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. 2017 Mar;27(3):221-238.
doi: 10.1016/j.nmd.2016.12.007. Epub 2016 Dec 23.

Large family cohorts of lymphoblastoid cells provide a new cellular model for investigating facioscapulohumeral muscular dystrophy

Takako I Jones  1 , Charis L Himeda  1 , Daniel P Perez  2 , Peter L Jones  3
Affiliations

Large family cohorts of lymphoblastoid cells provide a new cellular model for investigating facioscapulohumeral muscular dystrophy

Takako I Jones et al. Neuromuscul Disord. 2017 Mar.

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is associated with aberrant epigenetic regulation of the chromosome 4q35 D4Z4 macrosatellite repeat. The resulting DNA hypomethylation and relaxation of epigenetic repression leads to increased expression of the deleterious DUX4-fl mRNA encoded within the distal D4Z4 repeat. With the typical late onset of muscle weakness, prevalence of asymptomatic individuals, and an autosomal dominant mode of inheritance, FSHD is often passed on from one generation to the next and affects multiple individuals within a family. Here we have characterized unique collections of 114 lymphoblastoid cell lines (LCLs) generated from 12 multigenerational FSHD families, including 56 LCLs from large, genetically homogeneous families in Utah. We found robust expression of DUX4-fl in most FSHD LCLs and a good correlation between DNA hypomethylation and repeat length. In addition, DUX4-fl levels can be manipulated using epigenetic drugs as in myocytes, suggesting that some epigenetic pathways regulating DUX4-fl in myocytes are maintained in LCLs. Overall, these FSHD LCLs provide an alternative cellular model in which to study many aspects of D4Z4, DUX4, and FSHD gene regulation in a background of low genetic variation. Significantly, these non-adherent immortal LCLs are amenable for high-throughput screening of potential therapeutics targeting DUX4-fl mRNA or protein expression.

Keywords: D4Z4; DNA methylation; DUX4; Disease model; Epigenetic; FSHD.

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Figures

Fig. 1
Fig. 1
Positions of restriction enzyme sites, probe, and PCR primers used to distinguish D4Z4 sequence. Scheme depicting the chromosome 4q35 D4Z4 macrosatellite array, which is contracted in FSHD1, leading to epigenetic changes specific to the contracted chromosome. De-repression of the pathogenic allele leads to aberrant expression of the DUX4-fl mRNA from the distal-most D4Z4 repeat unit (RU). The 4q35 and 10q26 D4Z4 arrays are distinguished from each other by combined EcoRI (E) + AvrII (B) digestion followed by Southern blotting and probing with p13E-11 (probe). DNA methylation changes were assayed by BSS specific for the distal D4Z4 RU of 4qA and its allelic variant 4qA-L [12] (gray bars) or all D4Z4 RUs (black bars).
Fig. 2
Fig. 2
Pedigree for Family 2 from southern Utah. Families 7 and 11 (Fig. 3) are related as indicated. Those subjects determined by Southern blotting to be FSHD1 are indicated in black, with the contracted chromosome in red. The clinical diagnosis of FSHD is labeled. Relations were confirmed by 4qSTR analysis and 4q subtelomere haplotyping (4A, 4L, 4B, 4C) as indicated for each chromosome. When samples were not available (NA), the predicted 4qSTR based on the offspring is within an open oval. DNA methylation levels for each chromosome are indicated as % methylation above the number of D4Z4 RUs, when known.
Fig. 3
Fig. 3
Pedigrees for Families 7 and 11, direct relatives of Family 2 where indicated, from southern Utah. See legend for Fig 2. Those subjects determined to be genetically permissive for FSHD1 but reported as clinically healthy at the time of evaluation are designated as asymptomatic (ASYM).
Fig. 4
Fig. 4
Pedigrees for unrelated Families 6, 12, and 15. See Figure legends for Figs 2 and 3.
Fig. 5
Fig. 5
Pedigrees for unrelated Families 4, 18, and 25. See legend for Fig. 2.
Fig. 6
Fig. 6
Pedigrees for unrelated Families 32 and 36. See legend for Fig 2.
Fig. 7
Fig. 7
Pedigree for Family 33 from northern Utah. See legend for Fig 3. This family contains two different FSHD1 contracted chromosomes, one with 6RUs (red) and one with 9RUs (blue).
Fig. 8
Fig. 8
FSHD1 lymphoblastoid cell lines express variable levels of DUX4-fl. DUX4-fl mRNA levels were determined by qRT-PCR and normalized to levels of 18S RNA. The lowest expressing FSHD sample in each family or group was set to 1 and relative expression levels are shown in each graph. See Table 1 for a relative comparison of DUX4-fl expression across all LCLs.
Fig. 9
Fig. 9
Expression of DUX4-fl in FSHD1 lymphoblastoid cell lines correlates with increased expression of DUX4-FL target genes. mRNA levels of DUX4-fl and three of its downstream target genes, MBD3L2, TRIM43, and ZSCAN4, were determined by qRT-PCR and normalized to levels of 18S RNA.
Fig. 10
Fig. 10
DUX4-fl mRNA is induced in FSHD1 LCLs, but not in healthy controls, by treatment with epigenetic drugs. LCLs from Family 33 were treated (+) or not treated (NT) with 5-aza-2’-deoxycytidine (ADC) and Trichostatin A (TSA). DUX4-fl mRNA levels were determined by qRT-PCR and normalized to levels of 18S RNA. The lowest expressing NT FSHD1 sample (GM16126) is set to 1.
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