DOI: https://doi.org/10.21203/rs.3.rs-324151/v1
Pantothenate kinase-associated neurodegeneration (PKAN) is a rare hereditary neurodegenerative disease characterized by an accumulation of iron within the brain. In the present report, we describe a family with 4 affected siblings presenting with variable clinical manifestations, e.g., parkinsonian features, dystonia and slow disease progression over 5 years. Exome sequencing revealed a causative variant in the pantothenate kinase 2 gene (PANK2). Variant NM_024960.6:c.710C > T was homozygous in all affected subjects. Our report describes the first genetically confirmed cases of PKAN in the Egyptian population. Studying genetics of neurodegenerative diseases in different ethnicities is very important for determining clinical phenotypes and understanding pathomechanisms of these diseases.
Pantothenate kinase-associated neurodegeneration (PKAN) is a rare neurodegenerative disease characterized by abnormal accumulation of iron in distinct brain areas. It has an estimated worldwide incidence of 2 affected individuals in 1 million which is even lower among the African population (Brezavar and Bonnen 2019). However, in the heterogenous group of neurodegenerative disorders with brain iron accumulation (NBIA), it is considered to be the most common (Hayflick et al. 2003;Hartig et al. 2006). PKAN is a genetically inheritable disease following an autosomal recessive pattern with causative mutations in the pantothenate kinase 2 (PANK2) gene. Pantothenate kinases control the biosynthesis of the coenzyme A (Zhou et al. 2001).
Clinically, PKAN shows a wide range of signs and symptoms including motor manifestations, e.g., dystonia, parkinsonism, dysarthria, dysphagia and spasticity, psychiatric and cognitive impairment as well as oculomotor disturbances (Lee et al. 2016). According to age of onset and disease progression, PKAN is classified into typical and atypical types. Atypical PKAN is characterized by later onset and slower progression (Hartig et al. 2006). Genetic determinants of PKAN, however, are not well characterized. Additionally, the current knowledge about PKAN is mainly built on individual case reports and case series (Marshall et al. 2019). The disease progression, severity of symptoms and even correlation between specific mutations and the disease phenotype is highly variable (Tomic et al., 2015).
Neurogenetic studies about PKAN are scarce in North Africa. So far, only one case report described a female patient with PKAN in a consanguineous Moroccan family, identifying a causative homozygous deletion in PANK2 (Efthymiou et al., 2020). In the present study, we describe an Egyptian family with 4 affected members revealing slow progression of atypical PKAN. The patients had different ages of onset and showed variable clinical presentations (parkinsonism, dystonia), normal serum ferritin levels, absence of acanthocytes, the brain MRI characteristic eye-of-the-tiger sign, all caused by the same homozygous mutation in PANK2.
The family presented to the Movement Disorders Clinic, Department of Neurology at Ain Shams University Cairo, where six members (affected and healthy) were examined by three movement disorders experts (A.S.S., F.H. and G.U.H.). Blood samples were collected from healthy and affected members. Standard brain MRI at 1.5 Tesla with T1-, T2-, and FLAIR-sequences of patients II.3 and II.10 was performed.
Features | Patient II.3 | Patient II.4 | Patient II.10 |
|---|---|---|---|
Sex | Female | Female | Male |
Age at presentation (y) | 38 | 32 | 17 |
Age of onset (y) | 30 | 15 | 7 |
Disease duration (y) | 8 | 17 | 10 |
Symptoms at onset | Dysarthria, UL bradykinesia | Tongue protrusion, open jaw dystonia | Behavioral changes, open jaw dystonia |
Open jaw dystonia | Mild, sensory tricks | Present | Severe |
Tongue protrusion | - | Repeated | Occasional |
Limb dystonia | - | Present; more in UL (generalized dystonia) | Present; more in UL (generalized dystonia) |
Dysarthria | Present | Present | Severe (anarthria) |
Dysphagia | - | Present | Present |
Stuttering | Present | - | - |
Choking | - | Present | Present |
Sialorrhea | - | - | Present |
Parkinsonism | Present; symmetrical bradykinesia, rigidity, kinetic tremor, mainly UL, no response to levodopa | Present; mild rigidity of UL | - |
Pyramidal weakness | Present | Present | - |
Spasticity | Present | Present | - |
Plantar reflex | Extensor | Extensor | Flexor |
Deep tendon reflexes | Exaggerated | Exaggerated | Exaggerated |
Depression | Present | Present | - |
Anxiety | Present | Present | - |
Agitation | Occasional | Occasional | Present |
Behavioral changes | - | - | Present |
Cognition | Average (MMSE 26/30) | Impaired (MMSE 14/27) | No reported impairment |
Fundus | Normal | Normal | Normal |
Serum ferritin | Normal (188 ng/mL) | Normal (210 ng/mL) | Normal (199.5 ng/mL) |
Blood acanthocytosis | Absent | Absent | Absent |
Brain MRI scan | Eye-of-the-tiger-sign | Not available | Eye-of-the-tiger-sign |
Course of disease | Slowly progressing, mild worsening of OJD | Slowly progressing | Slowly progressing, mild worsening of LL dystonia |
OJD, Open jaw dystonia; UL, upper limbs; LL, lower limbs | |||
All patients had normal serum ferritin levels and did not show blood acanthocytosis. Brain magnetic resonance imaging (MRI) was done for patients II.3 and II.10 revealing the characteristic eye-of-the-tiger sign (see Fig. 2a, b).
Exome sequencing was performed in the two affected individuals II:4 and II:10. Genomic DNA libraries were captured using the Nextera Rapid Capture Expanded Exome Kit (Illumina, San Diego, CA), and DNA fragments were sequenced on an Illumina HiSeq2000 system with an average coverage of 80x. Variants were identified by a standard analysis pipeline and annotated using ANNOVAR (Wang et al. 2010). We discarded variants with a minor allele frequency (MAF) > 0.01 in gnomAD (Genome Aggregation Database, https://gnomad.broadinstitute.org) “all” exome as well as genome data. We discarded variants without an annotated exonic or splicing function and variants with a CADD (Combined Annotation Dependent Depletion) score below 15 (Kircher et al. 2014). Since the parents are related, we assumed autosomal recessive inheritance and homozygosity of the causative variant. However, to guarantee of not missing any variants of importance, we filtered for variants present in both II:4 and II:10 but not for homozygosity. We filtered for specific genes with the symbols ATP13A2, C19orf12, COASY, CP, DCAF17, FAH2, FTL, PANK2, PLA2G6, WDR45 known to harbor variants causing NBIA and related phenotypes. The filtering resulted in only one homozygous variant in the PANK2 gene, NM_024960.6:c.710C > T causing the amino acid change NP_079236.3:p.Thr237Met in the PANK2 protein in both exome sequenced affected family members (see Table 2 and Fig. 1). Segregation of this variant was ascertained by Sanger sequencing of all family members of whom DNA was available. We confirmed the presence of the variant and showed that patient II:3 also carries this variant in the homozygous state. The mother I:1 is a heterozygous carrier of the variant and her healthy son II:2 did not show this variant (see Fig. 1). DNA samples of the father I:2, the deceased affected brother II:9 and other siblings were not available for analysis.
Genetic finding | Variant |
|---|---|
Chromosome level | chr20.hg38:g.3918717C > T |
Genomic level | NC_000020.11:g.3918717C > T |
Coding sequence level | NM_024960.6:c.710C > T |
Protein level | NP_079236.3:p.Thr237Met |
CADD (Phred-scaled) | 22 |
MutationTaster (score/ class) | 0.926/deleterious |
PolyPhen-2 HVAR (score/ class) | 0.242/benign |
gnomAD exomes (MAF, nr. of alleles analyzed) | 0.000012 |
dbSNP (153 all) | rs137852967 |
HGMD (public 01.08.21) | Not listed |
ClinVar | Pathogenic, multiple submitters, no conflict |
CADD, Combined Annotation Dependent Depletion; dbSNP, database of single nucleotide polymorphism; gnomAD, Genome Aggregation Database; HGMD, Human Gene Mutation Database; MAF, minor allele frequency | |
The effects of amino acid substitutions on protein function were predicted using MutationTaster (Schwarz et al. 2014), PolyPhen-2 (Adzhubei et al. 2010), and CADD (Rentzsch et al. 2019). Furthermore, we searched the public version of the Human Gene Mutation Database (Stenson et al. 2020) and ClinVar (Landrum et al. 2018) for the variant (see Table 2).
In the last two decades, genetic research in neurodegenerative diseases has tremendously progressed, especially through focused analyses of families with Mendelian mode of inheritance in different populations. However, such progress is not globally oriented. Including more genetic findings from understudied populations such as Africans, will help to identify variable phenotypes, enhance discoveries and offer better understanding of the diseases’ pathophysiology and genotype-phenotype correlation.
Our report confirms the pathogenicity of the PANK2 variant NM_024960.6:c.710C > T which causes PKAN. Although this variant has been described before, this is the first report of a pathogenic PANK2 variant in the Egyptian population. Homozygous and compound heterozygous variants in PANK2 have been identified as the most common cause of NBIA (Zhou et al. 2001;Hayflick et al. 2003). The here identified variant has been reported by multiple submitters to the ClinVar database (Landrum et al. 2018) as pathogenic and can be found in other published genetic studies of NBIA (Hartig et al. 2006) but is not listed in the public version of the HGMD (Human Gene Mutation Database). Because the homozygous state of this variant segregates in the examined Egyptian family with the disease and has already been shown to be pathogenic, it is clearly the cause of NBIA in this Egyptian family.
Several variants in PANK2 have been identified in PKAN cases. The most common is the PANK2 variant G521R which accounts for approximately 30% of cases (Zhou et al. 2001). This variant leads to a catalytically inactive Pank2 protein due to improper folding (Zhang et al. 2006). As Pank2 is localized to the mitochondria where it is a key enzyme in the biosynthesis of coenzyme A (Leonardi et al. 2007), influencing important metabolic processes, nonfunctional Pank2 proteins have a tremendous influence on cell energy processes. However, the PANK2 variant (T528M) which we have identified in the Egyptian family is less common for PKAN and does not induce catalytic or regulatory deficits (Zhang et al. 2006). This implies that other presently unknown effects and functions of this variant might contribute to its pathogenicity.
Remarkably, the identified PANK2 mutation led to variable phenotypes and ages of onset within the same family. Previous studies reported the mutation to cause predominant open jaw dystonia, limb dystonia, and dysarthria (Hartig et al. 2006;Tomic et al. 2015;Yapici et al. 2016). Predominant parkinsonism was less commonly reported (Chang et al. 2020). However, in the present family we could observe combinations of these symptoms. The affected members showed early cranial symptoms and a slow course of disease progression compared to previous reports (Tomic et al. 2015). This milder course of late onset PKAN revealed no contractures and preserved ambulation, however, further follow-up is required. Our finding of a clinical variability within the reported family confirms the relevance of atypical PKAN as a differential diagnosis for familial movement disorders with variable phenotypes such as Wilson’s disease (Schneider et al. 2006;Shalash et al. 2014).
The current report describes variable clinical phenotypes and disease progression of atypical PKAN in affected members of an Egyptian family. To the best of our knowledge, it is the second report of a family with PKAN in Africa. These findings add to our knowledge about the genetics of this disease in North Africa.
AUTHOR CONTRIBUTIONS
All authors contributed to the study concept and design. ASS, MS, GK, TWR, FH, GUH and SHM collected and analyzed data. ASS, GUH, HSA, FH and GK carried out medical examinations. MS, ASS, TWR and GK drafted the manuscript. All authors corrected and approved the final manuscript.
FUNDING
This work was supported by the German Academic Exchange Service (DAAD) through the funding programme “Higher Education Dialogue with the Muslim World”, project “GeneFINDER”.
ACKNOWLEDGEMENTS
We would like to thank Lena Jaschkowitz for technical assistance.
CONFLICT OF INTEREST
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
DATA AVAILABILITY
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.