Silencing Chromosome 21

A treatment for Down syndromeHari Prakash Bharathi

Faculty of MedicineTSMU

Down Syndrome

Trisomy 21. Associated with physical growth delays, characteristic facial features,

and mild to moderate intellectual disability. The most common chromosome abnormality, 1:1000 birth per year. Trisomy 21 results in overexpression of a portion of the 310 genes

located on chromosome 21. Down syndrome region is located at bands 21q22.1–q22.3,with this

area including genes for amyloid, superoxide dismutase, and likely the ETS-2 proto oncogene.

Down syndrome - Trisomy 21 Trisomy 21 (also known by the karyotype 47,XX,+21 for females and

47,XY,+21 for males) is caused by a failure of the 21st chromosome to separate during egg or sperm development.

As a result, a sperm or egg cell is produced with an extra copy of chromosome 21; this cell thus has 24 chromosomes.

When combined with a normal cell from the other parent, the baby has 47 chromosomes, with three copies of chromosome 21.

About 88% of cases of trisomy 21 result from nonseparation of the chromosomes in the mother, 8% from nonseparation in the father, and 3% after the egg and sperm have merged.

Down syndrome - Epidemiology Globally, as of 2010, Down syndrome occurs in about 1 per 1000

births[2] and results in about 17,000 deaths. More children are born with Down syndrome in countries where

abortion is not allowed and in countries where pregnancy more commonly occurs at a later age.

Maternal age affects the chances of having a pregnancy with Down syndrome. At age 20, the chance is one in 1441; at age 40, it is one in 84; and at age 50 it is one in 44. 

Although the probability increases with maternal age, 70% of children with Down syndrome are born to women 35 years of age and younger, because younger people have more children. 

Silencing Chromosome 21 - Mechanism Insertion of XIST into trisomic chromosome 21. XIST RNA induces a chromosome 21 Barr body. Allele specific silencing across chromosome 21.

XIST X-Inactive Specific Transcript

XIST is an RNA gene on the X chromosome of the placental mammals that acts as major effector of the X inactivation process.

It is a component of the Xic - X-chromosome inactivation center - along with two other RNA genes (Jpx and Ftx) and two protein genes (Tsx and Cnbp2).

XIST RNA, a large (17 kb in humans)transcript, is expressed on the inactive chromosome and not on the active one and is processed in a similar way to mRNAs, through splicing and polyadenylation.

It has been suggested that this RNA gene evolved at least partly from a protein coding gene that became a pseudogene.

The inactive X chromosome is coated with this transcript, which is essential for the inactivation. X chromosomes lacking Xist will not be inactivated, while duplication of the Xist gene on another chromosome causes inactivation of that chromosome.

Insertion of XIST into trisomic chromosome 21 Because of the large size neither the XIST gene nor cDNA can be

inserted in the targeted region, hence ZFN (zinc finger nucleases) is used.

Using ZFNs to a 36 bp sequence in the intron 1 of the DYRKIA locus of chromosome 21q22 and validated their robust activity.

This is then technically tried on iPS cells of Down syndrome patients which shows developmental and therapeutical potential in those cells.

245 colonies from the pooled transformants from the interphase RNA/DNA FISH (fluorescence in situ hybridization) to test whether XIST is present and over lapped on one of three DYSKRIA alleles.

Insertion of XIST into trisomic chromosome 21 Six sub clones were for further investigation:

An XIST transgene on one of three chromosome 21 copies. Pluripotent colony morphology OCT4 staining Formation of embroyoid bodies FISH to metaphase chromosomes Southern Blotting.

High resolution cytogenic banding or/and array comparative genomic hybridization on selected clones showed no significant abnormalities other than trisomy 21.

XIST RNA induces a chromosome 21 Barr body In the panel of 6 independent genome edited clones, transgene

expression is induced and XIST RNA is detected by FISH. A localized XIST RNA territory over 85% is seen in one chromosome

21. The natural inactivated X chromosome forms a Barr body which

carries repressive histone mark. Similarly after 5days, the edited chromosome 21 is enriched in all hetero chromatin marks including H3K27me3, UbH2A and H4K20me in 90 – 100% cells.

Chromosome 21 is condensed in more nuclei and forms a heterochromatic chromosome.

Allele-specific silencing chromosome 21 By using broad assay heterogeneous nuclear RNA in situ hybridization

to CoT-1 DNA, the inactive x chromosome is distinguished from active X chromosome and measured overall transcription across XIST targeted chromosome 21.

This showed that the chromosome 21 XIST RNA territory is depleted for hnRNA detected by CoT-1 DNA similar to X chromosome.

Short term XIST expression resulted in incomplete repression of the targeted allele, which after 20 days was completely silenced.

Complete silencing of each allele on the edited chromosome 21 was seen in 100% of cells accumulating XIST RNA.

Allele specific silencing chromosome was further valided using single nucleotide polymorphism analysis.

Reference

Translating dosage compensation to trisomy 21 Jun Jiang, Yuanchun Jing, Gregory J. Cost, Jen-Chieh Chiang,Heather J. Kolpa, Allison M. Cotton,

Mégarbané, A. et al. The 50th anniversary of the discovery of trisomy 21: the past, present, and future of research and treatment of Down syndrome. Genet. Med. 11, 611–616(2009) Gardiner, K. J. Molecular basis of pharmacotherapies for cognition in Down syndrome.Trends Pharmacol. Sci. 31, 66–73 (2010)CAS

Prandini, P. et al. Natural gene-expression variation in Down syndrome modulates the outcome of gene-dosage imbalance. Am. J. Hum. Genet. 81, 252–263 (2007)

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