Gene therapy for monogenic disorders: challenges, strategies, and perspectives

Monogenic disorders refer to a group of human diseases caused by mutations in single genes. While disease-modifying therapies have offered some relief from symptoms and delayed progression for some monogenic diseases, most of these diseases still lack effective treatments. In recent decades, gene therapy has emerged as a promising therapeutic strategy for genetic disorders. Researchers have developed various gene manipulation tools and gene delivery systems to treat monogenic diseases. Despite this progress, concerns about inefficient delivery, persistent expression, immunogenicity, toxicity, capacity limitation, genomic integration, and limited tissue specificity still need to be addressed. This review gives an overview of commonly used gene therapy and delivery tools, along with the challenges they face and potential strategies to counter them.

Both gain-and loss-of-function variants of KCNA1 are associated with paroxysmal kinesigenic dyskinesia

KCNA1 is the coding gene for Kv1.1 voltage-gated potassium-channelαsubunit.Three variants of KCNA1 have been reported to manifest as paroxysmal kinesigenic dyskinesia(PKD),but the correlation between them remains unclear due to the phenotypic complexity of KCNA1 variants as well as the rarity of PKD cases.Using the whole exome sequencing followed by Sanger sequencing,we screen for potential pathogenic KCNA1 variants in patients clinically diagnosed with paroxysmal movement disorders and identify three previously unreported missense variants of KCNA1 in three unrelated Chinese families.The proband of one family(c.496G>A,p.A166T)manifests as episodic ataxia type 1,and the other two(c.877G>A,p.V293I and c.1112C>A,p.T371A)manifest as PKD.The pathogenicity of these variants is confirmed by functional studies,suggesting that p.A166T and p.T371A cause a loss-of-function of the channel,while p.V293I leads to a gain-of-function with the property of voltage-dependent gating and activation kinetic affected.By reviewing the locations of PKD-manifested KCNA1 variants in Kv1.1 protein,we find that these variants tend to cluster around the pore domain,which is similar to epilepsy.Thus,our study strengthens the correlation between KCNA1 variants and PKD and provides more information on genotype–phenotype correlations of KCNA1 channelopathy.

Paroxysmal Kinesigenic Dyskinesia:Genetics and Pathophysiological Mechanisms

Paroxysmal kinesigenic dyskinesia(PKD),the most common type of paroxysmal movement disorder,is characterized by sudden and brief attacks of choreoathetosis or dystonia triggered by sudden voluntary movements.PKD is mainly caused by mutations in the PRRT2 or TMEM151A gene.The exact pathophysiological mechanisms of PKD remain unclear,although the function of PRRT2 protein has been well characterized in the last decade.Based on abnormal ion channels and disturbed synaptic transmission in the absence of PRRT2,PKD may be channelopathy or synaptopathy,or both.In addition,the cerebellum is regarded as the key pathogenic area.Spreading depolarization in the cerebellum is tightly associated with dyskinetic episodes.Whereas,in PKD,other than the cerebellum,the role of the cerebrum including the cortex and thalamus needs to be further investigated.

Alzheimer’s disease early diagnostic and staging biomarkers revealed by large-scale cerebrospinal fluid and serum proteomic profiling

Amyloid-b,tau pathology,and biomarkers of neurodegeneration make up the core diagnostic biomarkers of Alzheimer disease(AD).However,these proteins represent only a fraction of the complex biological processes underlying AD,and individuals with other brain diseases in which AD pathology is a comorbidity also test positive for these diagnostic biomarkers.More ADspecific early diagnostic and disease staging biomarkers are needed.In this study,we performed tandem mass tag proteomic analysis of paired cerebrospinal fluid(CSF)and serum samples in a discovery cohort comprising 98 participants.Candidate biomarkers were validated by parallel reaction monitoring–based targeted proteomic assays in an independent multicenter cohort comprising 288 participants.We quantified 3,238 CSF and 1,702 serum proteins in the discovery cohort,identifying 171 and 860 CSF proteins and 37 and 323 serum proteins as potential early diagnostic and staging biomarkers,respectively.In the validation cohort,58 and 21 CSF proteins,as well as 12 and 18 serum proteins,were verified as early diagnostic and staging biomarkers,respectively.Separate 19-protein CSF and an 8-protein serum biomarker panels were built by machine learning to accurately classify mild cognitive impairment(MCI)due to AD from normal cognition with areas under the curve of 0.984 and 0.881,respectively.The 19-protein CSF biomarker panel also effectively discriminated patients with MCI due to AD from patients with other neurodegenerative diseases.Moreover,we identified 21 CSF and 18 serum stage-associated proteins re-flecting AD stages.Our findings provide a foundation for developing bloodbased tests for AD screening and staging in clinical practice.

Chinese patients with adult onset leukodystrophy caused by CST3 variants

Leukodystrophies represent a group of cerebral white matter disorders that mainly affect axon-glia units.The disorders are clinically diverse and display significant genetic variability.It is challenging to differentiate hereditary leukodystrophies,particularly those that present in adulthood,from acquired leukodystrophies and other genetic disorders,such as multiple sclerosis(MS)or hereditary spastic paraplegia(HSP)(Wei et al.,2021).Over the past few decades,a series of causative genes associated with leukodystrophies have been identified(Kohler et al.,2018).Nevertheless,a significant proportion of patients still lack a precise molecular diagnosis.

Mutation Analysis of MR-1, SLC2A 1, and CLCN1 in 28 PRRT2-negative Paroxysmal Kinesigenic Dyskinesia Patients

Background： Paroxysmal kinesigenic dyskinesia （PKD） is the most common subtype of paroxysmal dyskinesias and is caused by mutations in PRRT2 gene. The majority of familial PKD was identified to harbor PRRT2 mutations. However, over two-third of sporadic PKD patients did not carry anyPRRT2 mutation, suggesting an existence of additional genetic mutations or possible misdiagnosis due to clinical overlap. Methods： A cohort of 28 Chinese patients clinically diagnosed with sporadic PKD and excluded PRRT2 mutations were recruited, Clinical features were evaluated, and all subjects were screened for MR-l, SLC2A1, and CLCN1 genes, which are the causative genes of paroxysmal nonkinesigenic dyskinesia （PNKD）, paroxysmal exertion-induced dyskinesia, and myotonia congenita （MC）, respectively, In addition, 200 genetically matched healthy individuals were recruited as controls. Results： A total of 16 genetic variants including 4 in MR-1 gene, 8 in SLC2A1 gene, and 4 in CLCN1 gene were detected. Among them, SLC2A1 c.363G〉A mutation was detected in one case, and CLCN1 c. 1205C〉T mutation was detected in other two cases. Neither of them was found in 200 controls as well as 1000 Genomes database and ExAC database. Both mutations were predicted to be pathogenic by SIFT and PolyPhen2. The SLC2A 1 c.363G〉A mutation was novel. Conclusions： The phenotypic overlap may lead to the difficulty in distinguishing PKD from PNKD and MC. For those PRRT2-negative PKD cases, screening of SLC2A1 and CLCN1 genes are useful in confirming the diagnosis.

Challenges and suggestions for precise diagnosis and treatment of Wilson’s disease

Hepatolenticular degeneration,also known as Wilson's disease(WD),is an autosomal recessive disorder of copper dysfunction.The causative gene ATP7B encodes a copper transporting P-type ATPase,and the pathogenic mutations within ATP7B may lead to impaired copper excretion via the biliary tract and to massive copper accumulation in the liver,brain,kidney,bone and joints,cornea and other tissues and organs[1-3].Therefore,individuals with Wilson's disease may suffer liver damage,neuropsychiatric expressions,renal damage and osteoarthropathy.In general,the clinical manifestations of WD are complex and diverse,and early diagnosis is difficult.

Variants of Interleukin-7/Interleukin-7 Receptor Alpha are Associated with Both Neuromyelitis Optica and Multiple Sclerosis Among Chinese Han Population in Southeastern China

Background： Neuromyelitis optica （NMO） and multiple sclerosis （MS） are autoimmune demyelinating diseases of the central nerve system, lnterleukin-7 （IL-7） and interleukin-7 receptor alpha （IL-7Rα） were proved to be important in the pathogenesis of both diseases because of the roles they played in the differentiations of autoimmune lymphocytes. The variants of both genes had been identified to be associated with MS susceptibility in Caucasian, Japanese and Korean populations. However, the association of these variants with NMO and MS has not been well studied in Chinese Southeastern Han population. Here, we aimed to evaluate the association of six IL-7 variants （rsl520333, rs1545298, rs4739140, rs6993386, rs7816065, and rs2887502） and one variant of IL-7RA （rs6897932） with NMO and MS among Chinese Han population in southeastern China. Methods： Matrix-assisted laser desorption/ionization time of flight mass spectrometry （MassARRAY system） and Sanger sequencing were used to determine the variants of IL-7 and IL-7RA in 167 NMO patients, 159 MS patients and 479 healthy controls among Chinese Han population in southeastern China. Samples were excluded if the genotyping success rate 〈90%. Results： Statistical differences were observed in the genotypes of IL-7 rs1520333 in MS patients and IL-7RA rs6897932 in NMO patients, compared with healthy controls （P = 0.035 and 0.034, respectively）. There was a statistically significant difference in the genotypes of IL-7 rs2887502 between MS and NMO patients （P = 0.014）. And there were statistically significant differences in the rs6897932 genotypes （P = 0.004） and alleles （P = 0.042） between NMO-IgG positive patients and healthy controls. Conclusions： The study suggested that among Chinese Hart population in southeastern China, the variant of IL-7RA （rs6897932） was associated with NMO especially NMO-IgG positive patients while the variant of IL-7 （rs1520333） with MS patients. And the genotypic differences of IL-7 rs2887502 between MS and NMO indicated the different genetic backgrounds of these two diseases.

Paroxysmal kinesigenic dyskinesia and myotonia congenita in the same family:coexistence of a PRRT2 mutation and two CLCN1 mutations

Paroxysmal kinesigenic dyskinesia（PKD） and myotonia congenita（MC） are independent disorders that share some clinical features. We aimed to investigate the sequences of PRRT2 and CLCN1 in a proband diagnosed with PKD and suspected MC. Clinical evaluation and auxiliary examinations were performed. Direct sequencing of the entire coding regions of the PRRT2 and CLCN1 genes was conducted. Haplotype analysis confirmed the relationships among the family members. The proband suffered choreoathetosis attacks triggered by sudden movements, and lower-limb weakness a n d s t i ff n e s s t h a t w o r s e n e d i n c o l d w e a t h e r. Carbamazepine monotherapy completely controlled his choreoathetosis and significantly relieved his limb weakness and stiffness. His father, when young, had similar limb stiffness, while his mother and brother were asymptomatic. Genetic analysis revealed that the proband and his father harbored a PRRT2 c.649 dup C mutation, and CLCN1 c.1723C〉T and c.2492A〉G mutations. His brother carried only the two CLCN1 mutations. None of these mutations were identified in his mother and 150 unrelated controls. This is the first report showing the coexistence ofPRRT2 and CLCN1 mutations. Our results also indicate that both the PRRT2 and CLCN1 genes need to be screened if we fail to identify PRRT2 mutations in PKD patients or CLCN1 mutations in MC patients.

Wilson's Disease in China

Wilson＇s disease（WD） is an autosomal recessive disorder of copper metabolism. Its incidence is higher in China than in western countries. ATP7 B is the causative gene and encodes a P-type ATPase, which participates in the synthesis of holoceruloplasmin and copper excretion. Disease-causing variants of ATP7 B disrupt the normal structure or function of the enzyme and cause copper deposition in multiple organs,leading to diverse clinical manifestations. Given the variety of presentations, misdiagnosis is not rare. Genetic diagnosis plays an important role and has gradually become a routine test in China. The first Chinese spectrum of disease-causing mutations of ATP7 B has been established. As a remediable hereditary disorder, most WD patients have a good prognosis with an early diagnosis and chelation treatment. However, clinical trials are relatively few in China, and most treatments are based on the experience of experts and evidences from other countries. It is necessary to study and develop appropriate regimens specific for Chinese WD patients.

Genotype-phenotype correlations of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis(ALS)is a devastating neurodegenerative disease characterized by progressive neuronal loss and degeneration of upper motor neuron(UMN)and lower motor neuron(LMN).The clinical presentations of ALS are heterogeneous and there is no single test or procedure to establish the diagnosis of ALS.Most cases are diagnosed based on symptoms,physical signs,progression,EMG,and tests to exclude the overlapping conditions.Familial ALS represents about 5~10% of ALS cases,whereas the vast majority of patients are sporadic.To date,more than 20 causative genes have been identified in hereditary ALS.Detecting the pathogenic mutations or risk variants for each ALS individual is challenging.However,ALS patients carrying some specific mutations or variant may exhibit subtly distinct clinical features.Unraveling the respective genotype-phenotype correlation has important implications for the genetic explanations.In this review,we will delineate the clinical features of ALS,outline the major ALS-related genes,and summarize the possible genotype-phenotype correlations of ALS.

Penetrance estimation of PRRT2 variants in paroxysmal kinesigenic dyskinesia and infantile convulsions

Proline-rich transmembrane protein 2(PRRT2)is the leading cause of paroxysmal kinesigenic dyskinesia(PKD),benign familial infantile epilepsy(BFIE),and infantile convulsions with choreoathetosis(ICCA).Reduced penetrance of PRRT2 has been observed in previous studies,whereas the exact penetrance has not been evaluated well.The objective of this study was to estimate the penetrance of PRRT2 and determine its influencing factors.We screened 222 PKD index patients and their available relatives,identified 39 families with pathogenic or likely pathogenic(P/LP)PRRT2 variants via Sanger sequencing,and obtained 184 PKD/BFIE/ICCA families with P/LP PRRT2 variants from the literature.Penetrance was estimated as the proportion of affected variant carriers.PRRT2 penetrance estimate was 77.6%(95%confidence interval(CI)74.5%–80.7%)in relatives and 74.5%(95%CI 70.2%–78.8%)in obligate carriers.In addition,we first observed that penetrance was higher in truncated than in non-truncated variants(75.8%versus 50.0%,P=0.01),higher in Asian than in Caucasian carriers(81.5%versus 68.5%,P=0.004),and exhibited no difference in gender or parental transmission.Our results are meaningful for genetic counseling,implying that approximately three-quarters of PRRT2 variant carriers will develop PRRT2-related disorders,with patients from Asia or carrying truncated variants at a higher risk.

Three-Dimensional Heterogeneity of Cerebellar Interposed Nucleus-Recipient Zones in the Thalamic Nuclei

The cerebellum is conceptualized as a processor of complex movements and is also endowed with roles in cognitive and emotional behaviors.Although the axons of deep cerebellar nuclei are known to project to primary thalamic nuclei,macroscopic investigation of the characteristics of these projections,such as the spatial distribution of recipient zones,is lacking.Here,we studied the output of the cerebellar interposed nucleus(IpN)to the ventrolateral(VL)and centrolateral(CL)thalamic nuclei using electrophysiological recording in vivo and trans-synaptic viral tracing.We found that IpN stimulation induced mono-synaptic evoked potentials(EPs)in the VL but not the CL region.Furthermore,both the EPs induced by the IpN and the innervation of IpN projections displayed substantial heterogeneity across the VL region in three-dimensional space.These findings indicate that the recipient zones of IpN inputs vary between and within thalamic nuclei and may differentially control thalamo-cortical networks.

A De novo Mutation in Dystrophin Causing Muscular Dystrophy in a Female Patient

Background： Duchenne muscular dystrophy （DMD） and Becker muscular dystrophy （BMD） are X-linked recessive neuromuscular diseases resulting from dystrophin （DMD） gene mutations. It has been known that the carrier of DMD mutations may also have symptoms of the disease. While de novo mutation is quite common in BMD/DMD patients, it is rarely reported in the female carriers. Methods： Two sporadic Chinese patients with progressive muscular dystrophy and their familial members were recruited. The targeted next-generation sequencing （NGS） and the multiplex ligation-dependent probe analysis （MLPA） were performed in the proband. Blood tests, electrocardiography, echocardiography, and electromyography were also evaluated. Results： Two novel mutations of DMD gene were identified, c.7318C〉T（p.Q2440＊） in the male proband and c.4983dupA（p.A1662Sfs＊24） in the female carrier. The MLPA analysis did not detect any large rearrangements. The haplotype analysis indicated that the two mutations were derived from de novo mutagenesis. Conclusions： We identified two novel de novo mutations of DMD gene in two Chinese pedigrees, one of which caused a female patient with muscular dystrophy. The mutational analysis is important for DMD patients and carriers in the absence of a family history. The NGS can help detect the mutations in MLPA-negative patients.

Effect of Apolipoprotein E Genotypes on Huntington’s Disease Phenotypes in a Han Chinese Population

Huntington's disease(HD)is an autosomal dominant degenerative disease that mainly encompasses movement,cognition,and behavioral symptoms.The apolipoprotein E(APOE)gene is thought to be associated with many neurodegenerative diseases.Here,we enrolled a cohort of 223 unrelated Han Chinese patients with HD and1241 unrelated healthy controls in Southeastern China and analyzed the correlation between APOE genotypes and HD phenotypes.The results showed that the frequency of the E4 allele(7.1%)in HD patients was statistically less than that in controls(12.0%)(P =0.004).In addition,we divided patients into motor-onset and non-motor-onset groups,and analyzed the relationship with APOE genotypes.The results,however,were negative.Furthermore,the age at onset(AAO),defined as the age at the onset of motor symptoms,was compared in each APOE genotype subgroup and multivariate regression analysis was used to exclude the interference of CAG repeat length on AAO,but no association was found between APOE genotypes and AAO.Finally,we analyzed adult-onset HD to exclude the interference caused by juvenile HD(n = 13),and the results were negative.Therefore,our study suggests that APOE may not be a genetic modifier for HD,especially for adult-onset HD among Chinese of Han ethnicity.To the best of our knowledge,this is the first study of the correlation between APOE genotypes and HD phenotypes in a Han Chinese population.

A Novel Missense Mutation in Peripheral Myelin Protein-22 Causes Charcot-Marie-Tooth Disease

Background：Charcot-Marie-Tooth disease （CMT） is the most common inherited peripheral neuropathy.A great number of causative genes have been described in CMT,and among them,the heterozygous duplication of peripheral myelin protein-22 （PMP22） is the major cause.Although the missense mutation in PMP22 is rarely reported,it has been demonstrated to be associated with CMT.This study described a novel missense mutation of PMP22 in a Chinese family with CMT phenotype.Methods：Targeted next-generation sequencing （NGS） was used to screen the causative genes in a family featured with an autosomal dominant demyelinating form of CMT.The potential variants identified by targeted NGS were verified by Sanger sequencing and classified according to the American College of Medical Genetics and Genomics standards and guidelines.Further cell transfection studies were performed to characterize the function of the novel variant.Results：Using targeted NGS,a novel heterozygous missense variant in PMP22 （c.320G〉A,p.G107D） was identified.In vitro cell functional studies revealed that mutant PMP22 protein carrying p.G 107D mutation lost the ability to reach the plasma membrane,was mainly retained in the endoplasmic reticulum,and induced cell apoptosis.Conclusions：This study supported the notion that missense mutations in PMP22 give rise to a CMT phenotype,possibly through a toxic gain-of-function mechanism.

Advance in the pathogenesis and treatment of Wilson disease

Wilson disease is an autosomal recessive disorder of copper metabolism.Diagnosis depends primarily on clinical features,biochemical parameters and the presence of the Kayser-Fleischer ring.Genetic analysis for mutations within ATP7B is a convincing diagnostic tool.The traditional treatment for WD includes chelation of excessive copper accumulation and reduction of copper intake.Medical therapy is effective but WD is not yet curable.Liver transplantation is especially helpful for patients who fail to respond to medical therapy or present with fulminant liver failure,although evaluation of its long-term effect are still in need.

Huntington Disease in Asia

Objective： The objective was to review the major di fferences of Huntington disease （HD） in Asian population fiom those in the Caucasian population. Data Sources： Data cited in this review were obtained from PubMed database and China National Knowledge Infrastructure （CN KI） fiom 1994 to 2014. All the papers were written in English or Chinese languages, with the terms of Asia/Asian, H D, genotype, epidemiology, phenotype, and treatment used for the literature search. Study Selection： From the PubMed database, we included the articles and reviews which contained the HD patients＇ data from Asian countries. From the CNKI, we excluded the papers which were not original research. Due to tile language＇s restrictions, those data published in other languages were not included. Results： In total, 50 papers were cited in this review, authors of which were from tile mainland of China, .lapan, India, Thailand, Taiwan （China）, Korea, and western countries. Conclusions： The lower epidemiology in Asians can be partly explained by the less cytosine-adenine-guanine repeats, different haplotypes, and CCG polymorphisms. For the physicians, atypical clinical profiles such as the initial symptom of ataxia, movement abnormalities of Parkinsonism, dystonia, or tics need to be paid more attention to and suggest gene testing if necessary. Moreover, some pathogenesis studies may help progress some new advanced treatments. The clinicians in Asian especially in China should promote the usage of genetic testing and put more effects in rehabilitation, palliative care, and offer comfort of patients and their families. The unified HD rating scale also needs to be popularized in Asia to assist in evaluating the progression of HD.

No association between identified multiple sclerosis non-MHC risk loci and neuromyelitis optica

Neuromyelitis optica（NMO） and multiple sclerosis（ M S） a r e b o t h a u t o i m m u n e i n f l a m m a t o r y a n d demyelinating disorders of the central nervous system. Recently, more than 50 MS-susceptibility single-nucleotide polymorphisms（SNPs） have been detected outside the major histocompatibility complex（MHC） region. In this study, we aimed to evaluate the association of these identified non-MHC MS risk loci with Chinese patients with NMO. Thirtyfive non-MHC SNPs were selected and genotyped by matrix-assisted laser desorption/ionization timeof-fl ight mass spectrometry（MALDI-TOF MS） in 110 NMO patients and 332 controls from southeastern China. Among the 35 SNPs, only one, rs1800693 in the TNFRSF1 A locus, was nominally associated with NMO（P = 0.045, OR = 1.550, 95% CI = 1.007 – 2.384）. However, none of the 35 SNPs was associated with NMO after Bonferroni correction. Our results showed no association between these identified non-MHC MS risk loci and NMO, suggesting there are genetic differences in the etiology of NMO and MS.

Amyotrophic Lateral Sclerosis： Precise Diagnosis and Individualized Treatment

INTRODUCTION Amyotrophic lateral sclerosis （ALS） is a progressive neurodegenerative disease characterized by selective death of upper motor neurons （UMNs） and lower motor neurons （LMNs）, typically may die from respiratory failure within 2-5 years of symptorn onset）H About 10% orALS patients are familial whereas the remaining patients are sporadic. ALS is highly heterogeneous in genetic and clinical phenotype, with lack of definitive diagnostic tools, making it extremely difficult to make early diagnosis.