Facioscapulohumeral Dystrophy (FSHD) Information (FSHD1 and FSHD2)
Blood Samples: Adults - whole blood in lavender and pink top tubes (EDTA)
Children - whole blood in lavender and pink top tubes (EDTA)

The optimum sample is 15 mL whole blood in EDTA tubes, any combination of pink or lavender top tubes. Southern blots for 4q35 deletion detection and 4qA/4qB allele determination utilize peripheral blood leukocytes embedded into agarose plugs. Methylation testing and SMCHD1 gene sequencing for FSHD2 require isolated DNA. The minimum blood volume for testing depends on the medical circumstances and the assays requested.
Prenatal Cell Culture Samples: Prenatal amniotic fluid or chorionic villus samples are utilized to establish cell cultures. Six, confluent, T25 flasks of cells are required for testing. Testing is only available for FSHD1. Parental and prenatal samples are compared using standard Southern blots for 4q35 deletion detection.
FSHD Diagnostic Workflow Diagram
Turn Around Time: 21 days for routine FSHD1 testing or subsets of FSHD2 testing; at least 6 weeks for complete FSHD1 and FSHD2 testing; 6-8 weeks for prenatal samples, because cell cultures may require time to reach confluence or may need to be expanded.
CPT Codes: Standard FSHD - 81404
4qA/4qB Allele Testing - 81404
Methylation Testing - 81479
SMCHD1 Gene Sequencing - 81479
Background: Approximately 90% of individuals affected with FSHD have a chromosome 4q35 deletion. The identification of a characteristic 4q35 deletion is more than 90% specific for the disease. Furthermore, patients with FSHD have 4qA alleles. A deleted 4q35/4qA allele is diagnostic of FSHD type 1 (FSHD1). Approximately 5% of FSHD patients have FSHD type 2. These patients have non-deleted 4qA alleles, hypomethylation of D4Z4 repeats, and a heterozygous dominant mutation in the SMCHD1 gene.

Peripheral blood leukocytes (or cultured prenatal cells) embedded in agarose plugs are digested with specific restriction enzymes. The restriction fragments are separated by pulsed field gel electrophoresis and detected by Southern blot probed with p13E-11. As needed, the Southern blot is rehybridized with a D4Z4 probe. To determine the 4qA/4qB allele status of each restriction fragment, similar Southern blots are hybridized with 4qA or 4qB probes. Additional testing for FSHD2 involves determining the methylation status of the D4Z4 repeats, another Southern blot assay that uses isolated DNA. In patients with a permissive 4qA allele and an appropriate level of hypomethylation, mutation analysis of the SMCHD1 gene is performed on a next generation sequencing platform.
References: GeneReviews - Lemmers RJLF, Miller DG, and van der Maarel SM. Facioscapulohumeral Muscular Dystrophy.
Contact Information: Laboratory Procedures and Test Interpretation
Steven A. Moore, MD, PhD
Molecular Pathology Laboratory