Margaret's Story: The unfolding discovery of the Fragile X gene

Margaret’s Story The unfolding discovery of the Fragile X gene

Prof Gillian Turner ao & contributors

Margaret’s Story The unfolding discovery of the Fragile X gene

Prof. Gillian Turner ao & contributors

Margaret’s Story First published August 2021 by Fragile X Association of Australia Revised November 2022 All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording or by any information storage and retrieval system, without prior permission in writing from the publisher. The Australian Copyright Act 1968 (the Act) allows a maximum of one chapter or 10% of this book, whichever is the greater, to be photocopied by any educational institution for its educational purposes provided that the educational institution (or body that administers it) has given a remuneration notice to the Copyright Agency (Australia) under the Act. ISBN 978-0-6488878-0-5 Paperback ISBN 978-0-6488878-1-2 Epub

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Contents Introduction Professor Gillian Turner ao Margaret Cunningham

iv viii x

CHAPTER 1

Margaret’s story Margaret Cunningham

1

CHAPTER 2

X & Y – a brief historical perspective Prof. Gillian Turner ao

9

CHAPTER 3

The X-linked inheritance factor Prof. Gillian Turner ao

19

CHAPTER 4

My personal journey with Fragile X syndrome Prof. Grant R Sutherland

25

CHAPTER 5

Fragile X premutation carriers Prof. Randi Hagerman, with Prof. Paul Hagerman and Prof. Flora Tassone

35

CHAPTER 6

Carrier screening for Fragile X Prof. W Ted Brown MD

45

Acknowledgements

50

About Fragile X Association of Australia

53

Introduction Prof. Gillian Turner ao Margaret Cunningham (nee Hughes) was born in 1940. Unbeknown to her parents or doctors, she was a carrier of an X-linked inherited condition called Fragile X syndrome. Nobody could possibly have known she was a carrier because human chromosomes would not be examined for the first time until the late 1950s. It wasn’t until 1969 that American geneticist, Dr Herbert Lubs, discovered the link between the fragile site on the X chromosome and X-linked intellectual disability (ID). A further twenty-two years passed before the discovery of the Fragile X (FMR1) gene mutation was made in 1991. The reason it took so long to make the discovery was because the mechanism for disrupting the function of the gene remained unknown over that time. During the years inbetween, it was Australian research that led the way in building an understanding of the impact of changes to the FMR1 gene and Fragile X syndrome. Margaret has lived every day of her life coping with the effects of Fragile X on her family, and now also on herself. For many years, she did not know what was wrong with her second child, Sean. As medical science gradually recognised and could diagnose Fragile X syndrome, she and her husband John adapted to each new phase of discovery and its consequences. I first met Margaret in about 1970 at the Sydney Children’s Hospital when she took part in research my genetic team and I were conducting. Little did we know at the time how significant that meeting and her | iv |

involvement in our research would be. However, my team and I weren’t the only ones pursuing research in this area. Not long after we had started, Professor Grant Sutherland began studying fragile sites on various chromosomes in Adelaide, where he was working as Head of Cytogenetics at Adelaide Children’s Hospital (now the Women’s and Children’s Hospital, Adelaide). A breakthrough discovery Professor Sutherland made in 1977 sparked a friendly competition between the two of us and we later collaborated, publishing a joint paper in 1985. We made a formidable team, jointly discovering the FMR1 gene mutation in 1991, a major scientific breakthrough. I am proud to say, Professor Sutherland and I led the world in both clinical and genetic aspects of X-linked intellectual disability and, most notably, Fragile X syndrome from the late 1970s until the early 1990s. The research we pioneered was continued by Professor Randi Hagerman in the United States, and several others. Many advancements in the knowledge about Fragile X syndrome occurred over the years since I first met Margaret, and some of how this occurred is described in this booklet by a few of the key people involved, including Professor Sutherland, Professor Hagerman and myself. While we were motivated at the time by the need to find answers for families, we were also fuelled by the pure excitement of discovery. As mentioned, much of the work began here in Australia before eventually becoming a worldwide endeavour. Every human being carries the Fragile X gene, also known as the FMR1 gene. The gene is responsible for producing a protein called FMR1-Protein (FMRP), which is necessary for normal brain development and function. Fragile X-associated conditions are caused by a lengthening (expansion) of a promoter upstream of the FMR1 gene on the X chromosome, which can reduce or shut down the production of FMRP. The changes in the FMR1 gene can go through stages as that gene is passed down in a family. These | v |

stages start with the normal FMR1 gene and can expand to the premutation (Fragile X carrier) and then the full mutation (Fragile X syndrome). Fragile X syndrome is the leading cause of inherited intellectual disability and the most common single gene cause of autism spectrum disorder. Both males and females can be carriers of the Fragile X gene premutation. It is estimated that around one in 250 females and one in 800 males are carriers. A female carrier has a 50% chance of passing on the Fragile X gene change, either as a premutation or a full mutation, to a child of either sex, while male carriers pass the Fragile X gene premutation to all daughters but not to any sons. Around one in 3,600 males and one in 4,000 to 6,000 females are born with Fragile X syndrome. It affects individuals differently. People with Fragile X syndrome can experience delayed development, anxiety, and learning, speech and language difficulties. They can also present with autism spectrum disorder and epilepsy. The effects can range from mild to severe, however, males tend to be more severely affected than females. That being said, some females are also severely affected. In 2019, the Australian Federal Government funded an important research study known as ‘Mackenzie’s Mission’, named after Mackenzie Casella, who was diagnosed at ten weeks of age with spinal muscular atrophy (SMA) – a severe, inherited neuro­ muscular condition for which there is no cure. Mackenzie’s parents, Rachael and Jonathon, were unaware that they were both genetic carriers for the condition. After Mackenzie passed away at just seven months of age, the Casellas launched a campaign calling for reproductive genetic carrier screening to be made freely available for all prospective parents in Australia. This built on many years of advocacy in the SMA community for accessible genetic carrier screening for the condition. | vi |

The research study has offered reproductive carrier screening to around 10,000 couples across Australia between 2019 and 2022, with a view to investigating the best way to deliver a national reproductive carrier screening program to all couples who want it in Australia. I find it remarkable how one family’s story has become the stimulus for action, in contrast to written applications made through genetic services over a number of years, which were unsuccessful. Work on a potential cure for Fragile X syndrome using new techniques has also been very promising in recent years. So, a new era is dawning involving prevention, treatments and even a potential cure for Fragile X syndrome, which is very exciting. Margaret and John have generously provided the Fragile X Association of Australia, which Margaret co-founded in 1989, with the funding to tell this story. It begins with Margaret describing the long-term effect Fragile X has had on her family, followed by unique insights of the discoverers, written by some of the people who led the way to finding answers.

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Professor Gillian Turner ao Professor Gillian Turner ao is a distinguished physician, an inter­ nationally acclaimed geneticist and a worldwide authority on the X chromosome. Gillian contributed fifty-four years of service to the field of paediatrics and genetics before retiring in 2010. Her work led to new screening techniques for genetic disabilities. Among her most important work in the 1970s was contributing to the discovery of the gene which causes Fragile X syndrome and defining the importance of X-linked intellectual disability. Gillian studied medicine at St Andrews University, Scotland, graduating in 1956. She trained in Paediatrics in Canada and London before emigrating to Australia. She became interested in developmental disability and genetics while working with her husband, Dr Brian Turner, at Johns Hopkins Hospital, Baltimore, Maryland in the US. She was a research fellow with the Children’s Medical Research Foundation in Sydney from 1970 to 1976 and the Director of the Tumbatin Clinic for Assessing Children with Disability at the Sydney Children’s Hospital for a decade. As Associate Professor of Medical Genetics at the Prince of Wales Hospital, Sydney between 1986 and 1994, Gillian established a genetic outreach service for regional areas of New South Wales and ran the NSW Fragile X screening program. In 1994 she became Director of Hunter Genetics and the GOLD (Genetics of Learning | viii |

Disability) service and was appointed the first Professor of Medical Genetics at the University of Newcastle. Gillian’s publications include Y the X? Unravelling intellectual disability and autism (2012) and Finding the Sapiens genes (2019). She was recognised for her outstanding contribution to the field of medicine in being made an Officer of the Order of Australia (AO) for her services to genetics in 1990. She is an honorary member of the Human Genetics Society of Australia.

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Margaret Cunningham Margaret Cunningham has had significant involvement in the support of families affected by Fragile X syndrome and other disabilities for over forty years. As the mother of a son with an intellectual disability of unknown cause, she was a pivotal participant in the first Fragile X discovery group in Australia during the 1970s. Margaret and her husband John had known something was significantly different about their son Sean’s development and behaviours, however, the cause of his intellectual impairment remained a mystery for many years, despite seeing numerous specialists. That all changed when Margaret met Doctor (now Professor) Gillian Turner and became part of Dr Turner’s discovery group, which comprised a number of families whose children all had an intellectual disability of unknown cause. Key findings from the group significantly influenced Australian and international interest in and further research into Fragile X syndrome. As a proactive member of the Fragile X discovery group, Margaret engaged regularly with Dr Turner and her research, which later confirmed the diagnosis of Fragile X syndrome for Sean. They worked together on a key publication in 1985 and Dr Turner and Professor Grant Sutherland were subsequently instrumental in the discovery of the Fragile X gene in 1991. | x |

Since that time, Margaret has discovered that she too is affected by Fragile X. She decided to use her experience to help others. In 1985, she founded the Eastern Respite and Recreation organisation in Sydney, which grew to thirty employees and 150 volunteers. She served as President of that organisation for nineteen years and was made a Life Member in recognition of her service. Margaret also co-founded a parent support group that evolved into the Fragile X Association of Australia (FXAA). FXAA is a registered charity and the primary contact for supporting Fragile X families across Australia, educating health professionals to promote early and accurate diagnosis and advocating for research and public policy change in areas impacting the Fragile X community. In addition to her work as founder and her dedication to providing personal support, Margaret has been a major annual donor for the organisation. In recognition of her significant contribution to FXAA over a twenty-five year period, Margaret was awarded the inaugural Life Membership in 2012.

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CHAPTER 1

Margaret’s story Margaret Cunningham I am writing as the mother of a fifty-six-year-old son affected by Fragile X syndrome. While my husband John and I have been through some difficult times in raising our son, we are proud of the man he has become. I am sharing my story in the hope that it will help other families affected by Fragile X syndrome and to shed light on the part we played in the discovery process of the Fragile X gene, something you will learn a lot more about when you read the subsequent chapters in this booklet, as they have been written by pioneers and experts in the field. To paint the picture, I need to begin this story by briefly taking you back to my own family history. I was born in Sydney in 1940, the eldest of three daughters. My middle sister and I were very bright and went to Sydney Girls High School, a selective state school. Both of us were awarded Commonwealth scholarships to go to university. I considered the idea of studying medicine but decided to commence employment in the legal profession. I later met John, whom I subsequently married at the age of twenty. Looking back now, I can see there was evidence of Fragile X in my family history long before the breakthrough genetic discovery was made. My father was a dentist who had symptoms of Fragile X-Associated Tremor Ataxia syndrome (FXTAS), an adult-onset | 1 |

neurodegenerative disorder that can often be misdiagnosed as Parkinson’s disease due to the unsteadiness and hand tremors sufferers commonly present with. I can still recall my father’s shaky hands and Parkinson’s-like movements before he died. In later years, my mother was tested and found not to be a carrier. John’s and my second child, a son we named Sean, was a large baby who smiled a lot and had a huge appetite. The first sign that something wasn’t quite right was the way he would occasionally stiffen and throw himself back when picked up as an infant. This would happen completely without warning and for no apparent reason. He also seemed to be late at reaching certain milestones. I initially thought he may have just been a little slow when he made no attempt to roll over or sit up in those early months. However, he eventually did sit up at ten months. Then a couple of weeks later he started crawling and pulling himself up to stand, so I thought nothing more of it. An adventurous child with no sense of danger, Sean always seemed to have bruises on his body. At about eighteen months of age, while our GP was visiting – back when home GP visits were the norm – Sean displayed one of his stiffening attacks. Our GP suggested I take him to see Professor Stephens, a paediatrician at Prince of Wales Hospital, Sydney. The professor ran a series of tests and found nothing wrong. His conclusion was that Sean’s body was simply too big for his brain to cope and that things would right themselves by the age of five or six. However, my concerns that Sean was slow to reach the usual milestones were once again raised when I noticed he was very slow to talk. When Sean was close to turning three, my GP suggested we take him to an assessment centre at Grosvenor House in Sydney. He was seen by Dr Turner and Dr Maloney who diagnosed him with Cerebral Gigantism, now called Sotos syndrome, a non-progressive | 2 |

neurological disorder with intellectual disability characterised by a larger than normal head. He had IQ tests done which put him in the 80–100 range, however, it was his speech and comprehension which pulled him down. Blood tests were performed at Ryde Hospital to determine the presence of an X or Y chromosome in his blood. These were just a few of the many rounds of tests and assessments our son was to endure over the ensuing years. Sean attended preschool part-time at the age of three and mixed well with the children. However, when we moved him to a more structured kindergarten, he became frustrated and struggled to make friends. The worst part was when there were birthday parties and all the children were invited except Sean, which was very upsetting, both for Sean and for John and me as his parents. He desperately wanted to be like other children, and I can still remember how delighted he was when he was allowed to wear normal shoes rather than his usual orthopaedic boots! By this stage, John and I were well aware that our son had a problem and, while we accepted this, we wanted to know the cause and what we could do for his future education. So, we did our own research, asked a lot of questions and became known by some as ‘difficult parents’. As Sean grew older, he was placed in the special class at school but, realising he would benefit from activities that would help him become as independent as possible, we enrolled him in the local Cub Scouts group, which proved to be a great decision. Encouraged by the Cub leader, Sean earned badges for completing simple challenges, which he would wear on his shirt with pride, and went on camps, always returning exhausted but happy. At school, his teacher was determined to help Sean gain his independence, insisting that he travel to school on his own, which, in his case, meant catching two buses. Ten years old at the time, he took to | 3 |

commuting like a duck to water. He memorised all the bus route numbers, even managing to get back to school after accidentally getting onto the wrong bus one morning and ending up at Circular Quay. John and I were amazed at his capacity to do this all on his own. Little did we know how beneficial this skill would prove to be in later years as Sean has difficulty speaking and is unable to ask for help. After primary school, Sean attended Wairoa Special School in Bondi which he enjoyed as there was no pressure on him there. He loved playing sport and was part of the four-person relay swimming team which won the NSW Handicapped Schools Competition. When Sean was sixteen, we received some life-changing news. After years of wanting to know what was wrong with our son, I got a call from Dr Turner to say she had the answer. Sean had Fragile X syndrome. I vividly remember the sense of relief I felt to finally have a diagnosis and know the cause of his learning and behavioural problem. After finishing school, Sean went to work at Disability Services Australia in Redfern. He settled in well and enjoyed going there. As Sean entered adulthood, John and I realised we would have to make plans to gradually move him away from our influence and protection. We felt strongly that he should be assimilated into the community, so this was a very important step for us. We were pointed in the direction of a social worker from the local area health service, who guided us through the steps to prepare Sean to leave home and live independently. We psyched ourselves up for what we believed would be a lengthy process. Then a miracle happened. After just three visits with the social worker, Sean came to us and said he wanted to move out! He had not long moved when he called to let me know he had an earache and needed to see | 4 |

a doctor. This may seem trivial, but it was an enormous milestone for someone with limited communication skills who had never made a single phone call before. Sean has been working with the same disability services provider now for thirty-eight years, and for thirty-six of those years, he travelled there on his own by bus. Those early days of solo bus travel had prepared him well. These days he receives community transport through the NDIS. He even met a girl at work and was very keen on her, but unfortunately, she moved to Newcastle and they quickly lost touch. Like many people with an intellectual disability, Sean does not seem to have a special friend outside of work, which is sad as it means he stays home with us a lot despite having his own apartment close by. Don’t get me wrong, we love his company. It’s just sad for him because he should be enjoying his independence and the company of people his own age at this stage of his life. Fortunately, he genuinely loves going to work and it was difficult recently when he had to have an operation on his toe, which put him out of action for a while. Sean has been associated with Waverley Community Care for around thirty-six years – he was their first client – and receives carer help from the Waverley Community Living Program through the NDIS. This involves a one-on-one outing each Saturday and Sunday for four hours, a cooking session at his apartment every Thursday evening and participation in a group ‘breaking news’ session every Monday. He also goes to the gym three times a week. Because he has a speech problem, he spends a lot of time watching television. He particularly loves watching his favourite rugby league team, Parramatta, play as well as British TV shows. He is very aware of what is happening in the world and watches the news at least once a day. He can also emergency read bus timetables and is able to read the football fixtures. | 5 |

These days, my life is very different as I am no longer trying to be Sean’s mother, companion, friend and teacher. While our story may appear as though everything has gone very smoothly for us, I can assure you nothing could be further from the truth. I could fill pages telling you of the bad times, of our down moments, of the tears and emotions every parent of a Fragile X child has no doubt experienced. However, I have chosen to focus on the positives so that Sean may be seen as an example of what can be achieved by allowing a person to grow. You may be wondering at this point about our extended family. Like any family dealing with the complexities of a little-known inherited condition, the impacts are experienced differently by every family member, and I can openly say we endured some significant challenges over the years. The elder of my two sisters has three children, a son and two daughters, and her son has Fragile X syndrome. He too was seen by Dr Turner. My youngest sister, who recently passed away, was also a carrier but had no children. She was diagnosed with rheumatoid arthritis, which she assumed was a complication of being a carrier, having read about Fragile X-associated neuropsychiatric and other disorders (FXAND). Our story is a prime example of how Fragile X is carried from generation to generation and manifests itself in different ways. I am so pleased genetic testing is now available, sparing others the heartache we endured. I only hope it will be funded through Medicare one day, so it is truly available to everyone, not only to those who can afford it. Looking back, I believe that had we known what was ahead of us in the early years, we would have decided not to have our son. However, I am so glad we didn’t have to make that decision as he has grown into a great young man of whom we are very proud. Our life would not be the same without him. | 6 |

Our biggest concern now is what will happen to our son when we are no longer around. Right now, Sean fluctuates between his apartment and our house, which are only 300 metres apart. He is unable to make appointments for himself and, as he does not understand money, he can only shop with notes and bring the change home. He has used ATMs on his own for many years. We are very concerned for his future and know that he will need a practical living coordinator to assist him with daily living when we aren’t here to help. Fortunately, he has an outstanding financial coordinator through our family accountant. People like Sean are fine as long as someone organises them and is proactive. As for me, I wondered if I was developing the premutation FXTASrelated condition at the age of seventy-three and went to see a neurologist. He tested my walking and noted that I was unable to walk heel to toe, however, I had no tremors, so we decided to keep an eye on things. I saw the neurologist again two years later after having had a couple of falls, once falling forwards on my face and another time falling backwards. It was around this time that I also started using a walking stick. I’d had a knee replacement, but the stick was as much to assist with my deteriorating balance as it was for my knee. Dr Randi Hagerman confirmed my diagnosis of FXTAS. Unfortunately, I’m one of the 25% of female carriers affected by this condition. So far, I have developed ataxia and fibromyalgia. On the upside, I’m now back doing assisted gym work three times a week with a physiotherapist to improve my upper body strength. The only advice I can give anyone else in this situation is to stay positive and don’t give in to it, which is a philosophy I try to live my life by each and every day.

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Photo of Prof. Gillian Turner (on right) with Mrs Margaret Cunningham. Photo taken by Fragile X Association of Australia in 2019.

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CHAPTER 2

X & Y – a brief historical perspective Prof. Gillian Turner The fact that X and Y chromosomes determine the sex of offspring was first identified by a woman – American geneticist, Nettie Stevens, in the late 1900s. But it wasn’t until 1956, when human chromosomes could be grown and identified under the microscope, that the real connection between changes in chromosomes was identified as a major cause of intellectual disability. Each event that occurred along the way had a slice of history attached to it. This chapter briefly explores that history, beginning with Darwin’s Survival of the Fittest theory. In the 19th century, Charles Darwin educated the reading public on evolution by putting forward his Survival of the Fittest theory. He was writing at a time before chromosomes were known about. His theory of blended inheritance proposed that seminal material from the male and female contained tiny gemmules that developed into cells. This theory persisted for years. Meanwhile, thinner and thinner slices of tissue could be cut and examined under the new microscopes that were being developed. An idea considered extremely radical by the scientific community was proposed by Nettie Stevens at the turn of the 20th century. The daughter of a carpenter, Nettie spent her early life as a teacher | 9 |

but longed to conduct scientific research. By the age of thirty-five, she had saved enough money to attend university and, at thirtynine, had fulfilled her dream of becoming a research scientist. In 1900, Nettie examined the sperm of the meal worm and noted two different sizes of chromosomes; one carried a large chromosome and the other carried a much smaller one. The sperm carrying a small chromosome produced male offspring and the sperm carrying the larger one produced female offspring. This prompted Nettie to conclude that the sex of offspring was determined by the male. Her idea was not readily accepted by other scientists of the era.

Nettie Stevens (1861–1912), American geneticist. Source: Wikipedia

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The next breakthrough discovery happened in 1910 when Thomas Hunt Morgan, one of the scientists who refused to acknowledge Nettie’s work, discovered X-linked inheritance.

Thomas H Morgan (1866–1945), American biologist and geneticist. Source: Wikipedia

His eureka moment came when he was studying inheritance in the fruit fly. He observed one fruit fly that looked different to the others and, with the aid of his microscope, noted it was a male with white eyes, as opposed to the usual red eyes. After many crossbreeding experiments, Morgan deduced that no female flies with white eyes were being born because they did not survive. He was a strong believer in Darwin’s pangenesis theory, which describes the units of inheritance between parents and offspring and in which he coined the concept of gemmules. It was not until he rediscovered the work of Austrian monk, Gregor Mendel, which saw Mendel experiment with peas in his garden, crossing | 11 |

wrinkled or smooth peas with either green or yellow ones, that Morgan gradually accepted the idea of chromosomal inheritance. This prompted him to publish the results of his experiments, delineating X-linked inheritance. In 1933, Morgan was awarded a Nobel Prize for that work.

Fruit fly (Drosophila melanogaster). Illustration @_HETAKA

Significant time passed between Morgan’s breakthrough discovery and the next major advancement. It was 1943 when Purdon Martin and Julia Bell produced a joint paper describing a family with X-linked inheritance of intellectual disability. Highly regarded in their respective fields, Martin was a neurologist at Queen’s Square in London and Bell was a geneticist working in Lionel Penrose’s genetics institute, who had written a five-volume book on genetic diseases. Martin followed the family that was the subject of their paper for nineteen years before publishing. They were the first known family with Fragile X (proven in later tests) but described long before chromosomes were examined. Tracing their lineage, there were two grandfather brothers of normal intelligence who had each passed the genetic mutation down to their daughters | 12 |

who, in turn, each bore affected sons. Today we know how this can happen due to each grandfather being a premutation carrier.

Julia Bell (1879–1979).

James Purdon Martin FRCP (1893–1984).

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It was not until years later (1956), that human chromosomes could be examined. As chromosomes can only be seen when cells are dividing, bone marrow was used to examine the division of the white cells. It was subsequently discovered that if you grew white cells from a blood sample cultured in a medium containing colchicine, a drug used to treat gout, the division process was arrested and only an ordinary blood sample was needed. Down syndrome was the first condition in which a chromosome abnormality was detected, although it had been recognised for many years due to the distinctive facial features and markings on the hands observed in those with the condition.

An example of a karyotype.

I was a paediatrician in training in Canada and became interested in chromosomes after attending a scientific meeting in Toronto in 1958 where I attended talks about the new chromosome discoveries and viewed images of them. After completing my training, I was considering three job choices: a post in Nigeria, one in Alaska and a year’s fellowship at the Sydney Children’s Hospital, which included a six-month research stint. I accepted the Sydney offer. | 14 |

After settling down to work in Sydney, I enquired whether or not any labs had mastered the growth of chromosomes. There was one, run by Dr Brian Turner, a neuropathologist and director of a pathology lab based in a psychiatric hospital that serviced the needs of all institutions. I jumped at the chance to work there. Brian was full of ideas and energy. He had established newborn PKU (phenylketonuria) screening in New South Wales and also a service looking at chromosomes. Space was limited at the lab, so they set me up in one of the women’s toilets with the male toilet becoming unisex. My attempts at growing chromosomes did not go too well, however, on the bright side, Brian and I fell in love and we eventually married and had two children together! At that time, the advice given to parents of a child with an intellectual disability was, ‘Put him in an institution and have another baby’. However, to obtain admission, you needed to know somebody of importance. This all changed after a twenty-threeyear-old man with no speech, who had lived in an institution from the age of three, presented the medical superintendent with a perfect model replica of the Endeavour he had constructed from old matchsticks. The young man was found to be deaf but of normal intelligence. The story hit the newspapers. Government funding was found to open an assessment centre, the first in Sydney. Its goal was to determine the cause of the disability on a case-by-case basis and develop a list of criteria for admission based on need. At this time – the late sixties and seventies – chromosome studies had become routine and newer techniques could look for small deletions and duplication of chromosomes. I had some experience in assessing individuals with intellectual disability in the US and needed a part-time job, so I applied for a position at the new assessment centre. The NSW Health Department didn’t employ part-timers, so the job was unofficially | 15 |

split between myself and a psychiatrist by the name of Helen Maloney. It was at this time that my path first crossed with Margaret Cunningham. Margaret brought Sean in to be assessed and I initially diagnosed him as having Cerebral Gigantism, or Sotos syndrome, because of his size. He was a big lad and Sotos syndrome is now known to be due to a mutation on Chromosome 5. My diagnosis would prove to be wrong. Later, we worked on 2000 files at the centre to try to work out the rate of recurrence in order to answer a frequently asked question posed by parents: ‘Will it happen again if we have another baby?’ That study found that moderately handicapped males with no diagnosis had a one in ten chance of recurrence, whereas in females, the chance was much less, only one in forty-eight. This supported the idea that X-linkage might be important. While working at the centre, I met five families that had an X-linked pattern of intellectual disability inheritance. I was jokingly coerced by the team into presenting these five families at a scientific research meeting. I took the files home one evening, hoping to find that the fifteen affected males had features in common to make the presentation more interesting. I lined them up in columns with a list of possible distinctive physical features, but the physical features column remained disappointingly empty. They were all ‘normal’. Suddenly it dawned on me that to be normal in a handicapped group with disability was actually abnormal. My husband, Brian (Dr Turner) was equally excited by this discovery. On my return to work the next morning, I set out to analyse the file for all the males presenting with a moderate handicap but without a diagnosis. Of the 140 in that group, there were seventeen who met the criteria and eleven of those seventeen had a family tree which included other males connected through their mothers.

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I gave the talk, but it had no impact. To tell the audience that physical normality meant that the inheritance might be X-linked probably sounded too far-fetched at the time. I wrote up my finding, concluding that perhaps 10% of intellectual disabilities was X-linked, and submitted it as a letter to the medical journal, The Lancet. No response. But I was sure I was onto something big. I just needed to conduct further studies to prove it. I asked the clinic boss for an afternoon off a week to pursue the idea, but she knocked me back saying, ‘Too much work to do’. My response to her came without thinking, ‘Okay, I’ll resign and do it in my own time’, which is precisely what I did, with Brian’s full support. In conducting my studies, the first place I visited was Peat Island, an institution on the Hawkesbury River in New South Wales with 480 male residents. I asked the medical superintendent for permission to examine all pairs of brothers residing there and to be given access to their parents. My request was approved. There were eighteen pairs in total. Seven had notable physical features of their intellectual disability, while eleven were physically ‘normal’. Finding the parents proved to be an enormous challenge as most of the men had not been visited for years. Eventually, I was able to find most of them and these home visits proved to be very rewarding. They confirmed that those who were ‘normal’ in appearance often had an X-linked family history. I made a lot of friends at those home visits, once they knew I was not from the welfare department! They had some fascinating stories to tell. I went on to organise a postal survey supported by the NSW Department of Education. At that time, people with an intellectual disability were taught in special schools or classes, not assimilated as the majority are today. I asked each school principal to send me a list of all their pupils with an intellectual disability, recording whether they had affected brothers or sisters and also noting | 17 |

those with Down syndrome. Opening the principals’ lists became progressively more exciting. The results showed that there were fifty-eight pairs of brothers, but only twenty-two pairs of sisters. I assumed the number of pairs of sisters represented the other forms of inherited intellectual disorders, while the male excess must have been due to X-linkage. Knowing how many pairs of brothers were in the general population, I was able to calculate the prevalence of X-linkage from that data. I sent my report off to the British Journal of Medical Genetics for publication. The editor wrote back that some of my arithmetic calculations were wrong but that he would correct those errors and publish the report. The proof that X-linkage was of major importance was building up. Other groups in England, the US and Canada were reaching the same conclusions. Robert Lehrke, a psychologist from Wisconsin, wrote up his conclusions as a PhD thesis. In it, he suggested that the mutations causing intellectual disability were in the genes that developed the evolution of Homo sapiens. He was subsequently directed by his examiners to delete that conclusion. However, I’m sure he was correct. In fact, in my latest book, Finding the Sapiens genes (Exlibris 2020), and in general discussion, I refer to them as the ‘Sapiens genes’ to include the important contribution that the FMR1 gene makes.

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CHAPTER 3

The X-linked inheritance factor Prof. Gillian Turner ao In chapter 1, Margaret explains that she first learnt of Sean’s diagnosis from me when he was sixteen years old and what a relief it was to know, at last, the cause of his intellectual and behavioural problems. At that time, I was in a new job running an assessment clinic for those with intellectual disabilities at the new Sydney Children’s Hospital. Interestingly, Margaret’s sister also had a son with significant learning difficulties, and she and her sister may have wondered whether this was due to chance or the result of X-linked inheritance. To understand this, they would have had to know how X and Y chromosomes work. Put simply, the X chromosome and its partner, the Y chromosome, make up one of the twenty-three pairs of chromosomes in every cell. Females have a pair of X’s, one of which rests or is ‘inactivated’, while males have a single X whose partner is a very small Y chromosome. So, in families with an X-linked condition, only males are significantly affected, while women are partially protected as half of their cells carry an X without a mutation. It was the 1970s and Professor Grant Sutherland had published his work on how to grow chromosomes. His discovery of the low folic acid culture medium, TC199, was a significant breakthrough because the medium enabled fragile sites to become visible for the first time. In fact, our cytogenetics lab was using his technique | 19 |

to identify those with Fragile X. We finally had a tool we could use to screen males with intellectual disability (ID) for Fragile X syndrome! It is interesting to speculate today what would have happened had Grant (Professor Sutherland) not persisted in his experimentation with different media for growing the chromosomes and why finding the right medium was so important. Without a laboratory test for Fragile X syndrome, it might still be undefined and have remained to this day just another X-linked cause of intellectual disability. His significant finding speaks to the chance in discovery, but also to his extra­ ordinary persistence. This chapter explores the next part of the Fragile X journey from my perspective and explains how a relatively small screening program evolved into a state-wide research project that ended up painting a much bigger picture to reveal the X-linked inheritance factor. Our population screening program for Fragile X was the first of its kind in the world. We were fortunate to be successful in obtaining a grant to fund screening for all those with ID within the 1.2 million population of the area we serviced: the south-eastern suburbs of Sydney. However, we knew that only 15% of families affected by ID had used our clinic in the past. So, we started by reaching out to all principals and directors of special schools, workshops and group homes in the area, seeking their approval to send out letters to the parents of all their clients to ask for permission for our team to physically examine their children and collect blood samples. All agreed to do this and over 70% of parents sent back their signed permission slips. We ended up with a total of 1,977 individuals with ID to review. Our results found that forty participants had Fragile X syndrome, of which twelve were already aware of the diagnosis. The remaining twenty-eight families were given this news for the | 20 |

first time and almost all were very pleased to have this new information. One of the families involved in the study was the Cunningham family. Margaret must have given permission for Sean to be tested and he came back positive. Margaret’s extended family was then tested, and her mother was found not to be a carrier, so her deceased father, a dentist, must have been the carrier. This meant Margaret’s two sisters must have also been carriers, as males only have one X chromosome. Their father must have passed it to all three of his daughters, assuming he was their biological father, of course – a possibility you always need to be mindful of! One of Margaret’s two sisters had no children but was personally affected by painful muscles (myalgia) and her other sister had a son with Fragile X syndrome. Having completed the screening program, it became my responsi­ bility to expand this pattern of screening to the entire ID population of New South Wales. But that meant finding significant money. At the time, there was a major political movement underway to empty all the large ID institutions and rehouse their clients into group homes within the community. I attended the opening celebration of a new group home and the chief speaker at the launch was Dr David Richmond ao, the head of the Health Department. In his talk, Dr Richmond mentioned the importance of intellectual disability prevention without saying how he planned to achieve that. I chatted to him afterwards, telling him that perhaps I could help in the prevention aspect of his program. He asked me to send him a proposal, which I did, and then called his secretary to make an appointment to meet with him. Armed with my proposal and feeling confident in a smart suit I had purchased for the occasion, I was ushered into Dr Richmond’s office. He was on the phone, so he motioned for me to take a seat. I ended up going through the essentials of the proposal without him once putting the phone down. The meeting was over within five minutes. On the | 21 |

way back to the lift, my eyes filled with tears – tears of anger at being treated so dismissively. I turned around and marched back to Dr Richmond’s office requesting another meeting, which, to my surprise, was arranged without question. During that second meeting, Dr Richmond was attentive and charming. He agreed to fund the entire program without a murmur. I only wish I had asked for more funding! To run the screening program effectively, we needed additional resources including cytogeneticists, genetic counsellors, another medical genetics trainee and a good secretary. We also needed permission to use the Royal Flying Doctor Service and a doctor to take over some of my work running the clinic. Eventually, the team was ready and the program was underway. We met some wonderful people and families over the three years that it took to screen the entire state of New South Wales and to follow up branches of families in other states. I found that the GPs in rural areas, in particular, were quick to learn how to recognise Fragile X syndrome. Interestingly, most of the Fragile X families we worked with resided in rural areas. Of the 14,225 individuals surveyed, we identified and counselled 253 Fragile X families and a further ninety-five individuals with Fragile X in their extended families. We also identified 818 females with a 25-100% chance of being carriers. A secondary effect of our work was increased awareness amongst the medical fraternity and professional carers community. As more doctors, paediatricians and those involved in teaching and caring for people with ID became aware of Fragile X, more blood testing was organised, often before children started school. Our survey had clearly helped Fragile X become a well-known condition. Another positive effect of our work was a reduction in the rate of Fragile X births. We measured this in 1990 and the results showed that the frequency | 22 |

of Fragile X births in NSW had dropped from one in 4,000 to one in 10,000 births! It is interesting that Margaret writes that, as much as she loves Sean, in hindsight she would have requested prenatal diagnosis. It gradually dawned on us that the survey, which was funded to identify people in New South Wales with Fragile X, also revealed that X-linked inheritance of ID involved numerous families with affected sons that came up negative in Fragile X tests. A much bigger picture was being painted. The results set the wheels in motion for a larger investigation to uncover the other genes carried on the X chromosome and this developed into an international search. A total of 140 other genes have so far been found, of which the Fragile X gene accounted for 25%. This meant that the total contribution of X-linked genes causing ID had increased to roughly one in 1,000 births, the same frequency as Down syndrome. This side of the story is discussed in detail in my book, Finding the Sapiens genes. The search for those genes continues today. Now to speculation. What was, and is, the role of the FMR1 protein in evolution? We know that those genes on the X chromosome were vital to the mental development of Homo sapiens, but could it be that the FMR1 gene and its protein product were the main players? Was it essential for the neuronal sensory complex development in the early phases of the developing embryo? The changes in the number of DNA CGG triplet repeats increase in the female but not in the male. This was gradual at first, but when the CGG repeat numbers reached over fifty-nine, they increased more rapidly, and at 200 CGG repeats, the promoter was silenced and the FMR1 gene stopped production. This is a common mutation, probably starting as a mutation from an older sperm but then passed down to all daughters. In this way, it can spread rapidly in evolutional terms, aided by the occasional knockout of an AGG interruption in the | 23 |

Fragile X triple repeat, making exact replication more difficult. We know that using the CRISPR gene editing technique in mice with Fragile X to switch on the FMR1 gene, without being reliant on the promotor, had success in curing the mouse of its Fragile X symptoms and seeing FMR1 protein in mouse neurones. Will this technique eventually be the solution to improving the lives of individuals with Fragile X syndrome?

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CHAPTER 4

My personal journey with Fragile X syndrome Prof. Grant R Sutherland Preamble The study of fragile sites on human chromosomes, and in particular the Fragile X, has preoccupied me for more than two decades of my professional life. This narrative endeavours to create a record of how progress was made by my research group in this area and therefore does not acknowledge all the contributions from other groups.

The beginning I commenced work at the Adelaide Children’s Hospital (ACH) in 1975. I was a reasonably experienced cytogeneticist and had just obtained my PhD in Edinburgh on the properties of amniotic fluid cells in tissue culture, with an emphasis on prenatal diagnosis of genetic disease. Because I had previously worked in a cytogenetics laboratory in Melbourne, which served the medical practitioners assessing and managing the intellectually handicapped, and due to the fact that the main disorder that could be prenatally diagnosed was Down syndrome, I had developed (and maintain) a major interest in the genetics of mental retardation. | 25 |

On arrival at ACH, I was told by Rod Carter, who had recruited me, that half of the resources at my disposal in the Cytogenetics unit were for diagnostic cytogenetics and the remainder were for research. That left me with a team of just two. What to do? I was unsure. The records of the unit contained information on a number of individuals who had been found to have fragile sites on some of their chromosomes. At the time, most resided in a centre for the intellectually handicapped. Fragile sites had been described in the literature, but little was known about them. I decided to restudy the patients with fragile sites as this would give me a publication and provide time to think of a significant area of research. I restudied the patients but was unable to find the fragile sites that had previously been recorded. I knew I had a problem to study; the question was, how significant was it? In late 1975, I visited my old laboratory in Melbourne and the cytogeneticist there, Jill Harvey, showed me some families she had been working on who had the Fragile X chromosome. These were published by Jill and her colleagues in 1977 and their paper helped generate significant interest in the Fragile X. I thought the Fragile X was probably akin to the fragile sites on other chromosomes that I had begun to try and examine. I was keen to find some Fragile X families to work on but knew I would have to sort out my technology first. I recalled the original Fragile (then called ‘marker’) X family described by Dr Herbert Lubs in 1969. I had read his paper in detail with great fascination. He was a physician who, at the time, was working at Yale in the US when he identified a marker on the end of the long arm of the X chromosome, but it was only present in 20-30% of dividing cells. He studied a family with two affected boys, both of whom showed the marker. Interestingly though, the marker was not present in either parent. After considerable painstaking work, Dr Lubs recognised that it was likely to be the underlying cause of their disability. | 26 |

Back in Adelaide, I tried to work out what I needed to do to see the fragile sites. After several fruitless experiments with antibiotics added to culture medium, I decided to completely replicate the culture conditions that were still being used in the Melbourne laboratory. This involved obtaining culture medium TC199, as the Adelaide laboratory – along with many other laboratories – had progressed to more modern culture media, such as Ham’s f10. Lo and behold, the fragile sites I was studying reappeared! By studying patients with apparent X-linked mental retardation, I also rapidly found some Fragile X families. I announced the finding of the need to use TC199 during the discussion of a presentation by Malcolm Ferguson-Smith at the ACH Centenary Scientific Meeting in August 1976 and again at a presentation to the 182nd Meeting of the Genetical Society in London later that year. Those were times when unpublished findings were openly discussed in scientific meetings. This work was subsequently published as a letter to the New England Journal of Medicine and in Science (after being rejected by Nature).

What’s in TC199? Thoughts of other projects were on hold. I now had a way of seeing fragile sites, and one of them, the Fragile X, was of some relevance to the genetics of mental retardation. What was it about TC199 that allowed the fragile sites to be seen in lymphocyte culture? This medium contains many more components than newer culture media and it seemed reasonable to assume that one of these was responsible. It is a major undertaking to produce a series of culture media, each with a different component omitted. I conducted some experiments along these lines but nothing eventuated. With some degree of frustration, I regularly reviewed the recipe for TC199 in comparison to other media. One day, I noticed that TC199 contained much less folic acid than the other media. At last, | 27 |

an easy experiment! I added folic acid to TC199 and the fragile sites disappeared. I still remember the day I looked at the slides I had prepared. I published this work and a lot of experimental data on concentrations, times of effect, medium pH, cell types and the effects of folate metabolism inhibitors in two back-to-back papers in the American Journal of Human Genetics in 1979. As the Fragile X was slowly being found in other laboratories, the clinical aspects were beginning to emerge. Dr Gillian Turner’s work in New South Wales on X-linked mental retardation was gaining considerable attention. X-linked mental retardation with enlarged testes (macroorchidism) was described. A connec­ tion between the two was made when I enlisted the help of Histopathology Registrar, Peter Ashforth (now a child psychiatrist) to measure the testes of some Fragile X males I had identified. Controversy raged in the literature for a while about whether macroorchidism was indeed a feature of Fragile X syndrome, but the ‘measurers’ triumphed over the ‘visual inspectors’ and the now long list of clinical features associated with Fragile X syndrome emerged.

Molecular genetics By 1981, I had formed the view that we would learn little more about the fundamental nature of fragile sites unless we could clone them. I had thought that by understanding the molecular mechanisms of fragile site formation, it would have been possible to modify tissue culture conditions to allow fragile sites to be seen in close to 100% of cells, thereby greatly facilitating their cytogenetic detection. Over time, I established molecular genetics within the department by employing a series of young post-docs with molecular genetics experience and taking on PhD students with a grounding in molecular biology. | 28 |

In 1983, David Callen joined the group and we decided that we would positionally clone the fragile sites on Chromosome 16 and later, on the fragile site of the X chromosome. We built up a collection of fragile site families, including Fragile X syndrome families, that was unrivalled. These families have been and remain a unique resource for much of the fragile site research that we have performed. The Fragile X families allowed us to generate data that enabled virtually unequivocal diagnosis of non-fragile, siteexpressing carriers within families by computerised risk analysis before the Fragile X was cloned. Using our fragile site family material, I collaborated with Stephanie Sherman, a genetic epidemiologist working with Newton Morton and Pat Jacobs in Hawaii, on a segregation analysis of fragile sites. Stephanie had initially performed a segregation analysis on a group of Fragile X families, which was confirmed on an extended group of families with remarkable results. The Fragile X had the highest mutation rate of any locus that had been studied in the human genome. While asymptomatic, males could transmit an X-linked recessive gene. The risk of female carriers of the Fragile X having a child with the syndrome depended on their place within the pedigree (the Sherman Paradox) and whether or not they were mildly affected with the disorder themselves. We demonstrated that the autosomal folate sensitive fragile sites were also unusual because penetrance, when transmitted by females, was approximately 100%, but when transmitted by males, was only 50%. This offered a partial explanation for the puzzling observation that the carrier parent of new ascertainments with autosomal fragile sites was almost always the mother. Fragile sites had rapidly gained the reputation of being a unique genetic phenomenon.

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In 1987, Dr Graeme Suthers, a paediatrician who would eventually become a clinical geneticist at ACH, came to the group as a PhD student. He had collected blood samples from the thirty-two members of the first family described in Professor Turner’s original article on the importance of X-linked intellectual disability. They all agreed to meet and have blood collected on a Saturday afternoon at Grandma’s Place in Sydney. All thirty-two turned up and held out their arms. Dr Suthers’ project involved the detailed linkage and physical mapping of the region of the X chromosome around the fragile site. He organised an international consortium to put together a somatic cell hybrid panel to physically map probes within the region and determine linkage relationships. This work was highly successful. It put to rest claims made by others that the presence of the fragile site disturbed genetic distances and laid the foundation for the positional cloning of the Fragile X.

Cloning the Fragile X The final phases of our isolation of the Fragile X DNA sequences began with the concerted effort to build upon Dr Suthers’ mapping studies. The team included post-docs and a young Chinese clinical geneticist, Sui Yu, who had joined us as a PhD student. She was the first author on the final paper on finding the molecular cause of Fragile X syndrome. We initially collaborated and then competed with some of the other groups working in this area. David Schlessinger provided us with a YAC (Yeast Artificial Chromosome) containing the fragile site, Jean-Louis Mandel contributed some probes in the area and Steve Warren gave us access to somatic cell hybrids with breaks at the fragile site. The paper on the cloning of the Fragile X appeared in Science in 1991, the same issue in which Mandel’s group published their findings, which were similar to ours. Shortly afterwards, the other major groups – a consortium led by Tom Caskey at Baylor in Texas, Steve | 30 |

Warren in Atlanta and Ben Oostra in Holland – published the finding of the FMR1 gene in Cell. 1991 was a great year for fragile site aficionados. Although there were many competing groups, we had won the race to clone the Fragile X and held the patent on the use of the fragile site for diagnosis.

Early participants in the discovery of the Fragile X DNA repeats and FMR1 gene, Barossa, Adelaide, 2013. L–R: Dr Sui Yu, who was Prof. Sutherland’s PhD student and was critical to his work on the Fragile X DNA, Prof. Grant Sutherland, Prof. Jean-Louis Mandel from Strasbourg and Prof. David Nelson from Houston.

Trinucleotide repeats The cloning of the Fragile X resulted in the discovery of a novel mechanism of mutation – expansion of a normally occurring polymorphic trinucleotide (CCG) repeat sequence. Small increases in the number of repeats were unstable premutations without significant effect. But when these were transmitted by women, they could increase dramatically in copy number. These were the full mutations that resulted in Fragile X syndrome. This region of DNA was unstable, both somatically and when transmitted. | 31 |

With the cloning of the Fragile X, we finally had an explanation of the Sherman Paradox and a mechanism that could explain anticipation (when the effect seems to affect younger people in each generation). We published a hypothesis in The Lancet suggesting that a molecular mechanism similar to that found for Fragile X could also be responsible for myotonic dystrophy and explain the anticipation that had been observed (but discredited) in that disease. Shortly after this hypothesis was published, the myotonic dystrophy mutation was found to be an expansion of an AGC trinucleotide repeat. Trinucleotide and other repeat expansions have since been demonstrated as the mutational mechanisms in a number of neurological disorders. Diagnosis of Fragile X syndrome was now possible by direct assay of the mutation. Family members pre-diagnosed for Fragile X carrier status by linkage provided validation of the direct assay. On that basis, we were the first to use direct detection of the mutation for prenatal diagnosis and, in most centres, diagnosis of Fragile X syndrome has moved from the cytogenetics to the laboratory. Dr Rob Richards decided to haplotype the Fragile X chromosomes and, to my surprise, discovered the now well-recognised founder effects shown by X chromosomes with long stretches of perfect CCG repeat. These form a reservoir in the community for the generation of new premutations. From 1992 onwards, Rob and I regularly reviewed this area. In an early review, Rob coined the term ‘dynamic mutation’ to describe the mutational mechanism, and this has since gained widespread acceptance. Some Fragile X families had been identified without a FRAXA mutation. I had looked at the fragile site in these families and suspected that it was distal to FRAXA. Dr Liz Baker separated what we called FRAXE from FRAXA by FISH, with probes in the area, and confirmed my suspicions. Subsequently, what we | 32 |

had previously called FRAXE was further subdivided by others into FRAXE and FRAXF. Josef Gecz, a senior post-doc, together with geneticist, Dr John Mulley, was the first to isolate the gene (FMR2) associated with FRAXE. He demonstrated that FMR2 is inactivated by FRAXE and this results in Fragile XE mental retardation. The first gene for a non-dysmorphic form of X linked MR had been cloned.

Where to from here? There are still many unanswered questions about fragile sites and seeking the answers to these questions is the logical way forward. For example, the potential role of fragile sites in cancer has once again become something to consider. A common fragile site (FRA3B) is within a gene that produces abnormal transcripts in a range of solid tumours. There are individuals in whom rare fragile sites occur on more than one chromosome and there are also individuals with more than one fragile site on a single chromosome. These occur more often than would be expected by chance. Are there trans-acting factors that can generate instability at multiple loci that have the potential to give rise to multiple fragile sites? How do the DNA sequences that have been characterised at fragile sites actually give rise to the cytogenetic appearance of the fragile site? Why are fragile sites usually only seen in a proportion of cells? Not all types of fragile sites have been cloned. In particular, little is known about common fragile sites. Are they areas of late replication because they contain genes being transcribed late in the S phase of the cell cycle and are therefore under-replicated regions of DNA that cannot package for mitosis?

Perspective I have gained great satisfaction from the study of fragile sites. My initial work in the late 1970s provided the means for many | 33 |

groups to become interested, particularly in Fragile X syndrome, but also in the general phenomenon of fragile sites. To have been in a position to see this subject evolve, and to have participated in its evolution, from a cytogenetic curiosity to a significant area of genetics, has been gratifying. I have also greatly enjoyed building a research group, which now includes a relatively large number of individuals with a wide range of talents who have all been focused, at least for part of the time, on a single project with many facets. The only downside to this for me has been that inevitably over time, my direct input into any particular series of experiments has diminished. This progression has been reflected in the authorship of papers from the group on fragile sites. I am deeply grateful to the individuals in my former research group and to the organisation for their support which made my time at the Adelaide Women’s and Children’s Hospital so thoroughly enjoyable.

Acknowledgement I have been retired for many years. The substance of this narrative was written around 2000. Therefore, I take full responsibility for errors resulting from my incomplete familiarity with the current literature. I thank Prof. Gillian Turner for assisting with this chapter. Prof. Grant Sutherland AC is Emeritus Geneticist, Department of Cytogenetics and Molecular Genetics, Women’s and Children’s Hospital in Adelaide. His major work has been the cytogenetic and molecular characterisation of fragile sites on chromo­ somes, including the Fragile X site. He is a past President of the Human Genetics Society of Australasia and the Human Genome Organisation (HUGO).

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CHAPTER 5

Fragile X premutation carriers 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 | 35 |

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 immunocyto­ chemistry testing for the FMR1 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, | 36 |

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 | 37 |

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 f lights 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. | 38 |

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 neuro­ pathologists 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 | 39 |

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).

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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 | 41 |

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 | 42 |

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 | 43 |

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 neurodevelop­ mental 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, Australia, 2015.

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Prof. Flora Tassone.

CHAPTER 6

Carrier screening for Fragile X Prof. W Ted Brown MD, PhD Once the molecular basis for the Fragile X mutations was discovered in 1991 by laboratories in Australia (Sutherland), France (Mandel) and the US (Warren and Nelson), it became possible to screen for carrier status. Fragile X carriers were defined as persons carrying the number of CGG repeats in their FMR1 gene between fifty-six and 200. This repeat size was designated the premutation. It was soon discovered that male carriers would only pass on their premutation to all their daughters as a premutation, never as a full mutation. Since the FMR1 gene is on the X chromosome, they never pass their X chromosome to their sons. It was also discovered that the risk of a carrier mother passing on the mutation located on one of her two X chromosomes, was 50% by chance, and when passed on, the risk of it becoming a full mutation in her child was related to the number of repeats in her premutation X chromosome, and also by the number of AGG interruptions that commonly occur within the CGG repeat region. Data was developed in our laboratory at the New York State Institute for Basic Research in Developmental Disabilities to show that the risk for a full mutation to be passed on was close to 0% when the mother’s CGG repeat length was in the range of 55-59 repeats, less than 10% in the range of 60-69, around 33% when in the range of 70-79, around 70% when in the range of 80-90, and approached 100% when the repeat size was greater than 90. | 45 |

Percentage of maternal full mutation transmissions by total CGG repeat length (grey bars) and the number of AGG interruptions. Figure S1 from Nolin et al., 2015.

With the availability of a DNA test for the Fragile X gene and its number of repeats, it became possible to test females at risk of becoming carriers, and to provide accurate information about their risks of passing on the Fragile X mutation, either as a premutation or as a full mutation. In Australia, the cost of such testing of relatives of an affected child who has the full mutation is covered by Medicare funding. Often, when a child is diagnosed with Fragile X syndrome (FXS), there is no known family history of Fragile X. In my experience, there is no identifiable relative who would have had features of Fragile X or who had been previously diagnosed in about 50% of cases. Research by Dr Alison Archibald and colleagues (2017) carried out reproductive carrier screening on 12,000 couples for Fragile X along with two other common genetic conditions: Cystic Fibrosis (CF) and Spinal Muscular Atrophy (SMA) where there was no known previous family history. They identified thirty-five female carriers of the Fragile X mutation. | 46 |

Overall, they found that one in 240 couples were at risk of having a child with one of the three conditions. This study pointed out that the risk of having such a child was comparable to the risk of having one with Down syndrome, a condition that is usually screened for in early pregnancy. In the May 2018 Federal Budget, the Australian Government announced $500 million in funding towards genomics research: the Genomics Health Futures Mission, starting with a $20 million research study to offer reproductive genetic carrier screening to 10,000 couples across Australia who were either planning to have children or were in the early stages of pregnancy. The study would give couples information about their likelihood of having a child with a severely debilitating and/or life-limiting genetic condition occurring in childhood. It would screen couples for around 1,300 genes that are related to about 750 genetic conditions which can be passed on, including FXS, CF and SMA. The study is known as ‘Mackenzie’s Mission’ and was named for Mackenzie Casella, who died when she was seven months old due to SMA. Her parents were unaware that they were carriers and asked why they had not been screened. Advocacy by the Casellas and other parents led to government support of this project. Mackenzie’s Mission concluded in 2022. It is hoped that the results of the study will support the offer of this prenatal screening to all Australian couples in future. This approach, using modern DNA screening methods, including whole exome sequencing, will be used to evaluate screening uptake, frequency of carrier status, ethical issues, reproductive decisions when at increased risk and an economic cost-benefit analysis. Prof. Gillian Turner has reflected that she finds it remarkable how one family’s story has become the stimulus for action in contrast to written applications made through genetic services over a number of years, which were unsuccessful.

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In 2019, the Australian Medical Services Advisory Committee (MSAC), which has the role of appraising new medical services proposed for public funding, supported public funding for reproductive carrier testing to detect CF, SMA and FXS pathogenic variants in women early in pregnancy or intending to become pregnant, and in their reproductive partners as needed. MSAC’s recommendation for Medicare funding was provided to the Australian Government Department of Health. In March 2022, Hon Greg Hunt MP, Federal Health Minister at that time, announced that reproductive genetic carrier testing for cystic fibrosis, spinal muscular atrophy and Fragile X syndrome will be funded through Medicare from November 2023. This announcement was warmly welcomed by Fragile X Association of Australia, SMA Australia and Cystic Fibrosis Community Care, which had been advocating for public funding of this testing. The 2021 Federal Budget announcements included a measure that will benefit some women who are identified as Fragile X carriers and are considering pregnancy. Five Medicare Benefits Schedule items were added for Pre-implantation Genetic Testing of embryos for carriers of serious genetic disorders such as Fragile X. Fragile X Association of Australia welcomed this measure.

References Nolin SL, Glicksman A, Ersalesi N, Dobkin C, Brown WT, Cao R, Blatt E, Sah S, Latham GJ, Hadd AG. ‘Fragile X full mutation expansions are inhibited by one or more AGG interruptions in premutation carriers.’ Journal of Genetic Medicine. 2015, 17:358-364. Archibald AD, Smith MJ, Burgess T, Scarff KL, Elliott J, Hunt CE, McDonald Z, Barns-Jenkins C, Holt C, Sandoval K, Siva Kumar V, Ward L, Allen EC, Collis SV, Cowie S, Francis D, Delatycki MB, Yiu EM, Massie RJ, Pertile MD, du Sart D, Bruno D, Amor DJ. | 48 |

‘Reproductive genetic carrier screening for Cystic Fibrosis, Fragile X syndrome, and Spinal Muscular Atrophy in Australia: outcomes of 12,000 tests.’ Journal of Genetic Medicine. 2018, 20:513-523.

Author Prof. W Ted Brown MD, PhD is the recently retired Director of the New York State Institute for Basic Research (IBR) in Developmental Disabilities, Staten Island, NY. Much of his thirtyfive-year research career has focused on the Fragile X syndrome and autism. Prof. Brown was the first to describe a relationship between autism and the Fragile X syndrome. His team was the first to demonstrate the feasibility of prenatal diagnosis for Fragile X. Upon retiring, he relocated to Sydney. He has an appointment as a visiting professor at the University of Sydney and is President of the Fragile X Association of Australia.

Prof. W Ted Brown MD.

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Acknowledgements Fragile X Association of Australia wishes to acknowledge the pioneering work of Prof. Gillian Turner in the field of genetics and intellectual disability. Her dedication to scientific discovery and her tremendous commitment to families affected by developmental disorders, and in particular, Fragile X syndrome, continues to inspire many others working in this field. We also wish to acknowledge and thank the contributors for their work and the ongoing outcomes that have made such a difference to the lives of people affected by Fragile X. The Board and staff of Fragile X Association of Australia thank Margaret and John Cunningham for their significant contribution to the focus of the work we do, and for their generous support over more than thirty years.

Glossary of terms Fragile X-associated disorders Fragile X syndrome

Fragile X syndrome is the most common cause of inherited intellectual disability. People with Fragile X syndrome can have delayed development, learning difficulties, speech and language difficulties, anxiety, autism spectrum disorder and epilepsy. The effects of Fragile X syndrome on an individual vary and can range from mild to severe. Males commonly appear to be more severely affected by Fragile X syndrome, but females may also be severely affected. Educational, behavioural and medical interventions can improve outcomes for people who have Fragile X syndrome. Around one in 3,600 males and one in 4,000-6,000 females have Fragile X syndrome.

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Fragile X-associated Primary Ovarian Insufficiency (FXPOI)

Female Fragile X premutation carriers may experience FXPOI, a condition that can lead to infertility and early menopause. Studies show that approximately one in four female carriers of Fragile X experience FXPOI, and overall, the average age of menopause is reduced by about five years.

Fragile X-associated Tremor Ataxia syndrome (FXTAS)

FXTAS is an adult-onset neurological (brain and movement) disorder that affects some Fragile X premutation carriers later in life. Symptoms include unsteadiness (ataxia), intention tremor (shaking) and cognitive issues. FXTAS can often be misdiagnosed as Parkinson’s disease. It affects approximately 45% of men and 16% of women with the Fragile X premutation who are aged over forty. It was identified in 2001 by Profs Randi and Paul Hagerman following observation of a pattern of neurological symptoms present in older grandparents and parents of people with Fragile X syndrome and subsequent research by their teams.

Fragile X-associated Neuropsychiatric Disorders (FXAND)

FXAND is a term proposed to include a range of neuropsychiatric and physical conditions which have been associated with the Fragile X premutation in some people, including chronic pain, autoimmune issues, anxiety and depression.

Fragile X Premutation FXPAC is a term covering any condition linked to the Associated Fragile X premutation, including FXPOI, FXTAS, FXAND Conditions (FXPAC) and others. FMR1 gene (Fragile X gene)

The FMR1 gene is responsible for producing a protein called FMR1-Protein (FMRP), which is necessary for normal brain development and function. Fragile X-associated conditions are caused by a lengthening (expansion) in the FMR1 gene on the X chromosome, which can reduce or shut down the production of FMRP. The changes in the FMR1 gene can go through stages as that gene is passed down in a family. These stages start with the normal FMR1 gene and can expand to the premutation (Fragile X carrier) and then the full mutation (Fragile X syndrome).

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Premutation carrier

A carrier has an altered form of a gene that can lead to having a child or grandchild with a genetic disorder. Some carriers are full mutation carriers, while others are premutation carriers. In Fragile X, unlike some other X-chromosome linked disorders, both males and females can be carriers. It is estimated that about one in 250 females and one in 800 males are carriers of the Fragile X gene premutation. Female carriers have a 50% chance of having a child, male or female, with either the Fragile X premutation (carrier) or the Fragile X full mutation (Fragile X syndrome). They also have a 50% chance of having an unaffected child. Male carriers pass the Fragile X gene premutation to all daughters but none of their sons.

Reproductive genetic carrier testing

Reproductive genetic carrier testing is a genetic test that can tell people if they have an increased chance of having children with an inherited genetic condition, such as Fragile X syndrome. Reproductive genetic carrier testing can be undertaken pre-pregnancy or in the early stages of pregnancy. The Federal Government’s Mackenzie’s Mission research study offered reproductive carrier screening to up to 10,000 couples across Australia from 2019-2022. The study investigated the best way to deliver a national reproductive carrier screening program available to all couples in Australia.

CGG repeats

Fragile X-associated disorders are caused by a lengthening (expansion) of the FMR1 gene on the X chromosome. At the beginning of the FMR1 gene, there is a repeated sequence of chemical bases known by the letters ‘CGG’. These are repeated a number of times. Most people have less than fifty-five copies of this CGG repeat. Fragile X premutation carriers can have between fifty-five and 200 copies of the CGG repeat. People with the Fragile X full mutation (Fragile X syndrome) have over 200 copies of the CGG repeat. DNA testing can determine how many copies of the CGG repeat a person will have.

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About Fragile X Association of Australia Fragile X Association of Australia (FXAA) is a national memberbased organisation representing the Fragile X community. Our mission is to improve the health and wellbeing of those affected by Fragile X-associated disorders. We provide information and education, offer counselling support, raise awareness of Fragile X and advocate for public policy change in areas impacting the Fragile X community.

30 Years Strong Supporting, sharing, connecting, informing Help Line: 1300 394 636 | support@fragilex.org.au Website | fragilex.org.au Carrier screening website | carrierscreening.org.au

Registered office Suite 204, 20 Dale Street, Brookvale NSW 2100 ABN: 18 655 264 477 | ARBN: 626 478 966

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Finding the Sapiens genes Gillian Turner ao

During the 1960s, Gillian Turner ao recognised that the excess of

males with intellectual disability resulted from genes carried on the X chromosome. Males with only one X were affected while females having two X’s were partially protected but still carriers. The clue that these same genes, when functioning normally, were responsible for increasing intelligence during the evolution of Homo sapiens was that when mutated, the only thing affected was learning ability – these are our Sapiens genes. Finding the Sapiens genes covers both facts and speculation: •

The breakthrough of finding a laboratory test that confirmed X-linkage was the Fragile X syndrome with a visible marker on the end of the X, enabling the development of screening programs and a general acceptance of X-linkage.

•

This led to identifying which females in a family are carriers – they can now be offered help to have unaffected children.

•

Screening of women to identify carriers of both X-linked genes and also some recessively inherited conditions is now in the planning stages as part of the new federally funded McKenzie’s Mission.

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•

The mating of Homo sapiens women with Neanderthal men in Europe some 40,000 years ago led to females sharing skills of communication, fire making, cooking and food storage, opening up the possibility of change from nomadic to settlement societies.

•

Man’s Y-based testosterone production causes aggression, increased muscle strength and a desire to have many descendants – all important skills for success for nomadic tribes. Although these inbuilt urges persist, they are no longer advantageous in many of today’s societies.

•

The final chapters suggest answers to the increased incidence of autism and Asperger’s syndrome in males. What may prove to be a silver lining is that, by similar mechanisms, some women are becoming ‘super smart’.

•

The last chapter describes Ohno’s ideas on how the X chromosome enables the development of new species.

Finding the Sapiens genes is an easy read that will draw you into the excitement of research, and the part played by grit and stubbornness. For those involved in studying our evolution, there is a new set of tools to check the stages in our development. You will finish this book with a better grasp of the basics of human genetics.

ORDER SECURELY ONLINE AT

sapiensgenes.com ISBN 978-1-876231-50-7 Paperback, 152 pages illustrated RRP AUS$35

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