National Institute on Deafness and Other Communication Disorders (NIDCD) U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES National Institutes of Health Discovery of Causes of Stuttering Dennis Drayna, PhD July 8, 2010

Twin studies – Heritability estimates range from 50-70% Adoption studies – No evidence that stuttering is learned Family clusters of stuttering – Many small and a few large families Segregation analysis – Less successful Evidence for genetic factors in stuttering

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

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

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

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

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

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

Pakistani stuttering families PKST 72

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

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

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

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

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

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

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

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

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

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

Discussion Mutations in these 3 genes may account for 5-10% of familial stuttering worldwide, and stuttering in more than 100,000 individuals in the U.S. alone Lysosomal targeting disorders are clearly no longer rare Indicates that stuttering now overlaps the field of medicine Enzyme replacement therapy for lysosomal storage disorders is now well established

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

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 SLPs and physicians

How does a disorder of cell metabolism lead to stuttering?

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

Can we explain stuttering in more individuals?

Newly found Pakistani stuttering families

Linkage analysis in PKST77

Acknowledgments NIDCD – Changsoo Kang – M. Hashim Raza – Naveeda Riaz – Eduardo Sainz – Joe Kleinman – Alison Fedyna NHGRI/NISC – Alice Young – Jim Mullikan – Donna Krasnewich 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 Stuttering Foundation of America British Stammering Association National Stuttering Association Speak Clear Association of Cameroon – Joseph Lukong Stuttering research subjects worldwide

Research volunteers needed! Do you stutter? Have you stuttering for more than 6 months? Is there more than one person in your family who stutters? Join us immediately following this talk!