New genetic tools reveal insights into Huntington’s disease and Autism Marcy E. MacDonald, Ph.D. James F. Gusella, Ph.D. Freemasons Travelling Scholars CENTER FOR HUMAN GENETIC RESEARCH

Huntington’s Disease Affects about 1 in 10,000 people Inherited Brain cells die Mid-life onset of progressive movement disorder Intellectual decline, psychiatric symptoms Debilitation and early death No treatment to prevent or delay

The view from genetics: Huntington’s Disease is caused by a difference within the 3 billion letters of the DNA code that is always inherited by people who get the disease but not by people who do not get the disease

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HD mutation 4

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The HD Mutation AAGCCTTTC-----(CAG) CAACAGCCG TTCGGAAAG-----(GTC) CTTGTCGGC Normal HD Gene Mutant HD Gene AAGCCTTTC-----(CAG)> CAACAGCCG TTCGGAAAG-----(GTC)> CTTGTCGGC

The HD Mutation AAGCCTTTC-----(CAG) CAACAGCCG TTCGGAAAG-----(GTC) CTTGTCGGC Normal HD Gene Mutant HD Gene AAGCCTTTC-----(CAG)> CAACAGCCG TTCGGAAAG-----(GTC)> CTTGTCGGC Silence the mutant gene Safety trial IONIS Pharmaceuticals, Roche Pharmaceuticals

Conception Lifeline of a person with the HD mutation Clinical Diagnosis Death ConceptionDeath Lifeline of a person with a normal HD gene Disease as a lifelong process

Many people develop symptoms earlier or later than may be expected for their CAG mutation size Symptoms 20 years later than expected Symptoms 20 years earlier than expected

Conception With CAG43 Idealized Example of a Successful HD Clinical Trial Clinical Diagnosis Death Conception With CAG43 Intervention Clinical Diagnosis Death No Intervention

People are all different, largely due to differences in their DNA letters (sequence) All cases of HD are caused by CAG >36 mutation in the HD gene The HD mutation is expressed on the background of a different 3 billion letter genome in each individual so symptoms can differ even in individuals with the same size CAG mutation

New tools to query the differences in the 3 billion letters of your DNA (and thousands of other people too) all at once Allele Frequency Exome sequencing Whole genome sequencing Direct detection of functional variation High-frequency Common variant single nucleotide polymorphism (SNP) arrays: Indirect detection of functional variation Low-frequency Rare variant SNP arrays Exome sequencing Indirect and direct detection of functional variation Private Polymorphism Private Exome and genome sequencing

Search the 3 billion letters of the DNAs from 4,000 HD individuals to find the variant letters that appear together with earlier or later onset of symptoms, more often than can be expected by chance Significance Chromosome Genome wide association study - GWAS

HD mutation 4 Good modifier 3 Bad modifier 15 Bad modifier 8 Good modifier

Conception With CAG43 Idealized Example of a Successful HD Clinical Trial Clinical Diagnosis Death Conception With CAG43 Intervention Modifier genes – Chromosomes 3, 8 and 15 Need to find the exact letters that are different and how each difference alters the rate of the disease process that leads to symptoms Clinical Diagnosis Death No Intervention

Conception Clinical Diagnosis Death HD as a Genetic Disease – a lifelong process that is modifiable before symptoms appear Conception Clinical Diagnosis Death Conception Clinical Diagnosis Death

World-wide HD registry ENROLL-HD Investigators and HD family members in: HD MAPS PREDICT-HD COHORT REGISTRY

Neurodevelopmental disorders involving social deficits and communication difficulties, stereotyped or repetitive behaviors and interests, sensory issues, and in some cases, cognitive delays. Can vary in overall severity and in severity of individual features --- from mild to extreme. Often associated with other developmental disorders U.S. estimates as high as 1 in 68 children with males greatly exceeding girls Autism Spectrum Disorders

CHROMOSOMAL TRANSLOCATION

Two individual with extreme phenotypes on the autism spectrum DGAP123: Mentally impaired female with ritualized behaviors, vocal and motor mannerisms, limited eye contact, minimal verbal output, little change in affect or facial expression, and minimal seeking of interaction. DGAP200: Male with pervasive developmental delay- not otherwise specified (PDD_NOS), attention deficit hyperactivity disorder (ADHD), conduct disorder with early onset, and intermittent explosive disorder.

Balanced chromosome rearrangements can pinpoint genes causing autism spectrum disorders

Two individual with extreme phenotypes on the autism spectrum DGAP123: Mentally impaired female with ritualized behaviors, vocal and motor mannerisms, limited eye contact, minimal verbal output, little change in affect or facial expression, and minimal seeking of interaction. DGAP200: Male with pervasive developmental delay- not otherwise specified (PDD_NOS), attention deficit hyperactivity disorder (ADHD), conduct disorder with early onset, and intermittent explosive disorder. In both cases the chromosome rearrangement breaks the gene NRXN1, involved in interaction and communication between the surfaces of nerve cells in the brain

More examples of severely affected individuals NDR26867 (46,XX,t(3;14)(q25.31;q11.2)dn): Mentally impaired female with Autism Spectrum Disorder, precocious puberty, large head size, prominent forehead, shallow eye ridges, prominent eyes, and posteriorly rotated ears. Normal MRI. NDR27031 and NDR27032 (46,XY,t(3;18)(q13.32;q21.2)dn) : Identical twin boys with global developmental delay. Non- verbal at 27 months of age, could not crawl or stand, enlarged head size, prominent forehead, posteriorly rotated ears. Abnormal MRI.

More examples of severely affected individuals NDR26867 (46,XX,t(3;14)(q25.31;q11.2)dn): Mentally impaired female with Autism Spectrum Disorder, precocious puberty, macrocephaly, prominent forehead, shallow supraorbital ridges, prominent eyes, and posteriorly rotated ears. Normal MRI. The chromosome rearrangement breaks the gene CHD8 NDR27031 and NDR27032 (46,XY,t(3;18)(q13.32;q21.2)dn) : Identical twin boys with global developmental delay. Non- verbal at 27 months of age, could not crawl or stand, enlarged head size, prominent forehead, posteriorly rotated ears. Abnormal MRI. The chromosome rearrangement breaks the gene TCF4 Both CHD8 and TCF4 are involved in regulating the timing and degree of expression of large numbers of other genes

Another kind of rearrangement: microdeletion or microduplication

Gene discovery from single unique genomes Talkowski et al., 2012, Cell

Disease Trigger Steps in the Presymptomatic Disease Process Overt Clinical Disease Pathogenesis and clinical expression Steps in Disease Progression IndividualDiseaseFeatures End-stagedisease Factors contributing to disease susceptibility

Cryopreserved (Cell Bank) Reprogram to create pluripotent stem cells Develop mature cells grow skin cells Neural Differentiation 4-6 weeks 3-4 weeks2-3 weeks New Tools for Studying Human Disease Biology: Patient-Specific Stem Cells

CRISPR/Cas Genome Modification

Sequencing of Unique Individuals Structural analysis and genome sequencing have revealed many strong effect genes that contribute to autism spectrum disorders Discovery of specific sequence changes has offered new diagnostic potential for individual families Multiple genetic triggers discovered from individuals with overlapping manifestations offers hope of defining shared biological pathways that lead to individual features of the autism spectrum New genetic tools allow not only discovery of genetic changes but analysis of their effects in human cells in the laboratory

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