Clinical And Genetic Investigations Of Leukodystrophy Manifested As Late Onset Spastic Paraplegia

Background and PurposeSpastic paraplegia is a general term describing progressive stiffness and weakness in the lower limbs caused by pyramidal tract lesions which is frequently encountered in adult neurology.It contains both acquired and hereditary causes.Acquired factors include spinal cord compression,metabolic myelopathy,spinal vascular disease,spinal cord inflammation and infection,and so on.The most common hereditary factors are hereditary spastic paraplegia(hereditary spastic paraplegia,HSP).Late-onset leukodystrophies also account for a certain proportion.Several reports have proved that various leukodystrophies such as X-linked adrenoleukodystrophy/Adrenomyeloneuropathy(ALD/AMN),Krabbe disease(KD),metachromatic leukodystrophy(MLD)are a potential cause of late onset spasticity.Many of them may present with spasticity with or without additional neurological features,mimicking pure or complicated HSP.Therefore,both of HSP and leukodystrophies overlap in clinical and radiological manifestitions,and it is difficult to differentiate them in the clinical practice.Currently no effective treatment is available for HSP,but there have been many breakthroughs in the treatment of leukodystrophy,such as enzyme replacement and hematopoietic stem cell transplantation,which have provided more opportunities for improving the clinical outcome of leukodystrophy.Therefore,early differential diagnosis of leukodystrophy and HSP is of great significance in improving the prognosis of patients or winning early treatment opportunities for other pedigree members.The genetic mechanism of the leukodystrophy is complex,in combination with overlapping clinical and radiological phenotypes,it is difficult to identify the type of disease only by the clinical manifestations.Meanwhile,a large number of genetic and metabolic pathways involved means that the traditional approach including metabolic and biochemical testing is time consuming,costly,and often with low sensitivity.Recently the development of targeted next generation sequencing(Targeted-NGS)technology is very popular because of its high throughput and low consumption with satisfactory auxiliary diagnosis effect in clinical practice.So far there have been several studies on genotypes and phenotypes of late-onset leukodystrophy based on NGS technique in the foreign countries,but only case reports in our country,no large scale clinical studies can be found.The purpose of this study is to explore the proportion of leukodystrophies in patients with unknown causes of late onset spastic paraplegia by the NGS method and to summarize its genotypic and phenotypic characteristics in order to provide a theoretical basis for early clinical diagnosis and differential diagnosis with HSP.Materials and Methods1.PatientsA cohort of 112 patients with progressive lower limb spasticity of unknown causes that were referred to the Qilu Hospital of Shandong University for diagnosis or management from January 2016 to December 2018 were included in this study.Inclusion criteria:1.Only patients with age of onset of spastic gait greater than or equal to 14 years old were included in this study.2.Slowly progressive spasticity as the earliest manifestation or as the most significant clinical finding,with or without additional neurological features such as cognitive decline,epilepsy,peripheral neuropathy,and so on.3.Magnetic resonance imaging(MRI)combined with or without abnormal signals in the white matter of the brain 4.Detailed examination of routine blood test,blood amino acid and fatty carnitine screening,urine organic acid screening,cerebrospinal fluid and neuroimaging to exclude possible pathogenic factors such as metabolism(including vitamin B12 and folic acid deficiency,hepatic myelopathy,methylmalonic acidemia,urea cycle disorders,phenylketonuria and so on),poisoning,infection,inflammation,vascular abnormlities,spinal cord compression,etc.2.MethodsFirstly a custom-designed probe library which contains the known 130 pathogenic genes reported to be associated with hereditary leukoencephalopathies was synthetized.Genomic DNA was extracted from peripheral blood lymphocytes.After genomic DNA was sheared into fragments,we completed the library prepare and capture processes.Then the NGS was performed among them.We classified the sets of variants as "pathogenic","likely pathogenic","variants of uncertain significance(VUS)","likely benign" or "benign" according to the American College of Medical Genetics and Genomics(ACMG)guidelines,the "pathogenic" variants consistent with the clinical phenotypes were determined to be responsible pathogenic mutations,and the "likely benign" and "benign" variants were excluded because of their low pathogenic possibilities.Then four different Web-tools were used to predict the functional effects of the "likely pathogenic" and "VUS" variants:polyphen2(http://genetics.bwh.harvard.edu/pph2/),Mutation Taster(http://www.Mutation taster.org/),SIFT(http://sift.jcvi.org/www/SIFT_enst_submit.html),Mutation Assessor(http://mutationassesor.org/r2).Finally,candidate variants were confirmed through Sanger sequencing.Segregation test was performed if the DNA of the proband’s immediate family member was available,then the final pathogenic mutations were determined in combination with the clinical information.If the results were indicative of inborn metabolic disorders,additional biochemical examination such as lysosomal enzymatic activity and very long chain fatty acids(VLCFA)examination was conducted to confirm the diagnosis.Clinical characterization including neurological and psychiatric features,neuroimaging and neuroelectro-physiological data were collected to predict the genotypic and phenotypic relationship of them.ResultsTwenty-four patients(21.4%,24/112)were identifed pathogenic mutations in this study,including seven patients(29.2%,7/24)associated with X-linked adrenoleukodystrophy/adrenomyeloneuropathy(ALD/AMN),five(20.8%,5/24)associated with cerebrotendinous xanthomatosis(CTX),three(12.5%,3/24)associated with Krabbe disease(KD),three(12.5%,3/24)associated with alanyl-tRNA synthetase 2 mutation-related leukoencephalopathy(AARS2-L),one(4.2%,1/24)associated with leukoencephalopathy with brain stem and spinal cord involved and elevated lactate(LBSL),one(4.2%,1/24)associated with lysyl-tRNA synthetase mutation-related leukoencephalopathy(KARS-L),two(8.3%,2/24)associated with vanishing white matter leukoencephalopathy(VWM),one associated with hereditary diffuse leukoencephalopathy with spheroids(HDLS),one(4.2%,1/24)associated with metachromatic leukodystrophy(MLD).Six cases with positive family history were found in the family investigation.If the siblings were similarly affected in a family,only the proband patient was included to prevent skewing of the frequency analysis.1.Clinical characteristics of the total 24 patientsOf the 24 patients,16 were male and 8 were female,with a male-to-female ratio of 2:1.The age of onset of spasticity varied from16 to 51 years old and the average age was 30 years old.Two patients with AMN,three patients with KD and one patient with AARS2-L presented with only spasticity of the lower limbs at the onset of the disease,whilst the other patients showed additional complicating symptoms,including cognitive dysfunction(54.2%,13/24),epilepsy(8.3%,2/24),ataxia(45.8%,11/24),peripheral neuropathy(50%,12/24),extraneurological disorders including endocrine,skin,chronic diarrhea,cataract,hearing loss in 14 cases(58.3%,14/24).Plasma VLCFA analysis was detected in seven patients with ALD/AMN and lysosomal enzymes screening were performed in three patients with KD and one patient with MLD.The results were all consistent with the genetic diagnosis.A wide spectrum of MRI patterns were noted in our series.Four patients with AMN had no abnormalities on cerebral and spinal MRI.Three patients with KD,four patients with CTX,one patient with AMN had selective pyramidal tract involvement.All five patients with CTX had cerebellar dentate nucleus involvement.Two patients with ALD,two patients with CTX,three patients with AARS2-L,one patient with LBSL,one patient with KARS-L,two patients with VWM,one patient with HDLS,one patient with MLD presented with diffuse leukoencephalopathy which presented with dominantly periventricular white matter lesions.One case of KD complicated with abnormal signal of pyramidal tract in the lateral spinal cord,one case of LBSL complicated with longitudinal abnormalities along the lateral and posterior spinal cord.In addition,characteristic diffusion weighted imaging(DWI)patterns can be seen in our series.Hypersignal along the pyramidal tract was found in one KD patient in the early stage of the disease,but the hyperintense signal changes subside and demyelinated areas turn into hyposignal in more advanced stage of the disease.Spotted DWI hypersignals spread in the diffuse periventricular white matter abnormalities were seen in three AARS2-L and one HDLS patients.In LBSL and KARS-L,DWI shows prominent hyperintensities within the involved deep white matter structures and pyramidal tracts of brain stem.2.Gene analysis33 different variant loci associated with leukodystrophy were identified in 24 patients,all of them were confirmed by Sanger sequencing.Among them,19 loci were known disease-causing mutations which have been reported in the literature,14 loci have not yet been reported in Human Gene Mutation(HGMD),after predicted by the predictive Web-tools and consistency analysis with the clinical phenotype of the patients,they have been identified as novel disease-causing mutations,including c.1780G>A(p.G594S)in ABCD1 gene,c.1055C>A(p.S352X)、c.432T>G(p.Y144*)、c.472C>T(p.R158C)in CYP27A1 gene,c.965G>A(p.R322H)、c.334G>C(p.G112R)、c.2288T>C(p.L763P)、c.1703 1704del(p.Q568fs)、c.179C>A(p.P60H)in AARS2 gene,c.795T>A(p.N265K)、c.1610G>A(p.R537Q)in KARS gene,c.385C>T(p.R129*)、c.633G>T(p.R211S)in EIF2B5 gene,c.1979T>C(p.L660P)in CSFIR gene.Conclusions1.Late-onset spastic paraplegia is a highly heterogeneous disease,and leukodystrophy is one of its important causes,which should not be ignored by adult neurologists.After the elimination of common acquired causes for spastic paraplegia,other clues should be screened by performing brain MRI,nerve conduction studies,ophthalmological examination,endocrine screening,and so on.Lysosomal enzyme activities and VLCFA should be included in the routine diagnostic work-up of patients with spastic paraplegia.2.Clinicians should pay attention to the distribution patterns of cerebral MRI and the characteristics of DWI signals,which may provide valuable information in particular leukodystrophies and predict the progression of some cases.But normal brain MRI can not exclude the diagnosis of late-onset leukodystrophy.3.Targeted-NGS technology combined with clinical manifestitions,radiological characteristics and special biochemical tests is a promising method to improve the positive rate of diagnosis in clinical practice,but the selection of targeted genomic panel will directly affect the detection rate of pathogenic mutation.If the genes related to leukodystrophy with spastic paraplegia as the main manifestation are introduced into HSP panel,the discovery rate of genetic causes of spastic paraplegia can be further improved,the test cost will be reduced,and the test time will be shortened.