Assessing abnormal iron content in the deep gray matter of patients with multiple sclerosis versus healthy controls.

BACKGROUND AND PURPOSE: It is well known that patients with MS tend to have abnormal iron deposition in and around the MS plaques, in the basal ganglia and the THA. In this study, we used SWI to quantify iron content in patients with MS and healthy volunteers. MATERIALS AND METHODS: Fifty-two patients with MS were recruited to assess abnormal iron content in their basal ganglia and THA structures. One hundred twenty-two healthy subjects were recruited to establish a baseline of normal iron content in deep GM structures. Each structure was separated into 2 regions: a low-iron-content region and a high-iron-content region. The average phase, the percentage area, and the total phase of the high-iron-content region were evaluated. A weighting was also assigned to each subject depending on the level of iron content and its deviation from the normal range. RESULTS: A clear separation between iron content in healthy subjects versus patients with MS was seen. For healthy subjects 13% and for patients with MS 65% showed an iron-weighting factor >3 SDs from the normal mean (P < .05). The results for those patients younger than 40 years are even more impressive. In these cases, only 1% of healthy subjects and 67% of patients with RRMS showed abnormally high iron content. CONCLUSIONS: Iron-weighting factors in the basal ganglia, THA, and the midbrain appeared to be abnormal in roughly two-thirds of patients with MS as measured by SWI.

Iron stores and cerebral veins in MS studied by susceptibility weighted imaging.

AIM: In this paper, we seek to determine whether the iron deposition as seen by susceptibility weighted imaging (SWI) in the basal ganglia and thalamus of patients with multiple sclerosis is greater than the iron content measured in normal subjects (individuals unaffected by multiple sclerosis). As increased iron content may result from increased venous pressure, such information would add credence to the concept of Zamboni et al (1) that MS is caused by chronic cerebrospinal venous insufficiency. METHODS: Fourteen MS patients were recruited for this study with a mean age of 38 years ranging from 19 to 66 year-old. A velocity compensated 3D gradient echo sequence was used to generate SW images with a high sensitivity to iron content. We evaluated iron in the following structures: substantia nigra, red nucleus, globus pallidus, putamen, caudate nucleus, thalamus and pulvinar thalamus. Each structure was broken into two parts, a high iron content region and a low iron content region. The measured values were compared to previously established baseline iron content in these structures as a function of age. RESULTS: Twelve of fourteen patients had an increase in iron above normal levels and with a particular pattern of iron deposition in the medial venous drainage system that was associated with the confluence of the veins draining that structure. CONCLUSION: Iron may serve as a biomarker of venous vascular damage in multiple sclerosis. The backward iron accumulation pattern seen in the basal ganglia and thalamus of most MS patients is consistent with the hypothesis of venous hypertension.

X-linked distal hereditary motor neuropathy maps to the DSMAX locus on chromosome Xq13.1-q21.

OBJECTIVE: To clinically characterize and map the gene locus in a three-generation family with an X-linked adult-onset distal hereditary motor neuropathy. METHODS: Microsatellite markers spanning the juvenile distal spinal muscular atrophy (DSMAX) locus were genotyped and analyzed using genetic linkage analysis. The promoter, untranslated and coding region of the gap junction beta1 (GJB1) gene was sequenced. Nine positional candidate genes were screened for disease mutations using high-resolution melt (HRM) analysis. RESULTS: The family showed significant linkage to markers on chromosome Xq13.1-q21. Haplotype construction revealed a disease-associated haplotype between the markers DXS991 and DX5990. Sequence analysis excluded pathogenic changes in the coding and promoter regions of the GJB1 gene. Additional fine mapping in the family refined the DSMAX locus to a 1.44-cM interval between DXS8046 and DXS8114. HRM analysis did not identify disease-associated mutations in the coding region of nine candidate genes. CONCLUSION: We have identified a family with adult-onset distal hereditary motor neuropathy that refines the locus reported for juvenile distal spinal muscular atrophy (DSMAX) on chromosome Xq13.1-q21. Exclusion of mutations in the coding and regulatory region of the GJB1 gene eliminated the CMTX1 locus as a cause of disease in this family. Nine positional candidate genes in the refined interval underwent mutation analysis and were eliminated as the pathogenic cause of DSMAX in this family. The syndrome in this family may be allelic to the juvenile distal spinal muscular atrophy first reported at this locus.

Cerebellar leukoencephalopathy: most likely histiocytosis-related.

BACKGROUND: Histiocytosis, both Langerhans and non-Langerhans cell type, can be associated with cerebellar white matter abnormalities, thought to be paraneoplastic. The associated clinical picture consists of ataxia, spasticity, and cognitive decline. Hormonal dysfunction is frequent. MRI shows cerebellar white matter abnormalities, as well as brainstem and basal ganglia abnormalities. This so-called "neurodegenerative syndrome" may occur years before or during manifest histiocytosis and also years after cure. We discovered similar MRI abnormalities in 13 patients and wondered whether they could have the same syndrome. METHODS: We reviewed the clinical and laboratory information of these 13 patients and evaluated their brain MRIs. Seven patients underwent spinal cord MRI. RESULTS: All patients were isolated cases; 10 were male. They had signs of cerebellar and pyramidal dysfunction, behavioral problems, and cognitive decline. MRI showed abnormalities of the cerebellar white matter, brainstem, basal ganglia, and, to a lesser extent, cerebral white matter. Three patients had spinal cord lesions. Three patients had laboratory evidence of hormonal dysfunction. No evidence was found of an underlying metabolic defect. In two patients biopsy of nodular brain lesions revealed histiocytic infiltrates. CONCLUSIONS: Considering the striking clinical and MRI similarities between our patients and the patients with this neurodegenerative syndrome in the context of proven histiocytosis, it is likely that they share the same paraneoplastic syndrome, although we cannot exclude a genetic disorder with certainty. The fact that we found histiocytic lesions in two patients substantiates our conclusion. Patients with cerebellar white matter abnormalities should be monitored for histiocytosis.

17p11.2p12 triplication and del(17)q11.2q12 in a severely affected child with dup(17)p11.2p12 syndrome.

Multiple congenital anomalies/mental retardation syndromes due to genomic rearrangements involving chromosome 17p11.2 include deletion resulting in Smith-Magenis syndrome and a reciprocal duplication of the same region resulting in the 17p11.2 duplication syndrome. We present the clinical and molecular analysis of an 8-year-old male with a dup(17p11.2p12) who was evaluated for unusual severity of the phenotype. Fluorescent in situ hybridization (FISH) analysis not only confirmed the 17p duplication but also identified an approximately 25% mosaicism for tetrasomy 17p11.2p12. Whole-genome array comparative genomic hybridization (aCGH) was performed to identify other genomic rearrangements possibly contributing to the severe phenotype and the unusual features in the patient. The 17p duplication was determined by FISH and aCGH to encompass approximately 7.5 Mb, from COX10 to KCNJ12. An approximately 830 Kb deletion of 17q11.2q12, including exon 1 of an amiloride-sensitive cation channel neuronal gene, ACCN1, was also identified by aCGH; breakpoints of the deletion were confirmed by FISH. Sequencing the non-deleted allele of ACCN1 did not show any mutations. Western analysis of human tissue-specific proteins revealed that ACCN1 is expressed not only in the brain as previously reported but also in all tissues examined, including heart, liver, kidneys, and spleen. The large-sized 17p11.2p12 duplication, partial triplication of the same region, and the 17q11.2q12 deletion create a complex chromosome 17 rearrangement that has not been previously identified. This is the first case of triplication reported for this chromosome. Our study emphasizes the utility of whole-genome analysis for known cases with deletion/duplication syndromes with unusual or severe phenotypes.

Steroid-responsive neurologic relapses in a child with a proteolipid protein-1 mutation.

A 10-year-old boy developed corticosteroid-responsive relapsing neurologic signs, including nystagmus and ataxia. MRI revealed multifocal T2 white matter hyperintensities; several were gadolinium-enhancing. CSF contained oligoclonal bands. Although the patient met criteria for multiple sclerosis (MS), the proteolipid protein-1 gene (PLP1) contained a mutation in exon 3B (c.409C>T), predicting a tryptophan-for-arginine substitution. This case raises questions about the role of inflammation in PLP1-related disorders and, conversely, PLP1 mutations in MS.

Pelizaeus-Merzbacher disease: Genetic and cellular pathogenesis.

Pelizaeus-Merzbacher disease (PMD) and the allelic spastic paraplegia type 2 (SPG2) arise from mutations in the X-linked gene encoding myelin proteolipid protein (PLP). Analysis of mutations affecting PLP, the major protein in central nervous system myelin, has revealed previously unsuspected roles for myelinating glia in maintaining the integrity of the nervous system. The disease spectrum for PMD and SPG2 is extraordinarily broad and can be best understood by accounting not only for the wide range of mutations that can occur but also for the effects of PLP1 mutations on both cell autonomous and non-cell autonomous processes in myelinating cells. Appreciating the wide range of genetic and cellular effects of PLP1 mutations is important for patient and family counseling, understanding disease pathogenesis, and, ultimately, for developing future disease-specific therapies.

Alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-mediated excitotoxic axonal damage is attenuated in the absence of myelin proteolipid protein.

In vivo and in vitro studies have shown that alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-receptor-mediated excitotoxicity causes cytoskeletal damage to axons. AMPA/kainate receptors are present on oligodendrocytes and myelin, but currently there is no evidence to suggest that axon cylinders contain AMPA receptors. Proteolipid protein (PLP) and DM20 are integral membrane proteins expressed by CNS oligodendrocytes and located in compact myelin. Humans and mice lacking normal PLP/DM20 develop axonal swellings and degeneration, suggesting that local interactions between axons and the oligodendrocyte/myelin unit are important for the normal functioning of axons and that PLP/DM20 is involved in this process. To determine whether perturbed glial-axonal interaction affects AMPA-receptor-mediated axonal damage, AMPA (1.5 nmol) was injected into the caudate nucleus of anesthetized Plp knockout and wild-type male mice (n = 13). Twenty-four hours later, axonal damage was detected by using neurofilament 200 (NF 200) immunohistochemistry and neuronal damage detected via histology. AMPA-induced axonal damage, assessed with NF 200 immunohistochemistry, was significantly reduced in Plp knockout mice compared with wild-type mice (P = 0.015). There was no significant difference in the levels of neuronal perikaryal damage between the Plp knockout and wild-type mice. In addition, there was no significant difference in the levels of glutamate receptor subunits GluR1-4 or KA2 in Plp knockout compared with wild-type littermates. The present study suggests that PLP-mediated interactions among oligodendrocytes, myelin, and axons may be involved in AMPA-mediated axonal damage.

Juvenile onset Huntington disease resulting from a very large maternal expansion.

We report a 5(1/2)-year-old girl with a maternal family history of Huntington disease (HD), who presented clinically with unbalanced gait, impaired speech, and increasing difficulty with fine motor control. Onset of symptoms began at the age of 3(1/2) years. The suspected diagnosis of juvenile HD, based upon her family history, was confirmed by DNA analysis. At age 7, the patient died secondary to complications of her underlying disorder. Juvenile-onset Huntington disease is uncommon, predominantly transmitted by fathers and is always associated with very large expansions of the CAG repeat. Interestingly, this patient inherited a large CAG size expansion from her mother, who herself had symptoms of HD at the age of 18. Molecular analysis revealed that the mother had 70 CAG repeats whereas our patient had approximately 130 CAG repeats. This is the largest reported CAG expansion from a maternal transmission that has been confirmed molecularly and it demonstrates that very large expansions can also occur through the maternal lineage.

The myelinated axon is dependent on the myelinating cell for support and maintenance: molecules involved.

The myelin-forming cells, oligodendrocytes and Schwann cells, extend processes that spirally wrap axons and provide the insulation that allows rapid saltatory conduction. Recent data suggest a further role for the myelin-forming cells in axonal support and maintenance. This Mini-Review summarises some of the data that support this view and highlights the molecules involved.

Age-related axonal and myelin changes in the rumpshaker mutation of the Plp gene.

The PLP1/Plp gene encodes proteolipid protein (PLP) and DM20, the major central nervous system myelin proteins. Mutations in the PLP1/ Plp gene cause dysmyelinating disorders in man and animals. The rumpshaker mutation was first identified in mice and later linked to a family diagnosed with neurological deficits akin to spastic paraplegia. The dysmyelination in the young rumpshaker mouse is well characterised. Here we report evidence for an age-related increase in myelin due mainly to the myelination of small axons, many large axons remain dysmyelinated. Levels of PLP/DM20 and myelin basic protein are considerably greater in myelin fractions from older compared with younger mutants. Myelin in sheaths of larger axons remains poorly compacted and may account for levels of 2',3'-cyclic nucleotide 3'-phosphodiesterase and myelin-associated glycoprotein being elevated over wild type in older mutant mice. A late-onset distal degeneration of the axons of the longest spinal tract, the fasciculus gracilis, is also noted. This is the first report of Wallerian-type degeneration in mice with spontaneous mutations of the Plp gene.

Effect of monthly intravenous cyclophosphamide in rapidly deteriorating multiple sclerosis patients resistant to conventional therapy.

Fourteen consecutive clinically definite relapsing-remitting multiple sclerosis (MS) patients were treated with monthly intravenous cyclophosphomide (CTX) for 6 months. All had experienced severe dinical deterioration during the 12 months prior to treatment with CTX despite treatment with conventional immunomodulating agents and intravenous methylprednisolone. Treatment with CTX led to improvement and neurologic stability within 6 months which was sustained for at least 18 months after the onset of treatment with CTX. Therapy with CTX was well tolerated. CTX may be of benefit in MS patients who experience rapid clinical worsening and are resistant to conventional therapy.

Sterols in blood of normal and Smith-Lemli-Opitz subjects.

Smith-Lemli-Opitz syndrome (SLOS) is a hereditary disorder in which a defective gene encoding 7-dehydrocholesterol reductase causes the accumulation of noncholesterol sterols, such as 7- and 8-dehydrocholesterol. Using rigorous analytical methods in conjunction with a large collection of authentic standards, we unequivocally identified numerous noncholesterol sterols in 6 normal and 17 SLOS blood samples. Plasma or erythrocytes were saponified under oxygen-free conditions, followed by multiple chromatographic separations. Individual sterols were identified and quantitated by high performance liquid chromatography (HPLC), Ag(+)-HPLC, gas chromatography (GC), GC-mass spectrometry, and nuclear magnetic resonance. As a percentage of total sterol content, the major C(27) sterols observed in the SLOS blood samples were cholesterol (12;-98%), 7-dehydrocholesterol (0.4;-44%), 8-dehydrocholesterol (0.5;-22%), and cholesta-5,7,9(11)-trien-3beta-ol (0.02;-5%), whereas the normal blood samples contained <0.03% each of the three noncholesterol sterols. SLOS and normal blood contained similar amounts of lathosterol (0.05;-0.6%) and cholestanol (0.1;-0.4%) and approximately 0.003;-0.1% each of the Delta(8), Delta(8(14)), Delta(5,8(14)), Delta(5,24), Delta(6,8), Delta(6,8(14)), and Delta(7,24) sterols. The results are consistent with the hypothesis that the Delta(8(14)) sterol is an intermediate of cholesterol synthesis and indicate the existence of undescribed aberrant pathways that may explain the formation of the Delta(5,7,9(11)) sterol. 19-Norcholesta-5,7,9-trien-3beta-ol was absent in both SLOS and normal blood, although it was routinely observed as a GC artifact in fractions containing 8-dehydrocholesterol. The overall findings advance the understanding of SLOS and provide a methodological model for studying other metabolic disorders of cholesterol synthesis.

A prospective, open-label treatment trial to compare the effect of IFN beta-1a (Avonex), IFNbeta-1b (Betaseron), and glatiramer acetate (Copaxone) on the relapse rate in relapsing-remitting multiple sclerosis.

A prospective, non-randomized, open-label treatment trial was performed in patients with relapsing-remitting multiple sclerosis (RRMS), with follow up for 12 months. Our primary objective was to prospectively compare the effect of IFNbeta-1a (Avonex), IFNbeta-1b (Betaseron), and glatiramer acetate (GA, Copaxone) on the relapse rate in patients with RRMS. Between August 1996 and September 1999, 156 consecutive patients with clinically definite RRMS with a Kurtzke scale (EDSS) score of 4 or less were followed for 12 months, from the time of initiating therapy or electing to remain untreated. Prior 2-year relapse history and available chart information was carefully reviewed at the time of enrolment. Thirty-three of 156 elected no treatment (mean age 32.5 years; mean EDSS 2.64) at enrolment; 40 elected IFNbeta-1a (mean age 32.4 years; mean EDSS 2.69), 41 IFNbeta-1b (mean age 32.1 years; mean EDSS 2.56), and 42 chose GA (mean age 31.5 years; mean EDSS 2.57). Annual relapse rate based upon the 2 years prior to enrolment was 1.08 in the untreated group, 1.20 in the AV group, 1.21 in the BE group, and 1.10 in the GA group. There were no statistically significant differences among the four groups at enrolment. After 12 months of treatment, patients in the untreated groups had a relapse rate of 0.97, whereas patients in the IFNbeta-1a, IFNbeta-1b, and GA groups had relapse rate of 0.85, 0.61, and 0.62, respectively. Compared to the untreated group, reduction in the relapse rate was statistically significant only in the GA (P=0.003) and IFNbeta-1b (P=0.002) groups, in contrast to the IFNbeta-1a treated patients, who did not show a significant reduction (P=0.309). Compared to the untreated patients, mean EDSS was significantly reduced only in the GA (P=0.001) and IFNbeta-1b (P=0.01), in contrast to IFNbeta-1a treated patients (P=0.51). In this prospective, controlled, open-label, non-randomized 12-month study, treatment with only GA and IFNbeta-1b significantly reduced the relapse rate compared to untreated patients, supporting early treatment in RRMS. Our results are similar to the observations made after 12 months of therapy in phase III studies of IFNbeta-1a, IFNbeta-1b, and GA. Despite some limitations of the study design, the results provide helpful clinical information regarding the relative efficacy of each therapy in mildly affected treatment-naïve RRMS patients.

A prospective, open-label treatment trial to compare the effect of IFNbeta-1a (Avonex), IFNbeta-1b (Betaseron), and glatiramer acetate (Copaxone) on the relapse rate in relapsing--remitting multiple sclerosis: results after 18 months of therapy.

We previously reported results of a 12 month prospective, non-randomized, open-label treatment trial of immunomodulatory therapy in patients with relapsing-remitting multiple sclerosis (RRMS). We now report the results after 18 months of follow-up. Our primary objective was to compare the effect of IFNbeta-1a (Avonex), IFNbeta-1b (Betaseron), and Glatiramer Acetate (GA, Copaxone) to no treatment on the relapse rate in patients with RRMS. One hundred and fifty-six consecutive patients with clinically definite RRMS with a Kurtzke scale (EDSS) score of 4 or less were followed for 18 months. Prior 2-year relapse history and available chart information was carefully reviewed at the time of enrollment Thirty-three of 156 elected no treatment at enrollment; 40 elected IFNbeta-1a, 41 IFNbeta-1b, and 42 chose GA. There were no statistically significant differences among the four groups at enrollment. After 18 months of treatment 122 patients remained in their original treatment group. Compared to the untreated group (1.02), mean annualized number of relapses was significantly reduced only in the GA (0.49, P>0.0001) and IFNbeta-1b groups (0.55, P=0.001) in contrast to the IFNbeta-1a treated patients (0.81, P=0.106) who did not show a significant reduction. Despite limitations of the study design, the results provide helpful clinical information regarding the relative efficacy of each therapy in mildly affected treatment naive RRMS patients.

Gtx, an oligodendrocyte-specific homeodomain protein, has repressor activity.

Myelin, a multilamellar membrane structure that facilitates nerve conduction, is synthesized in the central nervous system (CNS) by oligodendrocytes. Gtx, a member of the homeodomain family of transcriptional factors, is a candidate regulator of myelin gene expression, because it is uniquely expressed in myelinating oligodendrocytes in postnatal rodent brain. To analyze the regulatory activity of Gtx, we first identified the optimal Gtx-binding sequence using an in vitro DNA-binding assay. This sequence, (A/T)TTAATGA, contains a TAAT core and is similar, but not identical, to that of other homeodomain protein binding sites. When coexpressed in cultured cells along with a minimal promoter containing five tandem repeats of this optimal Gtx-binding sequence, Gtx demonstrated repressor activity, which was also present when Gtx was tethered to DNA by way of the strong GAL4 DNA-binding domain. Truncations of the GAL4-Gtx fusion identified a portable repressor domain within a relatively proline/alanine-rich region N-terminal to the Gtx homeodomain. Cotransfection of a Gtx expression vector into a variety of cell lines, including oligodendrocytes, along with constructs containing portions of the PLP, MBP, or Gtx promoters fused to a reporter gene, however, did not modulate transcription from any of these promoter constructs. These data support the notion that the oligodendrocyte-specific homeodomain protein Gtx can act as a transcriptional repressor. In addition, they suggest that interaction of Gtx with other, as yet undefined, transcriptional regulators modifies Gtx activity in oligodendrocytes.

Proteolipid protein mRNA stability is regulated by axonal contact in the rodent peripheral nervous system.

Proteolipid protein (PLP) and its alternatively spliced isoform, DM20, are the main intrinsic membrane proteins of compact myelin in the CNS. PLP and DM20 are also expressed by Schwann cells, the myelin-forming cells in the PNS, and are necessary for normal PNS function in humans. We have investigated the expression of PLP in the PNS by examining transgenic mice expressing a LacZ transgene under the control of the PLP promoter. In these animals, myelinating Schwann cells expressed beta-galactosidase more prominently than nonmyelinating Schwann cells. PLP/DM20 mRNA levels, but not those of LacZ mRNA, increased during sciatic nerve development and decreased after axotomy, with resultant Wallerian degeneration. PLP/DM20 transcription rates, in nuclear run off experiments, however, did not increase in developing rat sciatic nerve despite robust increases in PLP/DM20 mRNA levels during the same period. In RNAse protection studies, PLP mRNA levels fell to undetectable levels following nerve transection whereas levels of DM20 were essentially unchanged despite both being transcribed from the same promoter. Finally, cotransfection studies demonstrated that PLP-GFP, but not DM20-GFP mRNA is down-regulated in Schwann cells cultured in the absence of forskolin. Taken together these data demonstrate that steady state levels of PLP mRNA are regulated at a posttranscriptional level in Schwann cells, and that this regulation is mediated by Schwann cell-axonal contact. Since the difference between these two mRNAs is a 105-bp sequence in PLP and not in DM20, this sequence is likely to play a role in the regulation of PLP mRNA.

Neurological dysfunction and axonal degeneration in Charcot-Marie-Tooth disease type 1A.

Charcot-Marie-Tooth disease type 1A (CMT1A), the most frequent form of CMT, is caused by a 1.5 Mb duplication on the short arm of chromosome 17. Patients with CMT1A typically have slowed nerve conduction velocities (NCVs), reduced compound motor and sensory nerve action potentials (CMAPs and SNAPs), distal weakness, sensory loss and decreased reflexes. In order to understand further the molecular pathogenesis of CMT1A, as well as to determine which features correlate with neurological dysfunction and might thus be amenable to treatment, we evaluated the clinical and electrophysiological phenotype in 42 patients with CMT1A. In these patients, muscle weakness, CMAP amplitudes and motor unit number estimates correlated with clinical disability, while motor NCV did not. In addition, loss of joint position sense and reduction in SNAP amplitudes also correlated with clinical disability, while sensory NCV did not. Taken together, these data strongly support the hypothesis that neurological dysfunction and clinical disability in CMT1A are caused by loss or damage to large calibre motor and sensory axons. Therapeutic approaches to ameliorate disability in CMT1A, as in amyotrophic lateral sclerosis and other neurodegenerative diseases, should thus be directed towards preventing axonal degeneration and/or promoting axonal regeneration.

Charcot-Marie-Tooth disease type 1: molecular pathogenesis to gene therapy.

Charcot-Marie-Tooth disease type 1 (CMT1) is caused by mutations in the peripheral myelin protein, 22 kDa (PMP22) gene, protein zero (P0) gene, early growth response gene 2 (EGR-2) and connexin-32 gene, which are expressed in Schwann cells, the myelinating cells of the peripheral nervous system. Although the clinical and pathological phenotypes of the various forms of CMT1 are similar, including distal muscle weakness and sensory loss, their molecular pathogenesis is likely to be quite distinct. In addition, while demyelination is the hallmark of CMT1, the clinical signs and symptoms of the disease are probably produced by axonal degeneration, not demyelination itself. In this review we discuss the molecular pathogenesis of CMT1, as well as approaches to an effective gene therapy for this disease.