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The 21st Century Imitation Game


High-throughput gene sequencing is now much more affordable and accessible. The real challenge is identifying which genetic variations are responsible for disease.

By Elena Tucker

New sequencing technologies are enabling scientists to crack the genetic code of rare mitochondrial diseases and disorders of sex development.

Imagine a child who appears healthy and happy for the first years of life but then develops a fever, starts experiencing seizures, muscle pain and weakness, and dies within the year.

Imagine another child who fails to thrive from birth, frequently vomits, never gains sufficient weight and often has trouble breathing.

Imagine a child born looking neither like a girl nor a boy. How would the family of this child feel when answering the typical first question: “Is it a girl or boy”?

These children are all likely to have inherited rare genetic diseases such as mitochondrial disorders or, in the case of the third child, a disorder or difference of sex development (DSD).

Our genetic code contains about three billion bases and encodes more than 20,000 proteins, any of which may potentially cause disease if faulty, so discovering which gene underlies a rare genetic disease is often a slow and daunting task. Without a genetic diagnosis, these affected children are often subjected to ongoing tests that can be painful and are often risky. Families experience confusion and frustration, not knowing what is wrong with their child and fear that their other children could be affected.

Although each rare genetic disease affects only a small number of families, collectively they have a large impact on the health of our community. There are more than 6000 rare diseases such as cystic fibrosis (which causes a build-up of mucus on the lung surface), Huntington’s disease (a neurodegenerative disease leading to problems with movement and cognition) and achondroplasia (a form of dwarfism). The rare genetic diseases that I have focused my research on are mitochondrial diseases, but I will soon be shifting my focus to DSD.

Mitochondrial diseases are rare diseases that affect our ability to convert food into energy. Any tissue or organ can be affected because every part of our body needs energy to function. For example, our heart needs energy to pump, our brains need energy to think, our muscles need energy to move and our guts need energy to digest. As a consequence, patients can present with almost any symptom and at any age of onset.

In many cases, children with mitochondrial disease die within the first years of life. In other cases, mitochondrial disease presents in adulthood and may have a milder course.

DSD is an umbrella term used to describe a range of conditions. At the rare and severe end of the spectrum, individuals may be born with ambiguous genitalia that look neither female nor male, or may have complete sex reversal whereby they are genetically one sex but physically the other.

DSD are sometimes associated with intellectual impairment, facial dysmorphia, infertility or susceptibility to cancer. The severe forms of DSD affect about one in 4500 births. At the mild end of the spectrum are anomalies of genital structure, such as the aberrant positioning of the urethral opening along the penile shaft.

Both mitochondrial diseases and DSD are difficult to diagnose at the genetic level. In the case of mitochondrial diseases well over 100 genes can be responsible, and there are many “disease genes” yet to be discovered.

A further complication is that mitochondrial diseases can have any mode of inheritance. Most genes are inherited as DNA in the nucleus of our cells. We get two copies of each gene – one from our mother and one from our father – but in addition to the ~20,000 genes in the nucleus we also inherit 37 genes as DNA in the mitochondria. These are always inherited from our mother.

Mitochondrial disease can therefore be caused by faults in either the regular nuclear DNA or in the mitochondrial DNA. This means that they can be inherited from a mother by all her children, or as dominant conditions when only one copy of a nuclear gene is faulty but causes disease, or as recessive conditions when both copies of a nuclear gene must be faulty before disease is evident.

Like mitochondrial diseases, DSD have a heterogeneous genetic basis – many genes can be responsible. And just like mitochondrial diseases, the genetic basis of DSD is not fully understood.

More than half of patients who are genetically male (XY) but present as female or intermediate sex have no known genetic basis for their condition. Complicating matters further, DSD can be caused by faults in “non-coding” DNA.

Only about 1.5% of human DNA encodes a physical product (protein) while the remaining 98.5% is mostly poorly understood. Although this DNA does not encode proteins, it can have an important role in when and how protein-coding genes are activated. This non-coding DNA is particularly important for processes that rely on signalling pathways to activate development, such as the development of the sex organs.

Unlike mitochondrial function, which is a highly conserved process occurring in a similar manner in all species, sex development is highly divergent, making its genetics harder to study in model organisms. For example, humans have a key male-determining gene, SRY, whereas a number of mammals such as moles and some rodents lack this gene so they clearly have different sex development pathways. While male mammals have a non-homologous set of sex chromosomes (XY) and females have a homologous set (XX), in birds the females have the non-homologous set (designated ZW) while males have the homologous set (ZZ).

This inability to easily use model organisms has made it difficult for scientists to understand the genetics of human sex development.

Next-Generation Sequencing

Before I started my PhD in 2008, gene sequencing was routinely done on a gene-by-gene basis and the sequencing of each new gene took weeks or months. Considering there are well over 100 different genes that can be responsible for mitochondrial diseases, it was often a slow process to identify the specific gene responsible for the disease in a particular family. Because we only know about half of the genes that might cause these diseases, in many cases families were left without answers altogether.

Now, however, Next Generation Sequencing enables simultaneous sequencing of many genes. In collaboration with a group at the Broad Institute in Boston, our group at the Murdoch Childrens Research Institute, under the supervision of Prof David Thorburn and Dr Alison Compton, carefully selected 103 genes that we suspected might cause a particular form of mitochondrial disease called complex I deficiency. We then sequenced these genes in 103 patients who had this form of the disease.

We were searching for sequence variants that might make a gene dysfunctional, leading to disease. This was one of the first times Next-Generation Sequencing was used in a medical setting, and we were able to provide new diagnoses to many families and discover two new genetic causes of disease.

The success of this first project gave us the confidence and motivation to pursue a larger project – sequencing more than 1000 genes in 44 patients with severe mitochondrial diseases. This time we selected any gene predicted to encode a protein that is found in mitochondria. This project was also very successful, discovering another five new disease genes and providing more families with answers and options for the future. Our research has since focused on how and why the newly-discovered disease genes influence disease.

The DSD Challenge

High-throughput gene sequencing is now much more affordable and accessible. Generating sequencing data is no longer the road-block but it is still a challenge to interpret it. There are many sequencing variations between individuals, but most are benign differences that merely contribute to our uniqueness – such as dark hair, a toothy grin, long legs, a flair for music or a talent for mathematics – and do not contribute to any form of disease or dysfunction. The real challenge is identifying which genetic variations are responsible for disease.

In the case of mitochondrial diseases, a defect in mitochondrial enzyme activity can sometimes be measured in a patient’s skin. Because a patient’s skin cells can be cultured, we can insert a normal copy of the gene suspected to be causing disease into a virus and then introduce this into the patient’s cells in the lab. We can then measure mitochondrial enzyme activity and see whether mitochondrial function is restored. If it can, this would prove that a fault in the gene was causing disease.

Proving the causation of genes responsible for DSD may be more challenging because these genes often only function during foetal development, so the window for studying them is over before the child is even born. It will likely be necessary to use models to study this developmental process, as well as performing indirect studies in vitro, so a lot of work is still required to interpret high-throughput gene sequencing results.

The Ethics of Next-Generation Sequencing

The lowering cost of gene sequencing and its wider availability means that it is becoming more commonly used. This brings with it many ethical questions and concerns.

If a whole genome is being sequenced to investigate a particular disease, sometimes additional information relating to the health of the individual is revealed. Is there an obligation to report it to the patient? If there is nothing that can be done to treat that condition, would the patient even want to know?

What if we discover that the supposed father of a child is not actually the father? Or we find an abnormality in a gene but are not certain about its consequences? Do insurance companies have the right to access this information?

And what is an actual disorder that warrants genetic attention and genetic action? What will we do with all this new information?

Wider Application of Next Generation Sequencing

Next Generation Sequencing technologies are not limited to studying mitochondrial diseases, but can be applied to the diagnosis of any genetic condition including DSD, epilepsy, cardiac dysfunction, intellectual disability and more. We are now undergoing a revolution in the rate of genetic discoveries, and this ought to accelerate the development of treatments and cures for rare conditions that have so far received little attention. I hope that, armed with this technology, we will soon be able to quickly and ethically diagnose any genetic disorder and promptly offer suitable treatments and cures.

Elena Tucker is a Peter Doherty Fellow at the Murdoch Childrens Research Institute, and an Honorary Fellow of the University of Melbourne’s Department of Paediatrics.