Relentless desire to help Rett syndrome patients
As told by Huda Zoghbi

2022 KAVLI PRIZE IN NEUROSCIENCE

MORE ABOUT HUDA Y. ZOGHBI

I was born and raised in the beautiful city of Beirut, Lebanon. My father owned a business making olive oil and olive oil soap, and my mother was a homemaker, but they valued knowledge. The house I grew up in had an enormous library stacked floor to ceiling with books. My father loved reading history and sharing what he learned, while my mother insisted that her children take their studies seriously. She often kicked me out of the kitchen because she felt that was a distraction from studying. They clearly set a good example: my oldest brother became an electrical engineer and businessman, the second brother became a chemical engineer and researcher, and the third a professor of history. My older sister became a philosopher and professor, then a lawyer, and I entered academic medicine.

As a child I was drawn to my biology and math classes, but in high school I grew to love literature – both Arabic and English. I decided to major in literature at the American University of Beirut (AUB). My mother, however, had a practical bent and insisted that I study medicine. She said medicine would allow me to be independent and able to take care of myself. She also said, “It’s a much simpler career” and writing could be my hobby. I resisted initially, but eventually yielded and majored in biology.

Civil war in Lebanon
I continued on to medical school at AUB. This changed the course of my life in every way. I met the love of my life, William Zoghbi, who has been my great joy for the past 46 years. Then, halfway through the first year of medical school, civil war broke out in Lebanon. Students could no longer commute to campus because of the bombing and sniper attacks. The students and faculty debated whether to finish the year or cancel the term and go home. We thought the war couldn't last long and made the decision to keep going. Our lives became centered on avoiding bombs. We went to classes during the day and stayed together at night. Those of us who had been commuting and didn't have dorm rooms had to find safe places to stay below ground on campus. I found a nook inside the ladies’ room, put a sleeping bag on the floor, and made it my home until the end of the semester.

That spring, my younger brother, who was 16 years old at the time, was hit by shrapnel. This brought home the danger. My parents could not leave but they planned for my younger brothers and me to go abroad and visit relatives for a month or two in the summer, expecting that the war would end and we could return to normal by the time school started again in October. The airport was closed, so we drove to Syria, and from there flew to Europe where we stayed with my uncle for a few days, then flew on to the U.S. to stay with my sister, who was in Austin, Texas at the time. I arrived in Austin July 4, 1976, during the U.S. bicentennial celebration. I recall when I heard the fireworks, I thought bombs were going off and I burst into tears.

Meharry Medical College
The civil war in Lebanon only grew worse. When we tried to return home, the borders were closed. It was already October, so I started looking for a medical school to attend in the U.S. All my applications were rejected – only then did I learn that medical school starts in August in the States. A friend of the family in Nashville invited me to visit, and together we went to Vanderbilt to see if they would allow me transfer. They said they did not accept transfer students, but recommended another nearby medical school, Meharry Medical College, an historically African-American institution. I told the Dean and the head of admissions my story and gave them my transcript that my family had sent by telex. They agreed to take me on the spot, despite the fact that the semester had started two months earlier. I will forever be grateful to Meharry.
Although I was excited about being accepted into an American medical school, I was terribly homesick, constantly worrying about my family, but the students were kind-hearted and looked out for me. I coped that year by studying all the time, still hoping I could go back to Lebanon. I managed to return to Lebanon the following summer, but my professors at AUB, particularly Drs. Adel Afifi and Ronald Bergman, who inspired my love of neuroscience, said the war would continue for a long time, and I should not return to AUB. I was sad to leave home again but grateful when William transferred to Meharry the year after I.

Fell in love with pediatric cardiology
During the fourth year of medical school, I took electives at Stanford, Emory, and Baylor College of Medicine. During my rotation at Baylor College of Medicine, I fell in love with pediatric cardiology. I placed Baylor College of Medicine at the top of my list, and on Match Day learned that I would be in Houston for my residency training. The highlight of my pediatric training was being mentored by Dr. Ralph Feigin, the chair of pediatrics. He was the quintessential clinical scholar and a most compassionate physician. “Feigin Rounds” were an exercise in using every clinical detail to solve a medical mystery, and a great reminder of the importance of mastering the literature. He taught with passion, ultimately influencing the lives of thousands of patients through his many sterling trainees.

Tug-of-war
As a pediatric resident, I rotated in many sub-specialties. I wanted to study pediatric cardiology, but when I rotated in pediatric neurology, I met Dr. Marvin Fishman, the chief of that section. Marv is a fantastic clinician and teacher, and he was the next big influence on the course of my life. On rounds, we often saw children who were in the hospital for heart defect, but who also had neurological problems. For the entire month of my rotation, there was a tug-of-war between heart and head: I would go on about how fascinating the heart was, and Marv would tease me about how the brain was so much more interesting. The rotation ended and I thought that was the end of neurology for me. But Marv had gotten to me. I missed the challenge of neurology, the detective work of taking a history and solving the puzzle of a patient through a detailed history, physical exam, and rigorous logic, so I decided to become a child neurologist.

Emotional pain
What I did not realize at the time was the emotional pain I would feel in that specialty. Even in the early 80’s, Texas Children’s Hospital was a major referral center. It always drew the most difficult cases. Every day I sat with parents and had to tell them that their child had a disorder whose cause was probably genetic but we could not be certain. What was certain was that their child would suffer and perhaps die prematurely, and there was nothing we could do to help. Many nights, I went home and cried over the bad news I had to deliver.

My career path was further defined by one of my patients. Ashley had appeared healthy at birth but lost her language and motor skills around the age of two, and she could only wring her hands incessantly. Ashley was referred by her pediatrician, Dr. Merlene McAlevy, who suspected Rett syndrome based on a paper describing the syndrome for the first time in English, just published by Bengt Hagberg (October 1983). (Rett syndrome is a rare neurological and developmental disorder that affects the way the brain functions after birth, causing a progressive loss of motor skills and speech). I saw Ashley with Drs. Alan Percy and Vincent Riccardi, the attending neurologist and geneticist. I was intrigued by Ashley’s diagnosis, but a serendipitous meeting a week later sealed my relationship with Rett syndrome. As residents, we could look over patient charts and choose which patients to evaluate, and I chose to see a 12-year-old girl with cerebral palsy. When she walked into the exam room wringing her hands, I immediately realized it wasn’t cerebral palsy, rather Rett syndrome. (For many years, Rett girls were commonly misdiagnosed as having cerebral palsy.) Having seen two girls in one week – despite no one having reported the disorder in the U.S. – I concluded there must be more patients who were simply being missed. I reached out to the volunteers in our pediatric neurology outpatient clinic, the Blue Bird Circle Clinic, to help me. These volunteers, whom we affectionately called “Blue Birds,” had dedicated their time and resources to help patients with neurological disorders and assist the physicians in the clinic. I gave them a list of key features and asked them to pull the records (these were paper records at the time) with such features. Within a few weeks, I identified and examined six girls with Rett syndrome. After seeing these girls, I could not continue as just a clinician; I had to figure out what was happening to these patients and do something to help.

Grant from the Blue Birds
I approached Dr. Art Beaudet, a renowned human and molecular geneticist at Baylor College of Medicine, to ask if he would allow me to undertake a postdoctoral research fellowship in his lab to learn molecular genetics. I wanted to work on Rett, and was convinced it was genetic because it almost exclusively occurred in girls, leading me to believe it resulted from a mutation on the X chromosome. With a $50,000 grant from the Blue Birds, I collected DNA samples from many families, but they all had just one child with the disorder. With the technology available in 1985, it was impossible to identify the causal mutation without other affected family members to help narrow down which genomic regions could harbor the gene. Art urged me to find another disease to study so that I could start my own independent career with a more tractable problem. While disappointed, I listened to him and told him I was also interested in dominantly-inherited neurodegenerative disorders because I was intrigued by their late onset and the lack of protection from the normal allele (the variant form of a gene). He introduced me to a family in Montgomery, Texas, with a dominantly-inherited spinocerebellar ataxia – a degenerative disease that causes progressive problems with movement and balance. I wrote my first National Institutes of Health (NIH) grant, known as a K08, while still finishing my pediatric neurology fellowship. The only preliminary data I had was a drawing of a large familial pedigree. I was a candidate with no research experience, but I possessed passion and promise, along with an incredible mentor. The NIH grant was awarded the first day I started in the lab. I share this story because finding support for physicians with little or no research experience is practically impossible today. If I had been held to today’s standard, my career in science never would have happened.

“Finding support for physicians with little or no research experience is practically impossible today.”

With funding secured for five years, I moved from the clinic and took basic science graduate courses at Baylor College of Medicine, immersing myself in cloning and linkage mapping. I traveled to Montgomery, Texas, for a few months to examine the extended family and collect blood for DNA. I then commenced my work on finding the genetic basis of their disease. Dr. William O’Brien, a collaborator of Dr. Beaudet’s on urea cycle studies, was a co-mentor. (The main purpose of the urea cycle is to eliminate toxic ammonia from the body.) The three of us became good friends and had coffee every Friday morning for years to discuss science and how best to solve the diseases I was interested in. Several computational geneticists helped me along the way. First, Steve Daiger at the University of Texas (Houston Health Science Center) collaborated to help me with data analysis, then Jürg Ott and his postdoctoral fellow, Lodewijk Sandkuyl, at Rockefeller University taught me how to perform linkage analysis. Art was very supportive despite the fact that his lab worked on a completely different set of disorders. He taught me how to perform rigorous research with well-controlled experiments and how to prepare the best possible slides when giving a talk – two traits he learned as a postdoc in Marshall Nirneberg’s lab. Art’s advice on choosing a clearly Mendelian disorder also proved sound. Studying ataxias led me to one of the highlights of my scientific career and ensured that I could be productive while I was still struggling to understand Rett.

Reaching out to Harry Orr
I read a paper by a scientist named Harry Orr about a family in Minnesota with ataxia that had its gene localized to chromosome 6 – the same chromosome my research pointed to. I wanted to reach out to him to see if we could collaborate, but I was petrified because he was a tenured, Associate Professor – an established scientist! – and I was just learning my way around the lab. Art met Harry at a conference and when he returned, he encouraged me to reach out to Harry. I gathered up my courage and called Harry on the phone, explaining that since we were each working with large families we could make faster progress if we joined forces. I found Harry easy to talk to and very kind. Curiously, because of where the genes mapped in our respective families, we were looking at two different parts of chromosome 6 despite being fairly certain we were studying the same disease. At that time, there weren’t many DNA markers, but a wonderful geneticist, David Cox, had developed an approach called radiation hybrid mapping to generate fragments of chromosomes that would help us pinpoint the disease-causing region of DNA. I read the protocol paper but wasn’t completely clear on it, so I called David. He was kind enough to walk me through the entire experiment. I developed radiation hybrid markers for chromosome 6 and was excited that the hybrids gave me something to offer Harry for a solid two-way collaboration, even if we were working on two differently-mapped ataxias.

Setting up a lab
By then it was 1988 and I had secured my first NIH grant (an R01) following the K08, so Art felt I was ready to start my own lab. I was concerned, however, about the location of my new lab. After all, my primary appointment was in pediatrics and neurology and the expectation was that I would be with more clinically-oriented researchers near the hospital. But I still had so much to learn and wanted to be with the basic scientitsts. I approached the Chair of Genetics, Dr. C. Thomas Caskey, for space. His vision for the department was that it would place researchers from diverse fields near each other to stimulate those ever-important serendipitous encounters in hallways and water-coolers that so often inspire new ideas, so I used his vision in my plea for space. He graciously gave me lab space, and although there would be no office, just open desks and lab benches, I was thrilled. In retrospect, not having an office was a gift because it meant that I was in the lab working at the bench alongside my trainees, having fun, encouraging them, and not being distracted by emails and other things.

“Not having an office was a gift because it meant that I was in the lab working at the bench alongside my trainees, having fun.”

OK, let’s do it
Harry and I worked on our different regions of chromosome 6, but it bothered me that the same clinical entity could map to two different regions of the same chromosome (this would not seem strange today, given our knowledge of genetic heterogeneity). I did a lot of detective work and figured out that in a small branch of my family, the disease did not come from the main bloodline but from a spouse who was likely to have the same mutation but died before developing symptoms. The odds of a disease with a prevalence of 1/100,000 running in the same family through two unrelated bloodlines were unfathomable, but I suspected exactly that and it turned out to be the case. I realized that by including this individual as the one passing on the disease to his three daughters, my gene mapped on top of the gene in Harry’s ataxia family. I was excited and telephoned Harry to tell him. His first reaction was, “Do you want your radiation hybrids back?” I said, “No, now we can collaborate more intensely because we’re working on the same gene!” He was silent for about 15 seconds, processing this new information, then said, “Okay, let’s do it.”

Between 1988 and 1993 we continued marching through genes. Then Dr. Caskey gave a noon conference describing the discovery of the myotonic dystrophy gene, which was the third gene to be identified with a triplet repeat expansion (after the androgen receptor and fragile X). He described how the repeat was bigger in the children than their mother, which explained the phenomenon of anticipation, wherein the disease strikes each subsequent generation at an earlier age with more severe symptoms. I realized this was exactly what I was seeing in my spinocerebellar ataxia (SCA) family. The last generation had a four-year-old who was affected, whereas the father didn’t show symptoms until his 30s. This raised the possibility that the ataxia was a triplet repeat disease, so I called Harry. We agreed this was very likely and designed an experiment to test the hypothesis. The region we were looking at was one million base pairs. We split the region in half and each of us started working from the outside in, searching for triplet repeats as we worked toward the middle. We would both screen a 70Kb region of overlap in the middle so we do not miss anything. Finally, on April 8, 1993, we both discovered the disease-causing gene on the same day, right in the middle of the candidate region. Harry was sending me a fax of the expansion he detected in his family, while I was sending him ours – both Southern blots made it into the paper. We had the pleasure of sharing the discovery and our data at an international ataxia meeting in Capri, Italy, that summer. After the gene discovery, we had to plan the next steps: do we continue to collaborate or do we go our different ways? We chose to continue our collaboration.

Friendship
Beyond that, Harry and I forged a decades-long friendship that we and our families cherish. When our daughter went to camp in Minnesota while in middle school, Harry was “the responsible adult” should she need help. Harry visited Lebanon as well, where he met my family and William’s family, visited my alma mater, enjoyed Lebanese food, and experienced driving on narrow mountain roads.

This collaboration inspired another, with the superb Drosophila geneticist Juan Botas (Baylor College of Medicine). Juan and his lab created fly models of SCA1 and identified many modifiers that helped us gain insight into Ataxin-1 biology. Collectively, our studies are continuing to help us understand the mechanisms driving disease and how the glutamine expansion stabilizes Ataxin-1 leading to its accumulation and toxicity. Our preclinical proof of concept studies revealed that lowering Ataxin-1 levels in SCA1 knockin-mice rescued disease features, providing promise for future interventional studies in people with SCA1. Moving forward, we are using our SCA1 knock-in mice that recapitulate SCA1 features and pathology to understand how a mutation in a broadly expressed protein can cause selective neuronal degeneration.

Not forgotten Rett
While SCA1 research was advancing, I had not forgotten Rett. I continued to collect Rett data and had DNA samples from over 200 families. The work, however, was one disappointment after another. One patient had a translocation on the X chromosome, but when my graduate student cloned the region spanning the translocation, there was no gene at the breakpoint site. Another family had two second cousins with Rett, but they shared no regions of the X chromosome. Yet another patient had a null allele in a gene involved in carnitine biosynthesis, but it turned out the father had the same null allele so we knew it could not be causing the disease. Despite the disappointments, I could not let go. The Rett symptoms were so distinctive and consistent that only an underlying genetic defect could cause such features. The children are born looking perfectly normal. They achieve developmental milestones and everything seems to be fine, but then they regress around the age of two. They lose the abilities they had gained in language and motor control and start moving their hands in stereotypic ways (wringing, flapping, etc.). Not only was this an unmistakable and puzzling constellation of symptoms, but the mechanism of disease was a mystery, too. At the time we thought there were two types of neurological disease: congenital and degenerative. But the girls were not like that: they were born normal, and the few neuropathology studies that had been done showed there was no degeneration of the brain even though there was loss of function.

Studying two families with two affected half-sisters helped eliminate two-thirds of the X chromosome based on discordance between the affected half-sisters. Carolyn Schannen and Uta Francke at Stanford studied another family with an aunt and a niece with Rett, so we combined our data and helped eliminate a few more megabases. After a decade of negative results, graduate students and fellows refused to work on Rett as they viewed it as a dead-end project with too much negative data. I could not obtain a grant to study Rett because it was hard for reviewers to imagine that a gene could underlie a sporadic disorder. Thankfully, a couple of fortuitous things happened: First, Ruthie Amir, a physician with no research experience, wanted to join my lab as a staff scientist. I was concerned this would not be a good title for her career, so I offered her a postdoctoral fellowship on the condition she work on Rett. She accepted. Second, Ruthie received a fellowship from the International Rett Syndrome Association and I was fortunate to obtain funding from the Howard Hughes Medical Institute (HHMI) that allowed us to keep going. (I confess I did not tell HHMI about Rett in my research proposal, fearing rejection.) At long last, in 1999 Ruthie found the genetic mutation that causes Rett. I had just opened the door of my house – returning from a trip to Lebanon – when the phone rang. I picked it up, and it was Ruthie. I asked her to bring her notebooks to my house, and she was there within the hour. She showed me patient after patient with a null mutation in MECP2 that was not in the parents. I knew this was it. People often ask me what kept me going despite years of negative data. Beyond my intuition that Rett was genetic and my feelings for the Rett girls, the support of family, friends, and colleagues was essential. Ralph Feigin encouraged me and always believed I would find the gene, even when many were doubtful.

“Ruthie showed me patient after patient with a null mutation in MECP2 that was not in the parents. I knew this was it.”

Humbled by the complexity
Since then, understanding how loss of this protein causes Rett syndrome has been a major focus of my lab. Adrian Bird showed that MeCP2 binds methylated DNA, and it's clear that it is important for neurons and that it somehow orchestrates gene expression. The Bird lab also showed that restoring normal MeCP2 levels rescues the disease phenotype in mice. Our animal model studies led us to patients with MECP2 duplication syndrome, which can also be ‘corrected’ in mice when MeCP2 protein levels are returned to normal. These results tell us that the brain architecture is still intact enough to regain function, when a treatment is found. Preclinical work using antisense oligonucleotides to reduce the concentrations of MeCP2 has provided proof of concept and led to the currently ongoing clinical trial readiness studies. After 23 years of research following the discovery of the gene, I am humbled by the complexity of this disorder, but believe we are beginning to understand it well enough to develop therapies that work through neuromodulation or manipulation of MeCP2 levels.

I am indebted to all the patients and families I have encountured for inspiring me and for persevering while we pursued gene discovery and the critical studies of mechanisms driving their diseases. We hope that the proof of concept preclinical studies will soon lead to clinical trials to help alleviate their symptoms.

HHMI support also allowed me to discover Atoh1, which took me into many interesting areas of biology. I asked my fellow HHMI and Baylor College of Medicine colleague, Hugo Bellen, about fruit fly genes important for balance. He told me about atonal, discovered by Yuh Nung Jan at UCSF to be critical for the formation of chordotonal organs and proprioreception in flies. I set out to identify the mammalian homolog with the help of my long-time technician Alanna McCall (this year marks our 35th anniversary working together). We succeeded and named it Mouse atonal homolog 1 (Math1), which is now known as Atoh1. We created mouse models that lack this gene, traced its lineage, and learned about its critical role for genesis of cerebellar granule neurons, inner ear hair cells, a variety of brain stem neurons critical for hearing, proprioception, interoception, and respiration, and cells in other parts of the body such as secretory cells in the intestines and Merkel cells in the skin.

The joy from the advances I have experienced collaborating with Harry, Juan, Hugo and others guided me in founding the Jan and Dan Duncan Neurological Research Institute (NRI) at Texas Children’s Hospital and Baylor College of Medicine. The NRI is dedicated to basic neurological disease research and incorporates all the ingredients that I have found most helpful in my career: collaboration, access to expertise outside our field, a culture of sharing, cross-species studies, and commitment to mentorship. It is rewarding to watch young faculty build their careers and work together to solve the unsolvable. The NRI has enabled my lab to broaden our studies to other neurodegenerative disorders and embark on collaborations within the Alzheimer’s disease JPB consortium to study regulators of tau levels in hope of revealing druggable targets that can reduce pathogenic tau accumulation in Alzheimer's disease and other tauopathies.

“The institute incorporates all the ingredients that I have found most helpful in my career: collaboration, access to expertise outside our field, a culture of sharing, cross-species studies, and commitment to mentorship”

Navigating busy lives
The collaborative environments at Baylor College of Medicine and Texas Children’s Hospital have been incredible for my growth as a geneticist and neuroscientist. My profoundest gratitude goes to my husband, William, who has been my partner every step of the way even while reaching great heights in his own career as a cardiologist. His unconditional love and support while we raised our two wonderful children, Roula and Anthony, allowed me to go to the lab on nights and weekends and kept me positive when experiments did not succeed. I am grateful to our children for their support, enduring dinner conversations about rejected papers and negative results, providing comforting words, and doing their part as we navigated busy lives with two parents in academia. Now my children are grown, and they have made me more proud than I could ever have imagined. Our family now includes their wonderful spouses, Tyler and Zena, whom we love dearly, and their wonderful children Camila, Tate, and Sienna. Being a “Tata” (the modified nickname for grandma in Lebanese) is one of my favorite and most enjoyable jobs. I love traveling with William, going on long nature walks together, and enjoying the opera. Besides reading, I love to cook. When the children were young, Sunday night was my “innovative cooking” night. Now that the family has grown, weekly Sunday dinners are a tradition and the grandkids have developed a sophisticated palate given the variety of foods I prepare. My favorite meals are those made with fish William has caught in the Pacific Northwest or with vegetables he grows in our garden.

“The Zoghbians”
My other extended family of trainees, technicians, and staff, “the Zoghbians” as they like to refer to themselves, made my science career a most rewarding one. I am grateful for their dedication, patience, trust, and friendship. They taught me so much and made me look forward to coming to work everyday. Their hard work, commitment and passion give me faith that neurobiological disease research will be in capable hands for decades to come.