# Presentation: 161846

## i2b2 HUNTINGTON’S DISEASEMicroarray Array Analysis of HD

- Jong-Min Lee1*, Tanya Cashorali 2*, MaryAnne Anderson 1, Yiping Shen 1, Isaac Kohane 2, Marcy MacDonald 1, and James Gusella 1
- 1 Center for Human Genetic Research, Massachusetts General Hospital, Boston MA 02114,  2 Children's Hospital Informatics Program, Boston, MA 02115

**ABSTRACT**
- Huntington’s disease (HD) is a dominant neurodegenerative disorder caused by expansion of a CAG trinucleotide repeat in the *HD* gene from about 6-35 repeats (non-HD range) to 36-100 repeats (HD range), extending a normally variable run of glutamines in the huntingtin protein. We have established genetic criteria from studies of human patients that define characteristics of the trigger mechanism (dominance, increasing severity with increased polyglutamine tract length in the non-HD and the HD range) but the biochemical nature of the trigger mechanism and the processes that lead to dysfunction and ultimate death of target neurons, the medium spiny neurons of the striatum, remain unknown. This project aims to test the hypothesis that microarray analyses of sets of cells and tissues with HD CAG repeats that span the non-HD and HD ranges, may uncover changes in gene expression patterns that conform to the HD genetic criteria and reveal the critical transcriptional regulators that determine the response to the HD trigger-mechanism.

**Fig. 3A     Transcriptome-based classification of HD CAG knock-in mice**

**Fig. 3B       Pathway analysis and hierarchical clustering**

**Table 1. *****HD***** CAG repeat lengths for each human lymphoblastoid cell line**** **
- ID                                   #               HD CAG 1         HD CAG 2          Gender           Age*          “Length”
- GUS3227	**1****	**70	19	F	na	L
- GUS19217	**2****	**35	17	M	24	S
- GUS115	**3****	**18	17	M	51	S
- GUS21273	**4****	**37	22	F	22	S
- GUS15213	**5****	**41	15	M	35	L
- GUS4606	**6****	**63	22	M	29	L
- GUS21250	**7****	**33	17	M	45	S
- GUS21521	**8****	**36	18	F	15	S
- GUS13402	**9****	**46	16	M	33	L
- GUS10438	**10****	**29	17	F	57	S
- GUS10928	**11****	**20	17	F	na	S
- GUS21565	**12****	**38	17	F	38	L
- GUS301	**13****	**49	20	F	51	S
- GUS21519	**14****	**24	15	M	9	L
- GUS13257	**15****	**43	21	M	10	S
- GUS1729	**16****	**66	21	F	24	L
- GM7356	17	18	17	M	13	S
- GUS21145	**18****	**31	17	F	10	S
- GUS1714	**19****	**52	19	M	31	L
- F, female; M, male; na, not available; L, long pathogenic range S, short normal range;  * Age at time sample collected

**METHODS**
- 	Total RNA isolated from the human lymphoblastoid cells or from the tissue dissected from 3 individual mutant or wild-type mice was used to synthesize cDNA / cRNA, and cRNA was hybridized to Affymetrix U133 Plus 2.0 and MG 430 2.0  microarray, respectively.
- 	The human and mouse expression datasets were then analyzed for significant gene changes using RMA, Bioconductor, clustering analysis, and significant gene pathways were detected using sigPathway and GSEA.
- 	The results of our preliminary analyses are presented in Figures 1 and 2 (Human) and in Table 2 and Figure 3 (mouse model).

- Fig. 1. *CAG*-length dependent expression signature            Fig. 2. Hierachical clustering of variable genes

**CAG length-dependent  gene expression profile**
- Unsupervised hierachical clustering  analysis of human lymphoblastoid cell lines revealed that  there was discrimination of the short (non-pathogenic) repeats from those in the disease-causing range (Fig. 1), and evidence of a genetic signature that varies by CAG repeat length (Fig. 2)

**Table 2. Gene expression changes in HD *****CAG***** knock-in mice**
- Tissue       Control                         HD                  Age                Increase         Decrease
- ST	7/7	111/111	3 weeks	8	10
- 	7/7	111/111	10 weeks	916	844
- 	7/7	7/50	5 months	0	0
- CB	7/7	111/111	3 weeks	0	0
- 	7/7	111/111	10 weeks	0	0
- 	7/7	7/50	5 months	0	0

**CONCLUSIONS**
- 1) The HD CAG repeat is associated with presymptomatic changes in gene expression that predate overt pathology, even in cells that are not the primary target of the HD mutation.
- 2) The response to the HD mutation *in vivo* is evident at an earlier age in striatum than in a non-target tissue (cerebellum).
- 3) Preliminary analysis suggests an HD CAG size dependent gene signature that may implicate several fundamental cellular pathways in the early response to mutant huntingtin - immune, mitochondria/energy, ribosome, RNA processing, transferase, and transport.
- 	This interpretation will be validated by performing additional microarray experiments with more lymphoblastoid cell lines and with non-brain HD mouse model tissues.
- 	Validated ‘response’ genes will then be assessed both for their potential involvement in presymptomatic HD and to reveal the underlying regulatory mechanisms.
- 	Gene changes that fulfill HD genetic criteria will provide optimal starting points for developing rational interventions for the earliest steps in the disease process.

- Immune                    Mitochondrial                     Ribosomal                            RNA                         Transferase                     Transport

**STRATEGY**
-  To determine global gene mRNA expression levels in response to the HD mutation in man, using Affymetrix U133 Plus 2.0 microarray, in a set of lymphoblastoid cell lines from 19 individuals, with HD repeats ranging from 18 to 70 CAGs (Table 1), looking for significant changes that fulfill HD genetic criteria.
- 2) To determine global gene mRNA expression levels in response to the HD mutation in accurate genetic mouse models with a juvenile onset (*Hdh**Q111*) or an adult onset (*Hdh**Q50*)* *CAG repeat, using Affymetrix MG 430 2.0 microarray, for brain tissues that are a target of the mutation (striatum) and a region that is spared in the disease (cerebellum).