The Clinical diagnosis of FSHD is frequently not an issue for a neurologist expert in neuromuscular disorders due to the typical pattern of muscle involvement. Nevertheless, the classical genetic approach based on identification of 4q35 D4Z4 contracted allele after EcoRI
digestion, LGE, Southern blotting and p13E-11 hybridization fails to offer the molecular confirmation in about 5–10% of cases[14
The present study was conducted to guide the clinician in the choice of additional genetic or epigenetic tests to confirm the clinical diagnosis, since several conditions can partially mimic the clinical phenotype of FSHD.
In clinical practice, when standard genetic testing does not confirm the clinical suspicion of FSHD1, EMG is required to confirm the myogenic pattern of muscle weakness and to rule out neurogenic involvement. For example, patients harboring deletions of PMP22
], or mutations in the TRPV4
], both transmitted as autosomal dominant traits, may present with a scapuloperoneal distribution of muscular involvement of neurogenic origin.
Other inherited myopathies may present with a clinical phenotype resembling FSHD: some limb girdle muscular dystrophies (LGMD) [36
], glycogenosis type II (Pompe disease) [23
] and glycogenosis type V (McArdle’s disease) [37
], myosin storage myopathies [38
], desminopathies [39
], other myofibrillar myopathies[24
], and mitochondrial myopathies [41
In most of the cases, the age of onset, the disease progression rate, the pattern of inheritance, the presence of respiratory muscle or cardiac involvement, the absence of facial weakness, the presence of contractures or ocular involvement, may aid the clinician in orientating the diagnosis toward these diseases.
In all these cases, muscle biopsy is mandatory to confirm the clinical suspicion. Indeed, histological, immunohistochemical and western blot studies on muscle samples are generally adequate to distinguish among different LGMDs due to lack of sarcolemmal protein, or to identify storage, vacuolar, mitochondrial, congenital or myofibrillar myopathies.
If EMG and muscle biopsy studies are not conclusive, the use of detailed PFGE based D4Z4 genotyping on agarose-embedded DNA plugs[42
] can be used to identify FSHD1 patients that could have been missed by the less detailed standard molecular diagnosis.
In our study we found two of such a patients among the 16 patients included in the study, P1 and P2. P1 was mosaic for the D4Z4 contraction. This condition results from a mitotic rearrangement of D4Z4 probably in early embryogenesis, is quite frequent in de novo
] and mosaicism often goes undetected[45
]. Their clinical phenotype appears to be milder compared to individuals carrying the same residual repeat size in all of their cells. In P2, the pathogenic hybrid allele was identified by PFGE and the distal D4Z4 fragment was analyzed by direct sequencing to confirm the pathogenicity[9
In all cases without a diagnosis, and with a permissive chromosome, FSHD2 should be suspected [10
]. Indeed, FSHD2 patients display: 1) a clinical phenotype identical to FSHD1 patients; 2) normal-sized repeats with at least one permissive chromosome (contrary to FSHD1 where the repeat is contracted on a permissive chromosome) and 3) reduced DNA methylation of the D4Z4 repeats on chromosomes 4q and 10q, as measured on the methylation-sensitive restriction sites FseI and BsaAI.
In our series six patients (P3 – P8) were found to meet these criteria. All of them were sporadic cases, and we studied the segregation of the permissive allele and the DNA methylation in all members available for the six families confirming that the FSHD clinical phenotype appears only in patients displaying hypomethylation on a permissive 4A161 background. Finally, when EMG studies, muscle biopsy studies and extensive D4Z4 repeat arrays genotyping and DNA methylation analysis are negative, we suggest reconsidering the possibility of a FSHD phenocopy.
In our series of patients, six out of the sixteen patients (P9 to P14) carried mutations in genes not related to FSHD, i.e. CAPN3
. These genetic defects had gone unnoticed by standard histological, immunohistochemical and Western blot studies on muscle biopsy. Actually, approximately 20% of CAPN3
mutations do not cause a reduction of the protein steady state levels, but affect its autocatalytic activity. A specific test is available to measure this activity in muscle, avoiding these misdiagnoses[22
]. Unfortunately, this test is done only in a restricted number of laboratories, and in the case of the present study, was not performed because no muscle sample was left. Furthermore, it has been reported that patients harboring VCP
mutation may display only minor and non-specific histopathological changes on muscle biopsies [14
Accordingly, in P13 and P14, carrying a mutation in the VCP
gene, no sign of Paget’s disease and no characteristic rimmed vacuoles on muscle biopsy were found. Nevertheless, after the molecular diagnosis, P14 underwent to a neuropsychological test demonstrating the presence of a mild dysexecutive syndrome. APA measurement, standard bone X ray (pelvis, hip, femur, humerus, vertebral bodies and skull) and adapted neuropsychological tests may help in detecting asymptomatic Paget’s disease or fronto-temporal dementia that are usually associated with a inclusion body myopathy and mutations in the VCP
Interestingly, CK levels in four out of the six patients displaying a phenotype resembling FSHD but carrying mutations in other genes, were more elevated then in classical FSHD1 patients, and five over six patients displayed no asymmetric muscular involvement. Concerning facial weakness, P9, P10, P11 and P12 (LGMD2A) and P14 (VCP) showed transversal smile and difficulty to blow, as P4 and P6 (FSHD2) while P2 (FSHD1), P3, P5, P7 and P8 (FSHD2) showed also weakness of orbicularis oculi
(Supplementary Figure 1
). Altogether, these clinical features, if present, may prompt clinicians to sequence CAPN3
gene, before undergoing more complex epigenetic studies.
Mutations in FHL1
have been described to cause a scapuloperoneal phenotype mimicking FSHD, inherited in an X dominant fashion[24
]. We could not find FHL1
mutations in our patients, possibly because they are less frequently associated with an FSHD-like phenotype. Nevertheless we cannot exclude that mutation in this gene may be associated to FSHD-like phenotype due to the small number of patients included in the present study.
In two out of sixteen patients (P15, P16) included in this study, no genetic or epigenetic defect could be found. In these two sporadic patients, CK values were elevated four to six times the upper limit of normal values, which may suggest the presence of other loci determining an FSHD-like phenotype. In the absence of an identified genetic or epigenetic defect, FSHD-like patients should undergo a complete clinical workout in order to identify additional signs that may be useful to orientate the diagnosis. Annual follow-up is also recommended to monitor the pattern of muscle weakness progression and, possibly, the appearance of signs of symptoms evocative of a specific muscular dystrophy. The possibility of a second muscle biopsy must be carefully discussed with the patient in case of worsening of clinical conditions.
In conclusion, in patients with a FSHD phenotype and no D4Z4 contraction on chromosome 4q35 detected by standard techniques it is important to consider these possibilities: 1) a false negative diagnosis; 2) FSHD2; and 3) a mutation in another gene resembling FSHD (especially if CK levels are consistently elevated, and there is evidence of symmetric muscle involvement); (see the Diagnostic Flowchart in ). Extensive D4Z4 genotyping by PFGE analysis and methylation studies of the D4Z4 repeat, which are currently available only in selected research laboratories, are required to diagnose the first two conditions.
Diagnostic flowchart for patients with FSHD-like phenotype.
Novel diagnostic approaches are currently being developed to simplify the diagnosis of FSHD and of other muscle disorders. Molecular combing is a promising new strategy which is apparently simpler and faster than classical Southern blot, and allows allele determination and the detection of mosaics and of complex alleles in a single step procedure [46
]. Furthermore, next-generation sequencing technology will allow to study large panels of genes for a fraction of the cost (and of the time) required by classic Sanger sequencing.