International Journal of Neuroscience, 2015; 125(1): 43–49 Copyright © 2015 Informa Healthcare USA, Inc. ISSN: 0020-7454 print / 1543-5245 online DOI: 10.3109/00207454.2014.904858
RESEARCH ARTICLE
Autosomal recessive posterior column ataxia with retinitis pigmentosa caused by novel mutations in the FLVCR1 gene
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Aziz Shaibani,1 Lee-Jun Wong,2 Victor Wei Zhang,2 Richard Alan Lewis,2,3 and Marwan Shinawi4 1
Nerve and Muscle Center of Texas, Houston, TX, USA; 2 Departments of Molecular and Human Genetics Baylor College of Medicine, Houston, TX, USA; 3 Department of Ophthalmology, Baylor College of Medicine, Houston, TX, USA; 4 Department of Pediatrics, Division of Genetics and Genomic Medicine, Washington University School of Medicine, St. Louis, MO, USA Posterior column ataxia with retinitis pigmentosa (PCARP) is an autosomal recessive disorder characterized by severe sensory ataxia, muscle weakness and atrophy, and progressive pigmentary retinopathy. Recently, mutations in the FLVCR1 gene were described in four families with this condition. We investigated the molecular basis and studied the phenotype of PCARP in a new family. The proband is a 33-year-old woman presented with sensory polyneuropathy and retinitis pigmentosa (RP). The constellation of clinical findings with normal metabolic and genetic evaluation, including mitochondrial DNA (mtDNA) analysis and normal levels of phytanic acid and vitamin E, prompted us to seek other causes of our patient’s condition. Sequencing of FLVCR1 in the proband and targeted mutation testing in her two affected siblings revealed two novel variants, c.1547G>A (p.R516Q) and c.1593+5 +8delGTAA predicted, respectively, to be highly conserved throughout evolution and affecting the normal splicing, therefore, deleterious. This study supports the pathogenic role of FLVCR1 in PCARP and expands the molecular and clinical spectra of PCARP. We show for the first time that nontransmembrane domain (TMD) mutations in the FLVCR1 can cause PCARP, suggesting different mechanisms for pathogenicity. Our clinical data reveal that impaired sensation can be part of the phenotypic spectrum of PCARP. This study along with previously reported cases suggests that targeted sequencing of the FLVCR1 gene should be considered in patients with severe sensory ataxia, RP, and peripheral sensory neuropathy. KEYWORDS: posterior column ataxia, neuropathy, autosomal recessive, retinitis pigmentosa, FLVCR1
Introduction Posterior column ataxia with retinitis pigmentosa (PCARP, MIM 609033) is an autosomal recessive Mendelian disorder characterized by retinitis pigmentosa (RP) and proprioceptive abnormalities manifest as severe sensory ataxia and muscle weakness and atrophy. Inversion recovery magnetic resonance imaging (MRI) of the spinal cord in affected individuals demonstrates hyperintense signal in the posterior columns [1]. The Received 26 November 2013; revised 11 March 2014; accepted 12 March 2014. Correspondence: Marwan Shinawi, M.D., F.A.C.M.G., Department of Pediatrics, Division of Genetics and Genomic Medicine, Washington University School of Medicine, One Children’s Place, Northwest Tower, 9132, Campus Box 8116, St. Louis, MO 63110, USA. Tel: 314.747.1837. Fax: 314.454.2075. E-mail: shinawi [email protected]
disorder begins during early childhood with progressive concentric constriction of the visual fields followed by proprioceptive loss. Affected surviving individuals have been reported to become blind and develop severe sensory ataxia, achalasia, gastrointestinal dysmotility, lean body habitus, and scoliosis [1, 2]. PCARP was described first by Higgins et al. [3] in six individuals from large American kindred of GermanSwiss descent. Two years later, the gene responsible for this condition was mapped to 1q31-q32 by genomewide linkage analysis and haplotype reconstruction [1]. With targeted DNA capture and high-throughput sequencing of a 4.2 Mb candidate region on chromosome 1q32, a homozygous missense mutation in the FLVCR1 (feline leukemia virus subgroup C receptor 1) gene was found in the original family described above (Family 1 in Table 1) [4]. Sanger sequencing of FLVCR1 identified 43
44 + Infancy −18 yr
Yes
Yes Absent 1–14 Intact N/A Absence of distal sensory potentials with normal motor conduction. Atrophy and loss of large myelinated fibers of sural nerve Cataract, kyphosis
+ 6 mo-early childhood
Yes
Yes Absent 3-childhood
Intact Yes Absence of sensory nerve conduction. Normal EMG
Atrophy & axonal degeneration of sural nerve. Normal muscle
Scoliosis, GI dysmotility, camptodactyly
Nerve/muscle biopsy
Miscellaneous
2
1
Muscle: normal
Hyperkeratosis of palms and soles, mild thoracic scoliosis
N/A
Intellectual disability
Atrophy & axonal degeneration of sural nerve. Normal muscle biopsy Seizures, scoliosis, finger Contractures, fecal incontinence
N/A
Absent N/A N/A
Yes Absent 9
Yes
No p.R516Q/c.1593 +5 +8delGTAA + 9 yr
1 Caucasian
II-2
5 (this report)
Osteomyelitis & left below-knee amputation. chronic pains & muscle cramps
N/A
Absent N/A N/A
Yes Absent 9
Yes
No p.R516Q/c.1593 +5 +8delGTAA + 9 yr
1 Caucasian
II-3
Abbreviations: DTRs, deep tendon reflexes; EMG, electromyography; GI, gastrointestinal; mo, month(s); N/A, not available or not performed; RP, retinitis pigmentosa; yr, year(s).
Absent N/A Absence responses of sensory nerves. Normal motor nerve velocity
Yes Absent 8
Yes
No p.R516Q/c.1593 +5 +8delGTAA + 5 yr
Caucasian
II-1 (Proband)
Intact Yes N/A
Yes Absent 5
Yes
+ 5 yr
Yes p.G493R/p.G493R
Japanese
4 (5)
Intact N/A N/A
Yes Absent 1–4
Yes
+ 9 mo–18 mo
Yes p.C192R/p.C192R
Yes p. A241T/p.A241T
RP Age of initial ophthalmological findings Proprioceptive (vibratory and position) deficit Ataxia DTRs Age (yr) of initial neurological findings Superficial sensations Abnormal Spine MRI Electrophysiology
Consanguinity Mutation
2 French-Canadian
3 (4)
4 Spanish (Gypsy)
8 German-Swiss (American) Yes p.N121D/p.N121D
# of patients evaluated Ethnicity
2 (4)
1 (4)
Clinical and ancillary tests findings in patients with PCARP.
Family # (reference)
Table 1.
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PCARP and novel FLVCR1 mutations
different homozygous missense mutations in two other, unrelated, Spanish and French-Canadian families with PCARP (Family 2&3 in Table 1) [4]. Subsequently, two siblings with PCARP from a Japanese consanguineous family (Family 4 in Table 1) were evaluated by massively parallel sequencing analysis and found to have a novel homozygous c.1477G>C (G493R) mutation in the same gene [5]. Only these four reported families have been characterized at the molecular level but the full phenotype of PCARP is not well characterized. Here we describe three siblings of Caucasian descent who were ultimately diagnosed with PCARP. The proband was a 33-year-old woman who presented with RP and sensory polyneuropathy. She has undergone an extensive metabolic and genetic evaluation including mtDNA analysis, phytanic acid levels, and vitamin E levels; all were normal. The constellation of clinical findings, negative prior genetic and metabolic evaluation, and the putative mode of inheritance suggested the diagnosis of PCARP, which was confirmed by targeted sequencing of the FLVCR1 gene. We describe the phenotype in her siblings and compare the phenotypic features in our patients with previously reported cases.
Materials and Methods Clinical report (Family 5, Table 1) The pedigree of the family with PCARP is shown as Figure 1. All subjects participated in this study after signing a consent form, which was approved by the Human Research Protection Office (HRPO) at Washington University in St. Louis. The proband (II-1) is a 33-year-old Caucasian female who was diagnosed with progressive peripheral visual loss and RP when she was 5 years old and at age 22 with the typical complicated cataracts (posterior precapsular and radial deep cortical linear opacities) commonly associated with progressive
A. Pedigree chart of a Caucasian family with PCARP. The arrow indicates the index patient.
Figure 1.
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2015 Informa Healthcare USA, Inc.
45
pigmentary retinal dystrophies. On presentation to us at age 28, her peripheral visual field (Goldmann) was constricted to less than 10 degrees in all meridians in each eye. Her gait started to be “wobbly” at age 8 years but became progressively unsteady after age 24 years. She became especially more ataxic in the dark when visual clues were limited. At about age 18 years, she developed numbness in her feet that progressed proximally to her knees and to her hands when she was 24 years old. She exhibited mild postural lightheadedness. However, she had no history of memory problems, hearing loss, dizziness, difficulty in speech or swallowing, musculoskeletal pain, or seizures. She has been diagnosed with mild, nonprogressive scoliosis. Her audiometry at age 27 was normal. On physical examination, she was healthy-looking Caucasian female, with thin body habitus. Her weight was 46.5 kg, height was 156.3 cm, and BMI was 19. Her uncorrected visual acuity was 20/25+2 bilaterally. External, adnexal and biomicroscopic examination showed typical complicated posterior cortical precapsular radial cataracts in each eye consistent with her minimally reduced central acuity and with her subjective complaints of glare in bright light. Dilated ophthalmoscopic evaluation showed vitreous syneresis and liquefaction with cells, more posteriorly than anteriorly, and the classic features of RP, including waxy pallor of the optic discs, widespread vascular attenuation, and extensive peripheral atrophy of the retinal pigment epithelium with bone spicule formations consistent with the constricted visual fields. Her neurological examination revealed intact cranial nerves and normal muscle mass, tone and strength. Her deep tendon reflexes were absent in lower extremities and reduced in her arms. She has severe impairment of sensation to all modalities in the feet up to knees and in the hands up to 10 cm proximal to the wrists. Romberg sign was positive. Her gait was ataxic but she was able to walk without support. Finger-to-nose test was normal, but she missed the target with closed eyes. There was mild thoracic scoliosis. Skin was dry on her legs and she had mild hyperkeratosis of palms and soles. Our initial diagnostic evaluation revealed normal levels of phytanic acid, very long chain fatty acids, plasma vitamin E, creatine phosphokinase, apolipoprotein B, plasma lactate and pyruvate, carbohydrate-deficient transferrin, plasma homocysteine, plasma methylmalonic acid, plasma amino acids and acylcarnitine profile. Her complete blood count was normal. The genetic testing for Neuropathy, Ataxia and Retinitis Pigmentosa (NARP) on blood was negative. The histology of muscle biopsy was unremarkable except for eight ragged red fibers in three blocks analyzed by modified Gomori trichrome stain but these were not felt to be abnormal for patient’s age. Screening of leukocyte lysosomal enzymes was normal. Nerve conduction studies
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A. Shaibani et al.
revealed absence of sensory responses of left sural, median and ulnar sensory nerves, normal motor nerve velocity in the left peroneal nerve and normal F wave. Brain MRI was normal but spine imaging was not performed. The proband’s 31-year-old sister (II-2) started complaining of poor vision and unsteady gait at 9 years of age. She has had no hearing problems, memory impairment, or bowel/ bladder dysfunction. On physical examination, her weight was 50.8 kg, height was 157.5 cm and BMI was 20.5. Her ophthalmological examination showed visual acuity at 20/50 uncorrected in the right eye and 20/20 uncorrected in the right eye. Biomicroscopy showed posterior cortical precapsular opacification centrally and paracentrally in each eye. Dilated fundus examination revealed classic features of vitreous syneresis with cells, vascular attenuation and symmetric progressive pigmentary retinopathy, all consistent with RP. Visual fields were constricted concentrically to less than 20 degrees in all meridians. She had diffuse areflexia and severe impairment of sensation to all modalities in the feet and positive Romberg sign. Her fast finger tapping was impaired. She showed no cerebellar abnormalities: normal heel-to-shin, finger to nose test and alternating hand movements were normal. She exhibited normal muscle strength. The proband’s 28-year-old brother (II-3) had numbness, unsteady gait and poor vision since 9 years of age. He had been diagnosed with Charcot–Marie–Tooth (CMT) disease. At age 24 years, he had osteomyelitis of the left foot resulting in left below-knee amputation. He exhibited chronic pain and muscle cramps in his legs partially controlled with Fentanyl and Duloxetine. He reported poor coordination, dizziness, headaches, anxiety and urinary frequency. On physical examination, his weight was 65.8 kg, height was 180 cm and BMI was 20.3. He had diffuse areflexia and severe impairment of sensation to all modalities in the right foot and hands. He had no formal ophthalmological evaluation but his visual fields were constricted concentrically to less than 20 degrees in all meridians. No other family members were known to have RP, neuropathy, or ataxia. The mother of the three siblings was of German ancestry, and the father was of German/Italian descent, with no known consanguinity.
Molecular Analyses Spectrophotometric analysis of the respiratory chain complexes was performed by traditional methods. mtDNA copy numbers were determined by real-time quantitative polymerase chain reaction (qPCR) and mtDNA sequencing was performed based on previously validated protocols [6]. The coding exons and the immediate flanking intronic sequences of the FLVCR1
gene were amplified by PCR and sequenced in the forward and reverse directions with automated fluorescence dideoxy-sequencing methods. GenBank (NCBI) ID NM 014053.3 served as the reference sequence.
Results DNA Analysis The mtDNA content in muscle specimen from the proband was approximately 111% of the mean value of age and tissue matched controls. The electron transport chain enzyme activities in muscle tissue were within normal range (70%–113% of mean value of age and tissue matched controls). Results of mtDNA sequencing in skeletal muscle were negative for deleterious point mutations. A homoplasmic rare variant, m.5074T>C (p.I202T, ND2), was detected, which was previously reported in Mitomap (http://www.mitomap.org/) as a polymorphism and in the Human Mitochondrial Genome Database (mtDB) (http://www.genpat.uu.se/mtDB/index.html) at a frequency of 2 (C) to 2702 (T). The data suggest that this nucleotide change likely represents a benign variant. Sequencing of FLVCR1 in the proband revealed two novel variants, c.1547G>A (p.R516Q) and c.1593+5 +8delGTAA (Figure 2A–C). The arginine (R) residue at amino acid position 516 of FLVCR1 is evolutionarily conserved as a positively charged amino acid residue [R or; lysine (K)] from zebrafish to human (Figure 2D). Both Sorting Intolerant From Tolerant (SIFT) and PolyPhen2 algorithms predicted the arginine to glutamine (Q) change at this position to be deleterious. The c.1593+5 +8delGTAA mutation causes a 4-base-pair deletion at nucleotide positions 5 to 8 from the donor intron-exon junction (Figure 2E). All five different algorithms used for in silico analysis predicted that the c.1593+5 +8delGTAA would affect the normal splicing at the donor site of intron 9 (Figure 2E). Either of the mutations has not been observed in 6500 subjects in the Exome Sequencing Project (ESP) database (http://evs.gs.washington.edu). A targeted mutation testing of the proband’s affected siblings was positive for the two mutations. Analysis of parental DNA samples confirmed that the identified mutations were in trans configuration.
Discussion Here, we describe three Caucasian siblings with PCARP, a disorder recently shown to be caused by mutations in the FLVCR1 gene. PCARP is an autosomalrecessive neurodegenerative condition associated with International Journal of Neuroscience
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PCARP and novel FLVCR1 mutations
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Figure 2. A. Schematic representation of FLVCR1 gene structure, mRNA transcript, and predicted secondary protein structure. The E1 to E10 represent the exon number 1 to 10, and UTR for untranslated region. The position of both mutations in the proband is indicated on the secondary structure as red bolt sign. The green star indicates the proposed PDZ [post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), zonula occludens-1 protein (Zo-1)] binding motif. B & C. Electropherograms show the sequence data for both mutations. The sequence trace was deconvoluted to indicate the heterozygous 4- base-pair deletion. D. Multiple sequence alignment of FLVCR1 protein across different species. The arginine (R) residue at amino acid position 516 is evolutionarily conserved as only positively charged amino acid residue [R or lysine (K)] from zebrafish to human. E. Four splice site effect prediction algorithms (SpliceSiteFinder-like, MaxEntScan, NNSPLICE, and GeneSplicer) predict the 4-bp deletion abolish the donor site. F. Ribbon diagram of structure model of human FLVCR1 transporter. The transmembrane helices are consistent with the secondary structure prediction. The N and C letters indicate the amino-terminus and carboxyl-terminus, respectively.
progressive sensory ataxia and RP. The initial signs of RP in these siblings became evident during childhood. The degeneration of the posterior columns of the spinal cord leads to central and peripheral axonal degeneration but without demyelination. Patients with PCARP exhibit sensory ganglionopathy and ataxia manifested as loss of proprioceptive sensation that is clinically evident in the first or second decade of life and progresses gradually. A phenotypic finding that distinguishes the three siblings in this family from previously reported cases is impaired sensation of all modalities in the distal extremities, expanding the neurological findings associated with FLVCR1 mutations (Table 1). Interestingly, in contrast to the three original families, two additional Western European families were reported with PCARPlike phenotype and decreased pain sensation but without mutations in FLVCR1 [4]. Other features described to C
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be variably associated with this condition include scoliosis, gastrointestinal dysmotility, intellectual disability and lean body habitus [1–3]. Neuroimaging studies are usually unremarkable, except for hyperintense signals in the posterior spinal cord on inversion recovery MRI, reflecting degeneration of the posterior columns [4]. The constellation of ataxia, neuropathy, and RP is seen in a spectrum of genetic conditions including Refsum disease, ataxia with isolated vitamin E deficiency, abetalipoproteinemia, PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa and cataract), spinocerebellar ataxia, NARP, congenital disorder of glycosylation type IA and spinocerebellar ataxia (Table 2). The clinical findings in our family are similar to the phenotype in Refsum disease, but the lack of demyelinating polyneuropathy and hearing loss and the normal phytanic acid levels do not support this diagnosis.
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A. Shaibani et al.
Table 2.
Genetic conditions associated with ataxia, neuropathy and retinitis pigmentosa.
Disease
OMIM#
Chromosome
Gene
Inheritance
NARP (neuropathy, Ataxia, and Retinitis Pigmentosa) PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract) Ataxia with isolated vitamin E deficiency Refsum disease Abetalipoproteinemia CDG1A (congenital disorder of glycosylation type IA) Spinocerebellar ataxia 2∗ PCARP (Ataxia, posterior column, with retinitis pigmentosa)
551 500 612 674
mtDNA 20p11.21
MT-ATP6 ABHD12
Maternal AR
277 460 266 500 200 100 118 200 183 090 609 033
8q12.3 10p13 4q23 16p13.2 12q24.12 1q32.2
TTPA PHYH MTP PMM2 SCA2 FLVCR1
AR AR AR AR AD AR
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(∗ ) Retinitis pigmentosa is rare finding
Mitochondrial disorders, including mitochondrial depletion syndromes, may overlap the clinical findings in this family but results of mtDNA sequencing, quantitative mtDNA analysis and respiratory chain complexes were unremarkable. The lack of cerebellar signs, vitamin E levels, cholesterol levels, transferrin isoelectric focusing, normal audiometry and negative NARP mutations in the proband, distinguish PCARP from the other conditions. Our data support the pathogenic role of FLVCR1 in PCARP and expand the molecular spectrum of mutations causing this condition. In contrast to our patients, all previous reports came from consanguineous families and expectedly had homozygous mutations. This is the first report of compound heterozygous mutations that do not involve the putative TMDs of FLVCR1 and occur at the C-terminal cytoplasmic domain. While the mutations in previous reports were identified by a whole genome approach with targeted capture and massively parallel sequencing technology, we confirmed the clinical diagnosis in our proband by targeted sequencing of the FLVCR1 gene. The FLVCR1 gene contains 10 exons, spans an approximately 40 Kb on chromosome 1q32, and encodes a 555 amino acid protein. Topology predictions suggest that the protein has 12 TMDs with intracellular Nand C-termini (∼108 amino acids and ∼43 amino acids, respectively) (Figure 2F) [7]. The C-terminus of FLVCR1 contains 4-amino acid sequence class I PDZ [post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), zonula occludens-1 protein (Zo-1)] domain-binding motif, which is important for its interaction with the PDZ domain-containing protein(s). All four previously reported mutations are located in the putative TMDs of FLVCR1. Recent evidence suggested that all previously reported TMD mutations lead to misfolding in the endoplasmic reticulum and rapid degradation in the lysosomes of the FLVCR1 protein and loss of its heme export activity [8]. The mutations in our family are located in the Cterminus of the protein and do not involve the putative
TMDs of FLVCR1 (Figure 2F). Although no functional studies were performed on these mutations, their location suggests different mechanisms for pathogenicity. The p.R516Q mutation involves a positively charged residue and possibly affects the interaction of FLVCR1 with other proteins. The second mutation is predicted to alter the splicing of last intron in the FLVCR1. mRNA nonsense-mediated decay surveillance pathway typically degrades only transcripts containing nonsense codons that are followed by at least one intron [9] and therefore the possibility of misfolded product cannot be excluded. FLVCR1 is a cell-membrane heme exporter involved in the regulation of intracellular heme levels and protects erythroid progenitors from heme toxicity [10]. Targeted deletion of FLVCR1 in mice results in severe anemia [11]. Mice data revealed abundant expression in the retina, posterior column of the spinal cord and the cerebellum [4] but the precise function of FLVCR1 in neurons and retina is unknown [8, 12]. Excessive amounts of free heme can cause lipid peroxidation and generation of reactive oxygen species, and was suggested to abolish the neuroprotective effects of neuroglobin (4), leading to enhanced apoptosis and to neuronal and retinal degeneration [7, 13]. Recently, Chiabrando, et al. [14] identified a mitochondrial Flvcr1 isoform, Flvcr1b, that facilitates heme efflux from the mitochondria and showed that its silencing causes termination of erythroid differentiation. The isoform encoded by the FLVCR1b contains a shortened N terminus (amino acids 277–555) [15] and the two mutations in our family are located in the coding region of the two different FLVCR1 isoforms. However, we did not find any hematological abnormalities in the three affected siblings.
Conclusions We present the first report of PCARP in a nonconsanguineous family with novel compound heterozygous mutations detected by targeted sequencing of the International Journal of Neuroscience
PCARP and novel FLVCR1 mutations
FLVCR1 gene. Our data expand the molecular spectrum of impaired FLVCR1 function and show that non-TMDs mutations in the FLVCR1 gene can cause PCARP. Our study also shows that impaired sensation can be part of the phenotypic spectrum of PCARP. Molecular and clinical studies of additional patients are needed to better define the molecular and phenotypic spectra of PCARP.
Acknowledgements
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Sincere appreciation is extended to all members of the family described herein for their willing and continuing cooperation in these pursuits.
Declaration of Interest Richard Alan Lewis received some unrestricted support for this investigation from Research to Prevent Blindness, New York, NY. Other authors report no conflict of interest. The authors alone are responsible for the content and writing of this paper.
References 1. Higgins JJ, Morton DH, Loveless JM. Posterior column ataxia with retinitis pigmentosa (AXPC1) maps to chromosome 1q31q32. Neurology 1999;52:146–50. 2. Berciano J, Polo JM. Autosomal recessive posterior column ataxia and retinitis pigmentosa. (Letter) Neurology 1998;5: 1772–3.
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3. Higgins JJ, Morton DH, Patronas N, Nee LE. An autosomal recessive disorder with posterior column ataxia and retinitis pigmentosa. Neurology 1997;49:1717–20. 4. Rajadhyaksha AM, Elemento O, Puffenberger EG, et al. Mutations in FLVCR1 cause posterior column ataxia and retinitis pigmentosa. Am J Hum Genet 2010;87:643–54. 5. Ishiura H, Fukuda Y, Mitsui J, et al. Posterior column ataxia with retinitis pigmentosa in a Japanese family with a novel mutation in FLVCR1. Neurogenetics 2011;12:117–21. 6. Shaibani A, Shchelochkov OA, Zhang S, et al. Mitochondrial neurogastrointestinal encephalopathy due to mutations in RRM2B. Arch Neurol 2009;66:1028–32. 7. Khan AA, Quigley JG. Heme and FLVCR-related transporter families SLC48 and SLC49. Mol Aspects Med 2013;34:669–82. 8. Yanatori I, Yasui Y, Miura K, Kishi F. Mutations of FLVCR1 in posterior column ataxia and retinitis pigmentosa result in the loss of heme export activity. Blood Cells Mol Dis 2012;49:60–6. 9. Nagy E, Maquat LE. A rule for termination-codon position within intron-containing genes: when nonsense affects RNA abundance. Trends Biochem Sci 1998;23:198–9. 10. Quigley JG, Yang Z, Worthington MT, et al. Identification of a human heme exporter that is essential for erythropoiesis. Cell 2004;118:757–66. 11. Keel SB, Doty RT, Yang Z, et al. A heme export protein is required for red blood cell differentiation and iron homeostasis. Science 2008;319:825–28. 12. Tailor CS, Willett BJ, Kabat D. A putative cell surface receptor for anemia-inducing feline leukemia virus subgroup C is a member of a transporter superfamily. J Virol 1999;73:6500–5. 13. Khan AA, Quigley JG. Control of intracellular heme levels: heme transporters and heme oxygenases. Biochim Biophys Acta 2011;1813:668–82. 14. Chiabrando D, Marro S, Mercurio S, et al. The mitochondrial heme exporter FLVCR1b mediates erythroid differentiation. J Clin Invest 2012;122:4569–79. 15. Fleming MD, Hamza I. Mitochondrial heme: an exit strategy at last. J Clin Invest 2012;122:4328–30.
RESEARCH ARTICLE
Autosomal recessive posterior column ataxia with retinitis pigmentosa caused by novel mutations in the FLVCR1 gene
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Aziz Shaibani,1 Lee-Jun Wong,2 Victor Wei Zhang,2 Richard Alan Lewis,2,3 and Marwan Shinawi4 1
Nerve and Muscle Center of Texas, Houston, TX, USA; 2 Departments of Molecular and Human Genetics Baylor College of Medicine, Houston, TX, USA; 3 Department of Ophthalmology, Baylor College of Medicine, Houston, TX, USA; 4 Department of Pediatrics, Division of Genetics and Genomic Medicine, Washington University School of Medicine, St. Louis, MO, USA Posterior column ataxia with retinitis pigmentosa (PCARP) is an autosomal recessive disorder characterized by severe sensory ataxia, muscle weakness and atrophy, and progressive pigmentary retinopathy. Recently, mutations in the FLVCR1 gene were described in four families with this condition. We investigated the molecular basis and studied the phenotype of PCARP in a new family. The proband is a 33-year-old woman presented with sensory polyneuropathy and retinitis pigmentosa (RP). The constellation of clinical findings with normal metabolic and genetic evaluation, including mitochondrial DNA (mtDNA) analysis and normal levels of phytanic acid and vitamin E, prompted us to seek other causes of our patient’s condition. Sequencing of FLVCR1 in the proband and targeted mutation testing in her two affected siblings revealed two novel variants, c.1547G>A (p.R516Q) and c.1593+5 +8delGTAA predicted, respectively, to be highly conserved throughout evolution and affecting the normal splicing, therefore, deleterious. This study supports the pathogenic role of FLVCR1 in PCARP and expands the molecular and clinical spectra of PCARP. We show for the first time that nontransmembrane domain (TMD) mutations in the FLVCR1 can cause PCARP, suggesting different mechanisms for pathogenicity. Our clinical data reveal that impaired sensation can be part of the phenotypic spectrum of PCARP. This study along with previously reported cases suggests that targeted sequencing of the FLVCR1 gene should be considered in patients with severe sensory ataxia, RP, and peripheral sensory neuropathy. KEYWORDS: posterior column ataxia, neuropathy, autosomal recessive, retinitis pigmentosa, FLVCR1
Introduction Posterior column ataxia with retinitis pigmentosa (PCARP, MIM 609033) is an autosomal recessive Mendelian disorder characterized by retinitis pigmentosa (RP) and proprioceptive abnormalities manifest as severe sensory ataxia and muscle weakness and atrophy. Inversion recovery magnetic resonance imaging (MRI) of the spinal cord in affected individuals demonstrates hyperintense signal in the posterior columns [1]. The Received 26 November 2013; revised 11 March 2014; accepted 12 March 2014. Correspondence: Marwan Shinawi, M.D., F.A.C.M.G., Department of Pediatrics, Division of Genetics and Genomic Medicine, Washington University School of Medicine, One Children’s Place, Northwest Tower, 9132, Campus Box 8116, St. Louis, MO 63110, USA. Tel: 314.747.1837. Fax: 314.454.2075. E-mail: shinawi [email protected]
disorder begins during early childhood with progressive concentric constriction of the visual fields followed by proprioceptive loss. Affected surviving individuals have been reported to become blind and develop severe sensory ataxia, achalasia, gastrointestinal dysmotility, lean body habitus, and scoliosis [1, 2]. PCARP was described first by Higgins et al. [3] in six individuals from large American kindred of GermanSwiss descent. Two years later, the gene responsible for this condition was mapped to 1q31-q32 by genomewide linkage analysis and haplotype reconstruction [1]. With targeted DNA capture and high-throughput sequencing of a 4.2 Mb candidate region on chromosome 1q32, a homozygous missense mutation in the FLVCR1 (feline leukemia virus subgroup C receptor 1) gene was found in the original family described above (Family 1 in Table 1) [4]. Sanger sequencing of FLVCR1 identified 43
44 + Infancy −18 yr
Yes
Yes Absent 1–14 Intact N/A Absence of distal sensory potentials with normal motor conduction. Atrophy and loss of large myelinated fibers of sural nerve Cataract, kyphosis
+ 6 mo-early childhood
Yes
Yes Absent 3-childhood
Intact Yes Absence of sensory nerve conduction. Normal EMG
Atrophy & axonal degeneration of sural nerve. Normal muscle
Scoliosis, GI dysmotility, camptodactyly
Nerve/muscle biopsy
Miscellaneous
2
1
Muscle: normal
Hyperkeratosis of palms and soles, mild thoracic scoliosis
N/A
Intellectual disability
Atrophy & axonal degeneration of sural nerve. Normal muscle biopsy Seizures, scoliosis, finger Contractures, fecal incontinence
N/A
Absent N/A N/A
Yes Absent 9
Yes
No p.R516Q/c.1593 +5 +8delGTAA + 9 yr
1 Caucasian
II-2
5 (this report)
Osteomyelitis & left below-knee amputation. chronic pains & muscle cramps
N/A
Absent N/A N/A
Yes Absent 9
Yes
No p.R516Q/c.1593 +5 +8delGTAA + 9 yr
1 Caucasian
II-3
Abbreviations: DTRs, deep tendon reflexes; EMG, electromyography; GI, gastrointestinal; mo, month(s); N/A, not available or not performed; RP, retinitis pigmentosa; yr, year(s).
Absent N/A Absence responses of sensory nerves. Normal motor nerve velocity
Yes Absent 8
Yes
No p.R516Q/c.1593 +5 +8delGTAA + 5 yr
Caucasian
II-1 (Proband)
Intact Yes N/A
Yes Absent 5
Yes
+ 5 yr
Yes p.G493R/p.G493R
Japanese
4 (5)
Intact N/A N/A
Yes Absent 1–4
Yes
+ 9 mo–18 mo
Yes p.C192R/p.C192R
Yes p. A241T/p.A241T
RP Age of initial ophthalmological findings Proprioceptive (vibratory and position) deficit Ataxia DTRs Age (yr) of initial neurological findings Superficial sensations Abnormal Spine MRI Electrophysiology
Consanguinity Mutation
2 French-Canadian
3 (4)
4 Spanish (Gypsy)
8 German-Swiss (American) Yes p.N121D/p.N121D
# of patients evaluated Ethnicity
2 (4)
1 (4)
Clinical and ancillary tests findings in patients with PCARP.
Family # (reference)
Table 1.
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PCARP and novel FLVCR1 mutations
different homozygous missense mutations in two other, unrelated, Spanish and French-Canadian families with PCARP (Family 2&3 in Table 1) [4]. Subsequently, two siblings with PCARP from a Japanese consanguineous family (Family 4 in Table 1) were evaluated by massively parallel sequencing analysis and found to have a novel homozygous c.1477G>C (G493R) mutation in the same gene [5]. Only these four reported families have been characterized at the molecular level but the full phenotype of PCARP is not well characterized. Here we describe three siblings of Caucasian descent who were ultimately diagnosed with PCARP. The proband was a 33-year-old woman who presented with RP and sensory polyneuropathy. She has undergone an extensive metabolic and genetic evaluation including mtDNA analysis, phytanic acid levels, and vitamin E levels; all were normal. The constellation of clinical findings, negative prior genetic and metabolic evaluation, and the putative mode of inheritance suggested the diagnosis of PCARP, which was confirmed by targeted sequencing of the FLVCR1 gene. We describe the phenotype in her siblings and compare the phenotypic features in our patients with previously reported cases.
Materials and Methods Clinical report (Family 5, Table 1) The pedigree of the family with PCARP is shown as Figure 1. All subjects participated in this study after signing a consent form, which was approved by the Human Research Protection Office (HRPO) at Washington University in St. Louis. The proband (II-1) is a 33-year-old Caucasian female who was diagnosed with progressive peripheral visual loss and RP when she was 5 years old and at age 22 with the typical complicated cataracts (posterior precapsular and radial deep cortical linear opacities) commonly associated with progressive
A. Pedigree chart of a Caucasian family with PCARP. The arrow indicates the index patient.
Figure 1.
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pigmentary retinal dystrophies. On presentation to us at age 28, her peripheral visual field (Goldmann) was constricted to less than 10 degrees in all meridians in each eye. Her gait started to be “wobbly” at age 8 years but became progressively unsteady after age 24 years. She became especially more ataxic in the dark when visual clues were limited. At about age 18 years, she developed numbness in her feet that progressed proximally to her knees and to her hands when she was 24 years old. She exhibited mild postural lightheadedness. However, she had no history of memory problems, hearing loss, dizziness, difficulty in speech or swallowing, musculoskeletal pain, or seizures. She has been diagnosed with mild, nonprogressive scoliosis. Her audiometry at age 27 was normal. On physical examination, she was healthy-looking Caucasian female, with thin body habitus. Her weight was 46.5 kg, height was 156.3 cm, and BMI was 19. Her uncorrected visual acuity was 20/25+2 bilaterally. External, adnexal and biomicroscopic examination showed typical complicated posterior cortical precapsular radial cataracts in each eye consistent with her minimally reduced central acuity and with her subjective complaints of glare in bright light. Dilated ophthalmoscopic evaluation showed vitreous syneresis and liquefaction with cells, more posteriorly than anteriorly, and the classic features of RP, including waxy pallor of the optic discs, widespread vascular attenuation, and extensive peripheral atrophy of the retinal pigment epithelium with bone spicule formations consistent with the constricted visual fields. Her neurological examination revealed intact cranial nerves and normal muscle mass, tone and strength. Her deep tendon reflexes were absent in lower extremities and reduced in her arms. She has severe impairment of sensation to all modalities in the feet up to knees and in the hands up to 10 cm proximal to the wrists. Romberg sign was positive. Her gait was ataxic but she was able to walk without support. Finger-to-nose test was normal, but she missed the target with closed eyes. There was mild thoracic scoliosis. Skin was dry on her legs and she had mild hyperkeratosis of palms and soles. Our initial diagnostic evaluation revealed normal levels of phytanic acid, very long chain fatty acids, plasma vitamin E, creatine phosphokinase, apolipoprotein B, plasma lactate and pyruvate, carbohydrate-deficient transferrin, plasma homocysteine, plasma methylmalonic acid, plasma amino acids and acylcarnitine profile. Her complete blood count was normal. The genetic testing for Neuropathy, Ataxia and Retinitis Pigmentosa (NARP) on blood was negative. The histology of muscle biopsy was unremarkable except for eight ragged red fibers in three blocks analyzed by modified Gomori trichrome stain but these were not felt to be abnormal for patient’s age. Screening of leukocyte lysosomal enzymes was normal. Nerve conduction studies
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revealed absence of sensory responses of left sural, median and ulnar sensory nerves, normal motor nerve velocity in the left peroneal nerve and normal F wave. Brain MRI was normal but spine imaging was not performed. The proband’s 31-year-old sister (II-2) started complaining of poor vision and unsteady gait at 9 years of age. She has had no hearing problems, memory impairment, or bowel/ bladder dysfunction. On physical examination, her weight was 50.8 kg, height was 157.5 cm and BMI was 20.5. Her ophthalmological examination showed visual acuity at 20/50 uncorrected in the right eye and 20/20 uncorrected in the right eye. Biomicroscopy showed posterior cortical precapsular opacification centrally and paracentrally in each eye. Dilated fundus examination revealed classic features of vitreous syneresis with cells, vascular attenuation and symmetric progressive pigmentary retinopathy, all consistent with RP. Visual fields were constricted concentrically to less than 20 degrees in all meridians. She had diffuse areflexia and severe impairment of sensation to all modalities in the feet and positive Romberg sign. Her fast finger tapping was impaired. She showed no cerebellar abnormalities: normal heel-to-shin, finger to nose test and alternating hand movements were normal. She exhibited normal muscle strength. The proband’s 28-year-old brother (II-3) had numbness, unsteady gait and poor vision since 9 years of age. He had been diagnosed with Charcot–Marie–Tooth (CMT) disease. At age 24 years, he had osteomyelitis of the left foot resulting in left below-knee amputation. He exhibited chronic pain and muscle cramps in his legs partially controlled with Fentanyl and Duloxetine. He reported poor coordination, dizziness, headaches, anxiety and urinary frequency. On physical examination, his weight was 65.8 kg, height was 180 cm and BMI was 20.3. He had diffuse areflexia and severe impairment of sensation to all modalities in the right foot and hands. He had no formal ophthalmological evaluation but his visual fields were constricted concentrically to less than 20 degrees in all meridians. No other family members were known to have RP, neuropathy, or ataxia. The mother of the three siblings was of German ancestry, and the father was of German/Italian descent, with no known consanguinity.
Molecular Analyses Spectrophotometric analysis of the respiratory chain complexes was performed by traditional methods. mtDNA copy numbers were determined by real-time quantitative polymerase chain reaction (qPCR) and mtDNA sequencing was performed based on previously validated protocols [6]. The coding exons and the immediate flanking intronic sequences of the FLVCR1
gene were amplified by PCR and sequenced in the forward and reverse directions with automated fluorescence dideoxy-sequencing methods. GenBank (NCBI) ID NM 014053.3 served as the reference sequence.
Results DNA Analysis The mtDNA content in muscle specimen from the proband was approximately 111% of the mean value of age and tissue matched controls. The electron transport chain enzyme activities in muscle tissue were within normal range (70%–113% of mean value of age and tissue matched controls). Results of mtDNA sequencing in skeletal muscle were negative for deleterious point mutations. A homoplasmic rare variant, m.5074T>C (p.I202T, ND2), was detected, which was previously reported in Mitomap (http://www.mitomap.org/) as a polymorphism and in the Human Mitochondrial Genome Database (mtDB) (http://www.genpat.uu.se/mtDB/index.html) at a frequency of 2 (C) to 2702 (T). The data suggest that this nucleotide change likely represents a benign variant. Sequencing of FLVCR1 in the proband revealed two novel variants, c.1547G>A (p.R516Q) and c.1593+5 +8delGTAA (Figure 2A–C). The arginine (R) residue at amino acid position 516 of FLVCR1 is evolutionarily conserved as a positively charged amino acid residue [R or; lysine (K)] from zebrafish to human (Figure 2D). Both Sorting Intolerant From Tolerant (SIFT) and PolyPhen2 algorithms predicted the arginine to glutamine (Q) change at this position to be deleterious. The c.1593+5 +8delGTAA mutation causes a 4-base-pair deletion at nucleotide positions 5 to 8 from the donor intron-exon junction (Figure 2E). All five different algorithms used for in silico analysis predicted that the c.1593+5 +8delGTAA would affect the normal splicing at the donor site of intron 9 (Figure 2E). Either of the mutations has not been observed in 6500 subjects in the Exome Sequencing Project (ESP) database (http://evs.gs.washington.edu). A targeted mutation testing of the proband’s affected siblings was positive for the two mutations. Analysis of parental DNA samples confirmed that the identified mutations were in trans configuration.
Discussion Here, we describe three Caucasian siblings with PCARP, a disorder recently shown to be caused by mutations in the FLVCR1 gene. PCARP is an autosomalrecessive neurodegenerative condition associated with International Journal of Neuroscience
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PCARP and novel FLVCR1 mutations
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Figure 2. A. Schematic representation of FLVCR1 gene structure, mRNA transcript, and predicted secondary protein structure. The E1 to E10 represent the exon number 1 to 10, and UTR for untranslated region. The position of both mutations in the proband is indicated on the secondary structure as red bolt sign. The green star indicates the proposed PDZ [post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), zonula occludens-1 protein (Zo-1)] binding motif. B & C. Electropherograms show the sequence data for both mutations. The sequence trace was deconvoluted to indicate the heterozygous 4- base-pair deletion. D. Multiple sequence alignment of FLVCR1 protein across different species. The arginine (R) residue at amino acid position 516 is evolutionarily conserved as only positively charged amino acid residue [R or lysine (K)] from zebrafish to human. E. Four splice site effect prediction algorithms (SpliceSiteFinder-like, MaxEntScan, NNSPLICE, and GeneSplicer) predict the 4-bp deletion abolish the donor site. F. Ribbon diagram of structure model of human FLVCR1 transporter. The transmembrane helices are consistent with the secondary structure prediction. The N and C letters indicate the amino-terminus and carboxyl-terminus, respectively.
progressive sensory ataxia and RP. The initial signs of RP in these siblings became evident during childhood. The degeneration of the posterior columns of the spinal cord leads to central and peripheral axonal degeneration but without demyelination. Patients with PCARP exhibit sensory ganglionopathy and ataxia manifested as loss of proprioceptive sensation that is clinically evident in the first or second decade of life and progresses gradually. A phenotypic finding that distinguishes the three siblings in this family from previously reported cases is impaired sensation of all modalities in the distal extremities, expanding the neurological findings associated with FLVCR1 mutations (Table 1). Interestingly, in contrast to the three original families, two additional Western European families were reported with PCARPlike phenotype and decreased pain sensation but without mutations in FLVCR1 [4]. Other features described to C
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be variably associated with this condition include scoliosis, gastrointestinal dysmotility, intellectual disability and lean body habitus [1–3]. Neuroimaging studies are usually unremarkable, except for hyperintense signals in the posterior spinal cord on inversion recovery MRI, reflecting degeneration of the posterior columns [4]. The constellation of ataxia, neuropathy, and RP is seen in a spectrum of genetic conditions including Refsum disease, ataxia with isolated vitamin E deficiency, abetalipoproteinemia, PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa and cataract), spinocerebellar ataxia, NARP, congenital disorder of glycosylation type IA and spinocerebellar ataxia (Table 2). The clinical findings in our family are similar to the phenotype in Refsum disease, but the lack of demyelinating polyneuropathy and hearing loss and the normal phytanic acid levels do not support this diagnosis.
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Table 2.
Genetic conditions associated with ataxia, neuropathy and retinitis pigmentosa.
Disease
OMIM#
Chromosome
Gene
Inheritance
NARP (neuropathy, Ataxia, and Retinitis Pigmentosa) PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract) Ataxia with isolated vitamin E deficiency Refsum disease Abetalipoproteinemia CDG1A (congenital disorder of glycosylation type IA) Spinocerebellar ataxia 2∗ PCARP (Ataxia, posterior column, with retinitis pigmentosa)
551 500 612 674
mtDNA 20p11.21
MT-ATP6 ABHD12
Maternal AR
277 460 266 500 200 100 118 200 183 090 609 033
8q12.3 10p13 4q23 16p13.2 12q24.12 1q32.2
TTPA PHYH MTP PMM2 SCA2 FLVCR1
AR AR AR AR AD AR
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(∗ ) Retinitis pigmentosa is rare finding
Mitochondrial disorders, including mitochondrial depletion syndromes, may overlap the clinical findings in this family but results of mtDNA sequencing, quantitative mtDNA analysis and respiratory chain complexes were unremarkable. The lack of cerebellar signs, vitamin E levels, cholesterol levels, transferrin isoelectric focusing, normal audiometry and negative NARP mutations in the proband, distinguish PCARP from the other conditions. Our data support the pathogenic role of FLVCR1 in PCARP and expand the molecular spectrum of mutations causing this condition. In contrast to our patients, all previous reports came from consanguineous families and expectedly had homozygous mutations. This is the first report of compound heterozygous mutations that do not involve the putative TMDs of FLVCR1 and occur at the C-terminal cytoplasmic domain. While the mutations in previous reports were identified by a whole genome approach with targeted capture and massively parallel sequencing technology, we confirmed the clinical diagnosis in our proband by targeted sequencing of the FLVCR1 gene. The FLVCR1 gene contains 10 exons, spans an approximately 40 Kb on chromosome 1q32, and encodes a 555 amino acid protein. Topology predictions suggest that the protein has 12 TMDs with intracellular Nand C-termini (∼108 amino acids and ∼43 amino acids, respectively) (Figure 2F) [7]. The C-terminus of FLVCR1 contains 4-amino acid sequence class I PDZ [post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), zonula occludens-1 protein (Zo-1)] domain-binding motif, which is important for its interaction with the PDZ domain-containing protein(s). All four previously reported mutations are located in the putative TMDs of FLVCR1. Recent evidence suggested that all previously reported TMD mutations lead to misfolding in the endoplasmic reticulum and rapid degradation in the lysosomes of the FLVCR1 protein and loss of its heme export activity [8]. The mutations in our family are located in the Cterminus of the protein and do not involve the putative
TMDs of FLVCR1 (Figure 2F). Although no functional studies were performed on these mutations, their location suggests different mechanisms for pathogenicity. The p.R516Q mutation involves a positively charged residue and possibly affects the interaction of FLVCR1 with other proteins. The second mutation is predicted to alter the splicing of last intron in the FLVCR1. mRNA nonsense-mediated decay surveillance pathway typically degrades only transcripts containing nonsense codons that are followed by at least one intron [9] and therefore the possibility of misfolded product cannot be excluded. FLVCR1 is a cell-membrane heme exporter involved in the regulation of intracellular heme levels and protects erythroid progenitors from heme toxicity [10]. Targeted deletion of FLVCR1 in mice results in severe anemia [11]. Mice data revealed abundant expression in the retina, posterior column of the spinal cord and the cerebellum [4] but the precise function of FLVCR1 in neurons and retina is unknown [8, 12]. Excessive amounts of free heme can cause lipid peroxidation and generation of reactive oxygen species, and was suggested to abolish the neuroprotective effects of neuroglobin (4), leading to enhanced apoptosis and to neuronal and retinal degeneration [7, 13]. Recently, Chiabrando, et al. [14] identified a mitochondrial Flvcr1 isoform, Flvcr1b, that facilitates heme efflux from the mitochondria and showed that its silencing causes termination of erythroid differentiation. The isoform encoded by the FLVCR1b contains a shortened N terminus (amino acids 277–555) [15] and the two mutations in our family are located in the coding region of the two different FLVCR1 isoforms. However, we did not find any hematological abnormalities in the three affected siblings.
Conclusions We present the first report of PCARP in a nonconsanguineous family with novel compound heterozygous mutations detected by targeted sequencing of the International Journal of Neuroscience
PCARP and novel FLVCR1 mutations
FLVCR1 gene. Our data expand the molecular spectrum of impaired FLVCR1 function and show that non-TMDs mutations in the FLVCR1 gene can cause PCARP. Our study also shows that impaired sensation can be part of the phenotypic spectrum of PCARP. Molecular and clinical studies of additional patients are needed to better define the molecular and phenotypic spectra of PCARP.
Acknowledgements
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Sincere appreciation is extended to all members of the family described herein for their willing and continuing cooperation in these pursuits.
Declaration of Interest Richard Alan Lewis received some unrestricted support for this investigation from Research to Prevent Blindness, New York, NY. Other authors report no conflict of interest. The authors alone are responsible for the content and writing of this paper.
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