In every generation, new mutations enter the human population for the first time. If a single copy of the new mutation causes a disease, its transmission may immediately affect the next generation. This grant challenges the idea that the frequency of male-derived mutations newly entering the population each generation is solely due to the frequency of new genetic lesions arising during the germline cell divisions. Another somewhat surprising possibility is that some new germline mutations alter the testis stem cell in which they arise so that this cell and its descendants acquire a proliferative advantage compared to wild type stem cells. This disproportionate increase in sperm carrying the new disease mutation, relative to unmutated sperm, increases the probability the disease will be transmitted to the next generation. A testis dissection/mutation detection approach coupled with computational modeling has shown five different disease-causing de novo mutations in four different genes produce the signature of germline selection. The diseases studied were Apert syndrome, multiple endocrine neoplasia 2B (MEN2B), achondroplasia, and Noonan syndrome. For these diseases, older fathers are at greater risk for having affected children than younger fathers (paternal age effect, PAE). As couples increasingly delay the age when they have children, this phenomenon becomes more important. Germline selection is a contributing factor to the PAE since it causes the disease mutation frequency in a man's sperm to increase exponentially as he ages. This application has four Aims. 1) Understand how the Apert syndrome, MEN2B, and Noonan syndrome mutated disease proteins alter the signaling pathways of spermatogonial stem cells. Cultures of undifferentiated spermatogonia from mouse models of each disease will be compared to controls in biochemical and stem cell transplantation experiments. 2) Use testis dissection in conjunction with a new deep next generation sequencing (NGS) method to test the hypothesis that the unusually high frequency of sporadic Noonan syndrome (one of the most common Mendelian diseases) is due to perhaps as many as 20 different gain-of-function mutations in the same gene with each providing a selective advantage. The idea that loss-of-function/dominant negative mutations can provide a germline selective advantage will be examined by studies on LEOPARD syndrome. Experiments on Rett syndrome will ask whether germline selection must always be accompanied by a marked PAE as has been the case in the previously studied diseases. 3) Further develop new NGS methods to measure the rate of very rare spontaneous mutations with both accuracy and high throughput. Such protocols could be useful in germline mutation studies as well as disease diagnosis. 4) Propose new computational models of germline selection based on the experimental work in both human and mouse and develop new statistical methods to test these models.
Surprisingly, some testis stem cells with a spontaneous disease mutation have a growth advantage that increases the chance an unaffected father will transmit the disease to one of his children, despite the fact that serious illness or early death can occur. In this grant, we explore the molecular basis of the mutation-induced stem cell growth advantage; the insights gained may eventually make it possible to reduce this advantage thereby lowering the chance a father will have an affected child with a new mutation. Some of the technologies we will develop in our project may also contribute to the early diagnosis of cancer as well as to many other areas of biomedical interest.
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