TRATAMIENTO DE LOS TRASTORNOS DE LA LECTURA…

Genes which were informative in at least one cell type, and where this corresponded with expression, were then analysed using non-quantitative RT-PCR on cDNA templates derived from each cell line. RNA was extracted from fMSC which

were undifferentiated and had been maintained at or below 70% confluency and from hES cells which were undifferentiated and karyotypically normal. Amplicons were sequenced or digested as for the genotyping. Results are summarised in Figure 5.6 and Figure 5.7 but complete analysis of each gene and cell line is included in Appendix II.

5.1.5.1 Allelic expression in fMSC (i)

(ii)

Figure 5.6. Allelic expression analysis: fMSC

= monoallelically expressed = biallelically expressed.

(i) No. of genes analysed against cell line. Each fMSC line displays both mono- and biallelic expression of some imprinted genes. Adult human fibroblast cells are used as a control, and show a similar pattern of both mono- and biallelic expression. (ii) No. of cell lines analysed against gene, genes are ordered according to their genomic sequence, running from NAP1L5 on hChr4 to GNAS EXON1A on hChr20. ZAC1 is

the imprinted P1 isoform, V2 = INPP5F_V2; A18 = SLC22A18, both SNPs are pooled*, A1LS = SLC22A1LS, LIT1 = KCNQ1OT1, rs231357 (Msp1) and rs231359 (Sac1) SNPs are pooled*. Certain genes are invariably either monoallelic or biallelic in fMSC. A subset show variable expression between cell lines. The overall ratio of biallelic to monoallelic SNPs is 3:4 respectively. *Except where informative samples overlapped.

According to the gene, variation in expression shows a specific pattern. Some genes are universally monoallelic, others always biallelic, the remainder show variable expression between mono- and biallelic depending on the cell line. More imprinted genes are monoallelically expressed in the fMSC lines than are biallelic, with a total count of 38 monoallelic SNPs and 31 biallelic SNPs, a ratio of almost 3:4.

There is inter-cell line variation but it is minor and not indicative of certain cell lines being more susceptible to biallelic expression of imprinted genes. Primary fibroblasts showed the same gene-specific pattern of expression as the fMSC. The minor inter-cell line variation is a result of the allelic expression character of the genes which happened to be informative in each cell line (see Table 5.3 below). In effect, these observations indicate that if all cell lines analysed were informative for the same genes, their overall expression profile would be identical.

An example comparison of two fMSC lines both derived from liver

Cell line fLiv10+4 fLiv11+5

Biallelic where variable NAP1L5 MEST Iso2, SLC22A1LS

Monoallelic where variabl MEST Iso1, SLC22A1LS MEST Iso 1 Always monoallelic INPP5F_V2, KCNQ1,

H19, NDN

KLF14, CDKN1C, EXON1A,

Table 5.3 Comparison between fMSC line expression status

fMSC line fLiv11+5 displays predominately biallelic expression of imprinted genes, at a ratio of four monoallelic genes to seven biallelic genes. This is due to the high number of genes which happen to be informative in fLiv11+5 which are always found to be biallelic. This is in contrast to fLiv10+4, which exhibits a majority of monoallelic expression, at a ratio of six monoallelic genes to two biallelic ones.

fLiv10+4 is only informative for one of the genes which are always biallelic (SLC22A18), but is informative for four of the genes which are always monoallelic.

Perhaps the most interesting comparisons are between the lines for genes which exhibit variable expression (see section 5.1.7).

The analysis of imprinting in fetal tissues of an equivalent ontological age to fMSC has been previously characterised within our lab (Bentley et al., 2003; Monk et al., 2006a; Monk et al., 2006b). It is important to note that several of the genes which are expressed biallelically in fMSC have a tissue-specific pattern of imprinting, i.e.

GNAS is only imprinted in kidney and GRB10 only in brain, so are likely to already be biallelic in bone marrow blood or liver before fMSC derivation.

5.1.5.2 Allelic expression in hES cells (i)

(ii)

Figure 5.7. Allelic expression analysis: hES cells

= monoallelically expressed = biallelically expressed.

(i)No. of informative genes analysed against cell line. Similarly to the fMSC, most hES cell lines display both mono- and biallelic expression of some imprinted genes.

(ii) No of cell lines analysed against gene, ordered by genomic sequence. ZAC1 is the imprinted P1 isoform, V2 = INPP5F_V2; A18 = SLC22A18, both polys are pooled, A1LS = SLC22A1LS, LIT1 = KCNQ1OT1, rs231357 (Msp1) and rs231359 (Sac1) SNPs are pooled. Certain genes are invariably either monoallelic or bialleic in hES cells. A subset show variable expression between cell lines. The overall ratio of biallelic to monoallelic SNPs is 1:2

The trend seen in the fMSC of variation in expression between genes but not between cell lines is mirrored in the hES cells. Table 5.4 below outlines the contrast between the genes informative in WT4, which appears to maintain monoallelic expression, and SHEF5, which is always biallelic.

Cell line WT4 SHEF5

Status of gene expression Monoallelic Biallelic Genes informative and expressed

Biallelic where variable DLK1, GNAS

Monoallelic where variable MEST Iso2

Always monoallelic INPP5F_V2, CDKN1C Table 5.4 Comparison between hES line expression status

The two genes informative in SHEF5 were variable, but for WT4, two of the informative genes were universally monoallelically expressed, with just one being monoallelic where variable.