A novel MAPT variant (E342K) as a cause of familial progressive supranuclear palsy

Background MAPT variants are a known cause of frontotemporal dementia and Parkinsonian syndrome, of which progressive supranuclear palsy syndrome (PSP) is a rare manifestation. Objective To report a novel MAPT variant in a PSP pedigree with autosomal dominant inheritance pattern, and to produce a literature review of PSP patients with MAPT variants. Methods A comprehensive clinical, genetic, and molecular neuroimaging investigation was conducted on a 61 years-old female proband diagnosed with PSP. We also collected the clinical presentation data and history of the patient’s pedigree, and performed further genetic analysis of 4 relatives, from two generations, with and without symptoms. Results The proband exhibited typical clinical manifestation of PSP. A cranial MRI revealed midbrain atrophy, and an FDG-PET scan suggested hypo-metabolic changes in caudate nucleus, left prefrontal lobe, both temporal poles, and midbrain. 18F-florzolo-tau-PET revealed tau-protein deposits in the thalamus and brainstem bilaterally. A gene test by whole-exome sequencing identified a novel MAPT variant [NM_005910.6, exon 11, c.1024G > A (p.E342K)], and the same variant was also identified in one affected relative and one asymptomatic relative, a probable pre-symptomatic carrier. Conclusion The PSP pedigree caused by the novel MAPT (E342K) variant, expanded the mutational spectrum of MAPT.


Introduction
Progressive supranuclear palsy (PSP), a rare neurodegenerative disorder of unknown origin, is pathologically characterized by abnormal tau deposition in the form of globose neuro-fibrillary tangles, tufted astrocytes, coiled bodies, and threads, with a predominance of 4-repeat (4R) tau isoforms (1,2).Clinically, PSP is heterogeneous and may manifest various phenotypes, in which the most classical form is Richardson Syndrome (PSP-RS, also known as Steele-Richardson-Olszewski Syndrome), with symptoms such as vertical supranuclear gaze palsy, postural instability, and motor and cognitive deficits.Other forms of PSP have been increasingly recognized, including PSP with predominant parkinsonism (3), PSP with progressive gait freezing (4), PSP with predominant corticobasal syndrome (5), PSP with a predominant speech/language disorder (6), PSP with predominant frontal presentation (7), PSP with predominant cerebellar ataxia (5), and PSP with mixed pathology (8).Although PSP is generally considered sporadic, an increasing number of familial cases have drawn attention to its potential genetic basis (8)(9)(10).Among these, variants in the MAPT gene are most commonly associated with PSP (11,12).We identified a PSP family with an autosomal inheritance pattern, performed an exhaustive clinical, neuroimaging investigation of the proband, and further performed gene analysis of the proband and four relatives with or without PSP symptoms for the purpose of confirming the pathogenicity of potential gene variant.Additionally, we reviewed the literature on MAPT variants and their related disease.
2 Materials and methods

Ethics, consent, and permissions
All steps of the investigation, including approval for genetic testing, were approved by the Ethics Committee of the Peking Union Medical College Hospital.Written informed consent was obtained from subjects under investigation.

Clinical and family history investigations
The proband underwent a thorough clinical and familial history assessment, along with a neurological examination.A neuropsychologist administered a standardized battery of cognitive tests.The medical records of affected family members, both alive and deceased, made available by other centers, underwent a thorough review.

18 F-FDG positron emission tomography and 18 F-florzolo-tau-PET scan
The 18 F-FDG tracer was prepared at the Department of Nuclear Medicine, Peking Union Medical College Hospital, and had a radiochemical purity of over 95%.Before the administration of 18 F-FDG, patients were required to fast for a minimum of 6 h and maintain a blood glucose concentration <7 mmol/L.The tracer dosage was set at 3.7 MBq/kg, which was administered intravenously.After the injection, patients were advised to rest in a quiet, dimly lit environment for approximately 60 min.Then, PET/CT imaging was conducted using either a PoleStar m680 (SinoUnion Healthcare, Beijing, China) or a Siemens (Erlangen, Germany) scanner.A low-dose CT scan was used for attenuation correction, followed by a 10 min brain PET 3D scan.PET images were then reconstructed using the ordered subset expectation maximization method.
The other tracer used in the investigation ( 18 F-florzolotau) was prepared by the PET Center of the Department of Nuclear Medicine, Peking Union Medical College Hospital, with an injection dose of 370 MBq, time to collect after injection (90 min), and collecting time of 20 min.The patient remained with eyes closed in a quiet, lightavoidance environment after static injection of the tracer.After the tracer was injected, the brain images were acquired using a PET/CT machine (Polestar m680) in a quiet, light-protected environment, with the patent's eyes closed, for about 90 min.A low-dose head CT scan was used for attenuation correction; a 20 min three-dimensional (3D) PET scan of the brain was performed.PET-image reconstruction was performed using the ordered subsets expectation maximization (OSEM) method.

Genetics
Blood samples were collected from the proband (III-9) and from the other four family members (III-5, III-7, IV-9, IV-10).Whole-exome sequencing was conducted using standard procedures, with genomic DNA extracted from peripheral blood and purification carried out using established protocols.The GenCap Whole Exome Capture Kit from MyGenostics GenCap Enrichment Technologies was utilized for library preparation, and sequencing was performed on DNBSEQ-T7.After sequencing, the raw data were saved in FASTQ format and subjected to bioinformatic analysis.Initial steps involved filtering out sequencing adapters and low-quality reads (<80 bp) using Cutadapt.After quality control, the clean reads were mapped to the UCSC hg19 human reference genome through the Burrows-Wheeler Aligner (BWA), achieving an average sequencing depth exceeding 100×.The targeted region coverage was more than 20× for over 90% and 10× for over 95%.Picard tools were employed to remove duplicated reads, and the mapping reads were utilized for variation detection.Subsequently, Genome Analysis Toolkit (GATK) Haplotype Caller was used to detect SNP and InDel variants, followed by variant filtration using GATK Variant Filtration.The resulting data were transformed into VCF format.For further annotation, variants were associated with multiple databases such as gnomAD, 1,000 Genomes, dbSNP.Predictions were made using SIFT, PolyPhen-2, mutationTaster, GERP++, phyloP, and phastCons.Sequence-variation interpretation adhered to the American College of Medical Genetics and Genomics (ACMG) guidelines.The candidate's variant sites were confirmed by Sanger sequencing of all the members of the family.The target sequences were among those sequenced on an ABl3730 analyzer (Applied Biosystem).Sites of variation were identified by comparing DNA sequences with the corresponding GenBank reference sequences using mutation Survey software.

Literature review
We conducted a literature review by searching the PubMed database, with the latest search date being December 1, 2023.The keywords used included "tau, " "MAPT, " and "microtubule-associated protein tau, " each paired with "PSP" in various combinations.We included all patients who had been diagnosed with PSP or PSP-like conditions with MAPT variant.

Case report (Proband, III-9)
Patient III-9 was a 61 year-old right-handed female farmer.Five years ago, she began to have the symptoms as unexplained recurrent falls, reduced stride, and unsteady walking, which worsen with time.After 2 years, she began to gradually develop more symptoms such as choking while drinking water, limited mouth opening, and excessive salivation.For the past 6 months, she has been completely dependent on others for daily activities, walking, and urinary incontinence.Immediate memory decline, hand tremors, and mood changes such as irritability and emotional lability, were also noted.During neurological examination, the proband was in a wheelchair and had flat facial expressions, decreased, hesitant speech output, but no comprehension difficulty.She exhibited restricted vertical gaze on pursuit and saccades, notable bradykinesia with difficulty getting out of the chair, and a tendency to fall backwards.She had normal muscle strength and notable rigidity of her neck and limbs with bilateral pyramidal signs.She struggled with finger-to-nose and heel-to-shin coordination tests.

Family history
The family history was significant; it showed an autosomal inheritance pattern, as shown in the pedigree depicted in Figure 1.The medical histories of all patients in her family, both alive and deceased, were reviewed, including medical records and additional information from caregivers.The age at onset of symptoms in the three deceased patients in her family was between 50 and 60 years old.All three deceased patients had shown walking slowness, easy falling frequent falls, partial gaze palsy, and memory decline.The duration of the disease of the three deceased patients was about 12-15 years.
Patient III-2 started showing postural instability at about 58 years of age and is now 71.Patient III-7 is 62 years old.She began showing amnesia at about age 53.Deceased patient I-2 presented with postural instability at about age 50 and died at age 65 when she was unable to take care of herself.Patient II-1 developed symptoms of postural instability at about age 50 and died at age 63.Patient II-3 developed symptoms of postural instability at about age 55 and died at age 67.Individual IV-9 is now age 32 with no abnormalities on neurologic examination or neuropsychological evaluation.
The clinical manifestations of this pedigree for family members and genetic analysis results are shown in Table 1.

Genetic analysis
A novel variant E342 [NM_005910.6,exon 11, c.1024G > A (p.E342K)] located in exon 11 of MAPT, which was detected in the proband (III-9), III-7, and IV-9.IV-9, the proband's second daughter, could be an non-symptomatic carrier.III-5 and IV-10, as healthy controls, did not carry the variant.According to the Standards and Guidelines for the Interpretation of Sequence Variants, the variant is considered likely pathogenic.

Review of PSP-relevant cases with MAPT variant
There were 27 analyzed studies on MAPT variants in 40 PSP cases.These cases of clinically or neuro-pathologically diagnosed PSP or PSP-like conditions (Table 2) (13-39) had MAPT variants primarily in exon 10 and its stem-loop region.Of these, 28 patients (70%) had nine types of variants in exon 10, and 4 patients (10%) had three types of variants in stem-loop of exon 10.The geographic distribution of these cases was as follows: 18 cases from Japan, with a total of 6 different variants; and 20 cases from Western countries, with a total of 14 different variants.With regard to the demographics and clinical features, about 86.5% cases had a family background of neurodegenerative diseases, and there were more males (64.9%) than females.The mean onset age was 45.6 (SD = 7.95) years (range = 36-75 years), the disease duration was 7.8 (SD = 6.66) years (range = 1-27 years).It seemed that patients with the ΔN296 had an earlier age at onset (in their thirties), and two patients with R406W showed longer disease course (26 and 27 years).Broad heterogeneity of MAPT variants, predominantly in exon 10 and its stemloop region, was revealed in these studies.Although there was regional variability in the types of variants observed, the age at onset seemed consistent across geographical boundaries.Notably, those with a family history of neurodegenerative conditions were more commonly affected.

Discussion
We examined a newly identified Chinese family with a novel MAPT variant, displaying typical PSP features.Those features included recurrent falls, oculomotor challenges, and progressive cognitive impairment with short-term memory loss.Additionally, a literature review of previously reported MAPT-mutated PSP patients demonstrated the heterogeneity and complexity of the disease caused by different genetic mutations, which has broadened our knowledge of MAPT variants and disease with tau-pathology.
The MAPT gene, situated on chromosome 17q21, encodes the tau protein and comprises 16 exons.Exons 0 and 14 do not contribute to protein encoding.Inheritance is autosomal dominant (40,41).Over 100 families worldwide have been identified with 50 distinct variants in the tau gene.Tau proteins exist in six isoforms, resulting from selective splicing in exons 2, 3, and 10.Depending on the splicing of exons 2 and 3, isoforms with 0, 1, or 2 N-terminal repeats (0 N, 1 N, 2 N) are produced.Selective splicing of exon 10 results in tau isoforms with either 3 or 4 microtubule-binding repeats (40).Disorders involving MAPT are primarily characterized by frontotemporal lobe dementia and/or clinical Parkinson's syndrome, and less commonly by motor neuron disease (42).In case-control genome-wide association studies (GWAS), MAPT is the most significant risk locus for sporadic PSP, and is also the most common cause of familial PSP (11,12).
The novel MAPT variant is deemed likely pathogenic based on criteria set forth by the American College of Medical Genetics and Genomics (ACMG) and the Society for Molecular Pathology (43).These criteria are as follows: the mutation resides in a functional structural domain (PM1).It is absent in healthy populations (PM2).
The new missense alteration occurs at an amino acid residue (E342V) previously identified as pathogenic (PM5) (44, 45).The mutation co-segregates with the disease in multiple family lines (PP1).The affected gene is highly conserved across species, and computational analyses suggest the mutation is deleterious (PP3).Clinical presentation of patients supports a monogenic etiology for the disease (PP4).
The variant is situated in the fourth repeat, R4, of MAPT's exon 11, which encodes the functional microtubule-binding domain (MTBD).Spanning from the latter part of exon 9 to exon 12 (amino acids 244-368), the MTBD contains four highly conserved 18-aminoacid repeats, separated by either 13 or 14 residues.MTBD plays a critical role in the interaction of tau with microtubules, promoting their assembly and stability (46,47).The p.E342V variant in exon 11 has demonstrated an increase in 4R tau mRNA levels.This variant not only escalates the splicing of exon 10 but also decreases the inclusion of exons 2 and 3.At both RNA and protein levels, the E342V variant hampers the ability of tau to promote microtubule assembly.Additionally, the variant is hypothesized to diminish the affinity of tau for microtubules, thereby facilitating its release, self-assembly, and fibrillar aggregation (45).Pathological examination has unveiled notable frontotemporal lobe neuronal loss, intracytoplasmic tau aggregates, and paired helical tau filaments, thus confirming the variant's deleterious effects.Moreover, exon 11 appears to influence the alternative splicing of other exons in the tau gene (44).
The MAPT gene is the most frequently implicated gene in hereditary forms of PSP.As shown in Table 2 and Figure 1, which contain variants in this patient's genes, there were 20 different MAPT variants described in cases presenting with PSP (clinical or neuropathologic diagnosis).Variant prevalence varies regionally, potentially due to ethnic and environmental influences.The occurrence of MAPT variants in PSP patients ranges from 0.6 to 14.3% (25, 48-50).The most commonly observed MAPT variant in PSP is at codon 279, found in 10 patients.Families carrying the N296 variant tend to experience an earlier onset of the disease.Almost all patients with MAPT variants are heterozygous, with only one reported case of a homozygous N296 variant.That particular patient exhibited a more severe phenotype and disease progression than did those with heterozygous variants (23).The p.K298H299insQ variant in exon 10 represents the first reported insertion variant in the MAPT gene.Patients with MAPT variants typically exhibit a gradual disease onset and are more likely to present with Parkinsonian symptoms.Pathologically, these patients often manifest standard PSP or PSP-like conditions.Table 2 shows that the vast majority of patients with MAPT variants, with the exceptions of R5L, V363I, and V363A, have a family history of Parkinsonian syndrome, dementia, or other neurodegenerative diseases.This supports the previously published notion of familial clustering in PSP (27,51).Clinical heterogeneity has been observed even among patients carrying the same variant, including those within the same family, underscoring the complex interaction of genetic and environmental factors in PSP (33, 34, 37).
In the family we studied, the mean age at onset was 53.7 years.This is higher than the mean age at onset in the general PSP population with MAPT variants, which is approximately 45.8 years.The family's mean duration of the disease was 13.3 years.This is longer than the mean disease duration in the general PSP population with MAPT variants, which is approximately 8.4 years.The older age at onset and longer disease duration in this family could suggest the presence of family-specific genetic or environmental factors that modulate the course of the disease.Also of interest is the presence of a genetic variant in IV-9 without clinical manifestations, which may be related to the fact that the patient was very young at the time of the present study.Future follow-up of this patient is needed to further understand this disease.
The predominance of postural instability as the initial symptom in this family aligns with observations from the broader PSP population with MAPT variants.This could have implications for early diagnosis and intervention strategies.The presence of amnesia as an initial symptom in one individual emphasizes clinical heterogeneity, even within families carrying the same MAPT variants.Memory disorder is seldom found to be one of the main symptoms of PSP, but has been described in association with MAPT variants in PSP (Table 2).The differences in age at onset and disease duration between  this family and the general PSP population call for more in-depth genetic and environmental studies to uncover the reasons behind these discrepancies.
The hummingbird and morning-glory-flower signs on MRI, previously established as markers of midbrain pathology, effectively distinguish patients with PSP from those with Parkinson's disease and multiple-system atrophy.The hummingbird sign has a 99.5% diagnostic specificity, and the morning-glory sign a 97.7% diagnostic specificity for PSP.As depicted in Figure 2 (52), our patient exhibited pronounced hummingbird and morningglory signs.The patient's MRI also revealed a notably small midbrain, which is indicative of a classic PSP-RS phenotype.This significant midbrain atrophy aligns well with the clinical manifestations commonly observed in patients with sporadic PSP-RS.In summary, the patient's MRI findings are consistent with established literature, reinforcing their diagnosis as a case of PSP-RS (53)(54)(55).
Studies have indicated that 18 F-Fluorodeoxyglucose PET often reveals hypometabolism in specific regions like the midbrain, basal ganglia, thalamus, and frontal lobes in patients with PSP-RS (56,57).The most frequently observed sites of hypometabolism are the thalamus (100%), caudate nucleus (86%), midbrain (86%), and frontal lobes (71%) (58).In line with these findings, our patient also exhibited hypometabolism in these regions, as well as in the bilateral temporal poles.Uniquely, our patient exhibited hypometabolism in the bilateral temporal poles.Kanel et al. posited that a greater loss of cholinergic activity in the superior temporal poles could explain this observation in patients with PSP (59).
Although structural MRI did not reveal significant atrophy in our patient, the functional deficits were evident.Prior research has identified focal, bilateral cortical thinning in patients with PSP, predominantly in the prefrontal/precentral cortex and the temporal pole.Even in the absence of structurally significant changes in our case, the patient did present with clinically relevant cognitive deficits (60).These additional findings contribute to the complexity of the PSP clinical picture.The hypometabolism in the bilateral temporal poles could serve as another layer of diagnostic criteria, potentially distinguishing subsets of PSP patients or highlighting disease severity.Moreover, although the absence of significant cortical thinning may limit the utility of structural MRI in some PSP cases, functional markers such as hypometabolism can provide valuable insights into the disease process, especially when aligned with clinical manifestations like cognitive deficits.
Our proband demonstrated tau protein deposition in the bilateral thalamus and brainstem.The most commonly used tau tracer, 18 F-AV-1451, typically shows increased uptake in multiple brain regions in PSP-RS, including the pallidum, chiasma, caudate nucleus, and thalamus (61,62).Previous research has consistently noted increased 18 F-AV-1451 uptake in the globus pallidus, putamen, and caudate nucleus, among other regions, when comparing PSP-RS with controls.Quantitative analyses have found that the best differentiation between PSP-RS and control groups is achieved through evaluating globus pallidus retention and thalamic activity (63, 64).Our proband's tau-PET results, which showed tau protein deposits in the bilateral thalamus and brainstem, align with previous studies (65)

Limitations
The key constraints of our study include the limited size of our cohort and the relatively few documented cases of PSP and PSP-like conditions with MAPT variants in existing literature.Such limitations could introduce publication or selection bias, as well as inconsistencies in symptom reporting and grading across different studies.To fully grasp the complex clinical heterogeneity observed in patients with MAPT variants, larger sample sizes are needed.
Ethical constraints also prevented further genetic studies among other family members to clarify the gene's pathogenicity, though we plan to pursue this in future research.Additionally, the review may be subject to publication bias, particularly because genetic screening is not commonly performed in some rural areas of China.To affirm the prevalence of MAPT variants in China, broader multicenter genetic studies are necessary.

Conclusion
The PSP pedigree caused by the novel MAPT (E342K) variant have been expanding the mutational spectrum of MAPT.The findings in the present study may be helpful for the development of targeted therapeutic interventions of PSP.

FIGURE 2
FIGURE 2 Pedigree and gene variant of the family.(A) The proband is indicated (arrow).Squares indicate males; circles, females; filled symbols, affected subjects; half-filled symbols, variant carriers without any clinical symptoms; open symbols, subjects without any clinical symptoms and without genetic testing, black spots, subjects with genetic testing; diagonal line through a symbol, death.(B) Sanger sequence showing the variant of MAPT (p.E342K, c.1024G > A). (C) Schematic representation of the MAPT gene showing the sites of the variants reported so far.The c.1024G > A variant in this Chinese family is highlighted in red.

TABLE 1
The clinical manifestations of this pedigree.
. These imaging markers further substantiate the diagnosis of PSP and contribute to our understanding of this disorder.