title: MLPA and sequence analysis of DPY19L2 reveals point mutations causing globozoospermia.
abstract: OBJECTIVE
  Do DPY19L2 heterozygous deletions and point mutations account for some cases of globozoospermia?
  
  
  CONCLUSIONS
  Two DPY19L2 heterozygous deletions and three point mutations were identified, thus further confirming that genetic alterations of the DPY19L2 gene are the main cause of globozoospermia and indicating that DPY19L2 molecular diagnostics should not be stopped in the absence of a homozygous gene deletion.
  
  
  BACKGROUND
  Globozoospermia is a rare phenotype of primary male infertility characterized by the production of a majority of round-headed spermatozoa without acrosome. We demonstrated previously that most cases in man were caused by a recurrent homozygous deletion of the totality of the DPY19L2 gene, preventing sperm head elongation and acrosome formation. In mammals, DPY19L2 has three paralogs of yet unknown function and one highly homologous pseudogene showing >95% sequence identity with DPY19L2. Specific amplification and sequencing of DPY19L2 have so far been hampered by the presence of this pseudogene which has greatly complicated specific amplification and sequencing.
  
  
  METHODS
  In this cohort study, 34 patients presenting with globozoospermia were recruited during routine infertility treatment in infertility centers in France and Tunisia between January 2008 and December 2011. The molecular variants identified in patients were screened in 200 individuals from the general population to exclude frequent non-pathological polymorphisms.
  
  
  METHODS
  We developed a Multiplex Ligation-dependent Probe Amplification test to detect the presence of heterozygous deletions and identified the conditions to specifically amplify and sequence the 22 exons and intronic boundaries of the DPY19L2 gene. The pathogenicity of the identified mutations and their action on the protein were evaluated in silico.
  
  
  RESULTS
  There were 23 patients who were homozygous for the DPY19L2 deletion (67.6%). Only eight of the eleven non-homozygously deleted patients could be sequenced due to poor DNA quality of three patients. Two patients were compound heterozygous carrying one DPY19L2 deleted allele associated respectively with a nonsense (p.Q342*) and a missense mutation (p.R290H). One patient was homozygous for p.M358K, another missense mutation affecting a highly conserved amino acid. Due to the localization of this mutation and the physicochemical properties of the substituted amino acids, we believe that this variant is likely to disrupt one of the protein transmembrane domains and destabilize the protein. Overall, 84% of the fully analysed patients (n = 31) had a molecular alteration of DPY19L2. There was no clear phenotypic difference between the homozygous deleted individual, patients carrying a point mutation and undiagnosed patients.
  
  
  CONCLUSIONS
  Globally poor fertilization rates are observed after intracytoplasmic sperm injection of round spermatozoa. Further work is needed to assess whether DPY19L2 mutated patients present a better or worse prognostic than the non-diagnosed patients. Evaluation of the potential benefit of treatment with a calcium ionophore, described to improve fertilization, should be evaluated in these two groups.
  
  
  CONCLUSIONS
  In previous work, deletions of DPY19L2 had only been identified in North African patients. Here we have identified DPY19L2 deletions and point mutations in European patients, indicating that globozoospemia caused by a molecular defect of DPY19L2 can be expected in individuals from any ethnic background.
  
  
  BACKGROUND
  None of the authors have any competing interest. This work is part of the project 'Identification and Characterization of Genes Involved in Infertility (ICG2I)' funded by the program GENOPAT 2009 from the French Research Agency (ANR).
authors.affiliation: Laboratoire AGIM, CNRS FRE3405, Equipe Génétique, Infertilité et Thérapeutiques, La Tronche F-38700, France.
authors.forename: Charles
authors.initials: C
authors.lastname: Coutton
authors.forename: Raoudha
authors.initials: R
authors.lastname: Zouari
authors.forename: Farid
authors.initials: F
authors.lastname: Abada
authors.forename: Mariem
authors.initials: M
authors.lastname: Ben Khelifa
authors.forename: Ghaya
authors.initials: G
authors.lastname: Merdassi
authors.forename: Chema
authors.initials: C
authors.lastname: Triki
authors.forename: Denise
authors.initials: D
authors.lastname: Escalier
authors.forename: Laetitia
authors.initials: L
authors.lastname: Hesters
authors.forename: Valérie
authors.initials: V
authors.lastname: Mitchell
authors.forename: Rachel
authors.initials: R
authors.lastname: Levy
authors.forename: Nathalie
authors.initials: N
authors.lastname: Sermondade
authors.forename: Florence
authors.initials: F
authors.lastname: Boitrelle
authors.forename: François
authors.initials: F
authors.lastname: Vialard
authors.forename: Véronique
authors.initials: V
authors.lastname: Satre
authors.forename: Sylviane
authors.initials: S
authors.lastname: Hennebicq
authors.forename: Pierre-Simon
authors.initials: PS
authors.lastname: Jouk
authors.forename: Christophe
authors.initials: C
authors.lastname: Arnoult
authors.forename: Joël
authors.initials: J
authors.lastname: Lunardi
authors.forename: Pierre F
authors.initials: PF
authors.lastname: Ray
mesh_terms: D020101:Acrosome Reaction; D000483:Alleles; D015331:Cohort Studies; D005602:France; D017353:Gene Deletion; D005838:Genotype; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D007248:Infertility, Male / Q000235:genetics*; D008297:Male; D008565:Membrane Proteins / Q000235:genetics*; D008957:Models, Genetic; D017354:Point Mutation; D017422:Sequence Analysis, DNA / Q000379:methods; D013094:Spermatozoa / Q000002:abnormalities / Q000473:pathology; D014416:Tunisia
chemical_list: C511317:DPY19L2 protein, human; D008565:Membrane Proteins