The Clinical and Molecular Consequences of Mutations in KCTD7

by Kyle Metz

A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy

Baltimore, Maryland

September, 2015

© 2015 Kyle Metz All Rights Reserved Abstract

Autophagy is a well-conserved eukaryotic pathway that delivers damaged organelles and aggregates to lysosomes for degradation. Several recent studies have shown that defects in the autophagy pathway leads to human disease. Epilepsy afflicts around 50 million people worldwide and ~1% of

Americans; however, a strong connection between epilepsy and autophagy has not been made. The progressive myoclonic (EPM), a subset of epilepsy, are a group of rare genetic disorders characterized by myoclonic/ tonic- clonic and severe neurodegeneration, often resulting in a poor outcome.

My doctoral work focused studying a gene called tetramerization domain 7 (KCTD7). Mutations in KCTD7 were linked to progressive in 2007. Since the original publication, 16 clincial cases have been published describing patients with mutations in KCTD7.

Essentially nothing about this gene is known, nor of its disease progression. Our lab recently identified the KCTD protein family as the mammalian homologs of a yeast stress-response protein, Whi2. In yeast, our lab has shown a role of Whi2 in autophagy induction and mitochondrial homeostasis.

Based on our knowledge of Whi2, we hypothesized that KCTD7 may play a role in autophagy and mitochondrial homeostasis. To demonstrate this, I obtained patient fibroblasts and showed a block in the autophagy pathway and accumulation of abnormal mitochondria. I additionally coordinated 14 different

ii universities and sequencing centers that identified patients with mutations in

KCTD7 to understand what sets this disorder apart from other progressive myoclonic epilepsy diseases. Finally, I generated a mouse model that deletes the second exon of KCTD7 to study its in vivo function.

iii Acknowledgements

I have received an incredibly large amount of help and instruction from members of the Johns Hopkins Community. I am grateful to all who have taught me both in and out of the classroom from preschool to my last course at JHSPH.

It would take a significant amount of time to thank every person that has been apart of this journey and will thus leave it to the most significant parts of my training.

My committee members, Drs Mike Matunis, Adam Hartman, Charlotte

Sumner and Isabelle Coppens, were always a wealth of knowledge of both scientific facts and for career/life advice. Each committee meeting was a productive time of discussion and ideas that helped develop my project and scientific thinking. Of note, Adam Hartman contributed greatly to the clinical part of my project, both in intellectual contributions and networking with those who identified new patient cases. It has also been a pleasure to work closely with

Isabelle Coppens and learn how to produce “textbook quality” images.

I thank the following collaborators for their contribution to the clinical part of my project: Thomas Markello, Adam Hartman, Rachel Kneen, Daniel Crooks,

Santosh R Mordekar, Min T Ong, Michael J Parker, the Deciphering

Developmental Disorders Study, Edda Haberlandt, Michael Alber, Isabelle Prehl,

Satish Agadi, Thomas Burrow, Katrina Peariso, Haiying Meng, Hatha Gbedawo,

iv Dianalee McKnight, Christine Stanely, Adolfo Garnica, Noelle R. Danylchuk,

Tobias Loddenkemper, Jill Mokry.

None of these individuals/ groups possessed any samples; however, two physicians agreed to inquire if the patient families would consider donating fibroblasts for our studies. This work would not have been possible without the kind consideration of these patient families and the dermal fibroblasts prepared by Dr. Edda Haberlandt and Dr. Leshinsky-Silver. Finally, I thank all the patient families that agreed to contribute to this project and advance our understanding of KCTD7.

I also thank Dr. Christine Petersen, my undergraduate advisor. She first got me excited about public health and convinced me to apply to east coast schools for graduate school. Christy continues to offer both scientific and career advice that I value greatly.

My mentor, Marie Hardwick, was gracious enough to allow me to rotate and ultimately join her lab for my doctoral work. She has taught me many important qualities but the one I value most of all is persistence. Her constant support and enthusiasm for my project has been very appreciated during these last several years. In addition to Marie, I must thank the members of her lab for their constant support, both in and outside the lab.

v Finally, I must express my gratitude toward my family. Words cannot describe my appreciation for their never ending love and support and for making me into the person I am today.

vi

Table of Contents

Chapter 1: Introduction

Autophagy………………………………………………………………………………1

Mitophagy………………………………………………………………………...……..7

Human Autophagy and Disease………………………………………………9

Epilepsy

Treatments for epilepsy………………………………………………………14

Myoclonic epilepsies…………………………………………………………..16

KCTD7 mutations………………………………………………………………18

Citations………………………………………………………………………...... …….23

Chapter 2: Epilepsy and movement disorder observed with KCTD7 mutations appears to result from underlying neurodegeneration with evidence of a defective lysosomal pathway

Difficulty of Treating and Diagnosis Epilepsy……………………………….40

Types of Progressive Myoclonic Epilepsy…………………………………..42

vii Lysosomal Defects in the Progressive Myoclonic Epilepsies……………..43

Consanguineous Genetics and KCTD7……………………………………..44

Reported Mutations in KCTD7………………………………………………..56

The Conserved BTB Domain in KCTD ……………………………48

Results……………………………………………………………..……………53

Discussion………………………………………………………………………59

Figures…………………………………………………..………………………67

Methods…………………………………………………………………………77

Figure Legends ………………………………………………………….…….80

Citations…………………………………………………………………………82

Complete Clinical Data for Chapter 2………………………...... …………...91

Chapter 3: The Role of KCTD7 in Autophagy and Mitochondrial

Homeostasis

Regulation of Mitochondrial Dynamics……………………………....…….140

Regulation of mTOR Through Amino Acids at the Lysosome………………...... 143

From Yeast to the Epilepsy Gene KCTD7……………………………..………145

Results………………………………………………………………………….148

viii Discussion……………………………………………………………………...159

Methods………………………………………………………………………..162

Figure Legends……………………………………………………...…………169

Figures…………………………………………………………………………174

Citations…………………………………………………………..……………188

Chapter 4: Resource for the Creation of a KCTD7-Deficient Mouse Model

Background……………………………………..……………………………...201

Methods………………………………………………………………………..205

Figures………………………………………….………………………………207

Citations…………………………………………………………..……………213

Future Directions and Perspectives…………………………………………….214

Curriculum Vitae…………………………….……………………………………...216

ix

Chapter 1

Introduction

1

Autophagy

Autophagy is a well-conserved eukaryotic process of recycling cytoplasmic contents. The term “autophagy”, pronounced either aw-TAH-fa-gee, or auto- phagy, was first coined by Christian de Duve (discoverer of lysosomes and peroxisomes) in 1963 and derives its name from Greek auto- (self) and –phagy

(to eat) (1).The term was coined to describe a specific process by which a cell must consume and recycle specific cellular contents to survive periods of stress and overall turnover of defective proteins and organelles.

This process is thought to have been first observed using electron microscopy (EM) in the lab of Dr. Keith Porter when the addition of glucagon resulted in a large increase of lysosomes containing mitochondria (2). Indeed, more lysosomes were formed; however, Porter missed the process that preceded the step that delivered the contents to the lysosome. The following three decades were comprised mostly of morphological descriptions of inducers and inhibitors of autophagy (3), focusing on amino acid deprivation and rapamycin treatment that each induce autophagy (4,5), and 3-MA treatment, which inhibits autophagy (6).

These studies provided the early evidence that signaling events involving kinases were involved in autophagy regulation.

Until this time, specific autophagy genes were unknown. While the vast majority of these studies were performed in liver cells, another model organism was needed to better describe the more detailed steps that lead to

2 autophagosome biogenesis. Much of the genetic work of identifying the essential

autophagy genes was performed in Saccharomyces cerevisiae. In 1992, it was

observed that autophagy occurs in yeast and the following year a screen of

autophagy deficient yeast strains was reported (7,8). These studies set the stage

for the burgeoning era of molecular biology.

Subsequent yeast screens allowed for the grouping of different yeast

genes in specific processes of autophagy. The first yeast autophagy gene was

identified in 1997 and is now termed Autophagy Gene 1, or ATG1 (9). In this

study, homology was also discovered with C. elegans gene Unc-51,

demonstrating evolutionary conservation of the autophagy pathway (9). Atg1 is a kinase and a homolog of mammalian proteins Ulk1 and Ulk2 (10). Atg1 and

Ulk1/2 are considered the most upstream signaling autophagy proteins and are negatively regulated by another kinase, target of rapamycin (TOR) in both yeast and mammals (10). More than 37 different genes have been identified in yeast that lead to a total or partial block in autophagy (11).

The principal method for identification of autophagy defects was based on electron microscopy. Only in recent years, effective biochemical assays have been developed to begin to assess the autophagy pathway, initiated by the discovery of the Atg12-5 conjugation system and subsequent processing of Atg8.

Lead by Noburu Mizushima in the lab of Yoshinori Ohsumi, the identification of

Atg12 linked covalently to Atg5 were the first autophagy proteins identified in mammals (12). Shortly thereafter, the mammalian homolog of Atg8 was

3 identified, microtubule-associated protein1 light chain 3 (LC3) (13). While one yeast Atg8 protein exists, seven mammalian homologs are reported: LC3A-C,

GABARAP 1-4 (14,15). While specific roles of each mammalian homologs is unknown, growing evidence suggests substrate specificity will be the rationale for the expansion to seven Atg8 homologues in mammals (16).

Mammalian LC3 occurs in two forms, LC3-I and LC3-II, of which the later is the active form. LC3 becomes its active form through lipidation by phosphatidylethanolamine (PE) via a ubiquitin-like conjugation system (17). Atg4 cleaves LC3, exposing a C-terminal glycine residue to which PE is covalently attached. LC3 interacts with the E1-like enzyme, Atg7 (17). The E2-like enzyme

Atg3 then interacts with the E3-like enzyme complex Atg12-5-16L conjugation system, creating the active, lipidated form of LC3 and allowing it to be inserted into the growing autophagosome (17).

Shortly after these autophagy proteins were identified in mammals, another essential autophagy protein was identified in yeast and humans: Atg6/

Beclin1 (18,19). Beclin1 derives its name from “B-cell lymphoma 2 (Bcl-2)- interacting”, with the “1” added by the committee on gene naming, though protested by Dr. Levine as it implied more than one Beclin protein existed

(personal communication); however, a second Beclin protein was recently identified (20). Beclin1 belongs to the VPS34 complex, or Class III PI3K complex where Beclin1 positively regulates phosphatidylinositol 3 phosphate (PI(3)P)

4 production through interaction with vacuolar sorting protein 34 VPS34, p150,

vacuolar sorting protein 15 (VPS15) and Atg14 (21).

The VPS34 complex allows other proteins to be effectively targeted to the

growing autophagosome through PI(3)P production. Several autophagy proteins

possess PI(3)P binding domains, such as the Fab1 YOTB Vac1 EEA1 (FYVE)

domain found in mammalian Double FYVE Containing Protein 1 (DFCP1) or an

amino acid sequence motif, FRRG, found in yeast Atg18/ mammalian WD-repeat protein Interacting with PhosphoInositides (WIPI). While the role of DFCP1 in autophagy is unknown, Atg18/WIPI has been shown to play a critical role by recruiting Atg2 to the growing autophagosome (22). In turn, Atg2 recruits Atg9, which is involved in retrieving membrane to the growing autophagosome (23).

The origin of the membrane that forms the growing autophagosome remains somewhat controversial. Studies have suggested that the endoplasmic reticulum (ER) is the formation site of the autophagosome. While this hypothesis is still supported by many investigations, further evidence suggests several other sites including mitochondria, plasma membrane and Golgi apparatus (24,25,26).

When an autophagy signal is induced, such as limited nutrients or other cellular stressors, negative autophagy regulators are silenced, such as TOR. This allows Atg1 or Ulk1/2 to localize to the pre-autophagosomal site (PAS) located

near the yeast vacuole (equivalent to the mammalian lysosome) or to the

functionally equivalent ER subdomains in mammals, which is required for Vps34

5 complex recruitment (27). This allows generation of PI(3)P at the PAS or ER

subdomain, subsequently recruiting PI(3)P-binding protein Atg18/ WIPI, allowing

Atg2 to recruit Atg9 and expand the PAS or ER subdomain into an isolation membrane (IM), a precursor to the autophagosome (27).

The IM then expands around a substrate and becomes a mature autophagosome; however, the steps that regulate this process remain ill defined.

While much progress had been made understanding the steps needed to initiate autophagosome formation, the process of expanding the IM and closing the autophagosome remain a complete mystery. Once the mature autophagosome is formed, it is delivered to the vacuole of yeast (28) or travels along actin filaments via HDAC6 (29) to fuse with a lysosome. The mechanisms of fusion of the autophagosome with the lysosome has been an area of active interest, and the first autophagy snare was recently identified, Syntaxin17 (30). Thus, it was been demonstrated how the autophagosome forms, but a comprehensive biochemical understanding of autophagy is only beginning to be characterized.

Another area of intense research has been how the autophagosome targets specific cargo for degradation. Whereas autophagy was largely considered a nonspecific process, the discovery of defined receptors that degrade specific aggregates, organelles, or pathogens has shifted the field to a consider autophagy as a specific process. One of the principal mediators of selective autophagy is Atg8/LC3 and their respective binding partners. The LC3- interacting region (LIR) amino acid sequence motif is defined an aromatic

6 residue followed by a hydrophobic residue with two intervening residues (e.g.

W/YXXL) found on a growing number of LC3-interacting proteins (31). Recently, a noncanonical LIR in an antibacterial receptor specific for LC3C has been reported that lacks the aromatic residue (32). Acidic residues either before or between these residues enhance binding to LC3 (33). Consequently, the identification of different receptors for removal of cargo has created new fields of research, such as the autophagic clearance of mitochondria (mitophagy) and the autophagic clearance of pathogens (Xenophagy) (34,35,36).

Introduction: Mitophagy

The autophagic removal of damaged or defective mitochondria

(mitophagy) was described in the first autophagy experiments when glucagon was added to liver cells and mitochondria were observed in vacuoles thought to be lysosomes2. It was further shown that a core set of autophagy genes are required to degrade mitochondria. However, recently the specific receptors have been identified (37). In 2008, the first gene was discovered in yeast to be specific for mitochondrial degradation, Atg11 (38). It was hypothesized that Atg11 was a scaffold protein and was found that Atg11 binds Atg8 through a canonical LIR motif (38). Atg11 further binds a mitochondrial protein, Atg32 (38). Atg32 localizes to mitochondria on a constitutive basis and upon a mitophagy stimulus binds both Atg11 and Atg8 to support degradation of the mitochondrion39. Atg32 is not required for other types of autophagy (39). While Atg32 localizes to mitochondria, its normal function, or “day job”, is not known.

7 The phenomenon is not confined to yeast. Recently, a similar protein to

Atg32 was discovered on the mitochondrial outer membrane in mammals, Fun14

Domain containing 1 (FUNDC1) (26). This study shows another outer mitochondrial membrane protein with unknown function critical for defective mitochondria clearance. Following a similar theme, FUNDC1 possesses a LIR on its cytoplasmic N-terminus that binds LC3 after mitochondrial damage (26).

Overexpression of FUNDC1 alone is able to induce mitophagy, demonstrated by

LC3 accumulation on endogenous mitochondrial markers and engulfment of mitochondria by double-membraned vesicles shown by EM. Additionally, mutation of the LIR motif in FUNDC1 abolishes its ability to induce mitophagy upon overexpression. This study highlights the critical role of LC3 binding a specific receptor to complete organelle degradation.

While removal of damaged mitochondria is critical to cellular homeostasis, cells can remove mitochondria during normal maturation processes, for example red blood cells (RBCs) and oocytes after fertilization (41,42). RBCs undergo a type of maturation that removes virtually all components of a cell, including the nucleus, endoplasmic reticulum, ribosomes, and mitochondria (43). Amongst the autophagy genes required for mitochondrial removal, a gene specific for mitochondrial clearance in RBCs: BNIP3L/NIX (44). Supporting this line of evidence, Nix knockout mice were found to retain mitochondria in their RBCs and were anemic (45). It was further discovered that Nix binds to LC3 to degrade

8 mitochondria (46). Thus, higher eukaryotes have evolved specific processes to remove organelles in a tissue-restricted manner.

Human Autophagy Genes and Disease

While the removal of mitochondria appears critical for cell health, a strong association between autophagy proteins and human disease remain elusive. Two proteins were identified in 1998 and 2004 that would fulfill this role: Parkin and

Pten-induced kinase (Pink1), respectively (47,48). Mutations in Pink1 and Parkin have been shown to cause familial Parkinson’s disease (47,48), Pink1 localizes to mitochondria in a constitutive manner where it is imported and degraded by mitochondrial several proteases (48). In 2006, it was shown in Drosophila melanogaster that defects in Pink1 led to abnormal mitochondria and that Pink1 was linked to Parkin (49,50). In 2008, another connection with Parkin and mitochondria was made when Narenda et al. demonstrated that Parkin was recruited to damaged mitochondria and in 2010 the same group showed that

Parkin recruitment was dependent on Pink1 (51,52). Upon a decrease in mitochondrial membrane potential, which impairs protein import, Pink1 accumulates on the outer mitochondrial membrane (OMM). In contrast to Pink1, the E3 ubiquitin ligase Parkin localizes to the cytoplasm. For several years, it was thought Parkin recruitment to mitochondria was a direct interaction with Pink1; however, three recent reports have shown that Pink1 indirectly recruits Parkin by

9 phosphorylating ubiquitin itself on the OMM, to which Parkin binds (53,54,55).

Parkin then ubiquinates more mitochondrial proteins that become phosphorylated by Pink1, thus creating a feed-forward mechanism that enhances Parkin recruitment (53,54,55).

Ubiquinated proteins further allow for recognition by autophagy machinery, such as the sequestosome1 protein (SQSTM1/p62) that binds LC3 through a LIR motif, directing the growing autophagosome to damaged mitochondria for lysosomal degradation (56). SQSTM1/p62 has also been shown to be important for recognition of damaged mitochondria (57). In 2013, familial and sporadic forms of amyotrophic lateral sclerosis (ALS) have been linked to mutations in

SQSTM1/p62, again showing how another step in this process leads to a different human disease (58).

Mutations in the autophagy genes had not been associated with specific human diseases until 2012. The first human disease mutations identified in a component of the autophagy machinery was found in WDR45 (WIPI-4), a homolog of yeast Atg18, which is involved in expanding the growing autophagosome through its interaction with Atg9 (59). De novo mutations in

WDR45 were discovered to cause Static Encephalopathy of childhood with

Neurodegeneration in Adulthood (SENDA), which is a subset of

Neurodegeneration with Brain Iron Accumulation (NBIA) disorders. The onset of

SENDA begins in infancy with psychomotor retardation as the presenting symptom with nonprogressive cognitive dysfunction (59). Four out of five patients

10 have epileptic seizures (59). Additionally, SENDA patients have dystonia,

Parkinsonism (rigidity, akinesia, tremor) and iron deposition in the globus pallidus

and substantia nigra, two regions of the brain critical for movement control (60).

SENDA patients were found to have a defect in the autophagy pathway,

demonstrated through and autophagy flux assay, which adds lysosomal inhibitors

and observing a non-significant increase in the active form of LC3-II when

compared to controls (59). In addition to a defect in the autophagy pathway, these patients further have accumulation of both LC3/Atg9A-positive vesicles

(59). Atg9A is normally not present on autophagosomes, potentially suggesting that WDR45 promotes Atg9A to cycle off the autophagosome, as defects in

WDR45 cause accumulation of Atg9A.

A second autophagy gene discovered to cause human disease when mutated, EPG-5, leads to Vici Syndrome (61). EPG-5 was first identified in a screen for autophagy-defective mutants in Caenorhabditis elegans where it was shown to have a role in autolysosome maturation, defined as fusion of the autophagosome to the lysosome (62). Vici Syndrome is a multisystem disorder that involves cataracts, cardiomyopathy, immunodeficiency and hypopigmentation (62). Dyslocalized clustering of mitochondria with variable shape and abnormal cristae were reported in patient muscle biopsies. The authors further show the accumulation of intracellular puncta labeled with neighbor of BRCA1 gene 1 (NBR1) puncta, an autophagy receptor similar in

function to p62 but specific for removal of peroxisomes in muscle biopsies by

11 immunofluorescence61(63). SQSTM1/p62 and NBR1 levels did not change in an autophagy flux assay in patient fibroblasts, indicating a block in the autophagy pathway (61). Finally, the authors show a lack of subcellular colocalization between LAMP1, a lysosomal marker, and LC3 in patient fibroblasts, demonstrating an inability of autophagosomes to fuse with lysosomes and form mature autolysosomes (61). While this study does not shed light on the molecular mechanism of EPG-5, it nevertheless demonstrates the importance of autophagy

genes in human health.

From its humble origins as a simple morphological phenomenon,

autophagy has emerged as an essential process for eukaryotes and is continuing

to be implicated in human disease. Autophagy will likely continue to emerge as

an important pathway in human health as more sequencing studies identify

established and novel autophagy gene mutations in patients with idiopathic

maladies.

12 Introduction: Epilepsy

Epilepsy is a heterogeneous group of neurologic disorders that results in uncontrolled shaking from excessive brain activity (64). Epilepsy is a relatively common disorder that afflicts around 50 million people worldwide, including 1.8% of Americans (65,66). A wide range of factors can have causal roles in the development of epilepsy, including genetic mutations, brain injury, stroke, tumor, and infectious disease. Although 265 genes have been shown to have causal roles in epilepsy, the majority of epileptic cases remain idiopathic (67,68).

Epilepsy was described ~2000 BC (69). Early descriptions of epilepsy indicate that its origins were assumed to be spiritual possession. It was not until

400 BC that Hippocrates proposed the idea that epilepsy was a disorder of the brain. Interestingly, Hippocrates noted several other key aspects of epilepsy that still hold true today. For example, families with epileptic members tend to have a higher chance of developing epilepsy (genetic component), that tends to become quite severe, and that fasting may improve seizures

(69).

While these astute observations were made nearly 2500 years ago, they were largely dismissed, favoring the notion of spiritual possession until the advent of effective treatments, such as bromide and (70,71). Until this point, many epileptic patients were stigmatized by society and were often placed in asylums, labeling these patients as insane or mentally ill (72). These stigmas continue to the present day, as certain cultures still attribute epilepsy to

13 spiritual possession or a contagious disorder (73). Some western societies carry

the belief that epileptic patients are mentally ill, further perpetuated by the fact

most epileptic patients have an idiopathic origin of their epilepsy (74). Even well

into the genome-sequencing era, most focused non-genetic

causes over genetic etiologies of epilepsy, but in the past five years the concept

that genetic mutations may underlie the majority of epilepsy mechanisms has

begun to take hold.

Epilepsy Treatment

As epilepsy is not a single type of neurologic disorder, prescribing regimes is complicated. Although medicines can stop ongoing seizures or reduce the propensity to have a , there are no available that have not been shown to prevent the process leading to the development of epilepsy, (75). The first epilepsy medications developed were bromide and Phenobarbital, both of which act primarily as a depressant to the , as do the majority of epilepsy managing medications (76).

While the majority of patients can have their seizures controlled after one or two different medication regimens, roughly one-third fall into the medically intractable category (77). Once in this category, patients will undergo a myriad of different regimens and, if unsuccessful, may undergo surgery or metabolism- based therapies (78).

14 Surgery is an option to control seizures if a lesion can be identified as the

point of epileptogenesis (79). Additionally, severing the point of connection

between both hemispheres, a corpus callosotomy, can reduce seizure frequency

by preventing the spread of epileptic activity (80). Less frequently, removal of an

entire hemisphere is recommended when a patient has catastrophic epilepsy,

defined as intractable seizures in infancy that does not respond to high levels of

medications (81). As radical as this surgery seems, many patients have complete

elimination of their seizures, and some complete college (82); however, the

earlier the surgery is performed reduces the chances of long-term adverse

effects, as the brain is believed to adjust and “rewire” more easily in infants.

Metabolism-based therapies provide an alternative strategy to treat

intractable epilepsy. The most widely used of these therapies is the ketogenic

diet, which has low levels of carbohydrates and high fat content (83). Greek

physicians noted that fasting reduce seizures activity (84), the use of the

ketogenic diet was not defined until the early 20th century and was further

modified by neurologists at Johns Hopkins Hospital (85). The most compelling

evidence that the ketogenic diet provides significant benefit for pediatric patients

with intractable epilepsy is a randomized trial showing patients can have almost

complete relief of seizures (86); however, the molecular mechanism by which the ketogenic diet functions is completely unknown (87).

The diversity amongst these methods to treat epilepsy is consistent with the heterogeneity nature of the disorder. Furthermore, a lack of understanding

15 about the specific mechanisms by which metabolism based therapies work

underscore the need to elucidate the underlying molecular mechanisms of

epileptogenesis.

Introduction: The progressive myoclonic epilepsies

The progressive myoclonic epilepsies (EPM) are a group of rare genetic disorders caused by mutations in specific genes (88). These progressive myoclonic epilepsies are characterized by myoclonic/ tonic-clonic seizures and severe neurodegeneration, often resulting in a poor outcome (89). Myoclonic seizures are defined as involuntary twitching of muscles that can affect only certain parts of the body whereas tonic-clonic are severe that consume the entire body (90).

Heinrich Unverricht and Herman Lundborg identified the first type of progressive myoclonic epilepsy in 1891 and 1903, respectively (91,92).

Unverricht-Lundborg Disease, or EPM1, is caused by homozygous mutations in the cystatin B (CSTB) gene, a cysteine protease inhibitor, specifically proteases localized in the lysosome (93,94).

The symptoms of EPM1 begin between 6 and 16 years of age with the presenting symptom reported as seizures (95). The disease is progressive and wheelchair-bound patients die in their mid-twenties. Early detection with effective treatment using medications, such as valproic acid (VPA), levetiracetam, and clonazepam, has greatly improved the mortality rate of patients with EPM1, thus

16 demonstrating the importance of establishing early diagnostics and treatments

(95,96).

Lafora Disease, or EPM2, is another common syndrome of progressive

myoclonic epilepsy. EPM2 was first described by a Spanish neurologist, Gonzalo

Rodriguez Lafora, in the early 1900s (97). The defining characteristic of EPM2 is

the accumulation of Lafora Bodies, now known to be abnormal glycogen

deposits, present in the cytoplasm of patient cells (98). EPM2 is caused by mutations in two genes, Laforin (EPM2A), a poorly defined phosphatase, or Malin

(NHLRC1), an E3 ubiquitin ligase (99,100). Laforin has also been suggested to have a role in regulating autophagy through regulating mTOR activity (101).

Prognosis for patients with EPM2 is poor with patients experiencing severe dementia shortly after diagnosis and death occurring about 10 years later (102).

Myoclonus Epilepsy with Ragged Red Fibers (MERRF) is a condition that is caused by mutation of a gene encoded on the mitochondrial genome, tRNA-

Lys (103). Mitochondria with this mutation have inefficient oxidative phosphorylation, as mitochondria-encoded proteins in the electron transport chain complexes are translated in mitochondria (104,105).

The neuronal ceroid lipofuscinoses (NCLs) are a rare group of disorders caused by 14 different genes, CLN1-14, including KCTD7/ CLN14 (106). The

NCLs are best characterized by the presence of lipophillic accumulations that have distinct ultrastructural morphologies referred to as Granular Osmophilic

17 Deposits (GROD, observed in CLN1, 9 & 10), curvilinear profiles (CL, observed in

CLN2, 3, 5, 6, 8 & 9) and fingerprint bodies (FP, observed in CLN3-9)(107). A summary of these gene’s function and characteristics can be found in Table 1. In addition to the presence of lipofuscin, other NCL characteristics include retinal degeneration, movement disorder, and dementia (108,109).

Overall, the functions of the NCL genes are poorly defined but there is a

growing body of evidence to suggest a role in the ability to remove and recycle

damaged components in the cell (110). Supporting this hypothesis, the most

common components of the characteristic lipophillic granules is subunit c of the

mitochondrial ATP synthase, suggesting an inability to fully degrade mitochondria

(111). While the progressive myoclonic epilepsies reflect a very small percentage

of neurological disorders (1% of epilepsy) (112), their essential role in neuronal

function and early age of onset underscore the need to better understand their

functions so that we may better understand neurologic disorders as a whole.

KCTD7

KCTD7 belongs to the Potassium Channel Tetramerization Domain

(KCTD) family consisting of 26 different mammalian genes (113). The KCTD proteins share an N-terminal domain, the bric-à-brac, tramtrak, and broad complex domain, or BTB domain which can be found in a range of different proteins (114). KCTD proteins are named as such because the BTB domains of

18 the KCTD family share greatest homology with the T1 tetramerization domains of voltage-gated potassium channels (115). However, a strong connection between

KCTD proteins and potassium channels has not been established.

Our lab became interested in the KCTD protein family because of a yeast protein, Whi2. Wen-Chih Cheng in our lab discovered that yeast WHI2 was frequently mutated in several independently constructed yeast knockout strain of

FIS1, encoding a conserved protein required for normal mitochondrial fission.

Reoccurring spontaneous secondary mutations in WHI2 were responsible for the observed cell death-sensitive phenotype (116). Xinchen Teng in our lab found that WHI2 is mutated in eight different gene knockout strains (117). WHI2 was also found to play a role in regulating TOR activity in response to low amino acid levels and is required to activate the process of autophagy (Teng et al., unpublished). Although WHI2 was reported to be fungi-specific, working with Dr.

Sarah Wheelan, our lab found that yeast Whi2 is a homolog (predicted to share a common ancestor) with the KCTD protein family using an HMM pred search

(117). Thus, our lab now possessed unique information that I sought to apply to these previously unidentified mammalian homologs.

The KCTD protein family is quite poorly characterized. The best- characterized proteins are KCTD8, KCTD12, KCTD12b (mouse) and KCTD16.

Two groups identified these KCTD proteins as subunits of GABAB receptors and promote the receptor desensitization (110,112). KCTD11 was identified as a potential oncogene, adjacent to tumor suppressor TP53, and as a candidate

19 medulloblastoma gene (119). KCTD11 and KCTD7 are reported to bind Cullin3, an E3 ligase (120). KCTD13 was recently shown to control brain size of zebrafish in a copy number variant (CNV)-dependent manner, where overexpression results in microcephaly whereas deletion causes macrocephaly, potentially explaining why deletion or duplication of this gene results in ~1% of idiopathic autism (121).

KCTD7 is 289 amino acids and is encoded by four exons on human 7. At the start of my PhD training, there was only one report on

KCTD7 describing a single homozygous, nonsense mutation in 3 children suffering from a progressive neurodegenerative and epileptic disorder (122).

Several additional case reports followed, allowing KCTD7 to be established as the cause of a previously unidentified disease, progressive myoclonic epilepsy type 3 (EPM3) (123,124,125,126,127). In these reports, four medications have been consistently listed for the treatment of EPM3, valproic acid, levetiracetam, clonazepam, and clobazam. Unfortunately, their exact mechanisms are poorly understood beyond the fact that they suppress the central nervous system, and unfortunately do not inform us about the function of KCTD7.

One clinical study reported the presence of storage material in one patient’s lymphocytes and fibroblasts, allowing the classification of KCTD7 as a new neuronal ceroid lipofuscinoses gene (CLN14) (127). Another clinical described a patient mutant KCTD7 to have opsonoclonus syndrome

(OMS), a rare and similar condition that includes abnormal eye movements

20 (126). Overexpression studies have shown KCTD7 hyperpolarizes

(127,128).

Overall, there is a lack of cohesive understanding of the clinical aspects associated with mutations in KCTD7 and the molecular function of KCTD7 is undetermined. Therefore, I addressed three questions in my doctoral research.

(1) What are the defining characteristics of patients with mutations in KCTD7, and are these distinguishable from other disorders? (2) Does KCTD7 play a role in autophagy like its yeast homolog, Whi2? (3) Can a mouse model be generated to test different therapeutic regimens and understand KCTD7 function in vivo?

21

22

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38

Chapter 2

Epilepsy and movement disorder observed with KCTD7 mutations appears to result from underlying neurodegeneration with evidence

of a defective lysosomal pathway

39 Introduction

Epilepsy is Difficult to Diagnose and Treat

Defined by the international league against epilepsy (ILAE) in 2014,

epilepsy may be diagnosed if a patient meets one of three criteria: two

unprovoked seizures that occur more than 24 hours apart, one unprovoked

seizure with risk of developing recurrent seizures (due to lesion/stroke/infection)

occurring over the next 10 years, or diagnosis of an epilepsy syndrome (1).

Previously, epilepsy was defined as brain disorder that predisposes a patient to generate epileptic seizures and that patient must have >1 seizure for diagnosis

(1). The expanded diagnostic criteria now may include the discussion of potential treatment after one seizure if the patient is at a greater risk of developing more seizures (1). . Importantly, epilepsy does not have to be considered a life-long

disorder, being resolved if the patient is seizure free after 10 years with the last

five years being medication free (1). . An epileptic seizure is defined as symptoms that result from excessive neuronal activity in the brain (1). Of note, an epileptic seizure is considered unprovoked, as opposed to an acute symptomatic seizure that results in less than one week of trauma to the brain (2).

Epilepsy has remained a difficult to group of disorders to treat due to the fact that the etiologies of most epilepsy diagnoses are idiopathic, complicating a

physician’s ability of to treat disorders with unknown causes (3). Another problem to treating epilepsy is the slow development of new medications. The standard of

40 developing new epileptic medications has been in place for ~60 years and

involves using a medication of interest and determining its ability to influence

seizure activity in mice that are put through a battery of seizure-inducing tests (4).

Additional critical contributing factor that challenges treatment

development is the fact that epilepsy is not a single disease or even a cluster of

related disorders as many underlying defects in other processes can manifest as

epileptic seizures. Even when the cause or mutation is known, very few

examples with corresponding targeted therapy, such as everolimus for patients

with mutations in tuberous sclerosis complex 1 and 2(TSC1/2), exist (5).

To make significant breakthroughs in the treatment of epilepsy, two

hurdles must be overcome: identification of the causes of idiopathic epilepsy, and

development of methods to accurately screen for new efficacious seizure- influencing medications. Fortunately, advancements in genome sequencing have identified many new genetic causes of epilepsy, indicating that genetic mutations likely explain a larger portion of cases than thought at the outset of the genome- sequencing era. A 2013 study identified de novo mutations in children with epileptic encephalopathies, such as infantile spasms or Lennox-Gastaut syndrome (8). These disorders do not have known causes (genetic mutations)

and are diagnosed based from symptoms, making treatment quite difficult (6,7).

In this 2013 study, more than 329 de novo (not inherited) mutations were

identified and de novo mutations were identified in 9 genes in from >2 patients

with infantile spasms and Lennox-Gastaut syndrome, demonstrating that

41 deficiencies in these genes may be a more common cause of these disorders in

other patients (8). This study is unique in that the genomes from hundreds of epilepsy patients are being searched for pathogenic mutations to explain the disease pathogenesis. Importantly, pathogenic mutations are being discovered in these patients, highlighting the importance of genetic factors in the development of epilepsy. Alternative strategies combined with genome sequencing has also identified mutations in other epileptic disorder, including the KCTD7 epilepsy gene, the subject of my thesis (9,10).

Types of Progressive Myoclonic Epilepsy

A portion of epilepsies are classified as progressive myoclonic epilepsy, which constitute ~1% of the epilepsy diagnoses at specialty centers; however, not all myoclonic epilepsies have progressive neurologic decline (11,12).

Myoclonic seizures are brief jerking motions (clonic) due to muscle (myo) contractions and can occur sporadically or in series, and disease may or may not be progressive. Progressive myoclonic epilepsy (EPM, or sometimes PME) is an umbrella term applied to describe several epilepsy disorders, Unverricht-

Lundborg disease (EPM1), Lafora disease (EPM2), myoclonus epilepsy with ragged red fibers (MERRF), the neuronal ceroid lipofuscinoses (NCLs), and type

1 sialidosis (13). There are approximately 17 diseases described as progressive myoclonic epilepsy, including 13 NCL disorders and one each of EPM1, EPM2,

MERRF, and type 1 sialidosis. New disorders have recently been identified through advances in sequencing, including mutations in KCTD7. Each of these

42 diseases has involuntary jerking of muscles (myoclonus) and progressive

neurodegeneration. All progressive myoclonic epilepsy disorders are genetically

recessive, meaning inactivation of both copies of the gene are required for disease manifestation.

Lysosomal defects in the Progressive Myoclonic Epilepsies

Historically, the accumulation of lysosomal storage material, described as

“accumulation of waste products in the lysosome” (14), was a principal method for identification of different types of progressive myoclonic epilepsy (15).More recently, genome analyses, such as an epilepsy panel that will sequence known epilepsy-causing genes in a patient, are preferred to diagnose a patient. Storage

material is a generic term applied to describe the presence of abnormal

aggregations, often in lysosomes (14). In general, storage material increases with

age normally and in disease states can greatly increase in accumulation. The

type of storage material present in a patient’s cells (neurons, fibroblasts, muscle

biopsy) with progressive myoclonic epilepsy can help distinguish a particular

disease. With the exception of Unverricht-Lundborg disease/EPM1 (caused by

mutations in Cystatin B, a lysosomal protease inhibitor), have been found to

accumulate storage material of various types (16). MERFF patients accumulate

aggregates of mitochondria in muscle tissue that can be visualized with Gömöri

trichrome stain (17). Glycogen deposits (Lafora bodies) are found in the cells of

patients with Lafora disease and are caused by mutations in one of two genes,

Laforin, a phosphatase, and Malin, an E3 ubiquitin ligase (18,19). It is not entirely

43 understood how mutations in Laforin and Malin cause Lafora’s disease; however,

deficiencies in Laforin have been suggested to cause a defect in autophagy, potentially through an inability to regulate mTOR activity, though the exact mechanism is unknown (20). Autofluorescent storage material (lipofuscin) is found in neurons and many other cell types in NCL patients (21). Lipofuscin accumulation results from an inability to degrade proteins and lipids, as a major components of lipofuscin in NCL disorders is mitochondrial subunit c of the ATP synthase, sphingolipids, and sphingolipid-binding proteins (22,23). Several NCL genes are lysosomal hydrolases (Chapter 1, Table1). Finally, type 1 sialidosis is caused by a deficiency in the lysosomal neuraminidase gene, Neu1, and patients accumulate sialylated glycopeptides and oligosaccharides in their neurons (24).

Overall, there is an underlying theme of defects in the ability of lysosomes to degrade cytoplasmic contents in the progressive myoclonic epilepsies (25). As previously mentioned, these disorders accumulate undigested cellular material, as do several other neurodegenerative disorders, such as Huntington’s Disease,

Parkinson’s Disease, and Alzheimer’s Disease (25). While the causes of non- familial Parkinson’s and Alzheimer’s Disease are poorly understood, the causes of progressive myoclonic epilepsy are genetically defined, with known mutations causing defined neurodegenerative diseases. The scientific community is now entering an age where whole genomes, transcriptomes, and proteomes can be compared for populations of 100 related individuals (26). We can now begin to

understand how slight differences between related individuals’

44 genome/transcriptome/proteome can influence susceptibility to disease. As the

genes involved in progressive myoclonic epilepsy cause neurodegeneration early

in age, perhaps unknown variations in these genes can influence susceptibility to

other neurodegenerative diseases later in life, raising the need to better

understand the roles of these proteins to cellular health.

Consanguineous family genetics studies identify KCTD7 mutations

The terms “consanguineous” comes from Latin, com- (with) and sanguine

(blood) (Merriam-Webster dictionary). This word is implied to describe a situation

in which members of the same family conceive children, e.g. first cousins. Many

consanguineous families are found in isolated regions in which a founder

mutation is transmitted from a single individual who relocates to a new area and

passes the mutation along to its offspring (27). This founder effect leads to a new population with increased likelihood that two individuals with the same mutation will interbreed (27).

Consanguineous family genetics have greatly aided researchers in

elucidating the function of many disease-causing genes. For example, the

genetic causes of and other metabolism disorders have been

identified among the Amish, Hutterite, and Mennonite populations (28).

Consanguineous families also facilitated the identification of KCTD7 mutations in

six consanguineous families, including the first patients identified (9). This number is likely higher due to inadequate family pedigrees.

45 Reported Mutations in KCTD7

Individuals with KCTD7 mutations have received one of three different but related diagnoses. Mutations in KCTD7 were first associated with epilepsy and neurodegeneration in children in 2007 when homozygosity mapping in a consanguineous family identified a homozygous mutation in three siblings with a rare syndrome of pediatric epilepsy and regression, diagnosed as progressive myoclonic epilepsy type 3 (EPM3) (29).

The second case of a patient with mutations in KCTD7 came from a

Turkish boy suffering from myoclonic epilepsy and severe developmental delay.

Genome wide linkage mapping identified the region of 7q11.21, which contains the KCTD7 gene. Sequencing revealed a homozygous mutation in the second exon of KCTD7, demonstrating that the first report of mutations in KCTD7 was not an isolated case (30).

In 2012, homozygosity mapping identified a 1.5 megabase segment that

mapped to the region of the KCTD7 gene (7q11.21) in 8/18 Turkish patients with

progressive myoclonic epilepsy of unknown origin (idiopathic) (31). Further

screening revealed a homozygous missense mutation in two of these patients.

The same study screened KCTD7 for mutations in 132 patients with progressive

myoclonic epilepsy of unknown origin, revealing mutations in 6 patients and

consolidating KCTD7 as a progressive myoclonic epilepsy gene (31). This study

further raises the possibility that mutations in KCTD7 are an underappreciated

46 cause of progressive myoclonic epilepsy. Indeed, another study that sequenced

33 patients for 265 known epilepsy-causing genes identified presumed disease- causing mutations in 16 patients, including one patient with mutations in KCTD7 identified in Kousi et al. (32).

A 2014 study identified three children from a low German-speaking

Mennonite family of Durango, Mexico with a homozygous mutation in KCTD7 by

whole-exome sequencing (33). This case is unique as these children had

developmental delay prior to the onset of seizures and was the first indication

that epilepsy may not be the underlying neurologic disorder in patients with

mutations in KCTD7 (33).

Thus far only a single patient with a homozygous mutation in KCTD7

(R184C) has been reported to have electron-dense storage material by electron

microscopy in primary fibroblasts and in a lymphoblastoid cell line (LCL) (34). This

finding is noteworthy as it was the only finding of storage material in a patient

with mutations in KCTD7, which prompted the authors to designate KCTD7

mutations as the cause of a new subtype of NCL, neuronal ceroid lipofuscinosis

type 14 (CLN14) (34). One additional diagnosis has been given to KCTD7 mutations. Opsonoclonus-myoclonus syndrome (OMS) was diagnosed in a patient with two heterozygous KCTD7 mutations (R84W and Exon3/4), one on each chromosome/allele, known as compound heterozygosity (35). OMS is

typically described as a disorder in which the prominent feature is involuntary

jerky eye movements in children. OMS is often associated with autoimmunity, but

47 is also observed in children with neuroblastoma, but is also attributed to unknown

etiology (36). OMS is known as a severe autoimmune disorder in which auto-

reactive antibodies attack the central nervous system (37). The opsonoclonus is a condition in which there are rapid, multidirectional movements of the eyes, or

“dancing eyes” (38). Mutations in KCTD7 are therefore associated with three similar disease subtypes: EPM3, OMS, and CLN14 (39), although the OMS diagnosis was not definitive. As a result, there is not a consensus in the field about the disease course caused by mutations in KCTD7. A reason for debate on the disease course is partially due to the physicians focusing/ possessing expertise in different areas. For example, one of six clinical reports did not thoroughly examine the patients for storage material. The study that identified lipofuscin comes from a lab that studies several NCLs disorders. Another reason is that physicians did not have access to patient material beyond the patient DNA

(personal communication from Dr. Lehesjoski), and would have missed this characteristic. Therefore, I sought to establish a set of standard criteria to evaluate patients with mutations in KCTD7 will allow better understanding of how patients progress through this disease.

The Conserved BTB Domain in KCTD Proteins

Humans and other mammals encode ~25 KCTD (potassium channel tetramerization domain) proteins on 25 separate genes. KCTD family proteins are readily identifiable by a BLAST search because of their defining N-terminal BTB

(Bric a brac, tramtrack and Broad-Complex) domain, and homologs are found

48 throughout metazoans, including 7 KCTDs in Drosophila melanogaster and one

in Saccharomyces cerevisiae (40, 41). Although BTB domains are found in several

different human protein families, those of the KCTD family are most similar to the

T1 tetramerization domains of voltage-dependent potassium channels, hence

their name despite lacking any other characteristics of membrane proteins or of

channels (31). The C-terminus of KCTD family proteins can be conserved among

subgroups of KCTDs, for example the subgroup containing KCTD7 and KCTD14,

but different subgroups often lack recognizable sequence similarity in their C-

termini.

As the BTB domain is the only known domain in KCTD proteins, the one

exception being the KCTD9 N-terminal KHA domain and C-terminal pentapeptide

repeats of unknown function, both of which are rarely encountered in mammals.

BTB domains have been implicated in a number of processes, such as regulating

cell cycle dynamics (43). Because the BTB domains of other protein families are

known to bind cullin 3 and to serve as adaptors for targets of the cullin3 ubiquitin

ligase complex, e.g. the well-studied tetrameric SCF (Skp1-cullin/Rbx–F-box), several groups have tested and confirmed that some KCTD family proteins also bind Cullin3 via their BTB domains (44). However, attempts to rename the KCTD protein family as KCASH: KCTD containing, Cullin3 adaptor, suppressor of

Hedgehog (45), have not taken hold. While finding a conserved role for the KCTD

proteins is an appealing idea, the study of De Smaele et al. relied solely on

overexpression data from two KCTD proteins, KCTD6 and KCTD21, and lacked

49 more detailed mass spectrometry/ proteomic analysis of all KCTD proteins (45).

More recently, a more comprehensive study has shown that while KCTD BTB domains are quite conserved, not all bind Cullin3 (46). This study demonstrates

that while KCTD proteins possess a very well conserved domain, but more than

likely have divergent roles in the cell.

One KCTD crystal structure has been solved, KCTD5 (47). The crystal structure reveals that KCTD5 oligomerizes through the BTB domain and that

KCTD5 existed in a pentamer (47). Thus, BTB domains may exist in different multimeric forms, which could indicate that additional KCTD oligomeric states may exist to serve multiple functions. The structure of KCTD5 also revealed important information outside of the BTB domain, a “C-terminal module”

connected to the N-terminal BTB domain by a flexible linker (47). If a similar structure exists for all KCTD proteins, this potentially indicates two functional domains of these proteins, an N-terminus involved in oligomerization and cullin 3-

binding, and a unique C-terminus involved in identifying specific proteins for

degradation by Cullin3.

Overall, the KCTD proteins are understudied but a growing theme of

association with disease-related processes is emerging, with KCTD7 being the

only gene causally linked with disease (31). For example, many studies have

associated KCTD15 with obesity. KCTD13 was found to be a driver of brain size

in zebrafish in a copy number-dependent manner. The best biochemically-

characterized KCTD proteins, KCTD8/12/12b(murine-specific)/16, were found to

50 bind the cytoplasmic tail of GABAB receptors (48). GABAB receptors are the main inhibitory neurotransmitter found in the central nervous system and are the G- coupled protein receptors for gamma amino-butyric acid (GABA). Mutations in

GABAB receptors have been shown cause epilepsy, linking additional KCTD proteins to this neurologic disease (49).

Recent work in the lab of Bernard Bettler has connected GABAB receptor signaling with potassium channel desensitization (deactivation) via KCTD proteins. Mass spectrometry identified that KCTD8/12/16 proteins bind the βγ subunits of guanine nucleotide-binding proteins (G proteins) (50). When inactive,

G proteins are heterotrimers, consisting of α/β/γ subunits. Activation dissociates the G protein heterotrimer, with the βγ subunits activating a substrate. Upon binding GABA, GABAB receptors activate βγ subunits, which in turn activate a class of potassium channels (Kir3 channels), causing potassium efflux and hyperpolarization (more negative charge) of the cell membrane due to the decreased positive ions present inside the cell (51). Patch-clamp experiments in

Chinese hamster ovary (CHO) cells demonstrated that potassium channel desensitization was specific for KCTD12 overexpression and not KCTD8/16 (50).

KCTD12 further prevented the βγ subunits from associating with Kir3 channels,

offering an explanation for how KCTD12 desensitizes the potassium channel (50).

The first KCTD knockout mouse, KCTD12-/-, has also been recently published, with knockout mice demonstrating increased excitability in pyramidal neurons compared to wildtype control littermates (52). This study supports the

51 previous work that KCTD12 regulates membrane charge and further connects

KCTD proteins with neurologic disease.

The use of mass spectrometry was central in these studies to elucidate

the role of the KCTD8/12/16 complex: binding GABAB receptors and to the βγ

subunits (48,50). A similar result for membrane hyperpolarization was shown for

KCTD7 overexpession in neurons; however, the authors only showed an interaction with E3 ubiquitin ligase as an explanation for this finding (53). KCTD7 may interact with Cullin3 to degrade positive regulators of Kir3 channel activity, such as the βγ subunits, or by degrading unknown negative regulators of

KCTD12. KCTD7 therefore may fulfill a similar role as KCTD8/12/16 by regulating membrane hyperpolarization but through targeted protein degradation in a

Cullin3-dependent manner. Furthermore, a patient mutation, R184C, was shown to disrupted with Cullin3 (54). This demonstrates that a patient mutation is able to disrupt binding with a previously identified binding partner, offering the first insight into a disease mechanism. Overall, better biochemically analyses, such as mass spectrometry, are needed to move the

KCTD field forward and understand how alterations in these proteins (either copy number or mutation) lead to disease.

52

Results

Identification of New Patients with KCTD7 mutations

To build a more comprehensive understanding of how mutations in

KCTD7 cause disease in children, we queried professional forums at national

epilepsy meetings, discussing if other clinicians had identified any similar

patients, and contacted sequencing facilities, Baylor College of Medicine, Emory

School of Medicine, GeneDX, CeGat, Courtagen, and Athena Diagnostics, asking

if any patients with mutations in KCTD7 had been identified. This allowed us to

uncover new previously unpublished clinical cases of EPM3/CLN14 and, in

several cases, help confirm the diagnosis based on our growing knowledge of

this disease and clinical description of the patient, as most mutations were

previously undescribed and a comprehensive description of the disease course is

not available. From this range of different sources, I have identified 16 new

mutations in 12 new patients, bringing the total to 27 unique mutations in 37

patients, all with a similar disease course. These numbers will likely increase as sequencing becomes more available. In this chapter, I present an analysis of

both published and unreported clinical cases, identifying definitive shared clinical

symptoms. Furthermore, I show how these shared symptoms distinguish KCTD7

mutations from other progressive myoclonic epilepsy disorders, and finally I show

that location of the mutation in KCTD7 influences clinical phenotype.

53 Lipofuscin Discovered in Patient Fibroblasts and Brain Biopsy

Except for one report finding evidence of lysosomal storage material for an

EPM3 patient, several other reports failed to detect similar pathology

(55,54,33,9,30,31). Thus, EPM3 may differ from other progressive myoclonic epilepsies. However, based on results from our lab with yeast Whi2 and my findings reported in Chapter 3, I therefore hypothesized that patients with KCTD7 mutations are likely to accumulate storage material. This hypothesis was supported with the first report of lipofuscin in one patient. The accumulation of lipofuscin is a hallmark of neuronal ceroid lipofuscinoses (NCLs). Lipofuscin accumulations appear electron-dense in patient biopsy electron micrographs and are categorized according to their ultrastructure (22) Furthermore, the

ultrastructure of the lipofuscin indicates the subtype of NCL, which is described

as either granular osmophilic deposits (GROD), curvilinear bodies, or fingerprint

profiles (Chapter 1, Table 1) (13). The lipofuscin profiles are electron-dense when viewed by EM due to their osmophilic properties. GROD is described as generic, electron-dense deposits, curvilinear bodies are alternating light and dark stacks of membranes, and fingerprint profiles resemble a human fingerprint (56).

The presence of lipofuscin with GROD and fingerprint profiles was reported in cultured primary fibroblasts and lymphobastoid cell lines (LCLs) of a patient with mutations in KCTD7 (34).To determine if NCL-like lipofuscin was

present in our two KCTD7 mutant patient fibroblasts, ages 4-8 years and passage number <10, kindly provided by Drs. Haberlandt and Leshinsky-Silver,

54 were examined by electron microscopy. Two normal human primary fibroblast lines (GM05757 and 498, Corriel Institute) were selected to closely match the patient cells in both age of the donor and passage number to serve as control fibroblasts. Control and KCTD7 mutant patient fibroblasts were plated in parallel and were fixed for EM and imaged by Dr. Isabelle Coppens. Upon examination of the fibroblasts we found the presence of lipofuscin, with electron-dense material located in the cytoplasm, similar to a profile of GROD. While the GROD in

KCTD7 mutant fibroblasts looked similar to GROD, it did not have the same distribution. We found that the GROD was found next to vacuole-like structures

(stars) (Figure 1A). Dr. Ralph Nixon, an expert on lysosome function, suggested that this morphology was more than likely a fixation artifact. However, the clear presence of membrane along this vacuole, along with intact mitochondrial morphology, argues against a fixation artifact (Dr. Isabelle Coppens). A similar profile of dark material next to a vacuole was found in a brain biopsy from a patient enrolled in the Deciphering Developmental Disorders (DDD) project in the

United Kingdom (provided by the group of Dr. Michael Parker), further arguing against a fixation artifact, as two independent groups demonstrated a similar morphology in different patients (Figure 1B). This indicates that a similar lipofuscin structure is found in 3/3 patients, is found in more than one type of tissue (fibroblasts and brain), and offers support for they hypothesis that KCTD7 defects lead to the accumulation of storage material, as seen in other progressive myoclonic epilepsies.

55 After thoroughly searching the literature and consulting several experts

here at Johns Hopkins and at other institutions, I did not identify a profile similar

to the dark, electron-dense material next to vacuoles (57,58). Several experts on

progressive myoclonic epilepsy/ NCL also could not recognize these structures

(personal correspondence with Dr. Anna-Elina Lehesjoki of the University of

Helsinki and Dr. Angela Schulz of University Medical Center Hamburg-

Eppendorf). However, persistence eventually identified two researchers, Dr.

Rosa Puertollano (NIH, Bethesda), and Dr. Gustavo Maegawa (Johns Hopkins)

who independently referred me to the same paper of Dr. Puertollano describing a

very similar phenotype observed in EM images of fibroblasts from a patient with

mucolipidosis type IV (59). These dark (electron dense) structures that are next to

vacuoles are described to be the accumulation of electron-dense lysosomes

fusing with autophagosomes (vacuoles structure) (59).

The gene mutated in mucolipidosis type IV is Mucolipin 1 and is involved

in the trafficking of endosomes and autophagosomes to the lysosome (59). This

morphology is further suggests that Mucolipin 1 is involved in fusion of the

autophagosome and lysosome, and that defects in Mucolipin 1 stall this process,

creating a buildup of autophagosome-lysosome structures. Children with

mucolipidosis type IV suffer from psychomotor delay, progressive visual

impairment, and low levels of gastric acid in the stomach (achlorydria) (60). An accumulation of lysosomes fusing with autophagosomes would be consistent with KCTD7 being classified as a progressive myoclonic epilepsy gene, as an

56 inability to degrade cellular contents through autophagy would cause a buildup of

undegraded proteins and lipids.

Mutation Clusters in KCTD7 Correlate with Clinical Phenotype

Advances in sequencing have allowed for increased identification of patients with mutations in epilepsy-causing genes, including KCTD7 (32). Until

2007 when the first case was identified, patients with mutations in KCTD7 would

have been classified as idiopathic, as the source of their disease would have

been unknown. Even still, patients with mutations in KCTD7 must travel from

clinic to clinic in search of a diagnosis, as an effective method to identify these

patients does not exist (personal communication from several physicians) (61).

To build a better diagnostic tool, I needed to identify additional cases that

are not found in the literature. Using a number of different strategies, I have

identified another 12 previously unreported patients carrying 13 novel KCTD7

mutations associated with a similar disease course. By mapping all 27 known

mutations onto a linear diagram of the KCTD7 protein, it is apparent that all

mutations occur in three clusters, the N-terminal BTB domain common to all

KCTD family members (mutations ranging from amino acid 64-121), a middle

region (mutations ranging from amino acid 153-235), and a tight cluster of

mutations in the C-terminus (mutations ranging from amino acid 259-289) (Fig.

2). While it is plausible to speculate that mutations in the BTB would disrupt

57 protein-protein interactions, the mutations in clusters 2 and 3 are more difficult to

understand.

Cancer research has shown mutations in different parts of oncogenes,

such as epidermal growth factor receptor (EGFR), can affect the clinical

outcomes in patients (62). Based on this clustering of patient mutations into the three non-overlapping protein regions, I sought to correlate mutation positions with clinical characteristics obtained from the published literature and from de-

identified medical records obtained from 14 separate universities, clinics, and

sequencing centers (see methods for complete list of author and affiliations). I

found that the age of onset could distinguish a patient with mutations in KCTD7

from other progressive myoclonic epilepsies. Patients with mutations in KCTD7

have a relatively narrow window of onset, between 8-36 months with 62%

occurring between 1-2 years (Fig. 3A). Of the patients with a known specific date of onset (19/31); the average was approximately 16 months. Of the progressive myoclonic epilepsies, only CLN1 has a similar age of onset. CLN1 patients are born normally and develop disease after birth from 6-24 months (21). This could indicate that KCTD7 and CLN1 play an important role in the development of the neonatal brain. Patients with mutations in the BTB domain (cluster 1) tend to have an earlier age of onset, while patients with C-terminal cluster 3 mutations

tend to have later onset, though not statistically significant (Fig. 3B).

I also determined if seizure control could be influenced by the location of a

patient’s mutation. Control of seizures is achieved in roughly two-thirds of all

58 epilepsy patients (63). Following this trend, we found 19 of 29 patients with reported seizure control, despite progressive neurologic decline (Table 1).

Myoclonus was also reported in all patients. Developmental milestones of these patients are generally unremarkable until onset of symptoms, however, some patients were reported to have delays in crawling and walking, but only one patient was reported to never walk (31). These patients also frequently presented with fine motor impairments and speech deficits, which may have preexisted without notice for many months. Ocular findings were unremarkable at onset for all patients, with only two patients developing degeneration of the optic nerve

(optic atrophy) after the onset of symptoms (Table 2). Photic stimulation, demonstrating activity after light stimulation on an EEG, was reported in seven patients. In addition to these neurological symptoms, I also found evidence for a movement disorder in patients with KCTD7 mutations. Ataxia was reported in 16 of 19 patients and 13 of 13 or which information was available had at least one

extra-pyramidal sign of movement disorders, including tremors, dystonia,

dyskinesia or choreoathetosis, indicating dysfunction of parts of the brain that

control movement (Table 3). My findings expand the current phenotype of

mutations in KCTD7 to include a movement disorder in addition to epilepsy and

neurodegeneration. These are an important observation, and inclusion of such

criteria in a diagnostic algorithm may lead to much earlier diagnosis, thereby

increasing the potential to therapeutically intervene before the onset of

progressive decline.

59 We next wanted to determine if mutation cluster could influence presenting symptom. We classified each mutant allele/chromosome (2 alleles/patient) based on mutation cluster and found significant differences between the presenting symptoms of patients with mutations in each mutation cluster (Fig 4). The presenting symptoms typically include seizures (73.3% of cases), developmental delay (13.3%), or a movement disorder (13.3%). Eighteen of the 31 known patients have homozygous mutations and 13 of these 31 possessing compound heterozygous mutations. All alleles except three in cluster 1 presented with seizures, four of 16 alleles presented as non-seizure phenotypes in Cluster 2, and 13 of 27 alleles presented as non-seizure in Cluster 3. This is the first indication that the position of the KCTD7 mutation may be predictive of the clinical phenotype in patients with KCTD7 mutations.

As I found that the position of KCTD7 mutations might affect the presenting symptom, including seizure presentation, I next wanted to determine if seizure control could be influenced according to a patient’s mutation cluster. As previously mentioned, 19 of 29 patients were reported to achieve seizure control with medication. I found a trend for disproportionate seizure control across the mutation clusters, meaning that 10 of 22 alleles in Cluster 1, 11 of 18 alleles in cluster 2 and 15 of 18 alleles in cluster 3 had seizure control with medication (Fig

5A). Because some patients had mutations in two different clusters, I also examined patients with only homozygous mutations to specifically assess this correlation, and found a similar trend of greater seizure control in patients with

60 homozygous mutations in cluster 3 (Fig 5B). Valproic acid (VPA) was the most common medication administered to these patients. VPA is a carboxylic acid that was originally isolated from an herb, named Valerian (64). VPA failed to control seizures in any patient with homozygous mutations in Cluster 1, yet improved seizures in all patients with homozygous mutations in clusters 2 and 3 (Fig 6A and 6B). VPA was commonly used in combination with other medications.

Unfortunately, as the mechanisms by which VPA and other epileptic drugs work, it is difficult, if not impossible, to speculate how and why certain drugs work beyond the fact that different domains in KCTD7 may affect different processes

(Supplementary Fig 1).

When all the patient information described here is taken together, they suggest an algorithm for that distinguishes patients with

KCTD7 mutations from all other disorders (Fig 7). At the top level, all patients were found to present with developmental delay or seizures (Fig 4), and all also had myoclonic seizures (Table 1). All patients present between the ages of 8-36 months, allowing this disorder to be distinguished from other non-infantile progressive myoclonic epilepsy syndromes (Fig 3A). Finally, patients with

KCTD7 mutations can be distinguished from CLN1 from the lack of blindness at onset (Table 2). As there appear to be many defining characteristics amongst these patients, we propose to name patients with KCTD7 mutations Van-

Bogaert-Abramowicz’s (VBA) Disease after those who first reported the

61 association between KCTD7 mutations and this neurologic disorder, with different mutations causing related subtypes: OMS and CLN14 (Supplementary Fig 2).

62 Discussion

A better understanding of the causes of idiopathic epilepsy is paramount for the better treatment of these neurologic disorders. Here, we present a comprehensive examination of the clinical aspects of a previously unknown cause of epilepsy, which is now known to be caused by mutations in KCTD7. We present 10 new clinical cases and document 12 novel pathogenic mutations in

KCTD7 obtained from other investigators and from sequencing center databases.

Of all new and previously published cases, we had access to fibroblasts form two patients. In these two fibroblasts, we found lipofuscin. Additionally, the group of Dr. Michael Parker (Sheffield Children’s Hospital, UK) kindly provided an

EM image of a brain biopsy that had lipofuscin as well, indicating that fibroblasts could be presenting the same disease-phenotype as in the more disease- relevant brain. Combined, we found 3/3 patients that had accumulations of lipofuscin, and speculate that if tested, the remainder of KCTD7 patients are likely to have similar structures detectable by electron microscopy. This lipofuscin had a distinct profile that is similar to the storage material found in a patient fibroblast with mucolipidosis type IV. As the role of KCTD7 is unknown, this could provide insight into the function of KCTD7, indicating that KCTD7 may play a similar role as the gene that as Mucolipin 1. Mucolipin 1 is involved in trafficking endosomes and autophagosomes to fuse with the lysosome and defects lead to the accumulation of storage material (lipofuscin), as cellular contents cannot be delivered to the lysosome for degradation. As patients with mutations in KCTD7

63 develop a similar phenotype as mucolipidosis type IV patients, this could indicate

that KCTD7 is involved in trafficking endosomes or autophagosomes to the

lysosome as well. Alternatively, KCTD7 could interact with Cullin3 to degrade a

negative regulator of endosomes/ autophagosomes trafficking, which could result

in a similar phenotype.

We found that all patients presented between 8 and 36 months of age.

While many progressive myoclonic epilepsy disorders can present over the span

of many years, patients with mutations in KCTD7 have a very narrow window in

which disease symptoms present. Importantly, these children are described as

normal before the onset of symptoms. This could indicate that KCTD7 plays an

important role in the development of infants at this stage of development.

In addition to the already documented epilepsy and neurodegenerative

features, we found that patients are demonstrating signs of a movement disorder.

Among the patients for whom we have data, 16/19 patients have ataxia and

13/13 patients have extra-pyramidal signs (dystonia, tremor, dyskinesia, or

choreoathetosis). An extra-pyramidal sign is associated degeneration of parts of

the brain that control motor coordination, such as the basal ganglia and

cerebellum, whereas ataxia is not specific to degeneration of a specific part of the

brain (65). This expands the clinical phenotype of mutations in KCTD7 to include movement disorder in the clinical description. Several of the patients also present with movement disorders as opposed to presenting with epilepsy. Of note, one other of the 13 neuronal ceroid lipofusinosis genes, CLN12/ Atp13A2, is also a

64 Parkinson’s gene, Park9 (39). Thus, two of the 14 known CLN epilepsy disorders also cause movement disorders with extra-pyramidal signs. CLN12/ Atp13A2 is a lysosomal ATPase that functions in cation homeostasis (66). Human olfactory

neurosphere cultures from patients with mutations in Atp12A2 have low

intracellular zinc concentrations, fragmented mitochondria and low ATP levels

(66). Future studies and mouse models will determine the role that KCTD7 mutations plays in the development of movement disorders, and if KCTD7 has a similar function as CLN12/ Atp13A2 in cation homeostasis and mitochondrial health.

We also found that rather than falling into a random distribution, mutations fall into 3 clusters in KCTD7. We further found that the cluster to which a patient’s mutation(s) falls is able to influence the clinical phenotype. For example, statistically significant differences were noted for presenting symptom, age of onset, and medication response in regard to mutation cluster. Identification of more patients will determine if medication response is truly determined by the mutation cluster, which could offer the first insight into how to treat patients with

KCTD7 mutations. Additionally, the mechanism by which VPA controls seizures is not known. If cluster 1 mutations do not respond to VPA whereas cluster 2/3 mutations do respond, this could offer insight how VPA is able to control seizures by understanding the role of the two functional domains of KCTD7. Correlations between effective drug regimens was proposed and found effective based on

65 mutation location in several types of cancer but this could be the first description

in an epilepsy-causing gene (62).

In summary, we present for the first time a comprehensive examination of the pathologies associated with mutations in KCTD7. As we expand the phenotype to include a movement disorder consisting of ataxia/extra-pyramidal signs, we propose to provide an overarching name for syndromes that result from

KCTD7 mutations: Van-Bogaert-Abramowicz’s (VBA) Disease. Different mutations in KCTD7 may lead to varied clinical courses (EPM3/CLN14/OMS); however, sufficient commonalities exist amongst all patients to warrant an all- encompassing disease. In contrast to many other forms of NCL, we are finding that some patients with mutations in KCTD7 are surviving into their third decade of life. Additionally, despite some patients having effective seizure control, all patients still experience a progression of neurologic decline. This raises the need to find effective treatment regimens that can extend the quality of life for patients suffering from mutations in KCTD7.

66 67

68 69 70 71 72 73 74 75 76 Materials and Methods

Patient Identification and Phenotype Analysis

New cases were identified through professional forums, Google searches, and inquiries at difference sequencing facilities:

Thomas Markello (Undiagnosed Disease Program, NIH), Adam Hartman

(Department of Neurology, Johns Hopkins School of Medicine), Rachel Kneen

(Paediatric Neurology, Alder Hey Children’s NHS Foundation Trust, Liverpool,

UK), Daniel Crooks (Neuropathology, The Walton Centre NHS), Santosh R

Mordekar (Foundation Trust, Liverpool, UK), Min T Ong (Foundation Trust,

Liverpool, UK), Michael J Parker (Clinical Genetics, Sheffield Children’s Hospital,

UK), the Deciphering Developmental Disorders Study (Wellcome Trust Sanger

Institute, Cambridge, UK), Edda Haberlandt (Medical University of Innsbruk,

Austria), Michael Alber (University Children's Hospital Tübingen, Germany),

Isabelle Prehl (CeGaT GmbH Tübingen, Germany), Satish Agadi (Baylor College of Medicine), Thomas Burrow (Cincinnati Children's Hospital Medical Center),

Katrina Peariso (Cincinnati Children's Hospital Medical Center), Haiying Meng

(Cincinnati Children's Hospital Medical Center), Hatha Gbedawo (Vital Kids

Medicine, PLLC), Dianalee McKnight (GeneDX), Christine Stanely (Courtagen),

Adolfo Garnica (Arkansas Children's Hospital), Noelle R. Danylchuk (Department of Genetic Counseling, College of Health Professions, UAMS), Tobias

77 Loddenkemper (Boston Children's Hospital), Jill Mokry (Baylor College of

Medicine).

Clinicians were provided with a questionnaire based on data provided in previously published cases. Commonalities among patients were identified and scored via alleles or homozygous patient according to the mutation’s respective mutation cluster. Statistical significances were determined using a 2X2 Fisher’s exact test. Significance was considered at P<0.0167. Each clinician contributing a patient was asked to include a brief write-up describing the development of the patient.

Preparation of Patient Fibroblasts

A previously identified patient with mutations in KCTD7 had a 3 mm diameter skin punch biopsy taken in the axilla. To establish a fibroblast line, fibroblasts were cultured with standard methods and the cells grew without incident. Control fibroblasts originated from the Corriel Institute.

Primary fibroblasts were shipped in T25 flasks to Johns Hopkins

University and were maintained in Dulbecco’s modified Eagles medium (DMEM) supplemented with 10-20% fetal bovine serum (FBS), 1% penicillin and streptomycin at 37°C and 5% CO2. Fibroblasts were plated, fixed and prepared at

Johns Hopkins University Microscope Facility and imaged by Dr. Isabelle

Coppens with a Philips CM120 Electron Microscope (Eindhoven, the

78 Netherlands) under 80 kV. The group of Dr Michael J Parker from the Sheffield

Children’s Hospital, Sheffield, UK, provided the image of the brain biopsy.

79 Figure Legends

Figure 1. Electron microscopy reveals lipofuscin in patients with KCTD7 mutations. The presence of lipofuscin was found in two primary human patient fibroblast lines (A) and in the brain biopsy of another patient (B).

Figure 2. All known human KCTD7 mutations are located in three clusters in the

KCTD7 protein. These mutations can be grouped into three clusters spanning

58% of the protein. Cluster 1 ranges from amino acid 64-121, cluster 2 ranges from amino acid 153-235 and cluster 3 ranges from amino acid 259-289.

Figure 3. Onset of the progressive myoclonic epilepsies. (A) Range of disease onset in years of those progressive myoclonic epilepsy (EPM) disorders with the earliest average onset, average age of onset denoted by a black bar. (B) Age of onset presented for mutant alleles/ as 13 of 31 patients have compound heterozygous mutations. Statistical significances were determined using a 2X2 Fisher’s exact test. No significance was found. Significance was considered at P<0.05.

Figure 4. Presenting symptoms for individuals with KCTD7 mutations. (A)

Presenting symptoms of all patients with corresponding mutant allele/chromosomes in each cluster, and the number of patients in each category with homozygous mutations. (B) Statistical significances were determined using a 2X2 Fisher’s exact test. Significance was considered at P<0.05.

80 Figure 5. Medication efficacy in seizure control by mutation cluster. (A)

Medication efficacy in all patients. (B) Medication efficacy in only those patients with homozygous mutations. Statistical significances were determined using a

2X2 Fisher’s exact test. Significance was considered at P<0.05.

Figure 6. Medication efficacy of VPA in all patients (A), or in patients with homozygous mutations (B). Statistical significances were determined using a

2X2 Fisher’s exact test. Significance was considered at P<0.05.

Figure 7. Suggested algorithm to distinguish Van Bogaert-Abramowicz’s Disease from other disorders. All patients presented with developmental delay, seizures, or a movement disorder between 8-36 months of age. All patients further had myoclonus and did not have visual abnormalities at onset.

Supplementary Materials

Supplementary Figure 1. Other medications used in combination with VPA.

Medication efficacy in seizure control per allele (A), and in homozygous patients

(B). VPA was the most common drug listed in treating patients with KCTD7 mutations and may have been used in combination with other drugs.

Supplementary Figure 2. Proposed name of Van Bogaert-Abramowicz’s

Disease for patients with KCTD7 mutations. As all patients with KCTD7 mutations share clinical commonalities, we propose an over-arching name of

“Van Bogaert-Abramowicz’s Disease” for patients with KCTD7 mutations causing subtypes, OMS, EPM3, and CLN14.

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90 Complete Clinical Data for Chapter 2

Dr. Adam Hartman provided me with a list of different symptoms to investigate and see if any symptoms were common amongst patients with mutations in KCTD7. We had an idea that the age of onset and presenting symptoms were uniform amongst these patients based on the published data; however, he also told me ask physicians for newly identified cases if any of the patients have symptoms of dystonia, dyskinesia, tremors or choreoathetosis. At the time, I didn’t pay too much attention to these symptoms but included them in our questionnaire to physicians. It soon became apparent that these patients also had these symptoms in common as well, raising my curiosity about what these symptoms describe: a movement disorder. Therefore, with our foresight and persistence we were able to identify a new phenotype associated with this disease.

91

Patients 1 & 2 (D229Y/L271H).

The patient was a female born after an unremarkable pregnancy and a delivery only notable for meconium-stained amniotic fluid. At 19 months, she developed recurrent episodes of falling, unsteady gait and tremors. The tremor was noted particularly when she was reaching for objects. Six weeks prior to her initial clinical presentation, she had a mild illness consisting of , vomiting, and constipation. Her initial exam was remarkable for a wide-based gait and truncal ataxia, as well as tremor when reaching for objects. Initially, she was thought to have opsoclonus-myoclonus syndrome and was treated with prednisone and eventually IVIG. The family history was significant for learning difficulties in the patient’s father and migraine headaches and depression in the patient’s mother. A neurologically normal sister had recurrent glucosuria and recurrent . A younger brother had developmental delays. Around 2 years of age, she started having seizures, with electroencephalograms (EEGs) showing progressively worsening multifocal sharp waves (notable for relatively little involvement over the temporal regions) and excessive background slowing.

Nearly constant asynchronous myoclonus was noted by 6 yrs of age. Hearing and vision were normal initially. Developmentally, regression was noted after the seizures started. By 26 mos, she was not making language milestones. Over time, she was noted to have eczema, short stature and recurrent molluscum contagiosum. By 6 yrs of age, she had global developmental delays and signs of

92 decreased axial tone. By 9 yrs, she was noted to have lost visual fixation and increased appendicular tone.

Magnetic resonance imaging studies (MRIs) showed increasing cortical atrophy over time, dilated ventricles and volume loss in the corpus callosum. An electroretinogram (ERG) at 5 yrs was normal. Electromyography (EMG) at 10 yrs showed decreased insertional activity and some fibrillations. Nerve conduction studies were normal. A muscle biopsy at age 5 yrs showed small lipid droplets scattered throughout the muscle fibers, often close to the mitochondria. The patient died in her sleep at age 11 yrs.

The second patient is the younger brother of Patient 1, with a normal birth history. At 18 mos of age, he was noted to have an oral-motor dysphagia with decreased oral, lingual, and facial tone. There were significant feeding problems.

At 16 mos, he was noted to have decreased tone and extensor posturing with vertical suspension. His gait was wide-based. By 23 mos, he was noted to have dystonic movement when reaching for objects and hearing loss. At 2 yrs of age, he was noted to be unsteady and have some mild shaking (including the tongue).

At 20 mos, an MRI showed prominent ventricles, mildly decreased cortical volume and a thin corpus callosum. Starting at 3 yr of age, serial EEGs showed multifocal sharp waves and spikes (less prominent over the temporal regions) and background disorganization, associated with myoclonus. Sequencing revealed compound heterozygous mutations in these patients: D229Y/L271H

(10).

93

Patient 3 (R153H).

The patient is a female born after an unremarkable antenatal and perinatal course. She was growing normal until 15 months of age when parents started noticing recurrent episodes of falling, unsteady gait and head bobbing. The head bobbing episodes were noted particularly when she was tired and often preceded by eye flutter. The family history was significant for consanguinity with parents being first-degree cousins. The patient has an older brother with normal development. At initial presentation of 18 months she used to get at least fifty head bobbing episodes a day with each lasting about five seconds. Her exam was remarkable for a wide-based gait and truncal ataxia, as well as witnessed episodes of downward eye gaze with head bobbing and left upper extremity twitching. She was treated with an intravenous dose that markedly decreased the head bobbing events. A month later she started having more frequent seizures, with electroencephalograms (EEGs) showing progressively worsening of the background with bursts of high amplitude generalized spike and slow wave activity that clinically correlated with head bobbing episodes.

Her hearing and vision were noted to be normal initially. Developmentally, regression was reported around 20 months of age. By 2 year 4 months of age, she was admitted for decreased use of left with tonic clonic seizures involving the left side of the body and also choreiform movements involving the left. During her most recent exam at 3 years, she was nonverbal with choreiform movements

94 involving face and tongue with diffuse global hypotonia and recurrent myoclonic

seizures. Her seizures were deemed to be pharmacoresistant after they did not

respond substantially to six antiseizure medicines. The ketogenic diet and intravenous steroids were also unsuccessful in controlling seizures. Her initial brain MRI did not show any abnormalities. A magnetic resonance spectroscopy performed through basal ganglia did not reveal any abnormalities. Sequencing revealed a homozygous mutation: R153H.

Patient 4 (R70W/R84Q)

The patient is a seven-year-old female born vaginally at full term after a pregnancy complicated by gall bladder dysfunction requiring treatment with pain medications. Her parents were unrelated and there was no family history of significant medical issues. Her early developmental and medical course was unremarkable.

At age 20 months, the child presented with episodic ‘sudden falls’ that correlated with generalized seizures on EEG. Brain MRI demonstrated a confluent area of FLAIR hyperintensity within the left parietal subcortical white matter, which was believed to be benign. She was diagnosed with and started on antiseizure medicines but continued to have significant seizure burden over time, despite aggressive medical treatment. During this time, she also had increasing difficulty with walking, developing a wide-based,

95 steppage gait with significant foot drop in addition to hypotonia and truncal ataxia.

While these findings were present on a constant basis, the intensity of the symptoms appeared to wax and wane with her degree of seizure burden. Her developmental progress slowed dramatically over this time, confounded by the increased difficulty in both gross and fine motor tasks secondary to her hypotonia and ataxia. Her language and cognitive development were also delayed in that the patient had a limited vocabulary (<100 words) and had only progressed to short phrases by 5 years of age.

In addition to her baseline neurological symptoms, she experienced three periods over the course of four years that consisted of lower extremity hypertonia and loss of ability to ambulate independently for up to 2.5 months. EEG, MRI and metabolic studies obtained near the onset of these episodes were unrevealing of an etiology for her change in symptoms. The patient ultimately returned to her baseline skill level at the end of each episode. Over six years, her brain MRI has remained unchanged, with the consistently observed left parietal FLAIR hyperintensity felt to be most likely secondary to an atypical perivascular (i.e.,

Virchow-Robin) space.

Due to the intractable nature of her epilepsy, at age 6 years 11/12 months, the patient underwent a complete corpus callosotomy and has been seizure free since that time. Additionally, she experienced improvement in ataxia and tremors, with improvements in gross and fine motor skills. She is now able to walk independently, navigate stairs, with alternating feet while holding onto a rail, and

96 feed herself with utensils. Her language has not returned to her presurgical baseline, but was attributed to the corpus callosotomy. Sequencing revealed compound heterozygous mutations: R70W/R84Q.

Patient 5 (P205L/V259E)

The patient was born at term to healthy non-consanguineous parents after an uneventful pregnancy. Cranial circumference, height and weight were in normal ranges. At the age of 12 months, he had his first febrile seizure. His development was normal until 18 months of age when parents noticed asymmetric use of his hands with perforation of his right hand. At the same time, atonic seizures with drop-attacks started to disturb his motor function. He had an unsteady gait with truncal ataxia. Swallowing problems also were noted.

At the age of 20 months, myoclonic jerks were observed, especially when he was fatigued. Several febrile seizures led to the diagnosis of epilepsy, leading to treatment with Sultiame. Other antiseizure medicines tried without success included topiramate, valproic Acid, levetiracetam, oxcarbazepine, zonisamide,

Ethosuximide, Clonazepam, cortisone, ACTH, Lamotrigine, Phenobarbital,

Vigabatrine, Lacosamide and Retigabine. The EEG showed diffuse slowing and continuous multifocal epileptic activity. His development further regressed and he presented with expressive speech problems at the age of 3 years, finally he was unable to move. He had to be fed by gastrointestinal tube. His hearing and vision were normal.

97 A brain MRI of the brain showed some hypomyelination of bilateral parieto-occipital regions. Extensive metabolic screening was normal, including enzymatic testing for Neuronal Ceroid Lipofuscinoses. Whole-exome sequencing

showed compound-heterozygote variants in KCTD7: P205L/V259E.

Patient 6 (R177H/ A178V)

The patient was born. The patient’s milestones were all delayed: crawling at 5 months and walking at 20 months. She did not exhibit ataxia but she consistently demonstrated a slow, deliberate gait. She stopped babbling at 8 months of age when she was treated with antibiotics. She also had developmental regression, including losing the ability to write clearly (sometimes affected by her tremor) and read. She consistently has socially immature behaviors.

Myoclonic seizures developed at 2 years of age. Tonic-clonic seizures developed at age 7 years with a potential association with onset of allergic response to beans and barley. The tremor became more pronounced around 16 years of age. Valproic acid helped seizures but tremor worsened and the patient developed pancreatitis. She developed dyskinesia from Ethosuximide, including jaw lateralization and tongue thrusting. Carbamazepine increased myoclonus, which even after discontinuation has persisted. Rufinamide had no effect on seizures but had a negative impact on cognition. Lamotrigine increased myoclonus. The patient is currently taking Clonazepam and Levetiracetam. She

98 developed difficulty swallowing due a paralyzed left vocal cord after a vagus nerve stimulator implant.

The patient also has autoimmunity and immune dysregulation with frequent infections and sore throats. At age 18, she was diagnosed with cerebral folate deficiency, due to blocking antibodies present to cerebral folate receptors, as well as elevated antistreptolysin O and anti-DNAse B titers. A trial of intravenous immunoglobulin (IVIG) greatly increased myoclonus.

Her seizures are currently fairly well managed on the AED cocktail,

Leucovorin (calcium folinate) and a Modified Atkins diet. Her cognitive abilities continue to decline with further loss of social and communication skills. In the last year, she also presented with bilateral eye deviation. Extensive prior evaluations were performed, including muscle biopsy for mitochondrial myopathy (negative) and lymphonodular hyperplasia (positive). Sequencing performed by Courtagen

Life Sciences, Inc. revealed compound heterozygosity in the patient:

R177H/A178V.

99

Homozygous Last Known Cluster1 Mutation Origin Age of Onset Age

6 R94W Turkey 10 months 8 yrs (Edda)

8 years 7 R94W Turkey 1-2 yrs (pub)

4 yrs 11 14 L108M Pakistan 1-2 yrs mon(pub)

3 yrs 11 mon 15 L108M Pakistan 1-2 yrs (pub)

Heterozygous Cluster1

16 R84W, Δ3,4 Israel 5-10 months 4.5 yrs (pub)

20 R70W, R84Q Cincinatti 20 months ~7 (Burrow)

30 G105E/G114E Baylor <16 months 14 yrs

31 G105E/G114E Baylor ?

Nonsense Cluster1

24- Van 1 R99X Morocco 18 months Bogaert

17- Van 2 R99X Morocco 24 months Bogaert

3 R99X Morocco 16 months 3 (death)

Homozygous Cluster2

17 R184C Mexico 9 months 13 (death)

18 R184C Mexico 8 months 17 (death)

100 594delC, 9 I199X74 Turkey 6-12 months 14 yrs (pub)

594delC, 10 I199X74 Turkey 1-2 yrs 4 yrs (pub)

19 R153H Baylor 18 months 3 years

Homozygous Cluster3

12 W289X Turkey >2 yrs (3 yrs) 8 yrs (pub)

13 W289X Turkey 1-2 yrs 3.5 yrs (pub)

11 N273I Turkey 1-2 yrs 8 yrs (pub)

Mennonites in At least 14 23 Y276C Mexico 24 months (pub)

Mennonites in 24 Y276C Mexico 24 months ?

Mennonites in 25 Y276C Mexico 15 months 12 (pub)

Heterozygous Cluster2

German/Dutc h/English/Can 29 R177H/A178A adian 2 years 21

Cluster 1/3

8 D115Y, N273I Turkey 1-2 yrs 9.5 yrs (pub)

27 R121L/W235S USA 18-24 months 15

28 R121L/W235S USA 18 months 25

Cluster2/3

21 Boston 24 months 7 yrs R181W,

101 D229Y

22 P205L, V259E Germany 16 months 7 yrs (blog)

4 D229Y, L271H USA, Oregon 15 months 11 (death)

~7 5 D229Y, L271H USA, Oregon 15 months (newspaper)

Cluster 1/2

26 T64A/R211X UK 23 months

102

Homozygous Symptoms at Neurologic Cluster1 Mutation Presentation Development Findings

Continuous multifocal myoclonic seizures affecting all limbs and face, atypical absences and some additional tonic and tonic-clonic seizures. Photic stimulation provoked frequency dependent spike-wave activity. At 6 yrs, shows severe cerebral movement disorder Does not sit or with muscular walk, reach for hypotonia and toys. IQ below cognitive 50. No developmental delay. expressive Wheelchair bound. language and No expressive does not follow language and does verbal not follow verbal 6 R94W Epilepsy commands commands

Mental regression, Epilepsy motor (Myoclonic impairment, Normal retinal 7 R94W Atonic) ataxia findings, Ataxia

Mental regression, Epilepsy motor (Myoclonic, impairment, Normal retinal 14 L108M Febrile Initially) ataxia findings, Ataxia

Mental regression, Epilepsy motor (Myoclonic impairment, Normal retinal 15 L108M Atonic) ataxia findings, Ataxia

103

Heterozygous Cluster1

5 months mild generalized tremulous movements, 10 months began to fall forward, stopped attempting to Multifocal action stand or walk myoclonus, around minipolymyocholus, furniture. 16 dysmetria, brisk months, deep tendon reflexes continuous in lower extremities miltifocal and a flexor plantar myoclonous response. and could not Neurological walk nor sit with examination intermittent significant for mild abnormal eye truncal ataxia and movement. Did severe continuous not develop myoclnus especially speech but for the hands, which comprehension worsens during appeared motor activity and normal. Brief disappears during 16 R84W, Δ3,4 Epilepsy, Ataxia attention span. sleep.

Epilepsy, 20 R70W, R84Q generalized ? ?

30 G105E/G114E Delayed Milestones

31 G105E/G114E ? ? ?

Nonsense Cluster1

Severe dysarthria, Walked limited expressive independently language and 1 R99X Epilepsy at 14 months comprehension, impaired fine finger

104 movements, severe truncal ataxia, walking only with assistance

Severe dysarthria, Normal until 2 impaired fine finger years, then movements, posture- severe evoked negative cognitive myoclonus in upper 2 R99X Epilepsy decline limbs, normal gait

Normal until 16 months, then psychomotor Intermittent head delay, severe nodding, low feeding amplitude myoclonus problems, in all extremities, 3 R99X Epilepsy drooling ataxia

Homozygous Cluster2

Normal birth and infancy until seizures. Normal developental miletsontes were documemtned until ~18 months, when motor and speec regression were Developmental noted. At age regression, vision 12, patient had loss (normal retinal microcephaly, exam), cerebellar was nonverbal, cortical atrophy and and was thinning of the Epilepsy without corpus callosum with (Myoclonic, spontaneous loss of subcortical face/extremities) motoric white matter (12 17 R184C and spasticity function. years) Showed no

105 response to visual thread and had markedly dimished pupillary ligh reflexes.

Normal birth and infancy until seizures. Normal developental miletsontes were documemtned until ~18 months, when motor and speec regression were noted. At age 10, patient had microcephaly, was nonverbal, and was without spontaneous motoric function. Showed no response to visual thread and had Developmental markedly regression, vission dimished loss with mild optic pupillary ligh atrophy, moderate reflexes. generatlized brain Epilepsy Showed mild atrophy more (Myoclonic, bilateral optic pronounced in the face/extremities) atrophy without frontal lobes and Precipitated by apparent cerebellum (13 18 R184C retinopathy. years)

594delC, Epilepsy Mental Normal retinal 9 I199X74 (Myoclonic) regression, findings, Ataxia motor

106 impairment, ataxia

Mental regression, Epilepsy (GTCS, motor 594delC, Myoclonic, impairment, Normal retinal 10 I199X74 Hypomotor) ataxia findings, Ataxia

Sat at 8 months, crawled at 11 months, walked at 13 months. Wide based gait, fell Difficulty with First word at 13 backwards after 2 ambulation and months. Five to steps, unable to sit, unstready gait six words at 18 stand or walk without 19 R153H with falls months. falling/ stumbling

Homozygous Cluster3

Mental regression, motor impairment, Normal retinal 12 W289X Ataxia ataxia findings, Ataxia

Mental regression, motor impairment, Normal retinal 13 W289X Epilepsy ataxia findings, Ataxia

Mental regression, motor impairment **Ataxia not **(never present**, Normal 11 N273I Epilepsy walked)** retinal findings

Developmental The first neurologic delay for assessment walking at 19 disclosed the Developmental months and presence of 23 Y276C Delay speech limited significant hypotonia, to single words. motor incoordination,

107 Following the and gait ataxia. onset of There were no signs seizures, of ocular develpmental movements, regression was nystagmus, or noted with loss cranial nerve deficits. of expressiv elanguage skills and eventual progression to a nonverbal state, with limited comprehension of spoken language. Between age 9 and 14 years, there were additional signs of pregression, with loss of independent ambulation, incontinence, with inability to feed independently and manage activities of independent living.

Normal development of milestones until age 2. Began to show developmental regression after onset of seizures and Moderately lost expressive increased tone in speech and had lower extremities, significant hyperreflexia, and 24 Y276C Epilepsy sialorrhea gait ataxia. (drooling).

108 Retains ability to walk and feed herself.

Showed signs of developmental delay in walking but had a vocabulary of several words. She is cognitively delayed, and Mild neurologic has limited symptoms compared expressive to her sisters with speech, but respect to gait and unlike her older coordination. She is sibling she has capable of yet to show independent further decline ambulation and she Developmental in her can feed and dress 25 Y276C Delay functioning. herself.

Heterozygous Cluster2

Crawling 5 months. Walking 20 months, all other milestones 29 R177H/A178A Myoclonus delayed. Myoclonus

Cluster 1/3

Mental regression, Epilepsy (GTCS, motor Myoclonic, impairment, Normal retinal 8 D115Y, N273I Hypomotor) ataxia findings, Ataxia

Normal until Hypotonia, 27 R121L/W235S Seizure about 18 hyperflexia months and

109 never developed speech

Able to walk and talk, although not normally, until about age 4. The parents noted the symptoms in the female patient were less severe Hypotonia, 28 R121L/W235S Seizure than the male. hyperflexia

Cluster2/3

Ataxia, few volcalization but no clear words; Sat at 4-5 intermittent months; walked tremulousness at 10 months. throughout body; and Lost some fine more focal motor function tremulousness in due to tremor at perioral musculature; 2 years. Started trunk and left leg. to speak first Muscle tone, words at strength and reflexes around 12-18 normal; stands and months. walks independently Speech delay with bent knees; R181W, evident by 2 base not particularly 21 D229Y Epilepsy years. wide.

Crawling at 11 months, Bilateral cerebral walking 11 spastic tetraparesis months, with choreoathetosis. generalized Missing control of ataxia, speech- head movement. 22 P205L, V259E Epilepsy/ Ataxia some words Bulbar paralysis

110 Tremors (action?), dysmetria, progressively Sat at 8 unsteady gait, months, truncal ataxia, no crawled at 10 visual fixation, axial months, walked hypotonia, dystonia, at 14 months exaggerated but never stable reflexes, loss of walking; failed expressive and to gain receptive language, D229Y, language dysarthria, 4 L271H Tremors milestones choreoathetosis

Unsteadiness and shaking at 2 years, tongue fasciculations and tremor; hearing abnormal (type?); axial hypotonia, Sat at 8 increased extensor months, tone, areflexia, crawled at 16 dysphagia and oral- months, motor problems; D229Y, Developmental cruising at 18- dystonia; myoclonus; 5 L271H Delay 19 months. chorea

Cluster 1/2

Dystonic posturing of right food, 26 T64A/R211X Couldn't stand on right leg Neurologic Findings

111

Homozygous Myoclonus Cluster1 Mutation Regression Seizure Semiology Present

Continuous multifocal myoclonic seizures affecting limbs and face, atypical absences and some additional tonic and furthermore tonic-clonic seizures with Does not sit or corresponding 6 R94W walk epileptogenic activity. (+)

Mental Regression, Motor 7 R94W Impairment Myoclonic, Atonic (+)

Mental Regression, Motor 14 L108M Impairment Myoclonic (+)

Mental Regression, Motor 15 L108M Impairment Myoclonic, Atonic (+)

Heterozygous Cluster1

10 months stopped attempting to stand or walk around furniture. 16 months could not walk nor sit. Did not develop speech but comprehension Single episode of appeared myoclonic seizures, oral normal. Brief automatisms, cluster of 16 R84W, Δ3,4 attention span. cyanotic apneic spells (+)

112 Difficulty with walking, developing a wide-based, steppage fait with significant foot drop in addition to hypotonia and truncal ataxia. Surgery improved ataxia, tremors, gross/fine motor skills, now able to walk independently, navigate stairs, with alternating feet while holding onto a rail, and feed herself with Not 20 R70W, R84Q utensils. ? mentioned

30 G105E/G114E +

31 G105E/G114E ? ? ?

Nonsense Cluster1

Multifocal myoclonus of Walking not face, limbs, evolved into possible without myoclonic status 1 R99X assistance epilepticus (+)

Normal until 2 GTCS (precipitated by years, then fever); focal-onset severe cognitive seizures; myoclonic 2 R99X decline seizures (+)

Normal until 16 Head nodding, low months, then amplitude myoclonus in 3 R99X psychomotor all extremities, ataxia (+) delay, severe

113 feeding problems, drooling

Homozygous Cluster2

Seizure manifestations, confirmed by 18 months, point and video telemetry, motor and included frequent speech myoclonic movements regression involving mainly the face noted. 12 years and extremities, were nonverbal and often precipitated or without worsened by fevers, and spontaneous were refractory to multiple 17 R184C motor function. antiepileptic drugs. (+)

Seizure manifestations, confirmed by electroencephalography 18 months, point and video telemetry, motor and included frequent speech myoclonic movements regression involving mainly the face noted. 10 years and extremities, were nonverbal and often precipitated or without worsened by fevers, and spontaneous were refractory to multiple 18 R184C motor function. antiepileptic (+)

Mental Regression, 594delC, Motor 9 I199X74 Impairment Myoclonic (+)

Mental Regression, 594delC, Motor GTCS, Myoclonic, 10 I199X74 Impairment Hypomotor (+)

Regression was Episodes of downward 19 R153H presorted eye gaze, head bobbing, (+) around 20 and with left arm twitching

114 months of age for few seconds

Homozygous Cluster3

Mental Regression, Motor 12 W289X Impairment Myoclonic, GTCS (+)

Mental Regression, Motor 13 W289X Impairment Myoclonic, GTCS (+)

Mental Regression, Motor 11 N273I Impairment Myoclonic, GTCS (+)

Loss of independent ambulation, incontinence, with inability to feed independently Multifocal myoclonus, and and manage developments of activities of pyramidal signs independent (hyperreflexia, extensor 23 Y276C living. plantar responses). (+)

Generalized clonic seizure activity, head and shoulder drops, and staring spells associated Gait continues to with eyelid fluttering. 24 Y276C worsen Myoclonus. (+)

Capable of Generalized seizure, independent myoclonic jerks. Has both ambulation, febrile and afebrile events cognitive delay, and occasional head can feed/ dress drops, but no observed 25 Y276C herself staring spells. (+)

115

Heterozygous Cluster2

Tonic clonic, absence, 29 R177H/A178A Yes myoclonus (+)

Cluster 1/3

Mental Regression, Motor Myoclonic, GTCS, 8 D115Y, N273I Impairment Hypomotor (+)

Myoclonic, Tonic. June 2011. Frequent bipatietal- central sharp and wave activities, as well as rare left parietal sharp and 27 R121L/W235S Yes wave activities. (+)

28 R121L/W235S Yes Myoclonic, Tonic (+)

Cluster2/3

R181W, 21 D229Y ? ? (+)

Absences, Myoclonic atonic seizures, generalized tonic-clinic seizure provoked by fever, focal clonic seizures, tonic seizures, Cannot walk/ sit reflex tonic seizures, 22 P205L, V259E (blog) (+)

Changes in interaction or irritability, eye deviation, head nodding, focal motor signs (limb flexion or extension), hypermotor D229Y, Noted after signs, sweating, repetitive 4 L271H seizures began language (+)

116 Noted to be D229Y, unsteady at 2 5 L271H yrs of age ? (+)

Cluster 1/2

Mostly myoclonic seizures but gets infrequently generalized tonic clonic seizures. Needed recurrent admission in PICU with status 26 T64A/R211X Yes epilepticus. (+)

117

Homozygous Date Cluster1 Mutation MRI of MRI Interictal EEG

Diffuse slowing of background activity with continuous multifocal epileptic activity with Diffuse atrophy 5 years secondary 6 R94W without focal lesions. old generalization.

Spikes from posterior areas of both hemispheres. No high amplitude waves on 7 R94W Normal Onset photic stimulation.

Sequences of rhythmic high amplitude delta waves with superimposed spikes Discrete non-specific 2 yrs predominant in posterior focal subcortical after regions. No abnormal 14 L108M white matter lesions onset photic stimulation.

Sequences of rhythmic high amplitude delta waves with 2 superimposed spikes months predominant in posterior after regions. No abnormal 15 L108M Normal onset photic stimulation.

Heterozygous Cluster1

After plasma exchange shows bilateral spike and wave discharges most prominent in the posterior quadrant. The amplitude is higher on the right 4.5 side. Sleep EEG shows years continuous spike and 16 R84W, Δ3,4 Normal old wave discharges most prominent in the fronto-

118 temporal regions.

Left Parietal subcortical 20 20 R70W, R84Q hyperintensity months ?

30 G105E/G114E

31 G105E/G114E ? ? ?

Nonsense Cluster1

Destructured background, diffuse slow dysrhythmia, multifocal spikes and polyspikes. 5 yrs IPS: photic driving at 12- 1 R99X Normal old 18 Hz

Slowing multifocal or generalized epileptiform discharges. IPS: photoconvulsive response at 15 Hz (generalized spike-wave discharges with bilateral myoclonus followed by focal occipital secondarily N/A- Not mentioned/ generalized GTCS (off 2 R99X performed N/A CZP)

2-3 Hz slowing, bilateral 19 multifocal asynchronous months spikes. IPS: photic 3 R99X Normal (?) driving at 9-18 Hz.

Homozygous Cluster2

Global cortical atrophy, particularly 12 marked in the years 17 R184C cerebellum, as well old N/A as thinning of the

119 corpus callosum and some loss of subcortical white matter

Global cortical atrophy, particularly marked in the cerebellum, as well 13 as thinning of the years 18 R184C corpus callosum old N/A

Slow background activity. Epileptic discharges start Cerebral, cerebellar, 3 years from the left 594delC, and hippocampal after frontotemporal and right 9 I199X74 atrophy onset temporal regions.

2.5 years 594delC, Frontotemporal after Epileptiform activity in the 10 I199X74 cortical atrophy onset left posterior hemisphere

Slow background, no PDR, frequent bursts of generalized high voltage 2-3 Hz spike and slow wave associated with atonic seizures (head Left medial parieto- nods) lasting 1-3 occipital focal cortical seconds, excess fast malformation; activity. Frequent spike biparietal parnchymal and wave activity in right volume loss. Right centro-parietal region. posterior This activity becomes 19 R153H periventricular cyst. ? continuous during sleep.

Homozygous Cluster3

Sharp spike activity on Hyper-intensivity on parieto-occipital and posterior parieto-temporal lobes. periventiruclar region 5 years Background activity is on T2 weighted after composed of irregular 12 W289X images onset delta activity.

120 Hyper-intensivity on posterior Epileptiform activity starts periventiruclar region from the fronto-ceontral on T2 weighted region of the right 13 W289X images Onset hemisphere.

Volume loss in bilateral posterior sylvian fissure and frontotemporal cerebral region. Small nonspecific Multifocal spike wave focal lesion on the 2 years activity that is prominent left frontal posterior after in the posterior cranial 11 N273I white matter onset region.

Background rhythms show poor organization and diffuse nonspecific slowing. In the early stages, abundant paroxysmal bursts of generalized spike wave discharges were prominent, however, with improving seizure control and increasing serial recordings showed multiple independent spike foci and regional expression of epileptiform activity distributed over bilateral central, temporal and parietal electrode chains, often with shifting asymmetry. In the instance of a single staring spell associated with behavioral arrest captured during a recording, the EEG showed focal spikes, with a posterior distribution N/A- Not mentioned/ and ictal rhythms that 23 Y276C performed N/A spread progressively from the occipital regions

121 to become generalized with an abrupt onset and offset. All three siblings showed a photoparoxysmal response on photic stimulation, which was self-limited in nature.

Background rhythms show poor organization and diffuse nonspecific slowing. In the early stages, abundant paroxysmal bursts of generalized spike wave discharges were prominent, however, with improving seizure control and increasing serial recordings showed multiple independent spike foci and regional expression of epileptiform activity distributed over bilateral central, temporal and parietal electrode chains, often with shifting asymmetry. In the instance of a single staring spell associated with behavioral arrest captured during a recording, the EEG showed focal spikes, with a posterior distribution and ictal rhythms that spread progressively from the occipital regions to become generalized with an abrupt onset and offset. All three siblings showed a N/A- Not mentioned/ photoparoxysmal 24 Y276C performed N/A response on photic stimulation, which was

122 self-limited in nature.

Background rhythms show poor organization and diffuse nonspecific slowing. In the early stages, abundant paroxysmal bursts of generalized spike wave discharges were prominent, however, with improving seizure control and increasing serial recordings showed multiple independent spike foci and regional expression of epileptiform activity distributed over bilateral central, temporal and parietal electrode chains, often with shifting asymmetry. In the instance of a single staring spell associated with behavioral arrest captured during a recording, the EEG showed focal spikes, with a posterior distribution and ictal rhythms that spread progressively from the occipital regions to become generalized with an abrupt onset and offset. All three siblings showed a photoparoxysmal response on photic N/A- Not mentioned/ stimulation, which was 25 Y276C performed N/A self-limited in nature.

Heterozygous Cluster2

29 R177H/A178A N/A N/A primarily the most activity is in the occiput,

123 asychronous mycolonics that alternate sides of the body, then it spreads into generalized seizure- at different points other parts of the brain- has been active in the parietal and temporal- not been identified in the frontal but ALWAYS starting the occipital

Cluster 1/3

6 months after Continuous Generalized 8 D115Y, N273I Ventricular dilation onset and Multifocal spikes;

Diffuse cerebral and 27 R121L/W235S cerebellar atrophy Aug-11 See seizure semiology

28 R121L/W235S No MRI Not described

Cluster2/3

Absence of sleep R181W, features and posterior 21 D229Y Normal ? dominant rhythm.

22 P205L, V259E MRI: 21.1.2011

Cortical atrophy, Multifocal rhythmical D229Y, dilated ventricles, this spikes and sharp waves, 4 L271H corpus callosum ? diffuse slowing

Mild cortical atrophy, thin corpus callosum, no pathological findings but some hypomelinisation on Multifocal spikes, slow D229Y, both hemispheres posterior basic rhythm, 5 L271H pareitoccipital region. ? diffuse slowing

124 Cluster 1/2

MRI brain with Interictal EEG spectroscopy was demonstrating diffuse initially normal but at slow background and 5 and 7 years frequent independent showed signal spike and sharp waves changes in both bilaterally. EEG (March cerebellar 2013) suggestive of hemispheres with 5 & 7 asymmetric tonic 26 T64A/R211X mild global atrophy. years seizures in sleep

125

Any Homozygous Treatments Seizure Cluster1 Mutation Succeeded (Failed) Control? Comments

Levetiracetam (Phenobarbital, valproic acid, zonisamide, topiramate, dexamethasone, 6 R94W clonazepam) Yes

(VPA, CLZ, TPM, LEV) 7 R94W No seizure control No, intractable

(VPA, CLB Seizure Controled for 6m) (ETX, LEV, LMT, PDN, RFM, No, Seizure control for 14 L108M STP) intractable 6 months

(VPA, CLB, ETX, LEV, LMT, PDN, RFM, STP, 15 L108M MSM) No seizure control No, intractable

Heterozygous Cluster1

Steroids (Valproic acid, ACTH. ACTH levetiracetam, induced severe topiramate, sulthiame, side-effects, carbamazepine, switched to 16 R84W, Δ3,4 clonazepam) Yes Dexamethasone.

Surgery, VPA, 20 R70W, R84Q CZP/felbamate Yes, after surgery

30 G105E/G114E

31 G105E/G114E ? ? ?

Nonsense Cluster1

1 R99X (LTG, CZP) both gave transient but dramatic No (Only Almost relentless

126 imporvement (VPA, transient) myoclonus TPM, FBM, ESM, LEV; initial trial of hydrocortisone 5 mg/kg/day + VPA + CZP resulted in remarkable but transient improvement in walking and myoclonus; LEV also associated with transient improvement)

CZP (for myoclonus), Control for 5 (+) 2 R99X LTG Yes years

Transient clinical imporovement from (TPM, LTG, FBM; hydrocortisone, transient clinical then tapered and improvement with patient died from 3 R99X hydrocortisone No infection

Homozygous Cluster2

Refractory to multiple 17 R184C antiepileptic drugs No

Refractory to multiple 18 R184C antiepileptic drugs No

594delC, 9 I199X74 VPA, CLZ Yes

594delC, VPA, LEV, Seizures 10 I199X74 controlled at 75% Yes Controlled at 75%

19 R153H CZP, CBZ ,VPA, Yes

Homozygous Cluster3

12 W289X VPA, CLZ, LEV Yes

13 W289X VPA Yes

127 PH, VPA, CLZ, CLB, LEV. Seizures partially 11 N273I controlled Yes Partially Controlled

23 Y276C Lamotrigine, CZP, LEV Yes

24 Y276C VPA, CZP Yes

25 Y276C VPA Yes

Heterozygous Cluster2

VPA, CLZ, LEV 29 R177H/A178A **Modified Atkins Diet Yes

Cluster 1/3

VPA, LMT, VGB, DZM, PH, LEV, CLZ, steroid, ACTH, PYRP. Seizures 8 D115Y, N273I controlled Yes

27 R121L/W235S CZP, LEV Yes

CZP, LEV, Lamotrigine, 28 R121L/W235S Topamax Yes

Cluster2/3

R181W, LEV, (CZP, 21 D229Y VPA) Yes

ACTH, steroid pulse therapy, Phenobarbital, Sultiame, Topiramat, Levetiracetam, Oxcarbazepine, Zonisamide, Ethosuximide, Diazepam, Lamotrigine, Vigabatrine, 22 P205L, V259E Lacosamide, Retigabine No

4 (Carbidopa-levodopa: No D229Y, transiently improved

128 L271H dystonia, dysmetria, visual tracking, head control but worsened chorea; transient improvements with prednisolone, ACTH and IVIG; VPA, CZP, LEV, PRM, PHT, ZNS, PB, LZP, PGB, LTG, CLZ, clonidine, leucovorin, glycopyrrolate, pyridoxine, CoQ, ketogenic diet)

D229Y, (LEV, LZP, Diastat, 5 L271H clonidine) No

Cluster 1/2

26 T64A/R211X VPA, CLZ, LEV Yes

129

Homozygous Tone Cluster1 Mutation Crawling Walking (Gait) Ataxia (Hypotonia)

6 R94W no no Unknown +

7 R94W Unknown Unknown + Unknown

14 L108M Unknown Unknown + Unknown

15 L108M Unknown Unknown + Unknown

Heterozygous Cluster1

(+) Decreased 16 R84W, Δ3,4 no no + diffusely

20 R70W, R84Q normal normal + (+)

30 G105E/G114E

31 G105E/G114E ? ? ? ?

Nonsense Cluster1

1 R99X unknown 14 months (+) (+)

2 R99X unknown Unknown Unknown Unknown

3 R99X unknown Unknown (+) Unknown

Homozygous Cluster2

17 R184C normal normal Unknown Unknown

18 R184C normal normal Unknown Unknown

594delC, 9 I199X74 Unknown Unknown + Unknown

594delC, 10 I199X74 Unknown Unknown + Unknown

130 19 R153H normal normal Unknown Unknown

Homozygous Cluster3

12 W289X Unknown Unknown + Unknown

13 W289X Unknown Unknown + Unknown

11 N273I Unknown Never Walked - Unknown

23 Y276C Unknown 19 months + +

24 Y276C normal normal + Unknown

Delayed walking, 25 Y276C Unknown prior to 15 months Unknown Unknown

Heterozygous Cluster2

Not officially 29 R177H/A178A 5 months 20 months diagnosed (+)

Cluster 1/3

8 D115Y, N273I Unknown Unknown + Unknown

Normal until 27 R121L/W235S 18 months Normal until 18 months (+)

Could walk but abnormally until 28 R121L/W235S Unknown about 4 years (+)

Cluster2/3

21 R181W, D229Y yes yes normal no

22 P205L, V259E 11 months 18 months (+) (+)

Walked but never 4 D229Y, L271H stable (+) (+)

N/A, not 5 D229Y, L271H mentioned (+)

131

Cluster 1/2

26 T64A/R211X 10 months 16 months normal

132

Movement Homozygous Disorder Cluster1 Mutation Symptom Dystonia Dyskinesia Tremors

6 R94W (+) Unknown (+) Unknown

7 R94W Unknown Unknown Unknown Unknown

14 L108M Unknown Unknown Unknown Unknown

15 L108M Unknown Unknown Unknown Unknown

Heterozygous Cluster1

(+) Mild tremulous movements at 5 16 R84W, Δ3,4 (+) Unknown (+) months

20 R70W, R84Q (+) Unknown ? (+)

30 G105E/G114E (+)

31 G105E/G114E ? ? ? ?

Nonsense Cluster1

1 R99X Unknown Unknown Unknown Unknown

2 R99X Unknown Unknown Unknown Unknown

3 R99X Unknown Unknown Unknown Unknown

Homozygous Cluster2

17 R184C Unknown Unknown Unknown Unknown

18 R184C Unknown Unknown Unknown Unknown

594delC, 9 I199X74 Unknown Unknown Unknown Unknown

133 594delC, 10 I199X74 Unknown Unknown Unknown Unknown

19 R153H (+) Unknown Unknown Unknown

Homozygous Cluster3

12 W289X Unknown Unknown Unknown Unknown

13 W289X Unknown Unknown Unknown Unknown

11 N273I Unknown Unknown Unknown Unknown

(+) Motor 23 Y276C Incoordination Unknown Unknown Unknown

24 Y276C Unknown Unknown Unknown Unknown

25 Y276C Unknown Unknown Unknown Unknown

Heterozygous Cluster2

29 R177H/A178A (+) (+) (+)

Cluster 1/3

8 D115Y, N273I Unknown Unknown Unknown

27 R121L/W235S (+) Tic

28 R121L/W235S (+) Repetitive hand movement

Cluster2/3

R181W, 21 D229Y (+) (+) no

22 P205L, V259E (+) (+) (+) (+)

4 D229Y, L271H (+) Unknown (+)

5 D229Y, L271H (+) (+) Unknown (+) Dystonic movement

134

Cluster 1/2

unknown/not 26 T64A/R211X (+) (+) (+) mentioned

135

Homozygous Fine Motor Cluster1 Mutation Choreoathetosis Speech Swallowing Defect

(-) No expressive language/ does not follow verbal (+) Does not 6 R94W Unknown commands Unknown reach for toys

7 R94W Unknown Unknown Unknown Unknown

14 L108M Unknown Unknown Unknown Unknown

15 L108M Unknown Unknown Unknown Unknown

Heterozygous Cluster1

(+) Hands (-) Did not worsen during 16 R84W, Δ3,4 Unknown develop speech Unknown motor activity

20 R70W, R84Q ? ? ? (+)

30 G105E/G114E

31 G105E/G114E ? ? ? ?

Nonsense Cluster1

(-) Few 1 R99X Unknown intelligible words Unknown (+) Problem

2 R99X Unknown Not mentioned Unknown (+) Problem

3 R99X Unknown Not mentioned Unknown Unknown

Homozygous Cluster2

17 R184C Unknown (-) Nonverbal Unknown (+) W/o spontaneous

136 motoric funciton

(+) W/o spontaneous 18 R184C Unknown (-) Nonverbal Unknown motoric funciton

594delC, 9 I199X74 Unknown Unknown Unknown Unknown

594delC, 10 I199X74 Unknown Unknown Unknown Unknown

19 R153H (+) choreiform movements

Homozygous Cluster3

12 W289X Unknown Unknown Unknown Unknown

13 W289X Unknown Unknown Unknown Unknown

11 N273I Unknown Unknown Unknown Unknown

(-) Loss of expressive (+) Motor 23 Y276C Unknown language skills Unknown incoordination

(-) Lost expressive 24 Y276C Unknown speech Unknown Unknown

(-) Limited expressive 25 Y276C Unknown speech Unknown Unknown

Heterozygous Cluster2

Yes but due to vagus nerve Depends on 29 R177H/A178A implant tremor

Cluster 1/3

8 D115Y, N273I Unknown Unknown Unknown Unknown

137 Never developed 27 R121L/W235S speech Unknown Unknown

28 R121L/W235S Speech delay Unknown Unknown

Cluster2/3

yes, due to 21 R181W, D229Y some words tremor

(-) Few 22 P205L, V259E (+) intelligible words (+) (+) None

(-) Failed Language milestones, dysarthria, D229Y, repetitive (+) Focal motor 4 L271H (+) language (-) signs

D229Y, ? Tongue (+) Oral-motor 5 L271H (-) fasciculations (+) Dysphagia problems

Cluster 1/2

(+) Impaired swallow. Fed exclusively by Gastrostomy tube. No 26 T64A/R211X (+) drooling. (+)

138

Homozygous Eye Cluster1 Mutation Social Retina Abnormalities

Retinal abnormalities with partial optical atrophy (interpreted as neurodegnerative signs) without any further progress and occulmotoric movement Retina 6 R94W Unknown disorder on the right eye abnormalities

7 R94W Unknown Normal retina Unknown

14 L108M Unknown Normal retina Unknown

15 L108M Unknown Normal retina Unknown

Heterozygous Cluster1

? (+) (+) Abnormal Attention eye 16 R84W, Δ3,4 span brief N/A- Leshinsky-Silver movements

20 R70W, R84Q ? ?- Burrow ?

30 G105E/G114E + ? ?

31 G105E/G114E ? ? ?

Nonsense Cluster1

1 R99X Unknown Normal Unknown

2 R99X Unknown Not performed Unknown

3 R99X Unknown Not performed Unknown

Homozygous Cluster2

17 R184C n/a Normal (+) No response to

139 visual threat, vision loss

(+) No response to visual threat, bilateral potic atrophy, vision 18 R184C n/a optic atrophy loss

594delC, 9 I199X74 Unknown Normal retina Unknown

594delC, 10 I199X74 Unknown Normal retina Unknown

19 R153H N/A- Baylor

Homozygous Cluster3

12 W289X Unknown Normal retina Unknown

13 W289X Unknown Normal retina Unknown

11 N273I Unknown Normal retina Unknown

Normal (no visual complaints and no retinal abnormalities on routine fund scope. They did not (+) Uprolling of undergo any formal eyes. No signs ophthalmological of ocular assessments or tests of movements or 23 Y276C Unknown retinal function like an ERG) nystagmus

Normal (no visual complaints and no retinal abnormalities on routine fund scope. They did not undergo any formal ophthalmological assessments or tests of (+) Eyelid 24 Y276C Unknown retinal function like an ERG) flutteirng

Normal (no visual 25 Y276C Unknown complaints and no retinal Unknown abnormalities on routine

140 fund scope. They did not undergo any formal ophthalmological assessments or tests of retinal function like an ERG)

Heterozygous Cluster2

(+) Myoclonus in eyelids and developed Yes, very lateral socially deviation at 19 29 R177H/A178A immature Normal Vision yrs (2013)

Cluster 1/3

8 D115Y, N273I Unknown Normal retina Unknown

Not 27 R121L/W235S applicable Not specified Not specified

Not 28 R121L/W235S applicable Not specified Not specified

Cluster2/3

21 R181W, D229Y Boston

22 P205L, V259E (+) Normal retina

(+) Eye D229Y, Deviation, no 4 L271H (+) irritability visual fixation

D229Y, 5 L271H (-) (-)

Cluster 1/2

(+) severely 26 T64A/R211X intellectually Normal No disabled

141 with some awareness of surrounding

142

Other Homozygous Photic Comments/ Cluster1 Mutation Drooling Stimulation Observations

(+) Photic stimulation provoked 6 R94W (-) stimulation

7 R94W Unknown Unknown

14 L108M Unknown Unknown

15 L108M Unknown Unknown

Heterozygous Cluster1

(+) Severe drooling, from 16 R84W, Δ3,4 dexamethasone? Unknown

20 R70W, R84Q ? ?

30 G105E/G114E ? ?

31 G105E/G114E ? ?

Nonsense Cluster1

1 R99X (+) Excessive drooling (+) Photic stimulation

2 R99X Unknown (+) Photic stimulation

3 R99X (+) (+) Photic stimulation

Homozygous Cluster2

17 R184C Unknown Unknown

18 R184C Unknown Unknown

9 Unknown Unknown 594delC,

143 I199X74

594delC, 10 I199X74 Unknown Unknown

19 R153H

Homozygous Cluster3

12 W289X Unknown Unknown

13 W289X Unknown Unknown

11 N273I Unknown Unknown

23 Y276C Unknown (+) Photic stimulation

(+) Signifigant 24 Y276C sialorrhea (+) Photic stimulation

25 Y276C Unknown (+) Photic stimulation

Heterozygous Cluster2

29 R177H/A178A Unknown Unknown

Cluster 1/3

8 D115Y, N273I Unknown Unknown

27 R121L/W235S Yes Unknown

28 R121L/W235S Yes Unknown

Cluster2/3

21 R181W, D229Y

22 P205L, V259E (+)

4 D229Y, L271H (-) Unknown

5 D229Y, L271H (-) Unknown

144 Pyelectasia

Cluster 1/2

26 T64A/R211X No drooling unknown

145

Chapter 3

The Role of KCTD7 in

Autophagy and

Mitochondrial Homeostasis

146 Introduction

Regulation of Mitochondrial Networks

Mitochondria are organelles most famously known as the powerhouses of

the cell by their ability to supply large amounts of adenosine triphosphate (ATP)

generated by oxidative phosphorylation (1). However, mitochondria are much

more that ATP power plants. Even in the absence of oxidative phosphorylation,

they are required for iron-sulfur cluster biosynthesis, governing programmed

apoptotic cell death, producing metabolites for protein and nucleic acid synthesis,

and very recently for the biosynthesis of lipids mediators (1, 2).

Mitochondria arose from ancient prokaryotes around 2 billion years ago

resulting in a fruitful endosymbiotic relationship for both host cell and prokaryote

(3). This led to a phenomenon in which the ancient prokaryote was endocytosed,

creating an inner and outer membrane with an inner membrane space in addition

to the matrix located in the inner most section of the mitochondrion (3). Within the

inner membrane resides a protein, cytochrome c, that when released into the

cytoplasm by pro-apoptotic factors, such as Bcl-2-associated X protein (Bax) and

BCL2-antagonist/killer 1 (Bak), and initiates programmed cell death by binding apoptotic protease activating factor 1 (Apaf1) (4). This activates a complex, known as the apoptosome that activates caspases, which begins a once thought irreversible demise of the cell; however, recent studies have challenged this belief by showing caspases may be activated but cells recover by an unknown

147 mechanism in a process designated anastasis, Greek for rising from the dead

(5).

The mitochondrial inner membrane creates a structure essential for the production of ATP, cristae. Cristae, Latin for crest, are folds that are found in the inner membrane (6). Here is located the F1FO ATP synthase, which generates

ATP from a proton gradient between the inner membrane space (higher proton concentration) and matrix (lower proton concentration) (6). The FO portion contains a ring of c-subunits that form part of the proton path and accumulation of this c subunit is a hallmark of the neuronal ceroid lipofuscinoses (7). The cristae also possess the electron transport chain (ETC) to generate the proton gradient

(6). As a result, the cristae play a pivotal role in mitochondria, and abnormalities in cristae structure have been described in several diseases, such as Parkinson’s

Disease and Alzheimer’s Disease (8,9).

Central to mitochondrial health is the ability to fuse and divide, as organelle fusion allows a mitochondrion to share mitochondrial DNA (mtDNA), and fission is thought to facilitate removal and degradation of defective organelle sections (10). Important to this process are a group of conserved GTPases first demonstrated in yeast to regulate mitochondrial organelle fission and fusion, including the large GTPases, dynamin-related protein 1 (Dnm1) and mitofusins1/2 (Mfn1/2) (11). Ablation of these genes in mice results in embryonic lethality midgestation (12,13). Mouse embryonic fibroblasts generated from these embryos demonstrate remarkable changes in the mitochondrial network. The

148 mitochondrial network describes the connectivity of the mitochondria inside of a

cell (14). Dnm1-/- MEFs possess very long mitochondria due to defective mitochondrial fission, whereas Mfn1- and Mfn2-deficient MEFs both have very

fragmented mitochondria as the mitochondria are unable to properly refuse

(12,13). Fusogenic and fragmented mitochondrial morphologies represent

opposite ends of a spectrum that end in embryonic lethality in vivo, although the

detailed explanations are incompletely understood.

Eliminating damaged and defective mitochondria is essential to maintain a

healthy population of mitochondria. Having a means to “pinch off” these damaged

mitochondrial fragments for degradation is a mechanism to maintain this healthy

pool of mitochondria (15). As discussed in Chapter 1, mutations in Parkin and

PINK1 cause Parkinson’s disease due to defects in initiating mitophagy

specifically in response to physiological impairment of mitochondrial membrane

potential, and to experimental triggers that collapses mitochondrial membrane

potential (e.g. the ionophore CCCP, carbonyl cyanide m-chlorophenylhydrazone).

A role for mitophagy as a mechanism of mitochondrial homeostasis is also

conserved in yeast. The autophagy adaptor protein Atg11 directly interacts with

the C-terminus of mitochondrial division protein Dnm1, yeast homolog to

mammalian Drp1 (16). In a coordinated effort, these proteins interact to deliver

mitochondria to the yeast vacuole for degradation, and disruption of these

proteins blocks this process (16). This study was the first to biochemically

connect the mitochondrial division machinery with autophagic degradation of

149 mitochondria and much more work is needed to elucidate how this process occurs in mammals.

Given its importance in human disease, mechanisms of turnover of mitochondria are perhaps the biggest questions in the field of mitochondrial health and maintenance. However, we still know very little about mitophagy, and even less is know in perhaps the most relevant cells type, neurons. Recent studies have shown Parkin-mediated mitophagy does not occur in a robust manner in neurons, suggesting that alternative modes of mitochondrial clearance may occur (17). For example, neurons were shown to expel whole mitochondrial organelles for degradation by adjacent microglia (18). Overall, a greater understanding of mitochondrial degradation in disease-relevant cell types will further our comprehension of how defects in this important process cause disease.

Regulation of mTOR Through Amino Acids at the Lysosome

The mammalian target of rapamycin (mTOR) is a master kinase that integrates upstream signaling, like insulin growth factor and amino acids, with downstream processes such as autophagy, protein translation and cell growth

(19). mTOR was identified through the use of Rapamycin, which derives its name from its discovery on the island of Rapa Nui (Easter Island) (19). Rapamycin

150 negatively regulates the activity of mTOR and is able to arrest cellular growth

(19).

mTOR regulation has been an intense area of research and significant

progress has been made towards biochemical dissection of nutrient-sensing pathways that regulate mTOR1 complex (mTORC1) (20) For example, amino acid availability leads to activation of mTOR by GTPases, and a lysosomal protein complex known as the “ragulator” (21). The regulator complex interacts with the Rag GTPases (rag-ulator) that, depending of GTP-bound state, activate

mTOR (20,21). When RagA/B are bound to GDP and RagC/D are bound to GTP,

mTOR is active; however, when RagA/B bind GTP and RagC/D bind GDP mTOR

is recruited to the lysosome (21,22). A second parallel positive signal from the

Ras homolog enriched in brain (Rheb) is then able to phosphorylate and activate

mTOR (21). These studies provided the first compelling evidence that nutrient

status regulates mTOR activity at the lysosome.

The Ragulator-Rag complex is a pentameric complex, including Rag

GTPases, that acts as a guanine nucleotide exchange factor (GEF) for the Rag

GTPases (23). The Ragulator was further shown to bind the lysosomal v-ATPase

in response to amino acids (23). When amino acids are present, the v-ATPase

activates the GEF activity of the Ragulator, which in turn changes the GTP state

of RagA/B, inactivating them (23). This was the first demonstration that amino

acids regulate mTOR activity specifically through the lysosome and could

151 indicate that much of a cell’s nutrient status could be signaled through the lysosome.

Supporting this idea, arginine levels were found to regulate mTOR activity through a protein of unknown function, Solute carrier family 38 member 9

(SLC38A9) (24). SLC38A9 has amino acid similarity to other amino acid transporters and was able to regulate mTOR activity, specifically to arginine, through the Ragulator (24). Overexpression of SLC38A9 could compensate for the lack of amino acids in the cell and activate mTOR but not override dominant negative versions of the Rag GTPases, indicating that SLC38A9 acts upstream of the Ragulator (24). Overexpression of SLC38A9 also inhibited autophagy induction during amino acid starvation (24). SLC38A9 may be an important amino acid sensor, consistent with the long-standing assumption that amino acid transporters would fill this role, and its location on lysosomal membranes supports the idea that the lysosome can integrate upstream nutrition status of the cell to downstream signaling events via mTOR. As there are many other nutrients that control mTOR activity, it is plausible that several other unidentified proteins on the lysosome signal nutrient status to mTOR. These studies further highlight the autophagy-lysosomal pathway in regulating key signaling events throughout the cell.

From Yeast to the Epilepsy Gene KCTD7

152 Advances in sequencing have allowed for increased identification of patients with mutations in epilepsy-causing genes, including KCTD7 (25). Until the advancement of genome/exome sequencing, many patients with mutations in

KCTD7 have been classified as idiopathic and have had to endure travel from clinic to clinic in search of a diagnosis (personal communication from several physicians) (26). Currently, there are 19 published cases of patients with mutations in KCTD7 (Chapter 2). Over several years, I have identified an additional 18 unreported patients, bringing the current total to 37 patients with mutations in KCTD7. I inquired of each research group, two of which prepared and provided patient fibroblasts, allowing us to test hypotheses generated from our previous studies in yeast.

Our lab has shown that yeast Whi2 is critical for autophagy induction in response to low leucine, and further identified further identified previously unrecognized human homologs of Whi2, the KCTD family of unknown function

(Teng et al. unpublished and [27]). The accumulation of defective mitochondrial organelles results in a number of other cellular defects, including the accumulation of lipid droplets potentially because lipids are not being utilized by mitochondria. Consistent with this, mutations in genes associated with the autophagy pathway have been shown to lead to accumulation of abnormal mitochondria, lipid droplets, and protein aggregates in a variety of cells including fibroblasts, LCLs, neurons, and myocytes (28,29,30). Deletion of autophagy genes has also been shown to cause neurodegeneration in mouse models and

153 more recently mutations in autophagy proteins have been shown to cause neurological and multi-system disorders in humans (31,32,33,34). Autophagy has been implicated in a number of human diseases (35,36,37). More recently, mutations in specific autophagy genes have been found to cause Static encephalopathy of childhood with neurodegeneration in adulthood (SENDA) and

Vici Syndrome (31,32).

Based on these studies, I examined the autophagy pathway in these patient fibroblasts with age and passage-matched controls and found evidence suggesting a defect in autophagy. Using a murine neuroblastoma cell line, I found that endogenous KCTD7 co-localizes with GFP-LC3 after mitochondrial damage. We additionally discovered an abnormal mitochondrial network by fluorescence microscopy, and collapsed cristae in patient fibroblasts by electron microscopy, indicating defective mitochondrial homeostasis. In contrast to ectopically expressed WT KCTD7, KCTD7-ΔC does not co-localize with damaged mitochondria decorated with mCherry-Parkin. Rather, KCTD7-ΔC localizes to distinct crescent structures that associate in close proximity to damaged mCherry-Parkin-labeled mitochondria, potentially indicating that the N- terminal BTB domain of KCTD7 targets KCTD7 to the pre-autophagosome. We suggest that KCTD7 functions in the autophagy pathway and is required for mitochondria homeostasis.

154 Results

Role for KCTD7 in autophagy

KCTD7 is a human homolog of yeast Whi2 (27), and Whi2 is a stress

response gene that is required to activate autophagy (Teng et al. unpublished).

As the function of KCTD7 is unknown, we asked if KCTD7 could be involved in

the autophagy pathway similar to its homolog, Whi2. This could offer novel

insight into the function of the poorly characterized human KCTD7 and begin to

explain the causal role for KCTD7 mutations in patients.

To examine the role of KCTD7 in the autophagy pathway, we obtained low

passage, primary fibroblasts from two patients reported to have compound

heterozygous (R84W; DExon3/4, EPM3#1) and homozygous (R94W, EPM3#2)

KCTD7 mutations. These three distinct mutations disrupt the BTB domain and

are assumed to completely inactivate both copies of KCTD7 in each patient

(Figure 1A). To confirm the presence of these mutations, genomic DNA was prepared from the fibroblasts and sequenced. In contrast to control fibroblasts,

EPM3#1 was heterozygous for Arg84 as expected, and EPM3#2 was homozygous for the mutant tryptophan at codon 94, replacing the wild type

arginine (Figure 1B).

Autophagy is a well-conserved process that targets cytoplasmic contents

for degradation in the lysosome and the competency of this pathway can be

evaluated in an autophagy flux assay by determining the conversion of LC3 to its

155 active form LC3-II. If the autophagy pathway is intact, a large increase in the active form of LC3-II will occur when cells are treated with inhibitors of lysosome function, such as bafilomycin A1 and chloroquine. As the cell normally has a means to deliver LC3-II to the lysosome for degradation, an addition of a lysosome inhibitor blocks this degradation, causing an increase in LC3-II.

However, an inability to deliver LC3-II to the lysosome will result in a smaller increase in LC3-II, as the cell cannot deliver LC3-II to the lysosome for degradation.

To determine if the autophagy pathway is altered in patients with mutations in KCTD7, patient fibroblasts and control age- and passage-matched human fibroblasts were treated with 15 μM chloroquine for 1 hr. This assay is known as an autophagy flux assay, as it estimates the amount of LC3-II the cell degrades in the lysosome (38). We found that WT fibroblasts had significantly higher chloroquine-induced levels of LC3-II when compared to fibroblasts with

KCTD7 mutations after treatment with chloroquine, indicating an inability to turnover LC3-II in patients with KCTD7 mutations. We found that CQ treatment induced the accumulation of LC3-II approximately 3-fold compared to untreated wild type cells (Figure 2A). In contrast, CQ only induced LC3-II accumulation by

1.5-fold in KCTD7 mutant cells consistent with a slower autophagy flux in EPM3 cells (Figure 2A and B). This is the first evidence to suggest a molecular pathway for KCTD7 function. Similarly, patient fibroblasts with a mutation in

EPG5, which is required for fusion of autophagosomes with lysosomes by an

156 unknown mechanism, also resulted in lowered LC3-II accumulation when

compared to WT fibroblasts after lysosome inhibition, indicating a defect in the

autophagy pathway (19,31).

As KCTD7 appears to be involved in the autophagy pathway, we

asked if endogenous Kctd7 co-localizes with markers of autophagy. Therefore, it

was necessary to identify a cell line that expresses robust levels of KCTD7, and

to screen and validate newly available commercial antibodies against KCTD7 for

suitable reagents. While antibodies from Sigma (1408394) and Abcam (103339)

gave negative results, the Abcam antibody (83237) detected a band of the correct size in mouse neuroblastoma cell line, N2a, but not other cell lines tested

(HeLa, Cos7, Sy5y). Furthermore, partial knockdown of murine Kctd7 in N2a cells using specific siRNAs, but not scrambled control, further validated the specificity of this antibody (Figure 3A).

Immunostaining of the murine neuroblastoma cell line N2a for endogenous

KCTD7 revealed a diffuse and cytoplasmic localization in all cells examined. To

determine if endogenous KCTD7 associated with autophagosomes, the N2a cells

were transfect with GFP-LC3, the classic autophagosome marker. Both localized

in a diffuse manner (Figure 3B). A robust method to induce autophagy is to

collapse the mitochondria membrane potential, using an ionophore, carbonyl

cyanide m-chlorophenylhydrazone (CCCP). Using CCCP, I found that

endogenous KCTD7 co-localized with large perinuclear GPF-LC3 positive

puncta, supporting the idea that KCTD7 is involved in autophagy (Figure 3C). To

157 demonstrate that KCTD7 is not cargo in the autophagosome, I performed the same experiment but with a maker of growing autophagosomes, GFP-Atg2.

Endogenous KCTD7 also colocalized with GFP-Atg2, demonstrating that KCTD7 associates with autophagosomes after mitochondrial damage and supports the idea that KCTD7 is not merely cargo destined for degradation (Figure 3D).

Colocalization with GFP-Atg2 could further suggest that KCTD7 is involved in the processes of growing and expanding the autophagosome. Taken together, these experiments suggest that KCTD7 affects the autophagy pathway, either directly or indirectly, that defects in KCTD7 lead to an inability to degrade LC3-II and localizes to the autophagosome.

As KCTD7 is a neurodegenerative/ epilepsy-causing gene, we hypothesized that KCTD7 would be highly expressed in the mouse brain. We found that KCTD7 is highly expressed in the mouse brain (whole brain) (Figure

4A), consistent with two previous reports showing KCTD7 expression in mouse brain and neurons (39,40). Looking at whole brains of mice at 10 days, 3 months, and 4 months, I found a decrease in expression of KCTD7, indicating that KCTD7 may be developmentally down-regulated with age (Figure 4B). These data suggest that KCTD7 may play an important role during development.

Abnormalities of EPM3 Patient Mitochondria

158 The autophagy pathway is required to recycle defective cellular organelles. I therefore hypothesized that the patient fibroblasts could have mitochondrial alterations. To investigate this hypothesis, I stained human fibroblasts with the membrane potential-dependent dye MitoTracker, and noticed a clear difference between KCTD7 mutant and wild type fibroblasts (Figure 5A).

To provide a more quantitative analysis, I systematically collected microscopy images of EPM3 and control fibroblasts in three independent experiments immunostained for the endogenous outer mitochondrial membrane protein,

Tom20. These images (25 randomly selected cells per condition per experiment) were coded and given to Dr. Lucian Soane for computational analysis. Based on this analysis, we observed what appeared to be a less connected and more fragmented mitochondrial network in the patient fibroblasts (Figure 5A).

Patient fibroblasts had a smaller degree of mitochondrial branching and possessed a smaller percentage of long mitochondria (>15 μM) when compared to WT (Figure 5B), illustrated by a color code for the connectivity of each mitochondrion according to its length (Figure 5C). These results suggest that mutations in KCTD7 lead to a more fragmented mitochondrial network, as has been reported in other neurodegenerative diseases, and is the first implication of mitochondrial perturbations in these patients.

Since the mitochondrial networks are more fragmented in EPM3 patient fibroblasts, we performed electron microscopy to examine if the mitochondrial substructure is altered in the patient cells. Patient and control fibroblasts were

159 plated in 6-well plates and were fixed, prepared, and imaged by Dr. Isabelle

Coppens (Johns Hopkins). Every patient fibroblast examined possessed

mitochondria with altered structures amongst their cristae, consisting of empty

spaces in mitochondria that possess multi-membrane structures, possibly

collapsed cristae (Figure 6A and B). In contrast, these structures were not

observed in the control fibroblasts (Figure 6C). These data suggest that, in addition to a more fragmented network, patients with mutations in KCTD7 accumulate mitochondria with abnormal substructures, which could indicate decreased mitochondrial function. Supporting this idea, an accumulation of lipid droplets in the patient fibroblasts was observed, often tightly associated with mitochondria (Figure 7). This observation was additionally reported in the muscle biopsy of another EPM3 patient (Chapter 2). We then hypothesized that this could be caused by a defect in β-oxidation but did not observe any abnormalities when staining for peroxisomes (Pex14) (data not shown) (41). Accumulations of lipid droplets have been reported in cells with dysfunctional mitochondrial (42).

These findings support a role for mitochondrial defects in disease pathogenesis in patients with mutations in KCTD7.

Ectopically expressed KCTD7 Associates with Damaged Mitochondria

Autophagy is a process needed to degrade cytoplasmic contents, including damaged organelles, As we identified mitochondrial abnormalities (a

160 more fragmented mitochondrial network and in the patient fibroblasts, we wanted to determine if exogenous KCTD7 would preferentially associate with damaged mitochondria. To demonstrate association with damaged mitochondria, we asked if KCTD7 would co-localize with Parkin, which is recruited to damaged mitochondria after treatment with an ionophore that depolarizes mitochondria.

Overexpression of GFP-KCTD7 with mCherry-Parkin (or separately) results in diffuse cytoplasmic localization, with some punctate structures (not shown) occasionally observed for GFP-KCTD7 and coincide with Parkin puncta (Figure

8A). Treatment with CCCP to induce mitochondrial depolarization, a known trigger of Parkin translocation to mitochondria and subsequent mitophagy, resulted in co-localization of GFP-KCTD7 and mCherry-Parkin, indicating that

KCTD7 is able to localize to damage mitochondria (Figure 8B).

Fluorescence microscopy does not readily distinguish double- versus single-membrane structures, or distinguish two closely juxtaposed structures.

Therefore, to gain more detailed information about the subcellular and sub- organelle localization of KCTD7, immuno-gold labeling and electron microscopy was performed using antibodies against GFP to detect GFP-KCTD7. COS7 cells were transfected overnight with GFP-KCTD7 and treated 4h with CCCP before fixation and shipment to the Yale microscopy center. The fixation and imaging were performed by Dr. Isabelle Coppens. Interestingly, gold label was associated with membrane-bound vesicles with heterogeneous content (91% of gold labeling), cytosolic localization (5% of gold labeling), and other

161 organelles/nucleus (4% of gold labeling). Consistent with our fluorescent images, gold labeling was found on vesicles with heterogeneous content, consistent with an autophagosome (Figure 9). These data suggest that KCTD7 localizes to membranes similar to autophagosomes after mitochondrial damage, consistent with a role for KCTD7 in autophagy.

KCTD7-ΔC Localizes to Distinct Crescent-like Structures

To determine the role of the N-terminal BTB domain region of KCTD7 in subcellular and sub-organelle localizations, similar experiments were performed on COS7 cells transfected with KCTD7-ΔC (150x, amino acids 1-149) with an N- terminal GFP-tag. As opposed to the full-length protein, GFP-KCTD7-ΔC localizes to distinct crescent-like structures in the cytoplasm (Figure 10A). When co-transfected with mCherry-Parkin and treated with CCCP for 4 hours, these crescent structures localize adjacent to mCherry-Parkin. (Figure 10B)

Interestingly, these structures are reminiscent of illustrations of the isolation membrane wrapping around its cargo (Youle and Narendra, Figure 2) (30).

However, these structures have never before been demonstrated to exist by fluorescent microscopy, presumably because this is a transient event and could indicate the C-terminus of KCTD7 is involved in forming mature autophagosomes. We also found that the tag influences the length of these structures, with HA- and untagged versions resulting in much longer and larger

162 structures (Figure 10C). As a GFP tag is much larger than an HA tag, we hypothesized that the GFP tag could interfere with KCTD7-ΔC binding a protein/complex and therefore cause different morphologies.

For more detailed analyses, gold labeling and electron microscopy were performed on GFP-KCTD7-ΔC, revealing that gold label was present on membrane structures (73% of gold labeling) surrounding electron dense material, potentially a damaged mitochondrion (Figure 11A) and further mimics our fluorescent data (Figure 11B). Unlike the full-length protein, gold labeling was also found inside mitochondria (14% of gold labeling) and present on p62/SQSTM1-like structures (13% of gold labeling) very similar to structures observed for an autophagy receptor, p62/SQSTM1 (Figure 12A and B) (43).

These data provide evidence that KCTD7 localizes to membranes and supports the hypothesis that KCTD7-ΔC localizes to the growing autophagosome.

Deletion of the C-terminus also results in labeling inside mitochondria and structures that resemble the autophagy receptor p62/SQSTM1. Overall, these data suggest that the N-terminus containing the BTB domain could bind proteins on the autophagosome and in mitochondria.

Novel Metal Binding Domain

A number of BTB domain-containing proteins in other protein families also contain metal-binding domains (Chapter 2), such as zinc fingers. The

163 electronegative charges of cysteine and histidine residues, specifically the free electrons on nitrogen of histidine and the sulfur of cysteine, are able to coordinate the positive charge of zinc and of metal cations. KCTD7 lacks known sequence motif patterns of spaced Cys and His residues found in other metal-coordinating proteins (44). However, the C-terminus of KCTD7 contains 5 closely spaced pairs of Cys-His residues, potentially suggestive of non-canonical metal binding. 5 cysteines and 6 histidine residues (Figure 13A and B). The crystal structure of

KCTD5 includes an unidentified domain in the C-terminus (45). KCTD7 could therefore have a novel metal binding in this domain. Furthermore, several patient mutations (purple amino acids) occur near these cysteine and histidine residues, indicating an important role for this putative domain in KCTD7 function.

To determine if KCTD7 is able to bind metal, GST-KCTD7 fusion proteins were purified from E. coli and metal content was determined by inductively coupled plasma mass spectrometry (ICP-MS), a type of mass spectrometry that speciates different metals in biological samples. ICP-MS was performed at the

Elemental Analysis Core at Oregon Health & Science University (OHSU).

Consistent with metal coordination, copper and zinc were found at significant levels in molar ratios (Figure 13C). Approximately, 2 atoms of Cu, or 1 atom of Zn per KCTD7 molecule was predicted, less than expected but the proportion of protein in the correct confirmation is unknown. To determine if the conserved cysteine and histidine residues were required for metal binding, we generated two mutant constructs that change these residues to alanines, 222HHC224 to

164 222AAA224 (Mut1), and the most C-terminal three His/Cys residues, His270,

His274 and Cys277 were all changed to alanine (Mut2) (Figure 13A). Both

KCTD7 mutants essentially lost the ability to bind molar ratios of metal (Figure

13D and E). Complete loss of binding by both mutant proteins suggests that amino acids 222-277 may be part of a single protein domain. These data provide some support for the hypothesis that KCTD7 has a novel metal binding domain in its C-terminus. A crystal structure will provide insight to fully understand the how

KCTD7 coordinates metal. Future studies will determine the significance of this

domain, its binding partners, and how defects contribute to epilepsy and

neurodegeneration.

165 Discussion

Here we present for the first time an investigation into KCTD7 function.

Our lab previously found that yeast Whi2 was required for turning on autophagy.

Our lab also discovered that Whi2 has homology with the KCTD protein family, to which KCTD7 belongs. As the number of mutations in KCTD7 grew, we wanted to see if we could apply this unique information uncovered about the yeast homolog, Whi2, to understand the cause of a devastating pediatric disease.

To initiate these studies, we obtained primary fibroblasts from two previously identified patients with mutations in KCTD7. As yeast Whi2 and human diseases are implicated in autophagy, we wanted to determine if the autophagy pathway is affected in these patient fibroblasts. Using an autophagy flux assay, we found a significant difference between the patient fibroblasts and

WT controls in LC3-II levels after using a lysosomal inhibitor, suggesting a defect in the autophagy pathway.

As KCTD7 is expressed in the mouse brain, we sought to determine the subcellular localization in a murine neuroblastoma cell line. Building upon the autophagy defect in patient fibroblasts, we found that endogenous KCTD7 co- localizes with autophagy markers after mitochondrial damage, further supporting a role of KCTD7 in the autophagy pathway.

Autophagy is a process that degrades cytoplasmic contents, protein aggregates, and defective organelles. Since we demonstrated the patient

166 fibroblasts to have a defect in autophagy, we hypothesized that abnormal mitochondria should accumulate. This line of investigation is further supported by the fact that deletion of yeast Whi2 causes a defect in oxygen consumption.

Using different mitochondrial markers, we found significant alterations in the mitochondrial networks between patient and WT fibroblasts. We next wanted to examine the mitochondrial substructure and found abnormal structures in inside mitochondria that could be consistent with collapsed cristae, potentially indicating accumulation of defective mitochondria. We further observed the accumulation of lipid droplets next to mitochondria in the patient fibroblasts.

We found that WT GFP-KCTD7 co-localizes with the mitophagy marker, mCherry-Parkin after mitochondrial damage. In contrast, deletion of KCTD7’s C- terminus produces crescent-like structures that only associate with mCherry-

Parkin after mitochondrial damage. The shape and association with damaged mitochondria is consistent with localization to the isolation membrane, an initial step in the autophagic process. As a transient step in autophagosome production, this process has only been graphically illustrated and never demonstrated by fluorescent microscopy. As we are easily able to observe these structures in the absence of a stimulus, we hypothesize that deletion of KCTD7’s

C-terminus liberates the BTB domain to bind to and modulate expansion of the isolation membrane, potentially indicating the C-terminus auto-inhibits the BTB domain.

167 The C-terminus contains a high number of conserved histidine and cysteine residues, consistent with metal binding domains. Using ICP-MS, we found that WT KCTD7 binds nearly 2 copper atoms and almost 1 zinc atom, while mutation of these conserved residues abolishes this metal-binding ability.

While the purpose of this novel metal binding domain remains elusive, it offers an important clue to KCTD7’s function and potential self-regulation. The crystal structure of KCTD5 has an unidentified domain in the C-terminus, indicating that other KCTDs could have a novel metal binding domain in the C-terminus as well.

Overall, these data suggest that KCTD7, like its yeast homolog Whi2, functions in the autophagy pathway and that mutations in KCTD7 lead to defective autophagy (Figure 14A). Our data suggests that KCTD7 localizes to the isolation membrane where it could be involved in its expansion to a mature autophagosome (Figure 14B). Our data from patient fibroblasts further show that defects in KCTD7 lead to accumulation of abnormal mitochondria, lipid droplets, and atypical lipofuscin profiles, consistent with an inability to turnover cytoplasmic contents (Figure 14B).

168 Materials and Methods

Cell culture and transfections

Dermal fibroblasts obtained from two previously reported patients with mutations in KCTD7 (46, 47). Age- and passage-matched control fibroblasts were obtained from Coriell. N2a and Cos7 cells were obtained from ATCC. Cell lines and primary fibroblasts were maintained in Dulbecco’s modified Eagles medium (DMEM) supplemented with 10-20% fetal bovine serum (FBS), 1% penicillin and streptomycin. Cells were transfected using FuGENE® 6

Transfection Reagent and X-tremeGENE 9 DNA Transfection Reagent.

Autophagy flux assay

Autophagic flux in fibroblasts was performed as previously described (32).

Primary fibroblasts were grown to near confluence and plated in 12-well plates then grown overnight in a CO2 incubator at 37°C. The following day, either vehicle (70% ethanol) or 15 mM of chloroquine (CQ) in 70% ethanol was applied to the medium cells for 1 hr. The media was then aspirated and cells, washed 1x with PBS and were lysed in SDS loading buffer (62.5 mM Tris-HCL, 2% SDS,

0.01% Bromophenol Blue, 10% glycerol), boiled for 10 min before loading onto a

12% PAGE and blotted as described in the legend analyzed by western blot.

169 PCR

HEK 293 cDNA was prepared using Qiagen Reverse Transcription Kit and

PCR for KCTD7 was obtained using Invitrogen AccuPrime Pfx polymerase in an

Eppendorf thermocycler using the following protocol: 94°C for 10 min, 35 cycles of 95°C for 15 sec, 55°C for 30 sec, and 68°C for 1 min, followed by 68°C for 10 min. Primers for KCTD7 were obtained from Integrated DNA Technologies (IDT).

Exon2 of primary fibroblasts was amplified after DNA extraction using 5’ Primer

(TGGCACCAATCAGACCCCAGGGATTGAAGATGGAGCAGCCC) and 3’ Primer

(CCCATTTATTAAATTTCATCAATATGCTATCTCCTCTTCTAGG). PCR products were run on a 1% agarose gel and the products were extracted using Qiagen Gel

Extraction Kit. Sequencing was performed by MacrogenUSA using the 5’ primer.

Plasmids

KCTD7 was PCR amplified from HEK293 cDNA and cloned into a GFP containing pSG5 backbone to create an N-terminally tagged GFP-fusion protein.

KCTD7 was also cloned into GH413 backbone via BglII to create N-terminally tagged GST fusion proteins. mCherry-Parkin, GFP-Atg2, and GFP-LC3 were kind gifts from Dr. Richard Youle (NINDS), Dr. Noburu Mizushima (University of

Tokyo), and Dr. Li Yu (NIH/NIAID), respectively.

170 Western Blot analysis.

Cells and tissue were lysed in SDS loading buffer containing protease

inhibitors (Thermo Scientific) and 2% β-mercaptoethanol. Cell lysates were separated by SDS–PAGE on 12% Tris-glycine gels and transferred to nitrocellulose membranes. The indicated primary antibodies (KCTD7 Abcam ab83237, HSP90 BD Biosciences 610419, LC3B Cell Signaling 2775) were used at 1:1000 dilution followed by incubation with HRP-conjugated secondary

antibodies (GE Healthcare) at 1:20,000 dilution. The membranes were developed

using ECL-Prime (GE Healthcare). Westerns were visualized using a Bio-Rad

ChemiDoc MP system and analyzed by ImageLab 5.0 software.

Tissue Lysates

129 mice were humanely euthanized at the indicated ages following

ACUC-approved protocols. Whole brains were extracted and homogenized with a

dounce homogenizer in radio immunoprecipitation assay (RIPA) buffer (30 mM

Tris-HCl, pH 7.4, 0.15 M NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium

deoxycholate, 1 mM EDTA, 1 mM DTT, 2 mM MgCl2). Homogenized lysates

were added to SDS-loading buffer (62.5 mM Tris-HCL, 2% SDS, 0.01%

Bromophenol Blue, 10% glycerol), boiled for 10 min and indicated proteins

analyzed by Western blot.

171 Fluorescence microscopy and image analysis.

Primary fibroblasts and cell lines were grown on 12 mm diameter glass coverslips (FisherBrand 12mm Circular coverslips). The cells were washed three times with cold phosphate buffered saline (PBS), fixed for 10 min in cold 4% paraformaldehyde, permeabilized with 0.2% TritonX-100 for 5 minutes, blocked with 2% goat serum in cold PBS, then immunostained with anti-Tom20 antibody

(Santa Cruz) or anti-KCTD7 (Abcam ab83237) for 1 hr, washed with cold PBS three times, incubated with 2% goat serum in cold PBS, and incubated with either

Alexa Fluor® 488 or Alexa Fluor® 594 secondary antibodies (Santa Cruz). The slides were mounted in Prolong Gold mounting medium, then examined on a

Nikon 90i fluorescence microscope. Alternatively, the cells were incubated with

100 nM Mitotracker Red for 15 min, washed in PBS, fixed in 4% PFA as above, and examined by fluorescence microscopy.

Images were acquired at 60x magnification using 0.5 μM Z-stacks and deconvoluted using Volocity Software. For mitochondrial network analysis, blinded images were binarized using a custom ImageJ plugin, then skeletonized using the ImageJ software. Mitochondrial structure parameters were then quantified using the “Analyze Skeleton 2D/3D” ImageJ plugin by Dr. Lucian

Soane.

Electron Microscopy

172 Monolayers of COS7 cells were transiently transfected with either GFP-

KCTD7 WT or GFP-KCTD7-ΔC. The following day, the cells were treated with 20 mM CCCP for 4 hr. The cells were then fixed by Dr. Coppens as follows; cells were first in 4% paraformaldehyde (PFA; Electron Microscopy Sciences, PA) in

0.25 M HEPES (pH7.4) for 1 hr at room temperature, then in 8% PFA in the same buffer overnight at 4°C (48). Samples were sent to the Yale EM facility were infiltrated, frozen and sectioned as previously described (48). The sections were immunolabeled with rabbit anti-GFP antibody at 1/150 dilution in PBS/1% fish skin gelatin, then with IgG antibodies, followed directly by 10 nm protein A- gold particles before examination with a Philips CM120 Electron Microscope

(Eindhoven, the Netherlands) under 80 kV.

Protein purification and ICP-MS

KCTD7 constructs were cloned into GH413 backbone via BglII to create

N-terminally tagged GST fusion proteins. The constructs were transformed into

FTL bacterial strain and plated on ampicillin selection plates overnight. Single colonies were picked and inoculated into 500 mL LB medium and incubated overnight at 37°C. The following day, isopropyl β-D-1-thiogalactopyranoside

(IPTG) was added to a final concentration of 0.01 mM in the medium once reaching a density of OD600 and incubated at 37°C for 2 hrs. After two hours, samples were spun down at 12,000g, 15 minutes, 4°C and lysed in lysis buffer [4

173 mL PBS + 1% TX100 + 10 mL 25 mg/mL PMSF + 2 mL lysozyme (70000) U/mL)] and sonicated with a Misonix sonicator for 30 sec, at an output level of 40%, duty cycle of 40 for a total of 5 times. Lysates were spun at 12,000g, 15 minutes at

4°C and incubated with 500 mL GST agarose beads overnight at 4°C.

GST beads were then washed with10 mL of PBS with PMSF three times, spinning down at 1000 RPM for 5 minutes between each wash. The beads were then washed in 10mL PBS with 25 mg/mL PMSF 3 times, spun down at 1000

RPM for 5 minutes, then with 5 mL 20 mM Tris, pH 8.0 3 times, spun down at1000 RPM for 5 minutes at 4°C, collecting supernatant. 3 mL were then dialyzed overnight at 4°C. Concentration was measured by a Bradford protein assay or densitometry from a coomassie gel. 500 mL of dialyzed protein was shipped on dry ice overnight to Oregon Health and Science University Elemental

Analysis Core for determination of metal concentration.

Estimation for a predicted metal atom was calculated as follows:

[Metal atom] in GST-KCTD7 (500 mL) for 1 atom/protein 59 kDa (KCTD7 33 kDa

+ GST 26 kDa):

[GST-KCTD7 protein in mg/mL] * 500 mL = X mg protein => X * 10^-6 g protein

X * 10^-6 g protein/ (59,000 g mol^-1) = X * 10^-11 mol protein

X * 10^-11 mol protein => X * 10^-11 mol Metal Atom

(5.9 * 10^-11 mol Metal Atom) * (g/mol Metal Atom) = X * 10^-9 g Metal Atom

174 X g Metal Atom = X ug Metal Atom

X ug Metal Atom/(5 * 10^-4 L total volume) = X ppb predicted Metal Atom

175 Figure Legends

Figure 1. Diagram of KCTD7 mutations in patient fibroblasts. (A) Dermal fibroblasts from patients with KCTD7 mutations (provided by Drs. Haberlandt and

Leshinsky-Sliver) with the indicated mutations in KCTD7 (46, 47). Green arrows represent missense mutations and a red arrow indicates deletion. (B) Exon 2 was

PCR amplified and sequenced to reveal genotypes of indicated fibroblasts.

Figure 2. Autophagy flux assay indicates a role for KCTD7. (A) Western of endogenous LC3 in primary human fibroblasts treated with vehicle (70% ethanol) or 15 uM chloroquine for 1 hr and probed with HSP90 (BD Biosciences 610419) and LC3B (Cell Signaling 2775) and used at a 1:1000 dilution. (B) Quantification of 3 independent experiments as shown in panel A for two wild type and two

EPM3 fibroblast lines.

Figure 3. Kctd7 protein expression in mouse brain. (A) Immunoblot for endogenous Kctd7 in mouse whole brains harvested at indicated time points (10 days N=3; 3 months N=2; 4 months N=1). (B) Quantification of Kctd7 levels in mouse brains relative to loading control Hsp90.

176 Figure 4. Endogenous Kctd7 co-localizes with autophagy markers after mitochondrial damage. N2a cells were transiently transfected with GFP-LC3 or

GFP-Atg2 overnight, followed by either no treatment (A), or treatment with 20 mM

CCCP for 3 hours (B and C). Detection with antibody against KCTD7 (Abcam ab83237 at 1:1000 dilution) and anti-GFP.

Figure 5. Patient Mitochondrial Networks are Fragmented (A) Control and patient fibroblasts immunostained for Tom20 (3 independent experiments). (B)

Quantification of long-branch frequency and mitochondrial skeletal length. (C)

Color-coded mitochondrial networks in control and patient fibroblasts, showing more disrupted networks in patient fibroblasts. Analysis by Dr. L. Soane.

Figure 6. Abnormal mitochondrial cristae in EPM3 patient fibroblasts. (A and B)

Electron micrograph patient mitochondria showing inner-membrane alterations not present in the control. (C) Electron micrograph of Representative control fibroblast mitochondrion.

Figure 7. Accumulation of lipid droplets in EPM3 patient fibroblasts. Electron micrographs of patient fibroblasts were observed to accumulate lipid droplets that were often next to mitochondria (arrows).

177

Figure 8. Ectopically expressed KCTD7 co-localizes with damaged mitochondria.

COS7 cells transiently transfected for 24 hr with WT GFP-KCTD7 constructs and mCherry-Parkin were treated for 4 hrs with 20 mM CCCP.

Figure 9. KCTD7 localizes to membrane-bound vesicles. Transiently transfected

COS7 cells expressing GFP-KCTD7 were treated with CCCP for 4 hrs and

subjected to immuno-gold labeling electron microscopy and revealed gold

labeling on vesicles.

Figure 10. KCDT7-ΔC Localizes to Distinct Crescent-like Strucutres. (A)

Schematic representation of WT KCTD7 and KCDT7-ΔC. (B) COS7 cells

transiently transfected for 24 hrs with GFP-KCTD7-ΔC and mCherry-Parkin. The

cells were treated with 20 μM CCCP for 4 hrs. KCTD7 and Parkin were detected

by direct fluorescence. (C) HA-KCDT7-ΔC version of KCDT7-ΔC results in larger

and longer structures.

Figure 11. Correlation between fluorescence microscopy and Immuno-EM. (A)

Immunofluorescence microscopy of COS7 cells transiently transfected with GFP-

KCTD7-ΔC and mCherry-Parkin treated and treated with 20 mM CCCP for 4

178 hours/. GFP-KCTD7-ΔC localizes next to damaged mitochondria marked by mCherry-Parkin. (GFP and mCherry tagged constructs, color contrast adjusted for presentation). (B) Immuno-gold electron microscopy of COS7 cells Transiently transfected with GFP-KCTD7-ΔC, treated 4 hrs with 20 mM CCCP and subjected to immuno-gold labeling reveals label for GFP-KCTD7 on a membrane surrounding an electron-dense material.

Figure 12. KCTD7-ΔC Localizes to Mitochondria and .p62/SQSTM1structures.

Immuno-electron microscopy of GFP-KCTD7-ΔC. COS7 cells transiently transfected with GFP-KCTD7-ΔC overnight and treated with 20 mM CCCP for 4 hrs then subjected to immuno-gold labeling reveals. (A) Gold-staining inside mitochondrion, and (B) p62/SQSTM1structures.

Figure 13. KCTD7 has a novel C-terminal Metal Binding Domain. (A) Alignment of conserved cysteine and histidine residues (red amino acids). Mutant 1 (Mut1),

222-224: HHC to AAA; Mut2 His270Ala, His274Ala and Cys277Ala. (B)

Schematic of Mut1 (red) and Mut2 (yellow) location. (C and D) Metal concentration in purified protein determined by ICP-MS with WT and Mut1/2. (E)

Calculated number of metal atoms per protein.

179 Figure 14. Proposed mechanism of disease for patients with KCTD7 mutations.

(A) Schematic of autophagy pathway with disease-causing autophagy genes and

the affected process. Our data suggest that KCTD7 localizes to the isolation

membrane (IM) and participates in its expansion to a mature autophagosome

(green doughnut), allowing degradation of damaged mitochondria and lipid

droplets (yellow circles), which are identified by LC3 and p62 for delivery to the

lysosome (red doughnuts). (B) KCTD7 localizes to the isolation membrane (first

image) and deficiency leads to defective autophagy, resulting in accumulation of

abnormal mitochondria (second image) and lipid droplets (third image).

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200

Chapter 4

Resource for the Creation

of a KCTD7-Deficient

Mouse Model

201 Construction of KCTD7-deficient mice

The use of mouse models has allowed us to greatly expand our knowledge of the in vivo effects of genetic modifications. The first knockout mouse models were produced in the late 1980s123. The introduction of recombinase systems (Flp and Cre) have enabled the selective deletion of DNA at specific times and tissues to further elucidate the function of a gene at specific developmental time points and organs4.

Mouse models of epilepsy are used to test candidate antiepileptic drugs (AEDs) through a series of different seizure tests: maximal electroshock seizure (MES) test, pentylenetetrazole (PTZ) tests, and the 6-Hz test, among others5. These tests have been in place for at least 60 years6. Since implementation of these tests occurred before the era of molecular biology, a number of candidate AEDs may be ignored for lack of efficacy. This has been documented for the drug Levetiracetam, which failed to suppress

MES seizures in a mouse model but protected against partial seizures in the clinic6.

Consequently, more mouse models that recapitulate human disease, with known disease-causing pathways, will be of great use to test AEDs.

Mutations in KCTD7 have been causally linked to cause disease in humans7. We therefore reasoned that creating a mouse model that deletes the second exon of KCTD7 will serve as a valuable model to study KCTD7’s role in vivo in addition to creating a new model to test AEDs for this disease. This chapter will serve as a resource for the creation of this mouse model, as I began this project during the later part of my fourth year and do not yet possess firm results.

202

Figure Legend

Figure 1. 5’ long range PCR (LRPCR) primer optimization. (A) Schematic of primer location on the 5’ arm of the Kctd7 gene and within the targeting vector. (B) Schematic of constructed plasmid with primer locations and expected sizes. (C) Protocol for Pfx polymerase (D) DNA Agarose gel demonstrating different efficacy of the four different 5’ primers at a concentration of 1 picogram and 1 femtogram of the plasmid in shown in

1B. (E) DNA Agarose gel of the LF2/LAR3 primer pair with the addition of Pfx added immediately before placement in thermocycler as opposed to addition in a master mix.

Figure 2. LRPCR of ES Cells. (A) PCR gradient using LongAMP polymerase showing most efficient annealing temperature of correctly targeted ES cell F8. (B) Protocol used for screening of ES cells. (C) HindIII digestion of LRPCR product (D) Schematic of

LCPCR product with location of HindIII cut site (E) LF2/LAR3 primers

Figure 3. 5’ Southern Confirmation of LRPCR positive ES cells. (A) Schematic demonstrating the addition of an Sph1 cute site in the cassette. WT will have one band at 14,544 bp and correctly inserted ES cells will have an additional band at 10,656 bp.

(B) Southern showing two bands for positively interested ES cells and one band for WT control with 24 and 72 hour exposures.

203 Figure 4. 3’ Southern Confirmation of LRPCR positive ES cells. (A) Schematic demonstrating that the addition of the cassette will increase the size of the product when cut with Bsph1. WT alleles will have a band at 13,713 bp and correctly inserted alleles will have a band at 20,839 bp. (B) Southern showing two bands for correctly inserted ES cells and one band for WT control at a 72 hour exposure.

Figure 5. Germline transmission of cassette. (A) DNA Agarose gel showing germline transmission of an F1 litter of four pups. A band at 581 bp indicates the presence of the cassette and a band at 419 bp indicates presence of a WT allele. A single band at 419 bp indicates a WT animal, one band at 419 bp and one band at 581 bp indicates a heterozygous animal for a WT allele and an allele containing the cassette, and one band at 581 bp indicates a homozygous animal for the cassette and lack of a WT Kctd7 allele.

(B) DNA extraction protocol, PCR mixture and PCR reaction protocol used in Figure 5A.

(C) Primers used in Figure 5A/B.

Figure 6. Successful Flp recombination and removal of cassette to generate conditional mice. (A) DNA Agarose gel showing Flp recombination of an F1 litter of five pups. A band at 568 bp indicates the removal of the cassette and a band at 419 bp indicates presence of a WT allele. A single band at 419 bp indicates a WT animal, one band at

419 bp and one band at 568 bp indicates a heterozygous. (B) DNA extraction protocol,

PCR mixture and PCR reaction protocol used in Figure 6A. (C) Primers used in Figure

6A/B.

204 Materials and Methods

Purchase and Preparation of Kctd7-tm1a Construct for Electroporation

Targeting vector Kctd7 tm1a was purchased from the Knockout Mouse Project

(KOMP) and linearized using AsiS1. The vector was then run on a 1% agarose gel and purified using Qiagen Gel Puriifcation Kit and eluted with PBS. 25 ul of linearized vector at a concentration of 1275 ng/µl (requirement of 1000 ng/µl) was provided to the ES Cell

Targeting Core for electroporation. After electroporation, ES cell were incubated in neomycin selection media and individual colonies were picked to determine correct insertion by PCR and Southern Blot analysis.

ES Cell Screening by LRPCR and Southern Blot

KOMP provided designed primers for the screening of ES cells. To determine the most effective primer set, a plasmid was designed containing the 5’ genomic DNA portion for the 5’ LRPCR primers and the 3’ portion of the cassette. Per the requirement of the ES Cell Targeting Core, a primer pair was able to amplify the DNA at a concentration of 1 femtogram: LF2/LAR3. This primer set was then used to screen the

ES cells provided by the core. An expected ~5200 bp product was obtained, purified, and cut with HindIII to give the correct fragment size of 943 bp.

DNA of positive LRPCR was provided to Taconic Biosciences, Inc. for confirmation of correct 5’ and 3’ insertion by Southern Blot. For 5’ confirmation, DNA samples were digested with Sph I restriction enzyme for at least 7 hours at 37°C. For 3’ confirmation, DNA samples were digested with Bsph1. The DNA was transferred by

205 vacuum blot to a positively charged nylon membrane. The probe was radiolabeled with

32P-dCTP and hybridized to the membrane using the primers Kctd7-F GCC AGG TCA

GAC CAG CGA C and Kctd7-R AAG GTC AGC TCT GCA AGC ACC. The expected product size for the homozygote is ~10,656 bp. A ~14,544 bp product is expected for the endogenous (wild type) allele. The expected product size for the homozygote is ~20,000 bp. A ~14,000 bp product is expected for the endogenous (wild type) allele.

ES Cell Karyotyping, Blastocyst Injection, and Germline Transmission

ES cells were karyotyped by the JHU Transgenic Core. ES cells with normal karyotypes, correct insertion confirmed by LRPCR and 5’ and 3’ Southern Blot analysis were then injected into B6 blastocysts and implanted into pseudopregnant albino females. Chimeras were born and germline transmission was confirmed by mating chimeras to WT 129 mice and screening pups for positive PCR band for neomycin cassette using the following primers: 5’ WT: GTA CCT CGA TGC AGT CAC AAA GAG

C; 5’ Neo: GGG ATC TCA TGC TGG AGT TCT TCG; 3’ WT: GAT GTT CTT AAT GCT

CTC CTC CCG C. To remove the neomycin cassette, heterozygous mice were mated to a male Flp recombinase mouse obtained from Jackson labs (Jackson Stock #:003946).

Confirmation of cassette removal was determined using the following primers: 5’ WT:

GTA CCT CGA TGC AGT CAC AAA GAG C; 3’ WT: GAT GTT CTT AAT GCT CTC CTC

CCG C).

.

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209

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213 Future Directions and Perspective

When I began my doctoral project, only three patients with a homozygous nonsense mutation in KCTD7 from one family were known in the world. The molecular function was completely unknown. This number has expanded to 33 patients in 22 families with 27 unique mutations worldwide. Undoubtedly, we are just seeing the tip of an iceberg for the number of patients with mutations in KCTD7 and several more undiscovered clinical phenotypes likely exist as well. With time, and asking the correct questions, a better picture of disease progression will emerge. Moving forward, I believe there are two important clinical questions to address:

1) Does mutation location impact severity of disease?

We have only known about KCTD7 as a disease gene since 2007. We have provided evidence to suggest that mutations in cluster 1 cause more refractory seizures but we do not know if this corresponds to an overall more severe disease course.

Perhaps a means to measure this is identifying a patients’ age when they become wheelchair bound. Though our numbers are limited, physicians seem to report that patients are more “functional”, able to write and walk, when their mutations are in clusters 2/3 than in cluster 1.

2) Is there an immunologic consequence to defects in KCTD7?

Two patients were diagnosed with opsonoclonus-myoclonus epilepsy, a severe autoimmune disorder. Additionally, several patients were noted to have recurrent infections. This could indicate that mutations in KCTD7 may have a role in proper

214 immune function and should be included on future questionnaires for newly identified cases.

In addition to these clinical questions, an important question persists about the function of KCTD7:

3) Does KCTD7 play a role in mitochondria respiration?

We found that patient fibroblasts had abnormal mitochondrial networks and disrupted cristae. In a mass spec hit list provided by Dr. James Wohlschlegel, KCTD7 interacted with many components of the ETC, in addition to many other mitochondrial proteins.

These data could indicate that KCTD7 may be imported by an unknown mechanism into the mitochondria and interact with complexes that regulate energy status of the cell and perhaps maintains or supports quality control of mitochondria.

Finally, the mouse model of KCTD7 deficiency could serve as an important model for more than just pediatric epilepsy studies.

4) Does deficiency in KCTD7 lead to a movement disorder in mice?

We have found that all patients with mutations in KCTD7 have a movement disorder.

Therefore, determining if these mice exhibit evidence of a movement disorder seems logical. Additionally, one patient with KCTD7 mutations was positively treated with

Sinemet, a drug used for patients with Parkinson’s Disease. This could position the mouse model for KCTD7 deficiency as an important model for treatment of movement disorders.

215 Kyle Metz 101 West Read Street Apartment 412 Baltimore, MD 21201 (712) 250-4353 Email: [email protected] EDUCATION Spring 2015 (est.), PhD Molecular Microbiology & Immunology, Johns Hopkins University Spring 2009, B.S. Biology, Iowa State University Minor in Emerging Global Diseases Spring 2009, B.A. Spanish, Iowa State University LABORATORY EXPERIENCE Johns Hopkins University 2010 to present. Graduate student in the lab of Dr. Marie Hardwick. • Elucidation of KCTD7 gene and its role in progressive myoclonic epilepsy type 3 (EPM3) • Identified KCTD7 is required for mitochondrial homeostasis • Created conditional knockout Kctd7 mouse model of EPM3 • Establish collaborations with researchers in Austria and Israel to study EPM3 • Mentored undergraduate and graduate students in lab

Iowa State University Spring 2008 to summer 2009. Undergraduate research assistant for Dr. Christine Petersen (Immunoparasitology, emphasis on Leishmania spp.) Summer 2006 to spring 2009. Lab assistant for Dr. Steve Rodermel (Chloroplast biogenesis, emphasis on the Immutans gene) University of Extremadura, Spain. Spring 2007. Translator of selected texts for publication for Dr. José Ángel Padilla Peñas PUBLICATIONS

Osanya AO, Song EH, Metz K, Shimak RM, Boggiatto PM, Huffman EL, Hostetter JM, Pohl N, Petersen CA. (2011) “Pathogen-derived oligosaccharides improve innate immune response to intracellular parasite infection.” The American Journal of Pathology 179(3):1329-37.

Boggiatto PM, Gibson-Corley K, Metz K, Gallup JM, Hostetter JM, Mullin K, Petersen CA. (2011) "Transplacental transmission of Leishmania infantum as a means for continued disease incidence in North America.” PLoS Neglected Tropical Diseases 5(4):e1019.

216 Boggiatto PM, Ramer-Tait AE, Metz K, Kramer EE, Gibson-Corley K, Mullin K, Hostetter JM, Gallup JM, Jones DE, Petersen CA. (2010). "Immunologic indicators of clinical progression during canine Leishmania infantum infection." Clin Immunol 17(2): 267-273.

PRESENTATIONS

Soane L, Metz K, Tanaka B, Haberlandt ED, Leshinsky-Silver E, Hardwick JM. Mitochondrial function and structure are altered in KCTD7-mutant patient fibroblasts. Published abstract. Cold Spring Harbor Cell Death Meeting. October 8 – 12, 2013 Cold Spring Harbor, New York.

Metz K, Teng X, Hartman A, Wheelan S, Hardwick JM. Role of KCTD7 in autophagy & neurodegeneration. Poster presentation. Keystone Symposia: Autophagy, and Immunity. February 17-22, 2013. Montreal, QC, Canada.

Metz K, Teng X, Hartman A, Wheelan S, Hardwick JM. The role of KCTD7 in autophagy & neurodegeneration. Poster presentation. Maryland/Hopkins Mitochondrial Research Retreat. Oct 29, 2011. Baltimore, MD.

Teng X, Metz K, Hardwick JM. Tumor-like genome changes in yeast identify human disease genes. Publishedabstract. Cold Spring Harbor Cell Death Meeting. October 11 – 15, 2011 Cold Spring Harbor, New York.

Metz K, Teng X, Hartman A, Wheelan S, Hardwick JM. The role of KCTD7 in autophagy & neurodegeneration. Poster presentation. Johns Hopkins SPH MMI departmental retreat. Sept 18, 2011. Hershey, Penn.

HONORS AND AWARDS 2012 Frederick B. Bang Award for Research in Pathobiology (Johns Hopkins SPH)

2011 Keerti V. Shah Award for Translational Research (Departmental Award, Johns Hopkins SPH)

2010 Katharine E. Welsh Award (Departmental Award, Johns Hopkins SPH)

2009 Distinguished Achievement in Spanish (Iowa State University)

2009 B.A., B.S. Magna Cum Laude (Iowa State University)

2008 Phi Beta Kappa (Iowa State University) 2008 Undergraduate Research Assistantship, (Iowa State University) VOLUNTEER EXPERIENCE Catholic Charities of Baltimore (2012- Present) My Sister’s Place Women’s Shelter

217 Assists with: • Serving women and children coming into the shelter • Meal service and preparation

Proteus Inc. (2007-2009) Medical interpreter for migrant workers Assisted patients with: • Take patient vitals and explain procedures • Aid medical professionals during examination • Developed brochures in Spanish to augment doctor/patient communication of contagious diseases

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