Wednesday, May 29, 2024

Relentless desire to help Rett syndrome patients

2022 Kavli prize in neuroscience

As told by Huda 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.


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.
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 scientists. 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.

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.


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.

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.

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.

A new atlas of the human body

NPR's Ayesha Rascoe talks with Kalyanam Shivkumar, a cardiologist at UCLA, about his push to create a new anatomical atlas after discovering the one used by doctors for decades was made by the Nazis.


The images of "The Pernkopf Topographic Anatomy Of Man" are delicately complex, clearly diagrammed and full of detail. It's an illustrated atlas of the human body, showing layers of tissues, organs, and bones, and as helpful as the multivolume anatomical atlas has been for generations of doctors, it's no longer in print because it's problematic to say the very least. Eduard Pernkopf, the Austrian doctor who oversaw its creation, was a loyal supporter of Adolf Hitler. The bodies he used as models belong to prisoners who died in Nazi prisons. Many of them were dissidents, gay men, lesbians, and Jews. The LA Times reported last week about a project that's underway to create a new atlas of the human body, one without this horrific history. We're joined by Dr. Kalyanam Shivkumar, who is a cardiologist at UCLA behind that project. Welcome to the program.

KALYANAM SHIVKUMAR: Thank you so much.

RASCOE: How did you first learn about the Pernkopf atlas?

SHIVKUMAR: So the atlas came to my attention around the year 2012. This entire collection was gifted to me by a colleague. We were at that time searching for the very best resources on anatomy of the heart in our case, but obviously also to understand nerves in the human body. And when this entire collection arrived, I was almost, you know, taken aback by the quality of the work. But my colleague who gifted the book had already sort of indicated to me that it actually had a very disturbing link in historical background, and that's how my sort of introduction to the Pernkopf atlases came about.

RASCOE: And I understand that you, as well as many other doctors, have been incredibly conflicted about using this atlas. Why not just use a different one? Like, why is this one still in use?

SHIVKUMAR: So the answer is twofold. One is, the time and effort that was put into creating the original atlases shows in the quality of the work they had produced. It actually has fine details which you simply don't find in other books. And many bootleg copies were floating around after its sort of very depressing history came into being, and it went out of print. Of course, older copies were still acquired and so forth.

RASCOE: So tell us about your project, starting with the name.

SHIVKUMAR: Yes, so in medical school, one of our founding, you know, principles is to make sure that there are no ethical violations. We teach our students, you know, some of the huge violations that have happened. And of course, when you see this type of work, where you see that, you know, there were prisoners, and these were people who were murdered, and people just went on to say, OK, let's just acknowledge it and move on, but still continue to use it. That didn't sit well with me. And at that time, to answer you, the first part of your question, we coined the term "Amara Yad." And that is sort of a combination of two words. Amara in Sanskrit means eternal. Yad in the Hebrew language means hand. So we coined the term "Immortal Hand" for this project to say that we're going to surpass this and create completely new atlases that will be far more detailed, way more artistic, and something that will really inform what medicine needs today.

RASCOE: Well, how are you getting cadavers for this project?

SHIVKUMAR: This comes from a UCLA program, which is called the willed body program, where many people leave their body for medical research. And it's one of the most noble contributions people make.

RASCOE: Well, you talked about how detailed the Pernkopf atlas was. How does this one - and you want this one to surpass it. How do you feel like it's better?

SHIVKUMAR: We spent a lot of time, in fact, a few years, contemplating how we would go about doing it. We'd take various parts of the body, but especially the heart, since we started with it, and we use very powerful laser microscopes to get very fine structural details. I would say that this is almost like the space program, but it's the inner space. We're looking at the human body in a completely new light. And that is how we surpass Pernkopf, by completely leapfrogging it. It's ethically sourced, and it's also highly contemporary. And we are working with our collaborators around the world who have this type of expertise. So that is how Pernkopf can and will be beaten.

RASCOE: You know, think about the Hippocratic Oath, that - do no harm. In a way, is your project an attempt to undo a harm?

SHIVKUMAR: I think what Pernkopf and those types of people have done is they've harmed what should be very pure, which is medical education. What one rabbi very poignantly said, it's a fruit of a poisonous tree. So in that sense, it's a moral corrective. We are undoing a harm. And in doing so, we are also providing a vastly superior source of information for the world to use.

RASCOE: That's Dr. Kalyanam Shivkumar, talking about his ongoing project to create a better anatomical atlas. Thank you so much for talking with us.

SHIVKUMAR: Thank you so much for the interview, and we appreciate the opportunity to share our work with all your listeners.

Tuesday, May 28, 2024

Critical differences in the brains of girls diagnosed with autism

From the moment you were born until about age 2, your brains' outer layer – the cortex – rapidly thickened in a frenzy of neuron creation. After all that excitement, that dense hedge of nerve cells was trimmed back in a process called 'cortical thinning'.

Now, a new study has found some key differences in how this process occurs in autistic children, depending on their birth sex.

Previous studies have found variations in the way autistic children's brains undergo cortical thinning, but so far the picture is hazy and inconsistent. This is partly because historically, studies into autism spectrum disorder under-represent the female sex, and that goes for research into cortical development.

"It is clear that this sex bias is due, in part, to under-diagnosis of autism in females," says neuroscientist Christine Wu Nordahl from the University of California Davis. "But this study suggests that differences in diagnosis are not the full story – biological differences also exist."

Though the actual ratio is likely to be a lot lower, only one female is diagnosed with autism for roughly every four males that receive a diagnosis, hinting at the possibility of sex influencing the condition's development.

By including both autistic and non-autistic children in the study, the researchers could compare differences in cortical thickness associated with autism within each birth sex group (for instance, the difference between autistic females and non-autistic females), as well as comparing results for the autistic groups on the basis of birth sex alone.

The study included brain scans from 290 autistic children (202 males, 88 females) and 139 non-autistic children (79 males, 60 females) with typical development.

These scans were collected up to four times for each child, from ages 2 to 13, offering a detailed picture of the childrens' cortical development from the age when the cortex is at its thickest, up to the age where thinning is at its most rapid, usually around 14 years.

At age 3, certain regions of the cortex – about 9 percent of its total surface – were thicker among autistic females than peers without a diagnosis of the same age and sex. In the male group, at age 3 there were few significant differences in cortical thickness between autistic and non-autistic children.

By age 11, cortical differences between sexes were much harder to spot. The main distinctions revealed in the study were only visible as changes to the cortex across time.

Compared to their non-autistic counterparts, female children with autism had more rapid cortical thinning in certain regions across childhood, while autistic males had less rapid thinning than non-autistic males overall. These changes weren't consistent across the entire brain: only in certain cortical regions that make up less than 5 percent of its entirety, including the networks that plan and control motor tasks, sustain attention and solve problems, and the brain's 'radar' which helps us to pivot attention when our conditions change.

In other regions, like the limbic network, where behavioral and emotional responses arise, cortex thinning occurred more rapidly in autistic males compared to non-autistic males, and less rapidly in autistic females compared to non-autistic females.

There are many reasons a person's biology may relate to or reflect which sex they are assigned at birth, and it's not necessarily fixed: certain traits can be X- or Y-chromosome linked, while others are affected by different hormone levels, or can even be the result of cultural attitudes towards assigned sex and gender that lead to different behaviors and lifestyles.

And so while this study has found observable differences between the male and female groups, more detailed research will be required to understand exactly how these differences arise, and what it may mean for transgender, nonbinary, or intersex people with autism.

That nuance is particularly relevant here, given that gender diverse adults are up to six times more likely to be diagnosed autistic than cisgender adults (those who identify with their gender and sex assigned at birth).

"We typically think of sex differences as being larger after puberty. However, brain development around the ages of 2 to 4 is highly dynamic, so small changes in timing of development between the sexes could result in large differences that then converge later," says psychiatric researcher Derek Andrews from the University of California Davis.

"It's important to learn more about how sex differences in brain development may interact with autistic development and lead to different developmental outcomes in boys and girls."

This research was published in Molecular Psychiatry.

Andrews DS, Diers K, Lee JK, Harvey DJ, Heath B, Cordero D, Rogers SJ, Reuter M, Solomon M, Amaral DG, Nordahl CW. Sex differences in trajectories of cortical development in autistic children from 2-13 years of age. Mol Psychiatry. 2024 May 16. doi: 10.1038/s41380-024-02592-8. Epub ahead of print. PMID: 38755243.


Previous studies have reported alterations in cortical thickness in autism. However, few have included enough autistic females to determine if there are sex specific differences in cortical structure in autism. This longitudinal study aimed to investigate autistic sex differences in cortical thickness and trajectory of cortical thinning across childhood. Participants included 290 autistic (88 females) and 139 nonautistic (60 females) individuals assessed at up to 4 timepoints spanning ~2-13 years of age (918 total MRI timepoints). Estimates of cortical thickness in early and late childhood as well as the trajectory of cortical thinning were modeled using spatiotemporal linear mixed effects models of age-by-sex-by-diagnosis. Additionally, the spatial correspondence between cortical maps of sex-by-diagnosis differences and neurotypical sex differences were evaluated. Relative to their nonautistic peers, autistic females had more extensive cortical differences than autistic males. These differences involved multiple functional networks, and were mainly characterized by thicker cortex at ~3 years of age and faster cortical thinning in autistic females. Cortical regions in which autistic alterations were different between the sexes significantly overlapped with regions that differed by sex in neurotypical development. Autistic females and males demonstrated some shared differences in cortical thickness and rate of cortical thinning across childhood relative to their nonautistic peers, however these areas were relatively small compared to the widespread differences observed across the sexes. These results support evidence of sex-specific neurobiology in autism and suggest that processes that regulate sex differentiation in the neurotypical brain contribute to sex differences in the etiology of autism.

Sunday, May 26, 2024

UNC13A mutation and congenital myasthenia

Inspired by a patient

Ohno K, Ohkawara B, Shen XM, Selcen D, Engel AG. Clinical and Pathologic Features of Congenital Myasthenic Syndromes Caused by 35 Genes-A Comprehensive Review. Int J Mol Sci. 2023 Feb 13;24(4):3730. doi: 10.3390/ijms24043730. PMID: 36835142; PMCID: PMC9961056.


Congenital myasthenic syndromes (CMS) are a heterogeneous group of disorders characterized by impaired neuromuscular signal transmission due to germline pathogenic variants in genes expressed at the neuromuscular junction (NMJ). A total of 35 genes have been reported in CMS (AGRN, ALG14, ALG2, CHAT, CHD8, CHRNA1, CHRNB1, CHRND, CHRNE, CHRNG, COL13A1, COLQ, DOK7, DPAGT1, GFPT1, GMPPB, LAMA5, LAMB2, LRP4, MUSK, MYO9A, PLEC, PREPL, PURA, RAPSN, RPH3A, SCN4A, SLC18A3, SLC25A1, SLC5A7, SNAP25, SYT2, TOR1AIP1, UNC13A, VAMP1). The 35 genes can be classified into 14 groups according to the pathomechanical, clinical, and therapeutic features of CMS patients. Measurement of compound muscle action potentials elicited by repetitive nerve stimulation is required to diagnose CMS. Clinical and electrophysiological features are not sufficient to identify a defective molecule, and genetic studies are always required for accurate diagnosis. From a pharmacological point of view, cholinesterase inhibitors are effective in most groups of CMS, but are contraindicated in some groups of CMS. Similarly, ephedrine, salbutamol (albuterol), amifampridine are effective in most but not all groups of CMS. This review extensively covers pathomechanical and clinical features of CMS by citing 442 relevant articles.

Engel AG, Selcen D, Shen XM, Milone M, Harper CM. Loss of MUNC13-1 function causes microcephaly, cortical hyperexcitability, and fatal myasthenia. Neurol Genet. 2016 Sep 8;2(5):e105. doi: 10.1212/NXG.0000000000000105. PMID: 27648472; PMCID: PMC5017540.


Objective: To identify the molecular basis of a fatal syndrome of microcephaly, cortical hyperexcitability, and myasthenia.

Methods: We performed clinical and in vitro microelectrode studies of neuromuscular transmission, examined neuromuscular junctions cytochemically and by electron microscopy (EM), and searched for mutations by Sanger and exome sequencing.

Results: Neuromuscular transmission was severely compromised by marked depletion of the readily releasable pool of quanta, but the probability of quantal release was normal. Cytochemical and EM studies revealed normal endplate architecture. Exome sequencing identified a homozygous nonsense mutation in the N-terminal domain of MUNC13-1 (UNC13A) truncating the protein after 101 residues.

Conclusions: Loss of Munc13-1 function predicts that syntaxin 1B is consigned to a nonfunctional closed state; this inhibits cholinergic transmission at the neuromuscular junction and glutamatergic transmission in the brain. Inactivation of syntaxin 1B likely accounts for the patient's cortical hyperexcitability because mutations of syntaxin 1B cause febrile seizures with or without epilepsy, haploinsufficiency of the STX1B is associated with myoclonic astatic epilepsy, and antisense knockdown of stx1b in zebrafish larvae elicits epileptiform discharges. A very recent publication also shows that syntaxin 1B has a separate obligatory role for maintenance of developing and mature neurons and illustrates impaired brain development in syntaxin 1A/1B double knockout mice. We therefore attribute our patient's microcephaly to the truncating homozygous Munc13-1 mutation that consigns syntaxin 1B to a permanently closed nonfunctional state akin to a knockout.

Nicolau S, Milone M. The Electrophysiology of Presynaptic Congenital Myasthenic Syndromes With and Without Facilitation: From Electrodiagnostic Findings to Molecular Mechanisms. Front Neurol. 2019 Mar 19;10:257. doi: 10.3389/fneur.2019.00257. PMID: 30941097; PMCID: PMC6433874.


Congenital myasthenic syndromes (CMS) are a group of inherited disorders of neuromuscular transmission most commonly presenting with early onset fatigable weakness, ptosis, and ophthalmoparesis. CMS are classified according to the localization of the causative molecular defect. CMS with presynaptic dysfunction can be caused by mutations in several different genes, including those involved in acetylcholine synthesis, its packaging into synaptic vesicles, vesicle docking, and release from the presynaptic nerve terminal and neuromuscular junction development and maintenance. Electrodiagnostic testing is key in distinguishing CMS from other neuromuscular disorders with similar clinical features as well as for revealing features pointing to a specific molecular diagnosis. A decremental response on low-frequency repetitive nerve stimulation (RNS) is present in most presynaptic CMS. In CMS with deficits in acetylcholine resynthesis however, a decrement may only appear after conditioning with exercise or high-frequency RNS and characteristically displays a slow recovery. Facilitation occurs in CMS caused by mutations in VAMP1, UNC13A, SYT2, AGRN, LAMA5. By contrast, facilitation is absent in the other presynaptic CMS described to date. An understanding of the underlying molecular mechanisms therefore assists the interpretation of electrodiagnostic findings in patients with suspected CMS.

Thursday, May 23, 2024

Head transplant 8

Retraction record

Numbers are everywhere in retraction land lately: A record 10,000-plus retractions in 2023. 19 journals shut down at Wiley. Now here’s another.

Readers who have checked the Retraction Watch leaderboard lately may have picked up on something notable: One researcher, Joachim Boldt, has now been credited with 210 retractions – making him the first author (to our knowledge) with more than 200 retractions to his name.

Boldt’s new tally – representing about half of his roughly 400 publications – admittedly is an accounting change rather than new problems being identified. Some journals have only now come around to acting on the corrupt articles. In that sense, it reflects both progress and a frustrating lack of concern-slash-urgency on the part of the journals that have taken more than a decade to resolve the case.

As we and others have reported, the Boldt saga began about 14 years ago with emails to Steve Shafer, then the editor-in-chief of Anesthesia & Analgesia, pointing out suspicious data in a study the German anesthesiologist had published in the journal in late 2009. (The BMJ has a helpful timeline about the case, including a bright line linking Boldt’s fraudulent research to potential harm to patients.)

Shafer, who is now a member of our advisory board, eventually became dissatisfied with Boldt’s responses, and then lack of responses, to his questions about the data, and helped trigger an initial institutional investigation into the researcher’s work. Anesthesiology’s recognition that there were problems in the literature – which came earlier for the field than for the vast majority of others – is likely responsible for the fact that so many anesthesiology researchers are among the authors with the most retractions in the world.



Rahman AA, Dell’Aniello S, Moodie EEM, Durand M, Coulombe J, Boivin JF, Suissa S, Ernst P, Renoux C. Gabapentinoids and Risk for Severe Exacerbation in Chronic Obstructive Pulmonary Disease: A Population-Based Cohort Study. Ann Intern Med. 2024;177(2):144-154. doi:


North American and European health agencies recently warned of severe breathing problems associated with gabapentinoids, including in patients with chronic obstructive pulmonary disease (COPD), although supporting evidence is limited.


To assess whether gabapentinoid use is associated with severe exacerbation in patients with COPD. Design: Time-conditional propensity score-matched, new-user cohort study. Setting: Health insurance databases from the Régie de l’assurance maladie du Québec in Canada. Patients: Within a base cohort of patients with COPD between 1994 and 2015, patients initiating gabapentinoid therapy with an indication (epilepsy, neuropathic pain, or other chronic pain) were matched 1:1 with nonusers on COPD duration, indication for gabapentinoids, age, sex, calendar year, and time-conditional propensity score. Measurements: The primary outcome was severe COPD exacerbation requiring hospitalization. Hazard ratios (HRs) associated with gabapentinoid use were estimated in subcohorts according to gabapentinoid indication and in the overall cohort.


The cohort included 356 gabapentinoid users with epilepsy, 9411 with neuropathic pain, and 3737 with other chronic pain, matched 1:1 to nonusers. Compared with nonuse, gabapentinoid use was associated with increased risk for severe COPD exacerbation across the indications of epilepsy (HR, 1.58 [95% CI, 1.08 to 2.30]), neuropathic pain (HR, 1.35 [CI, 1.24 to 1.48]), and other chronic pain (HR, 1.49 [CI, 1.27 to 1.73]) and overall (HR, 1.39 [CI, 1.29 to 1.50]). Limitation: Residual confounding, including from lack of smoking information.


In patients with COPD, gabapentinoid use was associated with increased risk for severe exacerbation. This study supports the warnings from regulatory agencies and highlights the importance of considering this potential risk when prescribing gabapentin and pregabalin to patients with COPD.


As one of the first so-called “second generation” of anti-seizure medications (ASM), gabapentin appeared with promise on the not-yet-crowded field of epilepsy drugs when it was approved for use in the United States in 1994, based primarily on randomized control studies in which it was compared to placebo as an add-on therapy in pharmacoresistant focal epilepsy.1 Its established competitors were long in the tooth, and all had complex pharmacokinetics and interactions that complicated their use. There were a few other new arrivals such as lamotrigine, but none had quite the straightforward profile of gabapentin; a 1994 review noted that the “potential advantages of gabapentin are its pharmacokinetic and toxicity profiles.”1 Doctors like drugs that are safe and simple to prescribe, as shown by the subsequent success of levetiracetam despite its psychiatric side effects. Just as levetiracetam later did, gabapentin might have been expected to eventually emerge from its position as a promising add-on to become a dominant first-line seizure therapy.

In epilepsy, gabapentin never fulfilled its promise. Over time, studies and clinical experience found it to be less efficacious for seizure control than older and newer competitors. This was emphatically demonstrated in the Standard and New Antiepileptic Drugs (SANAD) study of newly diagnosed focal epilepsy.2 Patients were randomized to one of 5 ASMs; gabapentin performed the worst, primarily because of poor efficacy. The authors concluded that “We see no reasons to prefer gabapentin […] to the standard drug carbamazepine, except where there might be individual mitigating factors.”

With its dreams of dominance lost, gabapentin found a smaller role as a utility player, with its pharmacokinetic and safety profile allowing it to carve out a few niches in epilepsy. Most prominently, it was considered as an alternative for geriatric patients, who have a higher burden of comorbidities and medication interactions. An RCT comparing gabapentin, lamotrigine, and carbamazepine in new-onset epilepsy patients 65 years and older found it to be about as efficacious as lamotrigine in preventing seizures, and less frequently stopped than carbamazepine.3 This established gabapentin for a time as at least a reasonable alternative to lamotrigine in elderly patients, although clinical experience suggested that its side effect profile was less favorable. It has also maintained use as a (usually distant) alternative adjunctive therapy in pharmacoresistant therapy and as a choice for patients with epilepsy (or possible epilepsy) who also suffer from conditions such as tremor or neuropathic pain that might also respond to gabapentin.

Pregabalin, the other gabapentinoid which shares structural, mechanistic, and clinical characteristics with gapapentin, was approved by the FDA in 2005 and has carved out a similar profile as an ASM with some efficacity against focal seizures, usually as an adjunctive therapy in specific circumstances.4

But if gabapentinoids were a disappointment in the ASM field, they have become a booming success in the treatment of many other conditions, some with more evidence than others. American Academy of Neurology (AAN) guidelines cite gabapentin as having Level B evidence in support for the treatment of essential tremor5 and insufficient evidence to recommend for migraine prevention,6 and the gabapentinoid class as probably more likely than placebo to improve pain from diabetic polyneuropathy.7

With sometimes limited evidence, physicians seized on the apparent versatility of gabapentinoids to extend their use to many other conditions such as chronic pain, leading to a dramatic increase in off-label prescriptions. One suspects that its apparently simple pharmacokinetic profile and the perception that it can cause no dangerous adverse effects, the qualities that initially brought gabapentin to the ASM market with such hope, have played a crucial role in its expansion. I recall that when I attended my first AAN meeting as a medical student in 2008, one speaker jokingly described gabapentin as so safe it could be put in drinking water.

The present study by Rahman et al challenges this rationale for gabapentinoid use.8 Inspired by reports that gabapentinoids can be responsible for severe adverse events including respiratory distress leading to multiple warnings by regulatory agencies, the authors investigated the risk of exacerbation of chronic obstructive pulmonary disease (COPD) associated with gabapentinoid prescription.

Making use of several large health databases in Québec, Canada, including hospitalization data and prescription records from its public prescription drug insurance plan, the authors created a cohort of patients 55 years and over suffering from COPD, identified primarily based on prescriptions of respiratory drugs. Of these, patients who were prescribed gabapentinoids for epilepsy, neuropathic pain, or other chronic pain were identified and matched 1:1 across multiple demographic and health variables to other individuals from the cohort. Looking primarily at the outcome of hospitalization or death due to COPD exacerbation, they compared patients prescribed gabapentinoids to those without, using a time-conditional propensity score matched design.

The database identified 13 504 COPD patients prescribed gabapentinoids. Interestingly, less than 3% of these were for the indication of epilepsy, reflecting their limited role in epilepsy care. Across all indications, gabapentinoid prescription was associated with higher risk of severe COPD exacerbation, with a hazard ratio (HR) of 1.39. Within the small subset of epilepsy patients, the HR was highest, at 1.58. The authors conclude that, taken together with other forms of accumulating evidence on this topic, their study supports the notion that the risk of COPD exacerbation should be carefully considered when prescribing gabapentinoids.

As with all large database studies, the results should be interpreted with care. Many clinical variables including COPD were identified using definitions that, although often validated by other studies, remain surrogates for their gold standards. Inevitably, some patients will be mislabeled, although hopefully not enough to significantly affect the bottom line. Furthermore, it is possible that unmeasured but relevant factors could have made the compared groups systematically different, thus introducing bias in the results. In this vein, the authors cite the inability to determine whether patients were smokers as a potential limitation.

Nevertheless, the signal identified in the study is strong, and we cannot expect an RCT to help us resolve this question. These are the best data that we have on this question. At minimum, it is incumbent upon neurologists to seriously consider the risk of COPD exacerbation prior to gabapentinoid prescription. Even further, this study reminds us that perceived safety or ease of prescription is hardly justification for the selection of a medication. As use of a medication expands, unsuspected dangers may emerge that are not justified by the risk-reward ratio, especially in the absence of proven benefit.

In epilepsy, gabapentinoids have failed to gain a large foothold mostly due to their relative lack of efficacy. Now, with increasing understanding of their risks, even their niche as a simple and “safe” treatment of elderly patients or patients with polypharmacy will need to be reconsidered.

EHMT1 related Kleefstra syndrome

Inspired by a patient

Lewerissa EI, Nadif Kasri N, Linda K. Epigenetic regulation of autophagy-related genes: Implications for neurodevelopmental disorders. Autophagy. 2024 Jan;20(1):15-28. doi: 10.1080/15548627.2023.2250217. Epub 2023 Sep 6. PMID: 37674294; PMCID: PMC10761153.


Macroautophagy/autophagy is an evolutionarily highly conserved catabolic process that is important for the clearance of cytosolic contents to maintain cellular homeostasis and survival. Recent findings point toward a critical role for autophagy in brain function, not only by preserving neuronal health, but especially by controlling different aspects of neuronal development and functioning. In line with this, mutations in autophagy-related genes are linked to various key characteristics and symptoms of neurodevelopmental disorders (NDDs), including autism, micro-/macrocephaly, and epilepsy. However, the group of NDDs caused by mutations in autophagy-related genes is relatively small. A significant proportion of NDDs are associated with mutations in genes encoding epigenetic regulatory proteins that modulate gene expression, so-called chromatinopathies. Intriguingly, several of the NDD-linked chromatinopathy genes have been shown to regulate autophagy-related genes, albeit in non-neuronal contexts. From these studies it becomes evident that tight transcriptional regulation of autophagy-related genes is crucial to control autophagic activity. This opens the exciting possibility that aberrant autophagic regulation might underly nervous system impairments in NDDs with disturbed epigenetic regulation. We here summarize NDD-related chromatinopathy genes that are known to regulate transcriptional regulation of autophagy-related genes. Thereby, we want to highlight autophagy as a candidate key hub mechanism in NDD-related chromatinopathies.Abbreviations: ADNP: activity dependent neuroprotector homeobox; ASD: autism spectrum disorder; ATG: AutTophaGy related; CpG: cytosine-guanine dinucleotide; DNMT: DNA methyltransferase; EHMT: euchromatic histone lysine methyltransferase; EP300: E1A binding protein p300; EZH2: enhancer of zeste 2 polycomb repressive complex 2 subunit; H3K4me3: histone 3 lysine 4 trimethylation; H3K9me1/2/3: histone 3 lysine 9 mono-, di-, or trimethylation; H3K27me2/3: histone 3 lysine 27 di-, or trimethylation; hiPSCs: human induced pluripotent stem cells; HSP: hereditary spastic paraplegia; ID: intellectual disability; KANSL1: KAT8 regulatory NSL complex subunit 1; KAT8: lysine acetyltransferase 8; KDM1A/LSD1: lysine demethylase 1A; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin complex 1; NDD: neurodevelopmental disorder; PHF8: PHD finger protein 8; PHF8-XLID: PHF8-X linked intellectual disability syndrome; PTM: post-translational modification; SESN2: sestrin 2; YY1: YY1 transcription factor; YY1AP1: YY1 associated protein 1.

Koemans TS, Kleefstra T, Chubak MC, Stone MH, Reijnders MRF, de Munnik S, Willemsen MH, Fenckova M, Stumpel CTRM, Bok LA, Sifuentes Saenz M, Byerly KA, Baughn LB, Stegmann APA, Pfundt R, Zhou H, van Bokhoven H, Schenck A, Kramer JM. Functional convergence of histone methyltransferases EHMT1 and KMT2C involved in intellectual disability and autism spectrum disorder. PLoS Genet. 2017 Oct 25;13(10):e1006864. doi: 10.1371/journal.pgen.1006864. PMID: 29069077; PMCID: PMC5656305.


Kleefstra syndrome, caused by haploinsufficiency of euchromatin histone methyltransferase 1 (EHMT1), is characterized by intellectual disability (ID), autism spectrum disorder (ASD), characteristic facial dysmorphisms, and other variable clinical features. In addition to EHMT1 mutations, de novo variants were reported in four additional genes (MBD5, SMARCB1, NR1I3, and KMT2C), in single individuals with clinical characteristics overlapping Kleefstra syndrome. Here, we present a novel cohort of five patients with de novo loss of function mutations affecting the histone methyltransferase KMT2C. Our clinical data delineates the KMT2C phenotypic spectrum and reinforces the phenotypic overlap with Kleefstra syndrome and other related ID disorders. To elucidate the common molecular basis of the neuropathology associated with mutations in KMT2C and EHMT1, we characterized the role of the Drosophila KMT2C ortholog, trithorax related (trr), in the nervous system. Similar to the Drosophila EHMT1 ortholog, G9a, trr is required in the mushroom body for short term memory. Trr ChIP-seq identified 3371 binding sites, mainly in the promoter of genes involved in neuronal processes. Transcriptional profiling of pan-neuronal trr knockdown and G9a null mutant fly heads identified 613 and 1123 misregulated genes, respectively. These gene sets show a significant overlap and are associated with nearly identical gene ontology enrichments. The majority of the observed biological convergence is derived from predicted indirect target genes. However, trr and G9a also have common direct targets, including the Drosophila ortholog of Arc (Arc1), a key regulator of synaptic plasticity. Our data highlight the clinical and molecular convergence between the KMT2 and EHMT protein families, which may contribute to a molecular network underlying a larger group of ID/ASD-related disorders.

Bock I, Németh K, Pentelényi K, Balicza P, Balázs A, Molnár MJ, Román V, Nagy J, Lévay G, Kobolák J, Dinnyés A. Targeted next generation sequencing of a panel of autism-related genes identifies an EHMT1 mutation in a Kleefstra syndrome patient with autism and normal intellectual performance. Gene. 2016 Dec 31;595(2):131-141. doi: 10.1016/j.gene.2016.09.027. Epub 2016 Sep 17. PMID: 27651234.


Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder with unknown genetic and environmental causation in most of the affected individuals. On the other hand, there are a growing number of ASD-associated syndromes, where the exact genetic origin can be revealed. Here we report a method, which included the targeted next generation sequencing (NGS) and filtering of 101 ASD associated genes, followed by database search. Next, RNA sequencing was used to study the region of interest at the transcriptional level. Using this workflow, we identified a de novo mutation in the euchromatic histone-lysine N-methyltransferase 1 gene (EHMT1) of an autistic patient with dysmorphisms. Sequencing of EHMT1 transcripts showed that the premature termination codon (Trp1138Ter) created by a single nucleotide change elicited nonsense-mediated mRNA decay, which led to haploinsufficiency already at the transcriptional level. Database and literature search provided evidence that this mutation caused Kleefstra syndrome (KS), which was confirmed by the presence of the disorder-specific phenotype in the patient. We provide a proof of principle that the implemented method is capable to elucidate the genetic etiology of individuals with syndromic autism. The novel mutation detected in the EHMT1 gene is responsible for KS's symptoms. In addition, further genetic factors might be involved in the ASD pathogenesis of the patient including a missense DPP6 mutation (Arg322Cys), which segregated with the autistic phenotype within the family.

Blackburn PR, Tischer A, Zimmermann MT, Kemppainen JL, Sastry S, Knight Johnson AE, Cousin MA, Boczek NJ, Oliver G, Misra VK, Gavrilova RH, Lomberk G, Auton M, Urrutia R, Klee EW. A Novel Kleefstra Syndrome-associated Variant That Affects the Conserved TPLX Motif within the Ankyrin Repeat of EHMT1 Leads to Abnormal Protein Folding. J Biol Chem. 2017 Mar 3;292(9):3866-3876. doi: 10.1074/jbc.M116.770545. Epub 2017 Jan 5. PMID: 28057753; PMCID: PMC5339767.


Kleefstra syndrome (KS) (Mendelian Inheritance in Man (MIM) no. 610253), also known as 9q34 deletion syndrome, is an autosomal dominant disorder caused by haploinsufficiency of euchromatic histone methyltransferase-1 (EHMT1). The clinical phenotype of KS includes moderate to severe intellectual disability with absent speech, hypotonia, brachycephaly, congenital heart defects, and dysmorphic facial features with hypertelorism, synophrys, macroglossia, protruding tongue, and prognathism. Only a few cases of de novo missense mutations in EHMT1 giving rise to KS have been described. However, some EHMT1 variants have been described in individuals presenting with autism spectrum disorder or mild intellectual disability, suggesting that the phenotypic spectrum resulting from EHMT1 alterations may be quite broad. In this report, we describe two unrelated patients with complex medical histories consistent with KS in whom next generation sequencing identified the same novel c.2426C>T (p.P809L) missense variant in EHMT1 To examine the functional significance of this novel variant, we performed molecular dynamics simulations of the wild type and p.P809L variant, which predicted that the latter would have a propensity to misfold, leading to abnormal histone mark binding. Recombinant EHMT1 p.P809L was also studied using far UV circular dichroism spectroscopy and intrinsic protein fluorescence. These functional studies confirmed the model-based hypotheses and provided evidence for protein misfolding and aberrant target recognition as the underlying pathogenic mechanism for this novel KS-associated variant. This is the first report to suggest that missense variants in EHMT1 that lead to protein misfolding and disrupted histone mark binding can lead to KS.

A new gene-editing system tackles complex diseases

Cowan, Q.T., Gu, S., Gu, W. et al. Development of multiplexed orthogonal base editor (MOBE) systems. Nat Biotechnol (2024).


Base editors (BEs) enable efficient, programmable installation of point mutations while avoiding the use of double-strand breaks. Simultaneous application of two or more different BEs, such as an adenine BE (which converts A·T base pairs to G·C) and a cytosine BE (which converts C·G base pairs to T·A), is not feasible because guide RNA crosstalk results in non-orthogonal editing, with all BEs modifying all target loci. Here we engineer both adenine BEs and cytosine BEs that can be orthogonally multiplexed by using RNA aptamer–coat protein systems to recruit the DNA-modifying enzymes directly to the guide RNAs. We generate four multiplexed orthogonal BE systems that enable rates of precise co-occurring edits of up to 7.1% in the same DNA strand without enrichment or selection strategies. The addition of a fluorescent enrichment strategy increases co-occurring edit rates up to 24.8% in human cells. These systems are compatible with expanded protospacer adjacent motif and high-fidelity Cas9 variants, function well in multiple cell types, have equivalent or reduced off-target propensities compared with their parental systems and can model disease-relevant point mutation combinations.


The human genome consists of around 3 billion base pairs and humans are all 99.6% identical in their genetic makeup. That small 0.4% accounts for any difference between one person and another. Specific combinations of mutations in those base pairs hold important clues about the causes of complex health issues, including heart disease and neurodegenerative diseases like schizophrenia.

Current methods to model or correct mutations in live cells are inefficient, especially when multiplexing—installing multiple point mutations simultaneously across the genome. Researchers from the University of California San Diego have developed new, efficient genome editing tools called multiplexed orthogonal base editors (MOBEs) to install multiple point mutations at once.

Their work, led by Assistant Professor of Chemistry and Biochemistry Alexis Komor's lab, appears in Nature Biotechnology.

Komor's team was especially interested in comparing genomes that differ at a single letter change in the DNA. Those letters—C (cytosine), T (thymine), G (guanine), A (adenosine)—are known as bases. Where one person has a C base, another person might have a T base. These are single nucleotide variants (SNVs) or single point mutations. A person might have 4–5 million variants. Some variants are harmless; some are harmful; and often it is a combination of variants that confers disease.

One issue with using the genome in disease modeling is the sheer number of possible variations. If scientists were trying to determine which genetic mutations were responsible for heart disease, they could decode the genomes of a cohort that all had heart disease, but the number of variations between any two people makes it very hard to determine which combination of variations causes the disease.

"There is a problem interpreting genetic variants. In fact, most variants that are identified are unclassified clinically, so we don't even know if they're pathogenic or benign," stated Quinn T. Cowan, a recent Ph.D. graduate from the university's Department of Chemistry and Biochemistry and first author on the paper.

"Our goal was to make a tool that can be used in disease modeling by installing multiple variants in a controlled laboratory setting where they can be studied further."

An evolution in gene-editing

To understand why MOBEs were created, we have to understand the limitations of the traditional gene-editing tool CRISPR-Cas9. CRISPR-Cas9 uses a guide RNA, which acts like a GPS signal that goes straight to the genomic location you want to edit. Cas9 is the DNA-binding enzyme that cuts both strands of the DNA, making a complete break.

Although relatively straightforward, double-stranded breaks can be toxic to cells. This kind of gene-editing can also lead to indels—random insertions and deletions—where the cell is not able to perfectly repair itself. Editing multiple genes in CRISPR-Cas9 multiples the risks.

Instead of CRISPR, Komor's lab uses a base-editing technique she developed, which makes a chemical change to the DNA, although only one type of edit (C to T or A to G, for example) can be made at a time. So, rather than scissors that cut out an entire section at once, base-editing erases and replaces one letter at a time. It is slower, but more efficient and less harmful to cells.

Simultaneously applying two or more base editors (changing a C to T at one location, and an A to G at another location in the genome), allows for better modeling of polygenic diseases—those occurring due to more than one genetic variant. However, a technology didn't exist that could do this efficiently without guide RNA "crosstalk," which happens when base editors make unwanted changes.

Cowan's MOBEs use RNA structures called aptamers—small RNA loops that bind to specific proteins—to recruit base-modifying enzymes to specific genomic locations enabling simultaneous editing of multiple sites with high efficiency and a lower incidence of crosstalk.

This system is novel and is the first time someone used aptamers to recruit ABEs (adenosine base editors) in combination with CBEs (cytosine base editors) in an orthogonal pattern to make the MOBEs.

The differences are stark: when CBE and ABE are given together not using MOBE, crosstalk occurs up to 30% of the time. With MOBE, crosstalk is less than 5%, while achieving 30% conversion efficiency of the desired base changes.

The study was a proof of principle to test the feasibility of the MOBE system, which has been granted a provisional patent. To test them even further, the team conducted several case studies with real diseases, including Kallmann syndrome, a rare hormonal disorder. Their experiments revealed that MOBE systems could be used to efficiently edit relevant cell lines of certain polygenic diseases.

"We're in the process of putting the plasmids up on AddGene so anyone can freely access them. Our hope is that other researchers will use the MOBEs to model genetic diseases, learn how they manifest and then hopefully create effective therapies," stated Cowan.

Wednesday, May 22, 2024

Acute flaccid myelitis 6

Suddenly, in the middle of a blizzard on April 5, 2023, Kellen was rushed to Children’s Hospital in Minneapolis. Over the course of just a few days, the otherwise strong seventh grader lost his ability to walk and talk. Kellen was sedated for weeks while his parents were preparing for the strong possibility that their 13-year-old son might be paralyzed for the rest of his life.

"It was also very scary watching your child really struggle with things like breathing," says Heather Knutson, Kellen’s mom. "When nurses and doctors would all rush into the room to help, you could tell in their eyes and their actions that it was a scary moment for them as well."

Through weeks of testing, doctors determined Kellen had acute flaccid myelitis, a rare neurological condition that affects the spinal cord and nervous system. His parents were told a couple of unknown bug bites were likely to blame because Kellen tested positive for two infectious diseases. One was Jamestown Canyon Virus, which comes from mosquitoes, and the other was Powassan virus, which is tickborne. Both are known to be present around the time of early melt, which in Minnesota often happens in March, just before Kellen started feeling weak.

"Cases like Kellen’s are not the norm," says Dr. Matt Severson from Gillette Children’s Hospital. "His case is quite rare."

Then one day, Kellen moved a toe, and more and more physical and occupational rehab at Gillette Children’s Hospital followed. Dr. Severson has helped Kellen with his progress for months. He wants to remind parents that this rare situation isn’t to scare people from enjoying the many benefits of getting outdoors but to drive home the importance of tick checks and paying attention to any health changes, especially if you are aware of bug bites.

"Things like rashes, if they develop fevers, they feel really fatigued and tired. If they're complaining of any muscle or joint pain or headaches, then that would be a good time. If any of those symptoms come up, either talk to your local pediatrician or, if their gut sense says this is more urgent than that – we're really seeing changes happen quickly – take your child to the local emergency department," says Dr. Severson.

For outdoor experts like park ranger Jess Althoff, two layers of tick protection are required by the DNR. Whether it’s gators or tucking pants into socks to protect ankles, plus DEET bug spray or permethrin spray on clothing, Althoff doesn’t take any chances.

"Before I even leave my house to come to work for the day, or if I’m going to be out recreating, it's really important that I think about those things before I leave home. So I’m thinking about what clothes I’m wearing, what sort of forms of tick protection I’m going to wear, and what I can protect my body with," says Althoff. "For me, once a year, I send my clothes into a company that can treat all of my clothing so that I don't have to worry about spraying that on regularly."

In Kellen’s case, he was never aware of any sort of bug bite, so there’s nothing different the self-proclaimed "indoor kid" on the robotics and swim team could have done. His hard work over the past year is paying off, as he is recently back in the pool with his eighth-grade swim team.

"That felt more natural than on land. That felt good," says Kellen.

"I didn't care that he won or anything. It was just like the fact that he's there, he's competing against all of these able-bodied humans, as not completely himself, was pretty, pretty awesome," says Phil Knutson, Kellen’s dad.

And the race to return to his normal life gives everything a new perspective.

"I feel like with what I had, you know, you wouldn't really know how it turned out, but I feel like I got the luckiest end possible," says Kellen.

Tuesday, May 21, 2024


A Bothell family is suing Seattle Children’s hospital, alleging the death of 16-year-old Sahana Ramesh was caused by racial discrimination in her health care and negligence.

In February 2021, Ramesh, the daughter of Indian immigrants, died from myocarditis — inflammation of the heart muscle that can lead to heart failure — after months of suffering from a rare disease known as Drug Reaction with Eosinophilia and Systemic Symptoms, or DRESS.

At the center of the case: Ramesh was never tested for myocarditis and never admitted to the hospital when suffering from DRESS — treatment her family says she would have received had she not been of South Asian descent.

Judge Elizabeth Berns on Thursday denied the hospital’s request to dismiss the racial discrimination claim, writing in her order that the family has “plausibly alleged harm, including dignitary harm, resulting from unlawful discrimination — not from health care.”

In a statement, a Seattle Children’s hospital spokesperson said they could not comment on Ramesh’s case.

“Our hearts go out to any family mourning the loss of a child and we take our responsibility to provide equitable, high-quality care seriously, but cannot comment on this specific case due to pending litigation,” the statement read.

In August 2020, after experiencing recurring seizures, Ramesh was prescribed lamotrigine by a Seattle Children’s hospital neurologist to treat her seizures and underlying anxiety.

By mid-November 2020, Ramesh had broken out into a painful and intense rash all over her body, with her hands, feet and face swelling. Seattle Children’s physicians diagnosed her with DRESS — a severe allergic reaction to medication that has various reported mortality rates, ranging from 3.8% to 10-20%.

After the diagnosis, doctors discharged Ramesh. Her symptoms worsened.

Over the next three months, Ramesh’s parents brought her to the emergency room repeatedly as she experienced frequent chills, a fever, extreme pain, a swelling face and an elevated heart rate. But doctors declined to admit her.

“They’re watching her symptoms worsen and they keep being sent home,” said Steve Berman, an attorney representing the family.

Blood work and lab testing “showed increasing problems with her liver and other facets of her organ functioning,” according to the complaint, but Seattle Children’s hospital physicians told Ramesh’s parents she could be safely treated at home.

Doctors did not tell Ramesh’s parents of the potentially life-threatening complications related to DRESS, the suit alleged, and cardiac-specific testing was not performed.

The family contacted the hospital via email or phone over 50 times to ask for help, Berman said, with “very little response.”

“Oftentimes they were ignored. Calls would be made and no one would respond,” Berman said. “There was once a ten-day lag, and they got an apology for being so late.”

On February 12, 2021, Ramesh collapsed and died at home in front of her family.

“They were treated differently than white people would’ve been,” Berman said. “People know when they’re discriminated against, and looking back, the Ramesh [family] know they were discriminated against.”

Ramesh’s family first filed their lawsuit against the hospital alleging negligence in October 2022.

They amended their complaint last year after their lawyers found a pattern of racial discrimination allegations at the hospital, Berman said.

In November 2020, the same month Ramesh was diagnosed with DRESS by Seattle Children’s physicians, the former medical director of the Odessa Brown Children’s Clinic at Seattle Children’s Hospital resigned.

At the time, Dr. Benjamin Danielson pointed to institutional racism and other issues that he said jeopardized the safety of patients and staff as the cause for his departure. He sued the hospital for racial discrimination and retaliation in October.

In 2021, in the wake of Danielson’s resignation, Seattle Children’s announced it would take steps to dismantle systemic racism within its institution to improve health equity among patients and promote diversity and inclusion among staff.

One of the physicians who treated Ramesh, Dr. Emily Hartford, published a paper in June 2022 on racial disparities in migraine treatment for children at the hospital between 2016 and 2020.

Reviewing medical records of more than 800 children, she found Asian, Black and Hispanic children, and children who received care in a language other than English, were significantly less likely than white children to receive intravenous medication for pain relief, despite reporting similar pain levels.

The court had previously granted a protective order allowing Seattle Children’s not to disclose records detailing the racial demographics of other children treated for DRESS and whether they were admitted to the hospital. Attorneys for the hospital had argued producing the records while complying with laws on health care information disclosure would be too burdensome.

With the new Thursday order allowing the discrimination claim to move forward, Berman said his legal team will again request the release of those records.

Courtesy of a colleague

Monday, May 20, 2024

Niemann-Pick type C 2

Hosseini K, Fallahi J, Razban V, Sirat RZ, Varasteh M, Tarhriz V. Overview of clinical, molecular, and therapeutic features of Niemann-Pick disease (types A, B, and C): Focus on therapeutic approaches. Cell Biochem Funct. 2024 Jun;42(4):e4028. doi: 10.1002/cbf.4028. PMID: 38715125.


Niemann-Pick disease (NPD) is another type of metabolic disorder that is classified as lysosomal storage diseases (LSDs). The main cause of the disease is mutation in the SMPD1 (type A and B) or NPC1 or NPC2 (type C) genes, which lead to the accumulation of lipid substrates in the lysosomes of the liver, brain, spleen, lung, and bone marrow cells. This is followed by multiple cell damage, dysfunction of lysosomes, and finally dysfunction of body organs. So far, about 346, 575, and 30 mutations have been reported in SMPD1, NPC1, and NPC2 genes, respectively. Depending on the type of mutation and the clinical symptoms of the disease, the treatment will be different. The general aim of the current study is to review the clinical and molecular characteristics of patients with NPD and study various treatment methods for this disease with a focus on gene therapy approaches.

Servín Muñoz IV, Ortuño-Sahagún D, Griñán-Ferré C, Pallàs M, González-Castillo C. Alterations in Proteostasis Mechanisms in Niemann-Pick Type C Disease. Int J Mol Sci. 2024 Mar 29;25(7):3806. doi: 10.3390/ijms25073806. PMID: 38612616; PMCID: PMC11011983.


Niemann-Pick Type C (NPC) represents an autosomal recessive disorder with an incidence rate of 1 in 150,000 live births, classified within lysosomal storage diseases (LSDs). The abnormal accumulation of unesterified cholesterol characterizes the pathophysiology of NPC. This phenomenon is not unique to NPC, as analogous accumulations have also been observed in Alzheimer's disease, Parkinson's disease, and other neurodegenerative disorders. Interestingly, disturbances in the folding of the mutant protein NPC1 I1061T are accompanied by the aggregation of proteins such as hyperphosphorylated tau, α-synuclein, TDP-43, and β-amyloid peptide. These accumulations suggest potential disruptions in proteostasis, a regulatory process encompassing four principal mechanisms: synthesis, folding, maintenance of folding, and protein degradation. The dysregulation of these processes leads to excessive accumulation of abnormal proteins that impair cell function and trigger cytotoxicity. This comprehensive review delineates reported alterations across proteostasis mechanisms in NPC, encompassing changes in processes from synthesis to degradation. Additionally, it discusses therapeutic interventions targeting pharmacological facets of proteostasis in NPC. Noteworthy among these interventions is valproic acid, a histone deacetylase inhibitor (HDACi) that modulates acetylation during NPC1 synthesis. In addition, various therapeutic options addressing protein folding modulation, such as abiraterone acetate, DHBP, calnexin, and arimoclomol, are examined. Additionally, treatments impeding NPC1 degradation, exemplified by bortezomib and MG132, are explored as potential strategies. This review consolidates current knowledge on proteostasis dysregulation in NPC and underscores the therapeutic landscape targeting diverse facets of this intricate process.

Goicoechea L, Torres S, Fàbrega L, Barrios M, Núñez S, Casas J, Fabrias G, García-Ruiz C, Fernández-Checa JC. S-Adenosyl-l-methionine restores brain mitochondrial membrane fluidity and GSH content improving Niemann-Pick type C disease. Redox Biol. 2024 Jun;72:103150. doi: 10.1016/j.redox.2024.103150. Epub 2024 Apr 3. PMID: 38599016; PMCID: PMC11022094.


Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized by impaired motor coordination due to neurological defects and cerebellar dysfunction caused by the accumulation of cholesterol in endolysosomes. Besides the increase in lysosomal cholesterol, mitochondria are also enriched in cholesterol, which leads to decreased membrane fluidity, impaired mitochondrial function and loss of GSH, and has been shown to contribute to the progression of NPC disease. S-Adenosyl-l-methionine (SAM) regulates membrane physical properties through the generation of phosphatidylcholine (PC) from phosphatidylethanolamine (PE) methylation and functions as a GSH precursor by providing cysteine in the transsulfuration pathway. However, the role of SAM in NPC disease has not been investigated. Here we report that Npc1-/- mice exhibit decreased brain SAM levels but unchanged S-adenosyl-l-homocysteine content and lower expression of Mat2a. Brain mitochondria from Npc1-/- mice display decreased mitochondrial GSH levels and liquid chromatography-high resolution mass spectrometry analysis reveal a lower PC/PE ratio in mitochondria, contributing to increased mitochondrial membrane order. In vivo treatment of Npc1-/- mice with SAM restores SAM levels in mitochondria, resulting in increased PC/PE ratio, mitochondrial membrane fluidity and subsequent replenishment of mitochondrial GSH levels. In vivo SAM treatment improves the decline of locomotor activity, increases Purkinje cell survival in the cerebellum and extends the average and maximal life spam of Npc1-/- mice. These findings identify SAM as a potential therapeutic approach for the treatment of NPC disease.

Friday, May 17, 2024

Abnormality of early white matter development in tuberous sclerosis complex and autism spectrum disorder

Srivastava S, Yang F, Prohl AK, Davis PE, Capal JK, Filip-Dhima R, Bebin EM, Krueger DA, Northrup H, Wu JY, Warfield SK, Sahin M, Zhang B; TACERN Study Group. Abnormality of Early White Matter Development in Tuberous Sclerosis Complex and Autism Spectrum Disorder: Longitudinal Analysis of Diffusion Tensor Imaging Measures. J Child Neurol. 2024 May 15:8830738241248685. doi: 10.1177/08830738241248685. Epub ahead of print. PMID: 38751192.


Background: Abnormalities in white matter development may influence development of autism spectrum disorder in tuberous sclerosis complex (TSC). Our goals for this study were as follows: (1) use data from a longitudinal neuroimaging study of tuberous sclerosis complex (TACERN) to develop optimized linear mixed effects models for analyzing longitudinal, repeated diffusion tensor imaging metrics (fractional anisotropy, mean diffusivity) pertaining to select white matter tracts, in relation to positive Autism Diagnostic Observation Schedule-Second Edition classification at 36 months, and (2) perform an exploratory analysis using optimized models applied to all white matter tracts from these data. Methods: Eligible participants (3-12 months) underwent brain magnetic resonance imaging (MRI) at repeated time points from ages 3 to 36 months. Positive Autism Diagnostic Observation Schedule-Second Edition classification at 36 months was used. Linear mixed effects models were fine-tuned separately for fractional anisotropy values (using fractional anisotropy corpus callosum as test outcome) and mean diffusivity values (using mean diffusivity right posterior limb internal capsule as test outcome). Fixed effects included participant age, within-participant longitudinal age, and autism spectrum disorder diagnosis. Results: Analysis included data from n = 78. After selecting separate optimal models for fractional anisotropy and mean diffusivity values, we applied these models to fractional anisotropy and mean diffusivity of all 27 white matter tracts. Fractional anisotropy corpus callosum was related to positive Autism Diagnostic Observation Schedule-Second Edition classification (coefficient = 0.0093, P = .0612), and mean diffusivity right inferior cerebellar peduncle was related to positive Autism Diagnostic Observation Schedule-Second Edition classification (coefficient = -0.00002071, P = .0445), though these findings were not statistically significant after multiple comparisons correction. Conclusion: These optimized linear mixed effects models possibly implicate corpus callosum and cerebellar pathology in development of autism spectrum disorder in tuberous sclerosis complex, but future studies are needed to replicate these findings and explore contributors of heterogeneity in these models.

Preventative treatment of tuberous sclerosis complex with sirolimus

Capal, J.K., Ritter, D.M., Franz, D.N., Griffith, M., Currans, K., Kent, B., Martina Bebin, E., Northrup, H., Koenig, M.K., Mizuno, T., Vinks, A.A., Galandi, S.L., Zhang, W., Setchell, K.D.R., Kremer, K.M., Prada, C.M., Greiner, H.M., Holland-Bouley, K., Horn, P.S. and Krueger, D.A. (2024), Preventative treatment of tuberous sclerosis complex with sirolimus: Phase I safety and efficacy results. Ann Child Neurol Soc.



Tuberous sclerosis complex (TSC) results from overactivity of the mechanistic target of rapamycin (mTOR). Sirolimus and everolimus are mTOR inhibitors that treat most facets of TSC but are understudied in infants. We sought to understand the safety and potential efficacy of preventative sirolimus in infants with TSC.


We conducted a phase 1 clinical trial of sirolimus, treating five patients until 12 months of age. Enrolled infants had to be younger than 6 months of age with no history of seizures and no clinical indication for sirolimus treatment. Adverse events (AEs), tolerability, and blood concentrations of sirolimus measured by tandem mass spectrometry were tracked through 12 months of age, and clinical outcomes (seizure characteristics and developmental profiles) were tracked through 24 months of age.


There were 92 AEs, with 34 possibly, probably, or definitely related to treatment. Of those, only two were grade 3 (both elevated lipids) and all AEs were resolved by the age of 24 months. During the trial, 94% of blood sirolimus trough levels were in the target range (5–15 ng/mL). Treatment was well tolerated, with less than 8% of doses held because of an AE (241 of 2941). Of the five patients, three developed seizures (but were well controlled on medications) at 24 months of age. Of the five patients, four had normal cognitive development for age. One was diagnosed with possible autism spectrum disorder.


These results suggest that sirolimus is both safe and well tolerated by infants with TSC in the first year of life. Additionally, the preliminary work suggests a favorable efficacy profile compared with previous TSC cohorts not exposed to early sirolimus treatment. Results support sirolimus being studied as preventive treatment in TSC, which is now underway in a prospective phase 2 clinical trial (TSC-STEPS).

Depression and euthanasia

A physically healthy, 28-year-old Dutch woman has decided to legally end her life due to her struggles with crippling depression, autism and borderline personality disorder, according to a report.

Zoraya ter Beek, who lives in a small village in the Netherlands near the German border, is scheduled to be euthanized in May — despite being in love with her 40-year-old boyfriend and living with two cats.

Ter Beek, who once aspired to be a psychiatrist, has been dealing with mental health struggles throughout her life.

She said she decided to be euthanized after her doctors told her, “There’s nothing more we can do for you. It’s never gonna get any better,” according to the Free Press.

“I was always very clear that if it doesn’t get better, I can’t do this anymore,” ter Beek said.

She is just one of the growing number of people in the West who have decided to die rather than continue living in pain that, unlike a terminal illness, could be treated.

More people are deciding to end their lives while suffering from a slew of other mental health problems like depression or anxiety amplified by economic uncertainty, climate change, social media and other issues, the Free Press reported.

Ter Beek said she will be administered the life-ending drug on her couch with her boyfriend by her side.

“I’m seeing euthanasia as some sort of acceptable option brought to the table by physicians, by psychiatrists, when previously it was the ultimate last resort,” Stef Groenewoud, a health care ethicist at Theological University Kampen, in the Netherlands, told the outlet.

“I see the phenomenon especially in people with psychiatric diseases, and especially young people with psychiatric disorders, where the health care professional seems to give up on them more easily than before,” she added.

Ter Beek plans to be cremated after she’s euthanized on the couch in her living room.

“No music,” she said.

The genetic cause of spinocerebellar ataxia 4

Spinocerebellar ataxia 4 is a devastating progressive movement disease that can begin as early as the late teens. Now, a multinational research team led by University of Utah researchers has conclusively identified the genetic difference that causes the disease, bringing answers to families and opening the door to future treatments.

Some families call it a trial of faith. Others just call it a curse. The progressive neurological disease known as spinocerebellar ataxia 4 (SCA4) is a rare condition, but its effects on patients and their families can be severe. For most people, the first sign is difficulty walking and balancing, which gets worse as time progresses. The symptoms usually start in a person's forties or fifties but can begin as early as the late teens. There is no known cure. And, until now, there was no known cause.

Now, after 25 years of uncertainty, a multinational study led by Stefan Pulst, M.D., Dr. med., professor and chair of neurology, and K. Pattie Figueroa, a project manager in neurology, both in the Spencer Fox Eccles School of Medicine at University of Utah, has conclusively identified the genetic difference that causes SCA4, bringing answers to families and opening the door to future treatments. Their results are published in the peer-reviewed journal Nature Genetics.

Solving a genetic enigma

SCA4's pattern of inheritance had long made it clear that the disease was genetic, and previous research had located the gene responsible to a specific region of one chromosome. But that region proved extraordinarily difficult for researchers to analyze: full of repeated segments that look like parts of other chromosomes, and with an unusual chemical makeup that makes most genetic tests fail.

To pinpoint the change that causes SCA4, Figueroa and Pulst, along with the rest of the research team, used a recently developed advanced sequencing technology. By comparing DNA from affected and unaffected people from several Utah families, they found that in SCA4 patients, a section in a gene called ZFHX3 is much longer than it should be, containing an extra-long string of repetitive DNA.

Isolated human cells that have the extra-long version of ZFHX3 show signs of being sick -- they don't seem able to recycle proteins as well as they should, and some of them contain clumps of stuck-together protein.

"This mutation is a toxic expanded repeat and we think that it actually jams up how a cell deals with unfolded or misfolded proteins," says Pulst, the last author on the study. Healthy cells need to constantly break down non-functional proteins. Using cells from SCA4 patients, the group showed that the SCA4-causing mutation gums up the works of cells' protein-recycling machinery in a way that could poison nerve cells.

Hope for the future

Intriguingly, something similar seems to be happening in another form of ataxia, SCA2, which also interferes with protein recycling. The researchers are currently testing a potential therapy for SCA2 in clinical trials, and the similarities between the two conditions raise the possibility that the treatment might benefit patients with SCA4 as well.

Finding the genetic change that leads to SCA4 is essential to develop better treatments, Pulst says. "The only step to really improve the life of patients with inherited disease is to find out what the primary cause is. We now can attack the effects of this mutation potentially at multiple levels."

But while treatments will take a long time to develop, simply knowing the cause of the disease can be incredibly valuable for families affected by SCA4, says Figueroa, the first author on the study. People in affected families can learn whether they have the disease-causing genetic change or not, which can help inform life decisions such as family planning. "They can come and get tested and they can have an answer, for better or for worse," Figueroa says.

The researchers emphasize that their discoveries would not have been possible without the generosity of SCA4 patients and their families, whose sharing of family records and biological samples allowed them to compare the DNA of affected and unaffected individuals. "Different branches of the family opened up not just their homes but their history to us," Figueroa says. Family records were complete enough that the researchers were able to trace the origins of the disease in Utah back through history to a pioneer couple who moved to Salt Lake Valley in the 1840s.

Since meeting so many families with the disease, studying SCA4 has become a personal quest, Figueroa adds. "I've been working on SCA4 directly since 2010 when the first family approached me, and once you go to their homes and get to know them, they're no longer the number on the DNA vial. These are people you see every day… You can't walk away. This is not just science. This is somebody's life."

This work was performed in collaboration with researchers from University of Tübingen, University of Lübeck and Kiel University, University Hospital Hamburg-Eppendorf, and Veterans Administration Medical Center, Albany, NY.

The study was supported by the National Institute of Neurological Disorders and Stroke of the National Institutes of Health under award number R35127253 and the DFG-funded INST 37/1049-1.

Figueroa KP, Gross C, Buena-Atienza E, Paul S, Gandelman M, Kakar N, Sturm M, Casadei N, Admard J, Park J, Zühlke C, Hellenbroich Y, Pozojevic J, Balachandran S, Händler K, Zittel S, Timmann D, Erdlenbruch F, Herrmann L, Feindt T, Zenker M, Klopstock T, Dufke C, Scoles DR, Koeppen A, Spielmann M, Riess O, Ossowski S, Haack TB, Pulst SM. A GGC-repeat expansion in ZFHX3 encoding polyglycine causes spinocerebellar ataxia type 4 and impairs autophagy. Nat Genet. 2024 Apr 29. doi: 10.1038/s41588-024-01719-5. Epub ahead of print. PMID: 38684900.


Despite linkage to chromosome 16q in 1996, the mutation causing spinocerebellar ataxia type 4 (SCA4), a late-onset sensory and cerebellar ataxia, remained unknown. Here, using long-read single-strand whole-genome sequencing (LR-GS), we identified a heterozygous GGC-repeat expansion in a large Utah pedigree encoding polyglycine (polyG) in zinc finger homeobox protein 3 (ZFHX3), also known as AT-binding transcription factor 1 (ATBF1). We queried 6,495 genome sequencing datasets and identified the repeat expansion in seven additional pedigrees. Ultrarare DNA variants near the repeat expansion indicate a common distant founder event in Sweden. Intranuclear ZFHX3-p62-ubiquitin aggregates were abundant in SCA4 basis pontis neurons. In fibroblasts and induced pluripotent stem cells, the GGC expansion led to increased ZFHX3 protein levels and abnormal autophagy, which were normalized with small interfering RNA-mediated ZFHX3 knockdown in both cell types. Improving autophagy points to a therapeutic avenue for this novel polyG disease. The coding GGC-repeat expansion in an extremely G+C-rich region was not detectable by short-read whole-exome sequencing, which demonstrates the power of LR-GS for variant discovery.