The brain's serotonergic system centrally regulates several physiological processes and its dysfunction has been implicated in the pathophysiology of several neuropsychiatric disorders. While in the past our understanding of serotonergic neurotransmission has come mainly from mouse models, the development of pluripotent stem cell and induced fibroblast-to-neuron (iN) transdifferentiation technologies has revolutionized our ability to generate human neurons in vitro. Utilizing these techniques and a novel lentiviral reporter for serotonergic neurons, we identified and overexpressed key transcription factors to successfully generate human serotonergic neurons. We found that overexpressing the transcription factors NKX2.2, FEV, GATA2 and LMX1B in combination with ASCL1 and NGN2 directly and efficiently generated serotonergic neurons from human fibroblasts. Induced serotonergic neurons (iSNs) showed increased expression of specific serotonergic genes that are known to be expressed in raphe nuclei. iSNs displayed spontaneous action potentials, released serotonin in vitro and functionally responded to selective serotonin reuptake inhibitors (SSRIs). Here, we demonstrate the efficient generation of functional human serotonergic neurons from human fibroblasts as a novel tool for studying human serotonergic neurotransmission in health and disease.

The goals are to understand the primary genetic mechanisms that cause Sick Sinus Syndrome and to identify potential modifiers that may result in intrafamilial variability within a multigenerational family. The proband is a 63-year-old male with a family history of individuals (>10) with sinus node dysfunction, ventricular arrhythmia, cardiomyopathy, heart failure, and sudden death. We used exome sequencing of a single individual to identify a novel LMNA mutation and demonstrated the importance of Sanger validation and family studies when evaluating candidates. After initial single-gene studies were negative, we conducted exome sequencing for the proband which produced 9 gigabases of sequencing data. Bioinformatics analysis showed 94% of the reads mapped to the reference and identified 128,563 unique variants with 108,795 (85%) located in 16,319 genes of 19,056 target genes. We discovered multiple variants in known arrhythmia, cardiomyopathy, or ion channel associated genes that may serve as potential modifiers in disease expression. To identify candidate mutations, we focused on ~2,000 variants located in 237 genes of 283 known arrhythmia, cardiomyopathy, or ion channel associated genes. We filtered the candidates to 41 variants in 33 genes using zygosity, protein impact, database searches, and clinical association. Only 21 of 41 (51%) variants were validated by Sanger sequencing. We selected nine confirmed variants with minor allele frequencies <1% for family studies. The results identified LMNA c.357-2A>G, a novel heterozygous splice-site mutation as the primary mutation with rare or novel variants in HCN4, MYBPC3, PKP4, TMPO, TTN, DMPK and KCNJ10 as potential modifiers and a mechanism consistent with haploinsufficiency.

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Mutations in the SLC22A5 gene, which encodes for the plasma membrane carnitine transporter OCTN2, cause primary carnitine deficiency (PCD). After our first report of OCTN2 mutations in Chinese, three more Chinese PCD patients were identified. The parents of these families were non-consanguineous and these families were unrelated. Two novel truncating mutations were found: R254X, a single-base mutation at cDNA position 981 (c.981C>T); and Y387X (c.1382T>G). Two probands, one each from Taiwan and Macau, were homozygous for R254X. The other proband from Taiwan carried both R254X and Y387X. Two additional heterozygote carriers of R254X were also identified among 250 control samples, while none was detected for Y387X. The population carrier rate for R254X would be about 1 in 125. Haplotypes of R254X alleles were examined and patients homozygous for R254X were also homozygous for the same haplotype of intragenic and microsatellites markers. Analysis of population frequencies of haplotypes revealed that the chance of 4 chromosomes having arisen as independent events was 0.016. We conclude that R254X is probably a founder mutation in Chinese. Other previously reported mutations found in the Japanese population were also screening in 250 control samples but no carrier was identified, indicating that they were either very rare or not present in Southern Chinese.