See Table A. Genes and Databases for chromosome locus and protein.

See Molecular Genetics for information on allelic variants detected in this gene.

The ability of the test method used to detect a variant that is present in the indicated gene/locus

Molecular genetic testing to determine the length or number of repeat units of the D4Z4 locus has typically relied on Southern blot analysis, typically with a probe (e.g., p13E-11) immediately proximal to D4Z4. Standard DNA diagnostic testing (defined here as linear gel electrophoresis and Southern blot analysis) uses the restriction enzyme EcoRI, which recognizes the D4Z4 locus on chromosomes 4 and 10. Pulsed-field gel electrophoresis and Southern blot analysis requires EcoRI/HindIII double digestion for a better resolution of DNA fragments between 20 and 50 kb. An EcoRI/BlnI double digestion further fragments the chromosome 10 array, allowing one to distinguish D4Z4 arrays located on chromosome 4 from the similar benign arrays on chromosome 10. Molecular combing, which has a higher resolution than Southern blotting [Nguyen et al 2017, Lemmers et al 2018, Nguyen et al 2019] has also been described, but may not be clinically available.

Detection of the pathogenic contraction of the D4Z4 locus by Southern blot analysis requires high-quality DNA; a false negative test result can be caused by poor-quality DNA that was sheared into small fragments.

In approximately 3% of the European families with FSHD1 the D4Z4 contraction on chromosome 4q35 is not visible using the standard genetic test because a deletion encompasses the region of the molecular diagnostic probe p13E-11. These individuals require additional testing to visualize the contracted D4Z4 repeat and resolve the size of the repeat [Lemmers et al 2003, Ehrlich et al 2007].

A combination of Southern blotting and molecular combing detected complex rearrangements of 4q35 with duplication of D4Z4 array [Nguyen et al 2017, Lemmers et al 2018] and a 4q deletion proximal to D4Z4 [Nguyen et al 2019].

4A161 is most common permissive haplotype, but others are reported (4A159, 4A168, 4A166H) [Lemmers et al 2010a]. All individuals with FSHD carry a permissive haplotype. Because 66% of controls also carry a permissive haplotype, this analysis (without sizing of the repeat array) is often not informative. Lemmers et al developed a clinically available diagnostic test to discriminate both haplotype variants using HindIII-digested DNA and specific probes for 4A and 4B [Lemmers et al 2002, Lemmers et al 2007].

D4Z4 methylation values below the threshold of 25% are indicative of FSHD. However, the CpG methylation at the D4Z4 repeat array is also determined by the size of the D4Z4 arrays on chromosomes 4q and 10q. Contracted D4Z4 arrays on chromosomes 4q and 10 have a significantly lower level of methylation than normal-sized arrays. D4Z4 methylation levels should always be evaluated with respect to the repeat size. A Southern blot-based method has been developed that measures the total D4Z4 methylation at chromosomes 4q and 10q by using methylation-sensitive restriction enzyme (FseI) in the promoter region of DUX4 [Lemmers et al 2012b]. The average methylation of D4Z4 in control individuals is 45%, while in individuals with FSHD2 the methylation level drops below 25%, with an average of 11% [Lemmers et al 2012b].

Sequence analysis detects variants that are benign, likely benign, of uncertain significance, likely pathogenic, or pathogenic. Variants may include small intragenic deletions/insertions and missense, nonsense, and splice site variants; typically, exon or whole-gene deletions/duplications are not detected. For issues to consider in interpretation of sequence analysis results, click here.

51 families of 60 with FSHD2 were found to have an SMCHD1 pathogenic variant with D4Z4 DNA hypomethylation [Lemmers et al 2015].

Gene-targeted deletion/duplication analysis detects intragenic deletions or duplications. Methods used may include a range of techniques such as quantitative PCR, long-range PCR, multiplex ligation-dependent probe amplification (MLPA), and a gene-targeted microarray designed to detect single-exon deletions or duplications. Gene-targeted deletion/duplication analysis detects intragenic deletions or duplications.

Deletions including SMCHD1 and other genes have been reported as 18p- syndrome [Lemmers et al 2015].

van den Boogaard et al [2016]