Obesity, Adiposity, and Satiety in Mouse Models of Smith-Magenis Syndrome and Dup(17)(P11.2) Syndrome

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Obesity, Adiposity, and Satiety in mouse models of Smith-Magenis Syndrome and dup(17)(p11.2) Syndrome

Brooke Burns Virginia Commonwealth University

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This is to certify that the thesis prepared by Brooke Burns entitled Obesity, Adiposity, and Satiety in mouse models of Smith-Magenis Syndrome and dup(17)(p11.2) Syndrome has been approved by his or her committee as satisfactory completion of the thesis requirement for the degree of Master’s of Science in Human and Molecular Genetics.

Sarah Elsea VCU, Advisor

Joyce Lloyd VCU

Edmond Wickham VCU

Jenny Wiley VCU

Rita Shiang VCU, Department Representative

Jerome Strauss, Dean of VCU School of Medicine

Dr. F. Douglas Boudinot, Dean of the Graduate School

May 6, 2009

OBESITY, ADIPOSITY, AND SATIETY IN MOUSE MODELS OF SMITH-

MAGENIS SYNDROME AND DUP(17)(P11.2) SYNDROME

A thesis submitted in partial fulfillment of the requirements for the degree of Master’s of Science at Virginia Commonwealth University.

BROOKE BURNS Bachelor of Science in Biology and Anthropology: Cultural Resource Management, California State Polytechnic University, 2007

Director: Linda Corey GRADUATE PROGRAM DIRECTOR IN DEPARTMENT OF HUMAN AND MOLECULAR GENETICS

Virginia Commonwealth University Richmond, Virginia May, 2009

Acknowledgement

Throughout my research there have been a number of people who deserve acknowledgement in the work itself. First and foremost, I would like to thank my advisor, Dr. Sarah Elsea, for her support, encouragement, guidance, and patience. I would also like to thank my graduate committee, Dr. Joyce Lloyd, Dr. Edmond Wickham, and

Dr. Jenny Wiley. Their guidance was indispensable. Dr. Tim York assisted with my understanding of the microarray studies preformed in my research and Dr. Jessica

Ketchum assisted with the biostatistics used to interpret these results. Dr. Mario Dance assisted in the animal studies completed in my research.

I would like to thank the current and former members of the Elsea Lab: Dr.

Santhosh Girirajan, Stephen Williams, Eri Kamura, Ria Vyas, Heather Wright, Kristie

Schmidt, Sureni Mullegama, and Anam Bashir. Thank you for your help, friendship, and for putting up with me.

I would like to thank the entire faculty of the Human and Molecular Genetics

Department for their dedication to the instruction and guidance of their students. In particular, I would like to thank Rita Shiang who helped me through every crisis and was very generous with her time. I would also like to thank Dr. Walter Holmes and Dr.

Zendra Zehner for their assistance and instruction in biochemisty.

I would like to thank my family: Jim and Robertta Burns, Frances Burns, Gina

Soliz, Jacki Burns, Nathan Richards, and little Madeline. They made me realize that even with three thousand miles between us, our family could still be very close.

Finally, thank you to my friends in Richmond: Matthew Madre, Roseann

Peterson, Jackie Meyers, Mary Bazille, Holly Paddock, Jia Yan, and the other genetics students for making Richmond a home.

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Table of Contents Page

Acknowledgements...... ii

List of Tables ...... vi

List of Figures ...... vii

Chapter

1 CHAPTER 1: Background and literature review...... 1

Smith-Magenis Syndrome ...... 1

RAI1 and the Genetic Basis of SMS...... 1

Behavioral Abnormalities Circadian Rhythm...... 3

Obesity and SMS...... 4

Dup(17)p11.2 Syndrome ...... 5

SMS and dup(17)p11.2 Mouse Models...... 5

Summary...... 6

2 CHAPTER 2: Determination of feeding and satiation in Rai1+/- and Rai1-Tg mice

...... 8

Introduction...... 8

Materials and Methods ...... 10

Results ...... 13

Discussion...... 21

3 CHAPTER 3: Adiposity in Mouse Models of RAI1 Syndromes ...... 22

Introduction...... 22

Materials and Methods ...... 23

Results ...... 25

Discussion...... 32

4 CHAPTER 4: Microarray analysis of Rai1 Mice Hypothalami...... 34

Introduction...... 34

Materials and Methods ...... 35

Results ...... 37

Discussion...... 51

5 CHAPTER 5: Discussion...... 53

References ...... 57

Appendix: Patient Growth Survey...... 67

List of Tables Page

Table 1: Summary of Patient Fat Distribution Survey...... 26

Table 2: Top downregulated genes in Rai1+/- mice...... 38

Table 3: Upregulated genes in Rai1-Tg mouse hypothalamus...... 39

List of Figures Page

Figure 1: Food consumption in Rai1-Tg male and female mice ...... 14

Figure 2: Food consumption in Rai1+/-, Rai1-Tg, and Wt mice...... 15

Figure 3: Food consumed during the dark (active) phase...... 16

Figure 4: Food intake after 24 hr fast...... 18

Figure 5: Fasting blood glucose measurements in mice...... 19

Figure 6: Fasting serum level analysis...... 20

Figure 7: Body weights and total fat collected in Rai1-Tg mice...... 28

Figure 8: Fat distribution patterns in Rai1 mice...... 29

Figure 9: Fat distribution patterns of male Rai1-Tg mice at 14 weeks on a low fat diet.. 30

Figure 10: Blood serum levels of adiponectin...... 31

Figure 11: Heat map of genes affected by Rai1 dosage...... 41

Figure 12: Heat map of obesity related gene expression ...... 42

Figure 13: Psychological Disorders and Behavioral Pathway in Rai1+/- mice ...... 46

Figure 14: Nervous System Development and Cell Death Pathway in Rai1+/- mice...... 47

Figure 15: Protein Synthesis and DNA Replication Pathway in Rai-Tg mice...... 48

Figure 16: Development, Behavior, and Digestion Pathway in Rai-Tg mice...... 49

Figure 17: Confirmation of microarray results...... 50

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Abstract

OBESITY, ADIPOSITY, AND SATIETY IN MOUSE MODELS OF SMITH-

MAGENIS SYNDROME AND DUP(17)(P11.2) SYNDROME

By Brooke Burns, M.S.

A Thesis submitted in partial fulfillment of the requirements for the degree of Master’s of Science at Virginia Commonwealth University.

Virginia Commonwealth University, 2009

Major Director: Linda Corey GRADUATE PROGRAM DIRECTOR IN DEPARTMENT OF HUMAN AND MOLECULAR GENETICS

Smith-Magenis syndrome (SMS) is a complex disorder caused by haploinsufficiency of

RAI1 and characterized by sleep disturbances, behavioral abnormalities, mental retardation, and obesity in teens and adults. Rai1+/- mice are obese after 20 weeks. Dup(17)(p11.2) syndrome is a complex disorder associated with overexpression of RAI1. A transgenic mouse model of dup(17)(p11.2) syndrome overexpresses Rai1 and results in a mouse that is growth delayed. In order to characterize the obese phenotypes of mouse models of SMS and the role of RAI1 in obesity, daily food intake and serum levels of insulin, glucose, PPY, and leptin were measured;

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adiposity was studied by characterizing fat deposition; and gene expression was studied in the hypothalamus. These studies show that Rai1+/- mice are hyperphagic, consume more during the inactive light phase, and have altered satiety genes in the hypothalamus. Adiposity studies have shown WT females have a higher body fat content and visceral fat proportion than males, but

Rai1-Tg and Rai1+/- females have similar fat deposition patterns as WT males. Hypothalamic gene expression studies show that many genes and pathways are affected by Rai1 and Rai1 dosage, including many genes associated with obesity and satiety.

CHAPTER 1: Background and literature review

Smith-Magenis Syndrome

Smith-Magenis syndrome (SMS) is a complex disorder with multisystemic involvement caused by haploinsufficiency of RAI1, a gene located within chromosome 17p11.2 1. It is estimated that SMS occurs in approximately one in 25,000 live births, though as awareness of this disorder increases and diagnoses become more accurate, this frequency will likely increase 2.

SMS is characterized by sleep disturbances, self-injurious behaviors, craniofacial abnormalities, mild to severe mental retardation, brachydactyly, hoarse voice, and developmental delay 3. SMS patients also have sleep disturbances, which are attributed to an inverted circadian rhythm 4.

RAI1 and the Genetic Basis of SMS Retinoic Acid Induced 1 (RAI1) is the causative gene that is either deleted or mutated in SMS 5, 6. The gene encodes a 7.6 kb mRNA from six exons with a predicted protein molecular weight of 203 kD 7. RAI1 contains a bipartite nuclear localization signal and a zinc finger-like plant homeo domain (PHD) 7. The gene also contains a CAG repeat region, though anticipation is not associated with SMS and pathogenic CAG repeats are not associated with the SMS phenotype. Human RAI1 has 84% homology to the mouse gene and similar expression patterns 7.

Though the cellular function is unknown, RAI1 is likely a transcription factor. The protein shows a strong homology with the transcription factor TCF20, contains a bipartite nuclear localization signals, and ChIP-chip data has shown that it binds to the promoter regions

of other genes 7, 8 [Unpublished Elsea Lab data]. RAI1 mRNA is expressed ubiquitously throughout the body, though there is approximately three-fold higher expression in the heart and two-fold higher expression in the brain when compared with broad tissue expression 7, 9. The higher level of expression was observed throughout the brain except within the corpus callosum

7, 8, 10. The corpus callosum primarily consists of glial cells and has an absence of neurons. An early study concluded that RAI1 (then called GT1) was upregulated in differentiated neurons 10.

SMS may be caused by either a deletion or a mutation of RAI1 5, 11. While 75% of the patients with an RAI1 deletion share a common deletion on 17p11.2 of approximately 3.5 Mb, deletions may range from ~1.5 Mb to ~9 Mb 12, 13. Patients with RAI1 mutations were observed to carry de novo nucleotide changes which may lead to a truncated protein, nonsense mediated decay of mRNA, or amino acid changes in conserved regions of the RAI1 protein 6. There have been no reported cases of homozygous deletions or mutations of RAI1.

All known cases of SMS are due to either de novo mutation or deletion, though there are rare instances of parental mosaicism. The SMS region 17p11.2 is prone to recombination and flanked by low-copy number repeats 14. These repeats make the region highly prone to inter- and intra- chromosomal recombination. Intrachromatid nonallelic homologous recombination

(NAHR) results in a deletion, while interchromosomal and interchromatid NAHR typically results in both a deletion and a mutation 15. Unequal meiotic crossovers through NAHR between the proximal and distal copy number repeats is the cause of approximately 70% of SMS 17p11.2 deletions 16, 17. Parental imprinting of 17p11.2 genes has not been observed 18.

There have been a broad range of clinical features reported in individuals with SMS including: craniofacial abnormalities, skeletal and digit abnormalities, short stature, eye abnormalities, ear abnormalities, chronic otitis media, organ anomalies, velopharyngeal

insufficiency, and hoarse voice. Craniofacial abnormalities include: brachycephaly, a broad face, frontal bossing, synophrys, hypertelorism, upslanting eyes, midface hypoplasia with a depressed nasal bridge, a tented upper lip, prognathism, low-set or abnormally shaped ears, dental anomalies, cleft lip or palate, and eye abnormalities 11, 19, 20. Skeletal and digital abnormalities include: brachydactyly, persistent fetal finger pads, fifth-finger clinodactyly, 2/3 toe syndactyly, scoliosis, vertebral anomalies, and polydactyly 1, 2, 21. Chronic otitis media and sensorineural hearing loss was observed in 68% or patients 11. Heart and renal defects, and cleft lip/palate are highly variable in SMS patients. It is hypothesized that these phenotypes may be due to heterozygosity of genes other than RAI1 in the 17p11.2 region 5. Brain imaging and autopsy reports of SMS patients have revealed cases of ventriculomegaly, enlarged cisterna magnum, patial agenesis of vermis, frontal lobe calcification, Joubert syndrome, and decreases in gray matter of the incula and lenticular nucleus 1, 2, 22, 23. Seizures are seen in approximately 30% of patients 2, 11.

Most SMS patients have mild to severe mental retardation, including delayed motor and speech milestones. IQs may range from 20-78 for 17p11.2 deletion cases 2. Some patients have been classified as low normal as young children but considered to have mild mental retardation by adulthood 17.

Behavioral Abnormalities Circadian Rhythm Behavioral phenotypes are observed in the majority of SMS cases, with 92-98% exhibiting self-injurious behavior 24, 25. Self-injurious behavior may include: hand-biting, hitting, self-pinching, scratching, picking at sores, head- banging, pulling at nails, and polyembolokoilamania 1, 26. Stereotypies, short attention span and poor short term memory have also been observed 27, 28. Some SMS patients have also been diagnosed with ADHD, OCD, autism, and hyperactivity 29, 30.

Sleep disturbances are reported in 75-100% of SMS patients 11, 31. Hypersomnolence is observed in infants, while older children have difficulties falling asleep, reduced night sleep, short sleep cycles with nocturnal and early-morning awakening, and excessive daytime sleepiness 31, 32, 33, 34. These patterns are likely due in part to inverted circadian rhythm of melatonin, though other circadian rhythm hormones (cortisol, growth hormone, and prolactin) are not affected 30, 33. Melatonin is a ubiquitous hormone that regulates sleep induction through vasodilation in the extremities and seasonal phase shifting 35, 36. Melatonin is synthesized in the pinealocytes of the pineal gland via the acetylation of serotonin, which is then converted to its active form via hydroxyindole O-methyltransferase. Photosensitive melanopsin expressing retinal ganglion cells signal the suprachiasmatic nucleus (SCN) via the retinohypothalamic track 35. The SCN then releases an inhibitory signal to the paraventricular nucleus, which stimulates the production of melatonin in the pineal gland. Circulating melatonin has a half-life of 2-20 min and is metabolized in the liver 35, 37. In a normal circadian rhythm, melatonin plasma levels rise a few hours before sleep is peaks in the middle of the night. In SMS patients, melatonin plasma levels are high during the day but low at night. This altered melatonin pattern has been treated with !-adrenergic antagonists during the day and melatonin administration during the night. The combination treatment was observed to decrease daytime sleepiness, reduce the incidence of tantrums, and increase concentration. There were no cognitive improvements and problematic behavior decreased but was not completely eliminated 30.

Obesity and SMS In infants with SMS, there are feeding difficulties and hypotonia. In teens and adults, truncal obesity is common 17. The majority of SMS patients become obese by the time they reach adulthood, with an increased prevalence of hypercholesterolemia 38. A study of

54 children found that by age 12, SMS females were in the 90th weight percentile and SMS males were in the 90th percentile by age 14 38. An earlier study found that a majority of SMS patients had lipid values greater than the 95th percentile for age and gender for at least one or

more of the following: total cholesterol, triglycerides, and/or low density lipoproteins 39. SMS patients may develop an obsessive fixation on food and problems regulating intake, which may require caretakers to regulate intake or lock up food.

Dup(17)p11.2 Syndrome

A different genetic disorder, dup(17)(p11.2) syndrome, sometimes referred to as Potocki-

Lupski syndrome, results in subtle craniofacial abnormalities, growth delay in childhood, heart defects, mild to severe mental retardation, lean body mass, and hyperactivity 40, 41. There are no observed circadian rhythm anomalies in dup(17)(p11.2) syndrome 42. All individuals with this disorder have 3 copies of RAI1 42, 43. While the incidence of dup(17)(p11.2) syndrome is unknown, it is suspected to have a similar incidence to SMS, the reciprocal deletion syndrome 43.

SMS and dup(17)p11.2 Mouse Models

In a model of SMS, heterozygous Rai1-knockout mice were created on a mixed background and backcrossed to C57Bl/6. These mice are slightly smaller than their wild-type littermates at 5 weeks, comparable to control mice at 10 weeks, and overweight by 20 weeks 9.

They also have an altered sleep cycle, behavioral abnormalities, variable penetrance of craniofacial defects, and reduced fertility 44 [Elsea lab unpublished data]. These mice were created by an insertion of a Escherichia coli lacZ coding sequence and a neoR expression cassette which deleted 3910 bp nucleotides of the coding region of exon 2 in 129SvEv/Brd embryonic stem cells. These clones were inserted into C57BL/6Tyrc-Brd blastocysts 9. This insertion truncates the Rai1 protein, removing nuclear localization signals and the PHD domain, producing

9 an effectively null allele . Animals that are homozygous for the Rai1 mutation are embryonic

lethal by approximately 16 days post coitum 9. There is a distorted transmission of the mutant

Rai1 allele, with litters containing ~13% Rai1+/- pups compared to ~50% Mendelian expectations 45

In order to evaluate the effects of overexpression of RAI1 and to determine how it contributes to the human dup(17)(p11.2) syndrome phenotype, a transgenic mouse model was created that overexpresses Rai1 42. Both hemizygous (with 2 copies of the BAC transgene) and homozygous (4 copies of the BAC transgene) Rai1 overexpressing mice in a C57Bl/6 background have been evaluated 42. Previous members of the Elsea lab reported growth retardation in Rai1-transgenic mice up to 20 weeks when compared with wild-type littermates 42.

These mice also have poor maternal behavior, have an abnormal gait, display behavioral abnormalities, and are hyperactive 42.

Summary

RAI1 duplication and deletion syndromes are multisystemic and complex. This study aims to focus on the obesity and growth phenotypes associated with Smith-Magenis Syndrome and dup(17)(p11.2) syndrome. Both the overexpression and underexpression of Rai1 in mice produces phenotypes similar to those observed in dup(17)(p11.2) syndrome and SMS patients, making these mice ideal to study the effects of Rai1 dosage. In order to determine the role of

Rai1 on growth and obesity, we have conducted studies focused on feeding behavior, adiposity, and hypothalamic expression. We have conducted an anonymous survey to characterize the fat deposition patterns in patients with SMS and dup(17)(p11.2) syndrome. These studies reveal that Rai1 plays a role in feeding behavior and timing, fat distribution, and the expression of genes relating to satiation and obesity within the hypothalamus. A more thorough understanding

of the role of Rai1 in growth and obesity will allow us to understand the comorbidities that may affect patients of RAI1 syndromes and provide insight into the complex biology of obesity, growth, and behavior.

CHAPTER 2: Determination of feeding and satiation in Rai1+/- and

Rai1-Tg mice

INTRODUCTION

Simulation of human conditions in animals can be used to identify biological mechanisms that contribute to behavior in known disorders. Mouse models are particularly useful for studying human genetic disorders. Transgenic mice and gene targeting in mice allow for the study of dosage effects of genes, because genes may be added to mimic overexpression, and

“knocked-out” to mimic under expression or null expression. Rai1-Tg mice overexpress Rai1, while Rai1+/- mice have reduced expression-- modeling dup(17)(p11.2) syndrome and Smith-

Magenis syndromes, respectively.

In Smith-Magenis syndrome, obesity is a noted phenotype in teens and adults, and hyperphagic behavior is observed 11, 27, 38. Case studies in SMS patients have revealed obsessive preoccupation with food and hoarding 11. Likely due to circadian rhythm abnormalities, night eating is also observed in SMS patients. Noted therapies for patients with SMS include strict dietary control and restricting food access by locking cabinet or kitchen doors.

Mouse models have been useful in identifying genetic factors that contribute to hyperphagic behavior and obesity in humans disorders, including Bardet-Biedl syndrome 46,

Prohormone convertase 1 (Pc1) deficiency 47, leptin deficiency 48, and brain-derived neurotrophic

factor (Bdnf) haploinsufficiency 49. Circadian rhythm abnormalities have also been studied in mice, including Clock mutation 50, Baml1 mutation 51, and the Period (Per) genes 52. These studies demonstrate that circadian rhythm is essential for normal behavior, feeding behavior, blood pressure, weight, and fertility.

To characterize feeding in these mouse models, we conducted studies to determine overall food consumption, the proportion of food consumed during the active phase, and serum level analysis of satiety hormones. Overall food consumption can be used to determine hyperphagia. The amount of food consumed during the active phase can be used to determine if these animals are modeling “night eating” or eating during the inactive phase. Fasting studies and serum level studies can be used to determine the efficiency of the satiation response after a period of induced hunger. These studies may lead to more comprehensive understanding of the behavioral phenotypes of Smith-Magenis that contribute to obesity.

MATERIALS AND METHODS

Breeding and maintenance. Rai1+/- and Rai1-Tg mice were bred with C57Bl/6J mice. When breeding, Rai1+/- and Rai1-Tg males were in isolated cages with one or two C57Bl/6J females, while Rai1+/- and Rai1-Tg females were in isolated cages with one C57Bl/6J male. Due to the poor maternal behavior of the transgenic females, nontransgenic or C57Bl/6J females were added 2-3 days prior to birth 42. Pups were weaned, tail clipped, tagged, and separated by sex at

3 weeks. One to five mice were housed by sex and age in a cage.

Mice had access to water and a standard diet of high fat Lab Diet 5P06 and low fat Tekland

LM-485 7012 food ad libitum. A constant temperature (21°C), humidity (40%), and automatic twelve hour light/dark cycle were maintained at all times. All tests were preformed during the light portion of the cycle except for light phase feeding studies, which were done using a red light to avoid altering the dark period. All animals were housed in compliance with the guidelines of the IACUC at Virginia Commonwealth University. All procedures were submitted and approved by the IACUC at Virginia Commonwealth University.

Genotyping. The genotypes of the mice were confirmed using PCR analysis on DNA prepared from mouse tails using a salt extraction method and the Qiagen miniprep isolation protocol. The genotypes of Rai1-Tg mice were confirmed using unique BAC-end specific primers reported in

Girirajan, 2008. The genotypes of the Rai1+/- were confirmed using target allele and wild type primers reported in Bi et al., 2005.

Isolated feeding studies. In order to examine the average daily food consumption, males and females of Rai1-Tg and Rai1 +/- genotypes were placed in isolated cages and given a pre-

weighed amount of high fat food. Food was weighed every 24hrs for 5 days. Evaluations were preformed at 5, 10, 15, 20, and 25 weeks. Data were compared with sex and age matched wild- type littermates. These methods were developed based on techniques reported in the literature regarding mouse feeding behavior 46, 53, 54.

Light phase and fasting feeding studies. To determine the effect of Rai1 on night eating and satiation, male mice of each genotype (Rai1-Tg, Rai1+/-, and wild-type) were placed in isolated cages with a predetermined amount of high fat food and an unlimited amount of water. Food was weighed every 12 hrs for 3 days. Night measurements were taken using a red lamp. To fast animals, all food was removed for 24 hrs and animals were placed in a new cage to prevent hoarding. After 24 hrs a predetermined amount of food was returned at the beginning of the light phase. Food weight measurements were taken every 2 hrs for 6 hours after the fast.

Measurements continued every 12 hrs for 3 days. These evaluations were preformed at 15 and

20 wks of age. These methods were developed based on techniques found in the literature regarding mouse night eating behavior 55.

Blood glucose. Blood was drawn from a needle prick on the end of the tail and measured using capillary test strips and an Easy Pro glucometer. Glucose tests were done in duplicate at 20 weeks to ensure accuracy.

Serum analysis. Blood was collected from animals via cardiac puncture after CO2-induced unconsciousness. Serum was separated by allowing the blood to clot for twenty minutes followed by centrifugation at 2000 xg for 10 minutes. For samples that required fasting

conditions (leptin, insulin, PYY), blood was collected from the “facial vein” under general anesthesia with isofluorene after the previously described fasting studies. ELISA tests were performed at the University of Cincinnati Mouse Phenotyping Core.

Ketone analysis. Animals were isolated in small container with no bedding until they urinated.

After the animals were returned to their normal cages, an Alva Thinz Metabolism Strip #25 was dipped into the urine.

Statistical Analyses. All statistical methods were conducted in JMP software. Mean food consumption at 5, 10, 15, 20, and 25 weeks were compared using pooled data from 12 different isolated feeding studies that depended on genotype and age availability. These data were compared among the 3 genotypes (WT, Rai1+/-, and Rai1-Tg) using ANOVA.

Food intake after the 24 hr fast (taken at 2-48hrs) was evaluated using repeated measures

ANOVA, with effects for individual mouse, genotype, time point, and group time interaction.

These data were also evaluated separately for each time point using ANOVA.

The average proportion of food consumed at night was evaluated using repeat measures

ANOVA.

RESULTS

Isolated Feeding Studies. The feeding behavior of Rai1+/-, Rai1-Tg, and wild-type mice was observed using isolated feeding studies as described in the methods. These studies were sex and age matched at 5, 10, 15, 20, and 25 weeks for Rai1-Tg and wild-type mice. Rai1+/- mice were not included in all studies due to their drastically reduced fertility. Male Rai1+/- were studied at

15 and 20 weeks of age. A two way ANOVA analysis of the average amount of food per day (g) over the weight of the mouse (g) was used to compare each group. Food consumption indicates that there is no difference between wild-type and Rai1-Tg mice food intake at 5, 10, 15, 20, or 25 weeks. Rai1-Tg mice did not consume more or less than their wild-type littermates (Fig. 1A,

1B). However, using pooled data from isolated feeding experiments, we found that Rai1+/- male mice were hyperphagic at 15 weeks (p=. 0159) and 20 weeks (p<0.05) (Fig. 2A, 2B).

Light phase and fasting feeding studies. To determine if the mice were eating during the rest cycle (for nocturnal mice, this is the light phase of the 12 hr dark/light cycle), isolated feeding studies were conducted by taking measurements every 12 hours, shortly after the beginning of each light and dark phase. Male Rai1+/-, Rai1-Tg, and wild-type littermates were studied at 15 and 20 weeks of age. A two way ANOVA analysis of the proportion of total food consumed during the dark cycle was used to compare each group. At age 15 weeks, Rai1+/- mice consumed a significantly higher proportion of their food during the (inactive) light phase than wild-type mice (p=.0191) (Fig. 3A). At 20 weeks, this pattern was observed with Rai1+/- mice consuming ~57% of their food during the dark phase and wild type mice consuming ~65% of their food during the dark phase, though it did not achieve significance (Fig. 3B).

Figure 1: Food consumption in Rai1-Tg male and female mice. A. Daily food intake (g) over body weight (g) for male mice ages 5-25 weeks. B. Daily food intake (g) over body weight (g) for female mice ages 5-25 weeks. Data were analyzed using a two way ANOVA for genotype, gender, and interaction (n!5 of each gender and genotype). Graphs indicate mean±SEM.

Figure 2: Food consumption in Rai1+/-, Rai1-Tg, and Wt mice. A. Daily food intake (g) over body weight (g) for male mice at age 15 weeks. B. Daily food intake (g) over body weight (g) for male mice at age 20 weeks. Data were analyzed using a two way ANOVA for genotype, gender, and interaction (n!5 of each gender and genotype). Graphs indicate mean±SEM.

Figure 3: Food consumed during the dark (active) phase. A. Rai1+/- mice eat significantly less of their daily intake during the dark phase. (p<.05) B. Rai1+/- mice eat less than wild-type mice at night. However these data do not achieve significance.

Hunger was induced using a 24 hr. fast to test satiety. Male mice of each genotype were isolated in cages with no access to food. After 24 hours, serum was collected, blood glucose levels were assessed, and food was returned. When food was returned, measurements of food consumption were taken ever 2 hours for the first 10 hours, and every 12 hours afterwards. For the first 12 hrs after fasting conditions, Rai1+/- mice consume significantly more food than wild- type mice (p=.0023) (Fig. 4A). Also, Rai1+/- mice were observed to have a delayed satiation response. Rai1+/- consume significantly more food than wild-type mice at 36 hours post fast

(p=.0473) (Fig. 4B). Blood glucose tests did not indicate elevated fasting glucose levels in either

Rai1-Tg or Rai1+/- mice (Fig. 5).

Urine Ketone Test. In order to determine if mice had diabetic ketoacidosis, urine was tested for ketones using urinalysis dip strips. Non-fasting wild-type, Rai1-Tg, and Rai1+/- mice showed no evidence of ketone bodies in urine.

Serum Analysis. To determine the possible effects of the satiety signals leptin and PYY, and insulin on the phenotype of these mice, isolated serum samples from each genotype were evaluated at the University of Cincinnati Mouse Phenotyping Core (Fig. 6). Insulin levels were low in the Rai1-Tg mice and unaltered in the Rai1+/- mice. Leptin levels were high in the

Rai1+/- mice. PYY was also elevated in the Rai1+/- mice, but not significantly.

Figure 4: Food intake after 24 hr fast. A. Immediately after the fast, food consumption was measured at 2 hr intervals. At 8 and 12 hrs, Rai1+/- mice consume more food than Rai1-Tg or wild-type mice. B. For two days after the fast, food intake was measure every 12 hrs. At 12 and 36 hours after the fast, Rai1+/- mice consume more of their food than wild-type mice. The large SEM for Rai1+/- mice at 48 hrs indicates delayed satiety in only one of the mice; this did not achieve statistical significance. Graphs indicate mean±SEM.

Figure 5: Fasting blood glucose measurements in mice. A. At age 15 weeks. The large SEM observed is within normal range for fasting glucose level and is a consequence of our low sample size B. At age 20 weeks. There was no observed difference in fasting blood glucose in any of the genotypes. Graphs indicate mean±SEM.

Figure 6: Fasting serum level analysis. Fasting serum levels of mice for insulin, PYY, and leptin. Data indicate hypoinsulinemia in Rai1-Tg mice, a possible increase in PPY in Rai1+/- mice, and increased leptin in Rai1+/- mice. *p<.05. Graphs indicate mean±SEM.

DISCUSSION

These data show that Rai1+/- mice are hyperphagic and fasting studies indicate that these mice have a delayed satiation response after induced hunger. These mice do not exhibit glucosuria, increased insulin, or have an elevated fasting glucose level, so it is unlikely that they have some type of insulin resistance. The elevated leptin and PYY levels in these mice indicate that the gut satiety signals are high. If leptin and PYY signaling were functioning as we would expect, Rai1+/- mice with an increased serum leptin would have a sensitive satiation response and possibly hypophagia and resistance to obesity 56, 57. However, high serum leptin has been identified as an early indication of adult-onset obesity in children 58. Taken together, the obesity observed in these mice is likely due to hyperphagia caused by a muted satiation response possibly due to leptin resistance 59.

Rai1-Tg mice are not hyperphagic and do not have a delayed satiation response. These mice do have hypoinsulinemia. However, these mice do not have an elevated fasting blood glucose. Taken together, these results may indicate high insulin sensitivity.

Light phase studies may indicate increased feeding activity during the day in Rai1+/- mice, which may mimic night eating behavior in humans. Unfortunately the variability within the sets of mice and lack of significance at 20 weeks indicate that, while promising, the incidence of night eating within Rai1+/- mice will need to be confirmed with a larger sample of mice.

Night eating, increased leptin, and obesity have also been observed in the mice deficient in the prostaglandin E2 signaling gene, EP3R 55. The similarity of these phenotypes could indicate a role of adipose hormone signaling in the phenotype of Rai1+/- mice.

CHAPTER 3: Adiposity in Mouse Models of RAI1 Syndromes

INTRODUCTION

Clinician and geneticists’ notes indicate truncal obesity in individuals with Smith-

Magenis Syndrome 17. SMS patients also have an increased incidence of hypercholesterolemia, which can contribute to heart disease 39. This is of particular interest due to current research that demonstrates increased stores of visceral fat may be pathogenic for co-morbidities of obesity including: cardiovascular disease, insulin resistance, hypertension, and metabolic syndrome 60.

Adiponectin, an adipocytokine, has been associated with juvenile onset obesity and increased visceral obesity 61.

In order to characterize adiposity in SMS and dup(17)(p11.2) syndrome, we conducted an anonymous online survey to assess body shape, determined fat deposition patterns in Rai1 mouse models, and measured serum adiponectin levels in mice. The anonymous online survey can be used to determine body shape, including truncal obesity, in patients with SMS and dup(17)(p11.2) syndrome. Body fat deposition in mouse models can be used to assess visceral fat deposition in mouse models of SMS and dup(17)(p11.2) syndrome. Investigation of fat deposition patterns and adiponectin levels in Rai1 mice may help to characterize the cause and pathogenicity of the truncal obesity observed in Smith-Magenis patients.

MATERIALS AND METHODS

Survey. An anonymous online survey was designed to assess body shape in patients with RAI1 deletions, mutations, and duplications. The survey was distributed to caregivers of RAI1 patients via the SMS support website PRISMS, the SMS Yahoo! Listserv, and the dup(17)(p11.2) syndrome Yahoo! Listserv. The survey assessed body type by asking the caregiver to select a description which best described the fat deposition pattern for their child. The survey included a cartoon depiction of the type of fat deposition as well as a written description such as “My son/daughter tends to store extra weight on arms, legs, hips, and buttocks. Very little weight is stored around the waist.”

The survey addressed the patient’s backgrounds and included questions regarding gender, age, height, weight, formal diagnosis by a geneticist, and the most recent time the patient saw a physician. The patient’s clinical history of diabetes, high cholesterol, and high triglycerides were also assessed. Weight percentile was determined using BMI and CDC growth assessment charts for children ages 2 to 20 years. See Appendix 1 for the survey measure.

Fat Distribution Patterns in Rai1 mice. Fat distribution in Rai1+/- mice and Rai1-Tg mice was determined via dissection. Male and female mice of each genotype were selected at ~30 weeks, weighed, and euthanized via a CO2 chamber. Intra-abdominal fat pads (gonadal, retroperitoneal, and mesenteric) and subcutaneous fat pads (dorsal, inguinal and groin) were collected via dissection and weighed 55. Measurements of both abdominal and subcutaneous fat pads were combined to determine the amount of total fat collected.

Serum Analysis. Blood was collected from animals via cardiac puncture after CO2-induced unconsciousness. Serum was separated by allowing the blood to clot for 20 minutes followed by centrifugation at 2000 x g for 10 minutes. ELISA tests for blood serum levels of adiponectin were performed at the University of Cincinnati Mouse Phenotyping Core.

Statistics. Fat distribution p-values were determined using a one-way ANOVA fit model with corrections for sex, genotype, and weight. All tests were done using JMP statistical software.

RESULTS

Body shape and adiposity in RAI1 patients. An online survey of RAI1 patient caregivers was used to corroborate clinician’s notes regarding abdominal obesity in SMS patients (Table 1).

From a sample of 65 responses, there were 38 individuals with Smith-Magenis syndrome (36 with a RAI1 deletion and 2 individuals with a RAI1 mutation) and 12 individuals with dup(17)(p11.2) syndrome.

In SMS individuals with a RAI1 deletion, the mean age was 13.5 years (range = 3 to 51), and 58% were female. Of these patients, three were diabetic, five had high cholesterol, and four had high triglycerides. In the SMS deletion group, 41% had a BMI in the 95th percentile or higher for their age and sex. Seventeen individuals (45%) were described as having mostly truncal obesity with excess weight stored “around the waist and stomach areas- very little stored around the legs and buttocks”. Of these seventeen individuals, 13 (76%) were female.

In dup(17)(p11.2) syndrome individuals, the mean age was 6.5 years (range=2 to 12), and

33% were female. None of these individuals were diabetic or had high cholesterol or triglycerides. In the dup(17)(p11.2) syndrome group, two (16%) had a BMI in the 95th percentile or higher for their age and sex. Nine individuals (75%) were described as “very thin, has no excess weight, [and] veins are visible through the skin.”

Table 1: Summary of Patient Fat Distribution Survey

SMS Dup17p11.2

n 38 12

Mean age 13.5±9.0 6.5±3.0

Range 3-51 2- 12

Sex 42% M/ 58% F 67% M/33% F

BMI 23.6±7.9 16.9±4.4

>95 percentile 41.00% 16.70%

>95 percentile; >9 years 50% 0%

Diabetes 3 0

Hypercholesterolemia 5 0

High triglycerides 4 0

Subcutaneous only 0 0

Truncal only 9 (7f) 0

Uniform distribution 10 2 Fat Distribution No excess weight 3 9

Excess uniform distribution 8 0

Excess truncal only 8 (6 F) 1

Fat distribution patterns in Rai1 mice. Fat distribution was analyzed by dissection on Rai1-

Tg, Rai1+/-, and wild-type mice. Rai1-Tg and wild-type mice were analyzed at ~30 weeks, though Rai1+/- mice were analyzed at 45+ weeks due to problems procuring new mice. There was no difference in body weights or the amount of total fat extracted between sex-matched

Rai1-Tg and wild-type mice (Fig. 7A, Fig. 7B). However, there was a significant difference in the amount of abdominal fat in Rai1-Tg and Rai1+/- mice compared to wild-type mice (Fig. 8).

Wild-type male mice have a higher proportion of abdominal fat than subcutaneous fat, while

Rai1-Tg and Rai1+/- have higher proportions of subcutaneous fat (Fig. 8A). In females, the patterns are reversed. Wild-type female mice have a higher proportion of subcutaneous fat than abdominal fat, while Rai1-Tg and Rai1+/- have higher proportions of abdominal fat (Fig. 8B).

To determine that the previous results were attributable to the genotypes and not the diet or age of the mice, the same studies were performed on 14 week old male littermates fed a regular chow rather than a high fat chow. The results showed the 14 week old Rai1-Tg mice also have a higher proportion of subcutaneous fat than their wild-type littermates, indicating diet and age were not a factor (Fig. 9).

Blood serum adiponectin levels. To determine the possible effects of the fat secreted hormone adiponectin on the phenotype of these mice, isolated serum samples from each genotype were evaluated at the University of Cincinnati Mouse Phenotyping Core. There was no significant difference in serum levels of adiponectin in Rai+/- or Rai-Tg mice when compared to wild-type adiponectin levels (Fig. 10).

Figure 7: Body weights and total fat collected in Rai1-Tg mice. A. Comparison of body weights at the time of study between sex matched Rai1-Tg and wild-type mice. There was no observed difference between genotypes. B. Comparison of total fat extracted from sex matched Rai1-Tg and wild-type mice during the fat deposition study. There was no observed difference between genotypes. Total fat was defined as the weight of the sum of all extracted fat over body weight. Graphs indicate mean±SEM.

Figure 8: Fat distribution patterns in Rai1 mice. A. Fat distribution in males. Rai1+/- and Rai1-Tg mice have a significantly lower proportion of visceral fat than wild-type mice. B. Females have an opposite fat deposition pattern compared to males. Female WT mice have a lower proportion of visceral fat than males, while Rai1+/- and Rai1-Tg mice have a significantly higher proportion of visceral fat than wild-type female mice. (* p <.05, **p <.01, *** p <.001) Graphs indicate mean±SEM.

Figure 9: Fat distribution patterns of male Rai1-Tg mice at 14 weeks on a low fat diet. Rai1-Tg mice (n=2) have a significantly lower proportion of visceral fat than their wild-type littermates (n=2). (p<.05) Graphs indicate mean±SEM.

Figure 10: Blood serum levels of adiponectin. There was no significant difference in the blood serum levels of adiponectin in any of the genotypes. Graphs indicate mean±SEM.

DISCUSSION

Population estimates from the Centers for Disease Control show that the prevalence of obesity at ages 6-11 is 17% and at ages 12-19 is 17.3%. Based on our sample, the prevalence of obesity in SMS patients at ages 9 and up is 50%. Edelman, et al. (2007) previously reported a higher incidence of obesity in RAI1 mutation patients, likely due to hyperphagia 11. A disproportionate number of SMS females also have a high reported abdominal fat deposition pattern. Visceral obesity has a much higher prevalence in males 62. Visceral obesity is associated with an increased risk for obesity comorbidities, including hypertension, high triglycerides, and diabetes. The incidence of diabetes, hypercholesterolemia, and hypertension is not higher for individuals with SMS when compared to the general population. This may be due to a lack of testing in younger patients or due to a lack of data on older SMS individuals.

When we evaluate the fat distributions in Rai1+/- and Rai1-Tg mice, we find similar changes in body fat distribution. Rai1+/- and Rai1-Tg females have higher proportions of abdominal fat than wild-type females, while Rai1+/- and Rai1-Tg males have less abdominal fat than wild-type males. This is similar to the large proportions of SMS females with reported abdominal adiposity.

Adiponectin negatively correlates with high visceral fat deposition, insulin resistance, diabetes mellitus, coronary heart disease, and metabolic syndrome 63, 64, 65, 66. Serum adiponectin levels in Rai1+/- or Rai1-Tg mice do not decrease and the serum results in Chapter 2 do not indicate diabetes. These data indicate that RAI1 haploinsufficiency contributes to early-onset obesity and increased visceral fat deposition in females, but does not increase incidences of visceral fat associated insulin resistance and diabetes.

CHAPTER 4: Microarray analysis of Rai1 Mice Hypothalami

INTRODUCTION

The alteration in dosage of transcription factors may have widespread effects on other genes. The Rai1 protein contains a bipartite nuclear localization signal and a PHD/zinc finger domain 7. It also has a strong homology to the transcription factor TCF20 8. Given these characteristics, we believe that the RAI1 protein is a transcription factor and that variation in dosage of RAI1, such as those seen in dup(17)(p11.2) syndrome and Smith-Magenis syndromes, may contribute to the complexity of these disorders.

The hypothalamus is a region of the brain involved in satiety 67, 68, 69, 70, thirst 71, 72, thermoregulation 73, 74, and circadian rhythm 75. Alterations of gene expression in the hypothalamus, including Bdnf, Npy, and estrogen receptor genes, can affect feeding and hyperphagia-induced weight gain in mice. In order to evaluate the downstream effects of Rai1 dosage alterations in mice, we choose to evaluate gene expression levels in the mouse hypothalamus. Whole genome hypothalamic expression patterns may indicate obesity associated genes that are altered when Rai1 is overexpressed in Rai1-Tg mice or underexpressed in Rai1+/- mice. These studies may indicate downstream pathways which affected by Rai1 expression that contribute to the phenotypes of SMS or dup(17)(p11.2) syndrome.

MATERIALS AND METHODS

Hypothalamus Isolation- Male and female 30 week old wild-type and Rai1-Tg mice were euthanized by CO2 chamber. Rai1+/- mice were 52+ weeks at time of death and tissue collection. Hypothalami were immediately extracted from mouse brains and stored at -80°C.

RNA Isolation- RNA was isolated using the Trizol Reagent/chloroform isolation method. The quality of the isolated RNA was determined using a 260/280 absorbency ratio and assessed using a Thermo Scientific Nanodrop 1000. Equal quantities of RNA were pooled for a wild-type sample (6 male mice) and a Rai1-Tg sample (5 male mice). Only two 52+ week male mice were available for the Rai1+/- sample. These mice were used as individual samples so that the data may be combined with future microarray data.

Microarray- Microarray hybridization was performed in the Massey Cancer Center Nucleic

Acids Research Facility, DNA Microarray Core using an Illumina MouseWG-6 v2.0 expression beadchip. The array was read using an Illumina BeadScan confocal scanner and analyzed by

Illumina's BeadStudio software.

Microarray Data Analysis- Using the Illumina BeadStudio Software, differential analysis was performed between the Rai1+/- and wild type genotypes and the Rai1-Tg and wild-type genotypes. Data were normalized using the quantile method. Differential P-values were determined using the Illumina custom analysis. The software was also used to calculate a false discovery rate (FDR) and to adjust p-values accordingly. Detection p-values <.05 were used to

determine which probes were detected. Heat maps to visualize gene distribution were created using JMP statistical software.

Pathway Analysis- Pathway analysis was done using the Ingenuity Pathway analysis for gene and protein interaction. Significant interactions were determined using the Ingenuity Pathway

Knowledge Base and a Fisher’s exact test for significance for each pathway and biological function.

Real-time quantitative PCR- RNA from hypothalami from male and female mice of each genotype was isolated using the Trizol reagent/chloroform method and quality assessed by nanodrop. Some individual hypothalamus samples that were used in the pooled samples in the microarray were analyzed using Q-PCR. cDNA extraction was performed using 4 "g of RNA,

Invitrogen Superscript 2 reverse transcriptase and random primers according to the manufacturer’s instructions. ABI Taqman probes for mouse mRNA expression were used to detect gene expression for Bdnf, Cck, Npy, and Rai1. All samples were run in triplicate in a 10

"L reaction on a ABI Prism 7500 Fast System with Gapdh as an endogenous control.

RESULTS

Hypothalamic gene expression altered by Rai1 dosage. Using a microarray to test the global expression of genes in the hypothalami of Rai1+/- and Rai1-Tg mice, we found that Rai1+/- mice express a 1.6-fold decrease of Rai1 in the hypothalamus compared to wild-type mice, while

Rai1-Tg mice have a 1.2-fold increased expression of Rai1. These values are muted compared to previous data which suggest Rai1+/- mice express less than ~50% Rai1 than wild-type and Rai1-

Tg mice express a 1.5 fold increase in Rai1 over wildtype 42, 9. Q-PCR was used to confirm Rai1 expression.

To identify genes differentially expressed from wild-type, we used the Illumina Custom test for differentiation, which is more useful at estimating variance within a small sample size than the Wilcoxon signed-rank test or Student’s t-test. Our analysis detected 23,000 probes for

16,000 genes.

In Rai1+/- mice, 92 genes were significantly upregulated (p < .05); using a higher stringency (p < .01), 5 genes were significantly upregulated. In Rai1+/- mice, 397 genes were significantly downregulated (p < .05), while using a higher stringency (p < .01), 68 genes were significantly downregulated. The top genes downregulated by reduced Rai1 expression by differential p-value and fold change are shown in Table 2.

In Rai1-Tg mice, 1593 genes were significantly upregulated (p < .05). Using a higher stringency (p < .01) to determine differential expression, 899 genes were significantly upregulated. In Rai1-Tg mice, 1462 genes were significantly downregulated (p < .05). Using the more stringent criteria (p < .01), 864 genes were significantly downregulated. The top genes upregulated by Rai1 overexpression by differential p-value and fold change are shown in Table

Table 2: Top downregulated genes in Rai1+/- mice. Genes were identified using differential expression p-values and ranked fold change. Functional information from Entrezgene, RefSeq, and Ingenuity.

Gene Differential fold change Gene Name Functions Associated Phenotypes/Functions Symbol p-value HOXA5 <.0001 -223.2415 homeobox A5 chr7 Transcription Factor Tumorigenesis HOXB6 <.0001 -109.6210 homeobox B6 chr17 Transcription Factor Embryonic development chr17 somatotropin/ Growth, obesity, dwarfism, metabolic GH <.0001 -82.7727 Growth Hormone prolactin hormone disorder, hypercholesterolemia PCP2 <.0001 -75.9074 Purkinje cell protein 2 chr19 GTPase activator Purkinje cell degeneration Glucocorticoid Receptor Signaling, PRL <.0001 -36.0794 Prolactin chr6 Cytokine hormone hyperinsulinemia HOXB5 <.0001 -17.3255 homeobox B5 chr17 Transcription Factor Lung and gut development solute carrier family 22 chr11 Membrane Sodium-independent organic anion SLC22A6 <.0001 -15.0388 member 6 Transporter transport Elastic absorption and energy dissipation of PRG4 <.0001 -12.3189 proteoglycan 4 chr1 Proteoglycan synovial fluid solute carrier family 6, GABA receptor signaling, epilepsy, SLC6A12 <.0001 -7.1673 chr12 Neurotransmitter member 12 generalized anxiety disorder, Steroidgenesis, pain and energy POMC <.0001 -5.2112 propiomelanocortin chr2 Hormone precursor homeostasis, melanocyte stimulation, and immune modulation EN1 <.0001 -4.8849 engrailed homeobox 1 chr2 Transcription Factor Development: segmentation chr1 Glucocorticoid- Cytoskeletal function, regulation of MYOC <.0001 -4.3210 myocilin inducible response protein intraocular pressure chr2 Membrane Cleavage of X-proline dipeptides, diabetes, DPP4 0.0022 -4.2198 dipeptidyl- peptidase 4 glycoprotein hyperinsulinemia, obesity IRX2 <.0001 -3.6987 iroquois homeobox 2 chr5 Transcription Factor Embryonic development solute carrier family 38, chrX Amino acid Na-coupled transport of neutral amino SLC38A5 0.0017 -3.2475 member 5 transporter acids, mental retardation

Table 3: Upregulated genes in Rai1-Tg mouse hypothalamus. Genes were identified using differential expression p-values and ranked fold change. Functional information from Entrezgene, RefSeq, and Ingenuity

Differential Fold Gene Name Function Associated Phenotypes/Functions P-val Change GH <.0001 71.3 Growth Hormone chr17 somatotropin/ Growth, obesity, dwarfism, metabolic disorder, prolactin hormone hypercholesterolemia LHB <.0001 68.39 luteinizing hormone beta chr19 glycoprotein hormone Spermatogenesis and ovulation, obesity polypeptide PRL <.0001 62.86 Prolactin chr6 Cytokine hormone Glucocorticoid Receptor Signaling, hyperinsulinemia TSHB <.0001 33.03 thyroid stimulating chr1 pituitary hormone cell-cell signaling, anatomical structure hormone, beta morphogenesis, response to vitamin A CGA <.0001 28.64 glycoprotein hormones, chr6 Glycoprotein Hormone Thyroid hormone signaling, obesity alpha polypeptide HOXB7 <.0001 23.62 homeobox B7 chr17 transcription factor Tumorigenesis POMC <.0001 4.17 propiomelanocortin chr2 Hormone precursor Steroidgenesis, pain and energy homeostasis, melanocyte stimulation AP2B1 <.0001 2.39 adaptor-related protein chr17 subunit of adapter Clathrin linkage to receptors in coated vesicles complex2, beta 1 subunit protein 2 GRID2IP <.0001 2.39 glutamate receptor, chr 7 Control of GRID2 glutamate recpetor in ionotropic, delta 2 purkinje cells, purkinje cell degeneration interacting protein TNNT1 <.0001 2.24 troponin T type 1 chr19 component of Heart failure, myopathy troponin PRKCD <.0001 2.11 protein kinase C, delta chr3 Protein kinase C family B cell signaling, growth, tumorigenesis ACTB <.0001 2.09 actin, beta chr7 actin contractile apparatus and nonmuscle cytoskeletal actin CHMP4B <.0001 2.01 chromatin modifying chr chromatin-modifying MVB formation, cell cycle, cataract protein 4B protein RREB1 <.0001 1.81 ras responsive element Chr6 transcription factor cancer binding protein1 DCTN1 <.0001 1.77 dynactin 1 Chr2 subunit of dynactin interaction with dynein intermediate chain

Gene Expression Patterns Dependant on Rai1 Dosage. Rai1+/- and Rai1-Tg mice have dosage related Rai1 expression patterns in the hypothalamus- the Rai1+/- mouse hypothalamus exhibits reduced Rai1 expression compared to wild-type, while the Rai1-Tg mouse hypothalamus shows elevated expression. These expression patterns allow us to identify which genes may be affected by Rai1 dosage.

Using a differential p-value stringency of .05, 36 genes are underexpressed when

Rai1 is underexpressed and overexpressed when Rai1 is overexpressed (Fig. 11). Using the same stringency, 2 genes, SGK and RBM3 are overexpressed when Rai1 is underexpressed and underexpressed when Rai1 is overexpressed (Fig. 11).

Obesity Genes. Considering the obese phenotype observed in Rai1+/- mice and SMS individuals, we looked at genes related to an obese phenotype. Using Ingenuity software analysis for disease and phenotype, we found 20 genes associated with obesity that were differentially expressed in either Rai1-Tg or Rai1+/- mouse hypothalamus (Fig. 12).

Figure 11: Heat map of genes affected by Rai1 dosage. Most genes shown have a direct dosage relationship with Rai1. SGK and RBM3 have an inverse dosage relationship with Rai1. All genes have a differential p-value of <.05.

Figure 12: Heat map of obesity related gene expression. All genes have a significant differential p-value from WT mice. Obesity function was determined using Ingenuity disease phenotypes and Entrez Gene entries.

Phenotypic Pathways. Ingenuity Pathway Analysis software was used to determine which biological pathways involved genes differentially expressed in Rai1+/- and Rai1-Tg mice. A p-value for each pathway was determined using Fisher’s exact tests to determine the likelihood of those genes assigned by chance. Biological pathways altered in Rai1+/- mice include psychological disorders and behavior (Adbr2, Ap2b1, Arrb1, Bdnf, Btrc,

Crhr2, Dleu2, Ednra, Etv5, Gtf2h4, Hist1h2bj, Isg15, Kcnk2, Mthfd1, Npy, Pes1, Ripk4,

Rras, Slc16a6, Sytl4, Tnfaip8, Tnfrsf19, Traf2, Ucn)(Fig. 13) and neurological development (Aebp1, Bdnf, Fgfrl1, Fndc5, Kif17, Leng8, Mkks, Ncan, Olfml3, Prkcsh,

Ptprs, Sgk1,Srr, Trhde, Ube2e3) (Fig. 14).

Biological pathways altered in Rai1-Tg mice include Development, Behavior, and

Digestion (Bzrap1, Cck, Ckbr, Cebpg, Dbi, Ddit3, Dlk1, Dmap1, Dnttip1, Epc1, Fosl2,

Grn, Hsf4, Insig1, Mark2, Mmp17, Pmch, Prkch, Ptprz1, Rgs3, S100a1, Sh2b3, Slc12a6,

Slc5a5, Tacc1, Trio, Uba5, Yeats4, Znhit1)(Fig. 15) and DNA Replication and Repair

(Actb, Akap8l, Banf1, Coro2b, Eif1ax, Eif3eip, Eif4a2, Eif4a3, Eif4g1, Emd, Ensa,

Hsd11b1, Igf2, Insulin, Ogn, Pde, Pde1a, Pde1b, Pde4a, Plagl2, Ran, Rpl10, Rpl15,

Rpl18, Rps6, Rps7, Rps8, Rps1g, Sez6l2, Stau1, Stxbp4, Syne1, Vrk3, Znrd1) (Fig. 16).

Confirmation of expression using qRT-PCR. Using Ingenuity pathway analysis software, we selected three genes which were associated with satiety and differentially expressed for confirmation using qPCR: Cholecystokinin (Cck), brain-derived neurotrophic factor (Bdnf), and neuropeptide Y (Npy). Each gene was confirmed using

RNA from 4 Rai1+/- mice, 6 wild-type controls, and 3 Rai1-Tg mice (Fig. 17). There were no differences between male or female expression.

Cholecystokinin (Cck) is a brain/gut peptide involved in satiety that regulates food intake and weight gain 76, 77. It can decrease the expression levels of Npy, which are usually colocalized with Cck in the neurons of the arcuate nucleus 78. It can regulate ghrelin expression and has a similar effect as the satiety hormone, leptin 79, 80. Microarray data show that Cck was downregulated 1.5 fold in Rai1+/- mice, while upregulated 1.2 fold in Rai1-Tg mice. Q-PCR showed a less significant decrease in Rai1+/- mice, but a very strong upregulation in Rai1-Tg mice.

Neuropeptide Y (NPY) is a neurotransmitter located in the neurons of the arcuate nucleus of the hypothalamus and plays an important role in the stimulation of hunger.

2+ Ghrelin activates Npy containing neurons causing an increase in [Ca ]i via the phospholipase C and adenylate cyclase-PKA pathways, while leptin acts as an agonist via the phosphatidylinositol 3-kinase-PDE3 pathway 81, 82. Overexpression of Npy causes an increase in feeding and weight gain 83, 84. Loss of Npy expression has been shown to attenuate hyperphagic response to fasting and leptin deficiency 85, 86. Microarray data show that Npy was upregulated 1.6 fold in Rai1+/- mice and not significantly changed in

Rai1-Tg mice. Q-PCR showed a less significant increase in Rai1+/- mice, but a strong downregulation in Rai1-Tg mice.

Brain-derived neurotrophic factor (Bdnf) is a neurotrophic growth factor involved in the growth of striatal neurons 87. Reduced expression of BDNF has been identified in depression, schizophrenia, and obsessive-compulsive disorder 88, 89, 90, 91, 92, 93. Local

43 deletion of Bdnf in the ventromedial and dorsomedial hypothalamus was found to induce hyperphagia and obesity 49. Haploinsufficiency for Bdnf in mice was found to cause increased food intake, early-onset obesity, hyperactivity, and cognitive impairment 94.

Microarray data show that Bdnf was downregulated 2.5 fold in Rai1+/- mice and unchanged in the Rai1-Tg mice. Q-PCR confirmed the downregulation observed in

Rai1+/- mice and also showed a positive dosage effect in Rai1-Tg mice.

Figure 13: Psychological Disorders and Behavioral Pathway in Rai1+/- mice. Ingenuity Created Pathway showing a Psychological and Behavioral Pathway and genes which were altered in Rai1+/- mice. Genes that were differentially expressed from wild- type in the whole genome microarray have a fold change value listed below the gene name. Note the inclusion of confirmed genes BDNF and NPY. IPA score=39.

Figure 14: Nervous System Development and Cell Death Pathway in Rai1+/- mice. Ingenuity Created Pathway showing a Nervous System Development and Cell Death Pathway and genes which were altered in Rai1+/- mice. Genes that were differentially expressed from wild-type in the whole genome microarray have a fold change value listed below the gene name. Note the inclusion of BDNF. IPA score=20.

Figure 15: Protein Synthesis and DNA Replication Pathway in Rai-Tg mice. Ingenuity Created Pathway showing a Protein Synthesis and DNA Replication Pathway and genes which were altered in Rai1-Tg mice. Genes that were differentially expressed from wild- type in the whole genome microarray have a fold change value listed below the gene name. Note the central role of insulin. IPA score=41.

Figure 16: Development, Behavior, and Digestion Pathway in Rai-Tg mice. Ingenuity Created Pathway showing a Development, Behavior, and Digestion Pathway and genes which were altered in Rai1-Tg mice. Genes that were differentially expressed from wild- type in the whole genome microarray have a fold change value listed below the gene name. Note the inclusion of CCK. IPA score=34.

Figure 17: Confirmation of microarray results. Q-PCR confirmation of microarray results. Wild-type is set to 1 and the relative expression in Rai1+/- and Rai1-Tg is shown in arbitrary units. Cck is strongly overexpressed in wild-type, but there is no significant downregulation in Rai1+/- mice. Bdnf is downregulated in Rai1+/- mice and seems to have a direct relationship to dosage of Rai1. Npy is downregulated in Rai1-Tg mice, but not significantly upregulated in Rai1+/-. May have an inverse relationship with Rai1 dosage. Rai1 expression levels are confirmed and match known genotype expression patterns. Graphs represent mean±SEM. (* p <.05, **p <.01, *** p <.001)

DISCUSSION

We have provided evidence for the role of the hypothalamus in the obesity and satiation phenotypes seen in mouse models of SMS and dup(17)(p11.2) syndrome. A large number of genes were differentially expressed in both Rai1+/- and Rai1-Tg mouse hypothalami. Top upregulated and downregulated genes include transcription factors, hormones, and neurotransmitters. Many of the genes found to be differentially expressed are associated with obesity. There were 38 genes that were differentially expressed due to

Rai1 dosage.

Thirty-six genes are underexpressed when Rai1 is underexpressed and overexpressed when Rai1 is overexpressed. Some genes were found to have phenotypes which relate to those observed in SMS and dup(17)(p11.2) syndrome. These may warrant further confirmation and investigation. Growth hormone (GH) and prolactin (PRL) are associated with growth, but levels within SMS cohorts were identified as being within normal levels and following a normal circadian rhythm 95. Proopiomelanocortin, POMC1, has been associated with severe early-onset obesity 96. WNT9B is involved in midfacial development. Knockout mutations of Wnt9b can model cleft lips in mice 97. Lecithin cholesterol acyltransferase, LCAT, regulates cholesterol transport and deficiency in mice can cause hepatic overproduction of triglycerides 98. Gastrulation brain homeobox 2,

GBX2, is a transcription factor necessary for hindbrain development 99. Transcription factor 7–like 2 gene, TCF7L2, is involved with insulin signaling in the pancreas 100.

TCF7L2 variants are associated with Type II Diabetes 101.

Two genes were found to be downregulated when Rai1 is overexpressed and upregulated in Rai1+/-, Sgk and Rbm3. SGK, serum- and glucocorticoid-dependent protein kinase, is involved in mediating the epithelial sodium channel and glucose transport in the kidneys 102. It is also activated by insulin and involved in the regulation of insulin secretion 103, 104. Recent findings indicate that SGK is involved in energy homeostasis.

SGK expression is high in conditions of hyperphagia, weight gain, or fasting, while low in conditions of energy storage 105. Considering that Rai1-Tg are hypoinsulinemic and

Rai1+/- mice have hyperphagia induced obesity, the role of Sgk in Rai1 mice warrants further study. RBM3, RNA binding motif protein 3, is a cold-stress induced shock protein and a global transcription regulator 106. Like Rai1, RBM3 localizes to the neuronal rich areas of the brain and is not found in the corpus callosum 107. The dosage model of Rbm3 observed in Rai1 mice is inverse to the dosage of Rai1. Further investigation is needed to determine if this is a compensatory response to low Rai1.

Confirmation of Cck, Npy, and Bdnf demonstrate which satiety genes in the mouse hypothalamus contribute to the obesity phenotype in Rai1+/- mice. Cck is likely not causative of the hyperphagia observed in Rai1+/- mice. However, overexpression of Cck has been linked to renal abnormalities and could be investigated in the kidneys of Rai1-Tg mice. Npy expression shows a slight dosage related effect and might cause a compensation of PYY, which was elevated in Rai1+/- serum. Bdnf may be causative of the hyperphagia and obesity seen in Rai1+/- mice.

CHAPTER 5: Discussion

We have provided evidence that Rai1 dosage affects feeding and satiety, with involvement of hypothalamic satiety pathways. We have also shown that Rai1 dosage affects fat deposition patterns and female visceral fat in mice and humans.

Rai1-Tg mice are not hyperphagic but have decreased insulin levels. Many genes differentially expressed in these mice are involved in insulin secretion. The pathogenic effects of hypoinsulinemia have not been characterized in Rai1-Tg mice and should be investigated. Pathology of the pancreas, liver, adipose tissues, and kidneys may reveal physical abnormalities that affect insulin secretion in these mice. Tissue pathology is of particular interest considering the excess of growth hormone seen in the microarray data.

Previous studies indicate that growth hormone has tissue specific effects on insulin secretion and sensitivity 108, 109. These mice, in addition to leading to a greater understanding of dup(17)(p11.2) syndrome, may also further characterize the role of growth hormone in diabetes.

Rai1+/- mice are hyperphagic and obese. These data seem to model the hyperphagia and obesity seen in humans. Many of these tests, particularly feeding tests, were limited by the availability of age and sex matched Rai1+/- mice. As more mice

52 become available, replications of previous studies are necessary. Rai1+/- consume more food during the rest cycle, which may indicate more dramatic changes in circadian rhythm than has previously been shown in wheel studies. Infrared light analysis may provide better insight into the circadian rhythm and levels of light phase activity in these mice.

Some reports indicate that SMS patients seem to hoard and seek out salty foods 31. Food preference tests may be done to determine a potential genetic basis for this behavior. The metabolic rate and energy expenditure of Rai1+/- mice can be measured using an indirect calorimetry metabolism chamber. A better understanding of the metabolic phenotypes and feeding behaviors in these mice will lead to better understanding of obesity in SMS and genetic effects on energy homeostasis.

In addition to confirming studies in mice, hyperphagia can be better understood in

SMS patients. Familial and anecdotal evidence suggests night eating and food hoarding in

SMS may be discouraged by locking up kitchens and cabinets. However, this information was not found in the available literature. Confirming these data using a caretaker study, similar to the survey on truncal obesity, will lead to a greater understanding of SMS behaviors which may contribute to obesity.

RAI1 affects adiposity when overexpressed or underexpressed. However, there is no conclusive evidence of the pathogenecity associated with increased visceral fat in these mice. Comorbidities that should be assessed include cardiovascular risk, blood pressure, and metabolic syndrome. There is strong evidence that these comorbidities of visceral obesity are also affected by alterations in the circadian cycle. Myocardial cells and adipocytes have a circadian cycle, and Clock mutant mice have an altered circadian

53 rhythm and muted 24 hr cycle in systolic and diastolic blood pressures 110. These mice also have cardiovascular disease 50. Evaluating blood pressure and changes in the expression levels in the hearts of Rai1 mice may reveal the downstream effects of the observed adiposity changes on cardiovascular health, as well as the relationship between the circadian cycles and obesity comorbidities.

Rai1-Tg females and Rai1+/- females have an increased proportion of visceral fat when compared to wild-type mice. Increased visceral fat deposition is seen in females with polycystic ovary syndrome, an endocrine-metabolic disorder that results in increased risk for infertility, obesity, metabolic syndrome, cardiovascular disease 112,113. Evaluation of serum hormones in Rai1-Tg and Rai1+/- mice can be used to determine if Rai1 affects sex hormone levels.

Further studies are needed to determine the implications of increased obesity and metabolic changes in these mice. Fat expression and secretion studies may be useful in determining altered levels of adipokines in Rai1 mice. Considering the observed increase in abdominal fat in Rai1-Tg and Rai1+/- females, these mice may be used to determine the risks of visceral obesity in SMS patients and lead to a more comprehensive understanding of the pathogencity of visceral fat in females in the general population.

Microarray analysis successfully identified changes in satiety genes (Npy and Bdnf) in Rai1 mice and demonstrated altered expression of Rai1 in the hypothalamus. This study was limited in scope to genes related to satiety and obesity, but these data can be used to identify genes related to observed hypoinsulinemia, cognitive dysfunction, and altered molecular mechanisms. Of particular interest are the many genes involved in behavior and

54 psychological disorders. Considering the severe impact of the behavioral phenotypes on the quality of life of SMS patients, these data may be useful in identifying how cognitive function is altered in a complex disorder. Rai1+/- mice also underexpress Bdnf in the hypothalamus, which has been shown to cause obesity. BNDF is a neurotropic factor which can affect serotonergic neurotransmission 111. Considering the overlapping behavioral phenotypes of BDNF haploinsufficiency and RAI1 haploinsufficency, as well as the changes in the melatonin pathway in SMS, comprehensive analysis of the role to serotonin uptake and regulation should be studied in these mice. BDNF expression could also be characterized in SMS human cells to clarify the role of BDNF in SMS.

In addition to a more comprehensive understanding of SMS and dup(17)(p11.2) syndrome, the data presented in these studies are precursory to a greater characterization of the role of RAI1 in growth, obesity, adiposity, and behavior.

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APPENDIX

PATIENT GROWTH SURVEY

Consent: • The purpose of this study is to better characterize growth and metabolism in chromosome 17 disorders (including Smith-Magenis syndrome (SMS) and duplication 17p11.2 syndrome (Potocki-Lupski syndrome). • In this study, you will be asked to complete a brief online survey about your child’s weight and body type. You may not get any direct benefit from this survey, but the information we learn in this survey may help us better understand and provide care for children with 17p disorders. • If you do not have online access or would prefer to complete the questionnaire by mail or email, please contact: Brooke Burns, Study Coordinator at (804)628-1631 or by email at [email protected] • There are no costs for participating in this survey other than the time you will spend filling out the questionnaire. • You will receive an opportunity to win a $100 Amazon.com gift card if you provide an email address when you complete the questionnaire. Winners will be chosen by random drawing. • The questionnaire is anonymous and access to all data will be limited to study personnel. A data and safety monitoring plan is established. What we find from this survey may be presented at meetings or published in papers, but neither your name nor your child’s name will be used in these presentations or papers. • You do not have to participate in this survey. If you choose to participate, you may stop at any time without any penalty.

I have been given the chance to read this consent information. I understand the information about this survey. Completion of this survey indicates that I am willing to participate.

Questionnaire:

My child has been diagnosed by a geneticist with:

1. Smith-Magenis syndrome, del(17)(p11.2) 2. Smith-Mageni syndrome, RAI1 mutation

3. Potocki-Lupski syndrome, dup(17)(p11.2) 4. My child has not been diagnosed by a geneticist.

5. Current weight of child _____ kg or ______lbs. 6. Current height of child ______cm or ______in. 7. Current age of child ______years 8. My son/daughter is ______male ______female

Email address to receive notification of gift card: ______

9. Please select the single description that best matches your child’s body type:

A. My son/daughter tends to store extra weight on arms, legs, hips, and buttocks. Very little weight is stored around the waist.

B. My son/daughter tends to store extra weight around waist and stomach areas. Very little is stored around the legs and buttocks.

C. My son/daughter stores weight evenly throughout body- over stomach, arms, legs, and buttocks.

D. My son/daughter is very thin and has no excess weight. Veins are visible through skin.

E. My son/daughter has a lot of excess weight that is distributed throughout the stomach, arms, legs, and hips.

F. My son/daughter carries a lot of excess weight in the stomach. Very little weight is stored around hips, arms, and legs. Legs may be very thin.