Dysferlinopathy in Switzerland: clinical phenotypes and potential founder effects

To evaluate the hypothesis that the DYSF mutation c.3031 + 2T>C is due to a founder effect, we performed haplotype analysis, as described previously, with 6 polymorphic microsatellite markers on chromosome 2p13.2 flanking the DYSF gene (D2S327, D2S2113, D2S292, D2S291, D21S2110 and D2S2111) [23].

Thirteen patients were included in the study. Of those, 9 patients were from three non-related families from Central Switzerland (Canton of Schwyz). The clinical data of 10 patients from 6 different families are summarized in Tables 1 and 2. Three patients from central Switzerland (C-6, D-10, D-11) provided blood for haplotype analysis but refused clinical evaluation. Consanguinity was not reported and the parents of all patients were healthy. Mean disease onset was in late adolescence or early adulthood (18.7 ± 4.5 years). Early manifestations (Table 1) included leg pain, steppage gait, Gowers’ sign and difficulty running and jumping. Mean CK levels were 12500 ± 14400 IU/L (range 2200–46350, normal value for our laboratory < 167 U/l, Table 1).

The clinical phenotype could be determined in 10 patients (Table 2). In all patients, the facial muscles were spared. Four of the patients from Central Switzerland (C-4, C-5, C-7, F-13) presented without arm weakness a few years after the onset of symptoms (Table 2). All patients had distal lower limb flexor weakness and 2 (C-7, F-13) also showed distal lower limb extensor weakness. Three (C-4, C-5, C-7) patients had proximal lower limb weakness and therefore were classified as the “proximo-distal” (PD) phenotype [6]. Another patient (F-13) demonstrated isolated distal lower limb weakness (foot flexion > extension) and was classified as the MM phenotype. Three patients (C-4, C-5 and C-7) were followed for 11, 9 and 4 years, respectively (Table 2). All developed distal upper limb extensor weakness with 2 (DC-4, C-7) additionally showing distal upper limb flexor and proximal upper limb weakness. Two patients from central Switzerland (C-8, D-9) were examined 20 and 28 years after onset and demonstrated proximal and distal upper and lower limb flexor and extensor weakness (Table 2). Patient D-9 experienced a relatively “benign” course with muscle strength grade of at least 3 on the MRC scale (Table 2). One patient from the Canton Thurgau (E-12) presented 7 years after onset with proximal and distal lower limb flexor and extensor weakness, eventually developing distal flexor and extensor and proximal upper limb weakness (Table 2). A patient from the Canton Aargau (B-3) presented with severe distal lower limb flexor, proximal lower limb flexor and extensor, distal upper limb flexor and extensor and proximal upper limb weakness 15 years after onset. Two patients from the Canton Fribourg were initially examined 23 (A-1) and 17 (A-2) years after onset, respectively. Patient A-1 had generalized weakness, pronounced in the distal lower limb extensors, and over the course of 9 years developed proximal lower and upper limb palsies. Patient A-2 had generalized weakness, more pronounced in the distal lower limb flexors, that progressive minimally over 7 years. Interestingly, members of family C presented with a proximo-distal phenotype early in the course and members of family C and patient F-13 were notable for the lack of upper limb weakness during the early phases of the disease. Later in the course, all patients manifested a typical LGMD phenotype; however, the distribution of weakness was heterogeneous (Table 2). The DMAT phenotype was not observed in our cohort.

The use of mobility aids showed the following timeline: 2 patients required canes for walking assistance (A-1, A-2) at the age of 35 and 49 years, respectively (disease duration until first use of canes 12 and 21 years, respectively), and both were eventually wheelchair-bound (17 and 24 years after disease onset; 5 and 3 years after first cane use). Patient B-3, at age 29, 14 years after disease onset, used a wheelchair intermittently (B-3), and patient C-8 was wheelchair-bound, at age 43, 27 years after disease onset.

Electrocardiography and ECG were normal in all 9 patients who underwent examination, the longest disease duration being 35 years (Table 1). Pulmonary function was normal in 6 out of 7 patients, with restrictive lung disease documented in patient A-1 (Table 1; initial pulmonary function testing performed after 29 years of disease duration). In 3 patients, muscle MRI showed fatty replacement of the lower limb muscles. In all three patients (A-2, E-12 and F-13), the short head of the biceps femoris and the sartorius and the gracilis muscles were symmetrically spared or appeared less affected than other muscles. In patients E-12 and F-13, the semitendinosus, the tibialis anterior and the extensor digitorum longus muscles also seemed less affected, while edema was suspected in selected lower limb muscles. In another patient (C-7), the pelvic and the lower limb muscles appeared normal. EMG was myopathic with short and small potentials in all 8 patients examined, with spontaneous activity found in 5 cases (Table 1).

A total of 5 surgical muscle biopsies were available (Table 3) for evaluation. In 2 patients, biopsies were not available because they were taken in 1986 and 1977. In 6 patients, the genetic diagnosis was made without prior biopsy. The mean age at biopsy was 28.25 ± 13.7 years, while the mean disease duration at biopsy was 8.3 ± 9.8 years. Samples were removed from the trapezius, quadriceps and gracilis muscles. All patients exhibited dystrophic changes with an increased spectrum of myofiber diameter and variable fibrosis. In one case with a LGMD phenotype (B-3), striking inflammatory changes were observed within the endomysium. Western blots (WB) and/or immunohistochemistry (IHC) were performed in 6 samples (WB only in 2 samples, IHC only in 3 samples, WB and IHC in a single sample). On WB, dysferlin was totally absent in all samples tested, whereas by IHC, dysferlin was judged absent in 2 cases and reduced in two.

Genetic data are summarized in Table 4. In 13 patients, 2 disease-causing mutations on separate alleles were identified. Nine patients from 3 non-related families from Central Switzerland (Canton Schwyz) carried the identical splice site mutation, c.3031 + 2T>C, 7 were homozygous (Table 4). Three patients from 2 different families from the Cantons of Fribourg and Aargau carried a heterozygous frameshift mutation c.1064_1065delAA, and 2 members from family A, harbored the heterozygous nonsense mutation c.2217C>A (Table 4).

Two novel mutations were identified in this patient set. Patient C-6, who was compound heterozygous for the c.3031 + 2T >C mutation on one allele, showed a novel mutation, c.2869C>T on the other allele. Patient E-12 carried the novel homozygous c.5928G > A mutation.

Haplotype analyses using markers flanking the DYSF gene revealed homozygosity for at least one nearest marker (D2S291) in all patients from the two apparently unrelated C and D families carrying the homozygous c.3031 + 2T>C mutation, suggesting a possible founder mutation (See Fig. 1). In patient C-7, carrying a compound heterozygous DYSF mutation (c.3031 + 2 T >C and c.2869C>T), marker D2S291 shows a heterozygous genotype (Fig. 1).