Novel allele-specific quantification methods reveal no effects of adult onset CAG repeats on HTT mRNA and protein levels.
Huntington's disease (HD) reflects dominant consequences of a CAG repeat expansion mutation in HTT. Expanded CAG repeat size is the primary determinant of age at onset and age at death in HD. Although HD pathogenesis is driven by the expanded CAG repeat, whether the mutation influences the expression levels of mRNA and protein from the disease allele is not clear due to the lack of sensitive allele-specific quantification methods and the presence of confounding factors. To determine the impact of CAG expansion at the molecular level, we have developed novel allele-specific HTT mRNA and protein quantification methods based on principles of multiplex ligation-dependent probe amplification and targeted MS/MS parallel reaction monitoring, respectively. These assays, exhibiting high levels of specificity and sensitivity, were designed to distinguish allelic products based upon expressed polymorphic variants in HTT, including rs149 109 767. To control for other cis-haplotype variations, we applied allele-specific quantification assays to a panel of HD lymphoblastoid cell lines, each carrying the major European disease haplotype (i.e. hap.01) on the mutant chromosome. We found that steady state levels of HTT mRNA and protein were not associated with expanded CAG repeat length. Rather, the products of mutant and normal alleles, both mRNA and protein, were balanced, thereby arguing that a cis-regulatory effect of the expanded CAG repeat is not a critical component of the underlying mechanism of HD. These robust allele-specific assays could prove valuable for monitoring the impact of allele-specific gene silencing strategies currently being explored as therapeutic interventions in HD.
Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114, USA.
Aram
A
Shin
Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114, USA.|Department of Neurology, Harvard Medical School, Boston, MA 02115, USA.
Baehyun
B
Shin
Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114, USA.|Department of Neurology, Harvard Medical School, Boston, MA 02115, USA.
Jun Wan
JW
Shin
Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114, USA.|Department of Neurology, Harvard Medical School, Boston, MA 02115, USA.
Kyung-Hee
KH
Kim
Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114, USA.|Department of Neurology, Harvard Medical School, Boston, MA 02115, USA.
Ranjit S
RS
Atwal
Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114, USA.
Jennifer M
JM
Hope
Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114, USA.
Tammy
T
Gillis
Proteomics and Mass Spectrometry Facility, University of Massachusetts Medical School, Worcester, MA 01655, USA.
John D
JD
Leszyk
Proteomics and Mass Spectrometry Facility, University of Massachusetts Medical School, Worcester, MA 01655, USA.
Scott A
SA
Shaffer
CHDI Foundation, Princeton, NJ 08540, USA.
Ramee
R
Lee
CHDI Foundation, Princeton, NJ 08540, USA.
Seung
S
Kwak
Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114, USA.|Department of Neurology, Harvard Medical School, Boston, MA 02115, USA.|Medical and Population Genetics Program, The Broad Institute of M.I.T. and Harvard, Cambridge, MA 02142, USA.
Marcy E
ME
MacDonald
Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114, USA.|Medical and Population Genetics Program, The Broad Institute of M.I.T. and Harvard, Cambridge, MA 02142, USA.|Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
James F
JF
Gusella
Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114, USA.|Department of Neurology, Harvard Medical School, Boston, MA 02115, USA.
Ihn Sik
IS
Seong
Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114, USA.|Department of Neurology, Harvard Medical School, Boston, MA 02115, USA.|Medical and Population Genetics Program, The Broad Institute of M.I.T. and Harvard, Cambridge, MA 02142, USA.
Jong-Min
JM
Lee
D000328:Adult; D017668:Age of Onset; D000483:Alleles; D001344:Autopsy; D001921:Brain / Q000378:metabolism* / Q000473:pathology; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D000071058:Huntingtin Protein / Q000096:biosynthesis* / Q000235:genetics; D006816:Huntington Disease / Q000235:genetics* / Q000473:pathology; D008297:Male; D012333:RNA, Messenger / Q000096:biosynthesis; D019680:Trinucleotide Repeat Expansion / Q000235:genetics*
C086055:HTT protein, human; D000071058:Huntingtin Protein; D012333:RNA, Messenger