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Summary, Discussion, and Prospects regel 1 regel 2 regel 3 regel 4 regel 5 regel 6 regel 7 Summary, Discussion, and regel 8 regel 9 Prospects regel 10 regel 11 regel 12 regel 13 regel 14 regel 15 regel 16 regel 17 regel 18 regel 19 regel 20 regel 21 regel 22 regel 23 8 regel 24 regel 25 regel 26 regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 regel 37 regel 38 regel 39 Summary, Discussion, and Prospects regel 1 1. Clinical Description regel 2 regel 3 The studies described in this thesis all started when a ten-year-old boy with macrocephaly regel 4 and cerebellar ataxia visited the out-patient department of the VU University Medical T regel 5 Center in 1991. MRI analysis revealed that this boy had a disease involving the white matter regel 6 of the brain. Soon after this patient other patients with strikingly similar clinical and MRI 1 regel 7 features were detected, as reported by Van der Knaap et al . Since then several other 2-10 regel 8 groups have described similar cases . In none of these children a specifi c diagnosis regel 9 could be established or a basic biochemical defect could be identifi ed. The homogeneous regel 10 clinical picture and MRI fi ndings were suggestive of a ‘new’ disease entity, now known regel 11 as ‘Megalencephalic Leukoencephalopathy with subcortical Cysts’ (MLC). Characteristic regel 12 clinical features of the disease are a macrocephaly that arises in the course of the fi rst regel 13 year of life and a delayed onset of motor deterioration, dominated by cerebellar ataxia. regel 14 Characteristic MRI features include diff use cerebral white matter signal abnormalities, 1 regel 15 swelling of the aff ected white matter, and the typical subcortical cysts . regel 16 regel 17 regel 18 2. Discovery of the MLC1 gene regel 19 regel 20 The high frequency of consanguinity of the parents and occurrence of families with regel 21 two aff ected siblings suggested an autosomal recessive mode of inheritance. In the late regel 22 nineties we had collected enough informative families (four consanguineous families regel 23 and one family with two aff ected children) to start a genetic linkage study with micro- regel 24 satellite markers. In the mean time, a Turkish-French consortium detected the locus for 11 regel 25 the disease gene at the very end of the long arm of chromosome 22 (22qtel) . The critical regel 26 region, however, was too large to identify the disease gene. We used the information of regel 27 our families to limit the critical region to a DNA stretch that only contained four genes. regel 28 Mutation analysis showed mutations in the second gene investigated, KIAA0027, which regel 29 we renamed into MLC1. The discovery of this gene is described in chapter 2. regel 30 regel 31 regel 32 3. Characterization of the MLC1 gene T regel 33 regel 34 3a. Types of mutations 12-22 regel 35 Over the years many diff erent mutations in the MLC1 gene have been found . These regel 36 mutations are distributed along the whole gene and include all diff erent types: splice- regel 37 site mutations, nonsense mutations, missense mutations, deletions, and insertions. regel 38 regel 39 114 Novel mutations in our patient population are described in chapter 3. In chapter 4 an regel 1 update is given describing all mutations found up to 2005. Although most families have regel 2 13,16,18,20 unique mutations, evidence for a founder eff ect is present in four communities . regel 3 The strongest evidence for a founder eff ect is found in the Indian Agarwal community regel 4 13,20 . All patients within this community share the same insertion (c.135_136insC) causing a regel 5 frameshift (p.Cys46LeufsX34) and the appearance of a premature stop. regel 6 regel 7 3b. Extended mutation analysis regel 8 In about 20% of the typical MLC cases, diagnosed by clinical and MRI criteria, no or only regel 9 one MLC1 mutation can be found. One reason for not fi nding mutation(s) is the standard regel 10 mutation analysis at genomic level, which may miss heterozygous deletions, mutations regel 11 in the promoter or 3’- and 5’-untranslated regions, and intron mutations that may aff ect regel 12 splicing of the mRNA. For this reason, we performed extended mutation analysis in regel 13 patients that have only one or no mutations in the MLC1 gene. This analysis included regel 14 sequencing of cDNA derived from lymphoblasts of the patients to show any missed regel 15 splice-site mutations, and qPCR to elucidate diff erences in expression levels of the MLC1 regel 16 gene. Chapter 4 provides, in addition to the mutation update, information on mutations regel 17 found with the extended mutation analysis. regel 18 regel 19 3c. Evidence for additional genes involved in MLC regel 20 Several families, in which no mutations are found, also do not show linkage with the regel 21 MLC1 locus. This indicates that there must be more than one gene associated with MLC regel 22 14,21,23 . Therefore linkage analysis was performed in four informative families, without regel 23 mutations in the MLC1 gene and without linkage on the MLC1 locus, which resulted in regel 24 several candidate regions. Some promising genes in these regions were sequenced, regel 25 but unfortunately no mutations have been found. Since there are too many candidate regel 26 regions, this study will only be continued when DNA from additional informative families regel 27 will become available that can possibly reduce this number. It should be noted that the regel 28 identifi cation of multiple candidate regions may not only be due to the limited number of regel 29 informative families, but can also be the result of several additional genes being involved regel 30 in MLC. regel 31 The consequence of the above fi ndings is that the diagnosis remains primarily based on regel 32 clinical and MRI fi ndings. Thus, if the MRI shows the typical features of MLC in an ataxic 8 regel 33 patient with macrocephaly, the diagnosis is MLC even if no mutations are found in the regel 34 MLC1 gene. regel 35 regel 36 regel 37 regel 38 regel 39 115 Summary, Discussion, and Prospects regel 1 4. Genotype-phenotype correlation regel 2 regel 3 No genotype-phenotype correlation has been observed. So, the severity of the phenotype regel 4 does not appear to correlate with the type and position of the mutation(s) in the gene. regel 5 There is no phenotypic diff erence between MLC patients with mutations in MLC1 and regel 6 patients without. Patients from the Agarwal community, who are all homozygous for the regel 7 same mutation, exhibit a wide phenotypic variation. regel 8 regel 9 regel 10 5. Localization of the MLC1 protein regel 11 regel 12 The MLC1 gene encodes a 377 amino acid plasma membrane protein, called MLC1. In regel 13 chapter 5 we report that this protein contains eight transmembrane domains and that regel 14 it is highly conserved throughout evolution in a variety of myelin-producing vertebrates regel 15 (see Figure 1). Moreover we show that, in the brain, MLC1 is expressed in distal processes regel 16 of astrocytes at the blood-brain and CSF-brain barriers and in the cellular processes of regel 17 Bergmann glia. This specifi c localization of MLC1 strongly suggests that MLC1 is localized regel 18 in astroglial endfeet, but higher-resolution electron microscopic (EM) studies are necessary regel 19 to show the exact localization. In support of this suggestion, Teijdo et al. already showed 24 regel 20 that mouse MLC1 is expressed in astrocytic endfeet . Besides the expression in brain, regel 21 MLC1 is also expressed in all types of leukocytes, as shown in chapter 5. The signifi cance regel 22 of this expression remains unclear. There is no evidence that mutations in the MLC1 gene regel 23 cause a defect in leukocyte function in patients. 25 regel 24 Recently, Teijido et al. published a paper about the expression of MLC1 in mice. They regel 25 showed that MLC1 is expressed in neurons in the adult mouse brain. In addition, EM regel 26 studies showed a more precise localization of MLC1 in the plasma membrane and in regel 27 vesicular structures of neurons. In the developing brain, MLC1 was mainly expressed regel 28 in axonal tracts during early stages of development. So far, we have not been able to regel 29 confi rm these results in humans. We, however, did not immuno-stain the complete regel 30 human brain systematically yet. Another contradiction with the human situation is MLC1 regel 31 expression in the peripheral nervous system. The lack of expression in humans is in line regel 32 with the absence of ‘peripheral dysfunction’ in MLC patients. Teijido et al. also described regel 33 EM studies showing that MLC1 is localized in astrocyte-astrocyte junctions, and not in regel 34 the perivascular membrane. They therefore concluded that MLC1 can not be a part of the regel 35 dystrophin-associated glycoprotein complex (DGC) (see chapter 6 for further discussion). regel 36 To confi rm or reject these fi ndings between species it is necessary to perform EM-studies regel 37 in the human brain. regel 38 regel 39 116 It is important to note that MLC is the second genetic defect that specifi cally involves an regel 1 astrocytic protein. The fi rst genetic defect was described in 2001 by Brenner et al.
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