Diagnosis
FSHD can be presumptively diagnosed in many cases based on signs, symptoms, and/or non-genetic medical tests. Genetic testing can provide definitive diagnosis. In the absence of an established family history of FSHD, diagnosis can be difficult due to the variability in how FSHD manifests. Genetic testing
Genetic testing is the gold standard for FSHD diagnosis, as it is the most sensitive and specific test available. Commonly, FSHD1 is tested for first. A shortened D4Z4 array length (EcoRI length of 10 kb to 38 kb) with an adjacent 4qA allele supports FSHD1. If FSHD1 is not present, commonly FSHD2 is tested for next by assessing methylation at 4q35. Low methylation (less than 20%) in the context of a 4qA allele is sufficient for diagnosis. The specific mutation, usually one of various SMCHD1 mutations, can be identified with next-generation sequencing (NGS). Assessing D4Z4 length
Measuring D4Z4 length is technically challenging due to the D4Z4 repeat array consisting of long, repetitive elements. For example, NGS is not useful for assessing D4Z4 length, because it breaks DNA into fragments before reading them, and it is unclear from which D4Z4 repeat each sequenced fragment came. In 2020, optical mapping became available for measuring D4Z4 array length, which is more precise and less labor-intensive than southern blot.Molecular combing is also available for assessing D4Z4 array length. These methods, which physical measure the size of the D4Z4 repeat array, require specially prepared high quality and high molecular weight genomic DNA (gDNA) from serum, increasing cost and reducing accessibility to testing.
In those with 8 or fewer repeats, symptoms are more likely than in those with 9 - 10 repeats, leading to diagnosis regardless of an additional SMCHD1 mutation.The apparent frequency of FSHD1/FSHD2 cases in the 9 - 10 repeat range, combined with the FSHD2-like methylation pattern, suggest the 9 - 10 repeat size to be an overlap zone between FSHD1 and FSDH2. Pathophysiology
Molecular
As of 2020, there seems to be a consensus that aberrant expression of DUX4 in muscle is the cause of FSHD. DUX4 is expressed in extremely small amounts, detectable in 1 out of every 1000 immature muscle cells (myoblast), which appears to increase after myoblast maturation, in part because the cells fuse as they mature, and a single nucleus expressing DUX4 can provide DUX4 protein to neighboring nuclei from fused cells.It remains an area of active research how DUX4 protein causes muscle damage. DUX4 protein is a transcription factor that regulates many other genes. Some of these genes are involved in apoptosis, such as p53, p21, MYC, and β-catenin, and indeed it seems that DUX4 protein makes muscle cells more prone to apoptosis. Other DUX4 protein-regulated genes are involved in oxidative stress, and indeed it seems that DUX4 expression lowers muscle cell tolerance of oxidative stress. Variation in the ability of individual muscles to handle oxidative stress could partially explain the muscle involvement patterns of FSHD.