RESUMEN
The pathological huntingtin (HTT) trinucleotide repeat underlying Huntington disease (HD) continues to expand throughout life. Repeat length correlates both with earlier age at onset (AaO) and faster progression, making slowing its expansion an attractive therapeutic approach. Genome-wide association studies have identified candidate variants associated with altered AaO and progression, with many found in DNA mismatch repair (MMR)-associated genes. We examine whether lowering expression of these genes affects the rate of repeat expansion in human ex vivo models using HD iPSCs and HD iPSC-derived striatal medium spiny neuron-enriched cultures. We have generated a stable CRISPR interference HD iPSC line in which we can specifically and efficiently lower gene expression from a donor carrying over 125 CAG repeats. Lowering expression of each member of the MMR complexes MutS (MSH2, MSH3, and MSH6), MutL (MLH1, PMS1, PMS2, and MLH3), and LIG1 resulted in characteristic MMR deficiencies. Reduced MSH2, MSH3, and MLH1 slowed repeat expansion to the largest degree, while lowering either PMS1, PMS2, or MLH3 slowed it to a lesser degree. These effects were recapitulated in iPSC-derived striatal cultures where MutL factor expression was lowered. CRISPRi-mediated lowering of key MMR factor expression to levels feasibly achievable by current therapeutic approaches was able to effectively slow the expansion of the HTT CAG tract. We highlight members of the MutL family as potential targets to slow pathogenic repeat expansion with the aim to delay onset and progression of HD and potentially other repeat expansion disorders exhibiting somatic instability.
Asunto(s)
Reparación de la Incompatibilidad de ADN , Proteína Huntingtina , Enfermedad de Huntington , Células Madre Pluripotentes Inducidas , Expansión de Repetición de Trinucleótido , Humanos , Enfermedad de Huntington/genética , Enfermedad de Huntington/metabolismo , Reparación de la Incompatibilidad de ADN/genética , Células Madre Pluripotentes Inducidas/metabolismo , Expansión de Repetición de Trinucleótido/genética , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Homólogo 1 de la Proteína MutL/genética , Homólogo 1 de la Proteína MutL/metabolismo , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Genes Modificadores , Proteína 3 Homóloga de MutS/genética , Proteína 3 Homóloga de MutS/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas MutL/genética , Proteínas MutL/metabolismo , Sistemas CRISPR-Cas , Estudio de Asociación del Genoma CompletoRESUMEN
CAG repeat expansion in the HTT gene drives Huntington's disease (HD) pathogenesis and is modulated by DNA damage repair pathways. In this context, the interaction between FAN1, a DNA-structure-specific nuclease, and MLH1, member of the DNA mismatch repair pathway (MMR), is not defined. Here, we identify a highly conserved SPYF motif at the N terminus of FAN1 that binds to MLH1. Our data support a model where FAN1 has two distinct functions to stabilize CAG repeats. On one hand, it binds MLH1 to restrict its recruitment by MSH3, thus inhibiting the assembly of a functional MMR complex that would otherwise promote CAG repeat expansion. On the other hand, it promotes accurate repair via its nuclease activity. These data highlight a potential avenue for HD therapeutics in attenuating somatic expansion.
Asunto(s)
Encéfalo/enzimología , Daño del ADN , Reparación de la Incompatibilidad de ADN , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Proteína Huntingtina/genética , Enfermedad de Huntington/enzimología , Enzimas Multifuncionales/metabolismo , Homólogo 1 de la Proteína MutL/metabolismo , Expansión de Repetición de Trinucleótido , Animales , Unión Competitiva , Encéfalo/patología , Línea Celular Tumoral , Endodesoxirribonucleasas/genética , Exodesoxirribonucleasas/genética , Células HEK293 , Humanos , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Ratones , Enzimas Multifuncionales/genética , Homólogo 1 de la Proteína MutL/genética , Proteína 3 Homóloga de MutS/genética , Proteína 3 Homóloga de MutS/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de ProteínasRESUMEN
BACKGROUND: Huntington disease (HD) is caused by an unstable CAG/CAA repeat expansion encoding a toxic polyglutamine tract. Here, we tested the hypotheses that HD outcomes are impacted by somatic expansion of, and polymorphisms within, the HTT CAG/CAA glutamine-encoding repeat, and DNA repair genes. METHODS: The sequence of the glutamine-encoding repeat and the proportion of somatic CAG expansions in blood DNA from participants inheriting 40 to 50 CAG repeats within the TRACK-HD and Enroll-HD cohorts were determined using high-throughput ultra-deep-sequencing. Candidate gene polymorphisms were genotyped using kompetitive allele-specific PCR (KASP). Genotypic associations were assessed using time-to-event and regression analyses. FINDINGS: Using data from 203 TRACK-HD and 531 Enroll-HD participants, we show that individuals with higher blood DNA somatic CAG repeat expansion scores have worse HD outcomes: a one-unit increase in somatic expansion score was associated with a Cox hazard ratio for motor onset of 3·05 (95% CIâ¯=â¯1·94 to 4·80, pâ¯=â¯1·3 × 10-6). We also show that individual-specific somatic expansion scores are associated with variants in FAN1 (pFDRâ¯=â¯4·8â¯×â¯10-6), MLH3 (pFDRâ¯=â¯8·0â¯×â¯10-4), MLH1 (pFDRâ¯=â¯0·004) and MSH3 (pFDRâ¯=â¯0·009). We also show that HD outcomes are best predicted by the number of pure CAGs rather than total encoded-glutamines. INTERPRETATION: These data establish pure CAG length, rather than encoded-glutamine, as the key inherited determinant of downstream pathophysiology. These findings have implications for HD diagnostics, and support somatic expansion as a mechanistic link for genetic modifiers of clinical outcomes, a driver of disease, and potential therapeutic target in HD and related repeat expansion disorders. FUNDING: CHDI Foundation.