979 1.1717652 1.0902108 1.0457231 1.10 8155460 1.1175282 1.2837061 1.0188383 1.14 CNTNAP3|CNTNAP3B 8155497 1.0188043 1.0948122 1.2570777 1.12 FAM27A|LOC100132948 8155540 1.2362591 1.1833643 1.015799 1.15 CNTNAP3B|LOC389725

186

8155598 1.1204321 1.0864356 1.1086205 1.11 8155600 1.1169566 1.0855013 1.1123775 1.10 8155877 1.0932345 1.1415263 1.2444632 1.16 8156043 1.4255711 1.1374127 1.1120727 1.23 PSAT1 8156082 1.1451217 1.1260239 1.0527076 1.11 8156228 1.0688051 1.2513236 1.1683512 1.16 CTSL1 8156371 1.0922865 1.074653 1.2985209 1.16 8157012 1.0623168 1.1991055 1.220366 1.16 OR13F1 8157189 1.0887934 1.1837585 1.0562266 1.11 8157191 1.0851548 1.0594902 1.1714817 1.11 8157270 1.0131303 1.1245633 1.1500993 1.10 SLC31A1 8157605 1.1711727 1.0038005 1.1191801 1.10 8157671 1.097877 1.1070179 1.1861688 1.13 OR1J4 8157798 1.1326007 1.1113757 1.018777 1.09 8157922 1.0048217 1.3315533 1.105234 1.15 LMX1B 8158539 1.1149029 1.0944729 1.1405255 1.12 C9orf106 8158686 1.1107867 1.1915709 1.0793692 1.13 FUBP3|LOC158301 8159609 1.1124463 1.2225214 1.0130048 1.12 SSNA1 8159687 1.064707 1.1443086 1.1367707 1.12 MRPL41 8160020 1.0222918 1.31548 1.1643031 1.17 C9orf38 8160317 1.0323927 1.1096245 1.1743586 1.11 LOC392288 8160478 1.1105914 1.103501 1.0314271 1.08 C9orf82 8160602 1.1767827 1.1346997 1.0107266 1.11 APTX 8160898 1.0717897 1.2448257 1.2752609 1.20 C9orf144|LOC259308 8161288 1.1058009 1.203981 1.0927559 1.13 CNTNAP3|CNTNAP3B 8161353 1.0934535 1.153123 1.0206475 1.09 MGC21881 8161442 1.0167046 1.1378217 1.3235116 1.16 FAM27D1 8161460 1.2384274 1.1555728 1.0188305 1.14 CNTNAP3B 8162132 1.3132415 1.12505 1.0015169 1.15 C9orf153 8162466 1.0028349 1.1408199 1.1335175 1.09 FAM120AOS 8162827 1.1041037 1.1476842 1.0497096 1.10 ALG2 8162934 1.1101322 1.1277272 1.1195179 1.12 OR13C5 8162940 1.1773858 1.5541046 1.2218634 1.32 ABCA1 8163147 1.4007379 1.1030204 1.0935711 1.20 8163348 1.1374443 1.1916264 1.0480826 1.13 C9orf84 8163424 1.0118339 1.2537329 1.0881228 1.12 8163509 1.0402774 1.1159592 1.0985936 1.08 ALAD 8163825 1.1332594 1.0355532 1.0949364 1.09 TRAF1 8164062 1.1816832 1.034712 1.22171 1.15 LOC613206 8164087 1.0397525 1.0915239 1.1548264 1.10 NR6A1 8164848 1.413336 1.0088072 1.2414905 1.22 LCN1L1 8164872 1.0187488 1.1398008 1.1040639 1.09 MED22 8164937 1.2778836 1.0630168 1.1228067 1.15 C9orf166 8165295 1.1212502 1.0547701 2.0085402 1.39 LCN8 8165430 1.1394957 1.0988486 1.2136029 1.15 C9orf140 8165552 1.1050174 1.2065797 1.0174502 1.11 NELF 8165707 1.0996068 1.0145693 1.1622504 1.09 8166278 1.3462875 1.3991286 1.090374 1.28 SCML1 8166349 1.712062 1.089633 1.0301445 1.28 8166355 1.116527 1.0266292 1.2466373 1.13 CNKSR2

187

8166440 1.087573 1.1628759 1.0766613 1.11 DDX53 8166525 1.1839311 1.0038052 1.1290929 1.11 POLA1 8166571 1.2587866 1.1510382 1.0584205 1.16 MAGEB6 8166948 1.215748 1.1078088 1.2184867 1.18 8167040 1.0611787 1.1831851 1.200182 1.15 RNU12 8167150 1.0625 1.1290246 1.0995166 1.10 8167161 1.2201802 1.2086483 1.3234901 1.25 8167305 1.1492254 1.881344 1.1490464 1.39 EBP 8167603 1.161024 1.0968379 1.1741564 1.14 CLCN5 8167910 1.0995148 1.1201953 1.0608511 1.09 8168026 1.1084582 1.1610855 1.1355162 1.14 LOC100130130 8168373 1.0541637 1.1458191 1.0991002 1.10 8169044 1.0147802 1.1717137 1.1385403 1.11 TCEAL3 8169158 1.0949765 1.0818429 1.11144 1.10 CXorf57 8169699 1.0873847 1.225836 1.1403404 1.15 8170135 1.0824031 1.1775193 1.0716027 1.11 GPR112 8170260 1.0852451 1.1914855 1.0171967 1.10 MAGEC3 8170326 1.1273046 1.0001676 1.1015092 1.08 FMR1 8170590 1.2002372 1.3611622 1.101783 1.22 NSDHL 8170671 1.0272422 1.096225 1.1576896 1.09 ATP2B3 8171041 1.0517544 1.1232461 1.1504529 1.11 VAMP7 8171161 1.232324 1.075585 1.1013486 1.14 ARSE 8171418 1.0840124 1.0454328 1.1044213 1.08 PIGA 8171537 1.0525298 1.115656 1.1074822 1.09 8172197 1.0576162 1.1209471 1.1844865 1.12 PPP1R2P9 8172266 1.2021059 1.3201519 1.1337903 1.22 8172538 1.0563872 1.110355 1.1853751 1.12 WDR45 8172776 1.0506797 1.1514826 1.3181976 1.17 8173349 1.1156648 1.0888233 1.147041 1.12 DGAT2L4 8173930 1.022486 1.1397591 1.096913 1.09 EPAG 8174047 1.252137 1.1058059 1.1079828 1.16 TIMM8A 8174193 1.1944609 1.0799495 1.2134553 1.16 8174207 1.0874211 1.1407509 1.09877 1.11 NXF3 8174226 1.6339853 1.3694111 1.1574363 1.39 8174304 1.0790232 1.1778498 1.201995 1.15 SERPINA7 8174568 1.1073577 1.1070614 1.5185852 1.24 LHFPL1 8174670 1.1166018 1.095806 1.0320758 1.08 RP13‐347D8.3 8175209 1.057184 1.1090962 1.1644659 1.11 TFDP3 8175269 1.0250143 1.2382797 1.1209238 1.13 FAM122B 8175763 1.1456838 1.0340267 1.2429242 1.14 PNMA5 8176133 1.0985228 1.1873282 1.0221903 1.10 G6PD 8176400 1.0390643 1.1455426 1.0823226 1.09 PCDH11Y|PCDH11X 8176679 1.0459173 1.091029 1.1503589 1.10 8176962 1.0512643 1.1230724 1.1468902 1.11 VAMP7 8177212 1.046048 1.0933173 1.1566148 1.10 8177280 1.223822 1.1852745 1.078616 1.16 8177867 1.0209799 1.1932471 1.0828575 1.10 DDR1 8177983 1.1264933 1.0591383 1.210469 1.13 TNF 8178063 1.1283132 1.1287776 1.0599024 1.11 C6orf21|LY6G6D 8178754 1.078662 1.1413023 1.1123735 1.11 PRRT1

188

8178795 1.1400728 1.1015059 1.0288314 1.09 BTNL2 8179184 1.0208236 1.1925173 1.0901499 1.10 DDR1 8179263 1.1225313 1.0565687 1.2115452 1.13 TNF 8179950 1.0632282 1.1363646 1.1133192 1.10 PRRT1 8179996 1.135169 1.0851085 1.0311197 1.08 BTNL2

189