Figure 5.

A possible scheme for use of the sequencing SNP assay in clinical testing for FSHD1, which involves short 4q D4Z4 arrays and constitutes ~95% of FSHD cases.²⁷ Analysis paths are shown by arrows in series. Thick arrows indicate most likely outcomes. ^aCanonical 10q D4Z4 arrays in EcoRI/BlnI digests²⁸ and canonical 4q arrays in XapI digests are reduced to small fragments that electrophorese off the gel.⁹ PFGE is highly preferable to LGE, for example, PFGE can reveal somatic mosaics having five hybridising EcoRI fragments.^{17 18} If LGE is used, the so-called dosage test²⁵ should be done to determine if the most proximal D4Z4 repeat unit on both 4q alleles is 4q-type (BlnI^R) and on both 10q alleles is 10q-type (BlnI^S), and the blot should be re-probed with D4Z4. EcoRI/HindIII digests can be used instead of EcoRI digests to get cleaner bands, but fragments are ~2 kb smaller.¹² The maximum size of pathogenic D4Z4 alleles is only an approximate threshold due to incomplete penetrance, especially when EcoRI fragments are 35–41 kb; also, some patients may have very slightly larger D4Z4 arrays.^{33 b}Matching EcoRI/BlnI fragments are ~3 kb shorter and XapI fragments are ~5 kb shorter than the corresponding EcoRI fragment.^{12 c}The sequencing test is the recommended assay for 4qA161, the predominant haplotype of pathogenic D4Z4 alleles. It can serve as a surrogate for 4qA/4qB testing by PFGE on HindIII digests with 4qA- and 4qB-specific oligonucleotide probes.^{11 20 22 23} There can be pathogenic non-4qA161 alleles with 10q-type BlnI^S D4Z4 repeat units on 4q.³² However, categories I, III and VI in figure 6 do not involve such arrays; therefore, 4qA161 testing would be informative for samples giving these types of PFGE patterns, especially because ~40–50% of controls have no 4qA161 allele. ^dThe D4Z4 subfragment probe under stringent hybridisation conditions detects fragments with p13E-11 deletions and gives stronger signals than the p13E-11 probe from XapI digests.^{12 e}Categories IV, Va and Vb are non-standard D4Z4 arrays with canonical 10q-type repeat units on 4q or vice versa, as evidenced by a missing EcoRI/BlnI fragment (only one EcoRI/BlnI fragment instead of two, figure 6, Vb) or an EcoRI/BlnI fragment that is less than 3 kb shorter than the corresponding EcoRI fragment (figure 6, IV and Va). ^fSometimes, the chromosomal origin of a short EcoRI fragment has to be determined by PFGE on NotI digests blot-hybridised to a 4q35-specific probe (B31),^{13 22} for example, figure 6, category VI versus category VII, and unusual cases with all four arrays beginning with 10q-type D4Z4 units. The NotI fragment length will be the sum of 187 kb and the length of the corresponding EcoRI fragment.