Novel Compound Heterozygous PRKN Variants in a Han-Chinese Family with Early-Onset Parkinson's Disease

Genetic factors are thought to play an important role in the pathogenesis of Parkinson's disease (PD), particularly early-onset PD. The PRKN gene is the primary disease-causing gene for early-onset PD. The details of its functions remain unclear. This study identified novel compound heterozygous variants (p.T240K and p.L272R) of the PRKN gene in a Han-Chinese family with early-onset PD. This finding is helpful in the genetic diagnosis of PD and also the functional research of the PRKN gene.


Introduction
Parkinson's disease (PD) is the second most frequent neurodegenerative disease after Alzheimer's disease [1]. Parkinsonism, the core clinical feature of PD, is defined as slowly progressive bradykinesia combined with rest tremor or rigidity [2]. e etiology of PD remains enigmatic, while environmental and genetic factors are thought to be involved in [3]. Currently, 23 disease-causing loci and 19 genes have been identified for PD and recorded in the Online Mendelian Inheritance in Man (OMIM) [4]. Although PD mainly affects those over 50, early-onset PD (EOPD) patients, whose motor disorder symptoms appear before age 40, account for 3-5% of all PD patients worldwide [5]. EOPD's primary genetic type is autosomal recessive juvenile parkinsonism (AR-JP, OMIM 600116) caused by homozygous or compound heterozygous mutations in the parkin RBR E3 ubiquitin protein ligase gene (PRKN) [6]. It has parkinsonism symptoms, but may be a different disease entity from late-onset sporadic PD.
is is suggested by more dystonia at onset, better levodopa responsiveness, slower disease progression, sleep benefit, and lower frequencies of nonmotor symptoms and Lewy bodies [7][8][9].
is gene encodes the parkin protein which could ubiquitinate numerous mitochondrial outer membrane proteins resulting in autophagy of damaged mitochondria, and PRKN mutations caused mitochondrial quality control deficiencies and neuron death [10]. e parkin protein has been intensely studied due to its complex activation mechanisms and suppressive roles in various tumors [11,12]. is study reports on new compound heterozygous variants in the PRKN gene in a family with EOPD ( Figure 1).
is finding expands the PRKN-associated PD genetic spectrum and may provide new insights into parkin protein structures and functions.

Materials and Methods
A Han-Chinese family with EOPD was recruited from China's Shandong Province (Figure 1(a)). e proband, her brother, and son were examined and diagnosed by two experienced neurologists. Two hundred unrelated healthy Han-Chinese older than 40 were enrolled as controls. is study was approved by the Institutional Review Board of the ird Xiangya Hospital of Central South University, Changsha, Hunan, China. After written informed consents were obtained, peripheral venous blood samples were taken from all participants. gDNA isolated from the proband (II : 2) was target captured by a PD-associated gene panel. Subsequently, paired-end sequencing using Illumina HiSeq X-ten platform (Illumina Inc., USA) was performed. All potential variants were filtered according to the Single Nucleotide Polymorphism database, the 1000 Genomes Project, and the Exome Aggregation Consortium database.

Clinical Features.
e age at onset of the proband (II : 2) was 32 years. e initial symptoms were slowness and rest tremor in her right arm, and tightness appeared one year later. ese motor symptoms slowly progressed and spread to the right leg and contralateral limbs over a period of six years. Levodopa therapy significantly improved motor symptoms as of her first examination at age 35 years, but detailed evaluation was not performed. At her latest evaluation, at age 39 years, clinically established PD was diagnosed on the basis of prominent bradykinesia symptom and other PD-related symptoms, including rest tremor, rigidity, face masking, numbness, difficulty falling asleep, olfactory impairment, constipation, mild cognitive impairment, depression, and anxiety. Her MDS Unified Parkinson's Disease Rating Scale motor score was 9 (on)/14 (off), and Mini-Mental State Examination score was 22. Skull CT and MRI results, blood and urine copper levels, and ceruloplasmin were all normal. No movement disorders or other nervous system disorders were found in her son (III : 1) or older brother (II : 1).

Molecular Findings.
After target capture sequencing and filtering, only the p.T240K (c.719C > A, rs137853054) and p.L272R (c.815T > G) variants of PRKN gene were considered as pathogenic candidates in the known monogenic PD-causing genes. Subsequent Sanger sequencing confirmed both in the proband (Figures 1(b) and 1(c)). e p.T240K variant was found in her son (III :1). e two variants were absent from her older brother (II :1) and from the 200 normal controls. e p.T240K variant has a very low recorded heterozygous state frequency in the Genome Aggregation Database (gnomAD, 7.954 × 10 − 6 ). SIFT and CADD predicted the c.719C > A (p.T240K) variant as damaging (Table 1). e p.L272R variant has not been reported in the gnomAD and also has a damaging prediction in SIFT and CADD analysis ( Table 2). Multisequence alignment shows that the leucine at position 272 is phylogenetically conserved from fruit flies to humans (Figure 1(e)).
ese data indicate that the compound heterozygous variants, p.T240K and p.L272R, are probably disease-causing for EOPD in this family.

Discussion
e parkin protein is a 465-amino acid E3 ubiquitin ligase of the RING-between-RING (RBR) family, which could catalyse the transfer of ubiquitin from the E2 conjugating enzyme to substrate proteins [10]. Numerous recent studies attempted to determine the relationship between the parkin protein structure and its functions through in vitro and animal experiments. e most direct and compelling evidence for protein structure change effects remains the mutations, particularly missense mutations detected in patients. Approximately 25% of all PRKN gene mutations have been found in the RING1 domain, which is considered to have a binding site for the E2 conjugating enzyme and be important to parkin activation due to its interactions with Ser65-phosphorylated ubiquitin and the UbL domain ( Figure 2) [10,14,15,[21][22][23][24][25][26][27][28][29].
e detailed molecular structure of parkin remains unclear, which results in several different activation and catalysis models [30].
In this study, clinically established EOPD and extensive nervous system impairment were suggested by the presence of three cardinal motor symptoms and multiple nonmotor symptoms in the proband. Two missense variants (p.T240K   [6,[14][15][16][17][18][19][20][21]. is amino acid is in the first Znbinding loop of the RING1 domain which has been regarded as the E2 binding site of parkin [31]. e p.T240R mutation has been found to change the E2 binding interface and destroy the autoubiquitination activation of parkin protein and lead to EOPD [31,32]. e first α-helix (260-273) of the RING1 domain was reported as the binding site for the UbL domain, which adjoins the RING1 domain through hydrophobic interaction to block E2 access [10,33]. ree other missense variants (p.L272I, p.L272V, and p.L272F) and one synonymous variant in the 272nd codon have been recorded. However, no pathogenic evidence has been reported for these four variants, which may be due to low allele frequencies in generating populations and preservation of hydrophobicity (Table 2). e p.L272R, resulting in materially hydrophilic alternation, is more likely to disrupt protein folding and affect its function, especially in a compact protein stabilized by numerous hydrophobic interactions such as parkin (Table 2) [34,35]. Investigating the structural changes resulting from the p.L272R variant may contribute to understanding parkin domain interactions and their potential function. More evidence including segregation information and functional research is needed to classify the two variants as pathogenic or likely pathogenic variants according to the American College of Medical Genetics and Genomics guidelines for variants interpretation [36].
In conclusion, the novel compound heterozygous variants of PRKN gene, p.T240K and p.L272R, were identified as the probable genetic cause for EOPD in a family with clinically established EOPD. ese two missense variants  Figure 2: (a) Domain structure and missense variants associated with autosomal recessive parkinsonism detected in the RING1 domain of parkin protein [10,14,15,21,[23][24][25][26][27][28]. Two variants detected in this study are highlighted in bold. β-Strand and α-helix are in dark blue and pink, respectively. Zinc-binding cysteines are signed with orange dots. (b) Cartoon representation of the p.T240K and p.L272R variants in parkin protein by PyMOL. Parkin protein crystal structure (PDB code 5C1Z) was used as a template [29]. Variants are shown as red stick models, and Zn 2+ are shown as spheres. pUb, Ser65-phosphorylated ubiquitin.
both lead to amino acid changes in the RING1 domain. is finding has potential value for functional research of the PRKN gene and genetic diagnosis of PD. Further studies are warranted to clarify their pathogenicity and may offer deeper understanding of the detailed functional effects.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Disclosure
Kuan Fan and Pengzhi Hu should be considered co-first authors.

Conflicts of Interest
e authors declare that they have no conflicts of interest.