Linkage and physical mapping of X-linked lissencephaly/SBH (XLIS): a gene causing neuronal migration defects in human brain
Linkage and physical mapping of X-linked lissencephaly/SBH ( XLIS ): a gene causing neuronal migration defects in human brainM. ElizabethRoss1,*,+, Kristina M.Allen2,+, Anand K.Srivastava3,+, TerryFeatherstone4, Joseph G.Gleeson2, BetsyHirsch5, Brian N.Harding6, EvaAndermann7, RabiAbdullah2, MichaelBerg8, DesireéCzapansky-Bielman1, Dean J.Flanders9, RenzoGuerrini10, JacquesMotté11, A. PucheMira12, IngridScheffer13, SamuelBerkovic13, F.Scaravilli14, Richard A.King9, David H.Ledbetter15, DavidSchlessinger4, William B.Dobyns1,* and Christopher A.Walsh2
1Department of Neurology and5Department of Laboratory Medicine and Pathology, UMHC,Minneapolis, MN 55455,USA,2Beth Israel Hospital Boston Children's Hospital and Harvard University,Boston, MA 02115,USA,3J. C. Self Research Institute of Human Genetics Greenwood Genetic Center,Greenwood, SC 29646,USA,4Molecular Microbiology and Center for Genetics in Medicine, Washington University,St Louis, MO,USA,6Department of Histopathology, Hospital for Sick Children,London,UK,7Montreal Neurological Institute, McGill University,Montreal,Canada,8Department of Neurology, University of Rochester Medical Center,Rochester, NY,USA,9Departments of Pediatrics and Medicine, University of Minnesota Medical School,Minneapolis, MN,USA,10Division of Child Neurology and Psychiatry, Stella Maris Foundation and University of Pisa,Pisa,Italy,11Unité de Neuropédiatrie, American Memorial Hospital,Reims,France,12Hospital Universario V. Arrixaca,Murcia,Spain,13Austin Hospital,Heidelberg,Australia,14Neuropathology National Hospital for Neurology and Neurosurgery,London,UK and15Center for Medical Genetics, University of Chicago,Chicago, IL,USA
Received October 28, 1996;Revised and Accepted January 21, 1997
While disorders of neuronal migration are associated with as much as 25% of recurrent childhood seizures, few of the genes required to establish neuronal position in cerebral cortex are known. Subcortical band heterotopia (SBH) and lissencephaly (LIS), two distinct neuronal migration disorders producing epilepsy and variable cognitive impairment, can be inherited alone or together in a single pedigree. Here we report a new genetic locus,XLIS, mapped by linkage analysis of five families and physical mapping of a balanced X;2 translocation in a girl with LIS. Linkage places the critical region in Xq21-q24, containing the breakpoint that maps to Xq22.3-q23 by high-resolution chromosome analysis. Markers used for somatic cell hybrid and fluorescencein situhybridization analyses place theXLIS region within a 1 cM interval. These data suggest that SBH and X-linked lissencephaly are caused by mutation of a single gene,XLIS, that the milder SBH phenotype in females results from random X-inactivation (Lyonization), and that cloning of genes from the breakpoint region on X will yieldXLIS.
Although once thought to be rare, malformations of the cerebral cortex are increasingly implicated as a major cause of recurrent seizures in children and adults. Magnetic resonance imaging (MRI) has detected focal cortical dysplasia in 25% of children with intractable partial onset seizures, with a frequency in adult populations of at least 15% (1 ,2 ). Several malformations of human cortical development have been described in which the primary defect is incomplete migration of cerebral cortical neurons (3 ,4 ). These inherited malformations provide a unique opportunity to identify genes that orchestrate appropriate neuronal movement to the cerebral cortex and further understand the pathogenesis of this important class of human neurological disorders.
The diagnosis of LIS or SBH is definitively made by MRI or autopsy examination of brain. Shown in Figure1 are scans of a male with LIS and his mother with SBH (Family D in the linkage studies). In X-linked LIS (Fig.1 A), there are absent or decreased surface convolutions and abnormally thick cortical gray matter, while the cerebellum appears grossly normal. On MRI, SBH is characterized by symmetric, circumferential bands of gray matter located just beneath the cortex, and separated from it by a thin band of white matter (Fig.1 B). The heterotopic band varies in thickness among individuals.
Brain tissue was examined from a male with LIS (Fig.1 D, MRI in Fig.1 A) whose mother and sister manifested SBH (Family D). The brain pathology of X-linked LIS is similar to that seen in MDS (11 -13 ). Luxol fast blue-hematoxylin and eosin stained sections show thickened cortical gray matter with reduced volume of periventricular white matter. There is a marked reduction in number and depth of sulci. On microscopic examination (not shown), XLIS brain lacks the clear neuronal lamination of normal six layered cortex. Instead, it can be roughly demarcated into a marginal zone overlying superficial and deep cortical gray layers, which are separated by a relatively neuron-sparse layer. Heterotopic neurons are often found in the subcortical white matter, suggesting arrested neuronal migration (13 ,14 ).
The post-mortem appearance of SBH is shown in Figure1 E. Though all of the SBH patients in the present linkage study are living, their MRI scans are consistent with the pathology presented here. The cerebral cortex appears normal in its surface convolutions and thickness. Within the white matter is a heterotopic band of neurons extending from frontal to occipital regions, sparing only the cingulate, striate and medial temporal cortices. At higher magnification (not shown), true cortex appears normal in lamination while neurons within the band are scattered with apical dendrites oriented either toward the cortex or inverted.
X-linked inheritance of both LIS in males and SBH in females has been reported in two families (15 ). Blood was obtained for DNA isolation and linkage analysis from one of these (A) and four other families (C-E) whose pedigrees are shown in Figure2 . In two of these families, 30 polymorphic markers [sequence tagged site (STS), simple tandem repeat (STR), or microsatellite] that cover the entire X chromosome were tested. No evidence for linkage was found anywhere but Xq21-q26. In all five families, linkage analysis was performed using >25 markers concentrated in the Xq22-q24 region (16 ,17 ). Pertinent anchor loci include theCOL4A5 andCOL4A6genes,DXS1105 andDXS1072 which map to this relatively marker poor region (18 ).
The critical region determined by linkage analysis lies between Xq21.3 and Xq24, flanked by markersDXS990andDXS1001(Fig.4 B), with a maximum two-point LOD score of ~3.3. Since this is an X-linked disorder, a LOD score >= 2 is significant, because the prior probability of linkage between the trait and marker locus on X is higher than for an autosomal trait (22 ,21 ). No obligatory crossover in the Xq21-q24 region was found between the SBH and lissencephaly phenotypes, indicating that these two migration disorders co-segregate as would be expected if a single gene locus were involved. This region corresponds to a recombination map distance of ~22 cM (17 ).
To map the t(X;2) breakpoint, FISH was performed (Fig.3 ) with several YACs from the Xq21.3-q24 region (24 ). Biotin-labeled and digoxigenin-labeled inter-Alu PCR products were amplified from YACs for use as probes. YACs from the contig containingDXS1105 andCOL4A5 gave a signal at Xq22.3 on both the normal X and derivative X (Xpter-q22.3). A YAC from theDXS1072 contig gave signal at Xq23 on the normal X and the derivative autosome 2, which contains the X(q22.3-qter). These dual label FISH studies, exemplified in Figure3 , place the breakpoint in the region telomeric toCOL4A5 and centromeric toDXS1072.
In order to more accurately position the translocation breakpoint within the region, a human-hamster hybrid cell line, JFA6, was derived from cells of patient XLI-01. JFA6 contains the der(2), including the segment Xq22.3-Xqter, as the only human chromosome. JFA6 was analyzed with multiple STS markers from the Xq21-q24 region (Fig.4 ;16 ,17 ). Markers mapped to Xq22.3 that amplified no specific PCR product from JFA6 includedDXS1105andCOL4A5 (Fig.4 A). Therefore, these were placed centromeric to the breakpoint (Fig.4 B). The most centromeric anchor marker that produced a specific PCR product in the hybrid wasDXS1072, which was therefore placed telomeric to the breakpoint (Fig.4 B). Anchor markersDXS1105 andDXS1072, which flank the Xq22 breakpoint, are located within 1 cM of each other on the Généthon map (17 ).
Cumulative clinical and experimental data indicate that a relatively large number of genes must act to determine appropriate neuronal position. Early studies have established the importance of glial-guided mechanisms for the migration of cerebral cortical neuroblasts along radially oriented glial fibers (25 -27 ). More recent studies indicate that cerebral cortical cells follow both radial and tangential migration patterns to the cortical plate (28 ,29 ). Thus several mechanisms must exist that orchestrate neuronal migration in developing neocortex.
There are few molecules yet identified that are known to influence neuronal migration. The gene mutated in thereeler mouse appears to encode an extracellular matrix protein secreted by the Cajal-Retzius neurons of the marginal zone and early post migratory neurons (30 -32 ). It has been postulated that reelin protein provides an extracellular cue to migrating neuroblasts to promote early architectonic organization (33 ). A second cortical migration gene cloned encodes a neuronally expressed, glial-guidance molecule designated astrotactin (34 ). This is a neuronal membrane associated protein which forms the contact between migrating neuroblasts and radial glial fibers.Reelerhas been mapped to human chromosome 7q22 (31 ), whileastrotactin is located on 1q25 (35 ), thereby excluding these genes as the site of mutation inXLIS. Another LIS gene,LIS1, was identified on chromosome 17p13, based on consistent deletions of chromosome 17p13.3 in MDS and ILS (10 ,36 ,37 ), and identified as the 45K subunit of the brain isoform of platelet-activating factor acetylhydrolase (38 ). It has been postulated thatLIS1 acts in signal transduction in the leading process of migrating neurons.
In the present linkage analysis, a recombination in Xq21.3 was detected in two of the five families studied, firmly establishing the proximal boundary of theXLIS critical region with respect to the centromere. The distal boundary in Xq24 is based on a recombination event in the proband of Family E that occurred betweenDXS101a andDXS1001. This placement is supported by Family C, in which there is a single affected female with SBH and two normal sons, the second son presumably having avoided theXLIS mutant allele due to a crossover that occurred in Xq24, telomeric to the proposedXLIS locus.
Linkage analysis places theXLIS gene in the region of Xq21.3-Xq24. Due to the small number and size of XLIS families available for study, the potential region of interest based on the linkage data alone is large. Definition of the critical region has been narrowed using ade novoX;2 balanced translocation in a female with LIS. The translocation falls within the critical region established by linkage analysis, suggesting that the breakpoint at Xq22.3-q23 disruptsXLIS, and that cloning DNA from the breakpoint region will facilitate isolation of the gene.
Although unlikely in this instance, the breakpoint at Xq22.3 need not disrupt theXLIS gene in order for this female to be affected. Because of the skewed X-inactivation pattern caused by the X-autosome translocation, a gene mutation anywhere on the translocated, and therefore active, X chromosome will be expressed. Theoretically, theXLIS gene could be on Xp and she would still be affected. Strong arguments against this are: (i) the decreased likelihood of having two rare events in a single chromosome, e.g. ade novo mutation and a translocation, (ii) the lack of family history for XLIS arguing against an inherited mutation in this patient, and (iii) the corroboration of the linkage data that confirm the localization of theXLIS gene within the Xq21.3-q24 region.
The appearance of LIS in patient XLI-01 with her demonstrated 100% skewing of X-inactivation, suggests that XLIS is typically due to a loss of function, which may result from a null or dominant negative mutation. The phenotypic differences in the XLIS syndrome between affected males (LIS) and affected females (SBH) probably result from Lyonization. In hemizygous males, most mutations of theXLIS gene would result in absent gene function and thus a severe phenotype. In heterozygous females, the band heterotopia could arise from neurons which inactivate the normal X, do not express theXLIS gene product, and therefore fail to complete migration. SBH females with thin bands and mild symptoms presumably have favorable skewing of X inactivation with theXLIS mutation preferentially inactivated, while unfavorable skewing results in a thick band, more severe mental retardation and intractable seizures. We hypothesize that the few reported males with SBH have a somatic mutation of theXLIS gene (or perhaps theLIS1 gene) which produces the same effect as Lyonization, though homozygosity for a partially inactivating mutation cannot be ruled out.
The coordinate action of a relatively large number of genes must be required for the initiation and regulation of radial and tangential neuronal migration and establishment of appropriate neuronal position. The study of human inherited malformations provides a unique opportunity to identify genes that orchestrate neuronal migration in cerebral cortex. The isolation of theXLIS gene and investigation of its functional relationship with theLIS1,reelin andastrotactin gene products will provide insight not only into fundamental aspects of cerebral histogenesis, but also into genetic mechanisms leading to a major class of human developmental disorders.
Patients with classical lissencephaly or SBH were studied with informed consent. Clinical summaries of Families A (15 ) and B (39 ) have been reported previously. Patient XLI-01 and Family E were ascertained as part of an ongoing Lissencephaly Research Project (8 ) which includes referrals from two parent support organizations, the Lissencephaly Network in North America and the Lissencephaly Contact Group in the United Kingdom. Clinical information was gathered on all individuals used in the analyses, with attention to family history, mental development and epilepsy. Brain MRI scans were obtained for all females and all affected males in the study. Since the LIS phenotype is always associated with marked clinical symptoms, scans were not required of normal males.
PCR-based linkage analysis.Peripheral blood from each XLIS patient and family member was collected and used to isolate high molecular weight DNA (40 ). PCR-based linkage analysis was carried out using polymorphic STSs, dinucleotide microsatellite, and gene specific markers (Research Genetics or synthesized from published sequences). Fixed reference maps for the X-chromosome were obtained from the X Chromosome Workshops (16 ) and the Généthon map (17 ). Standard protocols were adapted from those available from Research Genetics, Inc. Forward STS primers were 5' end-labeled with32P or33P using T4-polynucleotide kinase (PNK) and used in a PCR reaction with 20 ng of genomic DNA/5 µl reaction. The typical PCR program was: 94oC * 3 min, then 25 cycles of 94oC * 15 s, 57oC * 2 min, 72oC * 15 s, then 72oC * 2 min. Polymorphisms were visualized autoradiographically on 6 or 8%, denaturing polyacrylamide gels.Computer software for data analysis.Linkage analysis was performed on the XLIS families using the analysis programs MLINK and ILINK from the LINKAGE package, version C (19 ), FASTLINK (20 ) and the utility programs MAKEPED LCP, LRP and UNKNOWN, from LINKAGE 5.1. Data were tested for linkage between polymorphic STS markers andXLIS by two point analysis (21 ,22 ). Maximum LOD values for each informative marker were summed to derive the cumulative scores. Linkage of various markers on X withXLIS were examined as for a qualitative trait (e.g., LIS or SBH) having 100% penetrance, since all females and affected males used in the analysis were MRI scanned.XLIS gene frequencies in the general population were based on previously reported estimates (41 ).
Peripheral blood lymphocytes from patient XLI-01 and her parents were cultured for high resolution cytogenetic analysis using standard techniques (42 ). Early- to pro-metaphase cells were analyzed using G-banding at 550-800 band level resolution in order to determine the breakpoints of the X;2 translocation in XLI-01. Replication banding studies were performed on metaphase cells from patient XLI-01 by sequential G-R banding (43 ) to identify the early and late-replicating X chromosomes.
The JA6 somatic cell hybrid containing the distal Xq was constructed using hypoxanthine-guanine phosphoribosyl transferase (HPRT) as a selectable marker, under standard conditions (44 ). The hybrid was made by fusing XLI-01 derived cells bearing an X;2 translocation and a Chinese hamster cell line lacking HPRT. Selected in media containing azaserine and hypoxanthine, a stable diploid line was isolated in which the only human chromosomal material was the der(2) that includes the translocated region from Xq22.3 to Xqter. The karyotype of the der(2) is: 2q13-2p25::Xq22.3-Xqter. JFA6 was used to determine which STS markers flank the translocation breakpoint. Control hybrid line Ben3B contains an Xq21-qter translocated X as its only human material. Typical PCR parameters were: 95oC * 4 min, (95oC * 30 s, 55oC * 45 s, 72oC * 45 s) * 35 cycles, extension at 72oC * 5 min.
Probes for FISH analysis were prepared by inter-Alu PCR on YACs (45 ) that contain human DNA from the Xq22-q24 region. Cycling conditions were used as previously described (18 ). Alu-PCR amplification products were labeled by incorporation of biotin-11-dUTP (Sigma) or digoxigenin-11-dUTP (Boehringer Mannheim) by nick translation. Prometaphase chromosome spreads were obtained from peripheral lymphocytes and lymphoblastoid cells established from patient XLI-01, using a BrdU cocktail instead of thymidine as a release, to permit replication banding.In situhybridization and washing procedures were performed as previously described (18 ). Double hybridizations were carried out with 100 ng each of biotin and digoxigenin labeled DNA from different YACs. Replication banding was performed simultaneously with FISH. Biotin signal was amplified using Extravidin-Cy3 (Sigma) and the digoxigenin signal detected at the same time with FITC-conjugated sheep anti-digoxigenin (Boehringer Mannheim) and amplified with FITC-conjugated rabbit anti-sheep IgG (Boehringer Mannheim). Finally, slides were stained in DAPI and mounted for examination under a Zeiss Axiovert fluorescent microscope. The DAPI replication banding was viewed with a `02' filter set, the FITC signal with a `09' filter set and the Cy3 signal with a `15' filter set. Images on a Macintosh computer (Apple) were processed as described (46 ), with chromosomes identified by the DAPI replication banding pattern.
We gratefully acknowledge the families who participated in this study and those from small pedigrees or individuals that were uninformative for linkage but who will participate in later aspects of the work. We thank Prof. F. Gullotta for providing tissue samples from an additional male with probable XLIS. Supported by NIH grants NS35515 (MER, AKS and WBD) and NS32457 (CAW), and grants from the Minnesota Medical Foundation (WBD and MER) and a Human Frontier Science Award (CAW and SB).
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*To whom correspondence should be addressed
+These authors contributed equally to this work
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