What Sex did to the X - and Why

A chromosome account of evolution and revolution

What Sex did to the X – and Why; A chromosome account of evolution and revolution

On Thursday 17 March 2005, an international team led by the Wellcome Trust Sanger Institute, Cambridge, UK publishes in Nature the most complete analysis of this remarkable chromosome. Other major contributions to the sequence came from groups at Baylor College of Medicine, Houston TX, USA, Institute for Molecular Biotechnology, Jena, Germany, Washington University Genome Sequencing Center, St Louis, MO, USA and Max-Planck-Institute for Molecular Genetics, Berlin, Germany.

The landmark study shows how we got an X chromosome and how it has been preserved (while the Y chromosome has degenerated). It also identifies new genes involved in disease and provides a gold-standard platform for studies to understand, to diagnose and, it is hoped, to treat a huge range of human disease.

The human X chromosome has a different biology to all others. Whereas females have two X chromosomes, males have only one X chromosome and a Y chromosome, which is an eroded version of the X chromosome, containing only a few genes.

The consequences are dramatic; any defects in genes on the X chromosome are often apparent in males because the Y chromosome does not carry corresponding genes to compensate. For mutations on the X chromosome, the diseases are, most often, diseases of males – and not of humankind.

More than 300 diseases have been mapped to the X chromosome – by far the highest proportion of any chromosome – including Duchenne Muscular Dystrophy (DMD) and haemophilia. The genome sequence has been used in the isolation of more than 40 genes that are involved in medical conditions, including cleft palate and blindness.

“From studying such genes, we can get remarkable insight into disease processes. From our study of one gene involved in an X-linked disease, a genetic test was developed and a new pathway that controls the workings of the immune system was discovered.”

“But the importance of the sequence goes beyond the biology of individual genes. We have also gained a deep insight into the evolution and biology of the whole chromosome. We can see the way evolution has shaped the chromosomes that determine our gender to give them their unique properties.”

Mark Ross Project Leader at the Wellcome Trust Sanger Institute

These remarkable chromosomes evolved from humble beginnings as an ‘ordinary’ pair of identical chromosomes. It is thought that changes to a gene on one of the pair created the key switch in the pathway to male development and set in train the degeneration of this chromosome. As this emerging Y chromosome eroded, maintaining the integrity of the X chromosome was essential. When the integrity is compromised in human males, disease often results.

“The X chromosome was pivotal in early human genetics because we were able to see clearly how mutations cause disease. There are many more genetic disorders on the X chromosome where the underlying gene is still to be found. Now we can make use of the finished sequence to find them. These discoveries will have a major impact on our understanding of many fundamental biological processes.”

Dr Bentley Head of Human Genetics at the Institute

The X chromosome played an important part in developing the methods of genetics in the molecular age. One of the first genes cloned using modern methods was that involved in Duchenne Muscular Dystrophy. For genes such as DMD, diagnosis has been transformed by genomic sequence and we are beginning to see hopes for new treatments based on that understanding.

“The X chromosome has a unique place in biology and medicine. It is the first chromosome to which a human trait was mapped. Its remarkable biology has played a key role in understanding many important genetic causes of human disease. The freely available sequence analysis is a landmark in our understanding of the role of this chromosome in health and disease. Our task now is to ensure that we translate these research findings into improved healthcare.”

Dr Mark Walport Director of the Wellcome Trust

There remain many conditions that are associated with genes on the X chromosome for which a genetic basis is lacking. In simple cases, a ‘candidate’ gene can be identified and tracked down. For more complex diseases, more than one gene may be involved and the hunt is much more difficult and the path littered with false clues and false dawns. However, new methods that make use of the sequence are being developed to identify genes involved in common disease.

“The detailed analysis of the sequence of the human X chromosome is a monumental achievement. This work represents yet another exciting example of what we can learn from the vast trove of sequence data produced by the Human Genome Project and made freely available to researchers around the world,.”

Francis S. Collins MD, PhD, director of the National Human Genome Research Institute, which along with the U.S. Department of Energy, led the Human Genome Project in the United States

The X chromosome has played its part in political history: X-linked human diseases include haemophilia, in which the blood fails to clot properly. Queen Victoria was a ‘carrier’ of haemophilia and passed it to her own children and, through them, to the Royal families of Europe. It has been argued that inheritance of haemophilia by Alexei, son of the last Tsar of Russia, led indirectly to the Russian Revolution.

This chromosome has also been vital to biomedical history: from the first gene mapped in the human – red-green colour blindness in 1911 – to searching for an understanding of human diseases, this unique chromosome has taught us an enormous amount about genetics and human biology. From the sequence, much knowledge has already been wrested, but there is much more of the X to understand.

• The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK
• Baylor College of Medicine Human Genome Sequencing Center, Department of Molecular and Human Genetics, One Baylor Plaza, Houston, Texas 77030, USA
• Genomanalyse, Institut fur Molekulare Biotechnologie, Beutenbergstr. 11, 07745 Jena, Germany
• Washington University Genome Sequencing Center, Box 8501, 4444 Forest Park Avenue, St. Louis, Missouri 63108, USA
• Max-Planck-Institute for Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany
• Institute for Clinical Molecular Biology, Christian-Albrechts-University, 24105 Kiel, Germany
• Medizinische Genetik, Ludwig-Maximilian-Universitat, Goethestr. 29, 80336 Munchen, Germany
• HUGO Gene Nomenclature Committee, The Galton Laboratory, Department of Biology, University College London, Wolfson House, 4 Stephenson Way, London NW1 2HE, UK
• Department of Biochemistry and Molecular Biology, Pennsylvania State College of Medicine, Hershey, Pennsylvania 17033, USA
• Advanced Center for Genetic Technology, PE-Applied Biosystems, Foster City, California 94404 USA
• European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK
• Institute of Genetics and Biophysics, Adriano Buzzati-Traverso, Via Marconi 12, 80100 Naples, Italy
• Medical Genetics Section, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, UK
• Laboratoire de Genetique et de Physiopathologie des Retards Mentaux, Institut Cochin. Inserm U567, Universite Paris V., 24 rue du Faubourg Saint Jacques, 75014 Paris, France
• BACPAC Resources, Children’s Hospital Oakland Research Institute, 747 52nd Street, Oakland California 94609, USA
• Molekulare Genomanalyse, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany
• Institute of Human Genetics, GSF National Research Center for Environment and Health, Ingolstadter Landstr. 1, 85764 Neuherberg, Germany
• RZPD Resource Center for Genome Research, 14059 Berlin, Germany
• National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892, USA
• Laboratory of Genetics, National Institute on Aging, NIH, 333 Cassell Drive, Baltimore, Maryland 21224, USA
• Institute for Genome Sciences & Policy, Duke University, Durham, North Carolina 27708, USA

Additional information

X in Numbers

Chromosome size (total base-pairs, bp) = 155,000,000

Base-pairs sequenced (bp) = 151,005,926

Gaps (in euchromatin*) = 14

Size of euchromatin gaps (total bp) = < 1,000,000

Size of heterochromatin gaps (bp) = ~3,000,000 single gap at centromere

Total count (number) = 1098

‘New’ genes (number) = 399

Genes per Mb = 7.1

Chromosome in exons (%) = 1.7%

Largest gene (bp) = 2,220,223 Duchenne Muscular Dystrophy

Smallest gene (bp) = 114

Cancer-testis antigen gene = 99 Active in testis and in cancer tissues

Non-coding RNA genes (number) = 173

Largest non-coding RNA gene (bp) = 32,103 XIST – gene essential for X chromosome inactivation

Pseudogenes = 700

Known genes found = 99.3% from RefSeq

Genes shared with Y chromosome = 54

Original X-Y genes = 7 Genes surviving on X and Y chromosomes from the original chromosome pair

Repeat content (%) = 56% Genome average 45%

Repeat family LINE content (%) = 29% Genome average 17%

Total mapped (number) = 153,146 one SNP per 1012 bp

Protein-changing SNPs (number) = 901

Notes:* Euchromatin is the gene-containing region of the chromosome; heterochromatin is repetitive DNA containing few or no genes

X and Disease

Our illnesses are never about genetics alone, nor about environment alone. The ‘Book of Life’ will transform medicine, but the context of being a human must not be ignored in our reading of that Book.

One of the first discoveries from the work at the Wellcome Trust Sanger Institute and their collaborators on the sequence of the X chromosome epitomizes that belief. A variant in a gene called SH2D1A leads to no overt symptoms in males, even though it is X-linked. With one environmental event – infection with Epstein-Barr virus – it becomes a lethal change.

Males, most often boys, carrying the SH2D1A mutation are unable to mount the appropriate defence to EBV infection and develop a massive increase in numbers of cells of the immune system, leading to damage of vital organs and often death. The disease is called XLP (X-linked Lymphoproliferative) or Duncan’s Syndrome.

Mutation detection is now used as a diagnostic tool and early treatment afforded by accurate diagnosis can be essential in treatment. Just as important, the discovery shed light on . SH2D1A encodes a previously unknown protein that was shown to be essential in the balance of our immune system.

“This gene was isolated in one of the first sequencing projects at the Wellcome Trust Sanger Institute. It is typical of many of these projects – the implications go far beyond the gene identification. From our studies of the gene came a new diagnosis for Duncan’s Disease as well as a new understanding of how the immune system works.”

Dr Alison Coffey Sanger Institute

The X chromosome is also home to genes that, when damaged, result in haemophilia, as well as to the largest gene in the human genome, called DMD. Mutations in the DMD gene are the cause of Duchenne Muscular Dystrophy, a debilitating and eventually fatal disease of males.

“There are still questions about the DMD locus which the sequence will help us answer. It has a very high rate of new mutations, and part of the gene is deleted with high frequency. We are still trying to understand the complex genetics of this, the largest gene in the human genome.”

Professor Kay Davies A leading DMD researcher and Dr Lee’s Professor of Anatomy at the University of Oxford

From its discovery, the DMD gene has been the focus of intense research leading, as with SH2D1A, to new understanding of the biology beneath disease and new hopes for treatment. However, genetic research most often brings new methods for accurate diagnosis: improvements in treatment take years of painstaking work.

“For the common X-linked diseases, such as DMD and haemophilia, diagnosis has improved totally beyond recognition as result of molecular techniques. Real hopes for treatment of previously untreatable diseases are now just beginning to emerge: new approaches for treatment for DMD, such as Myostatin, are in very early clinical trials.”

Professor Martin Bobrow of the Cambridge Institute for Medical Research

Professor Bobrow is a leader in the study of genetic disease and its diagnostics With a particular interest in muscular dystrophies, and other X-linked diseases, such as Emery-Dreifuss muscular dystrophy, Alport syndrome (kidney failure) and the work described above on XLP.

“Although we have known the genes for many of these conditions for a while we still do not know much about how these genes are controlled and this may be critical information if new therapies are to be developed. The sequence of the X chromosome will contribute directly to this and an accurate catalogue of genes will make that task much easier.”

Professor Martin Bobrow, Cambridge Institute for Medical Research

Most of our diseases have a complex genetic underpinning, in which many genes play a significant, but minor role. And some diseases affect complex organs in which we have little understanding of biological and molecular events. For these, a complete X chromosome sequence is crucial.

“Because X-linked genes leave such a definite signature, many of the common ones have been recognised and cloned – the difficult ones are the uncommon and those where there is genetic heterogeneity.”

Professor Martin Bobrow Cambridge Institute for Medical Research

Professor Davies has similar hopes for new research opportunities. “For me, the finished sequence provides an opportunity to look for genes involved in intellectual disability, many of which have been mapped to the X: researchers can examine all the candidate genes to analyse for mutations. The fragile X syndrome is well known, but there are others, such as FRAXE site associated with milder mental impairment. The sequence of this gene will be vital in understanding its role and that of the related ALF gene family which is involved in leukaemia and other disorders.”

The sequence of the X chromosome has been essential in elucidating many of the genetic diseases that have a relatively simple basis – they are due to mutation in one gene. The accurate gene description – annotation – and study of variants will similarly be a indispensable in our efforts to find treatments for the more common diseases that have a complex basis.

X and Sex

Birds do it using the letters Z and W. Bees – and people – do it using the letters X and Y (although sometimes bees don’t bother). Platypus, for reasons best known to themselves, do it using five Xs and five Ys. The human X chromosome is about many things – including sex and how it evolved.

There are many advantages, some of them genetic. We and many other organisms are diploid – we carry two copies of each chromosome. Sex brings mixing of chromosomes and the chance to produce the new variation that is one of the driving forces of evolution.

In order to have sex, we need to have two sexes. But how is sex determined? In some cases, such as crocodiles and turtles, temperature determines sex. In other cases, like ourselves, the determining factor is genetic, and it is in these cases that sex chromosomes are observed.

It is thought that our sex chromosomes evolved from ‘ordinary’ chromosomes when, far in our evolutionary past, one gene on one chromosome was recruited as the key switch in determining sex. We now know that this is the Y chromosome. Since that process began, the Y chromosome has degenerated.

“Genome sequence information has provided compelling evidence for this model. The X chromosome, which is the original partner of the Y, has remained largely intact. Our X chromosome is related ancestrally to chromosomes 1 and 4 of chicken and not to the chicken sex chromosomes Z and W. Sex chromosomes of birds and mammals have evolved independently from ordinary chromosomes.”

“Sequence comparison between the X and Y shows how extensive degeneration of the Y chromosome has been, with only a handful of shared genes remaining. Even these few look very different on the two chromosomes and have different roles.”

Dr Mark Ross

The consequences of this chromosomal divergence are profound for our health and our biology. In males There is a single copy of most of the genes on the X chromosome, and, therefore, damage to any of these genes will often result in disease. In females, there are two copies of each X chromosome gene and so a mechanism is needed to prevent overproduction of protein from these genes.

Our lives are dependent on carefully controlled levels of genetic activity. Just as a musical piece is arranged for certain instruments at certain times playing at certain volumes, so our cells require the activity of our genes to be orchestrated, their levels to be set.

So how do humans and other mammals cope with the dramatic difference of the sex chromosomes – two ‘doses’ of each gene in females and only one in males?

The leap of inspiration came in 1961 from a British mouse geneticist, Mary Lyon, who noticed that a mutation in a coat-colour gene on one X chromosome sometimes resulted in female animals with spotted or mottled coats. Because these females had one normal X chromosome, no effect should have been seen. Something very unusual was going on.

In a remarkable synthesis, Lyon reasoned that one of the X chromosomes was inactivated in normal female mice during early development. She argued that this occurred at random, leading to patches of cells in which one or other of the two X chromosomes had been ‘switched off’, resulting in normal coat colour or mutant coat colour. The same phenomenon explained some familiar observations such as why tortoiseshell cats are always female.

This X chromosome inactivation (XCI) explains how females avoid overproducing protein. But, decades on, we are still trying to understand its mechanism.

“In the early 1960s I had no idea that XCI would be of such importance in human clinical genetics. At that time knowledge of gene action was so limited that one could not begin to imagine what the mechanism might be.”

Dr Lyon, who has worked for the UK’s Medical Research Council for most of her career

It would take 30 years before a key gene in the process was identified.

“In both mouse and human, X inactivation is controlled by a gene in the inactivation control centre on the X chromosome called Xist. Much more is known about XCI in the mouse because extensive experimental work has been possible. The availability of the human X-chromosome sequence will enable much more detailed knowledge of human XCI.”

Dr Lyon UK Medical Research Council

We still don’t know how the signal spreads out from the control centre along the chromosome, but Dr Lyon has suggested that repetitive sequences, often referred to as ‘junk’ DNA, play a role in this process. The analysis of the X chromosome sequence provides additional support for this proposal.

“In humans XCI has important clinical implications. It enables understanding of the defects seen in patients with abnormal numbers of X chromosomes or with structurally abnormal X chromosomes.”

Dr Lyon UK Medical Research Council

With the completion of the X chromosome project, we have the sequences of a sex chromosome pair for the first time. Analysis of these sequences is beginning to give us a much greater insight into the unique behaviour of these chromosomes.

X in history

A revolution one century ago changed the face of humanity. It was not the revolution that brought down the might of Imperial Russia, although more of that later. The quiet revolution was in genetics, in humble flies and beetles, a revolution that began to show us how scraps of genetic material decide whether we are men or women.

The revolution brought the first maps of the undiscovered richness of our genome and led to a medical understanding of diseases such as haemophilia and muscular dystrophy. The limited inheritance of haemophilia – males are affected and females tend not to be – had been noted for hundreds of years, including by the writers of the Talmud. The sex-limited inheritance of colour-blindness also intrigued scientists such as John Dalton, a British chemist who developed the theory of the atom and who was probably colour-blind himself.

Why were these disorders passed on through females and apparent, most often, only in males? The revolution occurred 100 years ago, when in 1905, two papers were published on the role of chromosomes in determining sex.

Two US researchers, Nettie Stevens and Edmund Beecher Wilson, suggested that one chromosome in males was not found in females. Looking at chromosomes down the microscope, they noticed that half of the sperm cells in insects contained a chromosome not found in eggs. The conclusion – shocking to many – was that this scrap of material was important in determining sex.

Stevens and Wilson further proposed that, except in sperm and eggs, chromosomes exist in pairs and that the small chromosome seen in some sperm was, in fact, the partner of the recently described X chromosome. Females have two X chromosomes but males have an X and a Y chromosome. The effect of mutating a gene on the male X chromosome is readily apparent because there is no compensating copy on the Y chromosome.

By 1910, Thomas Hunt Morgan had used this property to map the first gene, for white eyes, on the X chromosome in the fruit fly Drosophila. Genetic mapping had been born. The next year, EB Wilson proposed that the characteristic of colour blindness was located on the X chromosome – the first gene to be mapped in the human genome.

This unique pattern of inheritance was also already bringing its social consequences. At some point in the lineage of the British Royal family, a mutation occurred in the haemophilia A gene on the X chromosome, possibly in Edward, Duke of Kent and father of Queen Victoria. Victoria was an unaffected carrier of the disease gene and passed it to her son, Leopold, who died of haemophilia, and to her daughters Alice and Beatrice.

One of Alice’s daughters was Alexandra, who married Nicholas, last Tsar of Imperial Russia. Their son and heir, Alexei, was affected by haemophilia. It is suggested that his illness led the family to fall under the influence of Rasputin, who was believed to have extraordinary powers to heal. While Nicholas was engaged in leading the fight against Germany in World War I, the Empress focused her energies on her ailing son. The Empire was weakened, the Tsar abdicated in February 1917, and the monarchy fell with the assassination of the family in July 1918.

The discovery of chromosomal sex determination and the means to map genes onto the X chromosome in humans and other organisms laid the groundwork for genetics and, ultimately, for the work of the Human Genome Project. This unique pattern of inheritance led to a revolution in biology. And its effects led to a revolution in Europe.

Additional Quotes

“We often describe the results of sequencing as a ‘catalogue of human genes’. The results of projects such as the finished X chromosome are so much more than that. They are the forces that will drive biomedical advance in the UK and around the world.”

“We are already seeing clinical benefits and the sequence will stimulate research into the unusual biology of the X chromosome. Mary Lyon’s pioneering work more than 40 years ago was a foundation, and now the sequence will be the framework on which we can build new understanding.”

Professor Allan Bradley Director, Wellcome Trust Sanger Institute

“One of our projects in the earliest days of the Sanger Centre was to sequence what seemed then a huge region of the X chromosome which today, of course, would be the task of a few weeks. Our efforts then were stimulated by the medical and biological interest of the X chromosome and the wish to establish unfettered release of genome sequence. The X chromosome occupies a unique place in biology and in the Wellcome Trust Sanger Institute. The biological and medical benefits from the X chromosome sequence, outlined in this publication, are testimony to the efforts of a dedicated group of international collaborators, which were led by the staff at Hinxton.”

Dr Jane Rogers Head of Sequencing at the Wellcome Trust Sanger Institute

“Fifteen years ago, the X chromosome was a proving ground and became an icon of the efforts of our new institute to prove that sequencing of the human genome was possible, affordable and worthwhile.”

“Of course, we need to know about all the genes, because most conditions depend upon interactions among many of them. It’s therefore wonderful that our international programme to sequence the entire genome, not just bits of it, won through. Having the X chromosome completed is both a practical and symbolic expression of that achievement. All those involved, at Hinxton and beyond, deserve tremendous applause.”

Sir John Sulston Former Director of the Wellcome Trust Sanger Institute

“To date, complex diseases that show a bias in males such as autism, dyslexia, specific language impairment and attention deficit/hyperactivity disorder have not shown strong evidence for major X-linked genes involved in susceptibility in genetic studies using families. However, as the complete sequence of the X chromosome is now available with a high density of SNPs, association studies for these neurodevelopmental disorders may have more power to identify genes on the X chromosome with variants that increase susceptibility.”

Dr Tony Monaco Director and Head of Neurogenetics Group, Wellcome Trust Centre for Human Genetics, Oxford