title: Whole-exome sequencing of familial cases of multiple morphological abnormalities of the sperm flagella (MMAF) reveals new DNAH1 mutations.
abstract: Can whole-exome sequencing (WES) of patients with multiple morphological abnormalities of the sperm flagella (MMAF) identify causal mutations in new genes or mutations in the previously identified dynein axonemal heavy chain 1 (DNAH1) gene?
  
  
  
  WES for six families with men affected by MMAF syndrome allowed the identification of DNAH1 mutations in four affected men distributed in two out of the six families but no new candidate genes were identified.
  
  
  
  Mutations in DNAH1, an axonemal inner dynein arm heavy chain gene, have been shown to be responsible for male infertility due to a characteristic form of asthenozoospermia called MMAF, defined by the presence in the ejaculate of spermatozoa with a mosaic of flagellar abnormalities including absent, coiled, bent, angulated, irregular and short flagella.
  
  
  
  This was a retrospective genetics study of patients presenting a MMAF phenotype. Patients were recruited in Iran and Italy between 2008 and 2015.
  
  
  
  WES was performed for a total of 10 subjects. All identified variants were confirmed by Sanger sequencing. Two additional affected family members were analyzed by direct Sanger sequencing. To establish the prevalence of the DNAH1 mutation identified in an Iranian family, we carried out targeted sequencing on 38 additional MMAF patients of the same geographical origin. RT-PCR and immunochemistry were performed on sperm samples to assess the effect of the identified mutation on RNA and protein.
  
  
  
  WES in six families identified a causal mutations in two families. Two additional affected family members were confirmed to hold the same homozygous mutation as their sibling. In total, DNAH1 mutations were identified in 5 out of 12 analyzed subjects (41.7%). If we only include index cases, we detected two mutated subjects out of six (33%) tested MMAF individuals. Furthermore we sequenced one DNAH1 exon found to be mutated (c.8626-1G > A) in an Iranian family in an additional 38 MMAF patients from Iran. One of these patients carried the variant confirming that this variant is relatively frequent in the Iranian population. The effect of the c.8626-1G > A variant was confirmed by RT-PCR and immunochemistry as no RNA or protein could be observed in sperm from the affected men.
  
  
  
  N/A.
  
  
  
  WES allows the amplification of 80-90% of all coding exons. It is possible that some DNAH1 exons may not have been sequenced and that we may have missed some additional mutations. Also, WES cannot identify deep intronic mutations and it is not efficient for detection of large genomic events (deletions, insertions, inversions). We did not identify any causal mutations in DNAH1 or in other candidate genes in four out of the six tested families. This indicates that the technique and/or the analysis of our data can be improved to increase the diagnosis efficiency.
  
  
  
  Our findings confirm that DNAH1 is one of the main genes involved in MMAF syndrome. It is a large gene with 78 exons making it challenging and expensive to sequence using the traditional Sanger sequencing methods. We show that WES sequencing is good alternative to Sanger sequencing to reach a genetic diagnosis in patients with severe male infertility phenotypes.
  
  
  
  This work was supported by following grants: the 'MAS-Flagella' project financed by the French ANR and the DGOS for the program PRTS 2014 and the 'Whole genome sequencing of patients with Flagellar Growth Defects (FGD)' project financed by the Fondation Maladies Rares for the program Séquençage à haut débit 2012. The authors have no conflict of interest.
authors: Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, PO Box 16635-148, Tehran, Iran.|Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, INSERM 1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble F38000, France.
authors: Amir
authors: A
authors: Amiri-Yekta
authors: Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, INSERM 1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble F38000, France.|CHU de Grenoble, UF de Génétique Chromosomique, Grenoble F-38000, France.
authors: Charles
authors: C
authors: Coutton
authors: Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, INSERM 1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble F38000, France.|CHU de Grenoble, UF de Biochimie Génétique et Moléculaire, Grenoble F-38000, France.
authors: Zine-Eddine
authors: ZE
authors: Kherraf
authors: Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, INSERM 1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble F38000, France.|CHU de Grenoble, UF de Biochimie Génétique et Moléculaire, Grenoble F-38000, France.
authors: Thomas
authors: T
authors: Karaouzène
authors: Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, INSERM 1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble F38000, France.|CHU de Grenoble, UF de Génétique Chromosomique, Grenoble F-38000, France.
authors: Pauline
authors: P
authors: Le Tanno
authors: Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, PO Box 16635-148, Tehran, Iran.|Department of Medical Genetics, Institute of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
authors: Mohammad Hossein
authors: MH
authors: Sanati
authors: Department of Andrology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, PO Box 16635-148, Tehran, Iran.
authors: Marjan
authors: M
authors: Sabbaghian
authors: Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, PO Box 16635-148, Tehran, Iran.
authors: Navid
authors: N
authors: Almadani
authors: Department of Andrology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, PO Box 16635-148, Tehran, Iran.
authors: Mohammad Ali
authors: MA
authors: Sadighi Gilani
authors: Center for the Study and Treatment of Fertility Disorders, Niguarda Ca' Granda Metropolitan Hospital, Milan, Italy.
authors: Seyedeh Hanieh
authors: SH
authors: Hosseini
authors: Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, PO Box 16635-148, Tehran, Iran.
authors: Salahadin
authors: S
authors: Bahrami
authors: Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, PO Box 16635-148, Tehran, Iran.
authors: Abbas
authors: A
authors: Daneshipour
authors: Center for the Study and Treatment of Fertility Disorders, Niguarda Ca' Granda Metropolitan Hospital, Milan, Italy.
authors: Maurizio
authors: M
authors: Bini
authors: Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, INSERM 1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble F38000, France.
authors: Christophe
authors: C
authors: Arnoult
authors: Faculty of Medicine, Institute of Clinical Biochemistry, Catholic University, Rome, Italy.|Center for the Study of Rare Hereditary Diseases, Niguarda Ca' Granda Metropolitan Hospital, Milan, Italy.
authors: Roberto
authors: R
authors: Colombo
authors: Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, PO Box 16635-148, Tehran, Iran.
authors: Hamid
authors: H
authors: Gourabi
authors: Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, INSERM 1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble F38000, France pray@chu-grenoble.fr.|CHU de Grenoble, UF de Biochimie Génétique et Moléculaire, Grenoble F-38000, France.
authors: Pierre F
authors: PF
authors: Ray
mesh_terms: D048430:Cell Shape / Q000235:genetics; D004398:Dyneins / Q000235:genetics*; D059472:Exome; D006801:Humans; D007248:Infertility, Male / Q000235:genetics*; D008297:Male; D009154:Mutation; D010375:Pedigree; D012189:Retrospective Studies; D017422:Sequence Analysis, DNA; D013082:Sperm Tail; D013094:Spermatozoa / Q000002:abnormalities* / Q000166:cytology; D000073359:Exome Sequencing
chemical_list: C000598989:DNAH1 protein, human; D004398:Dyneins
keywords: DNAH1; MMAF; exome sequencing; flagellum; gene mutations; genetic diagnosis; male infertility; teratozoospermia