Homozygous Deletion of the Very Low Density Lipoprotein Receptor Gene Causes Autosomal Recessive Cerebellar Hypoplasia with Cerebral Gyral Simplification
2005; Elsevier BV; Volume: 77; Issue: 3 Linguagem: Inglês
10.1086/444400
ISSN1537-6605
AutoresKym M. Boycott, Shauna Flavelle, Alexandre Bureau, Hannah C. Glass, Takuya Fujiwara, Elaine Wirrell, Krista Davey, Albert E. Chudley, James N. Scott, D. Ross McLeod, Jillian S. Parboosingh,
Tópico(s)Caveolin-1 and cellular processes
ResumoAn autosomal recessive syndrome of nonprogressive cerebellar ataxia and mental retardation is associated with inferior cerebellar hypoplasia and mild cerebral gyral simplification in the Hutterite population. An identity-by-descent mapping approach using eight patients from three interrelated Hutterite families localized the gene for this syndrome to chromosome region 9p24. Haplotype analysis identified familial and ancestral recombination events and refined the minimal region to a 2-Mb interval between markers D9S129 and D9S1871. A 199-kb homozygous deletion encompassing the entire very low density lipoprotein receptor (VLDLR) gene was present in all affected individuals. VLDLR is part of the reelin signaling pathway, which guides neuroblast migration in the cerebral cortex and cerebellum. To our knowledge, this syndrome represents the first human lipoprotein receptor malformation syndrome and the second human disease associated with a reelin pathway defect. An autosomal recessive syndrome of nonprogressive cerebellar ataxia and mental retardation is associated with inferior cerebellar hypoplasia and mild cerebral gyral simplification in the Hutterite population. An identity-by-descent mapping approach using eight patients from three interrelated Hutterite families localized the gene for this syndrome to chromosome region 9p24. Haplotype analysis identified familial and ancestral recombination events and refined the minimal region to a 2-Mb interval between markers D9S129 and D9S1871. A 199-kb homozygous deletion encompassing the entire very low density lipoprotein receptor (VLDLR) gene was present in all affected individuals. VLDLR is part of the reelin signaling pathway, which guides neuroblast migration in the cerebral cortex and cerebellum. To our knowledge, this syndrome represents the first human lipoprotein receptor malformation syndrome and the second human disease associated with a reelin pathway defect. An autosomal recessive syndrome of nonprogressive cerebellar ataxia, mental retardation, and cerebellar hypoplasia was recognized and described in the Hutterite population in the 1980s (Schurig et al. Schurig et al., 1981Schurig V Van Orman A Bowen P Nonprogressive cerebellar disorder with mental retardation and autosomal recessive inheritance in Hutterites.Am J Med Genet. 1981; 9: 43-53Crossref PubMed Scopus (28) Google Scholar; Pallister and Opitz Pallister and Opitz, 1986Pallister P Opitz J Disequilibrium syndrome in Montana Hutterites.Am J Med Genet. 1986; 22: 567-569Crossref Scopus (10) Google Scholar). This constellation of findings was referred to as "dysequilibrium syndrome" (DES [MIM 224050]) on the basis of the clinical similarity to a group of patients with syndromic nonprogressive cerebellar ataxia initially described by Hagberg et al. in 1972 (Hagberg et al. Hagberg et al., 1972Hagberg B Sanner G Steen M The dysequilibrium syndrome in cerebral palsy: clinical aspects and treatment.Acta Paediat Scand. 1972; 61: 1-63Crossref PubMed Scopus (14) Google Scholar). As part of a larger project to characterize cerebellar disorders in the Hutterite population, we identified 12 Hutterites with DES (hereafter, "DES-H" refers to DES in the Hutterite population) and further described the clinical and neuroimaging features (Glass et al., Glass et al., in pressGlass HC, Boycott KM, Adams C, Barlow K, Scott JN, Chudley AE, Fujiwara T, Morgan K, Wirrell E, McLeod DR. Autosomal recessive cerebellar hypoplasia in the Hutterite population: a syndrome of nonprogressive cerebellar ataxia with mental retardation. Dev Med Child Neurol (in press)Google Scholar). DES-H is a nonprogressive syndrome characterized by moderate-to-profound mental retardation, delayed ambulation, and predominantly truncal ataxia. Additional features include strabismus and pes planus in the majority of patients, seizures in 40% of patients, and short stature in 15% of patients. Magnetic resonance imaging (MRI) demonstrates inferior cerebellar hypoplasia and mild cortical gyral simplification (fig. 1). The Hutterites originated from one of several Anabaptist groups formed during the Protestant Reformation in the 16th century and have lived on the North American prairies since the late 1800s (Hostetler Hostetler, 1985Hostetler J History and relevance of the Hutterite population for genetic studies.Am J Med Genet. 1985; 22: 453-462Crossref PubMed Scopus (48) Google Scholar). The population now numbers >40,000 individuals, the majority of whom are descendants of 89 founders (Nimgaonkar et al. Nimgaonkar et al., 2000Nimgaonkar VL Fujiwara TM Dutta M Wood J Gentry K Maendel S Morgan K Eaton J Low prevalence of psychoses among the Hutterites, an isolated religious community.Am J Psychiatry. 2000; 157: 1065-1070Crossref PubMed Scopus (43) Google Scholar). The unique social characteristics of this population have resulted in virtual genetic isolation. In a reconstructed 1981 census of 25,223 Hutterites in North America, the average inbreeding coefficient was 0.038, with a range of 0–0.164 (T. M. Fujiwara and K. Morgan, unpublished data). Over 30 different autosomal recessive conditions have been described in the Hutterite population (Innes et al. Innes et al., 1999Innes AM Wrogemann K Zelinski T Coghlan G Maendel S Maendel M Evans J Greenberg CR Delivery of genetic services to the Manitoba Hutterites in the molecular era.Am J Hum Genet Suppl. 1999; 65: A216Google Scholar). Given the population's characteristics, it is likely that each of these rare alleles was introduced by a single ancestor. We used an identity-by-descent mapping approach to localize the gene for DES-H to chromosome region 9p24. DNA samples from the eight patients of families 1, 2, and 3 (fig. 2) were genotyped using a set of 400 polymorphic microsatellite markers (ABI PRISM Linkage Mapping Set version 2 [Applied Biosystems]) with an average spacing of 4.0 for 11 markers from D9S917 to D9S1810, with a maximum of 4.3, providing statistical evidence of linkage to the telomeric end of the short arm of chromosome 9, in region 9p24. This analysis was performed under the assumption of autosomal recessive inheritance with complete penetrance. Marker-allele frequencies were estimated from the observed alleles in all the genotyped individuals. Although the families were known to be related in multiple different ways (Glass et al., Glass et al., in pressGlass HC, Boycott KM, Adams C, Barlow K, Scott JN, Chudley AE, Fujiwara T, Morgan K, Wirrell E, McLeod DR. Autosomal recessive cerebellar hypoplasia in the Hutterite population: a syndrome of nonprogressive cerebellar ataxia with mental retardation. Dev Med Child Neurol (in press)Google Scholar), the four pedigrees shown in figure 2 were used to calculate linkage, since they were expected to provide sufficient power. The frequency of the DES-H allele was set at 0.03333, on the basis of an observed population frequency of 1/900 (Glass et al., Glass et al., in pressGlass HC, Boycott KM, Adams C, Barlow K, Scott JN, Chudley AE, Fujiwara T, Morgan K, Wirrell E, McLeod DR. Autosomal recessive cerebellar hypoplasia in the Hutterite population: a syndrome of nonprogressive cerebellar ataxia with mental retardation. Dev Med Child Neurol (in press)Google Scholar) and under the assumption of Hardy-Weinberg equilibrium. This is probably an overestimate of the true DES-H allele frequency, given the level of inbreeding in the Hutterites. The extended haplotypes demonstrated both familial and ancestral recombination events (fig. 2). On the basis of familial recombination events, the minimal region for the DES-H locus was 18.1 cM (11.0 and 25.6 cM on the female and male, respectively, Marshfield genetic maps), between the p telomere and D9S286. Under the assumption of identity by descent and inferred ancestral recombination, the DES-H locus was predicted to lie in a 5.5-cM interval between D9S129 and D9S1871 (genetic distance estimated from the physical map for D9S129 and from the Marshfield genetic map and the physical map for D9S1871), in which all patients were homozygous for the same allele at D9S143, D9S54, D9S228, and D9S939. The 5.5-cM DES-H candidate region between D9S129 and D9S1871 corresponded to a 2-Mb interval and contained 10 transcripts, of which 6 encoded characterized genes. The critical interval contained an obvious candidate, the VLDLR gene, which encodes a receptor for reelin. The reelin signaling pathway guides neuroblast migration in the cerebral cortex and cerebellum (D'Arcangelo et al. D'Arcangelo et al., 1995D'Arcangelo G Miao GG Chen SC Soares HD Morgan JI Curran T A protein related to extracellular matrix proteins deleted in the mouse mutant reeler.Nature. 1995; 374: 719-723Crossref PubMed Scopus (1423) Google Scholar; Rice and Curran Rice and Curran, 2001Rice DS Curran T Role of the reelin signaling pathway in central nervous system development.Annu Rev Neurosci. 2001; 24: 1005-1039Crossref PubMed Scopus (559) Google Scholar; Tissir and Goffinet Tissir and Goffinet, 2003Tissir F Goffinet AM Reelin and brain development.Nat Rev Neurosci. 2003; 4: 496-505Crossref PubMed Scopus (585) Google Scholar). VLDLR consists of 19 exons and spans ∼40 kb (Sakai et al. Sakai et al., 1994Sakai J Hoshino A Takahashi S Miura Y Ishii H Suzuki H Kawarabayasi Y Yamamoto T Structure, chromosome location, and expression of the human very low density lipoprotein receptor gene.J Biol Chem. 1994; 269: 2173-2182Abstract Full Text PDF PubMed Google Scholar). Attempts to amplify the VLDLR coding exons by PCR repeatedly failed in patients but not in controls or unaffected family members. Lack of amplification of exons 1A and 19 suggested a homozygous deletion of the entire VLDLR coding region (data not shown). Part of exon 1 of the KCNV2 gene (GenBank accession number AL354723), ∼70 kb proximal to VLDLR, was present in patients, as determined by PCR amplification. Exon 4 of the SMARCA2 gene (GenBank accession number AL138755), 430 kb distal to VLDLR, was identified in patients by PCR amplification of the trinucleotide repeat PMC303366P11 (UniSTS) (data not shown). These results were consistent with a deletion of <500 kb. To determine the extent of the deleted sequence encompassing VLDLR, we used a sequence-tagged site (STS) walking strategy (fig. 3A). This approach generated a 2.6-kb junction fragment, which was amplified and sequenced in patients with DES-H and was compared with sequences generated by amplifying the two STSs flanking the proximal and distal boundaries in control DNA (fig. 3B). A homozygous deletion of 199,163 bp was present in each patient. A three-primer reaction was designed to allow for coamplification of both the normal and deleted alleles. Genomic DNA from all patients, parents, and siblings was amplified, and results were consistent with the linkage and haplotype data; each parent was a carrier of the deletion (fig. 3C). An 8-bp direct repeat, GAAAACAA (fig. 3B), was present at the breakpoints. Such short direct repeats have been found to mark the endpoints of many pathogenic deletions, which suggests that the repeats predispose to slippage during DNA replication that results in deletion of the intervening sequence and one of the repeat sequences (Antonarakis et al. Antonarakis et al., 2001Antonarakis SE Krawczak M Cooper DN The nature and mechanisms of human gene mutation.in: Scriver CR Beaudet AL Sly WS Valle D The metabolic and molecular bases of inherited disease. 8th ed. McGraw-Hill, New York2001: 343-377Google Scholar). The 199-kb deletion contained the entire VLDLR gene and part of a hypothetical gene, LOC401491 (GenBank [accession number AK092343]) (fig. 3D). The latter gene is supported by the presence of a 3,167-bp transcript, which is widely expressed and is predicted to encode a 109-aa protein. The entire coding region of this gene falls within the DES-H deletion. The sequence shows 80% homology to the ALU6 human Alu subfamily. Given the extent of the deletion, it is also possible that the expression of flanking genes is also affected because of the absence of intergenic regulatory elements. Comparison of the clinical findings of the patients with DES-H with the Vldlr-deficient mouse model (see below) supports homozygous loss of VLDLR as the major contributor to the DES-H phenotype. VLDLR is part of the reelin signaling pathway, which guides neuroblast migration in the developing cerebral cortex and cerebellum (D'Arcangelo et al. D'Arcangelo et al., 1995D'Arcangelo G Miao GG Chen SC Soares HD Morgan JI Curran T A protein related to extracellular matrix proteins deleted in the mouse mutant reeler.Nature. 1995; 374: 719-723Crossref PubMed Scopus (1423) Google Scholar; Rice and Curran Rice and Curran, 2001Rice DS Curran T Role of the reelin signaling pathway in central nervous system development.Annu Rev Neurosci. 2001; 24: 1005-1039Crossref PubMed Scopus (559) Google Scholar; Tissir and Goffinet Tissir and Goffinet, 2003Tissir F Goffinet AM Reelin and brain development.Nat Rev Neurosci. 2003; 4: 496-505Crossref PubMed Scopus (585) Google Scholar). In the cortex, Cajal-Retzius cells in the preplate secrete reelin to guide the cells of the cortical plate. In the cerebellum, the granule cells secrete reelin, which guides migration of Purkinje cells. VLDLR and apolipoprotein E receptor type 2 (ApoER2) are transmembrane receptors for reelin. With reelin binding, the cytoplasmic adaptor protein disabled-1 (Dab1) docks to the NPxY sequence in the intracellular region of VLDLR and ApoER2 and becomes phosphorylated (Howell et al. Howell et al., 1997Howell BW Hawkes R Soriano P Cooper JA Neuronal position in the developing brain is regulated by mouse disabled-1.Nature. 1997; 389: 733-737Crossref PubMed Scopus (595) Google Scholar; Trommsdorff et al. Trommsdorff et al., 1999Trommsdorff M Gotthardt M Hiesberger T Shelton J Stockinger W Nimpf J Hammer RE Richardson JA Herz J Reeler/disabled-like disruption of neuronal migration in knockout mice lacking the VLDL receptor and ApoE receptor 2.Cell. 1999; 97: 689-701Abstract Full Text Full Text PDF PubMed Scopus (1045) Google Scholar), which triggers an intracellular signaling cascade that allows neuroblasts to complete migration and adopt their ultimate positions in laminar structures in the CNS. Much of what is known about the reelin pathway comes from the study of a number of mouse models (reviewed by Lambert de Rouvroit and Goffinet [Lambert de Rouvroit and Goffinet, 1998Lambert de Rouvroit C Goffinet AM The reeler mouse as a model of brain development.Adv Anat Embryol Cell Biol. 1998; 150: 1-106Crossref PubMed Google Scholar] and Tissir and Goffinet [Tissir and Goffinet, 2003Tissir F Goffinet AM Reelin and brain development.Nat Rev Neurosci. 2003; 4: 496-505Crossref PubMed Scopus (585) Google Scholar]). In mice, deficiency of reelin results in the reeler phenotype (D'Arcangelo et al. D'Arcangelo et al., 1995D'Arcangelo G Miao GG Chen SC Soares HD Morgan JI Curran T A protein related to extracellular matrix proteins deleted in the mouse mutant reeler.Nature. 1995; 374: 719-723Crossref PubMed Scopus (1423) Google Scholar), which is characterized by impaired motor coordination, tremors, and ataxia. Reeler-like mice result from mutations in Dab1 (Howell et al. Howell et al., 1997Howell BW Hawkes R Soriano P Cooper JA Neuronal position in the developing brain is regulated by mouse disabled-1.Nature. 1997; 389: 733-737Crossref PubMed Scopus (595) Google Scholar) and double mutations in the lipoprotein receptors, Vldlr and ApoER2 (Trommsdorff et al. Trommsdorff et al., 1999Trommsdorff M Gotthardt M Hiesberger T Shelton J Stockinger W Nimpf J Hammer RE Richardson JA Herz J Reeler/disabled-like disruption of neuronal migration in knockout mice lacking the VLDL receptor and ApoE receptor 2.Cell. 1999; 97: 689-701Abstract Full Text Full Text PDF PubMed Scopus (1045) Google Scholar). In reeler and reeler-like mice, the preplate forms normally, with migration of cortical neurons from the germinative zones. However, as the neurons approach their destination, they fail to recognize their proper location and orientation. Later-migrating neurons fail to split the preplate into the marginal zone and subplate and instead form layers below the preplate with a reverse "outside-in" pattern compared with normal cortex. Reelin is also involved in the radial glial scaffold in the hippocampus. In reeler mutants, the distinct "reeling" gait is caused by profound hypoplasia of the cerebellum, in which the normal cerebellar folia are missing. Vldlr knockout mice with intact ApoER2 appear neurologically normal (Frykman et al. Frykman et al., 1995Frykman PK Brown MS Yamamoto T Goldstein JL Herz J Normal plasma lipoproteins and fertility in gene-targeted mice homozygous for a disruption in the gene encoding very low density lipoprotein receptor.Proc Natl Acad Sci USA. 1995; 92: 8453-8457Crossref PubMed Scopus (226) Google Scholar). However, the Vldlr-deficient mouse cerebellum is small, with reduced foliation and heterotopic Purkinje cells (Trommsdorff et al. Trommsdorff et al., 1999Trommsdorff M Gotthardt M Hiesberger T Shelton J Stockinger W Nimpf J Hammer RE Richardson JA Herz J Reeler/disabled-like disruption of neuronal migration in knockout mice lacking the VLDL receptor and ApoE receptor 2.Cell. 1999; 97: 689-701Abstract Full Text Full Text PDF PubMed Scopus (1045) Google Scholar). Neurons in the Vldlr-deficient murine cortex fail to distribute normally after reaching their assigned layer. The clinical and pathological features of the Vldlr-deficient mouse are less severe than those seen in the reeler mouse. Like reeler, mutations in the human orthologue reelin (RELN) cause significant disruption of neuroblast migration and architecture in the cerebellum and cerebral cortex. A syndrome of autosomal recessive lissencephaly with cerebellar hypoplasia (LCH) has been shown to be caused by mutations in RELN in two families (Hong et al. Hong et al., 2000Hong SE Shugart YY Huang DT Shahwan SA Grant PE Hourihane JO Martin ND Walsh CA Autosomal recessive lissencephaly with cerebellar hypoplasia is associated with human RELN mutations.Nat Genet. 2000; 26: 93-96Crossref PubMed Scopus (663) Google Scholar). The phenotype in these patients was characterized by hypotonia, ataxia, and developmental delay, with lack of unsupported sitting and profound mental retardation with little or no language development. Seizures and congenital lymphedema were also present. In contrast, patients with DES-H have moderate motor delays, with most eventually walking unsupported, and predominantly moderate-to-severe mental retardation (Schurig et al. Schurig et al., 1981Schurig V Van Orman A Bowen P Nonprogressive cerebellar disorder with mental retardation and autosomal recessive inheritance in Hutterites.Am J Med Genet. 1981; 9: 43-53Crossref PubMed Scopus (28) Google Scholar; Glass et al., Glass et al., in pressGlass HC, Boycott KM, Adams C, Barlow K, Scott JN, Chudley AE, Fujiwara T, Morgan K, Wirrell E, McLeod DR. Autosomal recessive cerebellar hypoplasia in the Hutterite population: a syndrome of nonprogressive cerebellar ataxia with mental retardation. Dev Med Child Neurol (in press)Google Scholar). Neuroimaging of patients with a RELN mutation demonstrated moderate lissencephaly, with an anterior greater than posterior gradient, a malformed hippocampus, and profound cerebellar hypoplasia with complete absence of detectable cerebellar folia (Hong et al. Hong et al., 2000Hong SE Shugart YY Huang DT Shahwan SA Grant PE Hourihane JO Martin ND Walsh CA Autosomal recessive lissencephaly with cerebellar hypoplasia is associated with human RELN mutations.Nat Genet. 2000; 26: 93-96Crossref PubMed Scopus (663) Google Scholar). In DES-H, the primary abnormality found on neuroimaging was marked hypoplasia of the cerebellar hemispheres and inferior vermis (fig. 1). A simplification and thickening of the cerebral cortex was also present, but this was a much milder feature in comparison with that of patients with a RELN mutation. In DES-H, the hippocampi appeared to undergo normal developmental rotation compared with the fetal configuration of the hippocampi in the patients with a RELN mutation. Deficiency of VLDLR in both mice and humans is associated with a less-severe phenotype than that seen with deficiency of reelin. Six subtypes of LCH have been defined (Ross et al. Ross et al., 2001Ross ME Swanson K Dobyns WB Lissencephaly with cerebellar hypoplasia (LCH): a heterogeneous group of cortical malformations.Neuropediatrics. 2001; 32: 256-263Crossref PubMed Scopus (106) Google Scholar). In this group of syndromes, the term "lissencephaly" refers to both the classic pattern of pachygyria/agyria, with marked thickening of the gray matter, and to less-severe phenotypes, ranging from mild pachygyria to simplification of the cortical gyral pattern with near-normal gray-matter thickness. Cerebellar manifestations range from relatively preserved hemispheres to marked cerebellar hypoplasia with foliation defects. The human phenotype associated with mutations in RELN has been classified as "LCHb" (Ross et al. Ross et al., 2001Ross ME Swanson K Dobyns WB Lissencephaly with cerebellar hypoplasia (LCH): a heterogeneous group of cortical malformations.Neuropediatrics. 2001; 32: 256-263Crossref PubMed Scopus (106) Google Scholar). The malformations seen in DES-H also fall within the LCH spectrum, with mild cerebral gyral simplification and more-marked cerebellar hypoplasia. VLDLR also functions as a peripheral lipoprotein receptor for VLDL triglycerides in mice. The low density lipoprotein receptor (LDLR) can mask the effect of VLDLR on lipoprotein metabolism (Tacken et al. Tacken et al., 2000Tacken PJ Teusink B Jong MC Harats D Havekes LM van Dijk KW Hofker MH LDL receptor deficiency unmasks altered VLDL triglyceride metabolism in VLDL receptor transgenic and knockout mice.J Lipid Res. 2000; 41: 2055-2062Abstract Full Text Full Text PDF PubMed Google Scholar; reviewed by Takahashi et al. [Takahashi et al., 2004Takahashi S Sakai J Fujino T Hattori H Zenimaru Y Suzuki J Miyamori I Yamamoto TT The very low-density lipoprotein (VLDL) receptor: characterization and functions as a peripheral lipoprotein receptor.J Atheroscler Thromb. 2004; 11: 200-208Crossref PubMed Scopus (120) Google Scholar]). Vldlr-deficient mice are protected from obesity after exposure to a high-fat, high-calorie (HFC) diet and have increased plasma triglycerides after HFC feeding (Goudriaan et al. Goudriaan et al., 2001Goudriaan JR Tacken PJ Dahlmans VE Gijbels MJ van Dijk KW Havekes LM Jong MC Protection from obesity in mice lacking the VLDL receptor.Arterioscler Thromb Vasc Biol. 2001; 21: 1488-1493Crossref PubMed Scopus (106) Google Scholar). Of the patients with DES-H, 50% had a BMI <18.5 (underweight), which suggests that there may be some protection from obesity. In summary, a homozygous deletion of the very low density lipoprotein receptor causes an autosomal recessive syndrome of cerebellar hypoplasia in the Hutterite population. We propose that this condition now be referred to as "VLDLR-associated cerebellar hypoplasia" (VLDLR-CH) to recognize the molecular pathogenesis. To our knowledge, this condition represents the first human lipoprotein receptor malformation syndrome and the second human disease associated with a reelin pathway defect. The clinical manifestations and MRI findings observed in this group of patients provide insight into the effects of homozygous loss of VLDLR on the development of the human CNS. It will be important to determine whether mutations in VLDLR and/or other genes in the reelin pathway cause cerebellar hypoplasia in patients from other populations. We thank all of the Hutterite families for their active participation in this research. We appreciate the efforts and support of physicians Coleen Adams and Karen Barlow and genetic counselors Carol Farr, Jackie Morris, and Caroline Powell during the clinical characterization phase of this research. We thank Drs. Richard Hawkes, Harvey Sarnat, N. Torben Bech-Hansen, A. Micheil Innes, and Kenneth Morgan for critical review of the manuscript. This work was supported by a short-term project grant from the University of Calgary, an Alberta Children's Hospital Foundation grant, and the Canadian Genetic Diseases Network (Networks of Centres of Excellence Program). S.F. was supported by a summer studentship from the Alberta Heritage Foundation for Medical Research. Drs. McLeod and Parboosingh contributed equally to this work, as senior investigators.
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