case_images: PMC10867183_01
case_images: Pedigree and clinical features of two patients. (A) Pedigree of the family with FSHD1. The age in years, number of D4Z4 repeat units, and haplotype are shown for each subject. Subject III-9 was identified as a compound heterozygote because of a shortened FSHD1 alleles, whereas her affected brother (III-10) carried just one allele. The parents (II-7 and II-8) were asymptomatic; the father was a heterozygote for eight units of D4Z4 with 4qA, while the mother was a low-level mosaic for four units of D4Z4 with 4qA. All other members of this family were asymptomatic. (B) The proband (III-9) showed decreased facial expression and an inability to close her eyes completely (left). She showed false hypertrophy of the orbicularis oris, her lips were thickened and slightly distorted (left), and she presented with a winged scapula (middle) and limitations in lifting activities in the bilateral upper limbs (right). (C) Patient 2 (III-10) showed atrophy of the facial muscles (middle), shoulder, upper arm, and chest muscles (right). He presented with a winged scapula (middle) and limitations in lifting activities in the bilateral upper limbs (right). The photographs of the proband (III-9) and the proband's younger brother (III-10) were taken at ages 27 and 25, respectively.
case_images: fneur-15-1258831-g0001.jpg
case_images: PMC10867183_02_fneur-15-1258831-g0001.jpg
case_images: F1
case_images: The proband's younger brother (patient III-10, Figure 1A), was 25 years old when he began participating in this case study. He was unable to raise his arms above his shoulders (Figure 1C).
case_images: Imagological examination of two patients. (A) X-ray examination of patient 1 (III-9). She had a slightly scoliotic spine and a straightened cervical spine (left). MRI analysis of the limb muscles revealed changes in the bilateral buttocks and thighs and the right upper arm and paravertebral muscles (middle and right). These changes were consistent with the presence of muscular dystrophy. (B) X-ray examination of the spine of patient 2 (III-10). The left image shows increased physiological curvature of the lumbar spine and an upturned sacrum. The right image shows a slight curvature of the spine, a straight cervical spine.
case_images: fneur-15-1258831-g0002.jpg
case_images: PMC10867183_02_fneur-15-1258831-g0002.jpg
case_images: F2
case_images: Hyperlordosis of the lumbar spine was also observed (Figure 2B). In addition, OGM accurately identified the D4Z4 repeat units in both the 10qA and 10qB alleles in all family members, as shown in Supplementary Figure 2.
case_images: PMC10867183_02
case_images: Pedigree and clinical features of two patients. (A) Pedigree of the family with FSHD1. The age in years, number of D4Z4 repeat units, and haplotype are shown for each subject. Subject III-9 was identified as a compound heterozygote because of a shortened FSHD1 alleles, whereas her affected brother (III-10) carried just one allele. The parents (II-7 and II-8) were asymptomatic; the father was a heterozygote for eight units of D4Z4 with 4qA, while the mother was a low-level mosaic for four units of D4Z4 with 4qA. All other members of this family were asymptomatic. (B) The proband (III-9) showed decreased facial expression and an inability to close her eyes completely (left). She showed false hypertrophy of the orbicularis oris, her lips were thickened and slightly distorted (left), and she presented with a winged scapula (middle) and limitations in lifting activities in the bilateral upper limbs (right). (C) Patient 2 (III-10) showed atrophy of the facial muscles (middle), shoulder, upper arm, and chest muscles (right). He presented with a winged scapula (middle) and limitations in lifting activities in the bilateral upper limbs (right). The photographs of the proband (III-9) and the proband's younger brother (III-10) were taken at ages 27 and 25, respectively.
case_images: fneur-15-1258831-g0001.jpg
case_images: PMC10867183_02_fneur-15-1258831-g0001.jpg
case_images: F1
case_images: The proband's younger brother (patient III-10, Figure 1A), was 25 years old when he began participating in this case study. He was unable to raise his arms above his shoulders (Figure 1C).
case_images: Imagological examination of two patients. (A) X-ray examination of patient 1 (III-9). She had a slightly scoliotic spine and a straightened cervical spine (left). MRI analysis of the limb muscles revealed changes in the bilateral buttocks and thighs and the right upper arm and paravertebral muscles (middle and right). These changes were consistent with the presence of muscular dystrophy. (B) X-ray examination of the spine of patient 2 (III-10). The left image shows increased physiological curvature of the lumbar spine and an upturned sacrum. The right image shows a slight curvature of the spine, a straight cervical spine.
case_images: fneur-15-1258831-g0002.jpg
case_images: PMC10867183_02_fneur-15-1258831-g0002.jpg
case_images: F2
case_images: Hyperlordosis of the lumbar spine was also observed (Figure 2B). In addition, OGM accurately identified the D4Z4 repeat units in both the 10qA and 10qB alleles in all family members, as shown in Supplementary Figure 2.
case_images: OGM results for this family. (A) The proband (III-9) was identified as a compound heterozygote for the 4qA allele with four and eight D4Z4 repeat units. (B) The younger brother (III-10) was found to only have one 4qA allele with four D4Z4 repeats and a 4qB allele with 28 D4Z4 repeat units. (C) The proband's father (II-7) carried the 4qA allele with eight D4Z4 repeats and a 4qB allele with 28 D4Z4 repeat units. (D-F) The OGM results of the proband's mother (II-8). The green bar is the reference map of chromosome 4, and the blue bar is the assembled map of chromosome 4. (D) Chromosome 4 of subject II-8 was found to have a calculated repeat count of >=5 with an unknown haplotype via FSHD pipeline analysis. A high proportion of consistently truncated sequences (black boxes) might reflect terminal DNA deletions or insufficiently long DNA strands. Several sequences are followed by the 4qA haplotype (red box) with a D4Z4 array length (approximately four repeat units) shorter than that in each map containing five units (purple box). (E) Chromosome 4 of a subject whose haplotype was unknown had a calculated repeat count of >=8, as determined by FSHD pipeline analysis. (F) A 4qA allele and 49 D4Z4 repeat units were manually determined based on the consensus maps of the de novo pipeline analysis.
case_images: fneur-15-1258831-g0003.jpg
case_images: PMC10867183_02_fneur-15-1258831-g0003.jpg
case_images: F3
case_images: But OGM analysis of all four individuals showed a repetition of D4Z4 units for both the 4qA and 4qB alleles, enabling precise determination of their genotypes (Figure 3). The proband (III-9) was a compound heterozygote for the 4qA allele with four and eight separate D4Z4 repeat units (Figure 3A), as confirmed through haplotype analysis, whereas her affected younger brother (III-10) was a heterozygote who inherited only one 4qA allele with four D4Z4 repeat units (Figure 3B). The proband's father (II-7, aged 50 years), who was found to have a normal phenotype upon neurological examination, also carried the 4qA allele with eight D4Z4 repeat units (Figure 3C). It should be emphasized that the unaffected mother (II-8) had a 4qA allele with 49 D4Z4 repeat units and low-level mosaicism with four D4Z4 repeat units (Figures 3D, F), but the proportion of low-level mosaicism could not be confirmed. In the chromosome 4 D4Z4 regions, the map was truncated before the ends of the repeat units, and the haplotype was considered unknown because the sequences did not fully span the repeat units and haplotype-specific labels; thus, the pipeline generated lower-bound estimates of the repeat counts of >=5 and >=8 (Figures 3D, E). Repeat unit lengths were estimated based on the interval between the labels flanking the D4Z4 arrays, and the number of D4Z4 repeat units was manually calculated to be 49 for the mother (Figure 3F). Furthermore, when we manually checked the sequences supporting a given map, we found several sequences followed by the 4qA haplotype (Figure 3D [red box]) with a D4Z4 array length to be shorter than that of the given map (Figure 3D [purple box]). In addition, a large fraction of sequences was consistently truncated (Figure 3D [black boxes]), which could have been due to terminal deletion or an insufficient DNA sequence length. Methylation analysis showed that CpG6 methylation levels in four subjects (III-9, III-10, II-7 and II-8) were 60%, 20%, 90%, and 90%, respectively (Supplementary Table 1, Supplementary Figure 3).