1	Molecular Approach to Alternating Hemiplegia of Childhood AHC Workshop Salt Lake City, 2003 Kathryn J. Swoboda MD
2	Summary of Goals Establish an international DNA and cell line database of AHC patients and families to facilitate genetic studies Identify and utilize families (with > 1 affected) to identify/evaluate candidate loci Identify patients and families with AHC and associated cytogenetic abnormalities to provide clues to molecular etiology Sequence analysis of candidate genes in familial and sporadic patients
3	Criteria for Inclusion for Molecular Studies as “definitely affected” Recurrent weakness/paralysis of one side of the body, at some point affecting alternate sides Onset recurrent hemiparesis/hemiplegia < 18 months of age Progressive neurologic difficulties including ataxia, chorea, cognitive impairment Meets exclusion criteria specifications
4	Criteria for Inclusion in Database as “possibly affected” Onset of first episode, with recurrent alternating hemiparesis/hemiplegia > 18 months of age Near exclusive involvement of one side of the body with spells Associated atypical MRI abnormalities: i.e. “periventricular leukomalacia”
5	Absolute Exclusion Criteria Neuroimaging data to suggest vascular etiology or known metabolic disorder associated with recurrent stroke-like episodes (MELAS, PDH) Exclusive involvement of one side of the body with hemiparetic/plegic spells Persistent interictal (not during an episode) focal epileptiform EEG abnormality suggesting an epileptic locus
6	Evidence for Genetic Etiology Two sets of affected identical twins Family with dominant inheritance and AHC (3p26;9q34 chromosome translocation) 5 families with > 1 affected child excluding twins; Greek family 3 (4) affected children (Youroukos, Xaidara), 3 families with 2 or 3 affected children Affected child with mother with hemiplegic migraine Affected half-siblings (Kramer)
7	Disorders with complex phenotypes and high frequency of sporadic occurrence Angelman syndrome Williams syndrome “Microdeletion syndromes” due to new spontaneous deletions on a single chromosome Is AHC a microdeletion syndrome?
8	Disorders with high rate of new mutations Osteogenesis imperfecta Retinoblastoma Is AHC due to mutations in a large gene or conserved gene family with high rate of new mutation?
9	Clues From Similar Disorders: Familial Hemiplegic Migraine Autosomal dominant disorder with episodic recurrent hemiparesis/hemiplegia, migraine Progressive trouble with balance, coordination, memory in some families Genetically more than 1 gene associated: CACNA1A (19p) ATP1A2: (1q) Fusco and Casari, 2003 Is AHC a calcium or ion “channelopathy”?
10
11
12	Clues from Animal Models A number of mouse models have been identified (or created) with genetic defects, mostly deletions, disruptions or other knockouts, in key neuronally expressed genes, with complex features including some overlap with symptoms in AHC
13	Selected Animal Models with Complex Phenotypes involving Seizure/Movement Disorder/Cognitive Impairment Opisthotonus (inositol 1,4,5-triphosphate receptor 1) Stargazer/Waggler (CACNG2) Tottering/Leaner/Rocker/Rolling Nagoya (CACNA1A) Ducky (CACNA2D2) SCNA8 knockout Ampa receptor knockout Amphiphysin knockout Striatin antisense Rat model Hamster dtsz model AND others…..
14	Clinical and DNA Databases: We have 143 families in the database for which we have DNA and clinical information to permit appropriate affection status designation, including 6 familial cases: 136 patients are sporadic As of last count several months ago, we have DNA on 139 affected patients, 288 other family members; 256 confirmed permanent cell lines
15	Dominant AHC family
16	Other potentially informative familial AHC cases
17	Family with apparently balanced chromosomal translocation 3p26;9q34 Since all affected family members with AHC carry the chromosomal abnormality, is a causative gene somehow disrupted? Initial goal: clone the translocation breakpoint in this family which might lead us to a causative gene disruption/deletion/positional effect
18
19
20	Genetic Studies K4323/panel of selected sporadic cases: Other appealing candidate genes excluded via direct sequence analysis, intronic and exonic regions (3kb pieces): KCNT1: Calcium activated potassium channel 200kb distal to breakpoint on 9q (28 exons) CACNA1D: in vicinity of breakpoint on 3p (48 exons)
21	Genetic Studies K4323: exclusion of linkage to candidate loci with selected flanking and intragenic markers CACNA1A (19p) ATP2B2 (calcium transporting ATPase on 3p) CACNG2 (22q) CACNA1B (9q34)
22	Genetic Studies K4323: Genome-wide linkage analysis completed using 289 highly polymorphic markers approximately every 15 cM Simulated max Lod score using a 2 point system, autosomal dominant inheritance and 0.9 penetrance with a 5 allele system: LOD (max,sim) 1.2 7 loci with LOD > 0.5 additional fine mapping at each locus Locus specific search being undertaken for all potentially appealing gene candidates, particularly ion channels, neurotransmitter receptors
23	Simulated LOD scores in informative families for genome wide linkage analyses Combined maximum LOD score of three “recessive” families, K5148 (3 affected), K5545 (2 affected), K8308 (2 affected) using 4 allele system: 2.74 Combined max LOD score of two dominant families: K4323 (4 affected) and K7940 (5 affected) using 4 allele system: 2.30 caveat 1: germ line mosaicism for a new mutation possible in “recessive” families caveat 2: genetic heterogeneity
24	Results: Genome wide linkage analysis on additional informative families We have identified a possible locus on chromosome 1 where affected individuals in three “recessive” families appear to share a haplotype within each family (preliminary data) We have excluded known loci for FHM (ATP1A2,CACNA1A) in these families with the exception of one family for CACNA1A: sequencing analysis in progress We have excluded CACNA1A by linkage in Greek family. Sequencing for ATP1A2 in progress
25	Acknowledgements: the AHC team AHC families, their children, their support organizations, their physicians Alternating Hemiplegia Foundation, USA Association Française de l'Hémiplégie Alternante Associazione Italiana per la Sindrome di Emiplegia Alternante A special thank you to Mylynda Schlesinger-Massart and Lynn Egan for organization of this workshop
26	Acknowledgements Research Resources AHF-USA, Mark Leppert, Bob Weiss, and Support Diane Dunn, Jim Metherall, Louis Ptacek Clinical Coordinators Mark Wride, Victoria McMeen, Catherine McKenna, Jenny Kimball Laboratory Specialists Justine Milligan Johnson, Pernilla Turnert Postdoctoral fellow /Medical Student Mylynda Schlesinger Massart Neuropsychologic Studies Joshua Magleby, Janiece Pompa
27	Acknowledgements Our many physician collaborators and colleagues Kenneth Silver Marion Gerard-Blanluet Alexis Arzimanoglou Mohammed Mikati Marie Bourgeois Athina Xaidara Sotiris Youroukos Frederic and Eva Andermann Denis Lamblin Jean Aicardi Bernard Echenne Harry Chugani Sergei Perelman Jong Rho ……….AND many others