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Protocols and Procedures

In the spirit of scientific openness and transparency, the Fields Center strives to make public as many of their research protocols as possible. The list of procedures and protocols used by the Center for FSHD (FSH dystrophy) research will be regularly updated.

Muscle Histopathologic Scoring


The following is a simplified pathologic grading score of FSHD muscle biopsies collected for research studies. The main objective of this scoring system is not a detailed assessment of the pathologic changes seen in FSHD but an overall pathological severity scoring to be used to correlate with clinical features (muscle strength or mass) or results of molecular studies derived from that same muscle. The scoring is based on H&E and Trichrome staining and scores the muscle based on 4 features: 1. variability in fiber size, 2. extent of central nucleation, 3. necrosis and regeneration, 4. interstitial fibrosis. For each feature, the severity of the abnormality is grade from 0-3 (0 normal, 1 mild, 2 moderate, 3 severe). The combined score range is from 0 (normal muscle) to 12 (end stage muscle).
Muscle biopsy histopathologic scoring

Disease Severity Scoring


There are several ways of measuring disease severity but they can be broadly divided into physician/evaluator determined severity scales and patient self-reported scales. The former are usually dependent on measurements of motor function either by direct strength measurement or determination of functional abilities. Severity scales, in addition to being useful clinical tools for patient managment, are important in describing the natural history of the disease which, in turn, is critical in the design of future therapeutic trials. In addition, clinical severity scoring allows us to look at genotype-phenotype associations. There are no fully validated clinical severity scoring systems specific for FSHD. However, the scale used below has been used in several published studies.
Clinical Severity Scoring

FSHD2 Clinical Assessment

FSHD2 Clinical Assessment Form

Leiden FSHD (FSHDystrophy) Genotyping


The following procedures describe the detailed FSHD genotyping process of D4Z4 alleles by pulsed field gel electrophoresis (PFGE) performed at the Leiden University Medical Center. Detailed genotyping studies showed the presence of different haplotypes of the D4Z4 (the FSHD locus) alleles of which some are nonpathogenic (4B alleles and 4A166 alleles) while others are rare and difficult to identify (deletion of p13E-11). The latter situation may in fact result in a false negative DNA test on routine commercial FSHD DNA diagnostic testing. To obtain complete information on all chromosomes 4 and chromosomes 10 alleles it is essential to isolate high quality DNA and separate the digested DNA fragments by PFGE. This is particularly important for the identification of mosaic FSHD alleles. High quality DNA is obtained by the preparation of DNA plugs as outlined below. Standard hybridizations to identify repeat sizes on chromosomes 4 and 10 are performed using a p13E-11 probe. The identification of the allele type (A or B) requires hybridizations with A/B probes on HindIII digested DNA. To identify FSHD alleles with a deletion of the p13E-11 probe region (false negative) an alternative hybridization method is provided (probe D4Z4 and formaldehyde hybridization buffer). Finally, we provide a PCR method to identify the haplotype of the D4Z4 allele, this method can be very helpful to discriminate between pathogenic and non-pathogenic alleles, to identify translocated alleles and for genotyping of low quality (fetal) DNA when the genotype of parents is completely analyzed.

Relevant References

1: Lemmers RJ, Wohlgemuth M, van der Gaag KJ, van der Vliet PJ, van Teijlingen CM, de Knijff P, Padberg GW, Frants RR, van der Maarel SM. Specific sequence variations within the 4q35 region are associated withfacioscapulohumeral muscular dystrophy. Am J Hum Genet 2007; 81(5):884-94.

2: Ehrlich M, Jackson K, Tsumagari K, Camaño P, Lemmers RJ. Hybridization analysis of D4Z4 repeat arrays linked to FSHD.Chromosoma 2007; 116(2):107-16.

3: Lemmers RJ, van der Wielen MJ, Bakker E, Padberg GW, Frants RR, van der MaarelSM. Somatic mosaicism in FSHD often goes undetected.Ann Neurol 2004 Jun; 55(6):845-50.

4: Lemmers RJ, Osborn M, Haaf T, Rogers M, Frants RR, Padberg GW, Cooper DN, van der Maarel SM, Upadhyaya M. D4F104S1 deletion in facioscapulohumeral muscular dystrophy: phenotype, size, and detection. Neurology 2003 Jul 22; 61(2):178-83.

5: Lemmers RJ, de Kievit P, Sandkuijl L, Padberg GW, van Ommen GJ, Frants RR, van der Maarel SM. Facioscapulohumeral muscular dystrophy is uniquely associated with one of the two variants of the 4q subtelomere.Nat Genet 2002; 32(2):235-6.

6: Lemmers RJ, Wohlgemuth M, Frants RR, Padberg GW, Morava E, van der Maarel SM. Contractions of D4Z4 on 4qB subtelomeres do not cause facioscapulohumeral muscular dystrophy. Am J Hum Genet 2004; 75(6):1124-30.

7: Wijmenga C, Hewitt JE, Sandkuijl LA, Clark LN, Wright TJ, Dauwerse HG, Gruter AM, Hofker MH, Moerer P, Williamson R, et al. Chromosome 4q DNA rearrangements associated with facioscapulohumeral muscular dystrophy. Nat Genet 1992; 2(1):26-30.

Leiden DNA Methylation Analysis


The following procedure describes the detailed DNA methylation analysis process of liquid DNA samples by methylation-sensitive Southern blot performed at the Leiden University Medical Center. Detailed DNA methylation studies showed D4Z4 hypomethylation on chromosomes 4q and 10q at the FseI restriction site in patients with FSHD2.
Prior to the FseI digestion DNA samples need to be purified to guarantee a complete digestion using this methylation sensitive restriction enzyme (see protocol). This analysis simultaneously measures the D4Z4 methylation level on both chromosomes 4q and 10q. The methylation value at D4Z4 can be obtained by comparing the ratios of signal intensities between bands representing the unmethylated and methylated D4Z4.

Updated Methylation Protocol: New Methylation Protocol PDF

Relevant References

1. Lemmers RJ, Tawil R, Petek LM, et al. Digenic inheritance of an SMCHD1 mutation and an FSHD-permissive D4Z4 allele causes facioscapulohumeral muscular dystrophy type 2. Nat Genet. 2012 Nov 11;44(12):1370-4.

Myoblast Cell Cultures

The following describes the methods used in the Fields Center for initiation of myoblast cell cultures from FSHD (FSH dystrophy) and conrol individuals.


Myoblast cell cultures are established from samples of muscle obtained either through an needle or open biopsy sampling procedure. Typically a muscle sample of between 60-100mg is adequate for establishing a primary myoblast culture.

Needle Muscle Biopsy


Needle muscle biopsy sampling offers a less invasive, more rapid alternative to conventional open muscle biopsies for sampling muscle for research purposes. The smaller size of the sample obtained is no longer an important limitation as the sensitivity of emerging molecular technologies has improved exponentially and small amounts of substrate can go a long way. Using the sampling techniques described below, up to 300 mg of tissue, depending on muscle bulk, can be obtained with three passes into the muscle. Such a sample, divided into three, is more than adequate for flash freezing in liquid nitrogen for expression studies, placement in appropriate media for myoblast culture as well as for histologic studies.

The following file describes the procedure used by the Field's Center to collect muscle biopsy research samples, and provides information on the types of needles and suppliers.

Needle Muscle Biopsy Procedure

Quantitative Myometry


There a number of clinical methods for quantifying muscle strength. The classical method using the five point MRC scale is useful in clinical care settings and when averaged over a large number of muscle groups to give a measure of global clinical severity. However, the MRC is an ordinal scale is and is not suited for quantitative measurement of strength in a single muscle group. Quantitative myometry is currently being used in the Fields Center as a measure of disease severity of the muscle that is being sampled by needle biopsy. The methods described have been previously used to study the natural history of FSHD (FSH dystrophy) and in subsequent clinical trials.

Maximum Isometric Voluntary Contraction Testing (MVICT)

Skin-Derived Fibroblast Culture


Fibroblasts provide an alternative cell line to myoblasts for certain experiments such as detailed genotyping and methylation studies. In contrast to myoblasts, they are easier to grow in abundant quantities. All fibroblasts used in the Fields Center are derived from skin sampled by punch biopsy at the distal ulnar aspect of the forearm about 3 cm proximal to the wrist. Samples are collected from individuals with FSHD (FSH dystrophy) and normal controls.

Total RNA Isolation

Isolation of Total RNA with Trizol Reagent