Abstract
Limb weakness is an uncommon symptom in children, with multiple
factors contributing to related diseases, particularly genetic
disorders. A nine-year-old boy presented with slowly progressive muscle
weakness of the limb-girdle muscles. We evaluated the clinical symptoms,
laboratory tests, imaging examinations, and pathological examinations of
this proband. We combined whole-exome and Sanger sequencing to identify
the novel compound heterozygous pathogenic mutations NM 001849.3:
c.1970-10_1978 del CGGCTTGCAGGGACGCGTG and c.2462-3C>A inCOL6A2 in this proband inherited from the mother and father,
respectively. Mutational confirmation at the mRNA level demonstrated
that the proband carried a homozygous abnormal sequence with 23bp
deletions (c.2462-2484 del GGACGCGTGTGGGCGTGGTGCAG) at the beginning of
exon 26. In contrast, both parents and sibling have normal sequences
with no clinical symptoms. The results of this study further expand the
mutational spectrum and will be helpful for further molecular diagnosis.
Keywords: COL6A2; Ullrich
congenital muscular dystrophy
(UCMD); compound heterozygous mutations; whole exome sequencing; sanger
sequencing;
Congenital muscular dystrophies (CMDs) are a group of rare genetic
diseases that primarily affect the muscle and are characterized by
progressive degeneration and weakness(Kirschner, 2013). Clinical
symptoms typically manifest at birth or within the first few months of
life(Carakushansky et al., 2012). Ullrich congenital muscular dystrophy
(UCMD) is a rare type of autosomal dominant or recessive CMDs, mainly
caused by mutations in the related genes leading to loss of collagen VI
with an earlier onset time and progressive clinical symptoms(Kirschner,
2013; Park et al., 2014). Collagen type VI is consistently linked to the
development of congenital muscular dystrophy(Bushby et al., 2014;
Antoniel et al., 2020; Simsek-Kiper et al., 2020), composed of three
distinct alpha chains encoded by COL6A (Lampe & Bushby, 2005).
Most patients with UCMD carry
mutations in COL6A that result in the abnormal production of
collagen type VI(Bushby et al., 2014). However, the relationship between
genotype and phenotype remains unclear.
The nine-year-old boy (II-2, Figure 1 A ) was the second child
of healthy non-consanguineous
parents.
The patient presented myasthenia and
was initially diagnosed with CMD. His symptoms of gait instability had
not improved and became increasingly severe as the proband got older;
intellectual development was normal compared with peers. Physical
examination shows a clear mind, mental reactivity, normal nutritional
condition, bilateral lower extremity weakness, the inability to stand,
and the left positive Babinski sign. The proband presented equinus when
he stood with auxiliary assistance. The muscle strength of the upper
limbs was grade IV, and that of the lower limbs was III. Deep tendon
reflexes were decreased in the knees. Auxiliary examinations yield the
following results: Laboratory examinations revealed no obvious
abnormalities in the blood routine or coagulation parameters.
Biochemical assays were as follows: creatine kinase isoenzyme MB 12.03
(Reference value: 0–5 ng/ml); creatinine 19 (Reference value: 62–115
µmol/L); creatine kinase 370 (Reference value: 38–174 U/L). On the
biceps brachii, vastus medialis, and anterior muscle, needle
electromyography revealed that the duration of voluntary motor unit
potential on volition was reduced by 32%, 27%, and 38%, respectively.
There were no discernible abnormalities in the upper and lower limb
sensory nerve conduction. Similarly, no abnormalities were detected
during brain MRI or diffusion-weighted imaging (DWI). The left biceps
brachii muscle biopsy was performed Figure 1 B .
The pathological outcome was the
atrophy of both types of fibers. Part of the muscle fiber was replaced
by connective and adipose tissues, and the size of the remaining muscle
fiber was noticeably different. A small amount of regenerative and
moth-eaten muscle fiber also appeared. Immunohistochemistry results
indicated that dystrophin-Rod, dystrophin-N, and β-sarcoglycan were
reduced in muscle fiber membranes compared to the normal control. A
proportion of muscle fiber membranes were absent of dystrophin-C
reporter staining. Collectively, the proband is highly probable to have
muscular dystrophy, and genetic testing is extremely helpful for disease
diagnosis.
Our whole-exome sequencing (WES) results indicate that the proband
carries two novel compound heterozygous pathogenic mutations inCOL6A2 , one of which was a
frameshift mutation (NM 001849.3:
c.1970-17_1971 del ACGCGTGCGGCTTGCAGGG), and the other was a
splice site mutation (NM 001849.3:
c.2462-3C>A) (Figure 1 C ). Subsequently, the
mutations of COL6A2 in the proband, his parents, and his sibling
were validated by Sanger sequencing. Notably, the frameshift mutation
(c.1970-10_1978 del CGGCTTGCAGGGACGCGTG) was confirmed at the DNA level
in the proband and his mother by Sanger sequencing and was different
from the WES results Moreover, another site was observed in the father
and proband. The mode of inheritance
complies with the rules of autosomal dominant inheritance. The mRNA was
extracted from the peripheral blood to further verify the functional
effect of the genetic variants. RNA sequence analyses showed wild-type
sequences in the parents and sibling.
However, the proband has
a homozygous but unusual sequence, a
23bp (c.2462–2484 del GGACGCGTGTGGGCGTGGTGCAG) deletion at the
beginning of exon 26 compared to wild-type (Figure 1 D ). We
speculated that mutations affect the phenotype only when two mutations
are present together.
Zhang et al. reported two UCMD patients with different mutations inCOL6A2 ; one carried a homozygous c.1870G>A (p.E624K)
variant and the other a homozygous c.2626C>A (p.R876S).
Both patients presented with myasthenia, joint contractures, and joint
laxity(Zhang et al., 2010). Lucarini et al. reported a proband who
carried a homozygous A > G mutation at −10 of intron 12 inCOL6A2 that goes along with
generalized muscle weakness,
arthrogryposis, and mild spine rigidity(Lucarini et al., 2005). In our
study, we described a UCMD proband with slowly progressive muscle
weakness of the limb-girdle muscles and
identified novel
compound heterozygous pathogenic
mutations of COL6A2 in a Chinese family. Subsequently, we
directly extracted RNA from peripheral blood to verify the effect of the
two different novel types of COL6A2 mutations at the mRNA level.
Our results demonstrated that the proband carried a homozygous aberrant
mRNA sequence, suggesting that two different novel mutations may
together contribute to the partial deletion at the beginning sequences
of the same exon 26. This might be attributed to the defective pre-mRNA
splicing and phenotype caused only when both mutations are present,
indicating that RNA sequencing is also essential to diagnose the
disease. However, the action mechanism of those mutations is not
entirely clear. Therefore, an ideal method combining DNA and RNA
sequencing has been suggested as an optimal tool for prenatal diagnosis.
In summary, our research demonstrated the novel pathogenicity of
mutations and expanded the variant spectrum of COL6A2 , further
improving the accurate diagnosis of
UCMD.