IMPC Resources and Mouse Genetics Supports Rare Disease Research

Avatar

By IMPC

Published 28th February 2020

The International Mouse Phenotyping Consortium (IMPC) is a collaboration between world-leading research institutions that specialise in mouse genetics to identify the function of every protein-coding gene in the mouse genome. In recent years, many projects have aimed to sequence the entire genome of numerous species, such as the Human Genome Project, but for many of these projects, the function of these genes remains unknown. The IMPC aims to complete an open-access database that describes what each gene does and how it can affect a mouse physically when it doesn’t work properly.

A map of IMPC collaborators

All organisms have two versions of each gene called alleles. Genes control the production of proteins within organisms, which are vital for cell structure and function. Mutations can cause genes to stop working, which changes the function of the protein or the amount produced. These changes can disrupt development and cause physical and chemical abnormalities.

To find the function of each gene, the IMPC genetically alters mice by turning off one or both alleles for each protein-coding gene, creating a knockout mouse line. The IMPC then conducts a series of tests to find what effect the alteration has had, such as testing grip strength, body weight, blood content, behaviour, sleeping and eating patterns, sight, hearing and much more. The IMPC then analyses the test data for significance and uploads findings into the open-access database. The knockout mouse lines are deposited in publicly funded repositories so researchers can obtain mouse lines for their studies.

How Does Mouse Genetics Apply to Humans?

We share 97% of our DNA with mice, as well as being biologically and behaviourally very similar to humans. They are also very well understood, convenient, and reproduce quickly so that we can observe and study the genetics of several generations. This makes mice a reliable model for human disease and researchers have used them to study cancer and inherited diseases such as diabetes, heart disease, Parkinson’s and Alzheimer’s for decades.

Queen Mary University of London (QMUL) takes IMPC data and analyses it to find associations to human disease. One method used is the study of ‘orthologs.’ These are genes in different species that share an ancestor gene and are therefore more likely to have a similar function. Another method is through data analysis – comparisons are made between the physical changes seen in the mouse after a gene has been turned off to the disease characteristics listed in human rare disease databases, such as OMIM and Orphanet, to see if there are any significant similarities. Through this, genetic mutations in mice can be related to disease in humans.

In 2017, experts from the IMPC and QMUL analysed 3,328 genes in the database and identified models for 360 diseases, including possibly the first models for type C Bernard-Soulier, Bardet-Biedl-5 and Gordon Holmes syndromes.

A disease is usually considered rare if less than 1 in 2,000 people is affected within the population, meaning most genetic conditions are classed as rare diseases. Some of the more well-known genetic diseases, such as Cystic Fibrosis, Muscular Dystrophy and Multiple Sclerosis are well studied but others are not. The less researched a condition is, the harder it is to treat and manage. Some mutations, and the disease they cause, are so rare that there are only clinical records of one or a few families with shared symptoms.

A major aim of the IMPC database is to assist research into rare disease, building a starting point for novel research into the mechanisms of rare disease, new possible treatments and precision medicine – allowing doctors to choose personalised treatments on a genetic basis. Through this, patients can receive accurate and effective healthcare for their conditions.

IMPC Resource Use

Data experts within the IMPC find new ways of analysing the IMPC database (and other open access rare disease databases) to find new ‘candidate genes’ – genes that have not previously been related to disease but have a high chance of causing disease. Recent IMPC screens include finding possible genetic causes behind hearing disorders, metabolic diseases, integumentary and oculocutaneous (hair, skin, eyes and pigmentation) conditions, sleeping and eating disorders and neurodegenerative disease.

Externally, researchers are constantly using IMPC resources for their work. Recent examples of how IMPC alleles have been used are:

  • Clear cell sarcomas, a rare soft tissue cancer.
  • Bardet-Biedl syndrome (BBS) which causes vision loss, obesity, extra fingers and toes and learning problems, among other symptoms.
  • Primary familial brain calcification (PFBC), in which deposits of calcium accumulate in the basal ganglia – structures found deep within the centre of the brain. This can cause movement disorders and psychiatric problems like psychosis, dementia and vertigo.
  • Aicardi-Goutières syndrome (AGS) which causes severe brain dysfunction leading to fevers, seizures, developmental issues and muscle issues. Symptoms start very early, around one year of age, and due to the severity of the condition, most do not reach adulthood.
  • Gray platelet syndrome (GPS), a bleeding disorder that can cause easy bruising, nosebleeds and heavy bleeding after an injury.
  • Karyomegalic interstitial nephritis (KIN), a hereditary, progressive and chronic form of kidney disease.

Researchers can source mice, embryonic stem cells and data for each processed gene on the IMPC website and database. Start searching genes by using our search function above.

For the Future

Since data release 11, the IMPC has now fully tested 6,440 protein-coding genes, but there are still over 11,000 mouse orthologs to be processed and their association to disease still needs to be analysed. The IMPC’s key aim is to complete the analysis of every protein-coding gene in the mouse genome. Having a complete database will only further the use and applications of IMPC resources in rare disease research, opening possibilities for new treatments and better healthcare for patients.

CRISPR/Cas9 has revolutionised genetic research, with seemingly no end to its potential applications. Until recently genome engineering relied on the use of stem cells to target new mutations in mice but now researchers can perform genome editing directly in embryos or specific tissues. As with most projects that use gene editing technology, CRISPR/Cas9 has transformed the efficiency and enhanced the applications of the IMPC.

New CRISPR/Cas9-based techniques are constantly being developed, and existing systems adapted and improved, allowing increasingly sophisticated genetic changes to be made. Two papers published in the journal BMC Biology by Lanza, Gaspero, Lorenzo et al. (2018) and Codner, Mianné, Caulder et al. (2018) explore new advancements and highlight applications for these techniques.

The CRISPR/Cas9 system used in conjunction with single stranded DNA donors is revolutionising our ability to generate targeted mutations directly in the embryo. Whilst short synthetic DNA molecules facilitate this, the use of longer single-stranded DNA donors is a more recent addition to the genome editing toolbox. The two new articles summarised here compare long and short single-stranded donors in a high-throughput setting, both look at conditional knock-out mutants while also presenting advances for the generations of point mutations.

In the first study, led by researchers in Lydia Teboul’s group at the MRC Harwell institute, long single-stranded molecules are utilised to facilitate the generation of conditional alleles. They also apply the system to the introduction of point mutations remote from the recognition site of active Cas9/sgRNA complexes, which up to now has not been possible. This last technique is particularly valuable for human genomic sequencing since it enhances our ability to replicate human mutations in mice.

Alongside this breakthrough the researchers also highlight the unpredictability of this technique. As well as on-target integrations, the system can also produce an array of incorrect alleles. These include unintended point mutations, small or larger sequence rearrangements, and additional donor integrations. Such events are unpredictable by-products and therefore must be omitted in the process of validation of newly established mutant lines.

However, these by-products do not reduce the value of the new system. Instead, they illustrate the importance of a comprehensive validation of new mutants, including sequencing of the locus and copy counting of the number of copies of donor integrations. This process will likely become simplified over time, and issues overcome as new technological advancements are made. In the meantime, although unpredictable, the existing strategies remain efficient at generating desirable mutants.

Author on the paper Lydia Teboul said “An increasing body of evidence is being compiled to indicate that model validation is the newest challenge for the community. After all, the quality and reproducibility of research based on genome editing mutants depends entirely on the thorough characterisation of the mutant in question.”

The aim of the second study was to look at scaling production of conditional null alleles to create IMPC mouse lines. The research, led by Jason Heaney at Baylor College of Medicine, tested the feasibility of using CRISPR/Cas9 gene editing technology to generate conditional knockout mice using Cas9-initiated homology-driven repair (HDR), with both short oligonucleotides and longer single stranded DNA.

The results demonstrate that using pairs of short oligodeoxynucleotides can generate conditional null alleles at many loci; however, at scale there are inefficiencies with this process. On the other hand, long single stranded DNA donors may enable high-throughput production of conditional alleles. Although long single stranded DNA donors are most efficient at generating conditional alleles, pairs of short oligodeoxynucleotides are a viable alternative when use of a long single stranded donor is not feasible due to distance between loxP sites or complexity of sequence between loxP sites. Importantly, in agreement with the Teboul group, random integration of donor DNA and mutagenesis events at the target integration sites were detected when using either type of DNA donor.

 Author on the article Jason Heaney said “Single stranded DNA donors are a critical component of the CRISPR/Cas9 genome editing toolbox and are an invaluable resource for producing conditional knockout alleles in mice. But, given the preponderance for random integration and potential for mutagenesis at sites of HDR, new mouse models produced with these donor DNAs must be carefully screened.”

Both studies highlight that it is essential to screen the sequence errors to check for point mutations, rearrangements and additional donor integrations. Researchers involved in genome editing face several challenges when using CRISPR/CAS9 technology and this latest research show it is essential to understand the unpredictability of different systems. Additionally, standards for the validation and documentation of mutants would be extremely beneficial to the field, and help to ensure quality and research reproducibility.

Research articles:

Codner GF, Mianné J, Caulder A et al. (2018) Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants. BMC Biology 16: 70.

Lanza DG, Gaspero A, Lorenzo I et al. (2018) Comparative analysis of single-stranded DNA donors to generate conditional null mouse alleles. BMC Biology 16: 69.

Avatar

By IMPC

Published 12th July 2018

The IMPC Newsletter

Get highlights of the most important data releases, news and events, delivered straight to your email inbox

Subscribe to newsletter