1Weatherall Institute of Molecular Medicine and 2Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK.
Costello and Noonan syndromes are part of a small class of congenital syndromes called ‘paternal age-effect’ (PAE) disorders. Other members of this class include Apert and Crouzon syndromes (associated with FGFR2 mutations), achondroplasia, thanatophoric dysplasia (FGFR3 mutations) and MEN2a/b (RET mutations). In the vast majority of cases, these PAE disorders are caused by spontaneous point mutations and exhibit the collective properties of (1) very high apparent rates of germline base substitution (~500-1000-fold over background), (2) exclusive paternal origin, and (3) an increased average age of the unaffected father from whom the mutation originates (PAE). As somatic events, the same gain-of-function mutations have been associated with various human cancers.
These unusual features have led us to study some of these ultra-rare mutations (such as the Apert 755C>G FGFR2 and thanatophoric dysplasia 1949A>G FGFR3substitutions) directly in human sperm where we showed that they accumulate over time in the sperm of most men. We have also identified somatic FGFR3(1949A>G) and HRAS (181C>A and 182A>G, encoding Q61K and Q61R) mutations in spermatocytic seminoma, a rare type of testicular tumour. The combined evidence suggests that these PAE mutations, although occurring rarely, provide a selective growth advantage to the mutant spermatogonial stem cells, resulting in their clonal expansion and a relative enrichment in sperm over time, accounting for the PAE. To date, all examples of PAE mutations result in the activation of the growth factor receptor-RAS signalling pathway, which is a key determinant of spermatogonial stem cell proliferation and renewal.
To further characterise the mechanisms associated with PAE, we have recently studied most of the Costello syndrome-associated mutations located at the G12 codon of HRAS directly in human sperm, using massively parallel sequencing. These mutations were detected at significant levels (ranging from <1:1,000,000 to 1:8,000 across 92 samples) in the sperm of most men and shown to increase exponentially with the age of the donor, accounting for the PAE observed for Costello syndrome. Our results indicate that the G12S (34G>A) substitution is by far the most common and the most abundant HRAS mutation in sperm (average ~1:45,000), followed by the G12D (35G>A) (average ~1:125,000), while the G12C (34G>T) and the G12V (35G>T) mutations were observed in a smaller number of samples and at lower levels (average ~1:300,000-1:400,000, respectively). Unexpectedly, we also identified many events of tandem substitutions in sperm. In the case of the strongly activating G12V change, tandem mutations (35_36GC>TT and 35_36GC>TA, which have been associated with several Costello cases) are more common than single substitutions (35G>T, reported only in one Costello patient), suggesting an unusual mechanism of mutagenesis taking place within the testis. The relative abundance of HRAS mutations we observed in sperm is consistent with the documented prevalence of Costello alleles and suggests that the strongly activating alleles (such as G12V and G12D), although causing severe phenotypes, do not simply lead to foetal lethality.
These data are also useful for genetic counselling of families presenting with de novo cases of Costello syndrome (and likely extends to all other RASopathies) as the mechanisms associated with PAE mutations in sperm predicts a very low risk of recurrence, which can be usually estimated to be <1:1,000.
