Genetic variability in Iranian limb‐girdle muscular dystrophy type 2B patients: An evidence of a founder effect

Figure 1. (a–g) Pedigrees of eight families with dysferlinopathy. Squares—males, circles—females, filled symbols affected individuals, open symbols—unaffected individuals. Double bars represent consanguineous unions. All family members tested in this project were abbreviated as Homo, Hete, and NL. Homo represents homozygote, Hete represents Heterozygote, and NL stands for Normal regarding to the mutation found in the family. Sequencing results are corresponding to the individuals in the pedigree who were shown by Homo, Hete, and NL

Figure 2. Two families, 19,315 and 19,342, shared the same haplotypes. Electropherogram peaks are shown in different colors and alphabetic letters for simplicity. All affected individuals of both families have haplotype “A” in homozygous form. This represents markers D2DYSFSU14.7, D2S1389, D2S2977, and D2S1394 with resulting peaks of 297, 209, 291, and 435 in the above mentioned families. D2DYSFSU14.7 marker was not previously reported, so we gave it this new name. Number 2 in the markers' name is the number of the chromosome on which DYSF is located, S denotes STR, U means upstream, and the following numbers denote distance from the gene (e.g., 8.05 × 105 base pairs)

Figure 3. STR markers surrounding DYSF in family 16,941. D2DYSFSU14.7 is a novel STR marker, where U means upstream. The numbers denote distance from the gene (e.g., 8.05 × 105 base pairs). Marker D2S1389 is located in intron 13 which does not produce any peak (DNW = Does Not Work). This marker reveals a homozygous peak in DNA from parents and in the heterozygote sibling

Figure 4. Evaluating exon deletion of exon 12 to exon 15 in DYSF on agarose gel by multiplex PCR. The deleted exons are shown in red in the schematic image. The PCR product size of each exon (s) is as follows: exon 12: 527 bp, exon 13:458 bp, exon 14–15: 641 bp, exon 55: 730 bp. The ladder used in this figure is a 100 bp ladder. P: Father, M: Mother, AC: Affected child, Co‐: Negative control, PCR: Polymerase chain reaction

Figure 5. Evaluating deletion of exon 12 to exon 15 in DYSF on agarose gel by long‐range PCR. Arrows show the location of primers: a primer pair (shown in orange) is designed for amplifying exon 13, while the other primer set is localized in a sequence surrounding exons 11 and 16 (shown in light blue). P: Father, M: Mother, AC: Affected child, Co‐: Negative control, PCR: polymerase chain reaction

Figure 6. Immunohistochemical analysis of patient's muscle biopsy from family 16,941. (a) Dystrophic pattern of staining with hematoxylin and eosin (H & E). (b) Sarcolemmal labeling of almost all muscle fibers by beta‐spectrin. (c) The loss of sarcolemmal labeling of all muscle fibers by dysferlin antibody