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Keywords:

  • Adult-onset NBIA;
  • autozygosity mapping;
  • C19orf12;
  • rapid disease progression

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Neurodegeneration with brain iron accumulation (NBIA) comprises a clinically and genetically heterogeneous group of neurodegenerative diseases characterized by progressive degeneration of the central nervous system and high basal ganglia iron deposition. The list of identified causative genes for NBIA syndromes continues to expand and includes one autosomal dominant, one X-linked, and a number of recessive forms. Mitochondrial membrane protein-associated neurodegeneration is a recently described NBIA syndrome caused by C19orf12 mutations. In this study, we report two consanguineous families with a homozygous C19orf12 p.Thr11Met mutation. Our patients presented at a later age and had more rapid disease progression, leading to early death in two, than those previously reported. We conclude that C19orf12 mutation is associated with wide phenotypic heterogeneity, and that further research is needed to examine the role of C19orf12 in NBIA and related diseases and to elucidate its protein function as well as other factors that may affect disease progression and expression.

Neurodegeneration with brain iron accumulation (NBIA) comprises a clinically and genetically heterogeneous group of neurodegenerative diseases characterized by progressive degeneration of the central nervous system and iron accumulation, particularly within the globus pallidus [1]. Clinical manifestations of NBIA can include parkinsonism, dystonia, dysarthria, cerebellar ataxia, optic atrophy, lower extremity spasticity, behavioral problems, dementia, psychomotor delay, and peripheral neuropathy. The list of NBIA disorders continues to grow; it includes the autosomal dominant, adult-onset NBIA form, neuroferritinopathy (MIM #606159), caused by mutations within the FTL gene [2], the recently identified X-linked beta-propeller protein-associated neurodegeneration (BPAN) caused by de novo WDR45 mutations [3], and a number of autosomal recessive early-onset NBIA forms, including pantothenate kinase-associated neurodegeneration (PKAN; MIM #234200) due to PANK2 gene mutations, phospholipase-associated neurodegeneration (PLAN; MIM #610217) caused by PLA2G6 gene mutations, fatty acid hydroxylase-associated neurodegeneration (FAHN; MIM #611026) caused by FA2H gene mutations, and adult-onset aceruloplasminemia due to mutations of CP [4-8]. In addition to these, it is worth noting that high brain iron accumulation, similar to that identified in NBIA patients, has also been reported in patients with Kufor–Rakeb syndrome due to ATP13A2 mutations [9].

Mutations within C19orf12 have recently been identified in patients with NBIA, who showed hypointensity in the globus pallidus and substantia nigra on T2-weighted magnetic resonance (MR) images. The C19orf12-associated disease is clinically characterized by progressive spastic paraplegia or quadriplegia, optic atrophy, motor axonal neuropathy, and psychiatric signs. The age of onset ranges from 3 to 21 years, with a mean age of onset of 9 years. C19orf12 encodes a mitochondrial membrane protein of unknown function, and the associated disorder has been named MPAN (mitochondrial membrane protein-associated neurodegeneration; MIM #614298) [10, 11]. Intriguingly, neuropathological examination has demonstrated the presence of α-synuclein-positive Lewy bodies and Lewy neurites [10]. Most recently, C19orf12 mutations have also been reported in patients with predominant upper and lower motor neuron dysfunction resembling amyotrophic lateral sclerosis (ALS) [12]. T2-weighted magnetic resonance imaging (MRI) in these patients also demonstrated hypointensity of the globus pallidus and substantia nigra.

In this report, we present the clinical, radiological, and genetic findings of three cases with adult-onset NBIA, in whom C19orf12 mutations were identified though autozygosity mapping and conventional gene screening analyses. In summary, our patients presented with later age at onset and more severe disease progression than those previously reported, leading to early death in two. The pathogenic C19orf12 mutation we identified, p.Thr11Met, has been previously reported as a compound heterozygous mutation in patients with a phenotype similar to juvenile ALS [12], and in patients with a more typical NBIA phenotype as either homozygous or compound heterozygous mutations [10]. Thus, the phenotypic heterogeneity associated with this particular C19orf12 mutation is similar to the wide clinical spectrum seen with other NBIA disorders [13].

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Subjects

Two first-degree consanguineous Turkish families affected by an autosomal recessive form of NBIA were available for examination. Family 1 consisted of healthy parents as well as two affected and three unaffected siblings. Family 2 consisted of healthy parents as well as one affected and three unaffected siblings (Fig. 1b). The local ethics committee of the Department of Neurology at Mersin University approved this study, and blood samples were collected from all available family members (n = 10) after informed consent was obtained. DNA samples were isolated from peripheral blood using the Gentra Puregene Blood kit (Qiagen Inc., Valencia, CA).

image

Figure 1. (a) The chromosome 19 B-allele-frequency plots of the three affected cases are shown. The loss of heterozygosity (LOH) area, 19q12, shared among all cases is highlighted with a blue rectangule. (b) Pedigree structures of both NBIA4 families. Affected individuals are shown with filled rectangules. P.T11M/p.T11M: homozygous mutant carriers; p.T11M/−: heterozygous mutant carriers; −/−: homozygous wild-type carriers. (c) Sanger chromatograms of C19prf12 p.Thr11Met mutation showing both homozygous (upper sequence) and heterozygous (middle sequence) mutant alleles as well as homozygous wild-type alleles (bottom sequence).

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Cognitive and behavioral examination

The Mini-Mental State Examination (MMSE) was used for the assessment of cognition, whereas the Neuropsychiatric Inventory-Questionnaire (NPI-Q) was used to assess behavioral changes.

High-throughput SNP genotyping and autozygosity mapping

Single nucleotide polymorphism (SNP) genotyping was performed using the HumanOmniExpressExome beadchip (Illumina Inc., San Diego, CA) that contains >700,000 genome-wide markers plus additional >240,000 putative functional exonic variants. Scanning and genotyping data collection was carried out through the HiScanSQ system (Illumina Inc., San Diego, CA). A total of seven individuals were genotyped: all available family members from Family 1 (n = 5) and two family members from Family 2 (one unaffected and the only affected case) (Fig. 1b). After genotyping, genotyping quality assessments were undertaken according to the appropriate options within the Genome Studio (GS) program (Illumina Inc., San Diego, CA). plınk input reports were generated within the GS and uploaded to plınk v1.07 program [14]. Homozygous (autozygous) segments were identified using the runs of homozygosity tool within plınk, where a minimum physical size threshold of 1 Mb and at least 100 homozygous adjacent markers in length, including no more than two SNPs with missing genotypes and only one possible heterozygous genotype, were used as inclusion criteria. Subsequently, overlapping and potentially matching segments were also identified in plınk using an allelic matching of 0.99 as threshold. Homozygous segments were also visualized using the Illumina Genome Viewer within the GS program (Fig. 1a).

Gene screening analyses

The entire coding region and intron–exon boundaries of C19orf12 were polymerase chain reaction (PCR) amplified in all available DNA samples using primers previously designed through the public ExonPrimer design website (http://ihg.gsf.de/ihg/ExonPrimer.html) and FastStart PCR master mix (Roche Applied Science, Indianapolis, IN). All purified PCR products were then sequenced in both forward and reverse directions with Applied Biosystems BigDye terminator v3.1 sequencing chemistry as per the manufacturer's instructions. The resulting sequencing reactions were resolved on an ABI3130 genetic analyzer (Applied Biosytems, Foster city, CA) and analyzed using Sequencher 5.0 software (Gene Codes Corporation, Ann Arbor, MI).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Clinical examinations

Family 1, Case 1 (Fig. 1b): A 29-year-old man, who was born from healthy consanguineous parents, was admitted with gait difficulty and frequent falls. He had developed serious behavioral disturbances starting 9 months prior to examination, immediately after witnessing his sister's suicide by hanging. He subsequently developed symmetrical bradykinesia and hand tremor. A local neurologist initiated dopaminergic therapy, but as the symptoms progressed he was referred to our tertiary movement disorders clinic. Nine months after onset, neurological examination revealed dysarthria, dysphagia, slowed saccadic eye movements, hyperactive deep tendon reflexes, bilateral Babinski signs, right arm dystonia, shuffling gait, postural instability, and asymmetric bilateral bradykinesia and rigidity that were worse on the right side, likely in part due to the right arm dystonia. There was no response to levodopa/benserazide at the dose of 1500 mg/day. Cerebral MRI showed bilateral hypointensities in globus pallidus and substantia nigra in both T2-weighted and susceptibility-weighted (SW) images (Fig. 2a,d). His clinical symptoms progressed rapidly, developing severe dysphagia and respiratory insufficiency due to akinesia and aspiration; he died of pneumonia and respiratory arrest 3 months after admission.

image

Figure 2. Brain magnetic resonance imaging (MRI). (a) T2-weighted images for Case 1 of Family 1; (b) T2-weighted images for Case 2 of Family 1; (c) T2-weighted images for Case 1 of Family 2; (d) susceptibility-weighted (SW) images for Case 1 of Family 1; (e) SW images for Case 2 of Family 1; and (f) SW images for Case 1 of Family 2. All images showed decreased signal intensity in the globus pallidus and substantia nigra.

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Case 2: This 25-year-old man, the brother of Case 1, presented with a right hand tremor and bradykinesia for several months. Neurological examination revealed hypomimia, right hand resting tremor, bilateral symmetric bradykinesia and rigidity, hyperactive deep tendon reflexes, parkinsonian gait, and postural instability. There was no response to high doses of levodopa/benserazide at 1500 mg/day. Brain MRI was similar to that of Case 1 (Fig. 2b,e).

Family 2, Case 3 (Fig. 1b): A 32-year-old man, born from healthy consanguineous parents, was admitted with gait difficulty and bradykinesia. He had developed bilateral kinetic and resting tremors starting 4 years previously, followed by forgetfulness, compulsive shopping, and squandering money. He was unable to ride his bicycle after 1 year of symptoms. Subsequently, he developed bradykinesia and disturbances of gait and behavior. A local neurologist and a local psychiatrist initiated both dopaminergic and antipsychotic therapies; however, as the symptoms progressed he was referred to our tertiary movement disorders clinic. Neurological examination revealed dysarthria, hypophonia, slowed saccades, hyperactive deep tendon reflexes, bilateral Babinski signs, symmetric bilateral bradykinesia and rigidity, parkinsonian gait and postural instability. There was no response to levodopa/benserazide treatment at the dose of 1500 mg/day. Cerebral MRI showed bilateral hypointensity in globus pallidus and substantia nigra in both T2-weighted and SW images, similar to those identified in Case 1 (Fig. 2c,f). His symptoms progressed quickly and he died of respiratory arrest due to pneumonia 1 month after admission.

Complete blood count, serum biochemistry, including iron and creatine kinase, serum and 24-h urine copper, serum ceruloplasmin, and ferritin levels were normal for Cases 1 and 3. Saline-incubated peripheral blood smear repeated on three occasions revealed no acanthocytes in Cases 1 and 3. Electroneuromyography and ophthalmologic examination were normal, and neither neuropathy nor optical or retinal abnormalities were detected in Cases 1 and 3.

In summary, all three cases present with extrapyramidal and pyramidal symptoms as well as behavioral disturbances (Table 1). Cognitive impairment was also seen in Cases 1 and 3 but not in Case 2.

Table 1. Summary of the phenotypic characteristics identified in three Turkish NBIA patients with C19orf12 p.Thr11Met mutation
Phenotypic characteristicsFamily 1Family 2
Case 1Case 2Case 3
  1. MMSE, Mini-Mental State Examination; N.A, not applicable; NBIA, neurodegeneration with brain iron accumulation; NPI-Q, Neuropsychiatric Inventory-Questionnaire.

GenderMaleMaleMale
Age at onset (years)282529
Age at presentation (years)292532
Age of death (years)29N.A32
Pyramidal signs+++
Focal dystonia+
Parkinsonism+++
Dysphagia+
Dysarthria++
Gait difficulty++
Tremor+++
Ocular movements+
Optic atrophy
Polyneuropathy
Psychiatric/behavioral signs++
NPI-Q score33 39
Cognitive signs++
MMSE score202821
Response to dopaminergic therapy

Molecular analyses

To detect possible genetic mutations causing NBIA, DNA samples from all available family members were collected. Given the efficiency of homozygosity mapping (HM) in determining disease-associated genetic loci in inbred families [15, 16], HM through genome-wide SNP genotyping was first performed. As both families were from the same geographical region in the south of Turkey, these were analyzed together. These analyses led us to the identification of a single homozygous segment common to all three affected individuals but not shared by any of the genotyped unaffected individuals. This homozygous segment of 2 Mb and located on chromosome 19q12 was flanked by rs1297975 (29,732,516 bp) and rs4805669 (31,780,647 bp) SNPs. This disease-associated locus containing 613 consecutive SNPs in length only comprised eight different genes, including the C19orf12 gene, which is already known to be associated with NBIA [10]. Additional genes included were as follows: VSTM2B, POP4, PLEKHF1, CCNE1, C19orf2, ZNF536, and TSHZ3. None of these is known to be disease-associated genes. Subsequently, direct sequencing of the entire coding region of C19orf12 identified a disease-segregating mutation previously reported to be pathogenic [10, 11]. This mutation (p.Thr11Met) consists of a C to T transition that results in a threonine to methionine amino acid change (Fig. 1b,c).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In this study, we report two consanguineous Turkish families with NBIA due to C19orf12 mutations. As mutations in at least six genes may be responsible for NBIA disorders [1, 17] and some movement disorders genes are associated with multiple neurological phenotypes [18], HM, which has already proved its ability to identify disease-associated loci in consanguineous families with NBIA [7], was the method employed here for disease loci identification. HM led us to identify chromosome 19q12 as disease-associated locus and the subsequent gene screening analysis identified C19orf12 p.Thr11Met mutation as the pathogenic genetic defect (Fig. 1).

Since the discovery of C19orf12 in 2011, 29 cases and 11 different mutations have been reported (Fig. 3). Many subjects have typical basal ganglia-related symptoms [10, 11, 19], whereas others have upper and lower motor neuron dysfunction mimicking ALS [12]. These latter patients, who presented with neuropsychological abnormalities and optic atrophy, but not extrapyramidal signs, showed hypointensity on T2-weighted MRI not only in the globus pallidus and substantia nigra but also in the medial cerebral peduncles, which could explain their upper motor neuron dysfunction [12]. All C19orf12 mutation carriers reported to date, including the current cases, with either basal ganglia symptomatology or axonal neuropathy showed brain neuroimaging compatible with iron deposition in the globus pallidus and substantia nigra, while the typical eye-of-the-tiger sign of PKAN with central pallidal hyperintensity was not observed [10, 11, 19].

image

Figure 3. C19orf12 gene structure and known pathogenic mutations. C19orf12 encodes four different transcripts: transcript variant 1 (NM_001031726.3), transcript variant 2 (NM_031448.4), transcript variant 3 (NM_001256046.1), and transcript variant 4 (NM_001256047.1). The mutation reported in our cases, p.T11M, only occurs in transcript variant 1, which is shown here. The 11 known pathogenic mutations in C19orf12 are shown in red at their respective residues. Exons are outlined and coding regions are shaded. The predicted transmembrane domain (TMD) is shown as a purple wavy line; protein domain predictions were obtained through smart (smart.embl-heidelberg.de). AA, amino acids; bp, base pairs; cbp, coding base pairs; kb, kilobases.

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Extrapyramidal signs, such as dystonia and parkinsonism unresponsive to dopaminergic therapy, are present in ∼68% of MPAN patients, pyramidal signs in ∼76%, and motor axonal neuropathy present in ∼45%. Neuropsychiatric or behavioral disturbances, mental impairment, and optic atrophy are also reported in most cases [10, 11, 19]. In our patients, extrapyramidal and pyramidal signs were prominent but neither motor neuropathy nor optic atrophy was observed. Dopaminergic therapy was not beneficial.

Adult-onset PKAN is typically associated with slow disease progression [4]. Disease progression is typically even slower in the case of MPAN [10]. In PKAN, adult onset and slow disease progression may be attributable to the observation that the PANK2 mutations in these cases do not completely inactivate the protein. It is unclear why all our current MPAN cases, unlike those previously reported with the same mutation, presented with a later age at onset (25–29 years) than those previously reported [3-21], and why two of three showed very rapid disease progression, leading to death after 12 and 36 months of admission, respectively. This clearly suggests that probably other not-yet-identified genetic, epigenetic, or environmental factors may also play an important role in the phenotypic expression of MPAN, as has already been suggested for PANK and PLAN [4, 20].

Despite the wide phenotypic and genetic heterogeneity associated with NBIA syndromes, there are many phenotypic similarities shared among the recessive forms that may be explained by their protein functions and localizations. For instance, PANK2-, PLA2G6-, and C19orf12-encoding proteins share mitochondrial localization and their mitochondrial dysfunctions are probably triggered by the oxidative stress caused by high brain iron content. PANK2-, PLA2G6-, and FA2H-encoding proteins are associated with impairment of brain lipid metabolism, probably due to oxidative stress and neuronal apoptosis. In all conditions, aberrant iron homeostasis and accumulation result in neurodegeneration, possibly due to a common pathophysiologic mechanism [21, 22].

In conclusion, we present three new NBIA cases with C19orf12 mutations that presented with an adult-onset form without optic abnormalities and, in two cases, with a rapidly progressive extrapyramidal and pyramidal disorder. Further research is needed to examine the role of C19orf12 in NBIA and related diseases, which may further expand the phenotypic spectrum associated with its genetic defects and elucidate the function of its encoded protein. This information will provide critical insights into the pathological mechanisms by which these genetic defects lead to neurodegeneration, and direct progress into the development of effective therapeutic strategies for these devastating human disorders.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors would like to thank the patients and their relatives for their participation in this study, as well as Dr Fatma Kucukkeles and Dr Dilek Hamdanogullari for referring the families to our center. The authors also thank Parkinson's Disease Foundation for support (C. P.-R.).

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References