A revision of the lissencephaly and Miller-Dieker syndrome critical regions in chromosome 17p13.3
A revision of the lissencephaly and Miller-Dieker syndrome critical regions in chromosome 17p13.3Samuel S. Chong1,2, Svetlana D. Pack1, Anna V. Roschke1, Akira Tanigami1, Romeo Carrozzo4, Ann C. M. Smith1,3, William B. Dobyns5 and David H. Ledbetter1,*
1National Center for Human Genome Research, National Institutes of Health, Bethesda, MD 20892, USA, 2Institute for Molecular and Human Genetics and Department of Pediatrics, and 3Department of Obstetrics and Gynecology, Georgetown University Medical Center, Washington, DC 20007, USA, 4Servizio di Genetica Medica, Ospedale San Raffaele, Milano, Italy and 5Division of Pediatric Neurology, Departments of Neurology and Pediatrics, University of Minnesota Medical School, Minneapolis, MN 55455, USA
Received May 29, 1996; Revised and Accepted October 4, 1996
DDBJ/EMBL/GenBank accession no. U58678
Miller-Dieker syndrome (MDS) is a multiple malformation syndrome characterized by classical lissencephaly and a characteristic facies. It is associated with visible or submicroscopic deletions within chromosome band 17p13.3. Lissencephaly without facial dysmorphism has also been observed and is referred to as isolated lissencephaly sequence (ILS). Apparently partial and non-overlapping deletions of the 5' or 3' end of a candidate gene LIS1 in one ILS and one MDS patient had suggested that MDS was a single gene disorder, and that LIS1 spans in excess of 400 kb. However, the originally presumed 5' end of LIS1 was found to belong to the 14-3-3[epsilon] gene residing more distally on 17p13.3. We have now isolated the correct 5' end of LIS1, constructed a ~500 kb genomic contig encompassing LIS1, and estimated its gene extent to be ~80 kb. Fluorescence in situ hybridization analysis of an ILS patient with a de novo balanced translocation, as well as analysis of several other key MDS and ILS deletion patients, localizes the lissencephaly critical region within the LIS1 gene. Therefore, LIS1 remains the strongest candidate gene for the lissencephaly phenotype in ILS and MDS. Our analyses also suggest that additional genes distal to LIS1 may be responsible for the facial dysmorphology and other abnormalities seen in MDS but not in ILS patients, supporting our original concept of MDS as a contiguous gene deletion syndrome.
Classical lissencephaly or `smooth brain' is a severe malformation of the brain manifest by a smooth cerebral surface with lesser involvement of the cerebellum and other rhombic lip derivatives. It results from incomplete neuronal migration to the cerebral cortex and rhombic lip derivatives at 9-13 weeks of embryonic development (1 ). Affected children have severe or profound mental retardation, epilepsy, and subtle facial abnormalities especially bitemporal hollowing and small jaw (2 ).
Classical lissencephaly occurs in several malformation syndromes. Miller-Dieker syndrome (MDS) consists of classical lissencephaly, characteristic facial abnormalities and sometimes other birth defects (3 ). The facial changes consist of prominent forehead, bitemporal hollowing, short nose with upturned nares, flat midface, protuberant upper lip with thin vermillion border and small jaw. It is associated with visible or submicroscopic rearrangements within chromosome band 17p13.3 in almost all patients (4 ). Isolated lissencephaly sequence (ILS) consists of classical lissencephaly and its direct sequela with no other major anomalies (2 ). Submicroscopic deletions of chromosome 17p13.3 have been detected in almost 40% of patients (4 ). Classical lissencephaly also occurs in X-linked lissencephaly and subcortical band heterotopia, which has been mapped to chromosome Xq22.3-q23 (5 ).
We previously isolated a candidate gene for MDS (LIS1; 6 ) which was subsequently shown to be the human homologue of the 45K subunit of the brain isoform of platelet activating factor acetylhydrolase isolated from bovine cerebral cortex (7 ). One of the four LIS1 cDNA clones (8-1) was initially thought to contain the 5' end of the gene, such that apparently non-overlapping deletions in the 5' or 3' end of the gene were detected in one ILS and one MDS patient (although reported as two MDS patients). This suggested that partial or complete deletion of LIS1 was necessary and sufficient for expression of the complete MDS phenotype (6 ). We recently discovered that clone 8-1 does not contain the 5' end of LIS1, but instead is a chimera of downstream LIS1 sequences and another more distally located gene 14-3-3[epsilon] (8 ). Since clone 8-1 was used in initial characterization studies of LIS1 deletions in MDS patients (6 ), we reassessed the candidacy of LIS1 as the MDS causative gene. For this purpose, we have isolated the true 5' end of LIS1 including the promoter region, and generated a genomic contig encompassing LIS1.
Using these reagents, we performed fluorescence in situ hybridization analysis (FISH) on an ILS patient with a de novo 17;19 balanced translocation. As a result, we have localized his chromosome 17 breakpoint to a large intron within the 5' untranslated region (UTR) of LIS1. Additionally, our FISH analyses of patients with interstitial deletions have revealed one MDS and three ILS patients whose proximal or distal breakpoints also occur in the same interval within LIS1. Based on these findings, we have redefined the lissencephaly minimal critical region to a <100 kb region centromeric to D17S379 and telomeric to D17S1566, coinciding with the LIS1 gene. The ILS translocation breakpoint within LIS1, and the partial deletions of LIS1 in ILS and MDS deletion patients, strongly suggest that LIS1 is the lissencephaly causative gene. The data, however, do not support the hypothesis that LIS1 is also responsible for the facial dysmorphism and other anomalies of MDS patients.
Patient MDS-019 was previously shown to be deleted for the 5' end of the LIS1 gene by analysis of somatic cell hybrids using cDNA clone 8-1 as probe, as well as by fluorescence in situ hybridization (FISH) analysis of patient lymphoblasts using cosmids isolated with clone 8-1 (6 ). LIS1 clone 8-1 has since been determined to be chimeric and does not contain the 5' end of the LIS1 gene (8 ). We therefore reassessed patient MDS-019 by Southern blot analysis of a somatic cell hybrid containing her abnormal chromosome 17, using the largest non-chimeric LIS1 cDNA we had as probe (clone 71, Fig. 1 a). Using probe 24F/215R representing the 5' end of clone 71, a ~6.5 kb human chromosome 17 specific PstI fragment is present for the hybrid of MDS-019 and absent for the hybrids from patients MDS-009 and ILS-024 (Fig. 1 b). When the same blot was rehybridized with probe 4846F/5167R representing the 3' end of clone 71, a ~4 kb human 17-specific PstI fragment is again detected for patient MDS-019 but not for patients MDS-009 and ILS-024 (Fig. 1 c). These data indicate that MDS-019 is not deleted for the LIS1 gene portion represented by the 5.2 kb cDNA clone 71.
Since deletion of the promoter of any gene can effectively extinguish its expression, we wished to determine if the promoter and 5' UTR sequences of LIS1 could be deleted in MDS-019. The high homology of the 5' UTR between human LIS1(47) and mouse Lis1 cDNAs (9 ) strongly suggests that the entire 5' UTR of LIS1(47) is LIS1-specific. Nonetheless, based on the ~7.5 kb size of the largest LIS1 transcript (9 ), the lack of a polyadenylation signal close to the 3' end of the 5.2 kb LIS1(71) cDNA (6 ), and the presence of a long 5' UTR in the bovine homolog (7 ), we deduced that among other things a portion of the 5' UTR of LIS1 could still be unaccounted for.
Attempts to isolate additional 5' UTR sequences of LIS1 cDNA by reverse-transcription-PCR (RT-PCR) did not yield any distinct fragments (data not shown). We therefore chose a genomic PCR approach and isolated a 1.5 kb fragment extending upstream of the LIS1(47) 5' UTR. Within this fragment resides a TATAA box, as well as a CCAAT box ~10 nucleotides upstream of it (Fig. 2 ). Northern blot analysis using this 1.5 kb genomic fragment detects the characteristic double LIS1 transcripts of ~7.5 and ~5.5 kb (Fig. 3 ), confirming that it represents the 5' end of LIS1. RT-PCR analysis of human brain poly(A)+ RNA using primer pair CP2F and CP6R did not yield any product, nor was product obtained when either CP9F or CP7F was paired with LIS1(47)73R or LIS1(47)86R, whereas a ~200 bp fragment was obtained when CP8F was used as the forward primer against LIS1(47)73R or LIS1(47)86R (data not shown). These results suggest that LIS1 transcription initiates between the CP7F and CP8F primer sites (Fig. 2 ).
We retested patient MDS-019 and the two other patients described above for the presence of the LIS1 promoter by Southern blot analysis, and the results now show all three patients to be deleted for a ~3 kb chromosome 17-specific HindIII fragment containing the LIS1 promoter (Fig. 4 ). Thus, while patients MDS-009 and ILS-024 are deleted for all of LIS1 on the affected chromosome 17, patient MDS-019's deletion involves only part of the 5' UTR and sequences upstream. Based on the results using LIS1(71) cDNA and the LIS1 promoter fragment, we conclude that the proximal deletion breakpoint of MDS-019 must lie downstream of the promoter but upstream of the LIS1(71) 5' UTR sequence. Significantly, no altered fragment size is detected in MDS-019 using the LIS1(71) 24F/215R probe (Fig. 1 b) and no fragment of any size is detected with the LIS1 promoter probe (Fig. 4 ). These observations imply the presence of a large intron within the 5' UTR of LIS1, which is supported by our Southern analysis results and confirmed by exon-intron sequence analysis of LIS1-containing genomic clones (see below, 10 ).
To determine the extent of the LIS1 gene and its relationship to the lissencephaly and Miller-Dieker syndrome critical regions, we isolated cosmids, P1s and PACs and generated a contig encompassing LIS1 that extends centromeric for ~150-200 kb and telomeric by the same distance to the L132 (D17S379) locus (Fig. 5 a). While several cosmids, P1s and PACs contain portions of LIS1, only one PAC (95H6) contains the entire gene. Further screening of the LA17NC01 and L4/FS17 chromosome 17-specific libraries did not yield additional cosmids spanning the gap between cosmids 120A7 and 37E9. Based on restriction digestion analyses, we calculated the insert sizes of c120A7, c37E9, PAC-95H6 and PAC-308F9 to be ~35, ~35, ~110 and ~120 kb, respectively (data not shown). Hybridization analyses of NotI/EcoRI double-digests of PACs 95H6 and 308F9 were also performed using LIS1(71) cDNA, LIS1 promoter fragment, c154B4, c120A7, c37E9 and c135A6 as probes separately. Examination of these separate hybridization results revealed several internal fragments common to both PAC inserts that did not hybridize with any of the above probes, and that by deduction are derived from a large intron downstream of the 1.5 kb promoter fragment but upstream of the genomic region represented by LIS1(71) cDNA, i.e. within the 5' UTR (data not shown). We estimate the size of this intron to be ~50 kb from the combined sizes of all the non-hybridizing fragments. The presence of this intron within the 5' UTR has been confirmed by exon-intron sequencing of LIS1-containing genomic clones, as shown in the accompanying paper (10 ). Additionally, LIS1(71) cDNA probe detected fragments of ~13 and ~7.5 kb from PACs 95H6 and 308F9 as well a ~6 kb fragment from PAC-95H6 (data not shown). The portions of LIS1 containing the open reading frame (ORF) and 3' UTR thus constitute ~26.5 kb, and we therefore estimate the entire LIS1 gene to be ~80 kb in size. Two simple sequence repeats (SSRs), D17S1566 and D17S379, were recently developed and assigned to the Miller-Dieker syndrome chromosome region (11 ; A. Tanigami et al., in preparation), and we have mapped these markers within the contig to positions flanking the LIS1 gene (Fig. 5 a).
To further define the lissencephaly minimal critical region, we used these genomic clones as probes for FISH analysis on several MDS and ILS deletion patients as well as on a previously unreported ILS patient (ILS-096) who carries a 17p;19q balanced translocation. From these studies we have identified three ILS patients (ILS-035, ILS-103 and ILS-136), apart from MDS-019, who show partial deletions of LIS1, and have determined the chromosome 17 translocation breakpoint of ILS-096 (Fig. 5 b).
Figure 6 illustrates the FISH results for patients MDS-019 and ILS-103 using two cosmids, c37E9 which contains the promoter and partial 5' UTR of LIS1, and c120A7, containing the ORF and 3' UTR exons of LIS1. The presence of hybridization signal with c120A7 and lack of signal with c37E9 in the affected chromosome 17 from patient MDS-019 confirms our Southern blot data on the position of her proximal deletion breakpoint (Fig. 6 a and b). FISH results of patient ILS-035 showed the same results using c120A7 and c37E9 (data not shown). In contrast, no signal is detected on the affected chromosome from patient ILS-103 with c120A7 probe while a signal is present with c37E9, thus defining her distal deletion breakpoint (Fig. 6 c and d). FISH analysis of patient ILS-136 using these two probes yielded similar results (data not shown).
We have identified the correct 5' end of LIS1 and estimated the physical extent of this gene to be ~80 kb, oriented 5' to 3' from telomere to centromere. Collectively, the FISH analyses of several key ILS and MDS patients have enabled localization of the lissencephaly minimal critical region to the LIS1 gene locus, which lies between the SSR markers D17S1566 centromerically and D17S379 telomerically. These data thus strongly implicate LIS1 as the gene responsible for the lissencephaly phenotype in ILS and MDS. Curiously, there is an overlap in the deletions of ILS-136 and ILS-103 with those of MDS-019 and ILS-035, occurring where the ~50 kb 5' UTR intron resides (Fig. 5 b). There thus exists the possibility that a small gene resides within this intron, and that it may be deleted or disrupted in these deletion patients and in the translocation patient. However, as shown in the accompanying paper (10 ), we have recently identified point mutations in two coding exons as well as a small exon-intron deletion within the LIS1 gene in patients with ILS, thus ruling out that possibility.
Cytogenetic deletions of 17p13.3 have been observed in many MDS patients, while more recent FISH analyses using cosmid probes representing the D17S379 locus have revealed submicroscopic deletions within 17p13.3 in a significant percentage of ILS patients (4 ). These findings led us to originally hypothesize that MDS was caused by disruption of more than one gene. Our subsequent identification of apparently non-overlapping deletions of LIS1 in patients MDS-019 and MDS-021 had suggested otherwise, and instead implicated LIS1 in the etiology of the complete MDS phenotype (6 ). Patient MDS-021 had actually been reclassified as patient ILS-084 (by WBD) prior to detection of this abnormality.
We have since discovered that one of the cDNA clones used in that study and presumed to contain the 5' end of LIS1 (8-1) actually contains a fragment of another more distal gene 14-3-3[epsilon] (8 ). With the true 5' end of LIS1 now identified, we reassessed these two patients and have shown that patient MDS-019 is deleted for part of the 5' UTR and promoter region. Our FISH analysis of patient ILS-084 was inconclusive and suggestive of a complex combination of deletion and mosaicism in a lymphoblast cell line. For these reasons, we have not been able to confirm nor revise our previous findings of her deletions. Notwithstanding this patient, our analyses of all other key ILS and MDS deletion patients have enabled the definition of a small region of overlap representing the lissencephaly minimal critical region. Additionally, the co-incident localization of the distal breakpoint of ILS-103 and the proximal breakpoint of MDS-019 (Fig. 5 b) to the same region of LIS1 co-localizes the lissencephaly minimal critical region with the proximal boundary of the Miller-Dieker syndrome chromosome region (MDCR).
Furthermore, our results are consistent with the notion that a gene or genes in addition to LIS1 are necessary for expression of the full Miller-Dieker syndrome phenotype. For example, patients ILS-035 and MDS-019 have similar proximal deletion breakpoints. Also, complete deletions of LIS1 occur in ILS and MDS patients alike, for example in patients ILS-083 and MDS-051 (Fig. 5 b). Additionally, deletions that start from LIS1 and extend towards the centromere have only been documented in ILS patients (ILS-103 and ILS-136, Fig. 5 b). In contrast, deletions of MDS patients may or may not involve loci centromeric to LIS1, but always involve LIS1 and other telomeric loci in excess of 250 kb. Although deletions of ILS patients may also include more distal loci (e.g. ILS-024), FISH analyses using newly isolated cosmids within the MDCR have shown that the distal breakpoints of MDS patients are always more telomeric than those of ILS patients (our unpublished data). These observations are thus consistent with the concept of an additional gene or genes telomeric to LIS1 contributing to the facial dysmorphology and other abnormalities seen in MDS but not in ILS patients, supporting our original hypothesis of MDS as a contiguous gene deletion syndrome. The possibility of modifier loci outside of 17p13 being responsible for the MDS-specific phenotype is inconsistent with previous observations in familial cases of MDS cases involving balanced translocations or inversions, in which all unbalanced children with 17p monosomy have the full MDS phenotype.
We have evaluated 56 patients with MDS and 151 patients with ILS over the past 14 years. The presence of classical lissencephaly was confirmed by review of brain imaging studies or autopsy, and syndrome diagnosis was established prior to special laboratory studies in all but a few patients by either direct physical examination or review of medical records and photographs (by WBD). Those patients referred to in this study are listed in Table 1 .
LIS1 cDNA clones 71, 47, and 8-1 and the full-length human 14-3-3[epsilon] cDNA clone have been described (6 ,8 ) and sequences are available in GenBank through accession numbers L13385, L13386, L13388, and U54778, respectively. The sequence of the 1.5 kb genomic fragment containing the promoter (Fig. 2 ) has been deposited in GenBank under the accession number U58678.
Somatic cell hybrids KCB4 and BR8 constructed from patients MDS-019 and MDS-009 have been described elsewhere (12 ), while CBB5 is derived from patient ILS-024 who has an interstitial 17p13 deletion occurring from but not including D17S5 (YNZ22) distally and extending proximally towards but not including TP53 (unpublished data).
The 1.5 kb LIS1 promoter-containing fragment was isolated using the Human PromoterFindertm kit (Clontech, Palo Alto, CA) with modifications. Briefly, a primer specific to the 5' UTR of the LIS1 cDNA, LIS1(47)86R (5'-AGCCGTTCCGGGGCTCGAGTC-3') and an adaptor-specific primer AP1 (5'-GTAATACGACTCACTATAGGGC-3') were used in a first round amplification from a `library' of total human genomic DNA digested with DraI and ligated to adaptors. An aliquot from this reaction was subjected to a second round (nested) amplification using an overlapping LIS1-specific primer LIS1(47)73R (5'-CTCGAGTCCAAGCTCGGCGCTC-3') and an overlapping adaptor-specific primer AP2 (5'-ACTATAGGGCACGCGTGGT-3'). For both rounds of amplification, 1 µl of template was used in 50 µl reaction volumes, with final reagent concentrations of 20 mM Tris-HCl (pH 8.75), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% TritionR X-100, 0.1 mg/ml BSA, 10% DMSO, 0.25 mM of each dNTP, 0.2 µM of each primer, and 0.025 U/µl exo(-) Pfu polymerase (Stratagene, La Jolla, CA). Amplifications for both rounds were performed in a PTC-200 thermal cycler (MJ Research, Watertown, MA), beginning with 7 cycles of 94oC for 2 s and 72oC for 3 min, followed by 32 cycles of 94oC for 2 s and 67oC for 3 min, and ending with a final extension at 67oC for 4 min. The amplified product was separated across a 1% agarose, 0.5* TBE gel, then gel purified and subcloned, or used as probe in Southern or northern analyses. Complete sequencing of both strands of this fragment was performed commercially (SeqWright, Houston, TX). Sequence alignments and analyses were accomplished using the DNASTAR program (DNASTAR, Madison, WI).
Probes 24F/215R (192 bp) and 4846F/5167R (322 bp) were PCR-amplified from LIS1 cDNA clone 71 using the primer pairs 5'-TCCGGTGGAATGAATCTTAC-3'/5'-TGGCTGTAATGTCAAGCTTATC-3' and 5'-GTCACGGCTGGACTGAATG-3'/5'- GCAACAAATCTTACTGTAAAACACG-3', respectively. Standard Taq polymerase-mediated PCR reactions were performed in a PE480 thermal cycler (Perkin Elmer-ABI), with an initial denaturation at 94oC for 2 min, followed by 35 cycles of 94oC for 45 s, 57oC for 1 min and 72oC for 1 min. Amplified products were separated across a 1.5% agarose gel in 0.5* TBE at 10 V/cm, gel-purified using the Qiaquick-spin kit (Qiagen, Chatsworth, CA), and labelled to high specific activity with [[alpha]-32P]dCTP using the DECAprime II random-decamer labelling kit (Ambion, Austin, TX). The 1.5 kb genomic fragment containing the LIS1 promoter was similarly labelled, and preassociated with excess human CoT1 DNA (Gibco-BRL, Gaithersburg, MD) prior to blot hybridization.
DNA from somatic cell hybrids was isolated by routine methods and digested with restriction endonucleases according to supplier instructions. Digested DNA was separated across a 0.7% agarose, 1* TBE gel, followed by alkaline capillary transfer to Zeta-Probetm GT membrane (Bio-Rad, Hercules, CA) according to supplier instructions. Prehybridizations and hybridizations were performed in Rapid-hyb buffer (Amersham, Arlington Heights, IL) according to supplier instructions. Final wash was in 0.2* SSC, 0.1% SDS at 65oC for 30 min.
Multiple-tissue adult and fetal northern blots were obtained from a commercial source and probe hybridizations and blot washes were performed according to manufacturer instructions (Clontech, Palo Alto, CA).
Cosmids spanning the lissencephaly critical region were isolated by hybridization screening of a flow-sorted human chromosome 17 gridded library (LA17NC01) constructed at the Los Alamos National Laboratory, Los Alamos, NM and available from the Physical Mapping Core (PMC), National Center for Human Genome Research, NIH. Additional screenings were performed on a chromosome 17-specific cosmid library (L4/FS17) obtained from the Reference Library Database, Max-Planck-Institute for Molecular Genetics, Berlin, Germany. Several P1 clones were obtained by PCR screening of a P1 library available from the PMC. Other P1 and PAC clones were obtained by hybridization screening of gridded PAC and P1 libraries (Genome Systems, St. Louis, MO).
The genomic contig was assembled using a combination of STS/SSR PCR analysis and Southern blot analysis of cosmid, P1 and PAC digests using cDNAs, whole cosmids or partial cosmid fragments. cDNAs and cosmid DNA were isolated using the Qiagen plasmid kit, while P1 and PAC DNA was obtained from Genome Systems. Cosmids, cosmid fragments, and cDNAs were radio-labelled as above and preassociated with excess human CoT1 DNA prior to hybridization. Primers 24F, 215R, 4846F and 5167R are as described under `Probes', while LIS1(47)73R, CP2F, CP6R, CP7F, CP8F, and CP9F are indicated in Figure 2 . Other primers used were 269F (5'-TATCTTCGTTCAAATGGCTATGAAG-3'), 371R (5'-CCAAAAGACCAGCATACTTTTTATC-3'), 2387F (5'-GTGTGCCCATTTGAAAGGAGTG-3'), and 2510R (5'-GCACGCTCCATTAAACCCTG-3'). Amplification conditions were as described under `Probes' with the exception that annealing of the CP and LIS1(47) primers were at 55oC, and 60oC annealing was used for the remaining primers.
Chromosome preparations were made from lymphoblastoid cell lines by conventional methods and aged slightly (1-2 weeks) to maintain morphology during denaturation. Slides were washed in 2* SSC at 37oC for 30 min, serially dehydrated in 70%, 80%, and 95% ethanol at room temperature, denatured in 70% formamide/ 0.65* SSC at 72oC for 2 min, then serially dehydrated at -20oC in 70%, 80%, 90% and 100% ethanol.
Cosmid, P1 or PAC DNA was labeled with digoxigenin-11-dUTP by nick translation (Boehringer Mannheim, Indianapolis, IN), precipitated in ethanol with 50* excess of human CoT-1 and herring testis DNA (Gibco-BRL), and resuspended to a final concentration of 25 ng/µl in hybridization solution (50% formamide, 2* SSC, 10% dextran sulfate). Probes were denatured at 76oC for 10 min and preassociated at 37oC for 30 min prior to hybridization. Chromosome-specific [alpha]-satellite probes were selectively amplified from human-rodent monochromosomal hybrids as described (13 ). Chromosome 19 [alpha]-satellite was amplified in the presence of biotin-16-dUTP (Boehringer Mannheim) while chromosome 17 [alpha]-satellite was amplified separately using either biotin- or digoxigenin-dUTP, then mixed in a 1:1 ratio.
Probe-hybridization mix (10 µl) was applied under a 22 mm * 24 mm coverslip and slides were incubated in a moist chamber for 16 h at 37oC, then washed as previously described (14 ). Probes were detected in 50 µl of rhodamine anti-digoxigenin (Boehringer Mannheim) at 1 µg/µl and FITC-avidin D (Vector Laboratories, Burlingame, CA) at 5 µg/µl. Slides were washed three times in 4* SSC, 0.1% Tween®-20 at 45oC and mounted in antifade solution (Vector) containing DAPI. Analysis was performed using a Zeiss Axiophot microscope equipped with filters to detect DAPI, FITC and rhodamine separately, as well as dual and triple band pass filter sets to detect signals simultaneously. Images were collected and merged using a cooled CCD camera (KAF 1400, Photometrics) and IP Lab Spectrum software.
We thank the parents of patients examined in this study, as well as other parents of the Lissencephaly Network, without whose help and co-operation this work would not have been possible. We also thank T. Kubota for isolating some of the cosmids, C. Lo Nigro for some of the RT-PCR analyses, and M. Macha and K. Precht for expert technical assistance. This work was supported in part by a grant from the Italian Telethon (E.148) to RC.
1 Dobyns,W.B. (1987) Developmental aspects of lissencephaly and the lissencephaly syndromes. In Gilbert,E.F. and Opitz,J.M. (eds) Genetic aspects of developmental pathology. New York: Alan R. Liss. Birth Defects Orig. Artic. Ser., 23, 225-241.MEDLINE Abstract
2 Dobyns,W.B., Elias,E.R., Newlin,A.C., Pagon,R.A. and Ledbetter,D.H. (1992) Causal heterogeneity in isolated lissencephaly. Neurology, 42, 1375-1388.MEDLINE Abstract
3 Dobyns,W.B., Curry,C.J.R., Hoyme,H.E., Turlington,L. and Ledbetter,D.H. (1991) Clinical and molecular diagnosis of Miller-Dieker syndrome. Am. J. Hum. Genet., 48, 584-594.MEDLINE Abstract
4 Dobyns,W.B., Reiner,O., Carrozzo,R. and Ledbetter,D.H. (1993) Lissencephaly, a human brain malformation associated with deletion of the LIS1 gene located at chromosome 17p13. J. Am. Med. Assoc., 270, 2838-2842.
6 Reiner,O., Carrozzo,R., Shen,Y., Wehnert,M., Faustinella,F., Dobyns,W.B., Caskey,C.T. and Ledbetter,D.H. (1993) Isolation of a Miller-Dieker lissencephaly gene containing G protein [beta]-subunit-like repeats. Nature, 364, 717-721.MEDLINE Abstract
7 Hattori,M., Adachi,H., Tsujimoto,M., Arai,H. and Inoue,K. (1994) Miller-Dieker lissencephaly gene encodes a subunit of brain platelet-activating factor. Nature, 370, 216-218.MEDLINE Abstract
8 Chong,S.S., Tanigami,A., Roschke,A.V. and Ledbetter,D.H. (1996) 14-3-3[epsilon] has no homology to LIS1 and lies telomeric to it on chromosome 17p13.3 outside the Miller-Dieker syndrome chromosome region. Genome Res., 6,735-741.MEDLINE Abstract
9 Reiner,O., Albrecht,U., Gordon,M., Chianese,K.A., Wong,C., Gal-Gerber,O., Sapir,T., Siracusa,L.D., Buchberg,A.M., Caskey,C.T. and Eichele,G. (1995) Lissencephaly gene (LIS1) expression in the CNS suggests a role in neuronal migration. J. Neurosci., 15, 3730-3738.MEDLINE Abstract
10 Lo Nigro,C., Chong,S.S., Dobyns,W.B., Carrozzo,R. and Ledbetter,D.H. (1997) Point mutations and an intragenic in LIS1, the lissencephaly causative gene in isolated lissencephaly sequence and Miller-Dieker syndrome. Hum. Mol. Genet., 6, 157-164.MEDLINE Abstract
11 Carrozzo,R. and Ledbetter,D.H. (1993) Dinucleotide repeat polymorphism mapping to the critical region for lissencephaly (17p13.3). Hum. Mol. Genet., 2, 615.
12 Isomura,M., Tanigami,A., Saito,H., Harada,Y., Katagiri,T., Inazawa,J., Ledbetter,D.H. and Nakamura,Y. (1994) Detailed analysis of loss of heterozygosity on chromosome 17p13 in breast carcinoma on the basis of a high-resolution physical map with 29 markers. Genes Chrom. Cancer, 9, 173-179.MEDLINE Abstract
13 Weier,H-U, Kleine,H-D. and Gray,J.W. (1991) Labeling of the centromeric region on human chromosome 8 by in situ hybridization. Hum. Genet., 87, 489-494.MEDLINE Abstract
14 Kuwano,A., Ledbetter,S.A., Dobyns,W.B., Emanuel,B.S. and Ledbetter,D.H. (1991) Detection of deletions and cryptic translocations in Miller-Dieker syndrome by in situ hybridization. Am. J. Hum. Genet., 49, 707-714.MEDLINE Abstract
*To whom correspondence should be addressed at present address: Center for Medical Genetics, The University of Chicago, 5841 South Maryland Avenue, MC 2050, Chicago, IL 60637-1470, USA. Email: dhl@babies.bsd.uchicago.edu
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