Genetic studies of stuttering in international populations. Dennis Drayna, PhD NIDCD/National Institutes of Health US Public Health Service, DHHS

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1 Genetic studies of stuttering in international populations Dennis Drayna, PhD NIDCD/National Institutes of Health US Public Health Service, DHHS

2 Disclosures Stuttering Foundation of America Board of Directors Hollins Communications Research Institute Collaborator, Contract

3 Learning objectives Understand the motivation for identifying genes that underlie stuttering Understand the general features of genetic contributions to stuttering Learn what genes have so far been associated with persistent stuttering, how they are related, and what they do Identify the strengths and weaknesses of animal models of stuttering

4 Stuttering The medical researcher s view A problem in the voluntary control of speech production Affects all populations and languages Common 4% lifetime prevalence, 0.5-1% of adults Difficult to treat long term success, temporary success, no success Profound effects While persistence vs. recovery remains a fundamental question, we study only persistent stuttering

5 Evidence for genetic factors in stuttering Genes vs. Environment Twin studies 8 independent twin studies published over 30 years English, Dutch, Japanese, Finnish, Italian, Danish Estimates of heritability (the amount attributable to genes) have risen as studies have become larger and more sophisticated Heritability estimates now ~.85 Adoption studies - Two independent studies - Neither large enough to achieve strong statistical significance - No evidence stuttering is learned

6 Why find genes that cause stuttering? Finding such genes will help us identify underlying causes of stuttering at the level of cells and molecules Identifying such causes is critical to improving the diagnosis and treatment of this disorder

7 How genetics is used to identify causative genes Classically begins with a linkage study Performed in families Identifies the location of the gene that causes the disorder in that family Once a linkage location is obtained, the genes in the region are sequenced in affected and unaffected family members Candidate causative genes are confirmed by sequencing the gene in other families with the same disorder

8 Finding genes for stuttering Early linkage studies disappointing Lack of strong statistical significance Failed to replicate in independent studies Re-focus efforts using unusual families from unique populations Pakistan Cameroon, West Africa

9 Pakistani stuttering families PKST 072 II:1 II:2 II:3 II:4 III:1 III:2 III:3 III:4 III:5 III:6 III:7 Sooban M.Din Bibi Rani Nizam Din Ali Muhammad Bagh Bare Roshan Din IV:1 IV:2 IV:3 IV:4 Aysha M.Din Charagh bibi(hajan) V:4 V:5 V:1 Genotyped Zahoor ahmad V:2 mukhtar V:3 ghulam Fatima Raj bibi M.Din VI:6 VI:7 VI:8 VI:11 VI:9 VI:10 VI:1 VI:2 VII:4 VI:3 VI:4 VI:5 Amanat Rasheedan kalsoom Bashiran Imran Haneef haji Tufail haji yousaf VII:19 VII:20 VII:21 VII:22 VII:23 VII:1 Yasmeen VII:2 Abdul Ghaffar VII:3 Genotyped M waqas VII:5 M Iqbal VII:6 VII:7 Abdul Jabbar VII:8 Bushra VII:18 Sohail VII:17 VII:9 Akram Surriya VII:10 Aslam VII:11 Ilyas VII:12 VII:13 VII:14 Reehana shafeeq VII:15 Rukhsana VII:16 Jameel VIII:5 VIII:7 VIII:1 VIII:2 VIII:3 VIII:4 Asif VIII:6 Abdul Baree Usman Asad IX:1 Atif IX:18 IX:2 Tahir IX:3 IX:4 Firdos Shagufta IX:5 Genotyped Tashfeen IX:6 Genotyped Kashif IX:7 Genotyped Yaseen IX:8 Genotyped Zulfiqar Ali IX:9 Genotyped M Tayyab IX:10 robina IX:11 awais IX:12 Genotyped M Hussain IX:13 shahid IX:14 hafeez IX:15 IX:16 shazia Yasmeen IX:17 Nazia X:14 X:12 X:13X:15 X:16 X:1 Genotyped Abid X:2 Genotyped Haroon X:3 Genotyped Asif X:4 X:5 X:6 X:7 Amir X:8 Ahmad X:9 Fehmeeda X:10 Jawwad X:11 Hamza shan Iqra Tayyaba

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11 Contributions to stuttering A mutation in PKST 072 and three other Pakistani stuttering families found in GNPTAB gene Suggests that one mutation in this gene is responsible for about 10% of persistent stuttering families in Pakistan Other mutations in this gene found in individuals with persistent stuttering in Pakistan, England, U.S., Brazil Mutations in the functionally related GNPTG and NAGPA genes found in individuals with persistent stuttering worldwide

12 GNPTAB, GNPTG, NAGPA Encode 2 enzymes that act in succession (a 2- step pathway) to put a mannose-6-phosphate onto any one of ~ 50 enzymes The mannose-6-phosphate acts as a targeting signal that tells the cell to route these enzymes to their proper location in the cell the lysosome The lysosome is the cell s recycling bin

13 Medical genetics Mutations in GNPTAB and GNPTG have long been known to cause Mucolipidosis Types II and III Rare, fatal genetic disease of children and young adults Extensive work-ups of stuttering subjects carrying mutations in these genes have so far failed to identify any symptoms of mucolipidosis Mutations in found in stuttering subjects are all different from those found in mucolipidosis patients, with one exception 3 bp deletion eliminating valine 78 in GNPTAB, observed in a case of Mucolipidosis Type III

14 Medical implications of stuttering mutations The mutations in these genes that cause stuttering are different than the ones that cause Mucolipidosis Stuttering is not a mild form of Mucolipidosis Mutations in NAGPA have not previously been associated with any human disorder

15 Finding more genes

16 Cameroon

17 Large stuttering family from Cameroon

18 The many cases of stuttering are due to multiple genes in this family Stuttering due to genes on chromosomes 2, 3, 14, and 15

19 The gene on chromosome 15 AP4E1 Mutation causes stuttering in one branch of the Cameroonian family Same mutation in the same gene found in 2 unrelated Cameroonian individuals with persistent stuttering Many other mutations in this gene found in cases from U.S., England, Brazil, and Pakistan

20 What are these genes? Genes encode components that act to move things around to their proper locations in cells Intracellular trafficking Subject of the 2013 Nobel Prizes in Medicine or Physiology R. Schekman, J. Rothman, T. Sudhöf Deficits in intracellular trafficking are an emerging concept in neurological disorders Alzheimer s, Parkinson s, Huntington s

21 How much of stuttering do they cause? We now have identified 4 different genes that cause stuttering Approximately 20% of unrelated individuals who stutter carry a mutation in one of these genes

22 What populations do they cause stuttering in? Mutations in these genes are found in individuals who stutter from North America, South America, Europe, Asia, and Africa Most are rare (seen only once or twice), but a few are more common, typically in one population

23 What populations do they cause stuttering in? Mutations in these genes are found in individuals who stutter from North America, South America, Europe, Asia, and Africa Most are rare (seen only once or twice), but a few are common, typically in one population GNPTAB Glu1200Lys South Asians Single origin ~14,000 years ago NAGPA Arg328Cys Europeans

24 Implications today Many genetic causes of stuttering Some genes found, likely we can find more High (but not perfect) correlation between carrying a mutation and persistent stuttering Persistent vs. recovered predictive testing? Correlation between gene/mutation and therapy outcome? Stuttering and neurology

25 What do these mutations cause? Studies done in a test tube on the products of these genes show that stuttering mutations cause modest decrease in function (~50%) How do modest deficits in moving things around inside cells cause stuttering?

26 Working hypothesis A specific group of nerve cells in the brain are unique to speech production and also uniquely sensitive to this cellular deficit Goal Identify these cells, discover what they do, determine what they re connected to, and understand how this inherited deficit uniquely affects them

27 Why study mice? Mice have unparalleled genetic tools for understanding biomedical phenomena Knock-outs - mice with total loss of a gene Knock-ins - mice carrying a specific introduced mutation Conditional knock-outs/ins Mice expressing a genetic defect in just one organ or cell type in the body

28 Establishment of the mouse as a model for human communication Mice display rich vocal communication Much of it is ultrasonic and not fully characterized Mating Pup isolation Social interactions Not generated by vibration of vocal folds More analogous to a whistle

29 Pup isolation calls Generated by <10 day old pups upon removal from the nest Lead to maternal retrieval behavior Stereotyped Ballistic Disappear when pups acquire hearing Different inbred lines display different calls

30 Genetics of mouse ultrasonic vocalization Pup isolation calls appear to be innate Cross fostering study Kikusui et al, PLoS one 2011 Pup isolation call patterns can segregate as ~ Mendelian trait Choi, Park, and Kim, BMC Neuroscience 2011

31 Our collaboration Terra Barnes, PhD and Tim Holy, PhD Washington University in St. Louis Mouse vocalization recording Acoustic signal analysis Tae-Un Han, PhD NIDCD/National Institutes of Health Knock-in mouse construction Mouse vocalization recording

32 Mice Pups Vocalize in the Ultrasonic Range (P8) Terra Barnes, PhD

33 Mouse carrying homozygous equivalent of the GNPTAB Glu1200 Lys mutation (Glu1179Lys)

34 Intrabout intervals may be longer Terra Barnes, PhD

35 Additional stuttering mutations GNPTAB Ala455Ser GNPTAB Ser321Gly Mice carrying these mutations are largely normal Pup vocalization recording and analysis by Tae-Un Han, PhD, NIDCD Homozygous animals of both lines show evidence of longer inter-syllable intervals within vocalization bouts

36 Conclusions I. The complex genetics of stuttering can be overcome (in at least some cases) by studying unusual families with structures and mating patterns not found in the U.S. The genes found in these specialized populations carry other mutations that cause persistent stuttering in the U.S., Europe, South America, Asia, and Africa

37 Conclusions II. We re beginning to find genes in which mutations cause stuttering 4 genes found so far, and more (perhaps many more) remain to be identified Early results show a previously unsuspected cellular deficit in stuttering Path now open to identifying the neuropathology that leads to dysfluent speech

38 Conclusions III. Putting human stuttering mutations into mice results in largely normal animals While a number of aspects of pup ultrasonic vocalization do not appear to be affected, the timing of vocalizations differs from wild type littermates This is a major feature of human stuttering vs. normal speech analyzed for the same measures using the same analyses

39 Acknowledgments NIDCD Changsoo Kang M. Hashim Raza Tae-Un Han Carlos Dominguez Eduardo Sainz Joanne Gutierrez Rachel Rahn Alan Braun NINDS Mark Hallett NIH Clinical Center Penelope Friedman NCBI Alejandro Schaffer Hollins Communications Research Institute Jennifer Mundorff CEMB/University of the Punjab Jamil Ahmad Shahid Khan S. Riazuddin Washington University St. Louis Wang Sik Lee, Stuart Kornfeld Terra Barnes, Tim Holy Stuttering Foundation of America British Stammering Association National Stuttering Association Speak Clear Association of Cameroon Joseph Lukong Stuttering research subjects worldwide