- Email: [email protected]
Novel biallelic FA2H mutations in a Japanese boy with fatty acid hydroxylase-associated neurodegeneration
Brain & Development xxx (2019) xxx–xxx www.elsevier.com/locate/braindev
Case Report
Novel biallelic FA2H mutations in a Japanese boy with fatty acid hydroxylase-associated neurodegeneration Masahiro Kawaguchi a, Takayuki Sassa b, Hiroyuki Kidokoro a,⇑, Tomohiko Nakata a, Kohji Kato a,c, Hideki Muramatsu a, Yusuke Okuno d, Hiroyuki Yamamoto a, Tadashi Kaname e, Akio Kihara b, Jun Natsume a a Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, Japan Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan c Department of Pediatrics and Neonatology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan d Medical Genomics Center, Nagoya University Hospital, Nagoya, Japan e Department of Genome Medicine, National Center for Child Health and Development, Tokyo, Japan b
Received 26 August 2019; received in revised form 14 November 2019; accepted 21 November 2019
Abstract FA2H encodes fatty acid 2-hydroxylase, which plays a significant role in maintaining the neuronal myelin sheath. Previous reports have revealed that a FA2H mutation leads to spastic paraplegia, leukodystrophy, and neurodegeneration with brain iron accumulation, collectively referred to as fatty acid hydroxylase-associated neurodegeneration (FAHN). The disease severity of FAHN varies among individual patients and may be explained by the enzyme activity of FA2H mutant proteins. Here we report a 10-year-old Japanese boy with FAHN having novel heterozygous mutations in FA2H. The patient presented with a spastic gait since the age of 5 years and was unable to walk without a cane by the time he was 8 years old. Brain MRI demonstrated a partial thinning of the corpus callosum, slight reduction of cerebellar volume, and posterior dominant periventricular leukodystrophy. Whole exome sequencing revealed two novel missense mutations in FA2H with compound heterozygous inheritance (NM_024306, p.Val149Leu, and p.His260Gln mutations). The enzyme activities of the p.Val149Leu and p.His260Gln variants were 60%–80% and almost 0%, respectively. Our cell-based enzyme assay demonstrated partial functionality for one of the variants, indicating a milder phenotype. However, considered along with previous reports, there was no definite relationship between the disease severity and residual enzyme activity measured using a similar method. Further research is needed to precisely predict the phenotypic severity of this disorder. Ó 2019 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.
Keywords: Hereditary spastic paraplegia; Spastic paraplegia type 35; Fatty acid 2-hydroxylase; Fatty acid hydroxylase-associated neurodegeneration
1. Introduction
⇑ Corresponding author at: Department of Pediatrics, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showaku, Nagoya, Aichi, Japan. E-mail address: [email protected] (H. Kidokoro).
Hereditary spastic paraplegias (HSPs) are a heterogeneous group of neurodegenerative disorders characterized by progressive lower limb spasticity that is often accompanied by other neurological findings, such as ataxia, extrapyramidal disturbance, orthopedic abnor-
https://doi.org/10.1016/j.braindev.2019.11.006 0387-7604/Ó 2019 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Kawaguchi M et al. Novel biallelic FA2H mutations in a Japanese boy with fatty acid hydroxylase-associated neurodegeneration. Brain Dev (2019), https://doi.org/10.1016/j.braindev.2019.11.006
2
M. Kawaguchi et al. / Brain & Development xxx (2019) xxx–xxx
mality, and seizures. Mutations in more than 80 genes have been reported to cause HSPs [1]. Spastic paraplegia type 35 (SPG35) is an autosomal recessive HSP caused by compound heterozygous or homozygous mutations in the fatty acid 2-hydroxylase (FA2H, MIM 611026) gene. FA2H is included in the synthesis of 2-hydroxy fatty acid-containing galactolipids (sphingolipid, galactosylceramide, and sulfatide), which are major components of the myelin sheath. A previous study on Fa2h-deficient mice reported that structurally and functionally normal myelin can be formed in the absence of 2-hydroxylated sphingolipids; however, its long-term maintenance is impaired [2], suggesting that the FA2H gene plays an important role in maintaining the neuronal myelin sheath. Recently, FA2H mutation in humans has been shown to be associated with not only SPG35 but also leukodystrophy and neurodegeneration with brain iron accumulation. These phenotypic spectrums are collectively referred to as fatty acid hydroxylase-associated neurodegeneration (FAHN). To date, approximately 50 variants of FA2H have been reported as pathogenic [3]. However, only limited examples of missense mutations or deletions have been shown to reduce or eliminate the enzyme activity of FA2H [4,5]. Here we report a Japanese boy with FAHN who had novel heterozygous mutations in FA2H that demonstrated decreased, or nearly lost, enzyme activity. We compared the clinical, neuroimaging, and enzyme activity with those previously reported for FAHN.
of the corpus callosum and slight reduction in the cerebellar volume. Spine MRI revealed no abnormality. Nerve conduction study of both motor and sensory nerves was normal. Transcranial magnetic stimulation revealed a delay in central motor conduction time in the right upper limb and no evoked potentials in the lower limbs. Based on these findings, he was clinically diagnosed with HSP. Despite rehabilitation by physical therapists, his spastic paraplegia was progressive, and he was unable to walk without a cane by the age of 8 years. A followup brain MRI revealed posterior dominant high signal intensity in the periventricular white matter on T2weighted and FLAIR images (Fig. 1). At the age of 9, he underwent tenotomies of the hip, knee, and ankle joints. His full-scale IQ was 100 (Wechsler Intelligence Scale for Children, fourth edition). He had no behavioral abnormalities, including autistic spectrum disorder or attention-deficit hyperactivity disorder. He also had no cerebellar signs or epileptic seizures. 2.1. Genetic analysis After obtaining written informed consent from the parents of the patient, whole exome sequencing was carried out to determine the genetic cause of the disease in the parents–patient trio, as previously described [6]. The examination protocols were approved by the Central
2. Case report A 10-year-old Japanese boy presented with a history of gait abnormality since the age of 5 years. He was the second child born to healthy nonconsanguineous parents. There was no family history of neurological disease, except his elder brother’s mild color blindness. He was born at the gestational age of 38 weeks, and his birth weight was 3116 g (+0.95 SD). He reached normal developmental milestones during infancy and was able to walk at the age of 13 months. He developed a progressively noticeable gait disturbance at the age of 5 years. A local doctor identified his equinus foot, and he was referred to our hospital. Though he presented with a spastic gait, he was able to walk without support. A neurological examination revealed tendon hyperreflexia, ankle clonus, and a positive Babinski sign in the bilateral lower limbs. However, muscle weakness, sensory disturbance of the lower limbs, and abnormal signs in the upper limbs were not identified. On physical examination, he had no dysmorphic features such as the recently reported bristly hair [3]. No abnormality was found in his blood or cerebrospinal fluid. Brain MRI showed a partial thinning
Fig. 1. Brain MRI at the age of 8 years. Axial FLAIR images (A and B) demonstrate high signal intensity in the periventricular white matter (arrows). A T2-weighted coronal image (C) shows mild cerebellar atrophy, whereas a T1-weighted sagittal image (D) shows partial thinning in the body of the corpus callosum (arrow) and mild volume reduction in the cerebellar vermis.
Please cite this article in press as: Kawaguchi M et al. Novel biallelic FA2H mutations in a Japanese boy with fatty acid hydroxylase-associated neurodegeneration. Brain Dev (2019), https://doi.org/10.1016/j.braindev.2019.11.006
M. Kawaguchi et al. / Brain & Development xxx (2019) xxx–xxx
Ethics Committee of Tohoku University School of Medicine Hospital. Two novel missense mutations in FA2H with compound heterozygous inheritance were identified in the patient. No other variants predicted to be pathogenic for this patient were found. Father-derived c.445G > T (p.Val149Leu) mutation and motherderived c.780C > A (p.His260Gln) mutation in FA2H were detected (NM_024306). However, these mutations were not listed in public SNP databases, including ExAC (http://exac.broadinstitute.org/) and the Human Genetic Variation Database (http://www.hgvd.genome. med.kyoto-u.ac.jp/). 2.2. Functional analysis 3FLAG-tagged wild type FA2H, p.Val149Leu, and p.His260Gln variants were exogenously expressed in CHO-K1 cells, and their enzyme activities were measured using the method mentioned previously [7]. Immunoblot analysis of the total cell lysates with anti-FLAG antibody demonstrated that the expression levels of p. Val149Leu mutant were comparable to those of wild type FA2H protein, whereas those of p.His260Gln mutant were slightly lower (Fig. 2A). Compared with wild type FA2H protein, which exhibited high fatty acid 2-hydroxylase activities toward C16:0 and C24:0 fatty acids, p.Val149Leu mutant exhibited the activities reduced by 58% and 77% toward C16:0 and C24:0 fatty acids, respectively (Fig. 2B and C). The p.His260Glu mutant essentially exhibited no activities; it was 0.3% toward wild type and below the detection limit toward C16:0 and C24:0 fatty acids (Fig. 2B and C). 3. Discussion This is the first case report of a Japanese patient with FAHN having the characteristic features of spastic
3
paraplegia and white matter abnormality in brain MRI. Our cell-based enzyme assay demonstrated significantly decreased and almost lost enzyme activity of novel alleles, supporting that these alleles are pathogenic. So far, approximately 70 individuals with FAHN have been reported in the literature. Although it occurs during adolescence or adulthood in a few cases, the age of onset is typically during childhood, ranging mostly between the ages of 3 and 5 years. In addition to spasticity, approximately 90% of patients have intellectual decline and 30% have epileptic seizures [3,8]. There is no curative treatment for FAHN; only supportive treatment is available. The severity of FAHN phenotypes varies among affected individuals. This may be due to the type and location of the FA2H mutations. However, a recent large cohort study on FAHN patients showed no apparent correlations between genotype and phenotype [3]. Therefore, a functional analysis is expected to estimate the actual influence of each missense mutation. In a similar cell-based enzyme assay, enzyme activity was examined in four variants in the FA2H of one family and four patients from two reports (Table 1). Dick et al. investigated the enzyme activity of p.Arg235Cys and p.Arg53_Ile58del mutations in FA2H from two patients with FAHN [4]. They reported that the enzyme activities of p.Arg235Cys and p.Arg53-Ile58del mutants were 60%–80% and almost 0%, respectively. Additionally, Liao et al. reported that the enzyme activities of p.Leu130Phe and p.Leu77Arg mutants were 52% and 43%, respectively [5]. In this patient with typical but milder clinical features, the enzyme activities of p. Val149Leu and p.His260Gln variants were approximately 60%–80% and almost 0%, respectively. His260 of FA2H was located in a functional (fatty acid hydroxylase) domain. A previous study revealed that the residue of Scs7p, a yeast homolog of FA2H corresponding
Fig. 2. Measurement of FA2H activity. To measure FA2H activity, CHO-K1 cells expressing wild type (WT) and p.Val149Leu (V149L) and p. His260Gln (H260Q) mutants were incubated with 0.25 mM deuterium-labeled sphingosine at 37 °C for 4 h. (A) Lysates (15 mg protein) prepared from the cells were separated by SDS-PAGE, followed by immunoblotting with anti-FLAG antibodies. (B, C) Lipids were extracted from the cells, and the amount of deuterium-labeled ceramide, which comprises deuterium-labeled sphingosine and 2-hydroxy or nonhydroxy fatty acid, was quantified by liquid chromatography–tandem mass spectrometry. FA2H activity was defined as the ratio of 2-hydroxyceramide/nonhydroxyceramide, and the activities toward C16:0 (B) and C24:0 (C) Fatty acids are shown. Values are means ± SD (n = 3). Statistically significant differences compared with WT are indicated. **P < 0.01 (Dunnett’s test). 2-OH, 2-hydroxyceramide; non-OH, nonhydroxyceramide.
Please cite this article in press as: Kawaguchi M et al. Novel biallelic FA2H mutations in a Japanese boy with fatty acid hydroxylase-associated neurodegeneration. Brain Dev (2019), https://doi.org/10.1016/j.braindev.2019.11.006
Dick et al. [4] Family 1 (7 individuals) Age at reported (years) Sex
Clinical features Age at onset (years) Initial symptom Neurological symptoms
Cognitive decline Epilepsy Motor outcome
Liao et al. [5]
Present study
Patient 2
Patient 3
Patient 4 (sibling of patient 3)
Patient 5
Patient 6
8–25
31
26
31
17
10
males (n = 4), females (n = 3) Omani homozygous p.Arg235Cys 60–80%
female
male
female
male
male
Pakistani homozygous p.Arg53_Ile58del almost 0%
Chinese compound heterozygous p.Leu130Phe c.506 + 6C > G 52% almost 0%
Chinese compound heterozygous p.Leu130Phe c.506 + 6C > G 52% almost 0%
Chinese homozygous p.Leu77Arg 43%
Japanese compound heterozygous p.Val149Leu p.His260Gln 60–80% almost 0%
8–25
4
17
10
4
5
gait disturbance dysarthria (n = 5), nystagmus (n = 2) mild (n = 5)
gait disturbance
weakness of lower limbs
weakness of lower limbs
lower limb spasticity
gait disturbance
anarthria, hypertonia of upper limbs, ophthalmoplegia
mild dysarthria, mild dysphagia, urinary dysfunction
speech deficiency
pyramidal tract signs, cerebellar signs, abnormal ocular motility
mild dysmetria
mild
none
mild
mild
none
present
absent
ND
present
absent
wheel chair (10 years old)
autonomous ambulation (26 years old)
wheel chair (30 years old)
ND
wheel chair (10 years old)
present
present
present
ND
ND
present
ND
present
present
ND
ND
present
ND
present
absent
ND
ND
present
ND
ND
absent
ND
ND
absent
present (n = 2) autonomous ambulation (n = 3), wheel chair (n = 4)
MRI findings T2 hypointensity in the white matter Cerebellar atrophy Thin corpus callosum Iron deposits ND, not described.
M. Kawaguchi et al. / Brain & Development xxx (2019) xxx–xxx
Race Genotype Mutation Enzyme activity
4
Please cite this article in press as: Kawaguchi M et al. Novel biallelic FA2H mutations in a Japanese boy with fatty acid hydroxylase-associated neurodegeneration. Brain Dev (2019), https://doi.org/10.1016/j.braindev.2019.11.006
Table 1 Characteristics of patients whose enzyme activity was analyzed.
M. Kawaguchi et al. / Brain & Development xxx (2019) xxx–xxx
to His260 of FA2H in humans, was essential for enzyme activity [9]. This is consistent with our results that showed an almost absent enzyme activity of p. His260Gln. We compared the association among the clinical, neuroimaging, and enzyme activity in FAHN patients listed in Table 1. Enzyme activity greatly differed from approximately 0% to 60%–80%. However, the clinical features are generally similar, although the onset in patient 3 was exceptionally late. Thus, disease onset and its severity may not be explained only by residual enzyme activity with this cell-based enzyme assay. We cannot exclude the possibility of these being additional consequences of the FA2H mutations (e.g., alterations in transcript levels and processing or aberrant protein translocation). Kruer et al. proposed that the p. Arg154Cys mutation of FA2H reduces stability of the mRNA or translated protein because, in a case of p. Arg154Cys mutation, protein abundance measured by western blot decreased compared with a wild type whereas enzyme activity measured by gas chromatography–mass spectrometry demonstrated no significant difference [10]. This patient had a milder phenotype, which may be supported by our result of no or slightly decreased protein levels of each variant. 4. Conclusion This is the first report of a Japanese patient with FAHN having novel biallelic FA2H mutations. Our enzyme analysis demonstrated partial functionality of one of the variants, indicating a milder phenotype. However, disease severity in patients with FAHN may not be explained by residual enzyme activity measured with this method alone. Further research is needed to accurately predict the phenotypic severity of this disease and, more importantly, to develop a therapeutic intervention.
5
by the Initiative on Rare and Undiagnosed Diseases in Pediatrics from the Japanese Agency for Medical Research and Development. References [1] Faber I, Pereira ER, Martinez ARM, Franc¸a Jr M, Teive HAG. Hereditary spastic paraplegia from 1880 to 2017: an historical review. Arq Neuropsiquiatr 2017;75:813–8. [2] Zo¨ller I, Meixner M, Hartmann D, Bu¨ssow H, Meyer R, Gieselmann V, et al. Absence of 2-hydroxylated sphingolipids is compatible with normal neural development but causes late-onset axon and myelin sheath degeneration. J Neurosci 2008;28:9741–54. [3] Rattay TW, Lindig T, Baets J, Smets K, Deconinck T, So¨hn AS, et al. FAHN/SPG35: a narrow phenotypic spectrum across disease classifications. Brain 2019;142:1561–72. [4] Dick KJ, Eckhardt M, Paisa´n-Ruiz C, Alshehhi AA, Proukakis C, Sibtain NA, et al. Mutation of FA2H underlies a complicated form of hereditary spastic paraplegia (SPG35). Hum Mutat 2010;31:1251–60. [5] Liao X, Luo Y, Zhan Z, Du J, Hu Z, Wang J, et al. SPG35 contributes to the second common subtype of AR-HSP in China: frequency analysis and functional characterization of FA2H gene mutations. Clin Genet 2015;87:85–9. [6] Sasaki H, Yanagi K, Ugi S, Kobayashi K, Ohkubo K, Tajiri Y, et al. Definitive diagnosis of mandibular hypoplasia, deafness, progeroid features and lipodystrophy (MDPL) syndrome caused by a recurrent de novo mutation in the POLD1 gene. Endocr J 2018;65:227–38. [7] Kanetake T, Sassa T, Nojiri K, Sawai M, Hattori S, Miyakawa T, et al. Neural symptoms in a gene knockout mouse model of Sjo¨gren-Larsson syndrome are associated with a decrease in 2hydroxygalactosylceramide. FASEB J 2019;33:928–41. [8] Mari F, Berti B, Romano A, Baldacci J, Rizzi R, Grazia Alessandrı` M, et al. Clinical and neuroimaging features of autosomal recessive spastic paraplegia 35 (SPG35): case reports, new mutations, and brief literature review. Neurogenetics 2018;19:123–30. [9] Zhu G, Koszelak-Rosenblum M, Connelly SM, Dumont ME, Malkowski MG. The crystal structure of an integral membrane fatty acid a-hydroxylase. J Biol Chem 2015;50:29820–33. [10] Kruer MC, Paisa´n-Ruiz C, Boddaert N, Yoon MY, Hama H, Gregory A, et al. Defective FA2H leads to a novel form of neurodegeneration with brain iron accumulation (NBIA). Ann Neurol 2010;68:611–8.
Acknowledgments Whole exome sequencing was performed through TOKAI-IRUD-P. This study was partially supported
Please cite this article in press as: Kawaguchi M et al. Novel biallelic FA2H mutations in a Japanese boy with fatty acid hydroxylase-associated neurodegeneration. Brain Dev (2019), https://doi.org/10.1016/j.braindev.2019.11.006
Case Report
Novel biallelic FA2H mutations in a Japanese boy with fatty acid hydroxylase-associated neurodegeneration Masahiro Kawaguchi a, Takayuki Sassa b, Hiroyuki Kidokoro a,⇑, Tomohiko Nakata a, Kohji Kato a,c, Hideki Muramatsu a, Yusuke Okuno d, Hiroyuki Yamamoto a, Tadashi Kaname e, Akio Kihara b, Jun Natsume a a Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, Japan Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan c Department of Pediatrics and Neonatology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan d Medical Genomics Center, Nagoya University Hospital, Nagoya, Japan e Department of Genome Medicine, National Center for Child Health and Development, Tokyo, Japan b
Received 26 August 2019; received in revised form 14 November 2019; accepted 21 November 2019
Abstract FA2H encodes fatty acid 2-hydroxylase, which plays a significant role in maintaining the neuronal myelin sheath. Previous reports have revealed that a FA2H mutation leads to spastic paraplegia, leukodystrophy, and neurodegeneration with brain iron accumulation, collectively referred to as fatty acid hydroxylase-associated neurodegeneration (FAHN). The disease severity of FAHN varies among individual patients and may be explained by the enzyme activity of FA2H mutant proteins. Here we report a 10-year-old Japanese boy with FAHN having novel heterozygous mutations in FA2H. The patient presented with a spastic gait since the age of 5 years and was unable to walk without a cane by the time he was 8 years old. Brain MRI demonstrated a partial thinning of the corpus callosum, slight reduction of cerebellar volume, and posterior dominant periventricular leukodystrophy. Whole exome sequencing revealed two novel missense mutations in FA2H with compound heterozygous inheritance (NM_024306, p.Val149Leu, and p.His260Gln mutations). The enzyme activities of the p.Val149Leu and p.His260Gln variants were 60%–80% and almost 0%, respectively. Our cell-based enzyme assay demonstrated partial functionality for one of the variants, indicating a milder phenotype. However, considered along with previous reports, there was no definite relationship between the disease severity and residual enzyme activity measured using a similar method. Further research is needed to precisely predict the phenotypic severity of this disorder. Ó 2019 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.
Keywords: Hereditary spastic paraplegia; Spastic paraplegia type 35; Fatty acid 2-hydroxylase; Fatty acid hydroxylase-associated neurodegeneration
1. Introduction
⇑ Corresponding author at: Department of Pediatrics, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showaku, Nagoya, Aichi, Japan. E-mail address: [email protected] (H. Kidokoro).
Hereditary spastic paraplegias (HSPs) are a heterogeneous group of neurodegenerative disorders characterized by progressive lower limb spasticity that is often accompanied by other neurological findings, such as ataxia, extrapyramidal disturbance, orthopedic abnor-
https://doi.org/10.1016/j.braindev.2019.11.006 0387-7604/Ó 2019 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Kawaguchi M et al. Novel biallelic FA2H mutations in a Japanese boy with fatty acid hydroxylase-associated neurodegeneration. Brain Dev (2019), https://doi.org/10.1016/j.braindev.2019.11.006
2
M. Kawaguchi et al. / Brain & Development xxx (2019) xxx–xxx
mality, and seizures. Mutations in more than 80 genes have been reported to cause HSPs [1]. Spastic paraplegia type 35 (SPG35) is an autosomal recessive HSP caused by compound heterozygous or homozygous mutations in the fatty acid 2-hydroxylase (FA2H, MIM 611026) gene. FA2H is included in the synthesis of 2-hydroxy fatty acid-containing galactolipids (sphingolipid, galactosylceramide, and sulfatide), which are major components of the myelin sheath. A previous study on Fa2h-deficient mice reported that structurally and functionally normal myelin can be formed in the absence of 2-hydroxylated sphingolipids; however, its long-term maintenance is impaired [2], suggesting that the FA2H gene plays an important role in maintaining the neuronal myelin sheath. Recently, FA2H mutation in humans has been shown to be associated with not only SPG35 but also leukodystrophy and neurodegeneration with brain iron accumulation. These phenotypic spectrums are collectively referred to as fatty acid hydroxylase-associated neurodegeneration (FAHN). To date, approximately 50 variants of FA2H have been reported as pathogenic [3]. However, only limited examples of missense mutations or deletions have been shown to reduce or eliminate the enzyme activity of FA2H [4,5]. Here we report a Japanese boy with FAHN who had novel heterozygous mutations in FA2H that demonstrated decreased, or nearly lost, enzyme activity. We compared the clinical, neuroimaging, and enzyme activity with those previously reported for FAHN.
of the corpus callosum and slight reduction in the cerebellar volume. Spine MRI revealed no abnormality. Nerve conduction study of both motor and sensory nerves was normal. Transcranial magnetic stimulation revealed a delay in central motor conduction time in the right upper limb and no evoked potentials in the lower limbs. Based on these findings, he was clinically diagnosed with HSP. Despite rehabilitation by physical therapists, his spastic paraplegia was progressive, and he was unable to walk without a cane by the age of 8 years. A followup brain MRI revealed posterior dominant high signal intensity in the periventricular white matter on T2weighted and FLAIR images (Fig. 1). At the age of 9, he underwent tenotomies of the hip, knee, and ankle joints. His full-scale IQ was 100 (Wechsler Intelligence Scale for Children, fourth edition). He had no behavioral abnormalities, including autistic spectrum disorder or attention-deficit hyperactivity disorder. He also had no cerebellar signs or epileptic seizures. 2.1. Genetic analysis After obtaining written informed consent from the parents of the patient, whole exome sequencing was carried out to determine the genetic cause of the disease in the parents–patient trio, as previously described [6]. The examination protocols were approved by the Central
2. Case report A 10-year-old Japanese boy presented with a history of gait abnormality since the age of 5 years. He was the second child born to healthy nonconsanguineous parents. There was no family history of neurological disease, except his elder brother’s mild color blindness. He was born at the gestational age of 38 weeks, and his birth weight was 3116 g (+0.95 SD). He reached normal developmental milestones during infancy and was able to walk at the age of 13 months. He developed a progressively noticeable gait disturbance at the age of 5 years. A local doctor identified his equinus foot, and he was referred to our hospital. Though he presented with a spastic gait, he was able to walk without support. A neurological examination revealed tendon hyperreflexia, ankle clonus, and a positive Babinski sign in the bilateral lower limbs. However, muscle weakness, sensory disturbance of the lower limbs, and abnormal signs in the upper limbs were not identified. On physical examination, he had no dysmorphic features such as the recently reported bristly hair [3]. No abnormality was found in his blood or cerebrospinal fluid. Brain MRI showed a partial thinning
Fig. 1. Brain MRI at the age of 8 years. Axial FLAIR images (A and B) demonstrate high signal intensity in the periventricular white matter (arrows). A T2-weighted coronal image (C) shows mild cerebellar atrophy, whereas a T1-weighted sagittal image (D) shows partial thinning in the body of the corpus callosum (arrow) and mild volume reduction in the cerebellar vermis.
Please cite this article in press as: Kawaguchi M et al. Novel biallelic FA2H mutations in a Japanese boy with fatty acid hydroxylase-associated neurodegeneration. Brain Dev (2019), https://doi.org/10.1016/j.braindev.2019.11.006
M. Kawaguchi et al. / Brain & Development xxx (2019) xxx–xxx
Ethics Committee of Tohoku University School of Medicine Hospital. Two novel missense mutations in FA2H with compound heterozygous inheritance were identified in the patient. No other variants predicted to be pathogenic for this patient were found. Father-derived c.445G > T (p.Val149Leu) mutation and motherderived c.780C > A (p.His260Gln) mutation in FA2H were detected (NM_024306). However, these mutations were not listed in public SNP databases, including ExAC (http://exac.broadinstitute.org/) and the Human Genetic Variation Database (http://www.hgvd.genome. med.kyoto-u.ac.jp/). 2.2. Functional analysis 3FLAG-tagged wild type FA2H, p.Val149Leu, and p.His260Gln variants were exogenously expressed in CHO-K1 cells, and their enzyme activities were measured using the method mentioned previously [7]. Immunoblot analysis of the total cell lysates with anti-FLAG antibody demonstrated that the expression levels of p. Val149Leu mutant were comparable to those of wild type FA2H protein, whereas those of p.His260Gln mutant were slightly lower (Fig. 2A). Compared with wild type FA2H protein, which exhibited high fatty acid 2-hydroxylase activities toward C16:0 and C24:0 fatty acids, p.Val149Leu mutant exhibited the activities reduced by 58% and 77% toward C16:0 and C24:0 fatty acids, respectively (Fig. 2B and C). The p.His260Glu mutant essentially exhibited no activities; it was 0.3% toward wild type and below the detection limit toward C16:0 and C24:0 fatty acids (Fig. 2B and C). 3. Discussion This is the first case report of a Japanese patient with FAHN having the characteristic features of spastic
3
paraplegia and white matter abnormality in brain MRI. Our cell-based enzyme assay demonstrated significantly decreased and almost lost enzyme activity of novel alleles, supporting that these alleles are pathogenic. So far, approximately 70 individuals with FAHN have been reported in the literature. Although it occurs during adolescence or adulthood in a few cases, the age of onset is typically during childhood, ranging mostly between the ages of 3 and 5 years. In addition to spasticity, approximately 90% of patients have intellectual decline and 30% have epileptic seizures [3,8]. There is no curative treatment for FAHN; only supportive treatment is available. The severity of FAHN phenotypes varies among affected individuals. This may be due to the type and location of the FA2H mutations. However, a recent large cohort study on FAHN patients showed no apparent correlations between genotype and phenotype [3]. Therefore, a functional analysis is expected to estimate the actual influence of each missense mutation. In a similar cell-based enzyme assay, enzyme activity was examined in four variants in the FA2H of one family and four patients from two reports (Table 1). Dick et al. investigated the enzyme activity of p.Arg235Cys and p.Arg53_Ile58del mutations in FA2H from two patients with FAHN [4]. They reported that the enzyme activities of p.Arg235Cys and p.Arg53-Ile58del mutants were 60%–80% and almost 0%, respectively. Additionally, Liao et al. reported that the enzyme activities of p.Leu130Phe and p.Leu77Arg mutants were 52% and 43%, respectively [5]. In this patient with typical but milder clinical features, the enzyme activities of p. Val149Leu and p.His260Gln variants were approximately 60%–80% and almost 0%, respectively. His260 of FA2H was located in a functional (fatty acid hydroxylase) domain. A previous study revealed that the residue of Scs7p, a yeast homolog of FA2H corresponding
Fig. 2. Measurement of FA2H activity. To measure FA2H activity, CHO-K1 cells expressing wild type (WT) and p.Val149Leu (V149L) and p. His260Gln (H260Q) mutants were incubated with 0.25 mM deuterium-labeled sphingosine at 37 °C for 4 h. (A) Lysates (15 mg protein) prepared from the cells were separated by SDS-PAGE, followed by immunoblotting with anti-FLAG antibodies. (B, C) Lipids were extracted from the cells, and the amount of deuterium-labeled ceramide, which comprises deuterium-labeled sphingosine and 2-hydroxy or nonhydroxy fatty acid, was quantified by liquid chromatography–tandem mass spectrometry. FA2H activity was defined as the ratio of 2-hydroxyceramide/nonhydroxyceramide, and the activities toward C16:0 (B) and C24:0 (C) Fatty acids are shown. Values are means ± SD (n = 3). Statistically significant differences compared with WT are indicated. **P < 0.01 (Dunnett’s test). 2-OH, 2-hydroxyceramide; non-OH, nonhydroxyceramide.
Please cite this article in press as: Kawaguchi M et al. Novel biallelic FA2H mutations in a Japanese boy with fatty acid hydroxylase-associated neurodegeneration. Brain Dev (2019), https://doi.org/10.1016/j.braindev.2019.11.006
Dick et al. [4] Family 1 (7 individuals) Age at reported (years) Sex
Clinical features Age at onset (years) Initial symptom Neurological symptoms
Cognitive decline Epilepsy Motor outcome
Liao et al. [5]
Present study
Patient 2
Patient 3
Patient 4 (sibling of patient 3)
Patient 5
Patient 6
8–25
31
26
31
17
10
males (n = 4), females (n = 3) Omani homozygous p.Arg235Cys 60–80%
female
male
female
male
male
Pakistani homozygous p.Arg53_Ile58del almost 0%
Chinese compound heterozygous p.Leu130Phe c.506 + 6C > G 52% almost 0%
Chinese compound heterozygous p.Leu130Phe c.506 + 6C > G 52% almost 0%
Chinese homozygous p.Leu77Arg 43%
Japanese compound heterozygous p.Val149Leu p.His260Gln 60–80% almost 0%
8–25
4
17
10
4
5
gait disturbance dysarthria (n = 5), nystagmus (n = 2) mild (n = 5)
gait disturbance
weakness of lower limbs
weakness of lower limbs
lower limb spasticity
gait disturbance
anarthria, hypertonia of upper limbs, ophthalmoplegia
mild dysarthria, mild dysphagia, urinary dysfunction
speech deficiency
pyramidal tract signs, cerebellar signs, abnormal ocular motility
mild dysmetria
mild
none
mild
mild
none
present
absent
ND
present
absent
wheel chair (10 years old)
autonomous ambulation (26 years old)
wheel chair (30 years old)
ND
wheel chair (10 years old)
present
present
present
ND
ND
present
ND
present
present
ND
ND
present
ND
present
absent
ND
ND
present
ND
ND
absent
ND
ND
absent
present (n = 2) autonomous ambulation (n = 3), wheel chair (n = 4)
MRI findings T2 hypointensity in the white matter Cerebellar atrophy Thin corpus callosum Iron deposits ND, not described.
M. Kawaguchi et al. / Brain & Development xxx (2019) xxx–xxx
Race Genotype Mutation Enzyme activity
4
Please cite this article in press as: Kawaguchi M et al. Novel biallelic FA2H mutations in a Japanese boy with fatty acid hydroxylase-associated neurodegeneration. Brain Dev (2019), https://doi.org/10.1016/j.braindev.2019.11.006
Table 1 Characteristics of patients whose enzyme activity was analyzed.
M. Kawaguchi et al. / Brain & Development xxx (2019) xxx–xxx
to His260 of FA2H in humans, was essential for enzyme activity [9]. This is consistent with our results that showed an almost absent enzyme activity of p. His260Gln. We compared the association among the clinical, neuroimaging, and enzyme activity in FAHN patients listed in Table 1. Enzyme activity greatly differed from approximately 0% to 60%–80%. However, the clinical features are generally similar, although the onset in patient 3 was exceptionally late. Thus, disease onset and its severity may not be explained only by residual enzyme activity with this cell-based enzyme assay. We cannot exclude the possibility of these being additional consequences of the FA2H mutations (e.g., alterations in transcript levels and processing or aberrant protein translocation). Kruer et al. proposed that the p. Arg154Cys mutation of FA2H reduces stability of the mRNA or translated protein because, in a case of p. Arg154Cys mutation, protein abundance measured by western blot decreased compared with a wild type whereas enzyme activity measured by gas chromatography–mass spectrometry demonstrated no significant difference [10]. This patient had a milder phenotype, which may be supported by our result of no or slightly decreased protein levels of each variant. 4. Conclusion This is the first report of a Japanese patient with FAHN having novel biallelic FA2H mutations. Our enzyme analysis demonstrated partial functionality of one of the variants, indicating a milder phenotype. However, disease severity in patients with FAHN may not be explained by residual enzyme activity measured with this method alone. Further research is needed to accurately predict the phenotypic severity of this disease and, more importantly, to develop a therapeutic intervention.
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by the Initiative on Rare and Undiagnosed Diseases in Pediatrics from the Japanese Agency for Medical Research and Development. References [1] Faber I, Pereira ER, Martinez ARM, Franc¸a Jr M, Teive HAG. Hereditary spastic paraplegia from 1880 to 2017: an historical review. Arq Neuropsiquiatr 2017;75:813–8. [2] Zo¨ller I, Meixner M, Hartmann D, Bu¨ssow H, Meyer R, Gieselmann V, et al. Absence of 2-hydroxylated sphingolipids is compatible with normal neural development but causes late-onset axon and myelin sheath degeneration. J Neurosci 2008;28:9741–54. [3] Rattay TW, Lindig T, Baets J, Smets K, Deconinck T, So¨hn AS, et al. FAHN/SPG35: a narrow phenotypic spectrum across disease classifications. Brain 2019;142:1561–72. [4] Dick KJ, Eckhardt M, Paisa´n-Ruiz C, Alshehhi AA, Proukakis C, Sibtain NA, et al. Mutation of FA2H underlies a complicated form of hereditary spastic paraplegia (SPG35). Hum Mutat 2010;31:1251–60. [5] Liao X, Luo Y, Zhan Z, Du J, Hu Z, Wang J, et al. SPG35 contributes to the second common subtype of AR-HSP in China: frequency analysis and functional characterization of FA2H gene mutations. Clin Genet 2015;87:85–9. [6] Sasaki H, Yanagi K, Ugi S, Kobayashi K, Ohkubo K, Tajiri Y, et al. Definitive diagnosis of mandibular hypoplasia, deafness, progeroid features and lipodystrophy (MDPL) syndrome caused by a recurrent de novo mutation in the POLD1 gene. Endocr J 2018;65:227–38. [7] Kanetake T, Sassa T, Nojiri K, Sawai M, Hattori S, Miyakawa T, et al. Neural symptoms in a gene knockout mouse model of Sjo¨gren-Larsson syndrome are associated with a decrease in 2hydroxygalactosylceramide. FASEB J 2019;33:928–41. [8] Mari F, Berti B, Romano A, Baldacci J, Rizzi R, Grazia Alessandrı` M, et al. Clinical and neuroimaging features of autosomal recessive spastic paraplegia 35 (SPG35): case reports, new mutations, and brief literature review. Neurogenetics 2018;19:123–30. [9] Zhu G, Koszelak-Rosenblum M, Connelly SM, Dumont ME, Malkowski MG. The crystal structure of an integral membrane fatty acid a-hydroxylase. J Biol Chem 2015;50:29820–33. [10] Kruer MC, Paisa´n-Ruiz C, Boddaert N, Yoon MY, Hama H, Gregory A, et al. Defective FA2H leads to a novel form of neurodegeneration with brain iron accumulation (NBIA). Ann Neurol 2010;68:611–8.
Acknowledgments Whole exome sequencing was performed through TOKAI-IRUD-P. This study was partially supported
Please cite this article in press as: Kawaguchi M et al. Novel biallelic FA2H mutations in a Japanese boy with fatty acid hydroxylase-associated neurodegeneration. Brain Dev (2019), https://doi.org/10.1016/j.braindev.2019.11.006