Splicing defects, which are frequently overlooked because they do not affect amino acid production, can play a significant role in rare genetic pituitary gland diseases.

The pituitary gland plays a critical role in hormonal activity. It produces a variety of hormones that regulate the growth and function of other endocrine glands. POU1F1, also known as PIT1, is a pituitary-specific transcription factor that is required for the development of three different hormone-producing cell types: growth hormone, prolactin, and thyroid stimulating hormone. While most mutations in POU1F1 are recessive, some variants have a dominant effect. Typically, either genetic cause results in multiple hormone deficiencies.

Sally Camper and colleagues from the University of Michigan Medical School, recently discovered POU1F1 splicing defects in six families with children who had congenital hormone deficiency.

Splicing is the process by which the introns (noncoding regions of genes) are permanently removed from the primary messenger RNA transcript and the exons (coding regions) are grouped together to form the mature messenger RNA (mRNA) that will eventually produce the protein.

Two distinct forms of POU1F1 are produced in the pituitary gland via alternative splicing: the predominant one is a transcriptional activator (POU1F1-alpha), while the other, which is typically a rare form, is a repressor (POU1F1-beta). When splice enhancer or repressor sites are mutated, the POU1F1 gene predominantly encodes a transcriptional repressor that inhibits hormone production, resulting in extreme short stature and hypothyroidism.

Jacob Kitzman, an assistant professor of Human Genetics and Computational Medicine and Bioinformatics at the UMMS, was intrigued by Camper’s research.

Kitzman proposed creating and testing every possible mutation in order to identify others that could disrupt splicing similarly to the patient variants and to ascertain the common factors underlying the splice-disruptive variants.

Kitzman with his graduate student Cathy Smith used experimental mutagenesis techniques and bioinformatic analyses developed by Kitzman for cancer variant analysis.

“Splicing effects are often difficult to predict, so we thought this would be a great opportunity for some of the approaches my group has developed to systematically test thousands of variants at a time,” said Kitzman.

The team discovered several variants that could disrupt splicing without altering the protein’s amino acid sequence. The researchers compiled a list of variants in genes that affect how the transcription factor is spliced in the pituitary gland. This catalogue determines whether a particular variant is associated with a particular disease.

“We all carry many variants in our genes, and it is difficult to identify those that are deleterious from those that are benign. For precision medicine, we need to understand the possible effect of each variant, and this catalog is very useful for this,” explained Camper.

This team then requested a list of variants from the sequences of their patients from collaborators. They discovered two families with POU1F1 variants that matched a catalogue of experimentally determined splice disruptive changes that do not alter the amino acid sequence. This strongly demonstrates that the Camper and collaborators’ catalogue accurately identifies disease-causing variant splicing.

Additionally, this study allowed for comparison of several software packages that predict the outcome of splicing, with one standing out as the best predictor.

“This validation is more broadly interesting for people who work on any disease. It gives them more confidence in the predictions from state-of-the-art software, and in the quality of their data and results,” said Camper.

Finding patients is one of the difficulties associated with studying rare diseases, which necessitates extensive collaborations. For example, approximately 1 in 4,000 live births result from pituitary hormone deficiency. Approximately 3 percent of cases are caused by mutations in the POU1F1 alpha isoform. Camper’s collaborative study makes use of six unrelated families from France, Brazil, Germany, and Argentina.

“When working on uncommon disease, it is very helpful to establish strong collaborations to identify and enroll patients whose participation is essential. The results from our study are much more convincing thanks to the six unrelated families who presented the same type of genetic defect,” said Camper. “Nobody could do that by themselves.”

Another recent collaborative study found that only 15 percent of 170 Argentinean patients had a mutation in a known hypopituitarism gene. Camper’s team is now planning to sequence the entire DNA of these patients in the hope of identifying additional genes involved in these diseases.

Papers cited:

“High-throughput splicing assays identify missense and silent splice-disruptive POU1F1 variants underlying pituitary hormone deficiency,” The American Journal of Human Genetics. DOI: 10.1016/j.ajhg.2021.06.013

“Comprehensive identification of pathogenic gene variants in patients with neuroendocrine disorders,” Journal of Clinical Endocrinology and Metabolism. DOI: 10.1210/clinem/dgab177