Rosenthal Fibers Research Articles

Chapter 44 - Alexander disease

Alexander disease is a rare and generally fatal disorder of the central nervous system, originally defined by the distinctive neuropathology consisting of abundant Rosenthal fibers within the cytoplasm and processes of astrocytes. More recently, mutations in GFAP, encoding glial fibrillary acidic protein, the major intermediate filament protein of astrocytes, have been identified in nearly all patients. No other genetic causes have yet been identified. The precise mechanisms by which mutations lead to disease are poorly understood. Despite the genetic homogeneity, there are a wide range of clinical phenotypes. The genetic issues and the approach to diagnosis are the prime consideration in this chapter.

A novel GIT2-BRAF fusion in pilocytic astrocytoma

BackgroundKIAA1549-BRAF fusion is the most common genetic event in pilocytic astrocytoma (PA), and leads to activation of the mitogen activated protein kinase (MAPK) signaling pathway. Fusions of BRAF with other partner genes, as well as other genetic alterations not involving BRAF but also leading to MAPK pathway activation have been described rarely.Case presentationWe present a new fusion partner in the low-grade glioma of a 10-year-old male, who presented with headaches and recent episodes of seizures. Magnetic resonance imaging (MRI) demonstrated a right temporal lobe tumor. Histological and immunohistochemical evaluation, and a next generation sequencing assay (Oncopanel, Illumina, 500 genes) including breaKmer analysis for chromosomal rearrangements were performed.Histology was remarkable for a low-grade glioma composed of mildly atypical astrocytes with piloid processes, in a focally microcystic background. Mitoses were not seen; unequivocal Rosenthal fibers or eosinophilic granular bodies were absent. The tumor was positive for OLIG2 and GFAP and negative for BRAF V600E and IDH1 R132H mutant protein immunostains. Oncopanel showed low SOX2 (3q26.33) copy number gain, and no gains at 7q34. There were no significant single nucleotide variants. BreaKmer detected a GIT2-BRAF fusion with loss of BRAF exons 1–8. The integrated diagnosis was low-grade glioma with piloid features, most consistent with pilocytic astrocytoma, WHO grade I.ConclusionGIT2-BRAF fusion has not been reported in the literature in any tumor. Given that the BRAF sequence deleted is identical to that seen in other fusion events in PA, it most likely acts as tumor driver by activation of the MAPK pathway.

Cervical Intramedullary Schwannoma with Syrinx: Case Report and Review of the Literature

AbstractSchwannoma is a nerve sheath tumor originating from the Schwann cell. It is benign in nature and it arises from anywhere where Schwann cells can be found. It is rarely found in the parenchyma of the spinal cord. Intramedullary schwannomas (or neurilemmomas) without evidence of neurofibromatosis are rare spinal cord tumors. Intramedullary schwannoma was first reported in 1932 by Penfield. Our patient presented with neck pain, gradually worsening, weakness in the right upper and lower limbs, numbness in both shoulders, and a decrease in the grasping strength of both hands over a 4-year period. A magnetic resonance imaging of the spine showed a heterogeneously enhancing mass in the cervical spinal cord extending from the C2 to T1 levels with associated hemorrhagic changes. Histologically, the tumor was found to be composed of bland spindle cells with blunt-ended and sometimes wavy nuclei admixed with hyalinized vasculature. Surrounding reactive spinal cord parenchyma with frequent Rosenthal fibers was also observed. Focal Verocay bodies were evident, and with immunohistochemistry, there was diffuse and strong positivity for S100, which is confirmatory for the diagnosis of schwannoma. We report a case of cervical intramedullary schwannoma presented with syringobulbia in a young adult.

Rosenthal fiber encephalopathy and Alexander disease

Background: Numerous Rosenthal fibers were revealed both adult onset Alexander disease (AOAD) and Rosenthal fiber encephalopathy (RFE). Rosenthal fibers usually showed the area of perivascular, periventricular and brainstem. Alexander disease is associated with mutations in GFAP gene. However, RFE has not detected mutation of GFAP gene.It remains obscure in what condition Rosenthal fibers are formed and how different RF formation in both AOAD and RFE. We are made some special reference about this presentation.

Relationship between rosenthal fiber and excitatory amino acid transporter in familial Alexander disease

Relationship between rosenthal fiber and excitatory amino acid transporter in familial Alexander disease

Modeling Alexander disease with patient IPSCS reveals cellular and molecular pathology of astrocytes

Background: Alexander disease (AxD) is a fatal neurological disorder characterized by macrocephaly, leukodystrophy and a variety of developmental delays with epileptic seizures, dysphagia, or bulbar signs. The common neuropathological feature of AxD is the presence of Rosenthal fibers, a unique cytoplasmic inclusion within astrocytes. Rosenthal fibers contain glial fibrillary acidic protein (GFAP), major astrocytic intermediate filament protein and molecular chaperones, including alpha-B-crystallin and other heat shock proteins.

Aggregation-prone GFAP mutation in Alexander disease validated using a zebrafish model

BackgroundAlexander disease (AxD) is an astrogliopathy that predominantly affects the white matter of the central nervous system (CNS), and is caused by a mutation in the gene encoding the glial fibrillary acidic protein (GFAP), an intermediate filament primarily expressed in astrocytes and ependymal cells. The main pathologic feature of AxD is the presence of Rosenthal fibers (RFs), homogeneous eosinophilic inclusions found in astrocytes. Because of difficulties in procuring patient’ CNS tissues and the presence of RFs in other pathologic conditions, there is a need to develop an in vivo assay that can determine whether a mutation in the GFAP results in aggregation and is thus disease-causing.MethodsWe found a GFAP mutation (c.382G > A, p.Asp128Asn) in a 68-year-old man with slowly progressive gait disturbance with tendency to fall. The patient was tentatively diagnosed with AxD based on clinical and radiological findings. To develop a vertebrate model to assess the aggregation tendency of GFAP, we expressed several previously reported mutant GFAPs and p.Asp128Asn GFAP in zebrafish embryos.ResultsThe most common GFAP mutations in AxD, p.Arg79Cys, p.Arg79His, p.Arg239Cys and p.Arg239His, and p.Asp128Asn induced a significantly higher number of GFAP aggregates in zebrafish embryos than wild-type GFAP.ConclusionsThe p.Asp128Asn GFAP mutation is likely to be a disease-causing mutation. Although it needs to be tested more extensively in larger case series, the zebrafish assay system presented here would help clinicians determine whether GFAP mutations identified in putative AxD patients are disease-causing.

Astrocytoma and epilepsy. Clinical case

Abstract Background Pilocytic astrocytoma is a rare tumour, usually occurring in paediatric ages, and mainly located in the posterior fossa. It can cause hydrocephalus and intracranial hypertension and, less frequently, seizures, or a focal neurological deficit. The main imaging study by magnetic resonance imaging, which shows a tumour with solid and cystic components without peri-lesional swelling. The election treatment is surgical, and the patient is considered cured if a total resection is accomplished. Clinical case The case is presented of 22-year-old female patient with a supratentorial pilocytic astrocytoma and epilepsy. Histopathology reported a low grade glial proliferation, with an extensive fibrillar matrix, small cells without atypia, extensive calcifications and piloid areas consisting of bipolar fusiform cells, and some Rosenthal fibres. There were also spongiotic areas consisting of multipolar cells and associated microcysts. The final report was a pilocytic astrocytoma. Conclusions Pilocytic astrocytoma is more frequent in paediatric patients and in the posterior fossa. The case presented is of a young female adult with supratentorial location, making it a special case. The surgery achieved a total resection. The long-term prognosis is good, but it is necessary to perform a follow-up, particularly in adult patients because of a higher risk of recurrence.

Characterization of a panel of monoclonal antibodies recognizing specific epitopes on GFAP.

Alexander disease (AxD) is a neurodegenerative disease caused by heterozygous mutations in the GFAP gene, which encodes the major intermediate filament protein of astrocytes. This disease is characterized by the accumulation of cytoplasmic protein aggregates, known as Rosenthal fibers. Antibodies specific to GFAP could provide invaluable tools to facilitate studies of the normal biology of GFAP and to elucidate the pathologic role of this IF protein in disease. While a large number of antibodies to GFAP are available, few if any of them have defined epitopes. Here we described the characterization of a panel of commonly used anti-GFAP antibodies, which recognized epitopes at regions extending across the rod domain of GFAP. We show that all of the antibodies are useful for immunoblotting and immunostaining, and identify a subset that preferentially recognized human GFAP. Using these antibodies, we demonstrate the presence of biochemically modified forms of GFAP in brains of human AxD patients and mouse AxD models. These data suggest that this panel of anti-GFAP antibodies will be useful for studies of animal and cell-based models of AxD and related diseases in which cytoskeletal defects associated with GFAP modifications occur.

A novel familial mutation in Alexander disease with marked variation in clinical severity

Objective: Alexander disease is a rare, often lethal disorder characterized by white-matter degeneration and cytoplasmic inclusions in astrocytes known as Rosenthal fibers, which are immunohistochemically positive to glial fibrillary acidic protein (GFAP). The disease typically results from dominant mutations in GFAP that arise de novo. However, series of familial cases with variable clinical manifestations have been described suggesting a potential recessive inheritance or germline mosaicism. We report a male infant with typical Alexander disease, inherited by his asymptomatic father with positive neuroimaging markers of the disease. Methods: Clinical histories were obtained and whole exome sequencing (WES) analyses in blood samples were performed through next generation sequencing. Results: We report a 6-month male, born of an uneventful term pregnancy, who presented with generalized hypotonia, chorioathetosis and drug-resistant seizures, without macrocephaly. Brain MRI showed symmetrical abnormalities of the basal ganglia, subcortical white matter abnormalities, mainly of the frontal area with contrast enhancement along with high intensity periventricular rim on T1. WES analysis of the patient revealed a heterozygous missense mutation, c.1120G>A (p.Glu374Lys) in GFAP gene, resulting in the substitution of the aminoacid glutamic acid with lysine at the protein level. Although this genetic change is novel, it was considered as pathogenic from in silico analysis. We therefore proceeded to genetic analyses of the remaining members of the family that revealed the same mutation in the asymptomatic father. A subsequent brain MRI showed white matter abnormalities suggestive of adult-onset Alexander disease. Conclusion: We present a familial case of Alexander disease caused by a novel mutation on GFAP gene. There is a marked discrepancy on clinical presentation between the father and the affected child imposing that there is a striking variation on penetrance of the mutated gene of unknown etiology. We suggest genetic analysis on parents of patients with Alexander disease even if asymptomatic.

The origin of Rosenthal fibers and their contributions to astrocyte pathology in Alexander disease

Rosenthal fibers (RFs) are cytoplasmic, proteinaceous aggregates. They are the pathognomonic feature of the astrocyte pathology in Alexander Disease (AxD), a neurodegenerative disorder caused by heterozygous mutations in the GFAP gene, encoding glial fibrillary acidic protein (GFAP). Although RFs have been known for many years their origin and significance remain elusive issues. We have used mouse models of AxD based on the overexpression of human GFAP (transgenic, TG) and a point mutation in mouse GFAP (knock-in, KI) to examine the formation of RFs and to find astrocyte changes that correlate with the appearance of RFs. We found RFs of various sizes and shapes. The smallest ones appear as granular depositions on intermediate filaments. These contain GFAP and the small heat shock protein, alphaB-crystallin. Their aggregation appears to give rise to large RFs. The appearance of new RFs and the growth of previously formed RFs occur over time. We determined that DAPI is a reliable marker of RFs and in parallel with Fluoro-Jade B (FJB) staining defined a high variability in the appearance of RFs, even in neighboring astrocytes. Although many astrocytes in AxD with increased levels of GFAP and with or without RFs change their phenotype, only some cells with large numbers of RFs show a profound reconstruction of cellular processes, with a loss of fine distal processes and the appearance of large, lobulated nuclei, likely due to arrested mitosis. We conclude that 1) RFs appear to originate as small, osmiophilic masses containing both GFAP and alphaB-crystallin deposited on bundles of intermediate filaments. 2) RFs continue to form within AxD astrocytes over time. 3) DAPI is a reliable marker for RFs and can be used with immunolabeling. 4) RFs appear to interfere with the successful completion of astrocyte mitosis and cell division.

The role of gigaxonin in the degradation of the glial-specific intermediate filament protein GFAP.

Alexander disease (AxD) is a primary genetic disorder of astrocytes caused by dominant mutations in the gene encoding the intermediate filament (IF) protein GFAP. This disease is characterized by excessive accumulation of GFAP, known as Rosenthal fibers, within astrocytes. Abnormal GFAP aggregation also occurs in giant axon neuropathy (GAN), which is caused by recessive mutations in the gene encoding gigaxonin. Given that one of the functions of gigaxonin is to facilitate proteasomal degradation of several IF proteins, we sought to determine whether gigaxonin is involved in the degradation of GFAP. Using a lentiviral transduction system, we demonstrated that gigaxonin levels influence the degradation of GFAP in primary astrocytes and in cell lines that express this IF protein. Gigaxonin was similarly involved in the degradation of some but not all AxD-associated GFAP mutants. In addition, gigaxonin directly bound to GFAP, and inhibition of proteasome reversed the clearance of GFAP in cells achieved by overexpressing gigaxonin. These studies identify gigaxonin as an important factor that targets GFAP for degradation through the proteasome pathway. Our findings provide a critical foundation for future studies aimed at reducing or reversing pathological accumulation of GFAP as a potential therapeutic strategy for AxD and related diseases.

Modeling Alexander disease with patient iPSCs reveals cellular and molecular pathology of astrocytes.

Alexander disease is a fatal neurological illness characterized by white-matter degeneration and formation of Rosenthal fibers, which contain glial fibrillary acidic protein as astrocytic inclusion. Alexander disease is mainly caused by a gene mutation encoding glial fibrillary acidic protein, although the underlying pathomechanism remains unclear. We established induced pluripotent stem cells from Alexander disease patients, and differentiated induced pluripotent stem cells into astrocytes. Alexander disease patient astrocytes exhibited Rosenthal fiber-like structures, a key Alexander disease pathology, and increased inflammatory cytokine release compared to healthy control. These results suggested that Alexander disease astrocytes contribute to leukodystrophy and a variety of symptoms as an inflammatory source in the Alexander disease patient brain. Astrocytes, differentiated from induced pluripotent stem cells of Alexander disease, could be a cellular model for future translational medicine.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0337-0) contains supplementary material, which is available to authorized users.

Adult anaplastic pilocytic astrocytoma – a diagnostic challenge? A case series and literature review

IntroductionAnaplastic pilocytic astrocytoma (APA) is an exceptionally rare type of high-grade glioma in adults. Establishing histopathological diagnosis is challenging and its clinical and radiological appearance insidious. By this case series and first literature review we investigated the various clinical, neuroradiological, and histopathological features of APA in adults. MethodsAn in hospital screening of the database from the Institute of Pathology was conducted to identify cases of APA. Further, we performed a literature review in PubMed using the keywords “anaplastic/malignant/atypical AND pilocytic astrocytoma” and “anaplastic astrocytoma/glioblastoma AND Rosenthal fibers” and summarized the current knowledge about APA in adults. ResultsOver the last decade we were able to identify 3 adult patients with APA in our hospital. According to the pertinent literature, the prognosis of APA in adults (documented survival of up to 10 years) appears to be better than in other high-grade gliomas. Few cases were associated with neurofibromatosis type 1, which seems to predispose for development of APA. Although molecular genetics is still of limited value for differentiation of APA from other high-grade glioma, advanced neuroimaging techniques such as magnetic resonance perfusion imaging and spectroscopy allow improved differential work-up. In particular, APA in adults has the ability to mimic various neurological diseases such as tumefactive demyelinating lesions, low-, or high-grade gliomas. ConclusionsAlthough currently not explicitly recognized as a distinct clinico-pathologic entity it seems that adult APA behaves differently from conventional high-grade glioma and should be included in differential diagnostics to enable adequate patient care. However, further studies are needed to better understand this extremely rare disease.

Composition of Rosenthal Fibers, the Protein Aggregate Hallmark of Alexander Disease.

Alexander disease (AxD) is a neurodegenerative disorder characterized by astrocytic protein aggregates called Rosenthal fibers (RFs). We used mouse models of AxD to determine the protein composition of RFs to obtain information about disease mechanisms including the hypothesis that sequestration of proteins in RFs contributes to disease. A method was developed for RF enrichment, and analysis of the resulting fraction using isobaric tags for relative and absolute quantitation mass spectrometry identified 77 proteins not previously associated with RFs. Three of five proteins selected for follow-up were confirmed enriched in the RF fraction by immunobloting of both the AxD mouse models and human patients: receptor for activated protein C kinase 1 (RACK1), G1/S-specific cyclin D2, and ATP-dependent RNA helicase DDX3X. Immunohistochemistry validated cyclin D2 as a new RF component, but results for RACK1 and DDX3X were equivocal. None of these was decreased in the non-RF fractions compared to controls. A similar result was obtained for the previously known RF component, alphaB-crystallin, which had been a candidate for sequestration. Thus, no support was obtained for the sequestration hypothesis for AxD. Providing possible insight into disease progression, the association of several of the RF proteins with stress granules suggests a role for stress granules in the origin of RFs.

EPID-02AN UNUSUAL PEDIATRIC HYPOTHALAMIC PILOCYTIC ASTROCYTOMA

BACKGROUND: Hypothalamic/chiasmatic tumors (H/C) constitute 7-10% of pediatric primary CNS tumors and are typically pilocytic astrocytomas. Local extension is seen in 75% of patients but metastatic spinal cord involvement without intracranial dissemination is rare. We describe an unusual multifocal H/C and spinal cord pilocytic astrocytoma in an 8 year old boy. CASE REPORT: The patient was found to have a pineal cyst 1 year prior to diagnosis when he had a CT scan following minor head trauma. Ten months later he presented with lower back pain. MRI (sacrum) showed distortion of L5 nerve roots. Over 3 months his sacral pain worsened and he developed headaches, emesis and gait disturbance. MRI (brain) showed an enhancing hypothalamic mass and hydrocephalus. Spinal cord MRI revealed an intramedullary T2 hyperintense C2-C5 mass, an intramedullary T2 hyperintense focus at T6 and a cystic lesion bordering S1/S2 nerve roots. Spinal cord lesions enhanced less than the hypothalamic lesion. Additional T2 hyperintense foci of uncertain origin were seen in the midbrain tectum, superior cerebellar peduncles and cerebellar hemispheres. Optic chiasm was eccentrically thickened on the right but without abnormal enhancement. CSF was negative for tumor cells and NF1 studies were negative. The hypothalamic and cervical spinal cord lesions were biopsied/debulked respectively. Microscopic examination (both specimens) revealed low-grade astrocytoma without mitotic activity, necrosis or microvascular endothelial proliferation and only occasional Rosenthal fibers (hypothalamic specimen). Immunostain for neurofilament protein (both specimens) suggested an infiltrative growth pattern but IDH-1 immunostain was negative. Both specimens were BRAF(7q34) duplication(+). Ki67 proliferative index was 2-3%. The patient is currently receiving treatment with vincristine/carboplatin with partial improvement of symptoms. CONCLUSION: Since our patient's CSF was negative for tumor cells it is unclear whether this case represents two genetically distinct (multicentric) tumors or a metastatic pilocytic astrocytoma with a hypothalamic or spinal cord primary lesion.

Ultrastructural characterization of craniopharyngioma at the tumor boundary: A structural comparison with an experimental toxic model using “oil machinery” fluid, with emphasis on Rosenthal fibers

Craniopharyngiomas (CPs) are cystic, encapsulated, slow-growing epithelial tumors. CPs can be aggressive forms invading and resorting surrounding structures of adjacent brain tissue, where Rosenthal fibers (RFs) are expressed. The aim of this study was to investigate the ultrastructure of these fibers in human biopsies and compare it with an experimental toxic model produced by the cortical infusion of the oil cyst fluid (“Oil machinery” fluid or OMF) from CPs to rats. For this purpose, the CPs from ten patients were examined by light and electron microscopy. OMF was administered to rats intracortically. Immunohistochemical detection of glial fibrillary acidic protein (GFAP) and vimentin was assessed. In both freshly obtained CPs and rat brain tissue, the presence of abundant cellular debris, lipid-laden macrophages, reactive gliosis, inflammation and extracellular matrix destruction were seen. Ultrastructural results suggest focal pathological disturbances and an altered microenvironment surrounding the tumor–brain junction, with an enhanced presence of RFs in human tumors. In contrast, in the rat brain different degrees of cellular disorganization with aberrant filament–filament interactions and protein aggregation were seen, although RFs were absent. Our immunohistochemical findings in CPs also revealed an enhanced expression of GFAP and vimentin in RFs at the peripheral, but not at the central (body) level. Through these findings we hypothesize that the continuous OMF release at the CPs boundary may cause tissue alterations, including damaging of the extracellular matrix, and possibly contributing to RFs formation, a condition that was not possible to reproduce in the experimental model. The presence of RFs at the CPs boundary might be considered as a major criterion for the degree of CPs invasiveness to normal tissue. The lack of RFs reactivity in the experimental model reveals that the invasive component of CPs is not present in the OMF, although the fluid per se can exert tissue damage.

Lithium Decreases Glial Fibrillary Acidic Protein in a Mouse Model of Alexander Disease.

Alexander disease is a fatal neurodegenerative disease caused by mutations in the astrocyte intermediate filament glial fibrillary acidic protein (GFAP). The disease is characterized by elevated levels of GFAP and the formation of protein aggregates, known as Rosenthal fibers, within astrocytes. Lithium has previously been shown to decrease protein aggregates by increasing the autophagy pathway for protein degradation. In addition, lithium has also been reported to decrease activation of the transcription factor STAT3, which is a regulator of GFAP transcription and astrogliogenesis. Here we tested whether lithium treatment would decrease levels of GFAP in a mouse model of Alexander disease. Mice with the Gfap-R236H point mutation were fed lithium food pellets for 4 to 8 weeks. Four weeks of treatment with LiCl at 0.5% in food pellets decreased GFAP protein and transcripts in several brain regions, although with mild side effects and some mortality. Extending the duration of treatment to 8 weeks resulted in higher mortality, and again with a decrease in GFAP in the surviving animals. Indicators of autophagy, such as LC3, were not increased, suggesting that lithium may decrease levels of GFAP through other pathways. Lithium reduced the levels of phosphorylated STAT3, suggesting this as one pathway mediating the effects on GFAP. In conclusion, lithium has the potential to decrease GFAP levels in Alexander disease, but with a narrow therapeutic window separating efficacy and toxicity.

Alexander Disease in Israel: Megalencephaly and Leukoencephalopathy and Its Differential Diagnosis

Alexander disease (AD) is a rare leukodystrophy caused by overexpression of glial fibrillary acidic protein and heat shock proteins, which accumulate in the astrocytes and appear as Rosenthal fibers. Clinically, there are three main phenotypes: infantile, juvenile, and adult forms, with a highly variable clinical course. The classical, and most common phenotype, is the infantile form, which occurs during the first 2 years of life. The diagnosis of AD is based on clinical findings and, supported by magnetic resonance imaging, should be suspected in infants with leukoencephalopathy associated with progressive megalencephaly. In this article, we report two additional patients. Their mutations were already reported; however, while one of them is a common hotspot of the severe infantile-onset phenotype, the other is uncommon and was not yet reported in early infantile-onset AD. To the best of our knowledge, these are the first cases of AD reported from Israel. AD was reported anecdotally in a few other Middle Eastern countries but, since they are usually de novo sporadic mutations, they are not affected by consanguineous marriages, and do not tend to cluster in isolated ethnic populations.

Screening for GFAP rearrangements in a cohort of Alexander disease and undetermined leukoencephalopathy patients

Alexander disease (AxD), a fatal degenerative leukoencephalopathy, is caused by de novo heterozygous missense mutations in the Glial Fibrillary Acidic Protein (GFAP) gene. The pathological hallmark of the disease is the presence of Rosenthal fibers, cytoplasmic inclusions in astrocytes, composed mainly of GFAP, αB-crystallin and HSP27.To date, several patients with a typical presentation of the disease or displaying characteristic Rosenthal fibers in brain material have been reported with no GFAP mutation. Recently, several studies have demonstrated a correlation between Rosenthal fiber formation and wild-type GFAP overexpression, despite the absence of mutations.We tested the hypothesis that a GFAP gene rearrangement could modulate AxD severity or promote GFAP overexpression and aggregation, resulting in leukoencephalopathy. A QMPSF assay was validated for 11 exonic fragments: 3 in control genes (CFTR, DSCR1, F9) and 8 corresponding to GFAP exons. A total of 97 patients suspected of AxD were analyzed: 28 with a GFAP mutation; 69 with clinical and magnetic resonance imaging criteria compatible with the disease. Neither duplications nor deletions of GFAP were found, suggesting that GFAP coding-region rearrangements may not be involved in AxD or Alexander-related leukoencephalopathies.In addition, 80 patients with undetermined leukodystrophies, and negative for PLP1, GJA12, Sox10 and MCT8 mutations and PLP1 and Lamin B1 rearrangements, were tested. These patients were also negative for GFAP rearrangements.Other hypotheses should be investigated for a molecular diagnosis in patients with undetermined leukoencephalopathy: mutations in GFAP isoforms, splicing sites or regulatory regions, or defaults in genes encoding molecular partners of GFAP.