Genetic findings
In general, about 97.5% of the target bases were covered with at least 50X per individual, and the mean depth of coverage for all target regions was 185. After filtering and validation by Sanger sequencing, 5 probable pathogenic variants were identified in 4 probands, involving one variant previously reported as pathogenic variant and four novel variants. The known pathogenic variant c.1304C > T (p.Pro435Leu) was identified in SPAST gene. The identified four novel variants include two B4GALNT1 variants (p.Ser475Phe and c.1002 + 2T > G), a SPG11 p.Val1979Ter nonsense variant, and a splicing site variants in SPAST (c.1245 + 5G > A)(Fig. 1). All of these variants, being predicted as harmful effects by the SIFT, PolyPhen-2, dbscSNV and Human Splicing Finder (HSF) software, were absent in publicly available database as well as 200 normal controls. According to the ACMG standards and guidelines, all four variants were classified as likely pathogenic variants. The novel splicing mutation (c.1245 + 5G > A) in SPAST was co-segregated perfectly with the phenotypes observed in family 2. Moreover, pathogenic repeat expansions which are implicated in all SCA types (SCA types 1–3, 6, 7, 12, and 17) and DRPLA, as well as large deletions or duplications of HSP culprit genes (SPAST, ATL1, REEP1, PGN, and SPG11), was excluded in this family. The two novel B4GALNT1 variants identified in compound heterozygous state, including a splicing mutation site (c.1002 + 2T > G) and a missense mutation (p.Ser475Phe), were found to segregate with the disease in family 3. In family 4, co-segregation analysis showed that the father and mother are both heterozygous for the novel SPG11 p.Val1979Ter variant.
Splicing pattern analysis
A splicing reporter minigene with SPAST exon 9 was constructed (Fig. 3). Agarose gel electrophoresis of RT-PCR showed that the fragment obtained in mutant group was smaller than that obtained in control group (Fig. 3). Sanger sequencing revealed that the SPAST c.1245 + 5G > A mutation resulted in mRNAs with a loss of exon 9 (Fig. 3).
Clinical manifestations of probands
The clinical manifestations of all probands are summarized in Table 1, and pedigrees of the families are displayed in Fig. 2. In total we recruited 13 affected individuals from 5 independent families, include 8 males and 5 females. The mean age at onset was 30.23 years, ranging from 7 to 48 years. Among the five probands, two showed an AD inheritance pattern, two with an AR inheritance pattern, and one was a sporadic case. Apart from the most common symptoms of progressive lower limbs weakness and spasticity, additional presentations including dysarthria, dysphagia, urinary incontinence, constipation, and intelligence impairment were also observed in present study.
Table 1
The clinical presentations and genetic analysis results of 5 HSP probands.
Proband
no
|
Sex
|
AAO
(y)
|
Inheritance
|
LL weakness
|
LL plasticity
|
Reflexia
|
Intellectual disability
|
EMG/NCT
|
Brain MRI
|
Additional features
|
Gene
|
Pathogenic variants
|
1
|
F
|
48
|
AD
|
+
|
+
|
++++
|
-
|
normal
|
normal
|
dysarthria
|
SPAST
|
p.Pro435Leu
|
2
|
M
|
26
|
AD
|
+
|
+
|
++++
|
-
|
normal
|
normal
|
constipation
|
SPAST
|
c.1245 + 5G > A
|
3
|
M
|
7
|
AR
|
+
|
+
|
++++
|
+
|
normal
|
normal
|
poor social communication skills
|
B4GALNT1
|
p.Ser475Phe, c.1002 + 2T > G
|
4
|
M
|
12
|
sporadic
|
+
|
+
|
+++
|
+
|
normal
|
thin corpus callosum
|
urinary dysfunction, dysarthria, dysphagia
|
SPG11
|
p.Val1979Ter
|
5
|
M
|
41
|
AR
|
+
|
+
|
++++
|
-
|
normal
|
normal
|
-
|
-
|
-
|
F, female. M, Male. LL, Lower limb. AAO, Age at onset. AD, autosomal dominant. AR, autosomal recessive. EMG, electroneuromyography. NCT, nerve conduction test. -, absent. |
Characteristics of HSP patients with SPAST mutations
The proband (II:2) in family 1, carrying SPAST p.Pro435Leu variant, was a 56-year-old male and presented with lower limbs weakness and gait disturbance at the age of 48. As the disease progresses, he began to show dysarthria and severe spasticity in the lower limbs. He currently needs to move around with the aid of crutch. No signs of intellectual disability were observed in the proband. Neurological examination showed hyperactive deep tendon reflexes in the lower limbs, gait ataxia, pes cavus, and positive Babinski signs. The EMG results and Brain MRI were unremarkable. His mother (I:2) developed the illness at the age of 50 with a disease duration of 9 years.
The proband (III:4) in family 2,a 36-year-old female, was detected as the carrier of SPAST c.1245 + 5G > A variant. She presented with lower limbs weakness and an unsteady gait at the age of 26. As the disease progresses, her gait disturbance became more severe with frequent falling. She had constipation problems since age 31. Severe spasticity was marked in the lower limbs although the early-stage of baclofen treatment was taken to the proband. Her examination was further marked by hyperactive deep tendon reflexes and positive Babinski signs, with mild rigidity in all extremities especially in the lower limbs. She was bilateral pes cavus and ankle clonus. Her older sister (III:3) experienced the similar symptoms at the age of 26. The proband’s mother (II:6) and other three affected uncles (II:3, III:4, III:5) presented with gait disturbance or walking difficulty at the age of 42, 44, 45 and 44, respectively. The proband's grandmother (I:2) was diagnosed with HSP when she was 50 years old and died at the age of 70 years. The proband’s daughter (IIII:2) had initial symptom of gait disturbance at age 7 years༌and experienced mild cognitive impairment, with a Mini-Mental State Exam (MMSE) score of 22/30, eight years after symptom onset. She had poor concentration and social communication skills. No muscular atrophy or fasciculation was observed in all affected individuals within this family. Upon neurological examination, these patients revealed moderate or severe spasticity in the lower limbs, pyramidal-tract signs, and gait ataxia. The Brain and spinal cord MRI were normal. The EMG and nerve conduction test were also unremarkable.
Presentations of HSP patients with B4GALNT1 mutations
The proband (II:3) from family 3 carrying the B4GALNT1 c.1002 + 2T > G and c.1424C > T(p.Ser475Phe) variants presented with subtle gait abnormalities at the age of 7 years. His gait disturbance was gradually observed as disease progresses, when he began to experience severe spasticity in the lower limbs. He had learning disability with poor academic performance and social communication skills. Muscle weakness and atrophy was not obviously observed in proximal limbs, but with a slightly reduced power of 4 to 5/5 and atrophy in the distal areas. His sensory, cerebellar, and sphincter function were not affected. Neurological examination at the age of 15 years showed severe spasticity in the lower limbs, exaggerated reflexes, pes cavus and pyramidal tract signs. Cerebral and spinal MRI showed no significant changes in the brain and spinal cord. Nerve conduction tests and EMG were unremarkable. His older brother (II:2), aged 28 years old, experienced the similar symptoms at the age of 8 years. He developed gait abnormality and severe lower limb spasticity. He is currently ambulatory with wheelchair assistance. He was unable to interact normally with others. His cognitive function declines significantly, with a Mini-Mental State Exam(MMSE) score of 5/30. Examination showed severe spasticity and weakness in the lower limbs, severe muscle atrophy involving the whole body, exaggerated reflexes, pes cavus and severe pyramidal tract signs. Consanguinity was not reported in the family.
Characteristics of HSP patients with SPG11 mutations
The proband (II:2) from family 4 carrying the SPG11 mutation (p.Val1979Ter) initially experienced gait disturbance at the age of 12 years. She showed an ataxic gait and frequently fell down. Three years after these symptoms, she complained progressive lower limb weakness, dysarthria, dysphagia, intellectual disability and urinary dysfunction occasionally. She was wheelchair-bound by April 2018. Neurological examination showed spasticity in the lower limbs, atxia gait, increased muscle tone, hyperactive deep tendon reflexes, and positive Babinski signs. No muscular atrophy or fasciculation was observed. Brain MRI showed an abnormally thin corpus callosum. Nerve conduction tests and EMG revealed no evidence of neurological disturbance. No history of neurologic disease was found in this family. It was noteworthy that her parents were non-consanguineous.