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Late-onset spastic ataxia phenotype in a patient with a homozygous DDHD2 mutation

View Article: PubMed Central - PubMed

ABSTRACT

Autosomal recessive cerebellar ataxias and autosomal recessive hereditary spastic paraplegias (ARHSPs) are clinically and genetically heterogeneous neurological disorders. Herein we describe Japanese siblings with a midlife-onset, slowly progressive type of cerebellar ataxia and spastic paraplegia, without intellectual disability. Using whole exome sequencing, we identified a homozygous missense mutation in DDHD2, whose mutations were recently identified as the cause of early-onset ARHSP with intellectual disability. Brain MRI of the patient showed a thin corpus callosum. Cerebral proton magnetic resonance spectroscopy revealed an abnormal lipid peak in the basal ganglia, which has been reported as the hallmark of DDHD2-related ARHSP (SPG 54). The mutation caused a marked reduction of phospholipase A1 activity, supporting that this mutation is the cause of SPG54. Our cases indicate that the possibility of SPG54 should also be considered when patients show a combination of adult-onset spastic ataxia and a thin corpus callosum. Magnetic resonance spectroscopy may be helpful in the differential diagnosis of patients with spastic ataxia phenotype.

No MeSH data available.


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Familial pedigree, brain MRI and proton MRS of a patient with homozygous DDHD2 mutation.(A): Familial pedigree. * indicates members whose genomic DNA was available for this study (II-3 and II-6). Arrow indicates the proband (II-6). Homozygosity mapping and linkage analysis were performed using DNA from the proband and the unaffected sibling (II-3). (B): Brain MRI of II-6 at 69 years of age. Axial and sagittal sections of fluid-attenuated inversion recovery image are shown. Mild atrophy of the cerebellum and the thinness of the splenium of the corpus callosum (arrow) are observed. (C): Schematic presentation of DDHD2 and mutations. The thick arrow indicates the location of the mutation in the patient. (D): Proton MRS obtained from left thalamus, at a magnetic field of 3 Tesla (echo time 30 ms and 144 ms, respectively). Arrows indicate the pathologic lipid peak at 1.3 ppm. mI: myo-inositol, Cho: choline, Cr: creatine, NAA: N-acetylaspartate.
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f1: Familial pedigree, brain MRI and proton MRS of a patient with homozygous DDHD2 mutation.(A): Familial pedigree. * indicates members whose genomic DNA was available for this study (II-3 and II-6). Arrow indicates the proband (II-6). Homozygosity mapping and linkage analysis were performed using DNA from the proband and the unaffected sibling (II-3). (B): Brain MRI of II-6 at 69 years of age. Axial and sagittal sections of fluid-attenuated inversion recovery image are shown. Mild atrophy of the cerebellum and the thinness of the splenium of the corpus callosum (arrow) are observed. (C): Schematic presentation of DDHD2 and mutations. The thick arrow indicates the location of the mutation in the patient. (D): Proton MRS obtained from left thalamus, at a magnetic field of 3 Tesla (echo time 30 ms and 144 ms, respectively). Arrows indicate the pathologic lipid peak at 1.3 ppm. mI: myo-inositol, Cho: choline, Cr: creatine, NAA: N-acetylaspartate.

Mentions: Among the children of first-cousin parents, two sisters were affected (Figure 1A). The early developmental milestones of the proband (II-6) were normal. At the age of approximately 45 years old, she developed gait unsteadiness and dysarthria. She was 69 years old at the last examination, and could not stand without holding on to something. She had gaze-evoked horizontal nystagmus, dysarthria, extensor plantar reflexes, mild limb ataxia, moderate truncal ataxia, postural tremor in the head and upper extremities, decline of vibratory sense in the lower extremities, and urinary incontinence. Patellar tendon reflexes were increased, while Achilles tendon reflexes were absent. Cognitive impairments including callosal apraxia were not observed. Laboratory biochemistry results were normal, including serum liver enzymes, ammonia, thyroid hormones, copper, α-fetoprotein, vitamin E and very long-chain fatty acids. Serum antibody for Human T lymphotropic virus type 1 was negative. Galactocerebrosidase activity in leukocytes was normal. Blood amino-acid analysis and urinary organic acid analysis revealed no apparent deviance. A nerve conduction study disclosed a mild slowing of motor and sensory nerve conduction velocities (between 36.0 and 46.3 m/s) with reduced compound muscle action potentials. Brain magnetic resonance imaging revealed mild atrophy of the cerebellum, and a thinness of the splenium of the corpus callosum (Figure 1B). Neither atrophy nor cross sign were observed in the brainstem. The patient was negative for the genetic alterations associated with spinocerebellar ataxia (SCA)1, SCA2, SCA3, SCA6, SCA7, SCA12, SCA17 and dentatorubral pallidoluysian atrophy. Her elder sister (II-2) developed gait unsteadiness at the age of 38 years, and by age 55 years, could not walk independently. She showed saccadic eye pursuit, dysarthria, dysphagia, limb muscle weakness, extensor plantar reflexes, limb and truncal ataxia, and urinary incontinence. She died at 65, most likely because of hepatic encephalopathy with hyperammonemia (164 μg/dl, normal range 12–66 μg/dl), high serum lactate level (22.9 mg/dl, normal range 4–16 mg/dl) and pyruvate (2.3 mg/dl, normal range 0.3–0.9 mg/dl), and ketonuria. Serum copper and ceruloplasmin were normal. Eventually, a precise cause of hepatic insufficiency could not be detected. Another two siblings (II-4 and II-5) died at early ages (7 and 5 years, respectively) with unknown cause, but it is unlikely that the causes of their deaths were related to spastic ataxia, considering that they died in childhood. Homozygosity mapping and linkage analysis identified 11 candidate regions totalized to ~240 Mb, with the maximum LOD score equaling 1.32 (Table S2). When an adult patient exhibits both cerebellar ataxia and spasticity, the primary diagnostic considerations are autosomal recessive ataxia of Charlevoix-Saguenay, late-onset Friedreich ataxia or SPG76. However, none of the related genes, SACS, FRDA and SPG7, were located in the candidate regions of our patient. As a result of whole exome-sequencing of the proband, approximately 39.8 million paired-reads were mapped to the human reference genome. A coverage analysis revealed that 95.9% of the bases within the target regions were covered by 10 reads or more. In total, 37,553 variations, which were unregistered in dbSNP137 and registered as uncommon SNPs with minor allele frequency <1%, were detected. Among these, 2,986 variations (including 1,148 homozygous variants) were located in exons or splice sites (within 2 bp of the boundaries). Only the eight homozygous missense single nucleotide variations (SNVs) remained in the ~240-Mb candidate regions with the frequency <1% in exome data from 575 “in house” Japanese controls (Table 1). Sanger sequencing confirmed that all of these SNVs were homozygous in the proband and heterozygous in the unaffected sibling. Among the SNVs, the c.658G > T [p.Val220Phe] of DDHD2 (Figure 1C) was of interest, because mutations of DDHD1 and DDHD2, which code for members of the intracellular phospholipase A1 (PLA1), have recently been found to be the causative genes for ARHSPs (SPG28 and SPG54)78. Furthermore, only the SNVs of DDHD2 and FAM222A were consistently predicted to be disruptive in protein function when analyzed with multiple tools including Polyphen2, SIFT and Mutation Taster91011, while the predictions for the other six SNVs were benign or inconclusive (Table 1). Considering the allele frequency of FAM222A SNV in Japanese control exome data (4/575), it is unlikely that the SNV is the cause of extremely rare diseases. The potentially compound heterozygous SNVs detected in the proband are listed, indicating that none of the listed genes is likely to be the cause of the disease (Table S3). We further checked whether any other causative variations were present in known ARCA or ARHSP genes, which are listed in Table S1. We confirmed that no pathological homozygous or compound heterozygous SNVs were found in these genes. Patients with DDHD2 mutations have been reported to show very early-onset (before the age of 6 years) spastic paraplegia with intellectual disability (SPG54), occasionally associated with strabismus and/or hypoplasia of the optic nerve (Table 2)8121314. Brain MRIs of these patients showed a thin corpus callosum with periventricular white-matter hyperintensity8. As a unique finding, proton magnetic resonance spectroscopy (1H-MRS) revealed an abnormal lipid peak in the basal ganglia and thalamus area (Figure 1D). Considering the highly characteristic 1H-MRS findings, and the observations that the patient carried a novel homozygous p.Val220Phe of DDHD2 predicted as deleterious1516 (and not present in 575 Japanese controls by whole exome sequencing or 429 Japanese controls by Sanger sequencing), we thought that the DDHD2 mutation was the causative agent in this patient. Because most causative mutations of SPG54 were protein-truncating (Figure 1C), loss of DDHD2 function is plausible. We first checked intracellular distribution of p.Val220Phe and wild type (WT) in HEK293T cells, but found no difference (Figure S1). The result indicated that p.Val220Phe does not severely affect the conformation or stabilities of DDHD2. We then assessed an impact of the p.Val220Phe mutation by mapping the mutation on a 3D structure. Val220 is predicted to be involved in a hydrophobic core near the candidate catalytic site, suggesting that the p.Val220Phe mutation may impair lipase activity (Figure 2). Although SPG54-linked point mutations were reported (Table 2), whether the mutations affect enzymatic activity was not examined. We thus analyzed the PLA1 activity of the p.Val220Phe mutant as well as p.Trp103Arg and p.Asp660His mutants, both of which were reported to be linked to SPG5414. The results clearly demonstrated that the p.Val220Phe mutant as well as both the p.Trp103Arg and p.Asp660His mutants has a statistically significant reduction in their PLA1 activity (Figure 3A–C, lane 6–8). Notably, the p.Val220Phe mutant, but not other two mutants still retained a marginal PLA1 activity (Figure 3A–C, lane 6). We also co-transfected equal amounts of the WT- and each mutant-expressing plasmids, and then measured PLA1 activity. These conditions mimicked the heterozygous states of healthy carriers with both wild-type and mutant alleles. In these conditions, the PLA1 activity was not severely affected (Figure 3A–C, lane 3–5), indicating that all of the mutants did not have a dominant-negative effect on the WT DDHD2. These results strongly indicated that the p.Val220Phe mutation of DDHD2 was indeed a culprit mutation in this patient.


Late-onset spastic ataxia phenotype in a patient with a homozygous DDHD2 mutation
Familial pedigree, brain MRI and proton MRS of a patient with homozygous DDHD2 mutation.(A): Familial pedigree. * indicates members whose genomic DNA was available for this study (II-3 and II-6). Arrow indicates the proband (II-6). Homozygosity mapping and linkage analysis were performed using DNA from the proband and the unaffected sibling (II-3). (B): Brain MRI of II-6 at 69 years of age. Axial and sagittal sections of fluid-attenuated inversion recovery image are shown. Mild atrophy of the cerebellum and the thinness of the splenium of the corpus callosum (arrow) are observed. (C): Schematic presentation of DDHD2 and mutations. The thick arrow indicates the location of the mutation in the patient. (D): Proton MRS obtained from left thalamus, at a magnetic field of 3 Tesla (echo time 30 ms and 144 ms, respectively). Arrows indicate the pathologic lipid peak at 1.3 ppm. mI: myo-inositol, Cho: choline, Cr: creatine, NAA: N-acetylaspartate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384088&req=5

f1: Familial pedigree, brain MRI and proton MRS of a patient with homozygous DDHD2 mutation.(A): Familial pedigree. * indicates members whose genomic DNA was available for this study (II-3 and II-6). Arrow indicates the proband (II-6). Homozygosity mapping and linkage analysis were performed using DNA from the proband and the unaffected sibling (II-3). (B): Brain MRI of II-6 at 69 years of age. Axial and sagittal sections of fluid-attenuated inversion recovery image are shown. Mild atrophy of the cerebellum and the thinness of the splenium of the corpus callosum (arrow) are observed. (C): Schematic presentation of DDHD2 and mutations. The thick arrow indicates the location of the mutation in the patient. (D): Proton MRS obtained from left thalamus, at a magnetic field of 3 Tesla (echo time 30 ms and 144 ms, respectively). Arrows indicate the pathologic lipid peak at 1.3 ppm. mI: myo-inositol, Cho: choline, Cr: creatine, NAA: N-acetylaspartate.
Mentions: Among the children of first-cousin parents, two sisters were affected (Figure 1A). The early developmental milestones of the proband (II-6) were normal. At the age of approximately 45 years old, she developed gait unsteadiness and dysarthria. She was 69 years old at the last examination, and could not stand without holding on to something. She had gaze-evoked horizontal nystagmus, dysarthria, extensor plantar reflexes, mild limb ataxia, moderate truncal ataxia, postural tremor in the head and upper extremities, decline of vibratory sense in the lower extremities, and urinary incontinence. Patellar tendon reflexes were increased, while Achilles tendon reflexes were absent. Cognitive impairments including callosal apraxia were not observed. Laboratory biochemistry results were normal, including serum liver enzymes, ammonia, thyroid hormones, copper, α-fetoprotein, vitamin E and very long-chain fatty acids. Serum antibody for Human T lymphotropic virus type 1 was negative. Galactocerebrosidase activity in leukocytes was normal. Blood amino-acid analysis and urinary organic acid analysis revealed no apparent deviance. A nerve conduction study disclosed a mild slowing of motor and sensory nerve conduction velocities (between 36.0 and 46.3 m/s) with reduced compound muscle action potentials. Brain magnetic resonance imaging revealed mild atrophy of the cerebellum, and a thinness of the splenium of the corpus callosum (Figure 1B). Neither atrophy nor cross sign were observed in the brainstem. The patient was negative for the genetic alterations associated with spinocerebellar ataxia (SCA)1, SCA2, SCA3, SCA6, SCA7, SCA12, SCA17 and dentatorubral pallidoluysian atrophy. Her elder sister (II-2) developed gait unsteadiness at the age of 38 years, and by age 55 years, could not walk independently. She showed saccadic eye pursuit, dysarthria, dysphagia, limb muscle weakness, extensor plantar reflexes, limb and truncal ataxia, and urinary incontinence. She died at 65, most likely because of hepatic encephalopathy with hyperammonemia (164 μg/dl, normal range 12–66 μg/dl), high serum lactate level (22.9 mg/dl, normal range 4–16 mg/dl) and pyruvate (2.3 mg/dl, normal range 0.3–0.9 mg/dl), and ketonuria. Serum copper and ceruloplasmin were normal. Eventually, a precise cause of hepatic insufficiency could not be detected. Another two siblings (II-4 and II-5) died at early ages (7 and 5 years, respectively) with unknown cause, but it is unlikely that the causes of their deaths were related to spastic ataxia, considering that they died in childhood. Homozygosity mapping and linkage analysis identified 11 candidate regions totalized to ~240 Mb, with the maximum LOD score equaling 1.32 (Table S2). When an adult patient exhibits both cerebellar ataxia and spasticity, the primary diagnostic considerations are autosomal recessive ataxia of Charlevoix-Saguenay, late-onset Friedreich ataxia or SPG76. However, none of the related genes, SACS, FRDA and SPG7, were located in the candidate regions of our patient. As a result of whole exome-sequencing of the proband, approximately 39.8 million paired-reads were mapped to the human reference genome. A coverage analysis revealed that 95.9% of the bases within the target regions were covered by 10 reads or more. In total, 37,553 variations, which were unregistered in dbSNP137 and registered as uncommon SNPs with minor allele frequency <1%, were detected. Among these, 2,986 variations (including 1,148 homozygous variants) were located in exons or splice sites (within 2 bp of the boundaries). Only the eight homozygous missense single nucleotide variations (SNVs) remained in the ~240-Mb candidate regions with the frequency <1% in exome data from 575 “in house” Japanese controls (Table 1). Sanger sequencing confirmed that all of these SNVs were homozygous in the proband and heterozygous in the unaffected sibling. Among the SNVs, the c.658G > T [p.Val220Phe] of DDHD2 (Figure 1C) was of interest, because mutations of DDHD1 and DDHD2, which code for members of the intracellular phospholipase A1 (PLA1), have recently been found to be the causative genes for ARHSPs (SPG28 and SPG54)78. Furthermore, only the SNVs of DDHD2 and FAM222A were consistently predicted to be disruptive in protein function when analyzed with multiple tools including Polyphen2, SIFT and Mutation Taster91011, while the predictions for the other six SNVs were benign or inconclusive (Table 1). Considering the allele frequency of FAM222A SNV in Japanese control exome data (4/575), it is unlikely that the SNV is the cause of extremely rare diseases. The potentially compound heterozygous SNVs detected in the proband are listed, indicating that none of the listed genes is likely to be the cause of the disease (Table S3). We further checked whether any other causative variations were present in known ARCA or ARHSP genes, which are listed in Table S1. We confirmed that no pathological homozygous or compound heterozygous SNVs were found in these genes. Patients with DDHD2 mutations have been reported to show very early-onset (before the age of 6 years) spastic paraplegia with intellectual disability (SPG54), occasionally associated with strabismus and/or hypoplasia of the optic nerve (Table 2)8121314. Brain MRIs of these patients showed a thin corpus callosum with periventricular white-matter hyperintensity8. As a unique finding, proton magnetic resonance spectroscopy (1H-MRS) revealed an abnormal lipid peak in the basal ganglia and thalamus area (Figure 1D). Considering the highly characteristic 1H-MRS findings, and the observations that the patient carried a novel homozygous p.Val220Phe of DDHD2 predicted as deleterious1516 (and not present in 575 Japanese controls by whole exome sequencing or 429 Japanese controls by Sanger sequencing), we thought that the DDHD2 mutation was the causative agent in this patient. Because most causative mutations of SPG54 were protein-truncating (Figure 1C), loss of DDHD2 function is plausible. We first checked intracellular distribution of p.Val220Phe and wild type (WT) in HEK293T cells, but found no difference (Figure S1). The result indicated that p.Val220Phe does not severely affect the conformation or stabilities of DDHD2. We then assessed an impact of the p.Val220Phe mutation by mapping the mutation on a 3D structure. Val220 is predicted to be involved in a hydrophobic core near the candidate catalytic site, suggesting that the p.Val220Phe mutation may impair lipase activity (Figure 2). Although SPG54-linked point mutations were reported (Table 2), whether the mutations affect enzymatic activity was not examined. We thus analyzed the PLA1 activity of the p.Val220Phe mutant as well as p.Trp103Arg and p.Asp660His mutants, both of which were reported to be linked to SPG5414. The results clearly demonstrated that the p.Val220Phe mutant as well as both the p.Trp103Arg and p.Asp660His mutants has a statistically significant reduction in their PLA1 activity (Figure 3A–C, lane 6–8). Notably, the p.Val220Phe mutant, but not other two mutants still retained a marginal PLA1 activity (Figure 3A–C, lane 6). We also co-transfected equal amounts of the WT- and each mutant-expressing plasmids, and then measured PLA1 activity. These conditions mimicked the heterozygous states of healthy carriers with both wild-type and mutant alleles. In these conditions, the PLA1 activity was not severely affected (Figure 3A–C, lane 3–5), indicating that all of the mutants did not have a dominant-negative effect on the WT DDHD2. These results strongly indicated that the p.Val220Phe mutation of DDHD2 was indeed a culprit mutation in this patient.

View Article: PubMed Central - PubMed

ABSTRACT

Autosomal recessive cerebellar ataxias and autosomal recessive hereditary spastic paraplegias (ARHSPs) are clinically and genetically heterogeneous neurological disorders. Herein we describe Japanese siblings with a midlife-onset, slowly progressive type of cerebellar ataxia and spastic paraplegia, without intellectual disability. Using whole exome sequencing, we identified a homozygous missense mutation in DDHD2, whose mutations were recently identified as the cause of early-onset ARHSP with intellectual disability. Brain MRI of the patient showed a thin corpus callosum. Cerebral proton magnetic resonance spectroscopy revealed an abnormal lipid peak in the basal ganglia, which has been reported as the hallmark of DDHD2-related ARHSP (SPG 54). The mutation caused a marked reduction of phospholipase A1 activity, supporting that this mutation is the cause of SPG54. Our cases indicate that the possibility of SPG54 should also be considered when patients show a combination of adult-onset spastic ataxia and a thin corpus callosum. Magnetic resonance spectroscopy may be helpful in the differential diagnosis of patients with spastic ataxia phenotype.

No MeSH data available.


Related in: MedlinePlus