De novo copy-number variants (CNVs) can cause neuropsychiatric disease, but the degree to which they occur somatically, and during development, is unknown. Single-cell whole-genome sequencing (WGS) ...in >200 single cells, including >160 neurons from three normal and two pathological human brains, sensitively identified germline trisomy of chromosome 18 but found most (≥95%) neurons in normal brain tissue to be euploid. Analysis of a patient with hemimegalencephaly (HMG) due to a somatic CNV of chromosome 1q found unexpected tetrasomy 1q in ∼20% of neurons, suggesting that CNVs in a minority of cells can cause widespread brain dysfunction. Single-cell analysis identified large (>1 Mb) clonal CNVs in lymphoblasts and in single neurons from normal human brain tissue, suggesting that some CNVs occur during neurogenesis. Many neurons contained one or more large candidate private CNVs, including one at chromosome 15q13.2-13.3, a site of duplication in neuropsychiatric conditions. Large private and clonal somatic CNVs occur in normal and diseased human brains.
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•Single-neuron, whole-genome sequencing allows copy-number variant detection•Most human single neurons are euploid at the chromosome level•Large (>1 Mb) somatic copy-number variants occur approximately once per genome•Some somatic copy-number variants are clonally inherited and some cause disease
Cai et al. performed copy-number analysis on over 200 single cells, including glia and >160 neurons and glia isolated from primary human brain tissue. Although most neurons in normal brains are euploid, large subchromosomal CNVs were commonly identified, suggesting genetic heterogeneity within normal human brains. Clonal somatic CNVs were identified from both normal and diseased human brains, suggesting that CNVs occurring during neurogenesis can have a wide range of functional impacts on human cognitive function.
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.
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▸ Whole-genome amplification and sequencing of single neurons from human brains ▸ Single-neuron genome-wide analysis of somatic L1 retrotransposition ▸ Somatic L1 insertions are infrequent in cerebral cortex and caudate nucleus ▸ Single-cell analysis of a somatic mutation causing malformation of the brain
Whole-genome sequencing of individual neurons from human brain shows that retrotransposition of the L1 element is infrequent. L1 insertions may thus generate some somatic mutations but are unlikely to account for neuronal diversity.
Despite recent advances in understanding the genetic bases of microcephaly, a large number of cases of microcephaly remain unexplained, suggesting that many microcephaly syndromes and associated ...genes are yet to be identified. Here we report mutations in PYCR2, which encodes an enzyme in the proline biosynthesis pathway, as the cause of a unique syndrome characterized by postnatal microcephaly, hypomyelination, and reduced cerebral white matter volume. Linkage mapping and whole-exome sequencing identified homozygous mutations in PYCR2 (c.355C>T p.Arg119Cys and c.751C>T p.Arg251Cys) in the affected individuals of two consanguineous families. A lymphoblastoid cell line from one affected individual showed a strong reduction in PYCR2 level. When mutant cDNAs were transfected into HEK293FT cells, the mutant protein retained normal mitochondrial localization but the level was lower than the wild-type protein, suggesting that mutant protein is less stable. A PYCR2-deficient HEK293FT cell line generated by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome-editing showed that PYCR2 loss of function led to decreased mitochondrial membrane potential and increased susceptibility to apoptosis under oxidative stress. Morpholino-based knockdown of the zebrafish PYCR2 homolog, pycr1b, recapitulated the human microcephaly phenotype, which was rescued by wild-type human PYCR2 mRNA, but not by mutant mRNAs, further supporting the pathogenicity of the identified variants. Hypomyelination and the absence of lax, wrinkly skin distinguishes this condition from that caused by previously reported mutations in the gene encoding PYCR2’s isozyme, PYCR1, suggesting a unique and indispensable role for PYCR2 in the human central nervous system during development.
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
due to one of these noncoding variants, showing a causative role for
disruption in MMS. We show that
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
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.
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