The sonic hedgehog (SHH) gene is a human homologue of the drosophilagene encoding inductive signals involved in patterning the earlyembryo and which is highly functionally conserved in many species.1The gene was mapped to chromosome 7 (7q36), the locus for thegene involved in holoprosencephaly (HPE3).2SHH mutations maycause the failure of cerebral hemispheres to separate into distinctleft and right halves, leading to holoprosencephaly, which isone of the most common developmental anomalies of the forebrainand midface in humans.3 Although the majority of cases of holoprosencephalyare sporadic, familial cases are not rare, with a clear patternof autosomal dominant inheritance.
There is great clinical variability of holoprosencephaly withinfamilies, ranging from alobar holoprosencephaly and cyclopiato cleft lip and palate, microcephaly, ocular hypotelorism,and even to a normal phenotype. This variability suggests aninteraction between SHH and other genes expressed during craniofacialdevelopment and the possible involvement of environmental factors.Because almost one third of carriers of SHH mutations may beclinically unaffected, even in affected families, the prenataldetection of SHH mutations might not justify termination ofthe pregnancy. Preimplantation genetic diagnosis is thus a moreattractive option for couples at risk for having a child withholoprosencephaly.
We describe the use of preimplantation genetic diagnosis forSHH mutation in a family with holoprosencephaly. The use ofthis technique, followed by confirmation of mutation-free statusby amniocentesis, resulted in the birth of a healthy girl.
Case Report
A couple who had had two children with clinical signs of holoprosencephalypresented for preimplantation genetic diagnosis (Figure 1).Their second child, a girl with severe holoprosencephaly andcleft lip and palate, died shortly after birth. The resultsof chromosomal analysis of peripheral-blood lymphocytes fromthis child and the parents were normal. However, DNA analysisof samples obtained at autopsy showed that the girl had an SHHnonsense mutation — a change from GAG to TAG — leadingto the premature termination of the protein at position 256of exon 3 (Glu256stop)3 (Figure 2A). SHH protein is an intercellularsignaling molecule. This precursor is cleaved internally intoa highly conserved domain (SHH-N) with signaling activity anda more divergent domain (SHH-C), which in addition to precursorprocessing, acts as an intramolecular cholesterol transferasecrucial for proper patterning activity in animal development.1Although the effect on SHH function of the nonsense mutationidentified in the child is unknown, the resulting protein maynot have the expected signaling function in early morphogenesis.1,3
The father (Subject I-1) has a gonadal mosaicism for the sonic hedgehog (SHH) mutation (M), which is linked to the 156-bp dinucleotide CA repeat allele of the D7S550 polymorphic marker, whereas the mother (Subject I-2) is normal (N), with one normal allele linked to a 158-bp repeat (158) and the other to a 138-bp repeat (138). Three previous pregnancies resulted in the birth of a son with holoprosencephaly (Subject II-1), who carries the mutant gene; a daughter who died soon after birth (Subject II-2), who also carried the mutant gene; and a spontaneously aborted fetus with Turner's syndrome (Subject II-3), without the SHH mutation. A second daughter (Subject II-4) was born after preimplantation genetic diagnosis (PGD), as described in this article. Squares indicate male subjects, circles female subjects, the triangle a spontaneous abortion, solid symbols affected subjects (the spontaneously aborted fetus was unaffected by holoprosencephaly but had Turner's syndrome), open symbols unaffected subjects, and the slash a deceased subject. The square with the dot indicates that the father was an unaffected carrier of the mutation.
Figure 2. Preimplantation Diagnosis for Nonsense Mutation Glu256stop in Exon 3 of the Sonic Hedgehog (SHH) Gene.
Panel A shows the location of the mutation in SHH and the D7S550 linked marker on chromosome 7. The arrows demonstrate the positions of heminested primers. Panel B shows the restriction map for XbaI digestion. The mutant allele has an additional XbaI restriction site. Panel C shows the polyacrylamide-gel electrophoregram of XbaI-digested polymerase-chain-reaction (PCR) products of nine blastomeres from the second cycle of preimplantation genetic diagnosis, paternal DNA from sperm, maternal (normal) DNA, DNA from the couple's affected son, and undigested PCR product. The ladder is the 100-bp standard; ADO denotes allele dropout. Panel D shows the results of follow-up analysis of genomic DNA from five embryos predicted to have the SHH mutation on the basis of blastomere testing. Panels E, F, G, and H show capillary electrophoregrams of fluorescently labeled PCR products of tightly linked marker D7S550. Paternally derived 156-bp dinucleotide indicative of the SHH mutation ("Paternal") is evident in blastomeres of Embryos 2, 9, 10, and 17. A maternally derived 158-bp CA repeat ("Maternal") is also evident in blastomeres of Embryos 2, 9, and 10, and the other maternally derived 138-bp repeat is evident in the blastomere of Embryo 17 (Panel H).
The same mutation was found in the couple's five-year-old son,who had been born after a full-term, normal pregnancy, weighing2.7 kg (6 lb), with a length of 45.7 cm (18.3 in.). This childhad less severe facial dysmorphism than his sister, includingmicrocephaly, Rathke's pouch cyst, a single central incisor,and choanal stenosis (the latter was dilated surgically afterbirth). He also had clinodactyly of the fifth fingers and incurvedfourth toes bilaterally. His growth was slow during the firsttwo years but subsequently has been reasonably good, and hissocial and cognitive development are apparently normal.
The woman had had another pregnancy, which ended in spontaneousabortion owing to Turner's syndrome (45,X). There was no evidenceof inheritance of the SHH mutation. The observation that themutation was not found in either parent's genomic DNA, althoughpaternity testing showed that the father was the biologic fatherof both affected children, clearly suggested a new gonadal mutationin one of the parents.
Methods
Two cycles of preimplantation genetic diagnosis were performedwith use of a standard in vitro fertilization protocol coupledwith micromanipulation procedures, as described elsewhere.4Single blastomeres were removed from the eight-cell embryosand tested by multiplex nested polymerase-chain-reaction (PCR)analysis, involving simultaneous testing for a specific mutationand linked marker analysis.5 Of 15 embryos in the first cycle,12 were available for blastomere biopsy at the eight-cell stage.Blastomeres from four embryos failed to amplify, leaving eightwith data available for mutation analysis. Seven of these eightembryos appeared to contain the mutant allele; only one embryowas free of the mutation and was transferred back to the patient,yielding no clinical pregnancy.
The second cycle of preimplantation genetic diagnosis was performeda year later. Nineteen embryos were available, 10 of which wereacceptable for blastomere biopsy and DNA analysis. Of these10 single blastomeres, only 1 failed to amplify. The remainingnine had data available for SHH gene and linked marker analysis,to identify the mutation-free embryos appropriate for transfer(Figure 2C).
Before preimplantation genetic diagnosis cycles, single-spermtesting was performed that identified mosaicism for the SHHmutation in the father. The mutation led to the addition ofan XbaI restriction site3; the normal allele was thus identifiedon the basis of the undigested PCR product, and the mutant allelewas represented by two fragments, as a result of XbaI digestion(Figure 2B).
To avoid misdiagnosis in mutation analysis owing to preferentialamplification of certain alleles (also termed "allele dropout"),the rate of which exceeds 10 percent in single-blastomere DNAanalysis,6 a closely linked microsatellite DNA marker D7S550was used as an internal control. Table 1 lists the primers usedin the first- and second-round PCR for mutation and linked markeranalysis and the reaction conditions. A haplotype analysis showedthat the mutant allele was linked to a 156-bp dinucleotide CArepeat and that the normal gene was linked to a 152-bp repeatallele in 7q36 (Figure 1). Although other linked markers havealso been described,2 they were not informative.
Table 1. Primers for the Detection of the Glu256stop Mutation in the Sonic Hedgehog Gene (SHH) and Linked Marker D7S550 in Heminested Polymerase-Chain-Reaction Analysis.
The protocol was approved by the institutional review board,and the parents provided written informed consent. On the basisof both mutation and linked-marker analyses, unaffected embryoswere identified and selected to be transferred back to the woman,whereas those predicted to have the SHH mutation underwent confirmatoryanalysis with the use of whole-embryo DNA to evaluate the accuracyof preimplantation genetic diagnosis based on analysis of asingle cell.
Results
Four allele dropouts were identified in analysis of the mutantallele in Embryos 2, 9, and 17 and in the normal allele in Embryo10 (Figure 2C and Figure 2D and Table 2). This result was basedon the marker analysis, showing that all four of these embryoswere heterozygous for the mutation (Figure 2E, Figure 2F, Figure 2G,and Figure 2H). In other words, three of these four embryos(Embryos 2, 9, and 17) could have been misidentified as normalin the absence of linked marker analysis. In addition to thesethree embryos, Embryos 10 and 19 also contained the mutant gene.
Table 2. Results of Multiplex Polymerase-Chain-Reaction (PCR) Analysis, Involving Simultaneous Testing for the Sonic Hedgehog Gene (SHH) Mutation and Linked Marker Analysis and Confirmatory Embryo Analysis.
The remaining four embryos were free of the mutant gene, asconfirmed by marker analysis showing that all contained twonormal alleles, the paternal one linked to the 152-bp repeatand either the normal maternal allele linked to the 138-bp repeat(Embryos 4 and 5) or the normal maternal allele linked to the158-bp repeat (Embryos 8 and 16). Two of these embryos (Embryos4 and 5) were transferred back to the woman, resulting in asingleton pregnancy, followed by confirmation of the mutation-freestatus of the fetus by amniocentesis and the birth of a healthygirl. The other two mutation-free embryos (Embryos 8 and 16)were frozen for subsequent use by the couple.
Discussion
Our data demonstrate that multiplex PCR-based blastomere analysiscan accurately identify SHH mutations, despite the well-knownhigh rate of allele dropout in this type of single-cell analysis.6Allele-specific amplification failure is particularly commonin single-blastomere analysis, as compared with analysis ofsingle fibroblasts and polar bodies. The follow-up analysisof the mutant embryos confirmed the results of preimplantationgenetic diagnosis, consistent with previous data.5 Although,ideally, three linked markers are needed to eliminate the riskof misdiagnosis resulting from allele dropout,5 we found thatthe use of only one linked marker was reliable, probably becausethe identification of mutation-free embryos was based on thepresence not only of the paternally derived normal allele, butalso of the maternally derived normal allele. In this case,the absence of the mutant gene together with the presence ofthe two normal alleles, identified by different linked markers,led to the correct identification of embryos as either normalor carrying the SHH mutation.
Our findings demonstrate the clinical relevance of the use ofpreimplantation genetic diagnosis for familial holoprosencephaly.Because of the high prevalence of congenital craniofacial anomalies,preimplantation genetic diagnosis may have practical implicationsfor couples at risk. Initiated first for severe mendelian disorders,7,8,9,10,11,12preimplantation genetic diagnosis was then used to exclude aneuploidy13,14,15,16and has recently been used for diseases for which there is agenetic predisposition17,18 and for congenital malformations.19These data suggest that preimplantation genetic diagnosis maybe an option for a large group of couples at risk for havingchildren with congenital malformations.
Source Information
From the Reproductive Genetics Institute, Chicago.
Address reprint requests to Dr. Kuliev at 2825 N. Halsted St., Chicago, IL 60657, or at anverkuliev{at}hotmail.com.
References
Online Mendelian inheritance in man, OMIM. Baltimore: McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University, 2001. (Accessed February 19, 2003, at http://www.ncbi.nlm.nih.gov/Omim.)
Muenke M, Gurrieri F, Bay C, et al. Linkage of a human brain malformation, familial holoprosencephaly, to chromosome 7 and evidence for genetic heterogeneity. Proc Natl Acad Sci U S A 1994;91:8102-8106. [Free Full Text]
Nanni L, Ming JE, Bocian M, et al. The mutational spectrum of the sonic hedgehog gene in holoprosencephaly: SHH mutations cause a significant proportion of autosomal dominant holoprosencephaly. Hum Mol Genet 1999;8:2479-2488. [Free Full Text]
Verlinsky Y, Kuliev A. Atlas of preimplantation genetic diagnosis. Pearl River, N.Y.: Parthenon, 2000.
Rechitsky S, Verlinsky O, Strom C, et al. Experience with single-cell PCR in preimplantation genetic diagnosis: how to avoid pitfalls. In: Hahn S, Holzgreve W, eds. Fetal cells in maternal blood: new developments for a new millennium. Basel, Switzerland: Karger, 2000:8-15.
Rechitsky S, Strom C, Verlinsky O, et al. Allele drop out in polar bodies and blastomeres. J Assist Reprod Genet 1998;15:253-257. [CrossRef][Web of Science][Medline]
Handyside AH, Kontogianni EH, Hardy K, Winston RML. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature 1990;344:768-770. [CrossRef][Medline]
Verlinsky Y, Ginsberg N, Lifchez A, Valle J, Moise J, Strom CM. Analysis of the first polar body: preconception genetic diagnosis. Hum Reprod 1990;5:826-829. [Free Full Text]
Handyside AH, Lesko JG, Tarín JJ, Winston RML, Hughes MR. Birth of a normal girl after in vitro fertilization and preimplantation diagnostic testing for cystic fibrosis. N Engl J Med 1992;327:905-909. [Abstract]
Sermon K, Lissens W, Joris H, et al. Clinical application of preimplantation diagnosis for myotonic dystrophy. Prenat Diagn 1997;17:925-932. [CrossRef][Web of Science][Medline]
Kuliev A, Rechitsky S, Verlinsky O, et al. Preimplantation diagnosis of thalassemia. J Assist Reprod Genet 1998;15:219-225. [CrossRef][Web of Science][Medline]
Xu K, Shi ZM, Veeck LL, Hughes MR, Rosenwaks Z. First unaffected pregnancy using preimplantation genetic diagnosis for sickle cell anemia. JAMA 1999;281:1701-1706. [Free Full Text]
Munne S, Magli C, Cohen J, et al. Positive outcome after preimplantation diagnosis of aneuploidy in human embryos. Hum Reprod 1999;14:2191-2199. [Free Full Text]
Gianaroli L, Magli MC, Ferraretti AP, Munne S. Preimplantation diagnosis for aneuploidies in patients undergoing in vitro fertilization with poor prognosis: identification of the categories for which it should be proposed. Fertil Steril 1999;72:837-844. [CrossRef][Web of Science][Medline]
Wilton L, Williamson R, McBain J, Edgar D, Voullaire L. Birth of a healthy infant after preimplantation confirmation of euploidy by comparative genomic hybridization. N Engl J Med 2001;345:1537-1541. [Free Full Text]
Kuliev A, Verlinsky Y. Current features of preimplantation genetic diagnosis. Reprod Biomed Online 2002;5:294-9.
Verlinsky Y, Rechitsky S, Verlinsky O, Masciangelo C, Lederer K, Kuliev A. Preimplantation diagnosis for early-onset Alzheimer disease caused by V717L mutation. JAMA 2002;287:1018-1021. [Free Full Text]
Rechitsky S, Verlinsky O, Chistokhina A, et al. Preimplantation genetic diagnosis for cancer predisposition. Reprod Biomed Online 2002;5:148-55.
Abou-Sleiman PM, Apessos A, Harper JC, Serhal P, Delhanty JDA. Pregnancy following preimplantation genetic diagnosis for Crouzon syndrome. Mol Hum Reprod 2002;8:304-309. [Free Full Text]
Traiffort, E., Dubourg, C., Faure, H., Rognan, D., Odent, S., Durou, M.-R., David, V., Ruat, M.
(2004). Functional Characterization of Sonic Hedgehog Mutations Associated with Holoprosencephaly. J. Biol. Chem.
279: 42889-42897
[Abstract][Full Text]
Verlinsky, Y., Rechitsky, S., Sharapova, T., Morris, R., Taranissi, M., Kuliev, A.
(2004). Preimplantation HLA Testing. JAMA
291: 2079-2085
[Abstract][Full Text]
Previous
