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Single-Cell Analysis Elucidates Pathogenic Underpinnings of Klinefelter Syndrome

NEW YORK – Researchers in Oregon, Utah, and Missouri have conducted a single-cell analysis on testis samples from human donors in order to elucidate the pathogenic mechanisms underlying Klinefelter syndrome.

In a paper published on Friday in the American Journal of Human Genetics, the researchers said the molecular basis for KS — which is characterized by a set of physiological abnormalities such as infertility — is still unclear. For their study, they used a comparative methodology to analyze single-cell RNA-sequencing data from the biopsies of 12 human donors. By comparing donors from a range of ages and forms of infertility, they generated gene expression signatures that characterized normal testicular function and distinguished clinically distinct forms of male infertility.

Overall, the systematic assessment of known cell signaling pathways helped the researchers identify 72 pathways that are potentially active in testes, dozens of which appear upregulated in KS.

Unexpectedly, they also identified a subpopulation of Sertoli cells (the somatic cells of the testes) within multiple individuals with KS that lacked transcription from the XIST locus. This resulted in increased X-linked gene expression compared to all other KS cell populations.

"Altogether our data support a model of pathogenic changes in interstitial cells cascading from loss of X inactivation in pubertal Sertoli cells and nominate dosage-sensitive factors secreted by Sertoli cells that may contribute to the process," the authors wrote. "Our findings demonstrate the value of comparative patient analysis in mapping genetic mechanisms of disease and identify an epigenetic phenomenon in KS Sertoli cells that may prove important for understanding causes of infertility and sex chromosome evolution."

Alongside their study, the researchers provided a web-based interface for their dataset which they called the Human Infertility Single-cell Transcription Atlas, with features for exploring the data and results.

To dissect the pathological biology in KS, the researchers combined and analyzed 26,300 single-cell transcriptomes derived from the testes of 12 donors. Their primary analysis framework, sparse decomposition of arrays, was useful for summarizing large, multi-donor scRNA-seq data into manageable sets of co-regulated genes.

This approach, they said, enabled them to identify numerous sets of genes with expression patterns specific to men with a type of infertility called idiopathic non-obstructive azoospermia, men with KS, or both.

The key finding was that XIST expression was lost in the Sertoli cells of post-pubertal KS-affected individuals, the researchers said. They were also able to validate that this loss of expression was functional, and that X-linked genes showed a concomitant increase in expression.

Interestingly, they also estimated that loss of XIST expression only led to an 11 percent to 14 percent increase in expression output from the X chromosome in Sertoli cells, which was much less than the theoretical increase of 100 percent. This was consistent with prior observations in female mice lacking XIST, where the increase in X-linked expression was in the range of 14 percent to 36 percent.

"Taken together, these results add to a growing number of observations that define the unusual epigenetic plasticity of Sertoli cells," the authors wrote. "Conditional deletion of the transcription factor DMRT1 from adult Sertoli cells causes them to transform to granulosa cells, the female analog of Sertoli cells."

They also noted that conditional deletion of the transcription factor WT1 from embryonic Sertoli cells leads to a phenotype similar to fetal Leydig cells, which produce testosterone, and complete loss of seminiferous tubules, producing testis histology reminiscent of KS.

"The fact that X chromosome copy number influences the epigenetic state of the Sertoli cells but not other cells of the testis presents an interesting puzzle and suggests that X chromosome inactivation and sex determination may have a shared evolutionary history with the differentiation program of somatic cells of the gonads," the authors concluded. "These results bring clarification to the cell types that are involved in KS pathology and what the temporal sequence of events might be."

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