, Volume 20, Issue 3, pp 117–127 | Cite as

Linkage analysis and whole exome sequencing reveals AHNAK2 as a novel genetic cause for autosomal recessive CMT in a Malaysian family

  • Shelisa Tey
  • Nortina Shahrizaila
  • Alexander P. Drew
  • Sarimah Samulong
  • Khean-Jin Goh
  • Esra Battaloglu
  • Derek Atkinson
  • Yesim Parman
  • Albena Jordanova
  • Ki Wha Chung
  • Byung-Ok Choi
  • Yi-Chung Li
  • Michaela Auer-Grumbach
  • Garth A. Nicholson
  • Marina L. KennersonEmail author
  • Azlina Ahmad-AnnuarEmail author
Original Article


Charcot-Marie-Tooth (CMT) disease is a form of inherited peripheral neuropathy that affects motor and sensory neurons. To identify the causative gene in a consanguineous family with autosomal recessive CMT (AR-CMT), we employed a combination of linkage analysis and whole exome sequencing. After excluding known AR-CMT genes, genome-wide linkage analysis mapped the disease locus to a 7.48-Mb interval on chromosome 14q32.11–q32.33, flanked by the markers rs2124843 and rs4983409. Whole exome sequencing identified two non-synonymous variants (p.T40P and p.H915Y) in the AHNAK2 gene that segregated with the disease in the family. Pathogenic predictions indicated that p.T40P is the likely causative allele. Analysis of AHNAK2 expression in the AR-CMT patient fibroblasts showed significantly reduced mRNA and protein levels. AHNAK2 binds directly to periaxin which is encoded by the PRX gene, and PRX mutations are associated with another form of AR-CMT (CMT4F). The altered expression of mutant AHNAK2 may disrupt the AHNAK2-PRX interaction in which one of its known functions is to regulate myelination.


Inherited neuropathy AHNAK2 Autosomal recessive CMT 


Compliance with ethical standards

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Supplementary material

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  1. 1.
    Brennan KM, Bai Y, Shy ME (2015) Demyelinating CMT—what’s known, what’s new and what’s in store? Neurosci Lett 596:14–26CrossRefGoogle Scholar
  2. 2.
    Harding AE, Thomas PK (1980) The clinical features of hereditary motor and sensory neuropathy types I and II. Brain 103:259–280CrossRefGoogle Scholar
  3. 3.
    Timmerman V, Strickland A, Züchner S (2014) Genetics of Charcot-Marie-Tooth (CMT) disease within the frame of the human genome project success. Genes 5:13–32CrossRefGoogle Scholar
  4. 4.
    Zimon M, Baets J, Almeida-Souza L, De Vriendt E, Nikodinovic J, Parman Y, Battaloglu E, Matur Z, Guergueltcheva V, Tournev I, Auer-Grumbach M, De Rijk P, Petersen BS, Muller T, Fransen E, Van Damme P, Loscher WN, Barisic N, Mitrovic Z, Previtali SC, Topaloglu H, Bernert G, Beleza-Meireles A, Todorovic S, Savic-Pavicevic D, Ishpekova B, Lechner S, Peeters K, Ooms T, Hahn AF, Zuchner S, Timmerman V, Van Dijck P, Rasic VM, Janecke AR, De Jonghe P, Jordanova A (2012) Loss-of-function mutations in HINT1 cause axonal neuropathy with neuromyotonia. Nat Genet 44:1080–1083CrossRefGoogle Scholar
  5. 5.
    Cottenie E, Kochanski A, Jordanova A, Bansagi B, Zimon M, Horga A, Jaunmuktane Z, Saveri P, Rasic Vedrana M, Baets J, Bartsakoulia M, Ploski R, Teterycz P, Nikolic M, Quinlivan R, Laura M, Sweeney Mary G, Taroni F, Lunn Michael P, Moroni I, Gonzalez M, Hanna Michael G, Bettencourt C, Chabrol E, Franke A, von Au K, Schilhabel M, Kabzińska D, Hausmanowa-Petrusewicz I, Brandner S, Lim Siew C, Song H, Choi B-O, Horvath R, Chung K-W, Zuchner S, Pareyson D, Harms M, Reilly Mary M, Houlden H (2014) Truncating and missense mutations in IGHMBP2 cause Charcot-Marie-Tooth disease type 2. Am J Hum Genet 95:590–601CrossRefGoogle Scholar
  6. 6.
    Ylikallio E, Woldegebriel R, Tumiati M, Isohanni P, Ryan MM, Stark Z, Walsh M, Sawyer SL, Bell KM, Oshlack A, Lockhart PJ, Shcherbii M, Estrada-Cuzcano A, Atkinson D, Hartley T, Tetreault M, Cuppen I, Ludo van der Pol W, Candayan A, Battaloglu E, Parman Y, van Gassen KLI, van den Boogaard M-JH, Boycott KM, Kauppi L, Jordanova A, Lönnqvist T, Tyynismaa H (2017) MCM3AP in recessive Charcot-Marie-Tooth neuropathy and mild intellectual disability. Brain 140(8):2093–2103CrossRefGoogle Scholar
  7. 7.
    Lindner TH, Hoffmann K (2005) easyLINKAGE: a PERL script for easy and automated two-/multi-point linkage analyses. Bioinformatics 21:405–407CrossRefGoogle Scholar
  8. 8.
    Abecasis GR, Cherny SS, Cookson WO, Cardon LR (2002) Merlin--rapid analysis of dense genetic maps using sparse gene flow trees. Nat Genet 30:97–101CrossRefGoogle Scholar
  9. 9.
    Cottingham RW, Idury RM, Schäffer AA (1993) Faster sequential genetic linkage computations. Am J Hum Genet 53:252–263Google Scholar
  10. 10.
    Matise TC, Chen F, Chen W, De La Vega FM, Hansen M, He C, Hyland FC, Kennedy GC, Kong X, Murray SS, Ziegle JS, Stewart WC, Buyske S (2007) A second-generation combined linkage physical map of the human genome. Genome Res 17:1783–1786CrossRefGoogle Scholar
  11. 11.
    Drew AP, Zhu D, Kidambi A, Ly C, Tey S, Brewer MH, Ahmad-Annuar A, Nicholson GA, Kennerson ML (2015) Improved inherited peripheral neuropathy genetic diagnosis by whole-exome sequencing. Mol Genet Genomic Med 3:143–154CrossRefGoogle Scholar
  12. 12.
    Bahlo M, Bromhead CJ (2009) Generating linkage mapping files from Affymetrix SNP chip data. Bioinformatics 25:1961–1962CrossRefGoogle Scholar
  13. 13.
    Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MAR, Bender D, Maller J, Sklar P, de Bakker PIW, Daly MJ, Sham PC (2007) PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet 81:559–575CrossRefGoogle Scholar
  14. 14.
    Blankenberg D, Von Kuster G, Coraor N, Ananda G, Lazarus R, Mangan M, Nekrutenko A, Taylor J (2010) Galaxy, a web-based genome analysis tool for experimentalists. Curr Protoc Mol Biol 0(19):Unit-19.1021Google Scholar
  15. 15.
    Goecks J, Nekrutenko A, Taylor J, Team TG (2010) Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol 11:R86CrossRefGoogle Scholar
  16. 16.
    Tey S, Ahmad-Annuar A, Drew AP, Shahrizaila N, Nicholson GA, Kennerson ML (2016) Mutation analysis of genes within the dynactin complex in a cohort of hereditary peripheral neuropathies. Clin Genet 90:127–133CrossRefGoogle Scholar
  17. 17.
    Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protocols 3:1101–1108CrossRefGoogle Scholar
  18. 18.
    Shahrizaila N, Samulong S, Tey S, Suan LC, Meng LK, Goh KJ, Ahmad-Annuar A (2014) X-linked Charcot-Marie-Tooth disease predominates in a cohort of multiethnic Malaysian patients. Muscle Nerve 49:198–201CrossRefGoogle Scholar
  19. 19.
    Kircher M, Witten DM, Jain P, O'Roak BJ, Cooper GM, Shendure J (2014) A general framework for estimating the relative pathogenicity of human genetic variants. Nat Genet 46(3):310–315CrossRefGoogle Scholar
  20. 20.
    Narasimhan VM, Hunt KA, Mason D, Baker CL, Karczewski KJ, Barnes MR, Barnett AH, Bates C, Bellary S, Bockett NA, Giorda K, Griffiths CJ, Hemingway H, Jia Z, Kelly MA, Khawaja HA, Lek M, McCarthy S, McEachan R, O’Donnell-Luria A, Paigen K, Parisinos CA, Sheridan E, Southgate L, Tee L, Thomas M, Xue Y, Schnall-Levin M, Petkov PM, Tyler-Smith C, Maher ER, Trembath RC, MacArthur DG, Wright J, Durbin R, van Heel DA (2016) Health and population effects of rare gene knockouts in adult humans with related parents. Science 352:474–477CrossRefGoogle Scholar
  21. 21.
    Sano K, Miura S, Fujiwara T, Fujioka R, Yorita A, Noda K, Kida H, Azuma K, Kaieda S, Yamamoto K, Taniwaki T, Fukumaki Y, Hiroki S (2015) A novel missense mutation of RYR1 in familial idiopathis hyper CK-emia. J Neurol Sci 356(1–2):142–147CrossRefGoogle Scholar
  22. 22.
    Nagata K, Takahashi M, Kiryu-Seo S, Hiroshi K, Saido TC (2017) Distinct functional consequences of ECEL1/DINE missense mutations in the pathogenesis of congenital contracture disorders. Acta Neuropathol Commun 5:83CrossRefGoogle Scholar
  23. 23.
    Komuro A, Masuda Y, Kobayashi K, Babbitt R, Gunel M, Flavell RA, Marchesi VT (2004) The AHNAKs are a class of giant propeller-like proteins that associate with calcium channel proteins of cardiomyocytes and other cells. Proc Natl Acad Sci U S A 101:4053–4058CrossRefGoogle Scholar
  24. 24.
    Luzón-Toro B, Gui H, Ruiz-Ferrer M, Sze-Man Tang C, Fernández RM, Sham P-C, Torroglosa A, Kwong-Hang Tam P, Espino-Paisán L, Cherny SS, Bleda M, Enguix-Riego MV, Dopazo J, Antiñolo G, García-Barceló M-M, Borrego S (2015) Exome sequencing reveals a high genetic heterogeneity on familial Hirschsprung disease. Sci Rep 5:16473CrossRefGoogle Scholar
  25. 25.
    Monies D, Abouelhoda M, AlSayed M, Alhassnan Z, Alotaibi M, Kayyali H, Al-Owain M, Shah A, Rahbeeni Z, Al-Muhaizea MA, Alzaidan HI, Cupler E, Bohlega S, Faqeih E, Faden M, Alyounes B, Jaroudi D, Goljan E, Elbardisy H, Akilan A, Albar R, Aldhalaan H, Gulab S, Chedrawi A, Al Saud BK, Kurdi W, Makhseed N, Alqasim T, El Khashab HY, Al-Mousa H, Alhashem A, Kanaan I, Algoufi T, Alsaleem K, Basha TA, Al-Murshedi F, Khan S, Al-Kindy A, Alnemer M, Al-Hajjar S, Alyamani S, Aldhekri H, Al-Mehaidib A, Arnaout R, Dabbagh O, Shagrani M, Broering D, Tulbah M, Alqassmi A, Almugbel M, AlQuaiz M, Alsaman A, Al-Thihli K, Sulaiman RA, Al-Dekhail W, Alsaegh A, Bashiri FA, Qari A, Alhomadi S, Alkuraya H, Alsebayel M, Hamad MH, Szonyi L, Abaalkhail F, Al-Mayouf SM, Almojalli H, Alqadi KS, Elsiesy H, Shuaib TM, Seidahmed MZ, Abosoudah I, Akleh H, AlGhonaium A, Alkharfy TM, Al Mutairi F, Eyaid W, Alshanbary A, Sheikh FR, Alsohaibani FI, Alsonbul A, Al Tala S, Balkhy S, Bassiouni R, Alenizi AS, Hussein MH, Hassan S, Khalil M, Tabarki B, Alshahwan S, Oshi A, Sabr Y, Alsaadoun S, Salih MA, Mohamed S, Sultana H, Tamim A, El-Haj M, Alshahrani S, Bubshait DK, Alfadhel M, Faquih T, El-Kalioby M, Subhani S, Shah Z, Moghrabi N, Meyer BF, Alkuraya FS (2017) The landscape of genetic diseases in Saudi Arabia based on the first 1000 diagnostic panels and exomes. Hum Genet 136:921–939CrossRefGoogle Scholar
  26. 26.
    Marg A, Haase H, Neumann T, Kouno M, Morano I (2010) AHNAK1 and AHNAK2 are costameric proteins: AHNAK1 affects transverse skeletal muscle fiber stiffness. Biochem Biophys Res Commun 401:143–148CrossRefGoogle Scholar
  27. 27.
    Hohaus A, Person V, Behlke J, Schaper J, Morano I, Haase H (2002) The carboxyl-terminal region of ahnak provides a link between cardiac L-type Ca2+ channels and the actin-based cytoskeleton. FASEB J 16:1205–1216CrossRefGoogle Scholar
  28. 28.
    Huang Y, Laval SH, van Remoortere A, Baudier J, Benaud C, Anderson LVB, Straub V, Deelder A, Frants RR, den Dunnen JT, Bushby K, van der Maarel SM (2007) AHNAK, a novel component of the dysferlin protein complex, redistributes to the cytoplasm with dysferlin during skeletal muscle regeneration. FASEB J 21:732–742CrossRefGoogle Scholar
  29. 29.
    Davies TA, Loos B, Engelbrecht AM (2014 Dec) AHNAK: the giant jack of all trades. Cell Signal 26(12):2683–2693CrossRefGoogle Scholar
  30. 30.
    Han H, Kursula P (2014) Periaxin and AHNAK nucleoprotein 2 form intertwined homodimers through domain swapping. J Biol Chem 289:14121–14131CrossRefGoogle Scholar
  31. 31.
    Gillespie CS, Sherman DL, Blair GE, Brophy PJ (1994) Periaxin, a novel protein of myelinating Schwann cells with a possible role in axonal ensheathment. Neuron 12:497–508CrossRefGoogle Scholar
  32. 32.
    Guilbot A, Williams A, Ravise N, Verny C, Brice A, Sherman DL, Brophy PJ, LeGuern E, Delague V, Bareil C, Megarbane A, Claustres M (2001) A mutation in periaxin is responsible for CMT4F, an autosomal recessive form of Charcot-Marie-Tooth disease. Hum Mol Genet 10:415–421CrossRefGoogle Scholar
  33. 33.
    Boerkoel CF, Takashima H, Stankiewicz P, Garcia CA, Leber SM, Rhee-Morris L, Lupski JR (2001) Periaxin mutations cause recessive Dejerine-Sottas neuropathy. Am J Hum Genet 68:325–333CrossRefGoogle Scholar
  34. 34.
    Kirov A, Kacer D, Conley BA, Vary CP, Prudovsky I (2015) AHNAK2 participates in the stress-induced nonclassical FGF1 secretion pathway. J Cell Biochem 116:1522–1531CrossRefGoogle Scholar
  35. 35.
    Hossain WA, Morest DK (2000) Fibroblast growth factors (FGF-1, FGF-2) promote migration and neurite growth of mouse cochlear ganglion cells in vitro: immunohistochemistry and antibody perturbation. J Neurosci Res 62:40–55CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Shelisa Tey
    • 1
  • Nortina Shahrizaila
    • 2
  • Alexander P. Drew
    • 3
    • 4
  • Sarimah Samulong
    • 1
  • Khean-Jin Goh
    • 2
  • Esra Battaloglu
    • 5
  • Derek Atkinson
    • 6
  • Yesim Parman
    • 7
  • Albena Jordanova
    • 6
  • Ki Wha Chung
    • 8
  • Byung-Ok Choi
    • 9
  • Yi-Chung Li
    • 10
  • Michaela Auer-Grumbach
    • 11
  • Garth A. Nicholson
    • 3
    • 4
    • 12
  • Marina L. Kennerson
    • 3
    • 4
    • 12
    Email author
  • Azlina Ahmad-Annuar
    • 1
    Email author
  1. 1.Department of Biomedical Science, Faculty of MedicineUniversity of MalayaKuala LumpurMalaysia
  2. 2.Department of Medicine, Faculty of MedicineUniversity of MalayaKuala LumpurMalaysia
  3. 3.Northcott Neuroscience Laboratory, ANZAC Research InstituteUniversity of SydneyConcordAustralia
  4. 4.Sydney Medical SchoolUniversity of SydneySydneyAustralia
  5. 5.Department of Molecular Biology and GeneticsBogazici UniversityIstanbulTurkey
  6. 6.Molecular Neurogenomics Group, VIB-U Antwerp Center for Molecular NeurologyUniversity of AntwerpAntwerpenBelgium
  7. 7.Department of Neurology, Istanbul School of MedicineIstanbul UniversityIstanbulTurkey
  8. 8.Department of Biological SciencesKongju National UniversityGongjuSouth Korea
  9. 9.Department of Neurology, Samsung Medical CenterSeoulSouth Korea
  10. 10.Department of Neurology, Taipei Veterans General HospitalNational Yang-Ming University School of MedicineTaipeiTaiwan
  11. 11.Department of Orthopedics and Trauma SurgeryMedical University of ViennaViennaAustria
  12. 12.Molecular Medicine LaboratoryConcord HospitalConcordAustralia

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