Using genetics to understanding the causes of stuttering

Transcription

1 Using genetics to understanding the causes of stuttering CSHA Fluency SC21 - 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 Who we are National Institute on Deafness and Other Communication Disorders (NIDCD) One of 21 Institutes that make up the National Institutes of Health (NIH) U.S. Department of Health and Human Services Located in Bethesda, MD Porter Neuroscience Research Center

4 Stuttering The medical researcher s view Affects all populations and languages Common 4% lifetime prevalence, 0.5-1% of adults Difficult to treat 1/3 long term success, 1/3 temporary success, 1/3 unsuccessful Profound effects; causes unknown

5 Fact #1- Not all stuttering is genetic Of the people who come to stuttering therapy, about half report a family history and half have no family history So, perhaps half of stuttering is due to genetic factors The other half is due to unknown causes Low birth weight, perinatal hypoxia

6 Fact #2 - Genes do not entirely control stuttering in anyone Severity varies from hour to hour, day to day, and in children, from month to month Stuttering therapy can largely eliminate stuttering Their genes have not changed

7 Part I. Human genetics for speechlanguage pathologists

8 Genetics The study of inheritance Our genes Individual units that code for specific products Together these products comprise the structure and functions of the body 21,000 different genes in humans Reside in structures inside the cell called chromosomes

9 How do we use genetics to identify a gene that causes a disorder?

10 The traditional paradigm 1. Identify stable variation a trait or a disorder 2. Demonstrate that variation is due to genetic factors 3. Perform studies in families with many cases of the disorder to identify the location of the causative gene(s) a linkage study 4. Search the location to identify a genetic variant carried by the affected family members and not by unaffected family members positional cloning

11 Step 1 Stable variation If a disorder comes and goes in an individual, it can be difficult to perform a genetics study In our research, we focus on persistent stuttering

12 Step 2 Determine heritability Done with: Twin studies Adoption studies Family studies

13 Step 3 A linkage study Gather a large number of families in which the disorder occurs Test genetic markers the reside at locations along the length of each chromosome Search for a marker that is co-inherited with the disorder in a family Such co-inheritance is called linkage Linkage occurs because the marker and the disease gene are located close to each other

14 The human chromosomes

15 Step 4 identify the gene The house-to-house search Greatly aided by the existence of the DNA sequence of the human genome Involves looking at the DNA code of all the genes in the region in affected individuals and comparing it to the DNA code of those genes in unaffected individuals

16 Positional cloning Refers to the isolation (cloning) of a specific gene based solely on its position in the genome Does not require knowledge about the pathology, physiology, or biochemistry of the disorder Agnostic with respect to disease mechanism

17 Positional cloning Produced a long line of successes in human genetics Identified the genes underlying all of the common single-genedisorders Cystic fibrosis, Huntington disease, muscular dystrophy, many others Extended to studies of complex genetic disorders

18 Complex disorders Caused by a combination of genetic and non-genetic factors Include most major classes of medical disorders Cardiovascular disease, psychiatric disorders, metabolic disorders

19 Linkage studies of complex disorders Generally disappointing Low linkage scores Failures to replicate across independent studies Produced many suggestive locations for genes underlying such disorders, but few convincing discoveries of causative genes

20 A solution? A new paradigm Population based association studies Case-control study design Test a very large number of genetic markers distributed across the human genome (100,000 2,000,000) Try to find markers that are more common in cases compared to controls Genome wide association study (GWAS)

21 GWAS experience GWAS works Can identify genetic variants that explain a small fraction of the disorder Problems Very large study populations needed Marginal odds ratios, frequently < 1.5 Majority of associated markers are not within genes

22 Whole-genome sequencing The next emerging genetic analysis method Enabled by technology developments Provide a complete enumeration of all genetic variants in a single individual Will require advances in information analysis for broad application in human disease gene finding

23 How to interpret reports of genetic studies Found genes linked to X Found gene mutations linked to X Found genes that contribute to X Ask yourself: How much of the disorder is explained by their findings? How well does having the genetic variant predict the disorder?

24 Part II. Genetic studies of stuttering

25 Why are genetic approaches so powerful? Capable of finding the genes that cause the disorder Ideal approach for inherited disorders that are difficult to study in other ways Once we have the gene(s), we can see what the gene codes for, and what it s function is, both normally and in people who stutter.

26 Stuttering a tractable target? Is it genetic?

27 Genetic Factors in Stuttering - Familial Aggregation - Inherited disorders tend to cluster in families Incidence of stuttering in first degree relatives = 20-74% Incidence of stuttering in general population = %

28 Genetic factors in stuttering - Twin Studies - Twins reared together Share 100% of early environment Share either 100% of genes (identicals) or 50% of their genes (fraternals) Five twin studies of stuttering published

29 Twin study results Identical twins are always more alike than fraternal twins Identical twin concordance = 20-63% Fraternal twin concordance = 3-19% Modeling using twin data concluded up to 70% of stuttering is due to genetic effects

30 Genetic factors in stuttering - Adoption studies - 2 adoption studies of stuttering have been published Both too small to reach statistically significant conclusions, however No evidence stuttering is learned

31 Family X Genetics of stuttering - Large families - Studied in the 1940 s The Utah Family Descendents of single affected individual Other families Cameroon

32 Genetic factors in stuttering - Segregation analyses - A disorder clusters in families. Does this clustering follow the rules for inherited disorders? Mixed results for stuttering Not possible to assign a specific mode of inheritance

33 A complication most stuttering goes away spontaneously Recovery from childhood developmental stuttering is high, perhaps 75% or more Our strategy study persistent stuttering

34 A common question If it s likely that variants in lots of different genes can cause stuttering, don t you need to find all of them?

35 The goal Find a variant in the genes a particular region of a particular chromosome that occurs in family members who stutter but not in family members who don t stutter Observe that or other variants in the same gene in the affected members of other stuttering families

36 Research plan Begin with genetic linkage studies Applicable to any inherited disorder Don t need to know anything about the underlying cause Identify the location of the gene or genes that cause the disorder Genes reside on structures inside cells called chromosomes which chromosome? Performed in families

37 North American linkage study Studied 70 modest sized nuclear families Found weak evidence of linkage on chromosome 18 Conclusion - there is no single common gene that causes stuttering in the general North American population

38 Similar results from studies by others Suggestive evidence for linkage on chromosomes 2, 3, 5, 7, 9, 12, 13, 15, and 21 Typical of linkage results for human complex traits Weak support for the findings Failures to find the same location across studies No direct identification of disease genes

39 Solution? Specialized populations Take advantage of unusual population structure Pakistan

40 Advantageous population structure - Pakistan 70% of all marriages between either 1st or 2nd cousins This marriage pattern has persisted over centuries Results in a population structure with greatly increased incidence of genetic disorders

41 Finding stuttering families in Pakistan Collaborated with the National Centre of Excellence in Molecular Biology (CEMB), University of Punjab, Lahore Sought stuttering families through the school system Identified 100 families, chose 44 for our linkage study

42 Pakistani stuttering families PKST 72 I:1 I:2 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 VII:11 Aslam Ilyas VII:12 VII:13 VII:14 VII:15 Reehana shafeeq 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 FirdosShagufta 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:13 X: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 PKST 72

43

44 The human chromosomes

45 Gene identification strategy Focus on this region on chromosome 12 in Pakistani family PKST72 87 genes lie within this interval

46 Variant of interest Variant that went along with stuttering in family PKST72 and did not appear in the normal Pakistani population This variant was an apparent mutation in a gene called GNPTAB This mutation changes an important part of the gene Invariant across all species known

47 Mutation associated with stuttering in family PKST72 The same mutation occurs in affected individuals in Pakistani families PKST 05, 25, 41 4/41 families suggests this mutation could account for ~10% of stuttering families in Pakistan The same mutation occurs in unrelated people who stutter from Pakistan and India Mutation not observed in normal North American individuals

48 The goal Find a variant in the genes a particular region of a particular chromosome that occurs in family members who stutter but not in family members who don t stutter Observe that or other variants in the same gene in the affected members of other stuttering families

49 Three other mutations in GNPTAB identified Found in affected individuals of South Asian and European descent All are mutations that make a change at a place in the gene an important place in the gene None ever found in normal control individuals

50 GNPTAB Encodes part of an enzyme Enzyme involved in the normal metabolism of all cells Functions as part of the cell s recycling bin

51 GNPTG Encodes another part of the same enzyme Identified 3 different mutations in 4 unrelated affected individuals All affect important parts of the gene All not observed in normals

52 GNPTAB/G Performs the first step in the lysosomal targeting pathway, which is responsible for directing ~ 60 enzymes to the cell s recycling bin, known as the lysosome

53 NAGPA The uncovering enzyme Performs the next step in the lysosomal targeting pathway Identified 3 mutations in 6 unrelated individuals All of European descent All affect important parts of the enzyme None observed in normal control individuals

54 GNPTAB/G mutations in known disorders Mutations in GNPTAB and GNPTG are known to cause mucolipidosis II and III (ML II and ML III) MLII is a severe disorder, fatal in the first decade of life MLIII is a less serious disease Both are rare lysosomal storage disorders with primary problems displayed in the skeletal system, joints, brain, liver, spleen

55 NAGPA mutations? No disorder in humans has been associated with NAGPA mutations This is surprising, because these might be expected to result in medical symptoms similar to those observed in ML II and III We hypothesize that the primary manifestation of NAGPA mutations is persistent stuttering

56 Discussion Lysosomal targeting disorders are clearly no longer rare Indicates that stuttering now overlaps the field of medicine Pharmacologic therapies for lysosomal storage disorders now well established

57 Is stuttering a mild form of mucolipidosis? To date, we ve examined 4 affected individuals at the NIH Clinical Center No symptoms of ML II or ML III were observed in any of these individuals Other than stuttering, all 4 individuals were neurologically normal

58 Implications for Speech Language Pathology Our results explain a small fraction of stuttering Our results will allow us to ask questions about therapy Could underlying genetic differences explain differences in therapy outcomes? Our results suggest a coming partnership between SLP s and physicians

59 Can we explain stuttering in more individuals?

60 Cameroon

61 Large stuttering family from Cameroon

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

63 Identification of gene on chromosome 15 AP4E1 Encodes part of the so-called adaptor protein (AP) complex 4 Moves things around to their correct location in the cell What cellular component is trafficked by the AP-4 complex?

64 The product of the AP4E1 gene interacts with the product of the NAGPA gene The NAGPA gene product carries a signal that is recognized by the AP4 complex This recognition is abolished when we destroy this signal by creating an artificial mutation in it

65 An emerging theme in the genes identified in persistent stuttering 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

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

67 However, 8% of randomly ascertained individuals in these populations carry apparent mutations Normal rate of mutation in these genes vs. unknown speech phenotype Leads to an estimate of 12-20% of stuttering may be caused by mutations in these four genes

68 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 Single origin ~35,000 years ago

69 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

70 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?

71 How does a disorder of cell metabolism lead to stuttering?

72 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

73 An animal model for stuttering Mice display rich vocal communication Much is ultrasonic and not fully characterized Pup isolation calls are highly reproducible Extensive genetic resources are available Knock-out, knock-in, conditional knock-in Mice vocalization is not a model for human speech Could mice be a model for volitional control of vocalization?

74 Putting human stuttering mutations into mice Animals are born in normal numbers, with the expected distribution of sex and genotype Animals grow and reproduce normally Animals exhibit normal behavior on a wide variety of behavioral assays Much about their ultrasonic vocalization is normal

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

76 The effect of human stuttering mutations on mouse vocalization Mutant mice display less vocalization The duration of their vocalizations are unaltered The duration of the pauses is increased These are the same alterations seen using the same automated acoustical analysis of the speech of humans who carry this mutation Terra Barnes et al, Current Biology, in press

77 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 North America and Europe The genes found in these specialized populations carry other mutations that cause persistent stuttering in the U.S., Europe, South America, Asia, and Africa

78 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 Putting human stuttering mutations into mice results in vocalization deficits similar to those found in the humans who carry these mutations

79 Acknowledgments NIDCD Changsoo Kang M. Hashim Raza Carlos Domingues Naveeda Riaz Eduardo Sainz Joanne Gutierrez Alison Fedyna Jessica Root Allen Braun NHGRI/NISC Jim Mullikan Donna Krasnewich NCBI Alejandro Schaffer Hollins Communications Research Institute Jennifer Mundorff NIH Clinical Center Penelope Friedman CEMB/University of the Punjab Shaheen Khan S. Riazuddin Stuttering Foundation of America British Stammering Association National Stuttering Association Speak Clear Association of Cameroon Joseph Lukong Stuttering research subjects worldwide

80 Research volunteers needed! Individuals with: Persistent stuttering A family history of stuttering A willingness to provide saliva and speech samples See me immediately following this talk