Fragile X premutation carriers

CHAPTER 5

Prof. Randi Hagerman, with Prof. Paul Hagerman and Prof. Flora Tassone

Historical perspective The background for understanding clinical involvement in Fragile X premutation carriers dates back to the 1980s and highlights the importance of working closely with families and in an interdisciplinary scientific environment. After we started the National Fragile X Foundation – a US organisation – in 1984, we worked on planning conferences every other year. These conferences included both families and professionals because we knew each group could learn from the other. At one of these conferences in Denver in the late 1980s, a group of mothers who had children with Fragile X syndrome and were therefore premutation carriers, were sitting at the luncheon table with me and several other researchers. One of the mothers was lamenting that she had gone into early menopause before the age of forty. Subsequently, two of the other mothers at the table commented that the same had happened to them. This prompted my colleague and genetic counsellor, Amy Cronister, and me to assess our data on carrier mothers. We found that early menopause was a problem in a significantly larger number of these premutation carrier mothers (eight of sixty-one) compared to none in female controls

who were not carriers (Cronister et al., 1991, American Journal of Medical Genetics). This is a good example of how the experiences of families can guide research. Subsequently, early menopause – or Fragile X-associated Primary Ovarian Insufficiency (FXPOI), as it is now called – has been found to be a common problem, affecting about 20% of premutation carriers in studies from many other centres throughout the world.

Another important discovery came after seeing children affected with intellectual disability displaying many features of Fragile X syndrome, but DNA testing only showed the premutation. This was an unexpected result because, in the mid-1990s, carriers were thought to be ‘non-penetrant’, meaning they had no clinical problems, with the exception of FXPOI, which was only just becoming accepted at that time. My husband, Prof. Paul Hagerman, and I discussed this at length, and we thought that perhaps the gene was turning off or becoming methylated for shorter CGG repeat expansions than the 200 CGG repeat boundary of the Fragile X full mutation range.

Meanwhile, Dr Flora Tassone was running the immunocytochemistry testing for the Fragile X mental retardation protein (FMRP) at Kimball Genetics and testing many subjects. It turned out that a few of the premutation carriers showed a lower percentage of cells expressing FMRP. That was strange because we knew that they had the premutation that was unmethylated, so therefore it did not make sense at the time that FMRP was lower. We even collected additional blood samples and observed the same thing. Flora thought looking at the ribonucleic acid (RNA) strand would help us understand why FMRP was lower. At the time, Flora found out about TaqMan real-time PCR. However, the only machine available in the US was located in San Francisco and Flora was located in Paul’s lab in Denver. She contacted Prof. Tony

Godfrey, who managed the Genome Analysis Core Facility at the University of California, San Francisco campus (UCSF), and raised the idea of travelling to UCSF to test a number of subjects with Paul. He agreed. All the premutation carriers Flora tested turned out to have high FMR1 mRNA levels, and the higher the CGG repeat within the premutation range, the higher the mRNA levels. This finding was published in a landmark paper, the first edition of the American Journal of Human Genetics to be published in the new millennium (Tassone et al., 2000).

In the late 1990s, many premutation mothers of children with Fragile X syndrome shared their worries about their fathers having neurological problems. After reviewing many detailed pedigree assessments she had conducted, Louise Gane, my genetic counsellor, noticed and pointed out to me that one grandfather had cerebellar ataxia, another had atrophy of the cerebellum, and another had other neurological problems that looked somewhat like Parkinson’s disease. One of the mothers in the group who had two children with Fragile X syndrome brought her father to see me when I was conducting a clinic at the Children’s Hospital in Oakland. He was an electrician who had developed a tremor in his mid-fifties, followed by balance problems or ataxia a few years later. When I examined him with finger to nose testing, he was almost unable to touch my finger because his hand was shaking so much.

After seeing the first patient with FXTAS, who had tremor and balance problems, I subsequently saw many more grandfathers of patients with Fragile X syndrome, and I began to follow them clinically. They all had the same phenotype, both physically and behaviourally, including intention tremor followed by ataxia and gradual cognitive decline with White Matter Disease and brain atrophy shown on MRI results. My main research area was behavioural phenotypes and I realised that these men presented

with similar symptom progression. These findings were associated with the premutation, but not observed in those with Fragile X syndrome as older adults. I thought this was a rare finding until I presented the first five cases at a workshop for parents and family members during the National Fragile X Foundation Conference held in Los Angeles in July 2000. After presenting the five patients, together with Paul and the psychologists from Denver, I asked the audience if they had ever seen such problems in older family members who were carriers of a premutation. There was an overwhelming response, with about a third of the roughly 200 women in the audience raising their hands. It was in that moment that we realised these problems were not rare in families with the FMR1 mutation.

Following this presentation, I had asked the editor of the American Journal of Human Genetics if he would publish our findings, but he denied my request as he was of the belief that carrier males were ‘non-penetrant’. I subsequently submitted our paper to the Neurology Journal because they did not have a preconceived notion that carrier males were unaffected. Although the reviewers of the paper did not recommend acceptance, the editor liked the paper, and it was eventually published in 2001 (Hagerman et al., 2001, Neurology).

While waiting for our flights home from the conference, I remember telling Robby Miller, the Director of the National Fragile X Foundation at the time, ‘This revelation is big, really big for the field’. Indeed it was, and the field of premutation involvement became a new research endeavour in the subsequent decades that distinguished itself from the field of Fragile X syndrome. Almost twenty years later, hundreds of papers have been published by researchers around the world and several animal models have been generated to study the pathogenesis of the premutation disorders.

At the end of the year 2000, Paul and I and many of our team members, including Flora Tassone and Louise Gane, moved from Denver to the MIND Institute at the University of California Davis Medical Center in Sacramento. The grandfather of one of my patients had been in a nursing home and was wheelchairbound due to balance problems. Just before I left, the family told me that he had died, and I asked them if they would consider donating his brain to our research. They agreed, and we carried his brain with us, carefully stored in formaldehyde, all the way to California. Once settled in Davis, I asked one of the neuropathologists at the MIND Institute, Dr Claudia Greco, if she would examine his brain. I will never forget her excited phone call to tell me she had found intranuclear inclusions in his brain that were like no others she had ever seen! That was a very thrilling moment because we knew we were in the midst of discovering a marker of this new condition that had never been reported before. Fortunately, we were able to obtain other brains of those who had died from this disease and Dr Greco found the same unique inclusions in all of the premutation brains examined. She reported her findings in 2002 (Greco et al., 2002, Brain). Paul’s laboratory subsequently worked on isolating and characterising the intranuclear inclusions, or ‘molecular piñatas’, as he liked to call them, to discover its contents.

Dr Sebastien Jacquemont, one of the young researchers who had come to work with us at the MIND Institute in 2000, became fascinated with this new condition and began working with Louise Gane, travelling all over California to study the older carriers within the Fragile X families we had identified. It was a total ascertainment study whereby we assessed tremor and balance problems in all individuals within the families who were fifty or older (n=192). About half of the individuals assessed had the premutation, documented by Prof. Flora Tassone, while the others

were negative. However, the total ascertainment of all the families combined with the blinded assessment allowed us to demonstrate the prevalence of this tremor and ataxia condition as a function of age. We found that male carriers who were fifty years and older had a thirteen-fold increased risk of tremor and balance problems than those who weren’t carriers. The percentage of those with tremor and balance problems increased significantly with age. Seventeen per cent of male carriers in their fifties had tremor and balance problems, 38% in their sixties, 47% in their seventies and 75% for those aged eighty or over. Female carriers also had a higher risk of experiencing these problems than the controls, but none presented with clear FXTAS and their symptoms were milder than the males experienced.

It was not until 2004, after my nurse at the MIND Institute informed me that her grandmother, a Hispanic woman by the name of Amalia Guerrero, was a carrier, that we reported FXTAS in females. Amalia was a social worker and a pioneer who fought hard to start the Regional Centers for the Intellectually Disabled in California in the 1950s because two of her children had Fragile X syndrome. She was widely regarded as a local hero because, in those days, Hispanic women did not go against the wishes of their husbands to fight for social rights like she did. Amalia had developed tremor and ataxia in her ageing years as well as gastrointestinal complications following surgery. By this stage, she was terminally ill at the hospital and she and her family were keen to donate her brain for research after she passed. When she died, we were able to collect her brain immediately following the time of death. Her precious gift led to the documentation of intranuclear inclusions, one of the main inclusion criteria for FXTAS diagnosis, as well as the documentation of FXTAS in women (Hagerman et al., 2004, American Journal of Human Genetics).

As Paul and I were members of the Society for the Study of Behavioral Phenotypes (SSBP), we presented the results of our study at the annual conference. One of the attendees was the editor of JAMA (Journal of the American Medical Association), Dr Cathy DeAngeles, and she was intrigued by this new condition, which presented with a high frequency in premutation carriers, and asked us to write a paper for JAMA. The editor was renowned for being very demanding, but as the publication had one of the highest impact ratings of all journals, it was important to get it right. Paul spent hours meticulously assessing and revising each draft before the paper was finally accepted and published (Jacquemont et al., 2004, JAMA). Read all over the world, it was a keystone paper, a friend from Stockholm even telling us that the findings made it onto the front page of their local paper!

Paul and I tossed around many ideas to come up with a name for the condition. We did not want it to be named ‘Hagerman syndrome’ as so many genetic disorders are named after their discoverers. Instead, we thought about naming it FRAXA to differentiate it from Fragile X syndrome and because FRAXA was also another name for the FMR1 gene. However, FRAXA was also the name of the FRAXA Research Foundation which did not believe in premutation involvement. Therefore, we settled on the Fragile X-associated Tremor Ataxia syndrome (FXTAS – pronounced ‘fax-tass’) which is a mouthful but is an accurate description of the core features of the syndrome.

We worked with our neuroradiologist, Dr James Brunberg, to describe the characteristic White Matter Disease in 2002, as the middle cerebellar peduncle (MCP sign) was present in approximately 60% of males with FXTAS in addition to brain atrophy and periventricular White Matter Disease (Brunberg et al., 2002). Other collaborators, including Maureen Leehey and

Deborah Hall, have published on the age of onset, the progression and misdiagnosis of many with FXTAS, in addition to the medications that are helpful for the tremor and balance problems. Prof. Jim Grigsby, a professor of psychology from the University of Colorado, has always collaborated on the neuropsychological features of FXTAS and has written many papers on this. More recently, Prof. Flora Tassone and Dr Deborah Hall edited a book on FXTAS called FXTAS, FXPOI and other premutation disorders (2012; 2018) with all our collaborators and researchers from around the world contributing chapters on various aspects of the phenotype and treatment. Dr David Hessl and Dr Susan Rivera have spearheaded a grant to follow middle-aged, premutation male carriers to determine the earliest radiological and neurocognitive signs of FXTAS. Dr Jun Yi Wang has brilliantly assessed MRIs of those with the premutation from childhood to adulthood all the way through the ageing process to plot the trajectory of atrophy and white matter changes through the lifespan (Wang et al., 2018).

Few believed that FXTAS was a real entity after the initial report in 2001 and subsequent lectures detailing the inclusions and neuropathology in 2002. I vividly remember Dr Jean-Pierre Fryns, a famous geneticist from Belgium, raising his hand after my lecture at the Fragile X and X-linked ID meeting in Rome in 2001 and remarking that the inclusions I presented were ‘just a normal part of the ageing process in Belgium’ and therefore had no bearing on the content of my presentation. I was used to Dr Fryns’s scepticism from previous conferences, including his comments about there being ‘no treatment for FXS’, and in the past, I would have no comeback to such comments. This time, however, I was prepared. I simply responded, ‘The intranuclear inclusions may be normal in Belgium but they are not normal in the US and are characteristic of FXTAS’. I remember the email I received from Prof. Gillian Turner and Dr Michael Partington following the conference in which they

apologised for the poor reception my presentation on FXTAS had received. They also wrote that they were both very interested in this new disorder and in surveying their carrier population to find those with tremor and balance problems. They followed through and subsequently reported a significant percentage with FXTAS, as did many other centres in the world. Currently, there are many conditions that occur at a higher rate in premutation carriers compared to those without the premutation. Hypertension, migraines, insomnia, autoimmune disorders, fibromyalgia and psychiatric problems are more common in carriers than in controls. The most common carrier problems are anxiety and depression, with these conditions occurring in approximately 50% of carriers. To bring more attention to the psychiatric problems in both children and adults with the premutation, the umbrella term, ‘Fragile X-associated Neuropsychiatric Disorders’ (FXAND), was coined in an effort to stimulate more research into treatment options for the carriers presenting with these symptoms (Hagerman et al., 2018, Frontiers in Psychiatry). Much research is needed to understand how the elevated mRNA leads to toxicity and how to treat and reverse the disorders associated with the premutation, including FXTAS, FXPOI and FXAND.

Authors

Prof. Randi J Hagerman MD is the medical director of the MIND Institute and Director of the Fragile X Research and Treatment Center at the University of California, Davis, Sacramento and is internationally recognised as both a clinician and researcher in the Fragile X field. Together with her husband, Paul Hagerman, she discovered the Fragile X-associated Tremor Ataxia syndrome (FXTAS) in 2001. Prof. Paul Hagerman MD PhD is a professor in the Department of Biochemistry and Molecular Medicine and a MIND Institute

investigator at the University of California, Davis, School of Medicine, Sacramento. Dr Hagerman is a molecular geneticist with a principal interest in understanding the basis for neurodevelopmental and neurodegenerative disease.

Randi and Paul Hagerman are co-editors of Fragile X Syndrome and Premutation Disorders: New Developments and Treatments. (London, MacKeith Press, 2020)

Prof. Flora Tassone PhD is Professor-In-Residence at the MIND Institute Wet Lab, UC Davis, Sacramento. The main focus of Dr Tassone’s research is to understand the molecular mechanisms and the correlation with clinical involvements of neurodevelopmental and neurodegenerative disorders. Her expertise is in transcriptional and translational gene regulation, particularly of the Fragile X (FMR1) gene.

Profs Paul and Randi Hagerman, Prof. Flora Tassone. Australia, 2015.