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What's new in Neurogenetics? Amish microcephaly
European Journal of Paediatric Neurology (2003) 7, 393–394
www.elsevier.com/locate/ejpn
WHAT’S NEW IN?
What’s new in Neurogenetics? Amish microcephaly Bruce R. Korf* Department of Genetics, University of Alabama at Birmingham, Kaul Human Genetics Building Rm 230, 720 20th Street South, Birmingham, AL 35294-0024, USA
Microcephaly is a relatively common finding in children with developmental impairment, and can result from a wide range of etiologies. The terms ‘primary microcephaly’ and ‘microcephaly vera’ are used to denote microcephaly and slow development in the absence of other physical or neurological problems. Though etiologically heterogeneous, a high proportion of instances are inherited in an autosomal recessive manner. This past year has seen the discovery of three genes that are associated with microcephaly: ASPM,1 involved in function of the mitotic spindle, microcephalin,2 of unknown function, and DNC, a deoxynucleotide carrier protein.3 The recent identification of mutation in DNC as the cause of Amish microcephaly tells an important story at three levels: the value of careful clinical observation in the elucidation of etiology, the power of genomic and bioinformatic tools in gene identification, and the interrelationship of metabolic and developmental disorders. A recessive form of microcephaly has been known in the Amish population for some time. In 2002, Kelley et al.4 described the phenotype in detail, and reported a metabolic abnormality in affected children. Individuals with ‘Amish microcephaly’ have congenital microcephaly, sometimes identified by prenatal ultrasound, indicating a reduction in the rate of head growth beginning around 20 weeks. The microcephaly tends to be profound, but usually no other congenital anomalies are seen. Affected children have *Tel.: þ1-205-934-9411; fax: þ 1-205-934-9488. E-mail address: [email protected]
severely impaired development, become irritable after around 2 – 3 months of age, and die, usually in the first year of life in the setting of a respiratory infection. Some have been noted to have hepatomegaly, particularly at times of stress such as infection, which prompted metabolic studies. Urine organic acid studies revealed consistent and substantial elevations of 2-ketoglutarate, an intermediate of mitochondrial Krebs cycle metabolism. The relatively high incidence of this recessive disorder among the Amish (1:480 births) suggests a founder effect. Kelley et al. have earned the trust of this group, and were able to trace the inheritance of the condition to a couple through seven generations. This permitted a mapping study to be done, which was reported in abstract form in 1999, showing linkage to chromosome 17q25.5 Identification of the gene required more detailed mapping and scrutiny of candidate genes within the region.3 A haplotype was identified that cosegregated with the disease gene and then a physical map was assembled, comprising 3.7 Mb of DNA from the region. Analysis of genomic data revealed over 50 potential genes. Two are involved in mitochondrial function, making them plausible candidates for this disorder. These genes are ATP5H, which encodes an ATP synthase, and DNC, which is involved in the transport of dinucleotides into the mitochondrion. Both genes were sequenced in DNA from affected individuals. No sequence variants were found in ATP5H, but all affected individuals were found to be homozygous for a G to C change in DNC that results in substitution of alanine for glycine at codon 177 in the protein. The change was not found in 252 control chromosomes,
1090-3798/$ - see front matter Q 2003 Published by Elsevier Ltd. on behalf of European Paediatric Neurology Society. doi:10.1016/j.ejpn.2003.09.005
394
suggesting that it is not a polymorphism. To determine whether the mutation has functional impact, the wildtype or mutant sequences were expressed in bacteria and then incorporated into phospolipid vesicles and tested for their ability to support dinucleotide transport. The mutant protein was found to be deficient in transport, whereas nucleotide exchange could be demonstrated in this model system using the wildtype protein. The authors propose that defective mitochondrial nucleotide transport leads to deficient mitochondrial DNA synthesis, and consequently a failure of energy metabolism. It remains unclear why accumulation of 2-ketoglutarate is the major biochemical signature of this problem, without accumulation of other intermediates of mitochondrial metabolism, and why deficient brain growth is the sole phenotype. The finding reinforces the notion that there is no clear line of demarcation between inborn errors of metabolism and developmental disorders. This was first shown clearly when a defect in cholesterol biosynthesis was found to underlie Smith-Lemli-Opitz syndrome,6 which was previously conceptualized as a congenital anomaly syndrome. As the cellular pathogenesis of developmental disorders comes to light, the intersection of biochemical and developmental pathways will become increasingly clear. The three genes so far discovered that underlie primary microcephaly act through three distinct mechanisms. It makes sense that cellular defects that lead to deficient cell replication would, within the nervous system, cause inadequate brain growth and microcephaly. It is interesting, though, that these particular genes appear to single out the nervous system, suggesting perhaps that they function more or less exclusively there, or perhaps that their functions are supplemented by other genes in other tissues. There are several additional microcephaly loci that have been mapped, including loci at 1q35 – q32,7 15q15 – q21,8 9q34,9 and 19q13.10 The availability of genomic resources makes it likely that the responsible genes at these loci will be identified before long, and the complexity of processes of neuronal proliferation and differentiation make it probable that many additional loci are yet to be discovered. Finally, the discovery of these genes offers the possibility of genetic testing for families, who, in the past, could only be counseled in general terms about
B.R. Korf
a recurrence risk as high as 25%. This will present a challenge, as more genes are found, each of which is likely to be associated with multiple distinct mutations in different populations. A sporadically identified child with primary microcephaly will present no clues as to which gene is responsible, making it necessary to develop a strategy for diagnostic testing, where multiple genes are analyzed for mutation. As we move from an era of empirical counseling to one of more precise risk assessment this issue will need to be addressed not only in microcephaly, or in neurogenetics, but throughout the practice of medical genetics.
References 1. Bond J, Roberts E, Mochida GH, Hampshire DJ, Scott S, Askham JM, et al. ASPM is a major determinant of cerebral cortical size. Nat Genet 2002;32(2):316—20. 2. Jackson AP, Eastwood H, Bell SM, Adu J, Toomes C, Carr IM, et al. Identification of microcephalin, a protein implicated in determining the size of the human brain. Am J Hum Genet 2002;71(1):136—42. 3. Rosenberg MJ, Agarwala R, Bouffard G, Davis J, Fiermonte G, Hilliard MS, et al. Mutant deoxynucleotide carrier is associated with congenital microcephaly. Nat Genet 2002; 32(1):175—9. 4. Kelley RI, Robinson D, Puffenberger EG, Strauss KA, Morton DH. Amish lethal microcephaly: a new metabolic disorder with severe congenital microcephaly and 2-ketoglutaric aciduria. Am J Med Genet 2002;112(4):318—26. 5. Rosenberg MJ, Agarwala R, Hilliard MS, Weber JL, Morton DH, Schaeffer AA, et al. Genetic mapping of a locus for Amish microcephaly. Am J Hum Genet 1999;65:A443. 6. Irons M, Elias ER, Tint GS, Salen G, Frieden R, Buie TM, et al. Abnormal cholesterol metabolism in the Smith-Lemli-Opitz syndrome: report of clinical and biochemical findings in four patients and treatment in one patient. Am J Med Genet 1994;50:347—52. 7. Jamieson CR, Fryns JP, Jacobs J, Matthijs G, Abramowicz MJ. Primary autosomal recessive microcephaly: MCPH5 maps to 1q25—q32. Am J Hum Genet 2000;67(6):1575—7. 8. Jamieson CR, Govaerts C, Abramowicz MJ. Primary autosomal recessive microcephaly: homozygosity mapping of MCPH4 to chromosome 15. Am J Hum Genet 1999;65(5): 1465—9. 9. Moynihan L, Jackson AP, Roberts E, Karbani G, Lewis I, Corry P, et al. A third novel locus for primary autosomal recessive microcephaly maps to chromosome 9q34. Am J Hum Genet 2000;66(2):724—7. 10. Roberts E, Jackson AP, Carradice AC, Deeble VJ, Mannan J, Rashid Y, et al. The second locus for autosomal recessive primary microcephaly (MCPH2) maps to chromosome 19q13.1—13.2. Eur J Hum Genet 1999;7(7):815—20.
www.elsevier.com/locate/ejpn
WHAT’S NEW IN?
What’s new in Neurogenetics? Amish microcephaly Bruce R. Korf* Department of Genetics, University of Alabama at Birmingham, Kaul Human Genetics Building Rm 230, 720 20th Street South, Birmingham, AL 35294-0024, USA
Microcephaly is a relatively common finding in children with developmental impairment, and can result from a wide range of etiologies. The terms ‘primary microcephaly’ and ‘microcephaly vera’ are used to denote microcephaly and slow development in the absence of other physical or neurological problems. Though etiologically heterogeneous, a high proportion of instances are inherited in an autosomal recessive manner. This past year has seen the discovery of three genes that are associated with microcephaly: ASPM,1 involved in function of the mitotic spindle, microcephalin,2 of unknown function, and DNC, a deoxynucleotide carrier protein.3 The recent identification of mutation in DNC as the cause of Amish microcephaly tells an important story at three levels: the value of careful clinical observation in the elucidation of etiology, the power of genomic and bioinformatic tools in gene identification, and the interrelationship of metabolic and developmental disorders. A recessive form of microcephaly has been known in the Amish population for some time. In 2002, Kelley et al.4 described the phenotype in detail, and reported a metabolic abnormality in affected children. Individuals with ‘Amish microcephaly’ have congenital microcephaly, sometimes identified by prenatal ultrasound, indicating a reduction in the rate of head growth beginning around 20 weeks. The microcephaly tends to be profound, but usually no other congenital anomalies are seen. Affected children have *Tel.: þ1-205-934-9411; fax: þ 1-205-934-9488. E-mail address: [email protected]
severely impaired development, become irritable after around 2 – 3 months of age, and die, usually in the first year of life in the setting of a respiratory infection. Some have been noted to have hepatomegaly, particularly at times of stress such as infection, which prompted metabolic studies. Urine organic acid studies revealed consistent and substantial elevations of 2-ketoglutarate, an intermediate of mitochondrial Krebs cycle metabolism. The relatively high incidence of this recessive disorder among the Amish (1:480 births) suggests a founder effect. Kelley et al. have earned the trust of this group, and were able to trace the inheritance of the condition to a couple through seven generations. This permitted a mapping study to be done, which was reported in abstract form in 1999, showing linkage to chromosome 17q25.5 Identification of the gene required more detailed mapping and scrutiny of candidate genes within the region.3 A haplotype was identified that cosegregated with the disease gene and then a physical map was assembled, comprising 3.7 Mb of DNA from the region. Analysis of genomic data revealed over 50 potential genes. Two are involved in mitochondrial function, making them plausible candidates for this disorder. These genes are ATP5H, which encodes an ATP synthase, and DNC, which is involved in the transport of dinucleotides into the mitochondrion. Both genes were sequenced in DNA from affected individuals. No sequence variants were found in ATP5H, but all affected individuals were found to be homozygous for a G to C change in DNC that results in substitution of alanine for glycine at codon 177 in the protein. The change was not found in 252 control chromosomes,
1090-3798/$ - see front matter Q 2003 Published by Elsevier Ltd. on behalf of European Paediatric Neurology Society. doi:10.1016/j.ejpn.2003.09.005
394
suggesting that it is not a polymorphism. To determine whether the mutation has functional impact, the wildtype or mutant sequences were expressed in bacteria and then incorporated into phospolipid vesicles and tested for their ability to support dinucleotide transport. The mutant protein was found to be deficient in transport, whereas nucleotide exchange could be demonstrated in this model system using the wildtype protein. The authors propose that defective mitochondrial nucleotide transport leads to deficient mitochondrial DNA synthesis, and consequently a failure of energy metabolism. It remains unclear why accumulation of 2-ketoglutarate is the major biochemical signature of this problem, without accumulation of other intermediates of mitochondrial metabolism, and why deficient brain growth is the sole phenotype. The finding reinforces the notion that there is no clear line of demarcation between inborn errors of metabolism and developmental disorders. This was first shown clearly when a defect in cholesterol biosynthesis was found to underlie Smith-Lemli-Opitz syndrome,6 which was previously conceptualized as a congenital anomaly syndrome. As the cellular pathogenesis of developmental disorders comes to light, the intersection of biochemical and developmental pathways will become increasingly clear. The three genes so far discovered that underlie primary microcephaly act through three distinct mechanisms. It makes sense that cellular defects that lead to deficient cell replication would, within the nervous system, cause inadequate brain growth and microcephaly. It is interesting, though, that these particular genes appear to single out the nervous system, suggesting perhaps that they function more or less exclusively there, or perhaps that their functions are supplemented by other genes in other tissues. There are several additional microcephaly loci that have been mapped, including loci at 1q35 – q32,7 15q15 – q21,8 9q34,9 and 19q13.10 The availability of genomic resources makes it likely that the responsible genes at these loci will be identified before long, and the complexity of processes of neuronal proliferation and differentiation make it probable that many additional loci are yet to be discovered. Finally, the discovery of these genes offers the possibility of genetic testing for families, who, in the past, could only be counseled in general terms about
B.R. Korf
a recurrence risk as high as 25%. This will present a challenge, as more genes are found, each of which is likely to be associated with multiple distinct mutations in different populations. A sporadically identified child with primary microcephaly will present no clues as to which gene is responsible, making it necessary to develop a strategy for diagnostic testing, where multiple genes are analyzed for mutation. As we move from an era of empirical counseling to one of more precise risk assessment this issue will need to be addressed not only in microcephaly, or in neurogenetics, but throughout the practice of medical genetics.
References 1. Bond J, Roberts E, Mochida GH, Hampshire DJ, Scott S, Askham JM, et al. ASPM is a major determinant of cerebral cortical size. Nat Genet 2002;32(2):316—20. 2. Jackson AP, Eastwood H, Bell SM, Adu J, Toomes C, Carr IM, et al. Identification of microcephalin, a protein implicated in determining the size of the human brain. Am J Hum Genet 2002;71(1):136—42. 3. Rosenberg MJ, Agarwala R, Bouffard G, Davis J, Fiermonte G, Hilliard MS, et al. Mutant deoxynucleotide carrier is associated with congenital microcephaly. Nat Genet 2002; 32(1):175—9. 4. Kelley RI, Robinson D, Puffenberger EG, Strauss KA, Morton DH. Amish lethal microcephaly: a new metabolic disorder with severe congenital microcephaly and 2-ketoglutaric aciduria. Am J Med Genet 2002;112(4):318—26. 5. Rosenberg MJ, Agarwala R, Hilliard MS, Weber JL, Morton DH, Schaeffer AA, et al. Genetic mapping of a locus for Amish microcephaly. Am J Hum Genet 1999;65:A443. 6. Irons M, Elias ER, Tint GS, Salen G, Frieden R, Buie TM, et al. Abnormal cholesterol metabolism in the Smith-Lemli-Opitz syndrome: report of clinical and biochemical findings in four patients and treatment in one patient. Am J Med Genet 1994;50:347—52. 7. Jamieson CR, Fryns JP, Jacobs J, Matthijs G, Abramowicz MJ. Primary autosomal recessive microcephaly: MCPH5 maps to 1q25—q32. Am J Hum Genet 2000;67(6):1575—7. 8. Jamieson CR, Govaerts C, Abramowicz MJ. Primary autosomal recessive microcephaly: homozygosity mapping of MCPH4 to chromosome 15. Am J Hum Genet 1999;65(5): 1465—9. 9. Moynihan L, Jackson AP, Roberts E, Karbani G, Lewis I, Corry P, et al. A third novel locus for primary autosomal recessive microcephaly maps to chromosome 9q34. Am J Hum Genet 2000;66(2):724—7. 10. Roberts E, Jackson AP, Carradice AC, Deeble VJ, Mannan J, Rashid Y, et al. The second locus for autosomal recessive primary microcephaly (MCPH2) maps to chromosome 19q13.1—13.2. Eur J Hum Genet 1999;7(7):815—20.