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Subcortical band heterotopia in rare affected males can be caused by missense mutations in DCX (XLIS) or LIS1
Introduction
Results
Discussion
Materials And Methods
Subject selection and clinical review
Mutation analysis
Acknowledgements
References
Subcortical band heterotopia in rare affected males can be caused by missense mutations in DCX (XLIS) or LIS1
Received April 21, 1999; Revised and Accepted June18, 1999
Subcortical band heterotopia (SBH) are bilateral and symmetric ribbons of gray matter found in the central white matter between the cortex and the ventricular surface, which comprises the less severe end of the lissencephaly (agyria-pachygyria-band) spectrum of malformations. Mutations in DCX (also known as XLIS) have previously been described in females with SBH. We have now identified mutations in either the DCX or LIS1 gene in three of 11 boys studied, demonstrating for the first time that mutations of either DCX or LIS1 can cause SBH or mixed pachygyria-SBH (PCH-SBH) in males. All three changes detected are missense mutations, predicted to be of germline origin. They include a missense mutation in exon 4 of DCX in a boy with PCH-SBH (R78H), a different missense mutation in exon 4 of DCX in a boy with mild SBH and in his mildly affected mother (R89G) and a missense mutation in exon 6 of LIS1 in a boy with SBH (S169P). The missense mutations probably account for the less severe brain malformations, although other patients with missense mutations in the same exons have had diffuse lissencephaly. Therefore, it appears likely that the effect of the specific amino acid change on the protein determines the severity of the phenotype, with some mutations enabling residual protein function and allowing normal migration in a larger proportion of neurons. However, we expect that somatic mosaic mutations of both LIS1 and DCX will also prove to be an important mechanism in causing SBH in males.
INTRODUCTION
Subcortical band heterotopia (SBH) consist of ribbons of gray matter located within the central white matter between the cortex and the ventricular surface, which are usually bilateral and symmetrical (1,2). It comprises the less severe end of the classical lissencephaly or agyria-pachygyria-band spectrum of malformations, which results from deficient neuronal migration (2-4). Rare patients have been identified with a combination of less severe lissencephaly and SBH in different regions of the brain, usually frontal pachygyria and posterior SBH (PCH-SBH), classified as lissencephaly grade 5b. SBH has also been called `double cortex' syndrome, although the band does not represent a true cortex.
Classical lissencephaly has been associated with mutations of two recently cloned genes, DCX (also known as XLIS) and LIS1, while SBH has been associated with mutations of DCX in females. On reviewing brain imaging studies of patients with lissencephaly and known mutations of either gene, we observed a distinct gradient in the severity of lissencephaly which differed depending on the gene involved. The gyral malformation was consistently more severe anteriorly than posteriorly (A->P gradient) in children with DCX mutations, but more severe posteriorly than anteriorly (P->A gradient) in children with LIS1 mutations (5,6).
The great preponderance of patients with SBH are female, an observation attributed to the effects of a gene on the X chromosome (2). This prediction was supported by mapping (7,8) and cloning (9,10) of the DCX gene on chromosome Xq22.3-q23 in multiplex families with SBH in females and lissencephaly in males. The difference in phenotype between females and males with mutations of this gene was originally hypothesized to be due to Lyonization, such that cells with the normal allele active reach the cortex, while cells with the mutant allele active fail to migrate properly, thus forming the subcortical band. This would represent a form of functional mosaicism (2,9,10). Mutations in DCX have now been identified in all multiplex families with isolated lissencephaly sequence (ILS) and SBH (9,10), in ~38% of females with sporadic SBH (11) and in ~12% of males with sporadic ILS (5). The SBH series included several patients with partial and other atypical bands (11). A higher proportion of mutations (nine of 10) in sporadic females with SBH was reported in another series, probably due to smaller numbers and selective ascertainment of patients with more `typical' bands (12). Our experience suggests much more clinical and imaging heterogeneity among SBH patients than has yet been reported. Mutations of DCX have not been reported in boys with SBH or mixed PCH-SBH, although at least three males have been studied (12).
The LIS1 gene was mapped and cloned using structural rearrangements involving chromosome 17p13.3 in patients with either Miller-Dieker syndrome or ILS (13,14). Mutations of LIS1 have been found in many children with ILS (5,15,16), but have never been reported in patients with SBH. The proportion of males to females among patients with ILS due to LIS1 mutations is about equal (5,16).
Based on these data, we hypothesized that SBH in males could be caused by either somatic mosaic or less severe germline mutations in either DCX or LIS1. We further hypothesized that DCX mutations were more likely in individuals with a more severe cerebral malformation anteriorly, while LIS1 mutations were more likely in individuals with a more severe malformation posteriorly, based on our experience in patients with classical lissencephaly (5,6). This is the first report of DCX mutations causing SBH and PCH-SBH in males and of an LIS1 mutation causing SBH in any patient.
RESULTS
We performed direct DNA sequencing of DCX, LIS1 or both genes in 11 boys with SBH or PCH-SBH. Missense mutations were identified in three of the 11 boys studied. A composite of the cranial MRI scans and sequencing electropherograms in these three boys and the affected mother of the third boy is shown in Figure 1.
Figure 1. Cranial MRI scans and sequencing electropherograms of all four patients, with patient LP94-051 on the far left, LP98-046 second from the left, LP98-060a1 third from the left and his mother, LP98-060a2, on the far right. The top row shows axial T2-weighted MRI scans of the three male probands and an axial inverted-T2 MRI on the mother of the third boy on the far right. The arrows with the black border point to the subcortical bands while the solid white arrow on the scan of patient LP98-046 indicates the pachygyria. The bottom row shows sequencing electropherograms of LIS1 in patient LP94-051 on the far left and of DCX in patients LP98-046, LP98-060a1 and LP98-060a2 in the succeding panels.
Patient LP98-046 is a boy evaluated soon after birth because of borderline congenital microcephaly and plagiocephaly (cranial asymmetry). The MRI showed frontal lissencephaly (pachygyria) and posterior SBH. The frontal malformation comprised true pachygyria with a very thick cortex and not the simplified gyral pattern with shallow sulci which typically overlies SBH and has been called pachygyria in prior reports. This A->P gradient suggested a possible DCX mutation (Fig. 1). Patient LP98-060a1 is a boy with mild developmental delay and seizures. His MRI showed symmetrical thin SBH limited to the frontal lobe, with the remainder of the cortex being essentially normal. His mother (LP98-060a2) had asymmetrical frontal SBH with an appearance on the right side similar to the proband and a more subtle appearance on the left. In both, the A->P gradient suggested a DCX mutation (Fig. 1). Patient LP94-051 is a male with moderate to severe mental retardation in whom the diagnosis of SBH was made when he developed seizures at the age of 8 years. His MRI scan showed thick symmetrical bands extending from the mid-frontal region to the occipital lobe, with overlying simplified gyri and shallow sulci (Fig. 1). The anterior frontal region appeared normal, compatible with a P->A gradient which suggested a possible LIS1 mutation. He was included in a series of SBH patients reported previously (1).
We detected mutations in DCX in the first two boys. Sequencing of DCX in patient LP98-046 detected a missense mutation in exon 4, resulting in an amino acid change (G233->A, R78H). His mother was not found to carry the mutation and it is therefore predicted to be de novo. Sequencing of DCX in patients LP98-060a1 and LP98-060a2 revealed the same missense mutation (C264->G, R89G) in exon 4 in both mother and son. Chromosome analysis in the son was normal, excluding Klinefelter syndrome. We have sequenced exon 4 of DCX in 66 alleles and not previously seen either of the base pair changes found in LP98-046 and LP98-060a1/a2. The mutation detected in LP98-046 also abolishes an Fnu4HI restriction site in the amplicon and the mutation found in LP98-060a1/a2 creates a second XhoII restriction site. PCR-RFLP studies (5,17) confirmed the mutations in these patients and also excluded the presence of these base pair changes in an additional 56 and 38 alleles, respectively. Therefore, these changes are unlikely to be polymorphisms.
We found a missense mutation in exon 6 of the LIS1 gene in patient LP94-051, resulting in an amino acid change (T499->C, S169P). Sequencing of the same exon in his parents showed that the base pair change was de novo. This mutation is unlikely to be a polymorphism, as we have now sequenced >100 LIS1 alleles and have never observed this base pair change before. The mutation creates a novel NlaIV restriction site in the amplicon, which also allowed detection of the base pair change by PCR-RFLP (5,17) and exclusion of the presence of this restriction site in an additional 88 alleles.
DISCUSSION
All three mutations detected in these patients with SBH or mixed PCH-SBH are missense mutations, which probably accounts for the less severe cerebral malformations. However, other patients with missense mutations both in exon 4 of DCX and exon 6 of LIS1 have been observed with diffuse lissencephaly consisting of mixed agyria and pachygyria (5,15). Therefore, it appears likely that the effect of the specific amino acid change on the protein determines the severity of the phenotype, with some mutations enabling residual protein function and allowing normal migration in a larger proportion of neurons. In patient LP98-060a1, inheritance of a germline mutation of DCX from his mother in association with frontal SBH rather than generalized lissencephaly strongly supports this conclusion. Exon 4 of DCX contains an Abl substrate domain, which is a possible site for tyrosine phosphorylation and potentially important for protein function (9,10,18). The mutations found in patients LP98-046 and LP98-060a1/a2 were outside this domain, however, as were the missense mutations reported in two males with generalized LIS (5).
The LIS1 protein (PAFAH1B1) contains seven WD40 repeats. Proteins with WD40 repeat units, which start and end with two highly conserved elements (19,20), show a very specific folding structure (21). Exon 6 of LIS1 largely codes for the second WD40 repeat; the missense mutation seen in the patient with generalized LIS causes an amino acid change at the start of this repeat (15), whereas the mutation we found in patient LP94-051 with SBH is located in the center of the same repeat. Therefore, they are likely to have different effects on the protein, for example affecting protein folding.
The fact that a mother and son (LP98-060a1/a2) have the same mild frontal band with a mutation of DCX is particularly intriguing, as previous experience would suggest that the son's cerebral malformation should be much more severe than that seen in his mother. Possible explanations for this include a 47,XXY karyotype in the son, but this was excluded. Alternatively, this may comprise a very mild mutation which would normally not result in a phenotype in a heterozygous female unless there was highly skewed X inactivation with a large majority of the active X chromosomes carrying the mutation. PCR-based X inactivation studies (22,23) undertaken in the mother (LP98-060a2) did not reveal any significant skewing (data not shown). However, these studies were done on lymphocytes and may not reflect X inactivation status in the brain. The presence of the mutation in both mother and son makes mosaicism in the boy unlikely. One could argue that the phenotype in the son is unusual for a germline mutation of DCX inherited from his mother and may not be the causative mutation in this family. However, we have not seen this base pair change in over 100 additional alleles examined, which makes it unlikely to be a polymorphism. In addition it leads to a non-conservative amino acid substitution from an Arg to a Gly and is therefore likely to be significant.
Somatic mosaicism of the mutations found in LP94-051 and LP98-046 could explain the less severe cerebral malformations. Those cells containing the mutant allele would form the heterotopic layer, consisting of the subcortical band of SBH or the deep cellular layer of pachygyria. To address the possibility of detecting mosaicism by direct DNA sequencing, we performed dilution studies. DNA from patients with ILS and heterozygous mutations of LIS1 was mixed with DNA of a normal individual. The results showed that detection of a point mutation, indicated by a double peak on the sequencing electropherogram, was possible at a 1:2 ratio of mutated DNA to normal DNA, which would be equivalent to 33% mosaicism. However, the abnormal peak became increasingly smaller than the normal peak with less mutated DNA in the sample (data not shown). In patient LP94-051, repeated sequencing of exon 6 of LIS1 did show a smaller abnormal peak on two occasions, but only on the forward and not on the reverse sequence. The sequencing electropherograms (Fig. 1) and PCR-RFLP results did not suggest mosaicism in patient LP98-046. The inheritance of the mutation from his mother excludes mosaicism in patient LP98-060a1. However, sequencing and PCR-RFLP are not quantitative methods and therefore we cannot rule out somatic mosaicism in patient LP94-051 or LP98-046. In addition, only one tissue (lymphocytes) was examined and mosaicism can vary between tissues.
We have shown that mutations in either DCX or LIS1 can cause SBH and mixed PCH-SBH in males. In the patients reported here these are likely to be germline mutations. This suggests that less severe mutations of either lissencephaly gene can result in the milder SBH phenotype. However, we expect that somatic mosaic mutations of both DCX and LIS1 will also prove to be an important mechanism in causing SBH. This has been supported for DCX in at least six patients or families studied by our collaborating laboratories (J.G. Gleeson, W.B. Dobyns, M.E. Ross and C.A. Walsh, in preparation). X inactivation status (random or skewed) may also contribute to phenotypic variability in females with SBH, but has so far not been demonstrated. These studies are currently in progress.
MATERIALS AND METHODS
Subject selection and clinical review
Our patient group included eight males with SBH and three males with a combination of frontal pachygyria and posterior SBH (PCH-SBH). The clinical data and MRI scans were reviewed by two of the authors (D.T.P and W.B.D). The gradient of SBH or PCH-SBH was determined by comparing the severity of the malformation over the anterior and posterior brain regions. Blood from probands and, selectively, their parents were obtained with informed consent. All protocols were approved by the appropriate Institutional Review Board Human Subjects Committee. Among the four patients in whom mutations were identified, two live in the USA (LP94-051 and LP98-046), while the affected mother and son live in Italy (LP98-060a1/a2).
Mutation analysis
DNA isolation.
DNA was extracted from lymphoblast/fibroblast cell lines or peripheral blood using a Puregene DNA isolation kit (Gentra System) according to the manufacturer's protocol.
Sequencing and PCR-RFLP.
Direct sequencing of the coding regions of LIS1 and DCX was performed and all mutations were confirmed by sequencing the opposite strand. To exclude polymorphisms as the basis for the amino acid changes found in three of the probands, unrelated controls from an unselected US population (see Results) were also examined by the PCR-RFLP approach (17).
The specific sequencing conditions for LIS1 and DCX and the primer sequences used have been published previously (5,9).
X inactivation studies.
DNA (500 ng) from patient LP98-060a2, a normal control and a patient with known highly skewed X inactivation was digested overnight with HpaII at 37°C. PCR was performed as previously described (22,23). The samples were run for 3 h on a 12% polyacrylamide gel, which allowed adequate band separation.
ACKNOWLEDGEMENTS
We would like to thank the families who participated in this study for their help and enthusiasm and A.J. Barkovich for referral of patient LP94-051. We are grateful to Stephanie Mewborn, Laura Dudlicek and Judy Fantes for technical support and to Patti L. Mills for clinical coordination. This work was supported by NIH grants R01-NS35515 (to M. Elizabeth Ross and W.B.D.) and P01-NS39404 (to W.B.D. and D.H.L.).
REFERENCES
+To whom correspondence should be addressed at: Department of Human Genetics, The University of Chicago, 5841 South Maryland Avenue, Room L041, MC 2050, Chicago, IL 60637, USA. Tel: +1 773 834 0555; Fax: +1 773 834 0556; Email: wbd{at}genetics.uchicago.edu
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