Leukodystrophies: Pathogenesis, diagnosis, strategies, therapies, and future research directions

TY - JOUR

T1 - Leukodystrophies

T2 - Pathogenesis, diagnosis, strategies, therapies, and future research directions

AU - Maria, Bernard L.

AU - Deidrick, Kathleen Mc Cann

AU - Moser, Hugo

AU - Naidu, Sakkubai

N1 - Funding Information: Maria Bernard L. MD, MBA Department of Child Health, University of Missouri Health Care, [email protected] . McCann Deidrick Kathleen PhD Department of Health Psychology, University of Missouri Health Care, Columbia, MO Moser Hugo MD Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD Naidu Sakkubai MD Department of Neurology, Kennedy Krieger Institute and Johns Hopkins University School of Medicine, Baltimore, MD 09 2003 18 9 578 590 sagemeta-type Other search-text 578 DiscussionLeukodystrophies: Pathogenesis, Diagnosis, Strategies, Therapies, and Future Research Directions SAGE Publications, Inc.2003DOI: 10.1177/08830738030180090401 Bernard L.Maria MD, MBA Department of Child Health, University of Missouri Health Care, [email protected]. KathleenMcCann Deidrick PhD Department of Health Psychology, University of Missouri Health Care, Columbia, MO HugoMoser MD Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD SakkubaiNaidu MD Department of Neurology, Kennedy Krieger Institute and Johns Hopkins University School of Medicine, Baltimore, MD Leukodystrophies comprise a heterogeneous group of dis- orders characterized by destruction or failed development of central white matter. On October 9, 2002, we held a sym- posium to determine how research findings enhance clini- cal practice and to identify research needed to improve diagnostic accuracy and develop safe, effective therapies. Participants described current diagnostic strategies, reviewed pathogenesis, discussed current therapies and clinical trials, and defined future research directions. This article summarizes their presentations and includes the verbatim edited transcript of question-and-answer sessions. CURRENT APPROACHES TO DIAGNOSIS AND MOLECULAR TESTING Moderator: Sakkubai Naidu, MD Kennedy Krieger Institute and the Johns Hopkins University School of Medicine Baltimore, MD Definition, Pathology, and Classification of Leukodystrophies James Powers, MD University of Rochester Medical Center Rochester, NY Dr Powers defined leukodystrophies as progressive dis- eases of myelin in which a genetically determined molecu- lar abnormality is responsible for metabolic deficits in myelin sheaths or myelinating cells, resulting in the destruc- tion or failed development of central white matter. He fur- ther described leukodystrophies as one of three types of cen- tral nervous system myelin diseases (ie, dysmyelinative, demyelinative, or myelinolytic). Dr Powers differentiated leukodystrophies from leukoencephalopathies, which can mimic leukodystrophies but consist of white-matter lesions lacking the necessary genetic, progressive, or predomi- nantly white-matter qualities of the leukodystrophies. Exam- p l e s o f l e u k o e n c e p h a l o p a t h i e s i n c l u d e subcortical arteriosclerotic leukoencephalopathy and cerebral auto- somal dominant arteriopathy with subcortical infarcts and leukoencephalopathy. In addition, he noted that diseases of white matter are not always diseases of myelin (eg, vascu- lar problems resulting in white-matter lesions). The four types of leukodystrophies are classic dys- myelinative, hypomyelinative, spongiform, and miscella- neous. Examples of classic dysmyelinative diseases include adrenoleukodystrophy, globoid cell leukodystrophy (Krabbe's disease), metachromatic leukodystrophy, sudanophilic dystrophy, and neuroaxonal leukodystrophy. Dr Powers also provided examples of hypomyelinative dis- eases (Pelizaeus-Merzbacher disease and Alexander's dis- ease), spongiform diseases (Canavan's disease, Canavan-van Bogaert-Bertrand disease, adult-onset spongiform leukody- strophy, and vacuolating megalencephalic leukoen- cephalopathy with subcortical cysts), and miscellaneous diseases (vanishing white-matter disease/childhood ataxia with diffuse cerebral hypomyelination, Aicardi-Goutieres syndrome, and Cockayne's syndrome). Several gross neuropathologic features are common to all leukodystrophies, including reduced brain weight, optic atrophy, ventriculomegaly, and atrophy of the corpus callo- sum. Sparing of U fibers is characteristic of all leukodys- trophies except Canavan's disease and Pelizaeus-Merzbacher disease, whereas bilaterally symmetric loss of cerebral and cerebellar white matter is characteristic of all leukodystro- phies except Pelizaeus-Merzbacher disease. Common light microscopic features include reduced myelin staining, loss of oligodendrocytes, relative sparing of axons despite marked axonal loss, macrophages with myelin debris, reactive astro- cytosis in the early stages, and fibrillary astrogliosis and sclerosis in the late stages. An absence of inflammatory Received April 24, 2003. Accepted for publication April 25, 2003. Presented in part at the Leukodystrophies Symposium of the Child Neurology Society Annual Meeting, Washington, DC, October 9, 2002. Address correspondence to Dr Bernard L. Maria, Department of Child Health, University of Missouri Health Care, One Hospital Drive, 7W-12A DC058.00, Columbia, MO 65212. Tel: 573-884-2935; fax: 573-884-4277; e-mail: 579 cells is associated with all leukodystrophies except adrenoleukodystrophy. Despite these commonalities, Dr Powers described light microscopic, macrophage and myelin debris, ultra- structural, and anatomic site differences among leukodys- trophies that help practitioners formulate the proper diagnosis. At the light microscopic level, examples include the observation of lymphocytes in adrenoleukodystrophy, metachromasia in metachromatic leukodystrophy, globoid cells in Krabbe's disease, Rosenthal fibers in Alexander's dis- ease, spongy to vacuolated myelin in Canavan's disease, and tigroid myelinopathy in Pelizaeus-Merzbacher disease. In adrenoleukodystrophy, macrophages are vacuolated and striated, whereas in sudanophilic leukodystrophies, macrophages are vacuolated, and cholesterol asters are present. Metachromatic leukodystrophy results in metachro- matic products. Ultrastructural differences include spongy, vacuolar myelin in Canavan's disease owing to intermyelin edema and astrocytic swelling. In Pelizaeus-Merzbacher disease, a patchy pattern with perivascular presence of myelin is observed. In adrenoleukodystrophy, bilamella leaflets called crystalloids form, whereas in metachromatic leukodystrophy, prismatic structures form. Finally, differ- ences in topography are also described. Although some leukodystrophies are constricted to the central nervous system white matter (eg, Alexander's disease, Canavan's disease, and Pelizaeus-Merzbacher diseases), others include peripheral nervous system involvement (eg, globoid cell leukodystrophy, adrenoleukodystrophy, metachromatic leukodystrophy). Clinical Manifestations and Diagnostic Approaches for Unclassified Leukodystrophies Sakkubai Naidu, MD Kennedy Krieger Institute and the Johns Hopkins University School of Medicine Baltimore, MD Dr Naidu presented a diagnostic algorithm using clinical criteria. First, she indicated that the disease is neuronal if seizures occur. If spasticity and ataxia are present, white- matter disease is the likely diagnosis. Thus, if a child has only minimal white-matter changes on magnetic reso- nance imaging (MRI) but marked cognitive and muscular tone deficits, axonal disease is suspected. In contrast, if MRI shows devastating white-matter degradation in asso- ciation with neurologic deficits, demyelinating disease is suspected. Dr Naidu defined two types of myelin abnor- malities: immature myelination and hypomyelination, con- sisting of arrested myelination or delayed myelination. Immature myelination is defined as a reduced quantity of mature myelin but with age-appropriate distribution. Hypomyelination is defined as a reduced quantity of myelin for age; myelin distribution can be normal or abnormal and includes delayed or arrested myelination. Follow-up MRI is necessary to determine whether myelin growth has been arrested (follow-up evaluations show no evidence of new myelin deposition) or delayed (follow-up MRI would show an increase in myelin deposition both in amount and dis- tribution that would be appropriate for a younger age group). In addition, children with delayed myelination can achieve developmental milestones, unlike those with arrested myelination. The presence of dysmorphism can help narrow poten- tial diagnoses, eliminating the leukodystrophy diagnosis and suggesting other diseases. For example, in Salla disease (a deficit in the lysosomal transport of sialic acid), chil- dren exhibit dysmorphism, hypomyelination, increased N- acetylaspartate on magnetic resonance spectroscopy, organomegaly, and sialic acid in the urine. Salla disease can be differentiated from Canavan's disease by the absence of megalencephaly. The peroxisome biogenesis disorders (eg, Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum's disease) are also characterized by dys- morphism. The absence of dysmorphism can direct the physician to consider infectious disease (eg, acute dissem- inating encephalomyelitis, cytomegalovirus). In addition, Aicardi-Goutieres syndrome should be suspected in children with basal ganglia and white-matter changes, lymphocyto- sis, elevated α-interferon, and no dysmorphism. The presence of calcifications indicates diseases such as Aicardi-Goutieres syndrome, Labrune's disease, and Cockayne's syndrome. The location of white-matter abnor- malities can also be a useful discriminating factor. Exam- ples include the absence of U fiber involvement in Canavan's disease, localization of white-matter problems to the frontal lobe in metachromatic leukodystrophy, and posterior pre- ponderance in Krabbe's disease. Other clinical features include retinal abnormalities, infections, skin abnormalities, light sensitivity, and U fiber involvement. Dr Barker reviewed the imaging techniques useful in diag- nosing leukodystrophies, although he cautioned profes- sionals not to rely on imaging alone for diagnosis. Although computed tomographic (CT) scans help identify calcifica- tions, Dr Barker stated that MRI is preferable to CT because it provides greater soft tissue contrast and does not involve ionizing radiation. With MRI, an array of conventional imag- ing techniques (eg, proton density, T1-weighted and T2- weighted, fluid-attenuated inversion recovery and advanced magnetic resonance spectroscopy, magnetization transfer imaging, diffusion tensor imaging, perfusion imaging, func- tional imaging, and molecular imaging) are available. Regardless of the imaging techniques used, the ability to recognize the MRI patterns characteristic of specific diseases is critical in diagnosing leukodystrophies. Dr Barker reviewed several characteristic MRI patterns observed in patients with leukodystrophies. Patients with 580 metachromatic leukodystrophy present with radial stripes on MRI. Bilateral symmetric parietal involvement, includ- ing the splenium of the corpus callosum, is a characteris- tic pattern in patients with adrenoleukodystrophy. However, in 17% of patients with adrenoleukodystrophy, frontal involvement is noted, and in some cases, frontopontine and spinal tract involvement is observed. Patients with Alexander's disease frequently have an enlarged head, hyperintense white matter with more loss of myelin in the frontal lobes noted on T2-weighted MRI, enlarged and hyperintense caudate nuclei, a dark periventricular rim on T2-weighted MRI and a bright periventricular rim on T1- weighted MRI, and midbrain and periventricular rim enhancement. Patients with megalencephalic leukoen- cephalopathy with subcortical cysts typically have megal- encephaly and develop cysts in the temporal lobes and subcortical regions. Despite these general patterns, some cases reveal idiosyncratic patterns that make diagnosis difficult. In addition, Dr Barker introduced the possibility that T2-weighted hyperintensity on MRI could be a non- specific and overly sensitive finding and might not always provide helpful information for diagnosis. Dr Barker discussed the use of magnetic resonance spectroscopy, which can show choline, myo-inositol, N- acetylaspartate, or lactate in patients with demyelinating diseases. Multiple techniques are available using magnetic resonance spectroscopy, including single-voxel spec- troscopy (obtains spectrum from one area of the brain) and spectroscopic imaging (shows the distribution of com- pounds in the brain). Spectroscopy could help predict which areas of the brain will deteriorate over time, as seen in a study of patients with adrenoleukodystrophy. In this set of patients, areas of white matter that appeared nor- mal but had high choline signals demyelinated over time. Of the 25 patients in the study, those with a low N-acetyl- aspartate-to-choline ratio had progressive disease. Dr Barker also noted that differences in spectroscopy patterns are found between chronic (low choline) and active (high choline) lesions and that spectroscopy can help differen- tiate T2-weighted hyperintensity owing to edema from T2- weighted hyperintensity owing to demyelination. Finally, spectroscopy can provide additional diagnostic informa- tion as leukodystrophies are often associated with unique spectroscopic findings. Examples include an increased N-acetylaspartate signal respective to other metabolites in Canavan's disease and low N-acetylaspartate and choline levels in the white matter of patients with vanishing white- matter disease. Other advanced MRI techniques used to evaluate leukodystrophies include diffusion tensor imaging, which measures the microscopic motion of water molecules within the brain. As water diffuses faster among white-matter tracts, the technique can be used to determine whether fiber tracts are intact in patients with leukodystrophies. In addition, the higher magnetic field strength in MRI, which provides higher resolution, and molecular imaging might help in the future. Molecular Testing Eric Hoffman, PhD Children's National Medical Center Washington, DC Dr Hoffman reviewed current molecular testing procedures for leukodystrophies and emerging technologies under development at the Children's National Medical Center. He began by reviewing recent studies involving molecular test- ing for two leukodystrophies (Alexander's disease and megalencephalic leukoencephalopathy with subcortical cysts), reviewed the current state of molecular testing and concluded by reviewing new molecular technologies. The glial fibrillary acidic protein gene (GFAP), located at chromosome 17q21 and critical to astrocyte structure, is implicated in Alexander's disease. A recent study investi- gated mutations in GFAP among 13 children (4 girls, 9 boys) meeting these criteria: MRI findings consistent with a diag- nosis of Alexander's disease, a normal karyotype, negative metabolic screening, and a negative family history. Clinical presentations varied, with some children experiencing subtle symptoms discovered secondary to investigation of another concern (eg, head injury). Seven of the children exhibited infantile onset of the disease and five of the children exhib- ited juvenile onset of the disease. The denaturing high-per- formance liquid chromatography technique was used to identify mutations. In denaturing high-performance liquid chromatography, patient DNA or polymerase chain reaction (PCR) products are placed through a column and the column's temperature is increased until DNA breaks off of the column at a position that indicates the location of the mutation. Among these children, 12 exhibited mutations, with changes to arginine's charge being the most characteristic. Scientists suggest that the change in charge causes arginine to fall out of solution, resulting in the Rosenthal fibers observed in Alexander's disease. Thus, as in many dominant neurologic disorders, the affected protein is available, but it is toxic. Presymptomatic diagnosis could allow for removal of the toxic protein prior to the development of Rosenthal fibers. Dr Hoffman also described an extensive series of pedi- gree studies for megalencephalic leukoencephalopathy with subcortical cysts in the Agarwal Indian population. The MLC1 gene is implicated and is located at chromosome 22qtel. Of 33 screened patients, most were homozygous for one extra C. Thus, this is a recessive disorder in which a com- ponent is missing, resulting in an absent or nonfunctional protein. The goal of molecular diagnostics in such cases would be to replace the missing protein. Dr Hoffman reviewed the current state of molecular diagnostic testing and emerging technologies in DNA, mes- senger ribonucleic acid (RNA), and protein testing. Most dominant disorders have been identified through DNA link- age studies. In addition, studies of single-nucleotide poly- morphism, genetic variations that dictate individual differences, are emerging as a methodology for DNA testing. Investigators use denaturing high-performance liquid chro- matography to identify single-nucleotide polymorphisms and 581 third wave and nanogen technologies to genotype single- nucleotide polymorphisms, with the goal of discovering the polymorphisms predisposing people to various common dis- eases (eg, heart disease, type 2 diabetes) and identifying rel- evant environmental factors interacting with single-nucleotide polymorphisms. Although molecular testing techniques, including genotyping for common mutations, are currently quite common and inexpensive, such techniques continue to be difficult and expensive to perform for people with private or unique mutations. Because leukodystrophies involve many private mutations, DNA testing is challenging. Second, using messenger RNA, scientists can now take a very small tissue sample and accurately assess every gene in the genome to find out if it is being used and to what extent. Such analysis (ie, microarrays/expression profiling) helps detect molecular changes related to the primary defect. For example, do Rosenthal fibers directly cause Alexander's disease, or do they result in other reactions (eg, stress, shock) that cause the disease? Scientists are build- ing large databases of messenger RNA fingerprints diagnostic of known diseases. Third, assessments of proteins (proteomics) involve purifying and characterizing proteins. Unlike DNA and mes- senger RNA analysis, all proteins cannot be looked at and identified at once. However, a new machine called the TOF/TOF is in development and promises to sequence 40,000 proteins a day using small amounts of tissue. Dr Hoffman described two new platforms for genotyp- ing: third wave and nanogen. Third wave technology involves a two-color assay, specific cleavage by the cleavase enzyme, and linear signal amplification by fluorescence resonance energy transfer. Third wave technology is a cost-effective method, is time efficient, and is the first method that does not require PCR. In a sample of 350 patients with Rett syn- drome, the technique was 100% accurate. The nanogen chip is small and involves a two-color assay, thermal discrimi- nation, and electronic hybridization. Nanogen is also cost effective and time efficient, although it does use PCR. Question and Answer Session 1 Dr Naidu: We covered broad aspects of trying to diagnose leukodystrophies, and, as you can see, it's rather difficult and confusing. We need multiple spe- cialists to handle this, from the clinical to the imaging to the diagnostics of the genetic aspects. Dr Giridharan: Dr Barker, how helpful is diffusion tensor imaging in exclu- sion of leukodystrophies? Dr Barker: Yes, there are decreases in the diffusion, anisotropy. The clin- ical significance is unknown, but the technique holds promise in studying diffuse axonal injury. Dr Stephenson: I am projecting a slide for what might be called Labrune syndrome. The slide shows calcification and leukodystrophy compatible with Labrune's disease. However, these children were first described in 1988; they have angiomata and reaction in the retina and facial dysmorphism. Several people in this room have seen similar patients. Dr Naidu: Dr Stephenson's point is that, in addition, there is a retinopa- thy in these patients, and that should be borne in mind. In the three cases that Dr Lydia presented from our place, one of them did have an exudative retinopathy, a patient of Dr Philippart's. So I think there is a spectrum in this disease, and you're very right that, in addition to the calcifications in the basal ganglia region, the white-matter changes, and the cysts, there is this retinopathy that should be kept in mind. Dr Stephenson: It also affects the bones and the bone marrow, or may do. It's a multisystem disorder. Dr Naidu: Yes. Dr Stephenson: I think it's a very exciting disorder. Dr Naidu: Yes. Dr Stephenson: It might be something to do with dyskeratosis congenita. All sorts of possibilities. Dr Legido: At St. Christopher's in Philadelphia, we had an adult patient who was diagnosed with multiple sclerosis who also had phenylketonuria. Are white-matter changes in phenylketonuria similar to changes seen in leukodystrophies? Dr Naidu: From what I see, the untreated patient who develops white-mat- ter changes must have a leukoencephalopathy. But I would also ask Dr Pow- ers what he sees in pathology. Dr Powers: I think what has been reported is hypomyelination or spongy change for the aminoacidurias and for the organic acidurias. So I think if it's progressive, it should be considered a leukodystrophy. Such cases are now very rare in neuropathology. Dr Maria: I have a question about the imaging. When we're suspecting that a patient has a leukodystrophy now, has the science of spectroscopy advanced sufficiently for it to be considered standard imaging? That is, when MRI has defined areas of abnormality and you're trying to narrow down your differential, is magnetic resonance spectroscopy something that we should be expecting of our neuroradiologists? Dr Barker: I think the answer to that is yes. It's a widely available tech- nique now. It's reimbursable, or at least I think it's reimbursable. It provides a terrific amount of information in terms of the status of the axons and myeli- nation. It can be done quickly. So I absolutely think that if you see a patient with a suspected leukodystrophy, you should do spectroscopy in those patients. Do you want to comment? Dr Naidu: It's also a question about spectroscopy because you showed that in magnetic resonance spectroscopy imaging, you can see abnormalities in the voxels anterior to lesions. My question is does it make sense to do con- ventional spectroscopy even if you don't have any white-matter abnor- mality in an MRI, if you don't understand the disease of the patient? What is the chance of finding anything? Dr Barker: Well, certainly, I've seen quite a lot of patients who have nor- mal conventional MRI scans but may have some abnormality on their spec- troscopy study. For instance, in adrenomyeloneuropathy, I've seen several patients who have high choline signals but a completely normal conventional brain MRI. In general terms, should everybody who has a normal brain MRI have a spectroscopy study? I think you're going to have a very low yield for detecting something in general terms. But I think if you have some other information, some clinical information where you're suspecting a particu- lar diagnosis, such as adrenoleukodystrophy for instance, then it's worth doing the spectroscopy study even if the brain MRI is normal. 582 Dr Naidu: And where would you do the spectroscopy? Dr Barker: Well, it depends on what disease you suspect. For instance, for adrenoleukodystrophy, obviously, you would choose the parietal white mat- ter. If you suspect a mitochondrial disease,in general, I would saylook at the gray matter because most mitochondrial diseases really present more often in gray matter thanin the white matter. But probably you should do both gray and white matter, maybe even look at the cerebrospinal fluid also. FROM CELL BIOLOGY TO ANIMAL MODELS TO BEDSIDE AND THERAPY: UPDATES ON SPECIFIC LEUKODYSTROPHIES: PART 1 Moderator: Hugo Moser, MD Director of Neurogenetics, Kennedy Krieger Institute Professor of Neurology and Pediatrics Johns Hopkins University School of Medicine Baltimore, MD Adrenoleukodystrophy Kirby Smith, PhD; Hugo W. Moser, MD Kennedy Krieger Institute and the Johns Hopkins University School of Medicine Baltimore, MD Adrenoleukodystrophy is a genetic disorder characterized by accumulation of very-long-chain fatty acids in the white matter, adrenal cortex, and testes. It is caused by a defect in a gene located at chromosome Xp28 that codes for the per- oxisomal membrane protein that is part of the adenosine triphosphate–binding cassette transporter superfamily. It is unclear how the genetic defect causes very-long-chain fatty acids to accumulate. The observed increase invery-long-chain fatty acids has been associated with decreased very-long- chain fatty acid β-oxidation, reduced levels of very-long- chain acyl coenzyme A (CoA) synthetase enzyme, and activation of CoA derivatives. Researchers have speculated that an interaction between the adrenoleukodystrophy pro- tein and the very-long-chain acyl CoA synthetase enzyme is a mechanism for accumulation of very-long-chain fatty acids. However, research involving mouse knockout models for the adrenoleukodystrophy protein, the very-long-chain acyl CoA synthetase enzyme, and for both proteins suggests an addi- tive effect. Specifically, adrenoleukodystrophy knockout results in accumulation of very-long-chain fatty acids with- out a decreased rate of very-long-chain fatty acid β-oxi- dization. In contrast, very-long-chain acyl CoA synthetase knockout results in a decreased rate of very-long-chain fatty acid β-oxidization but no accumulation of very-long-chain fatty acids. In a double-knockout mouse, both effects are observed. Instead, research suggests that “an interaction between mitochondria and peroxisomes, perhaps mediated by [adrenoleukodystrophy protein] ALDP, can account for a lot of the observations seen in fibroblasts.”1 Furthermore, researchers suggest that mutations in the gene are specifi- cally related to adrenomyeloneuropathy and are the result of problems with mitochondria rather than very-long-chain fatty acids. The cerebral inflammatory type of adrenoleuko- dystrophy, however, is still likely to be related to very-long- chain fatty acids. The incidence of adrenoleukodystrophy is equivalent to that of phenylketonuria, 1 in 17,000. There are three major types of adrenoleukodystrophy. The first, adreno- myeloneuropathy, is a distal, progressive, noninflammatory axonopathy that affects the corticospinal tracts and dorsal columns, resulting in paraparesis in young adults. Adreno- myeloneuropathy occurs in 40 to 45% of all patients, with a typical onset in middle age. All men, boys, and male infants with adrenoleukodystrophy and 50% of heterozygotes even- tually develop adrenomyeloneuropathy. The second form is an inflammatory cerebral demyelination, which affects 50% of men, boys, and male infants with the disorder. Thirty-five to 40% of patients exhibit the subtype, and many die before developing adrenomyeloneuropathy. Symptoms are pro- gressive and predominantly affect the parieto-occipital (65%) areas of the brain. The age of onset ranges from 2 to 10 years, and early symptoms resemble attention-deficit hyperactivity disorder. Approximately 20 to 30% of patients experience the third type, Addison's disease, only; most later develop adrenomyeloneuropathy. More than one form of the disorder can be observed in a single family, and phe- notypical variability is common. Early identification of patients with adrenoleukodys- trophy is crucial to successful treatment. A plasma very-long- chain fatty acid assay is 100% accurate in identifying patients with adrenoleukodystrophy but has a 20% false-negative rate in girls. Thus, mutation analysis is recommended for girls. Prenatal testing is generally accurate, with 63 fetuses identified to date, and plasma very-long-chain fatty acids are present at birth in children identified prenatally. Extended family screening is also useful as adrenoleukodystrophies are predominantly familial in nature, with new mutations accounting for less than 5% of cases. In one study of extended family screening, of 4000 cases, 594 new patients and 1270 carriers were identified. In half of the patients identified with adrenoleukodystrophy, the disease had not been diagnosed before screening. Most mutations are unique, and more than 403 have been identified. Adrenal hormone replacement therapy is a life-saving, often-neglected treatment. However, hormone replacement does not affect neurologic function. Bone marrow trans- plantation might help prevent neurologic difficulties in chil- dren with the cerebral inflammatory subtype of the disease, before the brain is heavily affected. A preliminary study also indicates that Lorenzo oil therapy can be preventive. In 105 asymptomatic boys with normal MRI who were younger than 6 years of age, those whose very-long-chain fatty acids levels were adequately lowered by Lorenzo oil therapy were compared with those without reduction in very- long-chain fatty acid production. A 77% risk reduction was reported for boys who received the therapy and demon- strated a reduction in very-long-chain fatty acids. Thus, the therapy is recommended for asymptomatic boys younger than 6 years old, although children receiving the treatment should be carefully supervised for side effects by a multi- 583 disciplinary team. In addition, ongoing monitoring of boys receiving the therapy is necessary to identify appropriate can- didates for bone marrow transplantation. The National Institutes of Health (NIH), the National Institute of Child Health and Human Development, and the Office of Rare Diseases are developing a multicenter ther- apeutic trial for treatment of adrenoleukodystrophy. Future research focuses on the development of mass neonatal screening for boys, promotion of extended family screen- ing, and improved identification of heterozygotes through mutation analysis. Practitioners also seek to develop more sensitive measures for evaluating the effect of therapy. Metachromatic Leukodystrophy Volkmar Gieselmann, MD, PhD University of Bonn Bonn, Germany Metachromatic leukodystrophy is a rare (1 in 100,000), auto- somal recessive disease characterized by a deficiency in arylsulfatase A, resulting in accumulation of cerebroside-3- sulfate (sulfatide) and demyelination. Subtypes of metachro- matic leukodystrophy include early onset (age 2 years; 40% of cases), juvenile onset (age range 3–16 years; 40% of cases), and adult onset (age ≥ 16 years; 20% of cases). At least 80 dif- ferent mutations in the arylsulfatase A gene can cause metachromatic leukodystrophy. Three common mutations have been identified. One allele affects the exon 2 splice donor site, is associated with the early-onset subtype, and occurs in 25% of cases. The Pro426Leu amino-acid substitution accounts for another 25% of cases and is most often asso- ciated with the late-onset subtype (neuromotor phenotype). Finally, the Lle179Ser mutation, involving an additional allele with amino-acid substitution, is present in 6% of cases and is associated with the late-onset subtype (psychiatric phe- notype). Study of an arylsulfatase A–deficient mouse suggests that the disease is not confined to the glial cells but also occurs in neurons. The arylsulfatase A–deficient mouse shows sulfatide storage in the bile ducts, kidney, and brain; reduced axon diameter; degeneration of acoustic ganglion and peripheral nervous system fibers; and progressive neu- rologic difficulties. However, clinical presentation is milder in mice than in humans, involving less demyelination. In arylsulfatase A–deficient mice, down-regulation of the myelin- and lymphocyte-associated protein is observed, as well as unusual distribution of the proteolipid protein. A bone marrow stem cell gene therapy has been tested in mice. Bone marrow from an arylsulfatase A–deficient mouse is modified using a retroviral vector encoding human arylsulfatase A. Cells transplanted into an irradiated mouse overproduce arylsulfatase A in the bloodstream, and aryl- sulfatase A–deficient tissues (eg, liver, kidney, and arterial cells) take up the enzyme. In a study of 27 mice, transplan- tation was successful for 60% of animals, despite variabil- ity in the amount of enzyme production. In treated animals, 25% of the normal enzyme activity level is observed in the brain, and sulfatide storage is reduced in the liver. In addi- tion, lipid ratios of galactocerebroside to sulfatide decrease. Despite improvements at the cellular level, sulfatide stor- age in the kidney and brain does not significantly decrease, and symptoms improve only marginally. Preliminary data suggest that injection of lentiviral vectors carrying the enzyme's complementary DNA directly into the hippocam- pus of the mouse may protect the hippocampus from degen- eration, improve mouse learning, and increase arylsulfatase A distribution. However, as hippocampal degeneration was not observed in either treated or untreated mice, the results are somewhat uncertain. Globoid Cell Leukodystrophy (Krabbe's Disease) Krabbe's disease is a severe, progressive, familial, neurologic disease in which oligodendrocytes completely disappear and are replaced by astrocytic gliosis and globoid cells (altered macrophages or histiocytic cells). Among childhood-onset subtypes of the disease, death is common within 3 years. The enzyme galactocerebroside β-galactosidase is deficient in this disease. Neither galactocerebroside nor psychosine is properly degraded by the enzyme in Krabbe's disease. Evi- dence suggests that psychosine accumulates in the brains of patients with Krabbe's disease. Psychosine is a cytotoxic compound and therefore might be the cause of myelin degeneration in Krabbe's disease. Naturally occurring ani- mal models of the disease are available, including a dog model, the twitcher mouse, and a rhesus monkey model. In animal models, psychosine accumulates in the white mat- ter only as the disease progresses. Thus, two pathogenetic mechanisms are implicated: galactocerebroside elicits globoid cell reaction, and psychosine accumulation leads to the death of oligodendrocytes and demyelination. New findings indicate a third mechanism for Krabbe's disease. Specifically, a deficit in saposin A, an activator protein for galactocerebrosidase, can also result in globoid cell leukodystrophy. In mouse models, saposin A deficiency causes a late-onset, chronic globoid cell leukodystrophy. In a saposin A knockout mouse model, pathology is similar to that observed in the twitcher mouse, including myelin and oligodendrocyte loss, astrocytosis, presence of globoid cells, accumulation of substrates of galactosylceramidase substrates in the kidney, accumulation of psychosine and monogalactosyldiglyceride in the brain, and presence of a seminolipid precursor in the testes. Clinically, the knockout mouse displays more gradual progression of symptoms. Unexpectedly, pregnancy in the mouse increases life span and decreases the presence of psychosine accumulation and globoid cell production, perhaps owing to a decrease in inflammatory cytokine levels. Furthermore, treatment with supplemental estrogen produces the same result in male and female mice. Additional studies in mice are evaluating nerve grafting, bone marrow transplantation, use of a trans- gene, and in vitro gene therapy. 584 Canavan's Disease Reuben Matalon, MD, PhD University of Texas Medical Branch Galveston, TX Canavan's disease is an autosomal recessive disease caused by aspartoacylase deficiency, which leads to a 10- fold or greater increase in N-acetylaspartate levels in the urine, blood, and cerebrospinal fluid. Spongy degenera- tion of the white matter, including subcortical regions, is noted. Microscopically, astrocytic swelling and mitro- chondrial changes are observed. Hypotonia, macro- cephaly, and head lag are characteristic clinical features. The disease progresses slowly, leading to spasticity; many of these children receive a diagnosis of cerebral palsy as they grow older. Diffuse loss of white matter is apparent on imaging, and U fibers are not spared in Canavan's disease. Although the disease is most prevalent among Jewish people from the Ashkenazi region, only 2 of the 40 identi- fied mutations on the ASPA gene are predominant in this group. Thus, testing for these two mutations should iden- tify most carriers in the Ashkenazi Jewish population. Car- rier rates in this population range from 1 in 37 to 1 in 50. Most mutations among non-Jewish carriers are private, although one mutation is common in 40% of those of non- Jewish descent. Thus, carrier testing for non-Jewish fam- ilies is not routine. Some mutations can result in residual ASPA activity, hypothetically leading to milder clinical symptoms. ASPA has been well characterized in both humans and mice. It spans 39 kilobases and includes 6 exons and 5 introns. The complementary DNA is 1435 base pairs long, with a molecular weight of 36 kd and coding for an enzyme with 313 amino acids. Knockout mice, in which 10 base pairs from exon 4 are deleted, exhibit failure to thrive, ataxia, high urine levels of N-acetylaspartate, and reduced aspartoacylase activity in the brain. MRI indicates spongy degeneration with subcortical vacuolization in the white matter, cerebellum, hippocampus, and retina. Gene expression profiling in mice indicates that the dis- ease affects multiple genes (eg, expression of the gene that codes for one of the γ-aminobutyric acid receptors is low, glutamate transporter expression is low, and the expression of some genes associated with cell death is high). Prevention efforts include testing carriers and providing genetic counseling, diagnosing children prenatally, testing amniotic fluid for N-acetylaspartate, and performing molec- ular studies. Current treatments are designed to support patients, but a gene therapy trial is under way. In mice, [recombinant adeno-associated virus] rAAV-aspartoacylase treatment in the corpus striatum decreases spongy degen- eration locally but does not transfer to other areas of the brain. Pelizaeus-Merzbacher Disease Pelizaeus-Merzbacher disease is caused by mutations in the gene that produces proteolipid protein. Proteolipid pro- tein, the most prevalent protein in myelin, stabilizes the structure of compact myelin by forming the bulk of myelin and spanning myelin lipid bilayers. The gene associated with proteolipid protein is one of the most highly conserved hydrophobic genes. Mutations in the PLP gene also can cause spastic paraparesis type 2. Key features of Pelizaeus- Merzbacher disease/spastic paraparesis type 2 include neu- rologic signs, familial characteristics (ie, X-linked recessive inheritance), MRI findings (ie, diffuse myelin abnormali- ties), and molecular genetic screening results. The most severe form of the disorder is characterized by onset of hypotonia, respiratory distress, stridor, nystagmus, and pos- sible seizures during the first few weeks of life. Over time, spasticity, reduced movement, poor head control and growth, and limited language skills are observed. A moderately severe subtype of the disorder is first identified during the neonatal period, when nystagmus, weakness in the lower extremities, head titubation, and reduced muscle tone are observed in contrast to normal respiration. Patients pro- gressively develop spastic quadriparesis, ataxia, dystonic pos- turing/movements, delayed motor milestones, dysarthric speech, and some cognitive deficits. Finally, patients with spastic paraparesis type 2 develop mild to severe paraple- gia, occasional ataxia, and nystagmus in the first 5 years of life, although they can appear almost normal. Heterogeneity in disease presentation might reflect the effects of modifiers or of variability in types of mutations at the same gene locus. In Pelizaeus-Merzbacher disease, three different pathogenetic mechanisms have been iden- tified. Sixty to 70% of cases are caused by duplications in the gene. Duplication results in production of excess pro- teolipid protein, which is shunted from the cell along with the proteins and lipids necessary for proper myelin for- mation. Another 20% of cases are the result of a single-point mutation of PLP, resulting in a misfolded protein. The protein clogs the endoplasmic reticulum, causing cell death. Point mutations account for early-onset cases in male carriers with a rapid progression. Female carriers of the severe form of the disease resulting from point muta- tions typically do not express symptoms because only half of their cells die, leaving functioning cells that make up for the deficit. Less than 1% of cases are owing to deletions in the gene, which result in improper compaction of myelin, causing myelin to “wear out.” In this milder form of Pelizaeus-Merzbacher disease, female carriers tend to express symptoms because half of their cells produce inadequate myelin. 585 Gene therapy is challenging in Pelizaeus-Merzbacher dis- ease because the problem is dosage based and in some cases requires a decrease, rather than an increase, in pro- tein production. However, cell transplantation could prove more promising, although animal studies have yet to demon- strate improvement. Question and Answer Session 2 Dr Thurston: It was concluded that psychosine contributed to the pathol- ogy and the clinical manifestations in Krabbe's disease. In the slide that you showed, the enzymatic production of psychosine goes through a pathway through ceramide. Now ceramide is a death signal. It's an apoptotic mole- cule. How much of the pathology and clinical features is owing to ceramide accumulation as opposed to psychosine? Dr Suzuki: So, as far as I know, there is no clear ceramide abnormality in Krabbe's disease. Dr Dowling: Have you given any hormones to twitcher mice and seen any changes in their life span or symptoms? Also, have you looked at the effects of steroids instead of estrogens in these mice? Dr Suzuki: The problem with the twitcher model is that it is so acute, and it becomes symptomatic at 20 days. However, we did not observe any clear difference between untreated and treated mice. We thought there were two parallel mechanisms: in the saposin A–negative mouse, the major pathogenic mechanism is mediated through this globoid cell accumulation because of inability to degrade galactosylceramide because psychosine accumulation is very moderate, if at all. On the other hand, in the twitcher, perhaps psy- chosine accumulation is the major pathogenic mechanism, and high levels of hormones may not alter the disease. When we started hormone treatment in saposin A–negative mice at 60 days, it did not improve the phenotype (it needed to be started at least at 30 days). We are evaluating the effects of progesterone, varieties of estrogens or sex hormone equivalents, and arti- ficial compounds. Dr Jacobson: My question is for Dr Hudson. I wonder if the nystagmus that's associated with Pelizaeus-Merzbacher is related to a predilection for the optic pathways themselves. Since you've described how there is also a tendency toward a spinal component or a paraparetic component to this disorder or this gene abnormality, is there something that the optic path- ways and the spinal pathways share with regard to proteolipid protein expression or anything else about the pathway that might explain that predilection? Dr Hudson: Why the optic nerve is selectively affected in Pelizaeus- Merzbacher is a little bit hard to understand, in the sense that the optic nerve is definitely myelinated, so you would think wherever you have problems with myelin formation or maintenance, you would have some particular prob- lems with the optic nerve. I should also point out that this nystagmus is not something that is seen throughout the course of the disease. It's usually an early sign that is transient. Perhaps a third of people within the mild form have nystagmus. Dr Naidu: Actually, I was amazed when the nystagmus faded in these patients. Perhaps brainstem pathways are initially involved. They should be the earlier pathways that get myelinated. So my feeling is that this is prob- ably more brainstem, the parapontine reticular nucleus, and third cranial nerve nuclear pathways. Dr Maria: A question about early treatment. I certainly understand the prin- ciple that if we are identifying patients very early, they stand a better chance with intervention. How is “early” defined in adrenoleukodystrophy? Is it age? Is it imaging burden of disease? Is it functional burden independent of imaging? What are the actual data on Lorenzo oil with respect to early treatment? Dr Moser: Our experience (and that of the University of Minnesota and others) is that brain MRI abnormalities precede symptomatology, both cog- nitive and neurologic. There are extensive data on that. We have found that, for instance, if a patient continues to have a normal MRI until age 7 [years], his chances of developing the cerebral disease are greatly diminished. So there are a lot of data on MRI being a sentinel. The experience has been in the patients that both the MRI score and also the particular [Wechsler] Per- formance IQ are predictive of the benefit achieved from bone marrow transplantation. If the [Wechsler] Performance IQ is less than 75, then the outcome has not been satisfactory. Some correlation can also be made with MRI scores. So, on the basis of empirical observation, patients with early lesions do better. The benefit, the effect of Lorenzo oil at this moment, is based on comparison of patients who lowered their fatty acids substan- tially for long periods of time. Our follow-up data (mean of 3 years) show that when fatty acids were significantly lowered by Lorenzo oil, the risk of developing symptoms was significantly lower. We don't know if it will per- sist. I also need to emphasize that it's not an absolute preventive and that some patients who had lowered fatty acids developed disease. As a statis- tical phenomenon, we have no new information about the effect on older patients who are symptomatic. The published literature has not shown benefit in symptomatic patients. Dr Stephenson: Questions on Pelizaeus-Merzbacher. It used to be that stri- dor was a prominent feature. Is that still common in the presenting forms? And, if so, what is the mechanism? What's the percentage pick-up for muta- tions now? Dr Hudson: The prevalence of detectable mutations or duplications in Pelizaeus-Merzbacher is 95%. Dr Tabb: Why do some adrenoleukodystrophy patients progress more rapidly than others? What is the molecular basis, if it is known? Dr Moser: It does not correlate with the nature of the primary mutation. There is a great deal of interest to determine if a modifier gene exists. So far, it has not been demonstrated. But it appears likely that there are modifier genes in other parts of the genome. There also may be environ- mental factors that contribute. But that extremely important question has not been solved, and we cannot predict what phenotype a young patient or a fetus will have. So it is a crucial question that we don't have the answer to. Dr Fatemi: I was quite amazed by Professor Gieselmann's presentation about the ex vivo gene transfer in the metachromatic leukodystrophy mouse model. You said that 40% of the transplanted mice did not have pro- longed arylsulfatase A expression or activity in the plasma. Do you have any explanation for that? Dr Gieselmann: Well, we didn't look into that specifically, but the hypoth- esis simply is that the retrovirus probably integrated somewhere in the genome at a locus that's switched off during time. That may be one pos- sibility. The other possibility is that during the transduction process, we didn't really hit the stem cells, so that cells are not replenished from the stem cell pool. Dr Fatemi: Could that be caused by antibodies, for instance? Dr Gieselmann: We looked in about 10 of those animals, and there was no evidence of an immune response. 586 UPDATES ON SPECIFIC LEUKODYSTROPHIES: PART 2 Alexander's Disease Dr Johnson described three subtypes of Alexander's disease: (1) onset between infancy and 2 years of age with mega- lencephaly, rapid symptom progression, developmental dis- abilities, psychomotor retardation, seizure, and possible hydrocephalus; (2) onset between ages 2 and 12 years with b u l b a r a n d p s e u d o b u l b a r s y m p t o m s (e g , impaired speech/swallowing, vomiting, ataxia, spasticity of legs), preservation of cognitive skills, and inconsistent appearance of megalencephaly; and (3) adult onset with palatal myoclonus with ataxia and tetraparesis, often initially diag- nosed as multiple sclerosis. In all types of Alexander's disease, dense, oblong bod- ies surrounded by intermediate filaments (Rosenthal fibers) occur within the cytoplasm of the astrocytes, particularly in subpial and periventricular regions. Rosenthal fibers include glial fibrillary acidic protein (GFAP), and also the small heat shock proteins αβ-crystallin and heat shock protein 27. GFAP is an intermediate filament protein unique to astrocytes in the central nervous system, whereas the small heat shock proteins are chaperones that correct folding errors; levels of all three proteins are greatly increased in the disease. Rosen- thal fibers are also ubiquitinated, a process that marks pro- teins for degradation by the proteosome. Although Rosenthal fibers are not unique to Alexander's disease, they are diag- nostic by their abundance and unique distribution pattern. GFAP is indicated in many cases, although, in some cases, a GFAP mutation is not identifiable. The role of GFAP was suggested by the finding of the high GFAP content of Rosenthal fibers and by an experiment in which a clone of the gene was used to create transgenic mice that overpro- duced GFAP. Unexpectedly, the mice showed symptoms consistent with Alexander's disease, including Rosenthal fibers. These observations led to studies of GFAP in humans, which indicated that dominant, unique missense mutations in the gene caused the disorder in 85% of cases. Mutations are distributed throughout the protein, and specific muta- tions are not strongly associated with clinical presentation in the infantile and juvenile forms. Cerebrotendinous Xanthomatosis Antonio Federico, MD, PhD University of Siena Siena, Italy Cerebrotendinous xanthomatosis is a late-onset familial dis- order of bile acid metabolism. Symptoms are caused by a block in bile acid synthesis, resulting in incomplete oxi- dization of cholesterol and the accumulation of cholestanol. The culprit gene is located at chromosome 2q33, and several mutations have been identified. Progressive accumulation of cholestanol leads to cataracts. Other visual problems include optic disk paleness, premature retinal senescence with reti- nal vessel sclerosis, cholesterol-like deposits, and myeli- nated nerve fibers. Chronic urea is also present during infancy. In the second or third decade of life, xanthomas char- acterized by crystalline storage material are observed in the Achilles tendon, the extensor tendons of the elbow, the patella, and the brain, heart, or lung. Gallstones and liver abnormalities can be observed, as well as cardiovascular problems (eg, premature atherosclerosis, abnormal lipid levels). Bone fractures related to impaired calcium absorp- tion are also observed. Studies describe sensory-motor neu- ropathy and mitochondrial abnormalities in muscle tissue. In adults, central nervous system changes are marked and include dementia and psychiatric symptoms (eg, behav- ior changes, hallucinations, aggression). Pyramidal and cerebellar signs, seizures, and palatal myoclonus can also be observed. On MRI, diffuse cerebral and cerebellar atro- phy is common. Other MRI findings include bilateral, focal lesions in the cerebellum; brainstem atrophy; lesions in white matter and the globus pallidus; and periventricular white-matter changes. Spectroscopy indicates a decrease in N-acetylaspartate levels and an increase in lactate levels. In cerebrospinal fluid, high levels of cholesterol, cholestanol, and apolipoprotein B result in impaired blood–brain barrier permeability. In addition, higher levels of lactate are observed in cerebrospinal fluid. Treatments include the provision of chenodeoxycholic acid, which decreases formation of bile alcohols and cholestanol. Treatment reduces levels of cholestanol in the plasma and central nervous system, particularly when 3- hydroxy-3-methylglutaryl CoA is also administered. In a study of five patients who underwent 4 months of treatment, nerve conduction, evoked potentials, and clinical symptoms improved. However, chenodeoxycholic acid is no longer com- mercially available, creating significant barriers to treatment. Vanishing White-Matter Disease Dr van der Knaap described the problem posed by white-mat- ter abnormalities that are evident on MRI but cannot be classified (50% of cases). In cooperation with Dr Naidu of the Kennedy Krieger Institute, Dr van der Knapp is trying to use MRI to identify new diseases, one of which is vanishing white-matter disease. Fast spin-echo proton density–weighted imaging or fluid-attenuated inversion recovery imaging is nec- essary to identify the disease, which is confirmed by differ- entiating abnormal white matter from cerebrospinal fluid that has replaced white matter. In a presymptomatic patient, diffuse abnormalities are common, followed by gradual dis- appearance of white matter. Magnetic resonance spec- troscopy for vanishing white-matter disease indicates an absence of all metabolites and the presence of lactates and resonances of glucose, similar to the pattern noted in cys- tic leukoencephalopathies. 587 The age of onset varies, ranging from less than 1 year to more than 30 years of age. However, the disease results in chronic, episodic motor deterioration, particularly after head injury or infection. Motor difficulties occur first, fol- lowed by cognitive difficulties, and ataxia progresses to spasticity. Additional features include absence of seizures or presence of a mild seizure disorder, and variable optic atro- phy that ensues late in the course of the disease. On autopsy, these changes are found: white-matter rarefaction, micro- cystic to macrocystic degeneration, a commensurate loss of axons and myelin sheaths, and abnormalities of oligo- dendrocytes (eg, preservation or proliferation, foamy oligo- dendroglial cells). The disease occurs almost exclusively among Cau- casians, and inheritance is autosomal recessive. Genetic linkage studies implicate chromosome 3q27. The gene EIF2B, which initiates the translation of every protein in the body, was specifically indicated. Sixteen mutations were found in an initial sample of 29 patients (35 families), with mutations occurring in each subunit of EIF2B, EIF2B1, EIF2B3, and EIF2B4. A cell's response to stress has two functions that are regulated by EIF2B: shutting off protein synthesis and expression of selective heat shock proteins that help damaged proteins break down. Given the function of EIF2B, Dr van der Knapp provided several potential explanations for vanishing white-matter disease based on the hypothesis that an imbalance between protein produc- tion and heat shock proteins can cause the disease: (1) under normal conditions, there is insufficient production of proteins; (2) during fever, proteins do not decrease, thus coagulating and resulting in cell death; (3) during fever, EIF2B shuts down and heat shock proteins are not produced, leading to protein coagulation and cell death; or (4) after fever, adequate protein production does not resume. How- ever, it is unclear why brain tissue is selectively affected. Treatments include avoidance of stress and infection leading to fever. Future research efforts will focus on a severe variant of vanishing white-matter disease called Cree leukoencephalopathy, pathophysiologic studies, and devel- opment of a transgenic mouse. Megalencephalic Vacuolating Leukoencephalopathy Bhim Singhal, MD Institute of Medical Sciences Bombay, India Dr Singhal described collaborative research with Dr Eric Hoff- man and Dr Rafael Gorospe of the Children's National Med- ical Center and Dr Sakkubai Naidu of the Kennedy Krieger Institute. Currently, 70 patients (42 male, 28 female) have been identified, with a median age of onset of 6 months (range, birth to 25 years). For more than half, megalencephaly was the first identifiable symptom, whereas some children did not come to the attention of medical professionals until devel- opmental delay, seizure, or motor disability was noted. In this population, the ability to participate in an educational envi- ronment varied, with 18 children performing below average, 22 performing in the average range, 6 graduating from col- lege, and many unable to attend school at all. More than half of the children had recurrent seizures and experienced fre- quent falls because motor difficulties exacerbated seizures (n = 19). Some children experienced prolonged uncon- sciousness (n = 5). Many children with the disease developed spasticity (n = 41), cerebellar ataxia (n = 42), and dysarthria (n = 22), with varied severity. In contrast, no problems with dysmorphology, neuropathy, retinopathy, splenic enlarge- ment, or metabolic screening were observed. Electrophysiologic studies indicated generally normal null conduction, visual evoked response imaging, and brain- stem auditory evoked responses. In somatosensory evoked potentials recorded in15 patients, the spinal cord was largely normal, but 8 patients had cortical delay. CT scans and MRI showed symmetric white-matter changes, frequently in combination with temporal cysts. The internal capsule and corpus callosum were relatively spared, and the cerebellum was less affected. Subcortical cysts were most often tem- poral but also occurred in other brain regions. Histologically, white matter was spongy, and membrane-encased vacuoles were observed using an electron microscope. Inheritance is autosomal recessive. Among the 70 patients in this group, a positive family history was identified in 40. Furthermore, 63 of the 70 patients belong to a specific eth- nic group in India, the Agarwal community. The abnormal- ity has been mapped to chromosome 22 with various mutations on the MLC1 gene. However, heterogeneity exists, with many patients exhibiting nonidentifiable mutations. In addition, phenotypic variability is observed among patients with identical genetic defects and among patients within the same family, suggesting the effects of modifier genes and/or the environment. Current treatments are supportive in nature and can include use of antiseizure medication. Question and Answer Session 3: Panel Discussion Audience: Could you comment on this very common minor head trauma causing seizures and deterioration in patients with vanishing white-matter disease or megalencephalic leukoencephalopathy with subcortical cysts? Is it because of some common pathogenesis? Dr van der Knaap: In vanishing white-matter disease, hyperthermia has been examined in bovine culture, but physical stress on cells has not been studied. However, a cellular stress response is induced by physical stress. So, for vanishing white-matter disease, I can understand why physical stress is just as bad as thermal stress. For megalencephalic leukoen- cephalopathy with subcortical cysts, it's much more difficult, I guess. We are now studying the location of the protein because nothing is known about MLC1. It's just assumed that it's a transmembrane protein that is probably an ion channel. So we don't know enough of the function of the protein to understand anything about it. Dr Kennedy: Dr Singhal, in the disease that you described, is there a pro- gression to the cystic changesin that disease, or are they there from early on? Dr Singhal: Of the two or three patients that we have tracked, the last case I described, at the age of 1 year, did not show many cystic changes, only in the temporal area. But after about 11 years, the cystic changes were quite pronounced. That's not always been the case, though. 588 Dr Stephenson: Can you expand on the question of headinjury as a trigger? Dr Singhal: The head traumas are trivial, and the underlying ataxia is prob- ably contributory. We do not recommend that the children wear helmets. Dr Stephenson: Is it the same sort of degree of head bump that leads to some cases of familial hemiplegic migraine or calcium channelopathy? Dr Singhal: No. Dr van der Knaap: Well, in vanishing white-matter disease and in my expe- rience also in megalencephalic leukoencephalopathy with subcortical cysts, they are just little traumas of daily life. A child falls from a bicycle. You know, it's an accident that may happen in the backyard—nothing big. So it's just daily life, small traumas. I don't recommend helmets, even to vanishing white- matter patients.We just advise them to avoid physical contact sports. Dr Cruise: I was wondering if you could expand on the children with Alexan- der's disease that do not have macrocephaly. Is imaging different than in those with macrocephaly? Dr Johnson: Usually, the children that don't have megalencephaly are older at onset, and they have more brainstem symptoms and preservation of normal function. Dr van der Knaap: A lot of people ask me whether a patient can have mega- lencephalic leukoencephalopathy with subcortical cysts and a normal head size. I've never seen a megalencephalic leukoencephalopathy with sub- cortical cysts patient with a normal head circumference and a DNA-con- firmed diagnosis. Dr Singhal: No. Dr van der Knaap: No. So, actually, the bottom line is, if one sees exten- sive white-matter disease and a normal head size, then megalencephalic leukoencephalopathy with subcortical cysts is not really an option. Dr Trescher: When considering all the various mutations in Alexander's disease, are there differences between the infantile and the juvenile forms? I have a patient who actually first presented with symptoms at a year of age who had difficulty swallowing and was worked up extensively for mito- chondrial disorder. And it was only after we sent the GFAP that a mutation was discovered. So I just wondered why he had early onset of symptoms and a cause of disease compatible with the juvenile form. Dr Brenner: Well, I think the short answer is that we really don't have any idea. All the mutations shown, except for one adult case, all are arising de novo. So they're just arising randomly. Some of them give rise to the infan- tile form and some are juvenile, and we don't have any clear correlation yet. We really don't understand why a mutation in one place can cause either type, and we have other mutations there where there's a single case where they were juvenile. There just aren't enough data to make a clear correla- tion. Why are there hot spots? That must have something to do with the muta- tion mechanism because, again, they're all occurring randomly. It's not a matter that you'll find the disease with the mutation in one spot and not find it so often with the other. As far as we know, they're all 100% penetrant. So, again, the short answer is we just don't know. Dr Johnson: I'd just like to add that some of what we call juvenile cases actually have onset in early infancy, but they progress much more slowly. I really call them juvenile if they predominantly involve brainstem lesions and symptoms rather than the onset of either hydrocephalus or seizures. Dr Bingham: How much is known about the obligate heterozygotes with megalencephalic leukoencephalopathy with subcortical cysts? Do they have trouble with head injuries or imaging manifestations? Dr van der Knaap: No. Dr Stephenson: Can patients with cerebrotendinous xanthomatosis have isolated neurologic findings? Dr Federico: In our experience, the early signs are always ocular problems andlight to moderate mental retardation. But peripheral neuropathy appears later, around the second decade. So in a child with neuropathy alone, I think the probability of having cerebrotendinous xanthomatosis is very low. Audience: Do transaminases increase with fever in vanishing white-mat- ter disease? Dr van der Knaap: Vanishing white matter is rather prevalent in the Netherlands. There is no hepatic dysfunction because when a child is admitted in coma, as a pediatrician, you do a lot of studies on these patients, and nothing is ever found—no liver failure, no bone marrow failure. Also, we had a full autopsy on our first patient. She died after 6 weeks of coma, and the autopsy only showed ovarian dystrophy. All the other organs were normal. I say this is ovarian dystrophy because Dr Raffi Schiffmann has described a so-called ovarian leukodystrophy. FUTURE DIRECTIONS AND INNOVATIVE THERAPIES Prospects for Neonatal Screening for Leukodystrophies Stephanie J. Mihalik, PhD Neo Gen Screening, Inc. Pittsburgh, PA Dr Mihalik described typical procedures and basic con- cepts related to newborn screening for leukodystrophies, discussed the complexity of screening for lysosomal stor- age and peroxisomal disorders, and described multiplex metabolite screening. The current system is very efficient, using a multiplexing technique in which the same 7.6 mL of blood is used to perform assays for multiple disorders simul- taneously. Newborn screening uses specific cutoff points to identify infants who could have inherited a genetic disorder. Thus, screening does not provide diagnostic information but instead identifies children who require further evaluation. Screening tests must be able to identify a disorder in a new- born using a spot of dried blood and must have low false- positive and false-negative rates. In addition, for a newborn screening to be viable, a disorder must be clinically signif- icant, of reasonably high frequency, treatable or manageable, and testable using a low-cost, simple procedure. Of the leukodystrophies, lysosomal diseases can be candidates for screening. Challenges associated with screen- ing for lysosomal storage disorders include the high number of different disorders (n = 42) and their various substrates, each of which could be available in small abundance (eg, mucopolysaccharides, oligosaccharides, glycopeptides, glycogen, sphingolipids). Because so many disorders exist, properly interpreting screening results would be difficult. In addition, it is unclear whether substrates accumulate suffi- ciently during the newborn period to allow for screening. Attempts to develop screening methods for these disorders currently involve a multiplexing approach. Potential screen- 589 ing approaches include using specific enzymatic assays. However, if such a technique was used, only one or two assays could be multiplexed. Alternatively, John Hopwood is looking at gross changes in lysosomal protein quantities as a marker for these disorders, specifically looking for lyso- some-associated membrane protein and saposin C protein. If these proteins are found, another level of analysis is sought to determine which disorder is represented. Another possi- bility is substrate assays in blood using electrospray with tan- dem mass spectroscopy on the end. Massive urine screening using matrix-assisted laser desorption/ionization time-of- flight mass spectrometry might also be a useful approach. In peroxisomal disorders (eg, adrenoleukodystrophy, Zellweger syndrome), a common substrate is available (very-long-chain fatty acids) and is useful for initial screen- ing. Other substrates can be used in a second-tier analysis to identify which peroxisomal disorder is indicated (eg, bile acids for Zellweger syndrome and multifunctional enzyme type 2 [MFE2]). Prospects for Bone Marrow Transplantation Charles Peters, MD University of Minnesota School of Medicine Minneapolis, Minnesota Dr Peters reviewed information regarding bone marrow transplantation in patients who have adrenoleukodystrophy, metachromatic leukodystrophy, or Krabbe's disease. The goals of transplantation are to prolong survival, improve somatic and cognitive functioning, and preserve quality of life. With transplantation, the patient receives a new blood system, which includes monocytes and macrophages that take up residence throughout the body, including in the central nervous system as microglia. In 90 boys with adrenoleukodystrophy who were trans- planted at 9 years of age (76 bone marrow, 13 cord blood), a 58% survival rate was observed, with death occurring because of adrenoleukodystrophy progression and/or graft- versus-host disease (30%). Two-thirds of patients were neu- rologically intact pretransplantation, whereas only 40% were intact afterward. The degree of neurologic impair- ment as measured by MRI and Wechsler Performance IQ test- ing was the best predictor of outcome. In a sample of 12 boys who received transplants, long-term beneficial effects were reported for 5 to 10 years post-transplantation. Finally, at the University of Minnesota, 110 boys with adrenoleukody- strophy have been studied, 51 of whom underwent trans- plantation (10 who are being tracked have biochemical abnormalities, 10 who were not transplanted have mild cerebral disease, and 13 were too severely affected for transplantation). Of 23 with mild to moderate disease, 15 are alive and 7 died (all but one of these deaths was owing to complications from transplantation). Of 28 with severe dis- ease, 12 are alive, and their level of functioning is poor, with adrenoleukodystrophy the leading cause of death. Thus, boys who are asymptomatic should be monitored and evaluated for transplantation if early stages of demyeli- nation are observed. Criteria for transplantation currently include the presence of elevated very-long-chain fatty acids, MRI severity of 3 or above, and a Wechsler Performance IQ of 80 or above. Transplantation is not possible for children with the late infantile disease form of metachromatic leukodystrophy because of the rapidly progressive nature of the disease. Thus, transplantation has been an option only for patients with the juvenile- or adult-onset forms. An 80% survival rate is reported among patients with the adult form of the disease who underwent transplantation, which appears to arrest the central demyelinating process. Patients with the late-onset form of Krabbe's disease could respond the best to transplantation. Six patients received cord blood trans- plants during the first month of life and had normal neuro- logic and neuropsychologic functioning at follow-up. Prospects for Gene Therapy Dr Aubourg reviewed challenges associated with using gene therapy to treat leukodystrophies. In the leukodystrophies, it is important to think not only about replacing and stabi- lizing myelin but also about protecting neural axons. Thus, therapy should occur before the onset of severe axonal damage. In addition, if oligodendrocytes are to be targeted, they must still be present in the brain. The gene that is tar- geted for modification does not necessarily need to be the gene that caused the disease. Because the vectors used for gene therapy are currently limited in their ability to travel within the brain, localized demyelination is more likely to be affected than diffuse demyelination. Currently, research suggests that lentiviral and adenoviral vectors are the best candidates for future clinical trials as they target genes in the liver, muscle, retina, or neurons. However, none of these vectors have been shown to work for oligodendrocytes, so new vectors for this use must be developed. In choosing a gene product, both disease physiology and the target pro- tein must be considered. Ultimately, it will be possible to tar- get within the brain other molecules to correct directly missense mutations. In leukodystrophies, it might be use- ful to target genes that modify the disease indirectly. Because of the complexity of the leukodystrophies, it is necessary to obtain extensive data in animals to determine the feasi- bility of these techniques, as well as stem cell therapy. Prospects for Stem Cell Therapy John Gearhart, PhD Johns Hopkins University School of Medicine Baltimore, MD Dr Gearhart opened the talk by cautioning conference atten- dees that stem cell therapies appear promising, but cur- rent data are preliminary. Research indicates that human cells can be engineered successfully using lentiviral vectors 590 and that stem cells could someday be used to build parts of organs that can be grafted (“organoids”). Stem cells are classified into two groups: embryonic stem cells and tissue- or organ-specific stem cells. Cells are derived from preim- plantation-stage embryos or from the germ cells of cadav- eric fetal tissue. Stem cells and bone marrow–derived cells, including mesenchymal and hematopoietic cells, can share similar properties, although bone marrow–derived cells are somewhat more restricted in function. In addition, terato- carcinomas could be a potential source of stem cells. Although stem cells can produce unlimited numbers of cells, mutations can emerge over time. Inducing stem cells to produce cell types of interest is a challenge. Scientists have tried placing stem cells with cells like those they sought to develop, but many different cell types formed. In the central nervous system, directing stem cells to mature into neurons or glia has been somewhat successful. During experiments in which scientists induced a self-limited encephalomyelitis in mice or rats that kills the ventral motor neurons, infusion of human stem cells into cerebrospinal fluid caused stem cells to coat the spinal cord. Cells then passed into the ventral horn and motoneurons that sent processes into the periphery form. In addition, the cells provided a source of cytokines and transforming growth factor-α that affected the animals' nerve cells. In animal models for Parkinson's disease, injection of human stem cells into the striatum results in decreased signs of the dis- ease. In animal models of trauma-induced cerebral palsy, injection of stem cells has also shown potential. Although as many as 40% of grafted stem cells differ- entiate as expected, it is unclear what the remaining cells generate into. In human cell studies, no tumor develop- ment has been found, although some experiments using mouse stem cells result in teratocarcinoma. Thus, cautious optimism is encouraged when thinking about stem cell ther- apies in humans. IMPLEMENTATION OF ADVANCES FOR THE BENEFIT OF PATIENT AND FAMILY Moderators: Hugo Moser, MD; Sakkubai Naidu, MD Kennedy Krieger Institute and the Johns Hopkins University School of Medicine Baltimore, MD Dr Moser invited a panel of representatives from federal agencies and community groups to discuss the views of patients and their representatives and the agencies that support investigators and clinicians. Panelists highlighted ways in which researchers and scientists can work together to better care for patients with leukodystrophies: • Paula Brazeal, president of the United Leukodystrophy Foundation, recommended improving the identification of patients with leukodystrophy, suggested forming a “second opinion” program in which medical informa- tion from patients with uncertain diagnoses can be sum- marized on a standard form given to experts consulted for a second opinion, and called for a continuing close relationship between the foundation and scientists, with the foundation playing a central role in informing patients of research findings and contacting potential partici- pants for clinical trials. • Abbey Meyers, president of the National Organization for Rare Diseases, indicated the group's support for the research community and highlighted the need for increased translational research so that scientific find- ings can be more quickly applied to patient care; described the organization's role in passing the Orphan Drug Act, increasing the likelihood that medications for rare dis- orders will be manufactured; and expressed concern regarding reports of potential links between gene ther- apies and cancer as a threat to continued research in gene therapy. • Marlene Haffner, MD, director of the Office of Orphan Products Development at the US Food and Drug Admin- istration, said that her office works with researchers and clinicians by helping pharmaceutical companies nav- igate the Administration's approval process for orphan drug development, offering incentives to firms for man- ufacturing orphan drugs, and providing grant support to scientists engaged in translational research. • Deborah Hirtz, MD, program director for clinical trials and studies at National Institute of Neurological Disorders and Stroke, described the agency's support for research aimed at reducing the burden of neurologic disease through prevention and treatment. • Dr Hanson, from the National Institute of Child and Human Development, emphasized the importance of bringing information about the disease to research com- munities, focusing on the whole child, and encouraging collaboration among scientists from various backgrounds to promote translational research. Acknowledgments The authors thank the National Institutes of Health, the Child Neurology Soci- ety, and the United Leukodystrophy Foundation for cosponsoring the conference. They also recognize Meadow Maze and Danny Liu for assistance in planning the conference and preparing the proceedings for publication and Melanie Fridl Ross, MSJ, ELS, for editing assistance. Reference McGuiness MC , Lu JF, Zhang HP, et al: Role of ALDP (ABCD1) and mitochondria in X-linked adrenoleukodystrophy. Mol Cell Biol 2003;23:744—753. McGuiness MC , Lu JF , Zhang HP , et al:

PY - 2003/9/1

Y1 - 2003/9/1

UR - https://www.scopus.com/pages/publications/0141893614

UR - https://www.scopus.com/pages/publications/0141893614#tab=citedBy

U2 - 10.1177/08830738030180090401

DO - 10.1177/08830738030180090401

M3 - Comment/debate

C2 - 14572135

AN - SCOPUS:0141893614

SN - 0883-0738

VL - 18

SP - 578

EP - 590

JO - Journal of child neurology

JF - Journal of child neurology

IS - 9

ER -