Table 1:

Table showing the essential and desirable actions in the genetic confirmation of FSHD based on the patient’s phenotype. The top table is similar for the SB+PFGE, MC and OGM and the bottom for SB-LGE only. The separation of FSHD1 allele sizes (≤7U; 8-10U and >10U 4qA) is based on individuals with a European genetic background. SB: Southern blot; PFGE: pulsed field gel electrophoresis; MC: molecular combing; OGM: optical genome mapping; LGE: linear gel electrophoresis.

SB+PFGE6, MC and OGM
Clinical presentation	Result1,3	Action Essential	Result	Diagnosis	Additional action Recommended5
Typical/atypical FSHD Severity: moderate to severe	≤7U 4qA	no further testing needed		FSHD1	If early/childhood onset and >5U, consider FSHD2 methylation analysis
	8-10U 4qA	FSHD2 methylation analysis, or SMCHD1 analysis (or panel)	Hypomethylation, and/or pathogenic variant in SMCHD14	FSHD1,2	-
			Normal methylation	FSHD1	Test to exclude alternative or additional genetic cause
	>10U 4qA	FSHD2 methylation analysis, or SMCHD1 analysis (or panel) and check for DPED allele or cis dupliciation	Hypomethylation, and/or pathogenic variant in SMCHD14	FSHD2 (>10U)	-
			DPED ≤10U 4qA allele2	FSHD1	-
			cis-duplication allele	Probably FSHD1	Segregation analysis, if possible, to confirm pathogenicity
			Normal methylation No DPED or cis-duplication	No FSHD	Test for alternative diagnosis
Typical FSHD Severity: mild	≤7U 4qA	Analyze for possible mosaicism		FSHD1 (possible mosaic)	-
	8-10U 4qA	no further testing needed		FSHD1	Segregation analysis, if possible, to confirm pathogenicity
	>10U 4qA	FSHD2 methylation analysis, or SMCHD1 analysis (or panel) and check for DPED allele or cis duplication	Hypomethylation, and/or pathogenic variant in SMCHD14	FSHD2 (>10U)	-
			DPED ≤10U 4qA allele2	FSHD1	-
			cis-duplication allele	Probably FSHD1	Segregation analysis, if possible, to confirm pathogenicity
			Normal methylation No DPED or cis-duplication	No FSHD	Test for alternative diagnosis
Atypical FSHD Severity: mild	≤7U 4qA	Analyze for possible mosaicism		FSHD1 (possible mosaic)	-
	8-10U 4qA	FSHD2 methylation analysis, or SMCHD1 analysis (or panel)	Hypomethylation, and/or pathogenic variant in SMCHD14	FSHD1,2	-
			Normal methylation	Probably FSHD1	Segregation analysis, if possible, to confirm pathogenicity
	>10U 4qA	Check for DPED allele or cis duplication	DPED ≤10U 4qA allele2	FSHD1	-
			cis-duplication allele	Probably FSHD1	Segregation analysis, if possible, to confirm pathogenicity
			Normal methylation No DPED or cis-duplication	No FSHD	Test for alternative diagnosis
SB+LGE
Clinical presentation	Result1	1st Action Essential	Result1,3	2nd Action Essential	Result	Diagnosis	Additional action Recommended5
Typical/atypical FSHD Severity: moderate to severe	≤7U 4q	Not required (assuming ≤7U 4qA)		-		FSHD1	If early/childhood onset and >5U, consider FSHD2 methylation analysis
	8-10U 4q	4qA/4qB haplotyping	8-10U 4qA	FSHD2 methylation analysis, or SMCHD1 analysis (or panel)	Hypomethylation, and/or pathogenic variant in SMCHD14	FSHD1,2	-
					Normal methylation	FSHD1	Test to exclude alternative or additional genetic cause. Consider checking for DPED ≤10U allele or cis duplication.
	>10U 4q	go to 2nd Action Essential		FSHD2 methylation analysis, or SMCHD1 analysis (or panel)	Hypomethylation, and/or pathogenic variant in SMCHD14	FSHD2 (>10U)	4qA/4qB haplotyping by SSLP or distal 4qA PCR
					Normal methylation	possible complex haplotype	Check for DPED ≤10U allele, or cis dupliciation
						No FSHD	Test for alternative diagnosis
Typical FSHD Severity: mild	≤7U 4q	Not required (assuming ≤7U 4qA)		-		FSHD1 (possible mosaic)	Check for mosaisism
	8-10U 4q	4qA/4qB haplotyping	8-10U 4qA	no further testing needed		FSHD1	Segregation analysis, if possible, to confirm pathogenicity
	>10U 4q	go to 2nd Action Essential		FSHD2 methylation analysis, or SMCHD1 analysis (or panel)	Hypomethylation, and/or pathogenic variant in SMCHD14	FSHD2 (>10U)	4qA/4qB haplotyping by SSLP or distal 4qA PCR
					Normal methylation	possible complex haplotype	Check for DPED ≤10U allele, or cis dupliciation
						No FSHD	Test for alternative diagnosis
Atypical FSHD Severity: mild	≤7U 4q	4qA/4qB haplotyping	≤7U 4qA	no further testing needed		FSHD1 (possible mosaic)	Check for mosaicism
	8-10U 4q	4qA/4qB haplotyping	8-10U 4qA	FSHD2 methylation analysis, or SMCHD1 analysis (or panel)	Hypomethylation, and/or pathogenic variant in SMCHD14	FSHD2 (8-10U)	-
					Normal methylation	Probably FSHD1	Segregation analysis, if possible, to confirm pathogenicity
	>10U 4q	Not required, exclusion FSHD				No FSHD	Test for alternative diagnosis
All technologies
Clinical presentation	Result	Action Essential	Result	Diagnosis
1st degree relative(s) has genetically confirmed FSHD1	FSHD1 allele that matches relative	no further testing needed		FSHD1
	>10U 4q	no further testing needed		not likely to be FSHD

Remarks:

¹It is required to do further analysis if genotype-phenotype does not fit.

²The identification of an 1-7U DPED allele confirms FSHD1 and further methylation analysis is not required

³Absence of any 4qA allele excludes FSHD, unless 10qA ≤3U is observed

⁴If possible, also consider possible pathogenic variants in other FSHD2 genes

⁵It is recommended to perform genetic counseling when FSHD is genetically confirmed

⁶The standard SB-PFGE analysis in this table includes 4qA/4qB hybridization