Faculty Bibliography

While next-generation sequencing has accelerated the discovery of human disease genes, progress has been largely limited to the "low hanging fruit" of mutations with obvious exonic coding or canonical splice site impact. In contrast, the lack of high-throughput, unbiased approaches for functional assessment of most noncoding variants has bottlenecked gene discovery. We report the integration of transcriptome sequencing (RNA-seq), which surveys all mRNAs to reveal functional impacts of variants at the transcription level, into the gene discovery framework for a unique human disease, microcephaly-micromelia syndrome (MMS). MMS is an autosomal recessive condition described thus far in only a single First Nations population and causes intrauterine growth restriction, severe microcephaly, craniofacial anomalies, skeletal dysplasia, and neonatal lethality. Linkage analysis of affected families, including a very large pedigree, identified a single locus on Chromosome 21 linked to the disease (LOD > 9). Comprehensive genome sequencing did not reveal any pathogenic coding or canonical splicing mutations within the linkage region but identified several nonconserved noncoding variants. RNA-seq analysis detected aberrant splicing in DONSON due to one of these noncoding variants, showing a causative role for DONSON disruption in MMS. We show that DONSON is expressed in progenitor cells of embryonic human brain and other proliferating tissues, is co-expressed with components of the DNA replication machinery, and that Donson is essential for early embryonic development in mice as well, suggesting an essential conserved role for DONSON in the cell cycle. Our results demonstrate the utility of integrating transcriptomics into the study of human genetic disease when DNA sequencing alone is not sufficient to reveal the underlying pathogenic mutation.

Neurons live for decades in a postmitotic state, their genomes susceptible to DNA damage. Here we survey the landscape of somatic single-nucleotide variants (SNVs) in the human brain. We identified thousands of somatic SNVs by single-cell sequencing of 36 neurons from the cerebral cortex of three normal individuals. Unlike germline and cancer SNVs, which are often caused by errors in DNA replication, neuronal mutations appear to reflect damage during active transcription. Somatic mutations create nested lineage trees, allowing them to be dated relative to developmental landmarks and revealing a polyclonal architecture of the human cerebral cortex. Thus, somatic mutations in the brain represent a durable and ongoing record of neuronal life history, from development through postmitotic function.

Somatic mutations occur during brain development and are increasingly implicated as a cause of neurogenetic disease. However, the patterns in which somatic mutations distribute in the human brain are unknown. We used high-coverage whole-genome sequencing of single neurons from a normal individual to identify spontaneous somatic mutations as clonal marks to track cell lineages in human brain. Somatic mutation analyses in >30 locations throughout the nervous system identified multiple lineages and sublineages of cells marked by different LINE-1 (L1) retrotransposition events and subsequent mutation of poly-A microsatellites within L1. One clone contained thousands of cells limited to the left middle frontal gyrus, whereas a second distinct clone contained millions of cells distributed over the entire left hemisphere. These patterns mirror known somatic mutation disorders of brain development and suggest that focally distributed mutations are also prevalent in normal brains. Single-cell analysis of somatic mutation enables tracing of cell lineage clones in human brain.

Genetic mutations causing human disease are conventionally thought to be inherited through the germ line from one's parents and present in all somatic (body) cells, except for most cancer mutations, which arise somatically. Increasingly, somatic mutations are being identified in diseases other than cancer, including neurodevelopmental diseases. Somatic mutations can arise during the course of prenatal brain development and cause neurological disease-even when present at low levels of mosaicism, for example-resulting in brain malformations associated with epilepsy and intellectual disability. Novel, highly sensitive technologies will allow more accurate evaluation of somatic mutations in neurodevelopmental disorders and during normal brain development.

A major unanswered question in neuroscience is whether there exists genomic variability between individual neurons of the brain, contributing to functional diversity or to an unexplained burden of neurological disease. To address this question, we developed a method to amplify genomes of single neurons from human brains. Because recent reports suggest frequent LINE-1 (L1) retrotransposition in human brains, we performed genome-wide L1 insertion profiling of 300 single neurons from cerebral cortex and caudate nucleus of three normal individuals, recovering >80% of germline insertions from single neurons. While we find somatic L1 insertions, we estimate <0.6 unique somatic insertions per neuron, and most neurons lack detectable somatic insertions, suggesting that L1 is not a major generator of neuronal diversity in cortex and caudate. We then genotyped single cortical cells to characterize the mosaicism of a somatic AKT3 mutation identified in a child with hemimegalencephaly. Single-neuron sequencing allows systematic assessment of genomic diversity in the human brain.

Hemimegalencephaly (HMG) is a developmental brain disorder characterized by an enlarged, malformed cerebral hemisphere, typically causing epilepsy that requires surgical resection. We studied resected HMG tissue to test whether the condition might reflect somatic mutations affecting genes critical to brain development. We found that two out of eight HMG samples showed trisomy of chromosome 1q, which encompasses many genes, including AKT3, a gene known to regulate brain size. A third case showed a known activating mutation in AKT3 (c.49G-->A, creating p.E17K) that was not present in the patient's blood cells. Remarkably, the E17K mutation in AKT3 is exactly paralogous to E17K mutations in AKT1 and AKT2 recently discovered in somatic overgrowth syndromes. We show that AKT3 is the most abundant AKT paralog in the brain during neurogenesis and that phosphorylated AKT is abundant in cortical progenitor cells. Our data suggest that somatic mutations limited to the brain could represent an important cause of complex neurogenetic disease.

Mutations accumulate in the genome of every cell of the body throughout life, causing cancer and other diseases^1,2. Most mutations begin as nucleotide mismatches or damage in one of the two strands of the DNA before becoming double-strand mutations if unrepaired or misrepaired^3,4. However, current DNA-sequencing technologies cannot accurately resolve these initial single-strand events. Here we develop a single-molecule, long-read sequencing method (Hairpin Duplex Enhanced Fidelity sequencing (HiDEF-seq)) that achieves single-molecule fidelity for base substitutions when present in either one or both DNA strands. HiDEF-seq also detects cytosine deamination-a common type of DNA damage-with single-molecule fidelity. We profiled 134 samples from diverse tissues, including from individuals with cancer predisposition syndromes, and derive from them single-strand mismatch and damage signatures. We find correspondences between these single-strand signatures and known double-strand mutational signatures, which resolves the identity of the initiating lesions. Tumours deficient in both mismatch repair and replicative polymerase proofreading show distinct single-strand mismatch patterns compared to samples that are deficient in only polymerase proofreading. We also define a single-strand damage signature for APOBEC3A. In the mitochondrial genome, our findings support a mutagenic mechanism occurring primarily during replication. As double-strand DNA mutations are only the end point of the mutation process, our approach to detect the initiating single-strand events at single-molecule resolution will enable studies of how mutations arise in a variety of contexts, especially in cancer and ageing.

The diagnosis and treatment of tumors often depends on molecular-genetic data. However, rapid and iterative access to molecular data is not currently feasible during surgery, complicating intraoperative diagnosis and precluding measurement of tumor cell burdens at surgical margins to guide resections. To address this gap, we developed Ultra-Rapid droplet digital PCR (UR-ddPCR), which can be completed in 15 minutes from tissue to result with an accuracy comparable to standard ddPCR. We demonstrate UR-ddPCR assays for the IDH1 R132H and BRAF V600E clonal mutations that are present in many low-grade gliomas and melanomas, respectively. We illustrate the clinical feasibility of UR-ddPCR by performing it intraoperatively for 13 glioma cases. We further combine UR-ddPCR measurements with UR-stimulated Raman histology intraoperatively to estimate tumor cell densities in addition to tumor cell percentages. We anticipate that UR-ddPCR, along with future refinements in assay instrumentation, will enable novel point-of-care diagnostics and the development of molecularly-guided surgeries that improve clinical outcomes.