Leiden FSHD (FSHDystrophy) Genotyping

Background: 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.


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