Mutation analysis in patients with total sperm immotility
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Perform the genetic characterization of five patients with total sperm immotility using Sanger sequencing and Whole Exome Sequencing (WES), in order to increase the knowledge on the genetics of sperm immotility and, ultimately, allow the identification of potential genetic markers for infertility.
Prospective study at a University Medical school. We analysed five men with total sperm immotility, four with dysplasia of the fibrous sheath (DFS), associated with disruption of several axonemal structures, and one patient with situs inversus totalis, which showed absence of dynein arms (DA) and nexin bridges. We screened 7 genes by Sanger sequencing, involved in sperm motility and associated to ultrastructural defects found in these patients (CCDC39, CCDC40, DNAH5, DNAI1, RSPH1, AKAP3 and AKAP4). Additionally, we performed WES analysis in the patient with situs inversus.
We identified nine new DNA sequence variants by WES. Two of these variants were considered particularly relevant: a homozygous missense change in CCDC103 gene (c.104G > C, p.R35P) probably related with absence of dynein arms; the other in the INSL6 gene (c.262_263delCC) is thought to be also involved in sperm immotility.
Our work suggests that WES is an effective strategy, especially as compared with conventional sequencing, to study highly heterogenic genetic diseases, such as sperm immotility. For future work we expect to expand the analysis of WES to the other four patients and complement findings with expression analysis or functional studies to determine the impact of the novel variants.
KeywordsDysplasia of the fibrous sheath (DFS) Genetic diagnosis situs inversus totalis Sperm immotility Whole Exome Sequencing (WES)
We would like to acknowledge: Helena Oliveira, MSc, ESHRE Senior clinical embryologist; Ilda Pires, MSc, ESHRE Senior clinical embryologist and Madalena Cabral, BSc, Embryologist, for semen processing at CHVNG; Ana Gonçalves, BSc, and Cláudia Osório, BSc, for semen processing at CGR; Elsa Oliveira, 1st Class Technical Specialist of Pathology, Cytology and Thanatology in the Area of Diagnosis and Therapy and Ângela Alves, Technical assistant teaching and research, for semen processing for electron microscopy at ICBAS-UP.
We also would like to acknowledge Conceição Egas, PhD and Hugo Froufe, MSc (GenoInseq) for performing exome sequencing and assisting the initial variant filtering/analysis.
This work was financed by the Institutions of the authors and in part by UMIB, which is funded by National Funds through FCT-Foundation for Science and Technology, under the Pest-OE/SAU/UI0215/2014.
The authors have nothing to declare.
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