Human Molecular Genetics

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Human Molecular Genetics

Pages 2479-2488

©1999 Oxford University Press

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The mutational spectrum of the Sonic Hedgehog gene in holoprosencephaly: SHH mutations cause a significant proportion of autosomal dominant holoprosencephaly
Introduction
Results
   Missense mutations
   Deletions
   Nonsense mutations
   Insertion
   Predicted polymorphisms
Discussion
Materials And Methods
   Patient samples
   PCR methods, single strand conformational polymorphism (SSCP) analysis and sequencing
   Subcloning to confirm deletion sizes
   Paternity testing
Acknowledgements
References

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The mutational spectrum of the Sonic Hedgehog gene in holoprosencephaly: SHH mutations cause a significant proportion of autosomal dominant holoprosencephaly

Luisa Nanni¹, Jeffrey E. Ming¹, Maureen Bocian³, Kathryn Steinhaus³, Diana W. Bianchi⁴, Christine de Die-Smulders⁵, Aldo Giannotti⁶, Kiyoshi Imaizumi⁷, Kenneth L. Jones⁸, Miguel Del Campo⁸, Rick A. Martin⁹, Peter Meinecke¹⁰, Mary Ella M. Pierpont¹¹, Nathaniel H. Robin¹², Ian D. Young¹³, Erich Roessler², Maximilian Muenke^{1, 2, +}

¹Departments of Pediatrics and Genetics, The Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, 34th and Civic Center Boulevard, Philadelphia, PA 19104-4399, USA, ²Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892-1852, USA, ³University of California Irvine, Orange, CA, USA, ⁴Tufts University School of Medicine, Boston, MA, USA, ⁵University Hospital Maastricht, The Netherlands, ⁶Bambino Gesu' Hospital, Rome, Italy, ⁷Kanagawa Children's Medical Center, Yokohama City, Japan, ⁸University of California, San Diego, CA, USA, ⁹St Christopher's Hospital for Children, Philadelphia, PA, USA, ¹⁰Altonaer Kinder-Krankenhaus, Hamburg, Germany, ¹¹University of Minnesota Hospital, Minneapolis, MN, USA, ¹²Case Western Reserve University, Cleveland, OH, USA and ¹³Nottingham City Hospital, Nottingham, UK

Received July 26, 1999; Revised and Accepted September 28, 1999

Holoprosencephaly (HPE) is a common developmental anomaly of the human forebrain and midface where the cerebral hemispheres fail to separate into distinct left and right halves. We have previously reported haploinsufficiency for Sonic Hedgehog (SHH) as a cause for HPE. We have now performed mutational analysis of the complete coding region and intron-exon junctions of the SHH gene in 344 unrelated affected individuals. Herein, we describe 13 additional unrelated affected individuals with SHH mutations, including nonsense and missense mutations, deletions and an insertion. These mutations occur throughout the extent of the gene. No specific genotype-phenotype association is evident based on the correlation of the type or position of the mutations. In conjunction with our previous studies, we have identified a total of 23 mutations in 344 unrelated cases of HPE. They account for 14 cases of familial HPE and nine cases of sporadic HPE. Mutations in SHH were detected in 10 of 27 (37%) families showing autosomal dominant transmission of the HPE spectrum, based on structural anomalies. Interestingly, three of the patients with an SHH mutation also had abnormalities in another gene that is expressed during forebrain development. We suggest that the interactions of multiple gene products and/or environmental elements may determine the final phenotypic outcome for a given individual and that variations among these factors may cause the wide variability in the clinical features seen in HPE.

INTRODUCTION

Holoprosencephaly (HPE) is a clinically variable and genetically heterogeneous malformation in which the developing forebrain fails to correctly separate into right and left hemispheres. Clinical expression of HPE is highly variable even within a single family (1). In its most severe form (alobar HPE), there is no interhemispheric fissure, a single brain ventricle is present and there may be cyclopia and/or a proboscis-like nasal structure. Less severe facial dysmorphisms (considered HPE microforms) include microcephaly, ocular hypotelorism, impaired sense of smell or single central upper incisor (2,3). The majority of HPE cases are apparently sporadic, although clear examples of autosomal dominant (AD) inheritance have been described. Interestingly, up to 30% of obligate carriers of an HPE gene in AD pedigrees are clinically unaffected (4).

Based on recurrent cytogenetic abnormalities, at least 12 chromosomal regions are proposed to contain genes involved in HPE (5). The gene at the locus designated HPE3 (6) was identified as Sonic Hedgehog (SHH) and we proposed that haploinsufficiency for SHH causes HPE (7,8). Recently, mutations in two other genes have been identified in association with HPE: (i) ZIC2, which maps to an HPE critical region on 13q32 (9,10); and (ii) SIX3, which maps to the HPE2 locus on 2p21 (11).

Shh plays a critical role in early forebrain and central nervous system development (12,13). Mice homozygous for a disrupted Shh gene show defects in the development of midline neural structures, lack ventral cells in the brain and display craniofacial anomalies, including cyclopia and a proboscis-like nasal structure (14). This phenotype is consistent with the defects seen in human HPE.

The Shh protein is a secreted intercellular signaling molecule which is synthesized as a precursor that undergoes autocatalytic cleavage into a highly conserved N-terminal domain (Shh-N) and a more divergent C-terminal domain (Shh-C) (15). During the autoprocessing reaction, a cholesterol moiety is covalently attached to the C-terminus of Shh-N (16-18). Shh-N contains all the known signaling activities (16,19-21). In contrast, Shh-C mediates both the enzymatic cleavage and cholesterol modification of the protein (17,18,22). This modification is crucial for proper patterning activity.

We had previously performed mutational analysis of the first and second exons of SHH in 30 familial cases, as well as the third exon in 41 familial and 184 sporadic cases of HPE (8,23). Ten different mutations consistent with a loss of function of the mutated allele were found, accounting for nine cases of familial HPE and one case of sporadic HPE. Here we report mutational analysis of the entire coding region and exon-intron boundaries of SHH in 344 unrelated HPE patients. We report 13 additional affected individuals with SHH mutations and provide further evidence that both SHH-N and SHH-C are equally affected by mutations.

RESULTS

We screened 344 HPE cases (78 familial and 266 clinical sporadic) for the entire coding region and exon-intron boundaries of SHH using SSCP. This study includes the same 41 families and 184 sporadic cases that had portions of the SHH gene previously analyzed and confirms the original findings. We detected 13 previously unreported probands with a mutation in SHH (Table 1). Of these mutations, the sequence change was present in more than one family member in eight of the nine kindreds depicted in Figure 1. Interestingly, one of these mutations (the 21 bp deletion) was also present in a family previously reported (23). Taking into account the 10 mutations described previously by our group (8,23) and four additional mutations reported by another group (24), a total of 27 probands have been noted to have SHH mutations (Table 1 and Fig. 2A and B).

Figure 1. Pedigrees for the nine families carrying SHH mutations. Solid symbols, HPE phenotype; half-solid symbols, MC, OH, CLP, DD or SCI; symbols enclosing a dot, clinically unaffected mutation carriers; *, tested individuals with normal SHH sequence. The following individuals were not available for the study: III.7 in pedigree A; II.2 and III.4 in pedigree C; II.1 in pedigree 0; II.2, II.6 and II.7 in pedigree I. For clinical details and abbreviations, see Table 2.

Figure 2. (A) Location of mutations in SHH in individuals with HPE. The mutations are distributed evenly between the coding regions. SHH-N, signaling domain; SHH-C, autocatalytic cleavage and cholesterol transferase domain; #, insertion; star, frameshift deletion; solid circle, missense mutations; solid diamond, nonsense mutations; *, deletions; SP, signal peptide, removed after translation; [darr], cleavage/cholesterol addition. (B) Amino acid sequence alignment for human Sonic (hSHH), mouse Sonic (mShh), chicken Sonic (cShh), zebrafish Sonic (zShh), and Drosophila hedgehog (dhh). The locations of the predicted amino acid changes in HPE patients are shown in blue (8,23,24; present study). Amino acids conserved between species are shown in red. The positions of the splice sites of exons 1, 2 and 3 are indicated by arrows and a gap is introduced in the protein sequence to mark the position of the autocatalytic cleavage between Gly197 and Cys198 of the human protein. The residues essential for hedgehog autoprocessing activity in Drosophila are in green (see text).

Table 1. Summary of sequence variations in the SHH gene

Patients	Sequence change	Expected effect	Reference
Exon 1
Pedigree I	160-161 insGCTG	3-4 ins	Present study
Pedigree E	189-196 del	13-15 del with frameshift	Present study
ADHPE 10	GGG->AGG	Gly31->Arg	8
Pedigree A	GAT->GTT	Asp88->Val	Present study
ADHPE 6	CAG->TAG	Gln100->Stop	8
Sporadic HPE	CAG->CAC	Gln100->His	24
Exon 2
ADHPE 14	AAG->TAG	Lys105->Stop	8
Pedigree B	AAC->AAA	Asn115->Lys	Present study
ADHPE 15	TGG->GGG	Trp117->Gly	8
ADHPE 2	TGG->CGG	Trp117->Arg	8
Familial HPE	TAC->TAG	Tyr158->Stop	24
Exon 3
Familial HPE	GAG->CAG	Glu188->Gln	24
Patient M	CAG->TAG	Gln209->Stop	Present study
Familial HPE	GAC->AAC	Asp222->Asn	24
ADHPE 11	GTG->GAG	Val224->Glu	23
ADHPE 43	GCG->ACG	Ala226->Thr	23
Pedigree C	AGC->AGA	Ser236->Arg	Present study
Pedigree H	GAG->TAG	Glu256->stop	Present study
Patient L	939-959 del	263-269 del	Present study
ADHPE 4	939-959 del	263-269 del	23
ADHPE 3	GAG->TAG	Glu284->Stop	23
Patient J	GGC->GAC	Gly290->Asp	Present study
Pedigree F	1283-1291 del	378-380 del	Present study
Sporadic HPE	GCG->ACG	Ala383->Thr	23
Pedigree G	1361-1375 del	404-408 del	Present study
Pedigree D	CCG->GCG	Pro424->Ala	Present study
Patient K	TCG->TTG	Ser436->Leu	Present study

Missense mutations

Six missense mutations were noted. The first was observed in two affected children and their clinically unaffected mother. Her three sisters also carried the mutation: one is clinically unaffected with a child who also showed the mutation and has microcephaly and moderate learning disabilities; the other two have microcephaly and a high arched palate (Figs 1A and 3J). The GAT->GTT (Asp88->Val) sequence change occurs in the N-terminal signaling domain at an invariant position in the hedgehog family of proteins (Fig. 2B). This mutation was confirmed by the loss of an MboI restriction site (data not shown). The second missense mutation was detected in a child with HPE and his clinically unaffected mother (Fig. 1B). The AAC->AAA (Asn115->Lys) change occurs at an invariant position in the hedgehog proteins, in the N-terminal domain (Fig. 2B). The third missense mutation was detected in a child with HPE as well as his mother and his maternal grandmother, both of whom have a single central maxillary incisor, his great grandfather, who has ocular hypotelorism, and in a great uncle and his daughter, both clinically unaffected (Figs 1C and 3E and F). The AGC->AGA (Ser236->Arg) sequence change also predicts a change at an invariant position in the hedgehog proteins, in the C-terminal domain (Fig. 2B).

A - F

G - L

Figure 3. Holoprosencephaly (HPE) in individuals with Sonic Hedgehog (SHH) mutations. (A) Microcephaly, absence of nasal bones, midline cleft lip and palate and semilobar HPE in patient II.1 of pedigree F with SHH and TGIF mutations. (B) Semilobar HPE, premaxillary agenesis and midline cleft lip in patient II.1 in pedigree D with an SHH mutation and a TGIF deletion. (C) Patient J with severe semilobar HPE on brain scan (D) and relatively mild facial findings with SHH and ZIC2 mutations. (E) Patient IV.1 of pedigree C with semilobar RPE on MRI (F), microcephaly, prominent globes, premaxillary agenesis and cleft lip. (G) Microcephaly, ocular hypotelorism, flat nose with no palpable cartilage, midface and philtrum hypoplasia and normal intelligence in individual II.3 of pedigree I with an SHH mutation. (H) Normal brain magnetic resonance imaging (MRI) of the same individual. (I) One of the twins (II.6/II.7) of family I with alobar HPE and severe facial findings (synophthalmia and proboscis above the eye). (J) Microcephaly, ocular hypotelorism, bilateral inferior iris colobomata, repaired right-sided cleft lip and palate and a normal brain MRI in patient III.4 of pedigree A. (K) Microcephaly; philtrum hypoplasia and a normal brain MRI in individual II.1 of pedigree E. (L) Patient III.2 of family E with lobar HPE, microcephaly and philtrum hypoplasia.

A GGC->GAC (Gly290->Asp) sequence change was observed in a 20-year-old woman with HPE (Fig. 3C and D and Table 2, patient J) and is predicted to occur in the processing domain. This mutation was confirmed by loss of a BanII restriction site (data not shown). Her parents were not available for study. Of note, a mutation in the ZIC2 gene, predicting an alanine repeat expansion, was also found in this patient (9). The fifth change was seen in an affected child and her clinically unaffected mother (Figs 1D and 3B). The CCG->GCG (Pro424->Ala) sequence change occurs 39 amino acids from the end of the protein. In addition, this patient's karyotype showed loss of the terminal portion of 18p, derived from a maternal balanced cryptic translocation [t(1;18) (q43;p11.3)]. The del(18p) chromosome is deleted for TG-interacting factor (TGIF), a candidate gene for the HPE4 locus at 18p (25). The last missense mutation was detected in a patient with semilobar HPE (Table 2, patient K). This TCG->TTG (Ser436->Leu) change predicts an amino acid substitution 27 amino acids from the end of the protein. The parents were unavailable for analysis.

Table 2. Phenotype evaluation of HPE patients with an SHH mutation

Patients	Individual	Clinical findings	Additional information
Familial
Pedigree A	I.2	Clinically normal	Normal SHH
	II.1	Clinically normal	Normal SHH
	II.2	Clinically normal	SHH mutation carrier
	II.3	Clinically normal	SHH mutation carrier
	II.4	MC, high arched palate
	II.5	LD, MC, high arched palate	Normal brain (CT)
	III.4	MC, OH, bilateral inferior iris colobomata, repaired right side CL, fused lower central incisors	Normal brain (MRI); DD; bilateral hearing loss; 46,XY
	III.5	HPE, OH, PA, single nostril, bilateral CL, absent olfactory nerves, fused thalami	Alobar HPE; fetus 46,XY
	III.6	Mild MC, moderate LD
	III.7	Mild MC, moderate LD	Not available for study
Pedigree B	I.1	Clinically normal	Normal SHH
	I.2	Clinically normal	SHH mutation carrier
	II.2	HPE, MC, flat nose, bilateral CLP	Lobar HPE; 46,XY
Pedigree C	I.1	OH
	1.2	Clinically normal	Normal SHH
	11.1	SCI, mild OH
	11.2	Died at 2 years old, `open skull', CL	Not available for study
	11.4	Clinically normal	SHH mutation carrier
	111.2	SCI, mild OH
	111.4	DD	Not available for study
	111.5	Clinically normal	Normal SHH
	111.6	Clinically normal	SHH mutation carrier
	IV.1	HPE, MC, sloped forehead, OH, prominent globes, bilateral optic nerve colobomata, PA, CL	Semilobar HPE; severe micropenis; 46,XY
Pedigree D	I.2	Clinically normal	SHH mutation carrier; cryptic 46,XX, t(1;18)(q43;p11.3)
	II.1	HPE, PA, midline CL	Semilobar HPE; DI; l8p-
Pedigree E	I.1	LD	Not available for study
	I.2	Clinically normal	Normal SHH
	II.1	MC, SCI, anosmia, absent frenulum	Normal brain (MRI)
	III.1	Mild DD, CHD	Normal SHH; normal brain (MRI)
	III.2	Severe DD, MC, philtrum hypoplasia	Lobar HPE
	III.3	HPE, proboscis
Pedigree F	I.2	Clinically normal	SHH mutation carrier
	II.l	HPE, MC, OH, absent nasal bones, midline CLP	Semilobar HPE; TGIF mutation; 46,XX
Pedigree G	I.1	Clinically normal	Normal SHH
	I.2	Clinically normal	SHH mutation carrier
	II.1	HPE, MC, moderate hydrocephalus, OH, absent proboscis and nares, CLP	Semilobar HPE; fetus 46,XX; not available for study
Pedigree H	I.1	Clinically normal	Normal SHH
	I.2	Clinically normal	Normal SHH
	II.1	MC, SCI, choanal stenosis
	II.2	HPE, MC, PA, single nasal cavity, bilateral CL and absent anterior palate	Lobar HPE; fetus 46,XX
Pedigree I	I.1	Clinically normal	Normal SHH
	I.2	Mild OH	Normal SHH
	II.2	HPE, MC, OH, single nostril, CP	Alobar HPE; ambiguous genitalia; 46,XY; not available for study
	II.3	MC, OH, flat nose, no palpable nasal cartilage, midfacial and philtrum hypoplasia	Normal IQ; SHH mutation
	II.6/II.7	HPE, OH, cyclopia, proboscis	Alobar HPE; 46,XY; not available for study
	II.10	HPE, cyclopia with double orbitis, proboscis	Alobar HPE; SHH mutation
Sporadic
Patient J		HPE, MC	Semilobar HPE; DI; progressive spastic paraplegia; SS; ZIC2 mutation; 46,XX; 20 years old; parents not available
Patient K		HPE, MC, single nostril, midfacial hypoplasia, CL	Semilobar HPE; optic nerve hypoplasia; parents not available
Patient L		HPE, corpus callosum agenesis, pachygyria, ventricular dilatation	MCA; 46,XX; parents normal SHH
Patient M		HPE, hydrocephalus, occipital encephalocele, CLP	Semilobar HPE; 46,XX; parents normal SHH

CHD, congenital heart defect; CL, cleft lip; CLP, cleft lip and palate; DD, developmental delay; DI, diabetes insipidus; LD, learning disabilities; MC, microcephaly; MCA, multiple congenital anomalies; OH, ocular hypotelorism; PA, premaxillary agenesis; SCI, upper single central incisor; SS, short stature.

Deletions

Four different deletions were observed. An 8 bp deletion leading to a frameshift (bases 189-196) was detected in a fetus with HPE, as well as in a sibling with microcephaly and severe developmental delay and their mother, who has a single central incisor and anosmia (Figs 1E and 3K and L).

A 21 bp deletion in the C-terminal domain at codons 263-269 was detected in a sporadic patient (Table 2, patient L). This predicts a deletion of seven amino acids (RLLLTAA). This deletion immediately precedes a critical His residue (His270, which corresponds to His329 in the Drosophila hedgehog protein) required in the autoprocessing cleavage reaction (26). The identical deletion was previously found to segregate with the affected members of an AD HPE family (23).

A 9 bp deletion was detected in an affected child and her clinically normal mother (Figs 1F and 3A). It occurs at codons 378-380 and predicts the loss of three amino acids (APF). Of note, this patient also showed a missense mutation in TGIF, a candidate gene for the HPE4 locus at 18p (25).

A 15 bp deletion (codons 404-408) was detected in a clinically unaffected mother of an HPE fetus (Fig. 1G). The fetus was not available for study. This deletion is predicted to result in omission of the amino acids GDRGG in a minimally conserved region of SHH-C.

Nonsense mutations

Two nonsense mutations were detected. The first mutation, a de novo CAG->TAG change, was detected in a sporadic HPE patient (Table 2, patient M) and predicts premature termination of the protein at position 209, 12 amino acids after the cleavage site. Such a protein would be predicted to fail to undergo the autocatalytic cleavage reaction and would not be expected to be modified by cholesterol.

The second nonsense mutation was found in a female fetus with HPE and her brother with a single central incisor and microcephaly (Fig. 1H). The GAG->TAG change was confirmed by the gain of an XbaI restriction site (data not shown) and predicts termination of the protein at position 256. Neither parent carried the mutation. Paternity testing showed that the father was in fact the biological father of both of the two siblings. This pedigree is consistent with germline mosaicism in one of the parents.

Insertion

A 4 bp insertion (GCTG) after the third codon of the gene was found in a large autosomal dominant HPE family (Figs 1I and 3G-I). Two affected members in the kindred were available for the study and both showed the 4 bp insertion. The predicted resulting peptide would terminate at codon 62. Neither parent was a mutation carrier and haplotyping confirmed that they were indeed the biological parents. This pedigree is consistent with germline mosaicism in one of the parents.

Predicted polymorphisms

Five nucleotide changes that would not be predicted to cause a change in the SHH protein were detected: two putative polymorphisms in exon 3 (Ala275->Ala and Val335->Val); three in intronic sequences (49 bases upstream of exon 2, 71 bases downstream of exon 2 and 92 bases downstream of exon 2).

DISCUSSION

In order to refine our estimate of the frequency of mutations in SHH that cause HPE and to attempt to establish a genotype-phenotype correlation, we performed an extensive mutational analysis of the entire coding region and exon-intron boundaries of the SHH gene. We identified 13 additional patients with SHH gene mutations: five were in familial HPE cases and eight were in sporadic cases. `Familial' versus `sporadic' cases were categorized based on clinical and not molecular findings. We had previously identified SHH mutations in 10 HPE probands. Combining all our data, we estimate that mutations in SHH account for 14/78 (18%) of the clinical familial HPE cases and for 9/266 (3.4%) of the sporadic cases. Therefore, SHH mutations are present in 23/344 (6.7%) of our total cohort of HPE patients. Using broad criteria for inclusion as an AD pedigree, including features that are present to a significant degree in the general population (e.g. microcephaly, hypotelorism, mental retardation), the frequency of SHH mutations in AD pedigrees was 24% (11/46). When we limited criteria for inclusion in an AD pedigree to structural anomalies (e.g. anosmia, corpus callosum agenesis in association with HPE, cleft lip and palate, single central incisor), the frequency of SHH mutations was 10 out of 27 (37%). The finding of SHH mutations in only a minority of the total cohort of HPE patients underscores the significant etiological heterogeneity of this condition. These figures should be considered estimates. Seven of the 23 changes would predict an obvious loss of function due to a nonsense mutation or frameshift. However, the functional effect of many of the missense mutations is unknown. Thus, the calculations of SHH mutation frequency could change, depending on the results of functional studies.

Interestingly, as illustrated by four of our pedigrees (Fig. 1B, D, F and G), mutational analysis of the clinically unaffected parents showed evidence for clinically unaffected mutation carriers. This emphasizes that clinically normal parents of a child with HPE have some risk of carrying an SHH gene mutation, making counselling for recurrence risk more challenging. Furthermore, we previously reported a family with a nonsense mutation in SHH in which a clinically normal family member also carried the mutation (8). It has been previously estimated that 30% of obligate carriers of HPE are clinically normal (4). In addition, in two kindreds (pedigrees H and I) two siblings had an SHH mutation, but neither parent carried the mutation. Paternity testing showed that the father was in fact the biological father of affected sibs in both cases. This is strongly suggestive of germline mosaicism and would be the first such cases for the SHH gene. However, we should note that the effect of these mutations on SHH function are unknown. Although these alterations were not present in >200 normal control chromosomes, the changes could represent rare polymorphisms which do not affect SHH activity. Functional studies are in progress to determine whether the activity of the abnormal protein is decreased.

No specific genotype-phenotype correlation according to type or location of the mutations in SHH is observed. In addition, there were no clinical features unique to individuals with an SHH mutation compared with those without a detected mutation. The phenotype of carriers of an SHH mutation within a single family can vary from alobar HPE to clinically normal individuals (1). As another example, a 21 bp deletion was detected in a sporadic case (Table 2, patient L) who had pachygyria, which is not typically associated with HPE, and additional congenital anomalies including a two-vessel umbilical cord, ventricular septal defect of the heart, malrotation of the large bowel and bicornuate uterus. The same deletion was also detected in an AD HPE family (23). Affected members in this family showed only features frequently associated with HPE, such as microcephaly, single central incisor and cleft lip/palate. Thus, the same deletion can give rise to somewhat different phenotypes.

Based on known domains in the hedgehog family of proteins, we can speculate about the effects of the mutations we observe in our HPE patients. The association of chromosomal deletions including the SHH gene with HPE is consistent with a loss of SHH function (27). Mutations could affect any of the three principal functional regions of Shh: (i) changes in the SHH-N signaling region; (ii) interference with the autocatalytic cleavage reaction in SHH-C, since an intact precursor molecule has no patterning activity (21); and (iii) interference with the cholesterol transferase activity. We encounter likely examples of all three mechanisms in our sample of patients.

Combining the present data with previously published information (8,23,24), eight mutations predict premature termination of the protein: five cause truncation of SHH-N and three truncate within SHH-C (Fig. 2) and may lead to abnormal processing (26).

A total of seven missense mutations are observed in the signaling domain. In addition, certain of the residues were abnormal in two unrelated individuals. Both a nonsense and a missense (Q100H) mutation have been reported for Gln100 (8,24). Two different missense mutations (Trp117->Gly and Trp117->Arg) have been reported for nucleotide 500 (8). Trp117 is a residue located just distal to the first [alpha]-helix motif in the murine protein (28) that is invariant between species studied and mutations at this position may destabilize the SHH-N fragment.

The 263-269 deletion was found in two probands and occurs just before a key His residue (His270) that is essential for hedgehog autoprocessing activity in Drosophila (26). Furthermore, this deletion encompasses a Thr residue (Thr267) that is required for thioester formation in the Drosophila hedgehog protein, which is the first step in the autoprocessing reaction. The 263-269 deletion has also been shown to inhibit autoprocessing of the protein in vitro (E. Roessler et al., unpublished data).

Given the great intrafamilial clinical variability in kindreds carrying an SHH mutation, we speculate that other genes acting in the same or different developmental pathways might act as modifiers for expression of the HPE spectrum. We identified three cases in which individuals with an SHH mutation also had a base change in a second gene which acts in brain development.

The Gly290->Asp change in SHH was present in a patient (Table 2, patient J) who also had a mutation predicting an expansion of an alanine repeat in exon 2 of ZIC2. This alanine repeat extension has been described in other HPE patients (9,10). Her parents were not available for analysis. Since functional effects of these mutations are not known, we do not know whether the effects of one or the other mutation is causing HPE. Although there is no known interaction between the ZIC2 and SHH pathways, it is possible that their biological functions converge on a common pathway. Therefore, reduced amounts or activity of one protein might negatively affect the expression or function of the other. The relative roles of ZIC2 and SHH in the developing brain and face may be deduced from the frequency of associated craniofacial malformations in individuals with a mutation in one of the genes. As with most cases of HPE, affected individuals with SHH mutations often have facial anomalies. However, craniofacial anomalies appear to be less severe and less frequent in individuals with ZIC2 mutations (10).

Second, a Pro424->Ala change in SHH was noted in both a child who was deleted for 18pter and the putative HPE4 gene, TGIF (25), and her mother, who carried a balanced translocation involving chromosome 18 (Fig. 1D). The third example is a 9 bp deletion in SHH (378-380del) in a child with HPE (Fig. 1F) who also showed a missense mutation (Thr151Ala) in TGIF (25).

TGIF maps to the minimal critical region of HPE4 on chromosome 18p. This gene is expressed during early brain development in mice (29) and four missense mutations have been identified in patients with HPE (25). TGIF codes for a transcription factor which competitively inhibits binding of the retinoic acid receptor (RXR) to a retinoid-responsive promoter. Therefore, decreased TGIF levels may theoretically lead to enhanced binding of RXR, mimicking increased retinoic acid (RA) levels. Interestingly, prenatal RA exposure is associated with HPE-like malformations in animal models (30). In addition, high doses of RA down-regulate Shh expression in craniofacial primordia in the chicken (31,32). Thus, decreased TGIF could result in decreased SHH expression, which could accentuate the effects of an allele of SHH with reduced or no activity. Of note, only 10% of patients carrying a TGIF deletion show HPE. It is possible that either maternal RA levels or altered activity in another protein could modify the effect of TGIF.

In addition to potential modifier genes, environmental factors could also modulate the clinical expression of HPE (33). The importance of cholesterol in normal SHH function (18) raises the possibility that cholesterol levels in utero may contribute to the variable phenotype of HPE caused by SHH mutations. Maternal hypocholesterolemia in early gestation causes an HPE-like phenotype in rodent embryos (reviewed in ref. 5). Moreover, HPE is associated with severe cases of Smith-Lemli-Opitz syndrome, which is due to a defect in 7-dehydrocholesterol reductase, the final step in the cholesterol biosynthetic pathway (34,35).

These speculated interactions between genes might help to explain the clinical variability seen in HPE. Functional studies of the mutations detected in SHH, TGIF and ZIC2 will be essential in determining the significance of this hypothesis. As additional genes related to HPE are identified and the inter-action between their products is investigated, a more complete understanding of the numerous genetic and environmental factors which contribute to normal brain development and HPE will be elucidated.

MATERIALS AND METHODS

Patient samples

All of the patients were evaluated by a clinical geneticist prior to enrollment in this study and extensive pedigrees were taken. All had normal chromosomes [except for del(18p) in II.1 pedigree D]. The genomic DNA from HPE patients and family members (when available) was extracted from lymphocytes or established lymphoblastoid cell lines by routine methods. All samples were obtained by informed consent according to the guidelines of our institutional review board.

PCR methods, single strand conformational polymorphism (SSCP) analysis and sequencing

Human SHH is composed of three exons. We used six pairs of primers (exon 2 and exon 3 were split into two and three overlapping amplicons, respectively) that encompassed the intron-exon boundaries and coding regions of the entire SHH gene. Five primer pairs are published elsewhere (8,23). Amplification of amplicons 3a and 3b was performed using the primer pairs 3F1a/11 (3a) and 3F2/3R1 (3b). Because of the high GC content of the 3[prime]-end of exon 3 (3c), we had been unable to satisfactorily complete the mutation analysis of this exon (23). For the remainder of the 3[prime]-end of the gene we modified our original strategy (36) as follows: 3F3 (5[prime]-GCCCGGGCCAGCGCGTGTACGTGG-3[prime]) and 3R3 (5[prime]-CCCCTCCCCCGGCCCCCCGGCTTC-3[prime]) were used to amplify 3c under PCR conditions of 94°C for 5 min, 94°C for 45 s, 60°C for 30 s, 72°C for 45 s for 35 cycles, followed by 72°C for 6 min. This reaction yielded a 483 bp product that on digestion with BssHII generated two fragments of 249 and 234 bp. Amplification of 3c was performed in a 15 µl reaction volume, using 60-100 ng DNA template, 200 µM each dATP, dGTP and dTTP, 125 µM dCTP, 3.5 µCi [[alpha]-³²P]dCTP (800 Ci/mmol, 10 mCi/ml), 30 pmol each primer, 1.5 µl 10× PCR buffer (Gibco, Gaithersburg, MD), 1.25 µl 10× PCR Enhancer (Gibco), 1.5 mM MgSO₄ (Gibco) and 1 U AmpliTaq polymerase (Perkin Elmer, Foster City, CA). All of the PCR reactions were performed in a PTC-100 thermal cycler (MJ Research, Waltham, MA).

SSCP analysis was performed as described elsewhere (37). Sequencing of the amplicons demonstrating SSCP band shifts was performed by the Protein and DNA Core Facility of the Children's Hospital of Philadelphia on an ABI Prism 377 analyzer. None of the 13 detected SSCP alterations was found in >200 control chromosomes from unrelated normal Caucasian individuals or in >200 chromosomes from unrelated normal Hispanic individuals.

Subcloning to confirm deletion sizes

For the four deletions detected, we subcloned the PCR products from the affected individuals using a TA Cloning kit (Invitrogen, Carlsbad, CA). Plasmid DNA containing the mutated allele was extracted (spin miniprep; Qiagen, Valencia, CA) and sequenced.

Paternity testing

In two kindreds (Fig. 1H and I) paternity testing was performed to confirm the identity of the biological father, using 10 polymorphic microsatellite markers from 10 different chromosomes. After amplification, the samples were run on a denaturing (8.3 M urea) polyacrylamide gel and analyzed by autoradiography.

ACKNOWLEDGEMENTS

We are grateful to all of the families for their participation in this study. This work was supported in part by the Catholic University of Rome, Italy (L.N.), by NIH grant HD01218 (J.E.M.), by NIH grants HD28732 and HD29862 and by the Division of Intramural Research, NHGRI, NIH (M.M.).

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+To whom correspondence should be addressed at: Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, 10 Center Drive, MSC 1852, Building 10, 10C101, Bethesda, MD 20892-1852, USA. Tel: +1 301 402 8167; Fax: +1 301 496 7157; Email: mmuenke{at}nhgri.nih.gov

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