- Email: [email protected]
Gene analysis of L1 neural cell adhesion molecule in prenatal diagnosis of hydrocephalus
pain" of leprosy could be explained in this way, especially as loss of the axon-reflex flare occurs in this disease; finally, and the pathological pun cannot be avoided, burns are the original cause of "burning" pain. As with other cutaneous syndromes, recovery may have delayed our recognition of its causalgic nature. Replacement of neuropeptides may prove difficult, but local injection should be tried to test the hypothesis. Burns, and the pain of epidermal necrolysis, may allow therapeutic experiment because percutaneous absorption of neuropeptides would be enhanced. Because of the difficulty of neuropeptide delivery, inhibition of the consequences of neuropeptide depletion may prove more therapeutically profitable than replacement. Perversely, capsaicin itself should be tried. With repeated applications, capsaicin causalgia becomes less severe, perhaps because of a secondary change in concentration of, or sensitivity to, substance P. "nerve
SS receives
a
Leverhulme Emeritus
Fellowship.
References
1 Schott GD. Mechanisms of causalgia and related clinical conditions. Brain 1986; 109: 717-38. 2 Dotson M. Causalgia—reflex sympathetic dystrophy—sympathetically maintained pain: myth and reality. Muscle Nerve 1993; 16: 1049-55. 3 Walton J. Brain’s diseases of the nervous system. 10th ed. Oxford: Oxford University Press, 1993: 563. 4 Breneman DL, Cardone JS, Blumsack RF, Lather RM, Searle EA, Pillack WE. Topical capsaicin for treatment of hemodialysis-related itch. J Am Acad Dermatol 1992; 26: 91-94. 5 Simone DA, Bauman TK, LaMotte RH. Dose-dependent pain and mechanical hyperalgesia in humans after intradermal injection of capsaicin. Pain 1989; 38: 99-107. 6 Lynn B. Capsaicin: actions on nonreceptive C fibres and therapeutic potential. Pain 1990; 51: 61-69. 7 International Association for the Study of Pain (IASP), classification of chronic pain, subcommittee on taxonomy pain, 1986; 26 (suppl 3): S29-30. 8 Lewis T. The blood vessels of the human skin and their responses. London: Shaw & Shaw, 1927: 69-71. 9 Barbut D, Polak JM, Wall PD. Substance P in spinal cord decreases following peripheral nerve injury. Brain Res 1981; 205: 289-98. 10 Porter R, O’Connor M, eds. Substance P in the nervous system. London: Ciba Foundation (symposium 91), 1982.
Department of Dermatology, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4BW, UK (Prof S Shuster FRCP)
congenital hydrocephalus is a common malformation affecting up to 1 in 1000 births. X-linked hydrocephalus, the most common inherited form, occurs in 2-7% of cases. In addition to hydrocephalus, affected boys develop mental retardation and spastic paraparesis, Primary
often with an unusual fixed flexion and adduction of the thumbs.’1 Female carriers are unaffected. Prenatal diagnosis of hydrocephalus has been achieved only after 16 weeks’ gestation by fetal scanning. Although linkage of the disease locus to markers within Xq28 has been used for genetic counselling,2 linkage analysis can be used only in families showing a clear pattern of X-linked inheritance. The availability of a definitive method to detect the condition in early pregnancy would be desirable. We have shown that X-linked hydrocephalus is caused by diverse mutations in the gene that encodes the neural cell adhesion molecule Ll.3,4 This finding has provided the basis for accurate genetic counselling for families in which we have identified the pathological mutation. We report here the prenatal diagnosis of hydrocephalus by mutational analysis in two families with
apparently sporadic hydrocephalus. The first case was a 38-year-old woman with no hydrocephalic or mentally retarded relatives; she gave birth to a boy who has hydrocephalus, development delay, and adducted thumbs. These typical signs prompted the screening of his Ll gene by single-strand conformation polymorphism (SSCP).4 Direct sequencing (f-mole sequencing system, Promega) identified a novel mutation (g+ 1-7t) in the intron 1 donor splice site (figure, A). Mutation at this invariably conserved base has proved pathological in several disorders.5 Fetal sexing at 10 weeks’ gestation in a subsequent pregnancy with DNA from a chorionic villous sample (CVS) revealed that the fetus was male, and prenatal diagnosis was requested. As the mutation creates a novel recognition site for the enzyme Tsp 509 I, restriction enzyme digestion of PCR fragments containing the mutated region of the Ll gene was carried out for the mother and fetus (CVS). The patient carries the mutation but her fetus does not possess the Ll mutation identified in her affected son (figure, B). By detection of the mutation and testing of CVS DNA we able to confirm that the affected son is an isolated of X-linked hydrocephalus and that his mother has a 25% risk of having future affected children, and provide a definitive prenatal diagnosis at 10 weeks’ gestation. Since the test, this woman has given birth to a healthy boy. The second case was a 32-year-old woman who gave birth to a boy with hydrocephalus, ascertained at 37 weeks’ gestation, and severe developmental delay; at 25 months he neither speaks nor walks. His thumbs are adducted. No other family member has hydrocephalus or mental retardation and the nonare parents Ll mutation was done in this consanguineous. screening SSCP of individual exons. A onenovel analysis boy by basepair deletion was found in exon 22which results in a frameshift and produces a stop codon 49 aminoacids downstream. Translation of this new mRNA would create a truncated protein and abolish cell-surface expression of Ll. We have identified three other mutations with identical consequences for the Ll protein, which are also disease causing. Fetal sexing from CVS at 10 weeks’ in her second pregnancy revealed that her fetus was male. For prenatal diagnosis, maternal and CVS DNA were analysed for the characterised mutation by direct sequencing of exon 22. No mutation was found in the were case
Gene analysis of L1 neural cell adhesion molecule in prenatal diagnosis of
hydrocephalus
X-linked hydrocephalus is the most common form of inherited hydrocephalus, and is associated with severe neurological deficits and premature death. We have shown that mutations in the gene encoding L1 neural cell adhesion molecule result in X-linked hydrocephalus, which enables improved prenatal diagnosis and investigation of the role of this molecule in sporadic cases. Here we report two pedigrees with apparently sporadic hydrocephalus in which we demonstrated a disabling mutation in the L1 gene. This enabled us to provide definitive prenatal diagnosis at 10 weeks’ gestation.
161
Figure: Sequencing
and
linkage analysis
A=novel Ll mutation identified in son creates a novel Tsp 509 recognition site, primer Gl was used for sequencing B=restriction digest of purified PCR products (Magic PCR columns, Promega) amplified from exon 1-intron 1 of L1 gene; C=direct sequencing of products amplified from exon 22 of Ll gene, primer G37 was used for sequencing and D=linkage analysis with polymorphic marker DXS52. bp=basepair.
mother’s genomic DNA (figure, C), which indicates that a new mutation must have occurred in her germline. However, the possibility of gonadal mosaicism for the mutation represents a significant risk for future offspring. The fetus (CVS) was found not to carry the Ll mutation present in the affected son (figure, C). Genetic analysis with polymorphic marker DXS52 (Stl4),"located within one megabase of the Ll gene, showed that the mother was heterozygous at this locus, whereas the CVS possessed only a single allele (figure, D). Maternal contamination could then be safely excluded. This analysis was crucial because the result could be attributed unequivocally to fetal DNA. On June 20, 1994, the mother gave birth to a
been
suggested8 and thus prenatal misdiagnosis could by assuming Xq28 linkage. To date, ultrasound scanning during pregnancy was the only way to detect a fetus affected by X-linked hydrocephalus. This is rarely achieved before 16 weeks’ gestation and often results in late abortion. In both our isolated cases, Ll mutation screening enabled the clearcut diagnosis of X-linked hydrocephalus as well as a definitive prenatal diagnosis at 10 weeks’ gestation.
occur
We thank Dr Christine Garrett, Dr Joy Redman, and Dr Renata Laxova for referring the two families to us, and Professor Tim Cox for reading and commenting on the manuscript. This work was supported by the Medical Research Council.
healthy boy. Ll mutation screening in these two apparently sporadic male cases of hydrocephalus showed that they both have X-linked hydrocephalus characterised by a defect in this neural cell adhesion molecule. Similar isolated cases of Xlinked hydrocephalus may not be recognised as such because of the lack of antecedents and the often obscure clinical picture. The presence of hydrocephalus with mental retardation, spastic paraparesis, and adducted thumbs was vital for the preliminary diagnosis of X-linked hydrocephalus and should prove valuable in the assessment of future sporadic cases. Halliday et af reported that all their cases of X-linked hydrocephalus showed pyramidal tract lesions at the level of the medulla oblongata, which is therefore a reliable diagnostic feature for this disorder. The poor value of linkage analysis in families with isolated cases of X-linked hydrocephalus was reemphasised in our second case, in which the Stl4 genotype of the unaffected fetus was identical to that of the affected boy. Therefore, in this family, exclusion would not have been possible with polymorphic markers and the information provided by Stl4 analysis would have indicated an increased risk for the unborn fetus. Furthermore, genetic heterogeneity of this condition has 162
References
PJ, Brouwer OF, Dijkstra I, Wilmink J. X-linked hydrocephalus. Am J Med Genet 1987; 27: 921-28. Serville F, Benit P, Saugier P, et al. Prenatal exclusion of X-linked hydrocephalus-stenosis of the aqueduct of Sylvius sequence using closely linked DNA markers. Prenat Diagn 1993; 13: 435-39. Rosenthal A, Jouet M, Kenwrick S. Aberrant splicing of L1 CAM mRNA associated with X-linked hydrocephalus. Nature Genet 1992;
Willlems
1 2
3
2:
107-12. 4
Jouet M, Rosenthal A, Armstrong G, et al. X-linked spastic paraplegia (SPG1), MASA syndrome and X-linked hydrocephalus result from
mutations in the L1 gene. Nature Genet 1994; 7: 402-07. Cooper DN, Krawczak M. Single base-pair substitutions in human gene mRNA splice junctions and their phenotypic consequences. In: Cooper DN, Krawczak M , eds. Human gene mutation. Oxford: Bios Scientific Publishers, 1993: 239-60. 6 Richards B, Heilig R, Oberlé I, Sorjohann L, Horn GT. Rapid PCR analysis of the St14 (DXS52) VNTR. Nucleic Acids Res 1991; 19: 1944. 7 Halliday J, Chow CW, Wallace D, Danks DM. X-linked hydrocephalus: a survey of a 20 year period in Victoria, Australia. J Med Genet 1986; 23: 23-31. 8 Willems PJ, Vits L, Rayemaekers P, et al. Further localization of Xlinked hydrocephalus in the chromosomal region Xq28. Am J Hum Genet 1992; 51: 307-15.
5
Department of Medicine, Level 5, Addenbrooke’s Hospital, Cambridge, CB2 2QQ, UK (M Jouet MSc, S Kenwrick phD)
Correspondence to:
Dr Susan Kenwrick
SS receives
a
Leverhulme Emeritus
Fellowship.
References
1 Schott GD. Mechanisms of causalgia and related clinical conditions. Brain 1986; 109: 717-38. 2 Dotson M. Causalgia—reflex sympathetic dystrophy—sympathetically maintained pain: myth and reality. Muscle Nerve 1993; 16: 1049-55. 3 Walton J. Brain’s diseases of the nervous system. 10th ed. Oxford: Oxford University Press, 1993: 563. 4 Breneman DL, Cardone JS, Blumsack RF, Lather RM, Searle EA, Pillack WE. Topical capsaicin for treatment of hemodialysis-related itch. J Am Acad Dermatol 1992; 26: 91-94. 5 Simone DA, Bauman TK, LaMotte RH. Dose-dependent pain and mechanical hyperalgesia in humans after intradermal injection of capsaicin. Pain 1989; 38: 99-107. 6 Lynn B. Capsaicin: actions on nonreceptive C fibres and therapeutic potential. Pain 1990; 51: 61-69. 7 International Association for the Study of Pain (IASP), classification of chronic pain, subcommittee on taxonomy pain, 1986; 26 (suppl 3): S29-30. 8 Lewis T. The blood vessels of the human skin and their responses. London: Shaw & Shaw, 1927: 69-71. 9 Barbut D, Polak JM, Wall PD. Substance P in spinal cord decreases following peripheral nerve injury. Brain Res 1981; 205: 289-98. 10 Porter R, O’Connor M, eds. Substance P in the nervous system. London: Ciba Foundation (symposium 91), 1982.
Department of Dermatology, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4BW, UK (Prof S Shuster FRCP)
congenital hydrocephalus is a common malformation affecting up to 1 in 1000 births. X-linked hydrocephalus, the most common inherited form, occurs in 2-7% of cases. In addition to hydrocephalus, affected boys develop mental retardation and spastic paraparesis, Primary
often with an unusual fixed flexion and adduction of the thumbs.’1 Female carriers are unaffected. Prenatal diagnosis of hydrocephalus has been achieved only after 16 weeks’ gestation by fetal scanning. Although linkage of the disease locus to markers within Xq28 has been used for genetic counselling,2 linkage analysis can be used only in families showing a clear pattern of X-linked inheritance. The availability of a definitive method to detect the condition in early pregnancy would be desirable. We have shown that X-linked hydrocephalus is caused by diverse mutations in the gene that encodes the neural cell adhesion molecule Ll.3,4 This finding has provided the basis for accurate genetic counselling for families in which we have identified the pathological mutation. We report here the prenatal diagnosis of hydrocephalus by mutational analysis in two families with
apparently sporadic hydrocephalus. The first case was a 38-year-old woman with no hydrocephalic or mentally retarded relatives; she gave birth to a boy who has hydrocephalus, development delay, and adducted thumbs. These typical signs prompted the screening of his Ll gene by single-strand conformation polymorphism (SSCP).4 Direct sequencing (f-mole sequencing system, Promega) identified a novel mutation (g+ 1-7t) in the intron 1 donor splice site (figure, A). Mutation at this invariably conserved base has proved pathological in several disorders.5 Fetal sexing at 10 weeks’ gestation in a subsequent pregnancy with DNA from a chorionic villous sample (CVS) revealed that the fetus was male, and prenatal diagnosis was requested. As the mutation creates a novel recognition site for the enzyme Tsp 509 I, restriction enzyme digestion of PCR fragments containing the mutated region of the Ll gene was carried out for the mother and fetus (CVS). The patient carries the mutation but her fetus does not possess the Ll mutation identified in her affected son (figure, B). By detection of the mutation and testing of CVS DNA we able to confirm that the affected son is an isolated of X-linked hydrocephalus and that his mother has a 25% risk of having future affected children, and provide a definitive prenatal diagnosis at 10 weeks’ gestation. Since the test, this woman has given birth to a healthy boy. The second case was a 32-year-old woman who gave birth to a boy with hydrocephalus, ascertained at 37 weeks’ gestation, and severe developmental delay; at 25 months he neither speaks nor walks. His thumbs are adducted. No other family member has hydrocephalus or mental retardation and the nonare parents Ll mutation was done in this consanguineous. screening SSCP of individual exons. A onenovel analysis boy by basepair deletion was found in exon 22which results in a frameshift and produces a stop codon 49 aminoacids downstream. Translation of this new mRNA would create a truncated protein and abolish cell-surface expression of Ll. We have identified three other mutations with identical consequences for the Ll protein, which are also disease causing. Fetal sexing from CVS at 10 weeks’ in her second pregnancy revealed that her fetus was male. For prenatal diagnosis, maternal and CVS DNA were analysed for the characterised mutation by direct sequencing of exon 22. No mutation was found in the were case
Gene analysis of L1 neural cell adhesion molecule in prenatal diagnosis of
hydrocephalus
X-linked hydrocephalus is the most common form of inherited hydrocephalus, and is associated with severe neurological deficits and premature death. We have shown that mutations in the gene encoding L1 neural cell adhesion molecule result in X-linked hydrocephalus, which enables improved prenatal diagnosis and investigation of the role of this molecule in sporadic cases. Here we report two pedigrees with apparently sporadic hydrocephalus in which we demonstrated a disabling mutation in the L1 gene. This enabled us to provide definitive prenatal diagnosis at 10 weeks’ gestation.
161
Figure: Sequencing
and
linkage analysis
A=novel Ll mutation identified in son creates a novel Tsp 509 recognition site, primer Gl was used for sequencing B=restriction digest of purified PCR products (Magic PCR columns, Promega) amplified from exon 1-intron 1 of L1 gene; C=direct sequencing of products amplified from exon 22 of Ll gene, primer G37 was used for sequencing and D=linkage analysis with polymorphic marker DXS52. bp=basepair.
mother’s genomic DNA (figure, C), which indicates that a new mutation must have occurred in her germline. However, the possibility of gonadal mosaicism for the mutation represents a significant risk for future offspring. The fetus (CVS) was found not to carry the Ll mutation present in the affected son (figure, C). Genetic analysis with polymorphic marker DXS52 (Stl4),"located within one megabase of the Ll gene, showed that the mother was heterozygous at this locus, whereas the CVS possessed only a single allele (figure, D). Maternal contamination could then be safely excluded. This analysis was crucial because the result could be attributed unequivocally to fetal DNA. On June 20, 1994, the mother gave birth to a
been
suggested8 and thus prenatal misdiagnosis could by assuming Xq28 linkage. To date, ultrasound scanning during pregnancy was the only way to detect a fetus affected by X-linked hydrocephalus. This is rarely achieved before 16 weeks’ gestation and often results in late abortion. In both our isolated cases, Ll mutation screening enabled the clearcut diagnosis of X-linked hydrocephalus as well as a definitive prenatal diagnosis at 10 weeks’ gestation.
occur
We thank Dr Christine Garrett, Dr Joy Redman, and Dr Renata Laxova for referring the two families to us, and Professor Tim Cox for reading and commenting on the manuscript. This work was supported by the Medical Research Council.
healthy boy. Ll mutation screening in these two apparently sporadic male cases of hydrocephalus showed that they both have X-linked hydrocephalus characterised by a defect in this neural cell adhesion molecule. Similar isolated cases of Xlinked hydrocephalus may not be recognised as such because of the lack of antecedents and the often obscure clinical picture. The presence of hydrocephalus with mental retardation, spastic paraparesis, and adducted thumbs was vital for the preliminary diagnosis of X-linked hydrocephalus and should prove valuable in the assessment of future sporadic cases. Halliday et af reported that all their cases of X-linked hydrocephalus showed pyramidal tract lesions at the level of the medulla oblongata, which is therefore a reliable diagnostic feature for this disorder. The poor value of linkage analysis in families with isolated cases of X-linked hydrocephalus was reemphasised in our second case, in which the Stl4 genotype of the unaffected fetus was identical to that of the affected boy. Therefore, in this family, exclusion would not have been possible with polymorphic markers and the information provided by Stl4 analysis would have indicated an increased risk for the unborn fetus. Furthermore, genetic heterogeneity of this condition has 162
References
PJ, Brouwer OF, Dijkstra I, Wilmink J. X-linked hydrocephalus. Am J Med Genet 1987; 27: 921-28. Serville F, Benit P, Saugier P, et al. Prenatal exclusion of X-linked hydrocephalus-stenosis of the aqueduct of Sylvius sequence using closely linked DNA markers. Prenat Diagn 1993; 13: 435-39. Rosenthal A, Jouet M, Kenwrick S. Aberrant splicing of L1 CAM mRNA associated with X-linked hydrocephalus. Nature Genet 1992;
Willlems
1 2
3
2:
107-12. 4
Jouet M, Rosenthal A, Armstrong G, et al. X-linked spastic paraplegia (SPG1), MASA syndrome and X-linked hydrocephalus result from
mutations in the L1 gene. Nature Genet 1994; 7: 402-07. Cooper DN, Krawczak M. Single base-pair substitutions in human gene mRNA splice junctions and their phenotypic consequences. In: Cooper DN, Krawczak M , eds. Human gene mutation. Oxford: Bios Scientific Publishers, 1993: 239-60. 6 Richards B, Heilig R, Oberlé I, Sorjohann L, Horn GT. Rapid PCR analysis of the St14 (DXS52) VNTR. Nucleic Acids Res 1991; 19: 1944. 7 Halliday J, Chow CW, Wallace D, Danks DM. X-linked hydrocephalus: a survey of a 20 year period in Victoria, Australia. J Med Genet 1986; 23: 23-31. 8 Willems PJ, Vits L, Rayemaekers P, et al. Further localization of Xlinked hydrocephalus in the chromosomal region Xq28. Am J Hum Genet 1992; 51: 307-15.
5
Department of Medicine, Level 5, Addenbrooke’s Hospital, Cambridge, CB2 2QQ, UK (M Jouet MSc, S Kenwrick phD)
Correspondence to:
Dr Susan Kenwrick