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Whole-exome sequencing of familial cases of multiple morphological abnormalities of the sperm flagella (MMAF) reveals new DNAH1 mutations.
Hum Reprod. 2016 12; 31(12):2872-2880.HR

Abstract

STUDY QUESTION

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?

SUMMARY ANSWER

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.

WHAT IS KNOWN ALREADY

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.

STUDY DESIGN, SIZE, DURATION

This was a retrospective genetics study of patients presenting a MMAF phenotype. Patients were recruited in Iran and Italy between 2008 and 2015.

PARTICIPANTS/MATERIALS, SETTING, METHODS

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.

MAIN RESULTS AND THE ROLE OF CHANCE

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.

LARGE SCALE DATA

N/A.

LIMITATIONS, REASONS FOR CAUTION

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.

WIDER IMPLICATIONS OF THE FINDINGS

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.

STUDY FUNDING/COMPETING INTERESTS

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+Show Affiliations

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.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.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.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.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.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.Department of Andrology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, PO Box 16635-148, Tehran, Iran.Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, PO Box 16635-148, Tehran, Iran.Department of Andrology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, PO Box 16635-148, Tehran, Iran.Center for the Study and Treatment of Fertility Disorders, Niguarda Ca' Granda Metropolitan Hospital, Milan, Italy.Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, PO Box 16635-148, Tehran, Iran.Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, PO Box 16635-148, Tehran, Iran.Center for the Study and Treatment of Fertility Disorders, Niguarda Ca' Granda Metropolitan Hospital, Milan, Italy.Genetic Epigenetic and Therapies of Infertility, Institute for Advanced Biosciences, INSERM 1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble F38000, France.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.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 pray@chu-grenoble.fr. CHU de Grenoble, UF de Biochimie Génétique et Moléculaire, Grenoble F-38000, France.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

27798045

Citation

Amiri-Yekta, Amir, et al. "Whole-exome Sequencing of Familial Cases of Multiple Morphological Abnormalities of the Sperm Flagella (MMAF) Reveals New DNAH1 Mutations." Human Reproduction (Oxford, England), vol. 31, no. 12, 2016, pp. 2872-2880.
Amiri-Yekta A, Coutton C, Kherraf ZE, et al. Whole-exome sequencing of familial cases of multiple morphological abnormalities of the sperm flagella (MMAF) reveals new DNAH1 mutations. Hum Reprod. 2016;31(12):2872-2880.
Amiri-Yekta, A., Coutton, C., Kherraf, Z. E., Karaouzène, T., Le Tanno, P., Sanati, M. H., Sabbaghian, M., Almadani, N., Sadighi Gilani, M. A., Hosseini, S. H., Bahrami, S., Daneshipour, A., Bini, M., Arnoult, C., Colombo, R., Gourabi, H., & Ray, P. F. (2016). Whole-exome sequencing of familial cases of multiple morphological abnormalities of the sperm flagella (MMAF) reveals new DNAH1 mutations. Human Reproduction (Oxford, England), 31(12), 2872-2880.
Amiri-Yekta A, et al. Whole-exome Sequencing of Familial Cases of Multiple Morphological Abnormalities of the Sperm Flagella (MMAF) Reveals New DNAH1 Mutations. Hum Reprod. 2016;31(12):2872-2880. PubMed PMID: 27798045.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Whole-exome sequencing of familial cases of multiple morphological abnormalities of the sperm flagella (MMAF) reveals new DNAH1 mutations. AU - Amiri-Yekta,Amir, AU - Coutton,Charles, AU - Kherraf,Zine-Eddine, AU - Karaouzène,Thomas, AU - Le Tanno,Pauline, AU - Sanati,Mohammad Hossein, AU - Sabbaghian,Marjan, AU - Almadani,Navid, AU - Sadighi Gilani,Mohammad Ali, AU - Hosseini,Seyedeh Hanieh, AU - Bahrami,Salahadin, AU - Daneshipour,Abbas, AU - Bini,Maurizio, AU - Arnoult,Christophe, AU - Colombo,Roberto, AU - Gourabi,Hamid, AU - Ray,Pierre F, Y1 - 2016/10/26/ PY - 2016/07/15/received PY - 2016/09/09/revised PY - 2016/09/15/accepted PY - 2016/11/1/pubmed PY - 2018/1/18/medline PY - 2016/11/1/entrez KW - DNAH1 KW - MMAF KW - exome sequencing KW - flagellum KW - gene mutations KW - genetic diagnosis KW - male infertility KW - teratozoospermia SP - 2872 EP - 2880 JF - Human reproduction (Oxford, England) JO - Hum. Reprod. VL - 31 IS - 12 N2 - STUDY QUESTION: 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? SUMMARY ANSWER: 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. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: This was a retrospective genetics study of patients presenting a MMAF phenotype. Patients were recruited in Iran and Italy between 2008 and 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS: 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: 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. WIDER IMPLICATIONS OF THE FINDINGS: 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. STUDY FUNDING/COMPETING INTERESTS: 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. SN - 1460-2350 UR - https://www.unboundmedicine.com/medline/citation/27798045/Whole_exome_sequencing_of_familial_cases_of_multiple_morphological_abnormalities_of_the_sperm_flagella__MMAF__reveals_new_DNAH1_mutations_ L2 - https://academic.oup.com/humrep/article-lookup/doi/10.1093/humrep/dew262 DB - PRIME DP - Unbound Medicine ER -