Table 2.

Cytogenetics, cytogenomics, and molecular techniques used to study ring chromosome 20 [r(20)].

Method	Advantages/aim	Limitations	References
Karyotype on peripheral blood (Figures 1b–d)	r(20) identification Analysis extended up to 200 metaphases to detect low-level mosaicism	Unrecorded: - chromosomal aberration <10 Mb - low mosaicism level - tissue-specific mosaicism	Atkins et al. (4) (first description)
Karyotype on skin fibroblasts or other tissues	r(20) identification and/or confirmation of ring 20 syndrome in case of undetected ring 20 on peripheral blood Multi-tissue estimation of mosaicism. Analysis extended up to 200 metaphases to detect low-level mosaicism		Faed et al. (5) (first report); Back et al. (25), Zou et al. (26), Giardino et al. (12), Cappanera et al. (27), Elens et al. (22)
Prenatal karyotype analysis (chorionic villi, amniotic fluid)	Precocious diagnosis of ring chromosome 20 syndrome, with consequent genetic counseling and follow up		Giardino et al. (12), Cignini et al. (28)
FISH with pantelomeric probe (Figure 1h)	Assess if common telomeric sequences are present/absent.	Lack of signal on the ring does not determine deletion extent.	Zou et al. (26), Elghezal et al. (29), Giardino et al. (12), Unterberger et al. (30), Tayama et al. (31)
FISH with 20p-20q subtelomeric probes (Figures 1i,l)	Assess if a subtelomeric deletion is present/absent		Idem; de Falco et al. (23), Herrgård et al. (32), Cappanera et al. (27), Gahr et al. (33), Inal et al. (34)
FISH with probe specific for chromosome 20 centromeric sequences (Figures 1f,g)	Identification of chromosome origin of the RC Evaluation of alphoid-specific heteromorphism of r(20) and its linear homolog Detection of low chromosome 20 mosaicism for a monosomic cell line	Deletion/duplication cannot be detected	Giardino et al. (12), Kamoun et al. (35)
FISH with whole chromosome 20 painting probe	Detection of other chromosome regions on r(20) (low resolution)	Deletion/duplication cannot be detected	Elghezal et al. (29), Zou et al. (26), Cabras et al. (24), Tezer et al. (36)
BAC FISH on CHRNA4 and KCNQ2 candidate genes (Figures 1m–p)	Detection of deletions of candidate genes. (resolution higher than karyotype)	Limited to the targeted sequence(s). Resolution lower than CMA microarray	Zou et al. (26), Elghezal et al. (29), Giardino et al. (12); Cappanera et al. (27), Kamoun et al. (35)
Segregation analysis of polymorphic loci	Exclusion of whole or segmental UPD20	Tissue specific UPD and low-level mosaicism cannot be detected	Giardino et al. (12)
Chromosomal microarray
Array-CGH (Resolution from 30 to 0.6 Mb)	Identification of CNVs on chromosome 20 and in the whole genome	Tissue specific and low-level mosaicism cannot be detected	Giardino et al. (12); Cabras et al. (24), Rodan al. (37), Corrêa et al. (38)
SNP-array (Resolution from 4.2 to 8.2 kb)	Identification of CNVs, UPD, and homozygosity regions on chromosome 20 and in the whole genome		Conlin et al. (10), Unterberger et al. (30)
Array-based genome-wide methylation analysis array (Human Methylation450 BeadChip kit, Illumina)	Evaluation of the methylation level of CpGs in the whole genome in r(20) patients compared to normal controls	Tissue-specific and low-level epimutation mosaicism cannot be detected	Calzari L. [patients from Giardino et al. (12)]; present data (Figures 1q,r)