Iso-Seq Analysis Sheds Light on Splicing Associated with Schizophrenia

Neurexin genes, which have been associated with certain neuropsychiatric disorders, are known to make heavy use of alternative splicing. In a recent study, scientists used the Iso-Seq method with SMRT Sequencing to better understand splice variants in neurons derived from human induced pluripotent stem cells (hiPSCs).
The study, “Neuronal impact of patient-specific aberrant NRXN1α splicing,” was published in Nature Genetics. Lead authors Erin Flaherty (@erinkflaherty) and Shijia Zhu, senior author Kristen Brennand (@kristenbrennand), and collaborators at the Icahn School of Medicine at Mount Sinai and other institutions undertook the project to help shed light on disorders linked to exonic deletions in the neurexin-1 gene, including schizophrenia.
“Deletions occur non-recurrently (with different boundaries) between patients, and the mechanisms underlying variable penetrance and diverse clinical presentations remain unknown,” the scientists write, adding that mouse models have been of limited value in elucidating this biology. “To better understand the clinical impact of NRXN1+/− mutations, it is critical to evaluate how distinct patient-specific deletions alter the NRXN1 isoform repertoire and impact synaptic function in a human context.”
Central to the study are four patient samples with rare heterozygous intragenic deletions in NRXN1 with severe psychosis disorder. Two patients contained a 136 kb deletion in the 3’ region of NRXN1 (3’-NRXN1^+/-), while the other two shared a 115 kb deletion in the 5’ region (5’-NRXN1^+/-).
To understand how the deletions affect splicing, the authors incorporated long-read and short-read sequencing of the NRXN1 gene on hiPSC-derived cell types. In hiPSC and other human samples, the team identified more than 120 human NRXN1α isoforms that are predicted to be translated. A comparison showed that “hiPSC-neurons modeled well the NRXN1α alternative splicing diversity found in vivo, particularly the high-abundance isoforms,” the authors report.
They showed that patient-derived NRXN1^+/- hiPSC-neurons have a >2-fold reduction in wild type NRXN1α isoforms and an increase in novel isoforms from the mutant allele. “Across the two 3’-NRXN1^+/- cases, we observed reduced abundance of 50% of the wild-type isoforms,” they note. The authors add that they “further detected 31 mutant NRXN1α isoforms unique to the 3’-NRXN1^+/- hiPSC-neurons that resulted from splicing across the three deleted exons not found in controls.”
This alteration of isoform expression may be affecting neuronal maturation and activity. Compared to control, the 5’-NRXN1^+/- and 3’-NRXN1^+/- hiPSC-neurons had fewer mature neurons and decreased neuronal activity. Interestingly, over-expression of certain wild-type isoforms increased neuronal activity in the 5’-NRXN1^+/- hiPSC-neurons, while over-expression of other mutant NRXN1α isoforms decreased neuronal activity in control hiPSC-neurons. “Our data supports a model whereby functional deficits in 5’-NRXN1^+/-  neurons arise from NRXN1 haploinsufficiency and can therefore by rescued by overexpression of wild-type NRXN1α isoforms,” the authors write, “but unexpectedly, haploinsufficiency in  5’-NRXN1^+/-  neurons is exacerbated by novel dominant-negative acitivity of mutant splice isoforms, and so cannot be rescued by simply increasing wild-type NRXN1α  levels.”
“Our report links patient-specific, heterozygous intragenic deletions in NRXN1 to isoform dysregulation and impaired neuronal maturation and activity in a human and disease-relevant context,” the authors note. “Mutant NRXN1α isoforms may be particularly biologically relevant as our experimental data demonstrated that overexpression of even a single mutant isoform was sufficient to perturb neuronal activity in control neurons.”
Ultimately, the scientists believe their findings from this project could have significant benefits for understanding and potentially treating schizophrenia. “Evaluating how loss and/or gain of specific NRXN1 isoforms impact neuronal fate, maturation and function in a cell-type-specific and activity-dependent manner represents a critical first step towards a more genetics-based form of precision medicine,” they conclude. “Understanding how NRXN1+/− deletions perturb the splice repertoire and alter neuronal function could ultimately improve genetic diagnosis, prognosis and/or lead to new therapeutic targets.”